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Plasminogen in periodontitis and wound repair Rima Šulniūtė Department of Medical Biochemistry and Biophysics Umeå 2013

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Plasminogen in periodontitis and wound repair

Rima Šulniūtė

Department of Medical Biochemistry and Biophysics Umeå 2013

Responsible publisher under Swedish law: the Dean of the Medical FacultyThis work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7459-547-5 ISSN: 0346-6612 Ev. info om Omslag/sättning/omslagsbild: Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Servicecenter KBC Umeå, Sweden 2013

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Table of contents Table of contents 3 

Abstract 5 

List of papers 7 

Abbreviations 8 

Introduction 9 

1. The Plasminogen Activator System 10 

1.1. Plasminogen/plasmin 10 

1.2. Plasminogen activators 12 

1.2.1. Tissue-type plasminogen activator 12 

1.2.2. Urokinase-type plasminogen activator 12 

1.3. Inhibitors of the plasminogen activator system 13 

1.3.1. Plasmin inhibitors: α2-antiplasmin and α2-macroglobulin 13 

1.3.2. Plasminogen activator inhibitors 14 

1.4. Receptors of the plasminogen activator system 14 

1.4.1. Plasminogen receptors 15 

1.4.2. The urokinase receptor 17 

1.5. Plasminogen in cell signaling 17 

1.6. The plasminogen activator system in tissue remodeling 18 

1.6.1. Fibrinolysis 18 

1.6.2. Bone remodeling 19 

1.6.3. Other tissue remodeling processes requiring the plasminogen activator system 19 

1.7. The plasminogen activator system in infection 20 

1.8. The plasminogen activator system in inflammation 21 

2. Periodontitis 22 

2.1. The periodontium in health and disease 22 

2.1.1. Gingiva 23 

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2.1.2. Periodontal ligament and root cementum 24 

2.1.3. Alveolar bone in health and disease 25 

2.2. Pathogenesis of periodontitis 26 

2.3. The plasminogen activator system in periodontitis 27 

2.4. Animal models in periodontal research 28 

3. Wound Healing 30 

3.1. Phases of wound repair 30 

3.1.1. The inflammatory phase 30 

3.1.2. The proliferative phase 31 

3.1.3. The remodeling phase 32 

3.2. The plasminogen activator system in wound healing 32 

3.3. Cutaneous wound models in mice 33 

Results and Discussion 35 

Plasmin is essential in preventing periodontitis in mice (Paper I) 35 

Plasminogen is a critical regulator of cutaneous wound healing (Paper II) 36 

Plasminogen is a key pro-inflammatory regulator that accelerates the healing of acute and diabetic

wounds (Paper III) 37 

Concluding Remarks 39 

Acknowledgements 40 

References 42 

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Abstract

The plasminogen activator (PA) system plays a critical role in many physiological and

pathological processes, such as fibrinolysis, extracellular matrix (ECM) degradation, wound healing,

inflammation, and cancer. The key component of the PA system is plasmin, a broad-spectrum serine

protease that is derived from its inactive form, plasminogen. The first aim of this thesis research was to

determine the role of plasminogen in periodontitis, an inflammatory oral disease. The second aim was to

explore the molecular mechanism by which plasminogen contributes to wound healing in the skin. Finally,

the third aim was to investigate the possibility of using plasminogen as a treatment for skin wounds,

especially for chronic wounds, such as diabetic wounds.

Periodontitis is an oral disease that involves a bacterial infection, the inflammation of the

periodontium, and the degradation of gum tissue and alveolar bone. This disease is irreversible and, in

severe cases, can lead to loss of teeth due to the degradation of the periodontal ligament and alveolar

bone. To study the effects of the PA system on oral health, we monitored the development of periodontitis

in plasminogen-deficient mice and plasminogen activator-deficient mice. In control wild-type mice,

periodontitis did not occur. However, in plasminogen-deficient mice, periodontitis developed rapidly

within 20 weeks after birth. The morphological studies of plasminogen-deficient mice showed the

detachment of gingival tissues, resorption of the cementum layer, formation of necrotic tissue, and severe

alveolar bone degradation. Immunohistochemical staining showed the massive infiltration of neutrophils

into the periodontal tissues. Interestingly, doubly deficient mice lacking both tissue-type plasminogen

activator (tPA) and urokinase-type PA (uPA) developed periodontitis at a similar rate as the plasminogen-

deficient mice, but mice lacking only tPA or uPA remained healthy. The intravenous injection of human

plasminogen for 10 days into plasminogen-deficient mice led to the absorption of necrotic tissue, the

diminution of inflammation, and the full regeneration of gum tissues. Notably, there was also partial re-

growth of degraded alveolar bone.

The wound healing process consists of three overlapping phases: inflammatory,

proliferative, and remodeling. It has been postulated that the PA system plays an integral role in this

process, and a lack of plasminogen leads to delayed wound healing in mice. To study the role of the PA

system in wound healing, we monitored the responses of wild-type, plasminogen-deficient and diabetic

mice to incision and burn wounds. We found that in addition to being delayed, the wound healing process

in plasminogen-deficient mice was only superficial in nature. The plasminogen-deficient mice were unable

to clear the provisional matrix after the formation of granulation tissue, and an extensive fibrin

deposition. In addition, persistent inflammation was still present subcutaneously in these mice 60 days

after introduction of the wound.

The essential role of plasminogen in burn and incision wounds healing was further

confirmed by reconstitution experiments. Both intravenous and subcutaneous administrations of human

plasminogen to plasminogen-deficient mice led to a restored healing rate and wound maturation that was

comparable to those of wild-type mice. We also demonstrated that plasminogen supplementation of

plasminogen to wild-type and diabetic mice significantly improved the healing of cutaneous wounds.

Plasminogen levels were not only temporally increased during the inflammation phase but also spatially

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concentrated at the site of the wound. The wound-specific accumulation of plasminogen after systemic

supplementation is mainly due to the transportation of plasminogen by neutrophils and macrophages.

Furthermore, the increased expression of interleukin 6 and the enhanced phosphorylation of STAT3 were

observed in the wound after plasminogen treatment. These data indicate that plasminogen acts as a key

pro-inflammatory regulator. It enhances pro-inflammatory cytokines and activates intracellular signaling

events during wound healing.

Taken together, the data obtained during the course of this project indicate that

plasminogen is crucial for oral health in mice. We also demonstrate that supplementation of plasminogen

to mice with periodontitis results in healing of gum tissues and significant re-growth of alveolar bone.

Therefore, plasminogen may be a new drug that will be competitive to currently used oral health-related

procedures, such as implantations and surgeries. Furthermore, we demonstrate for the first time that, in

addition to its role in extracellular matrix degradation, plasminogen is a key pro-inflammatory factor that

accumulates at the wound and potentiates the early inflammatory response during wound healing. Based

on our findings, we propose the administration of plasminogen as a novel therapeutic strategy for the

treatment of different types of wounds, including chronic diabetic wounds.

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List of papers

I Sulniute R., Lindh T., Wilczynska M., Li J., and Ny T. Plasmin is essential in

preventing periodontitis in mice. Am. J. Path. 2011 179(2): 819-28.

II Sulniute R., Shen Y., Ahlskog N., Guo Y-Z., Li J., Wilczynska M, and Ny T.

Plasminogen is a critical regulator of cutaneous wound healing. Manuscript

III Shen Y., Guo Y-Z., Mikus P., Sulniute R., Wilczynska M., Ny T*, and Li J*.

Plasminogen is a key pro-inflammatory regulator that accelerates the healing of acute and diabetic

wounds. Blood. 2012 119(24): 5879-87.

* Corresponding authors

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Abbreviations

α2-AP α2-antiplasmin

α2-M α2-macroglobulin

aa amino acid

CEJ cemento-enamel junction

ECM extracellular matrix

EGF epidermal growth factor

FBN fibrinogen

K1-K5 kringle 1 to kringle 5

kDa kilodalton

MMP matrix metalloproteinase

PA plasminogen activator

PAI-1 plasminogen activator inhibitor type 1

PAI-2 plasminogen activator inhibitor type 2

PAP plasminogen N-terminal activation peptide

Plg-R plasminogen receptor

PMN polymorphonuclear leukocyte

PN-1 protease nexin-1

tPA tissue-type plasminogen activator

uPA urokinase-type plasminogen activator

uPAR urokinase-type plasminogen activator receptor

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Introduction

The key component of the plasminogen activator (PA) system is the broad-spectrum serine

protease plasmin, which is formed from its inactive form, plasminogen. Plasminogen is activated by either

of the two plasminogen activators (PAs), tissue-type PA (tPA) or urokinase-type PA (uPA). These

proteases are regulated by several specific inhibitors against both PAs and plasmin. The PA system plays a

key role in many physiological and pathological processes, such as fibrinolysis, extracellular matrix (ECM)

degradation, tissue remodeling, inflammation, and cell migration. These processes contribute to

development of periodontitis and occur during wound healing.

Periodontitis is an oral disease that involves a bacterial infection, the inflammation of the

periodontium, and the degradation of gum tissue and alveolar bone. It normally starts with formation of

gingivitis that is a mild, reversible inflammation of the gums caused by bacterial plaque that accumulates

on the teeth at the gingival margin. In some patients, gingivitis develops into periodontitis. Periodontitis is

an irreversible disease and, in severe cases, can lead to tooth loss due to degradation of the periodontal

ligament and the alveolar bone.

The wound healing process consists of three overlapping phases: inflammation,

proliferation, and remodeling. The PA system is involved in all phases of the healing process beginning

with clot formation and ending with the remodeling of granulation tissue. The improper or delayed

healing of wounds caused by either trauma or surgical procedures may lead to chronic infections and

severe scarring.

The aims of thesis were three-fold:

1) to study the role of plasminogen in oral health by monitoring the development of periodontitis in wild-

type mice, plasminogen-deficient mice, and mice lacking the plasminogen activators tPA and uPA;

2) to study plasminogen’s contribution to, and acceleration of, the healing of cutaneous wounds in

plasminogen-deficient and diabetic mice;

3) to investigate the molecular mechanism by which supplemental plasminogen improves the healing of

cutaneous wounds in diabetic mice.

Conclusion: The studies included in this thesis suggest that plasminogen has a potential as a new drug

to treat periodontal disease and to be an alternative for presently used oral health-related procedures,

such as implantations and surgeries. These studies also indicate that plasminogen is an important

regulator of wound healing, and it enhances the rate and quality of healing of acute and chronic cutaneous

wounds.

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1. The Plasminogen Activator System

The PA system, traditionally called the fibrinolytic system, is a well-balanced enzymatic

cascade. The central molecule of this system, plasmin, is formed by proteolytic activation of the

proenzyme, plasminogen, by tPA or uPA [1]. The activity of this system is regulated at many levels, which

includes inhibition by plasmin inhibitors such as α2-AP and α2-macroglobulin (α2-M) and PA inhibitors

(PAIs) such as PAI-1 and PAI-2 (Figure 1).

Plasmin is a serine protease that degrades a large number of ECM proteins, including

fibrin, gelatin, fibronectin, and proteoglycans [2]. The PA system has been implicated in important

physiological events such as embryogenesis, wound healing, and angiogenesis, as well as in a variety of

pathological conditions such as infection, tumor growth, and metastasis [3-7]. The development and

characterization of mice deficient in plasminogen and other members of the PA system has provided a

number of animal models that mimic human conditions and contribute to an understanding of the

mechanisms associated with many pathological events. Such animal models have been utilized in this

thesis to determine novel roles for plasminogen in the development of periodontitis and in healing of

cutaneous wounds.

Figure 1. A schematic representation of the PA system. The proenzyme plasminogen is converted to the

active enzyme plasmin by tPA or uPA. Plasmin degrades fibrin and can convert MMPs and TGF-β into

their active forms. Inhibition of plasmin can occur at the PA level by PAIs and at the plasmin level by α2-

AP or α2-M. Adapted from C. Moali and D.J. Hulmes [7].

1.1. Plasminogen/plasmin

Human plasminogen is synthesized as an 810 amino acid (aa) single polypeptide protein

that is cleaved during secretion from the liver to form a 791 aa protein with a molecular mass of 92 kDa

[8]. The concentration of plasminogen in blood plasma is approximately 2 µM (200 µg/ml), and its half-

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life is 2.2 days [9-11]. Plasminogen is composed of an amino-terminal domain containing five kringle

structures that are stabilized by three intra-chain disulfide bridges (the heavy chain) and a carboxyl-

terminal trypsin-like serine protease domain that contains the catalytic triad His603, Asp646, and Ser741 (the

light chain) (Figure 2) [12]. The five kringle domains (K1-K5) on the heavy chain contain lysine binding

sites, and K1 has the highest affinity for lysine [13]. The lysine binding sites are crucial for the specific

recognition of fibrin, cell surface receptors, and α2-AP [13].

Plasminogen exists in two different molecular forms: Glu-plasminogen and Lys-

plasminogen. Glu-plasminogen is the native, uncleaved plasminogen with an amino-terminal glutamic

acid residue. Upon cleavage of the Lys76-Lys77 bond, Lys-plasminogen is formed, which is 76-aa shorter

than Glu-plasminogen and has an amino-terminal lysine residue [14, 15]. Lys-plasminogen has a higher

binding affinity to fibrin but has a shorter half-life (0.8 days) compared to Glu-plasminogen (2.2 days) [13,

16]. Both forms of plasminogen can be activated by the physiological PAs [17]. When the Arg560-Val561

bond of the single-chain plasminogen is cleaved by tPA or uPA, a two-chain disulfide-linked active serine

protease is formed [18]. Plasminogen is primarily synthesized in the liver [19, 20], but several other

organs, including the adrenal gland, kidney, brain, testis, heart, lung, uterus, spleen, thymus, and gut,

have been identified as possible sources of plasminogen [21].

Figure 2. The structural features of plasminogen and PAs. Adapted from C. Moali and D.J. Hulmes [7].

Plasmin is responsible for the degradation of ECM components, which include fibrin,

fibronectin, and laminin, into soluble fragments [4, 5]. It can also indirectly degrade additional

components of the ECM by activating the precursors of the matrix metalloproteinases (MMPs) [6, 22].

Both the degradation and cell surface binding abilities of plasmin play a crucial role in cell migration

during angiogenesis, inflammation, wound healing and cancer. The existence of protease-activated

receptors (PARs) and the newly discovered plasminogen receptors (Plg-Rs) suggest an even broader

spectrum of action for plasminogen through cell signaling pathways [23-25].

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1.2. Plasminogen activators

Similar to plasminogen, the two main physiological PAs, tPA and uPA, are multidomain

serine proteases [26]. Although tPA and uPA catalyze the same reaction and share similar overall

structures in terms of kringle and epidermal growth factor-like (EGF) domains (Figure 2), tPA and uPA

have different biological roles. Traditionally, tPA is thought to activate plasminogen during fibrinolysis,

whereas uPA activates cell-associated plasminogen during cell migration [4]. However, functional studies

in PA-deficient mice have shown that tPA and uPA are involved in many other physiological reactions and

may also have partially overlapping physiological functions [27, 28].

1.2.1. Tissue-type plasminogen activator

Human tPA is secreted as a 527 aa single-chain, multidomain glycoprotein with a molecular

mass of approximately 70 kDa [29, 30]. The normal plasma concentration of tPA is 5-10 ng/ml. The non-

catalytic amino-terminal domain (the heavy chain) of tPA contains the fibrin-binding finger domain, the

EGF domain, and two kringle domains, where the second kringle domain has a high binding affinity for

lysine [1]. The carboxyl-terminal region (the light chain) contains the serine protease domain. The single-

chain form of human tPA is converted to the two-chain form upon cleavage of the Arg275 – Ile276 peptide

bond by plasmin or kallikrein [31]. Interestingly, both the single-chain and two-chain forms are active,

which makes tPA unique among serine proteases [32, 33].

tPA is involved in biological functions that are not related to the activation of plasminogen,

such as long-term potentiation in the hippocampus and excitotoxic damage [34]. Several tPA-binding sites

on different cell types have been described, but a specific receptor for tPA has not yet been identified [35-

37]. tPA is mainly produced by endothelial cells, but it is also produced by keratinocytes, neurons, and

melanocytes [38-40]. Additionally, tPA is broadly distributed in the brain and plays important roles

during physiological and pathological conditions [41]. Elevated levels of tPA lead to hyperfibrinolysis and

excessive bleeding disorders [42].

1.2.2. Urokinase-type plasminogen activator

Human uPA is synthesized as an inactive, single-chain, multidomain glycoprotein of 411 aa

(53 kDa). The concentration of uPA in plasma varies between 2 and 20 ng/ml [43, 44]. The single-chain

uPA is an inactive proenzyme that is converted to the active two-chain form of uPA upon cleavage of the

Lys158-Ile159 bond by proteases such as plasmin, kallikrein, coagulation factor XIIa, or cathepsin B [28, 45].

The amino-terminal region (the heavy chain) contains the EGF domain and the single kringle domain

(Figure 2), and the carboxyl-terminal region (the light chain) contains the active site with the classical

serine protease catalytic triad – His204, Asp255, and Ser356 [1, 45, 46]. In contrast to tPA, uPA has no fibrin-

binding capability. However, uPA does have a well-characterized receptor, the uPA receptor (uPAR), that

is expressed on the surface of many cell types. This receptor enables cell-localized accumulation of uPA

and the high local activation of plasminogen [3]. In vitro studies in cultured pre- and postnatal human

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tissues and cells have shown that uPA is expressed in lung and kidney cells, as well as in keratinocytes and

endothelial cells [47, 48]. Increased levels of expression of uPA and/or uPAR are seen in the cancer cells

from tumors of the breast, colon, ovary, stomach, kidney, brain, and other organs [49, 50].

1.3. Inhibitors of the plasminogen activator system

Tight regulation of proteolytic activity is fundamental in many biological processes. The

three major serine protease inhibitor (serpin) molecules, α2-AP, PAI-1, and PAI-2, regulate the activity of

plasmin and PAs. The native state of serpins, the stressed (S)-state, is metastable, and the proteolytic

cleavage of the reactive center loop (RCL) results in a dramatic conformational change to the relaxed (R)-

state along with an increase in the thermal stability of the serpin molecule. This family of inhibitors is

known as suicide inhibitors – the serpin-protease complex, in which inhibition of the protease is

maintained by covalent linkage to the serpin, is irreversible [51-53]. Serpins regulate very diverse

physiological processes that require proteolytic activity, including coagulation and fibrinolysis,

complement activation, inflammation, angiogenesis, apoptosis, neoplasia, and viral pathogenesis [54].

1.3.1. Plasmin inhibitors: α2-antiplasmin and α2-macroglobulin

α2-AP, also known as α2-plasmin inhibitor (A2PI), is the major physiological inhibitor of

plasmin, but it can also inhibit chymotrypsin and trypsin. Human α2-AP is a 452 aa single-chain

glycoprotein with a molecular mass of approximately 70 kDa. The concentration in plasma is 1 µM (70

µg/ml), and the half-life is 2.6 days [55, 56]. α2-AP rapidly binds to the active center and the lysine-

binding sites of plasmin, thereby inhibiting both its enzymatic activity and its lysine-binding capability.

Both of these sites must be free for α2-AP to inhibit plasmin, which means that plasmin bound to fibrin is

protected from inhibition by α2-AP [57-59]. An α2-AP deficiency in humans increases the chances of

excessive bleeding, especially during trauma, surgery or dental extraction [60]. In contrast, the lack of α2-

AP in mice increases platelet microaggregation, resulting in thrombus formation [61]. α2-AP is mainly

synthesized in the liver, but is also produced to some extent in the kidneys, intestines, and brain [62, 63].

Human α2-M is a disulfide-linked homotetramer of 1451 aa (725 kDa) with a relatively high

plasma concentration of 2.5 mg/ml [64-67]. It is a member of the I39 family of protease inhibitors, and it

inhibits proteases, regardless of their catalytic type, through a unique mechanism often referred to as the

‘trap mechanism’ [68]. Specific cleavage by the active protease in the ‘bait’ region of α2-M induces a large

conformational change that results in the trapping of the protease within the large central cavity of the

inhibitor. In this way, α2-M bound to a protease prevents the protease from reacting with high molecular

weight substrates such as fibrin and collagen, but leaves it free to interact with low molecular weight

substrates. The liver is the main producer of this inhibitor, but other organs may also produce it. A lack of

α2-M is associated with Alzheimer disease [69].

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1.3.2. Plasminogen activator inhibitors

PA inhibitor type 1 (PAI-1) is a single-chain glycoprotein of 379 aa (45 kDa) and has a

plasma concentration of approximately 20 ng/ml [70]. PAI-1 is in its active form when secreted but

spontaneously converts to its latent form [71, 72]. This serpin rapidly inhibits single-chain tPA, two-chain

tPA, and two-chain uPA, but not single-chain uPA [70, 73], by forming covalently bound one-to-one

complexes [53, 74, 75]. PAI-1 is produced at low levels by several different cell types in response to growth

factors, cytokines, phorbol esters, endotoxins, and hormones [76]. A primary function of PAI-1 in vivo is

to balance the proteolytic activity of the PAs during fibrinolysis [77]. Beside its role in regulating

hemostasis, PAI-1 is important for the regulation of cell adhesion, cell migration, and PA- or plasmin-

dependent tumor invasion [78, 79]. Studies in transgenic mice have also revealed a functional role for

PAI-1 in wound healing, atherosclerosis, metabolic disturbances such as obesity and insulin resistance,

tumor angiogenesis, chronic stress, bone remodeling, asthma, rheumatoid arthritis, fibrosis,

glomerulonephritis, and sepsis [80].

PA inhibitor type 2 (PAI-2) is a single-chain, 425 aa serpin that is detectable in plasma

only during pregnancy and reaches concentrations from 5 ng/ml up to 250 ng/ml [81-83]. PAI-2 is not

efficiently secreted and, therefore, accumulates intracellularly as a non-glycosylated 47 kDa protein [84].

A small portion of single-chain PAI-2 is secreted as a 60 kDa plasma glycoprotein [85]. PAI-2 can

spontaneously polymerize, and the CD-loop of PAI-2 functions as a redox-sensitive molecular switch that

converts PAI-2 between a stable, active, monomeric conformation and a polymeric conformation [86].

This serpin is produced by several cell types, including monocytes/macrophages, keratinocytes, and

epithelial cells. Extracellular PAI-2 inhibits both uPA and two-chain tPA, although PAI-2 is a poor

inhibitor of single-chain tPA, with up to 10,000-fold lower inhibition compared to uPA inhibition [87].

The extracellular form of PAI-2 is also considered to be a regulator of uPA activity in blood vessels and in

the ECM during the pregnancy, cancer formation, and inflammation [88, 89]. The role of intracellular

PAI-2 remains unclear, although several studies have suggested that it may play a role in protecting cells

from apoptosis [81, 90].

1.4. Receptors of the plasminogen activator system

Cell surface-associated proteolysis plays a crucial role in cell migration during

embryogenesis, angiogenesis, inflammation, healing, and invasion. There are receptors for uPA and

plasminogen, and co-localization of plasminogen and PAs on the cell surface ensures that sufficient local

proteolytic activity is available. Cell-bound plasmin is protected from its physiological inhibitors α2-AP

and α2-M [91]. The interaction between the cell surface and plasminogen is mediated by a heterogeneous

group of Plg-Rs. Many, but not all, Plg-Rs interact through their carboxy-terminal lysine with the lysine-

binding sites within the kringle domains of plasminogen [92, 93]. These receptors can be categorized into

two groups: tailless (those without a cytoplasmic tail) and tailed (those with a cytoplasmic tail) [94]. Most

Plg-Rs, including enolase-1, annexin 2, Pg-RKT, and histone H2B, belong to the tailless group, and the

tailed group contains protease-activated receptors and integrins (αMβ2, αIIbβ3, αvβ3, α4β1, and α5β1).

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Although β2 integrins do not contain carboxyl-terminal lysines, they are still able to interact with the

lysine binding site of plasminogen. Both tailless and tailed Plg-Rs promote plasminogen activation and

pericellular proteolysis.

1.4.1. Plasminogen receptors

Plasminogen receptors (Plg-R) are a diverse group of proteins that are expressed on the

surface of many types of cells. Plasminogen binding has been observed on B cells, smooth muscle cells,

fibroblasts, keratinocytes, endothelial cells, renal epithelial cells, leukocytes, platelets, monocytes or

monocytoid cell lines, and metastatic colorectal and mammary carcinoma cells [91, 93, 95-99]. The tailless

Plg-Rs are responsible for concentrating the local proteolytic activity of plasminogen on the cell surface,

and this promotes sufficient cell mobility (Figure 3). The tailed receptors, on other hand, might be

involved not only in cell surface-associated proteolytic activity but also in cell signaling.

Figure 3. Plg-R activity during monocyte/macrophage recruitment. Monocyte activation by external

stimuli leads to expression of Plg-Rs at the cell surface. Plasminogen binds to the Plg-R and is converted

to plasmin by PAs. When monocytes differentiate into macrophages, additional Plg-Rs are exposed on the

cell surface to maintain further migration and activation of intracellular signaling at the inflammation site.

Adapted from E.F. Plow and R. Das [100].

Enolase-1, also called α-enolase, is a 48 kDa glycolytic enzyme that acts as a 2-phospho-

D-glycerate hydrolase in the cytoplasm of prokaryotic and eukaryotic cells [101]. Enolase-1 was first

described as a plasminogen receptor on the surface of monocytoid cells [92] and was later detected on the

surfaces of other cell types [102]. The plasminogen-enolase-1 interaction is mediated by the binding of

plasminogen’s kringle domains to the carboxyl-terminal lysine residue of enolase-1 [92, 103]. On the cell

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surface, the interaction of plasminogen with enolase-1 is enhanced by tPA, and protects plasminogen from

inhibition by α2-antiplasmin [103]. Enolase-1 plays a crucial role in plasminogen-mediated recruitment of

monocytes to inflamed areas of the lung. Using a monocytoid cell line and isolated blood monocytes, cell-

surface expression of enolase-1 was also shown to increase dramatically upon lipopolysaccharide

stimulation [104].

Annexin 2 is a member of the annexin superfamily of calcium-dependent, phospholipid-

binding proteins. Annexin 2 is 38 kDa protein containing a large, conserved carboxyl-terminal ‘core’

domain (34.2 kDa) and a smaller (3.4 kDa) more variable amino-terminal ‘tail’ domain [105]. This

receptor does not contain a carboxyl-terminal lysine, but its binding partner, p11 (S100A10), does, and

this allows annexin 2 to bind plasminogen, tPA and plasmin [106]. The annexin 2 can form heterodimer,

which consists of two annexin 2 monomers and two p11 subunits, which have even greater plasmin

generation ability than monomeric annexin 2 [107]. Annexin 2 was first identified as a Plg-R on the

surface of endothelial cells, but it is also abundant on monocytoid, monocytes/macrophages, myeloid

cells, developing neuronal cells, and some tumor cells [108]. Mice lacking annexin 2 show an impaired

clearance of arterial thrombi, deposition of fibrin in the microvasculature, and angiogenic defects in a

variety of tissues. Interestingly, p11 is expressed at very low levels in the absence of annexin 2 [109, 110].

In rats, arterial thrombosis is diminished by intravenous treatment with recombinant annexin 2 [111].

Plg-RKT is the first transmembrane receptor for plasminogen that has been identified. This

membrane-integrated, 147 aa, and 17 kDa protein has a carboxyl-terminal lysine and is predicted to

contain two transmembrane helices. It is widely expressed on the surface of cells known to bind

plasminogen and is highly co-localized on the cell surface with urokinase receptor (uPAR) [25]. Plg-RKT

plays a key role in the plasminogen-dependent regulation of macrophage recruitment during an

inflammatory response [112]. This novel Plg-R is highly expressed in murine and human adrenal

medullary chromaffin cells, human pheochromocytoma tissue, and murine hippocampal cells [113]. The

expression of Plg-RKT in catecholaminergic tissues is responsible for the regulation of catecholamine

release through cell surface plasminogen activation [113].

Integrin αMβ2, a non-covalently linked heterodimer of αM and β2 subunits, belongs to a

large family of heterodimeric cell adhesion receptors that mediate a wide spectrum of biological functions

[114]. Integrin αMβ2 is a tailed Plg-Rs that does not contain a carboxyl-terminal lysine but still interacts

with the lysine binding sites in plasminogen’s kringle domains. This receptor binds both plasminogen and

uPA in a dose-dependent, saturable manner, and this binding leads to a 50-fold acceleration of plasmin

generation [115]. Integrin αMβ2 also forms a tight complex with uPAR that leads to enhanced integrin-

dependent leukocyte responses [116]. As a tailed Plg-R, αMβ2 takes part in cell signaling in which

plasminogen triggers activation of ERK1/2 and Akt through αMβ2 in neutrophils [117]. Integrin αMβ2 is

expressed in monocytes, granulocytes, macrophages, and natural killer cells, and it has been implicated in

the diverse functions of these cells, which include phagocytosis, cell-mediated killing, chemotaxis, and

cellular activation [118-120]. The characterization of αMβ2-deficient mice showed a variety compromised

leukocyte-dependent responses [118-120].

Protease-activated receptors (PARs) are G-protein-coupled receptors that are

activated by proteolytic cleavage of their extracellular amino-terminus. The cleaved part of the receptor

17  

interacts with its extracellular loop and leads to a transmembrane signal. There are four known human

PARs – PAR1, PAR2, PAR3, and PAR4 [121] – and these receptors are found on the surfaces of platelets,

endothelial cells, neurons, and myocytes. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce

activation of platelets and vascular cells, whereas PAR2 is preferentially cleaved by trypsin. Alternatively,

plasmin can activate PAR1 and PAR4 [122, 123]. Plasmin stimulates an inflammatory response in human

dental pulp cells by activating PAR1 [124], and it also mediates platelet aggregation through proteolytic

cleavage of PAR4 [123]. PAR1-mediated signaling is involved in cell growth and division, which suggests

additional biological roles for plasmin [125, 126]. In fibroblasts, plasmin is able to induce p44/42

mitogen-activated protein kinase (MAPK) activation and to upregulate Cyr61 gene expression through

PAR1 [122]. This expression of a growth factor-like gene can potentially contribute to fibroblast

proliferation and migration, especially during the wound healing process.

1.4.2. The urokinase receptor

uPAR is a 335 aa polypeptide that includes a 22 residue amino-terminal signal peptide.

The mature uPAR protein (283 aa) is highly glycosylated and composed of three similarly sized

homologous domains [127]. uPAR is anchored to the plasma membrane through

glycosylphosphatidylinositol (GPI) linkages [128] and is the primary, specific cellular receptor for uPA

[129]. uPAR-bound uPA is active and susceptible to inhibition by PAI-1 or PAI-2. Furthermore, uPAR co-

localizes with integrins at the leading edge of migrating cells. This receptor not only facilitates uPA-

dependent proteolytic activity at the cell surface but can also initiate intracellular signaling [130].

The plasma levels of PAs are tightly regulated in vivo to maintain a balance between

fibrinolysis and thrombosis. One of the mechanisms responsible for this balance is the rapid clearance of

PAs via receptor-mediated endocytosis. uPAR can function as one such clearance receptor in cooperation

with low density lipoprotein receptor-related protein (LRP) [131]. PAIs inhibit uPAR-bound uPA and

cause this complex to be internalized and undergo degradation in the lysosomes of hepatic and

monocytoid cells.

uPAR is expressed on monocytes, macrophages, fibroblasts, endothelial cells, and a variety

of tumor cells [129], and its expression is increased in many pathological conditions, including cancer,

inflammation, and infection [132]. uPAR-deficient mice are essentially healthy, with normal fertility,

development, and hemostasis, even though macrophages from these mice showed reduced activation by

plasminogen [133]. The only obvious health issue these mice have is an increased sensitivity to induced

urinary tract infections. Neutrophil recruitment is similar in both uPAR-deficient and wild-type mice, but

neutrophils of uPAR-deficient mice have impaired bacterial clearance [134].

1.5. Plasminogen in cell signaling

Cell-bound plasminogen has traditionally been regarded to only be responsible for cell migration through

its proteolytic degradation of the ECM. However, in vivo studies have demonstrated that plasmin is able

to trigger functional changes in cells, which suggests that plasminogen can activate receptor-mediated

18  

signaling pathways [23]. Several receptors in the PA system (annexin 2, integrins, PARs, and uPAR) also

induce intracellular signaling pathways that are critically important for the expression of many pro-

inflammatory mediators in various cells [135]. In monocytes, plasmin induces the expression of cytokines

(IL-1α, IL-β, and TNF-α) and tissue factor (TF) via the NF-κB pathway [136]. Plasmin also triggers pro-

inflammatory cytokine expression in macrophages via the annexin 2 receptor and is involved in the

activation of multiple signaling pathways, including the JAK/STAT, p38 MAPK, ERK1/2, and NF-κB

pathways [137]. Notably, the MAPK pathway is one of the major signaling pathways that control the

expression of different pro-inflammatory genes [138]. In Paper II, we also show that plasminogen

accumulates around wounds, induces the expression of IL-6, and activates STAT3. Additionally, plasmin

induces the expression of the angiogenic and wound-healing promoter Cyr61 in fibroblasts via the PAR1

receptor [122]. Furthermore, plasmin leads to a decrease in the expression of the pro-apoptotic protein

BimEL in primary hepatocytes via the ERK1/2 signaling pathway [139]. Taken together, these studies have

revealed a much broader range of functions for plasmin than was previously known.

1.6. The plasminogen activator system in tissue remodeling

The PA system has been implicated in several physiological and pathological processes that

require tissue remodeling. The most studied of these processes are fibrinolysis and the degradation of the

ECM. Plasmin has a proteolytic activity against various components of the ECM, which include laminin,

fibronectin, and collagens. Furthermore, plasmin may activate other ECM-degrading proteases, such as

MMPs, and in this way, it may promote additional degradation processes [4]. Several cell types can bind

plasminogen/plasmin and/or other components of the PA system on their surfaces to initiate cell

migration, tumor invasion, and angiogenesis [3, 140].

1.6.1. Fibrinolysis

The fibrinolytic activity of plasmin is crucial for the degradation of fibrin clots in the

vascular system. A localization of this proteolytic activity at the site of a developing thrombus, where

plasmin and PAs are protected from circulating inhibitors, is an essential process in regulating fibrinolysis

[1]. tPA is the main molecule responsible for activating fibrin-bound plasminogen because uPA lacks a

fibrin-binding domain. However, in the absence of tPA, uPA may take over the plasminogen-activating

function in fibrinolysis [27]. It is necessary to maintain a strict balance between plasminogen activation

and inhibition of the PAs. Patients with reduced concentrations of PA inhibitors suffer from excessive

bleeding, whereas elevated levels of PA inhibitors can lead to myocardial infarction, embolism, and

atherosclerosis [141-143].

The fibrinolytic activity of plasmin plays a critical role in tissue remodeling during wound

healing [144, 145]. In Paper II, we also have shown that fibrinogen deposition remained at the wound site

even after 60 days of healing in plasminogen-deficient mice. Bugge et al demonstrated that mice deficient

19  

in both plasminogen and fibrinogen have restored rates of re-epithelialization comparable to wild-type

mice, but the quality of the healed wound may not be the same as that of wild-type mice [146].

1.6.2. Bone remodeling

Bone remodeling includes bone formation and bone resorption. These processes are

regulated by a number of genetic, hormonal, nutritional, growth and other factors and continues during

the lifetime of the bone. During the bone remodeling process, mineralized bone is removed by osteoclasts

followed by the formation of a new bone matrix by osteoblasts [147]. The balance between osteoclast-

osteoblast functions has to be tightly regulated, as excessive bone resorption can lead to diseases such as

osteoporosis.

Both bone-producing and bone-degrading cells express components of the PA system

including uPA, tPA, uPAR, and PAI-1, indicating that the PA system is involved and tightly regulated in

bone remodeling [148]. A number of in vitro studies have shown that the PA system mainly participates in

bone resorption. Plasmin can, to some extent, activate TGF-β and release insulin-like growth factor (IGF-

1), which may stimulate osteoblast proliferation and activity and lead to bone formation processes [149].

Recently, Kanno et al have demonstrated the direct involvement of plasminogen in bone metabolism by

showing that the pre-osteoclast cell population in plasminogen-deficient mice was higher than that in

wild-type. The absence of plasminogen caused reduced expression of osteoprotegerin, also known as

osteoclastogenesis inhibitory factor, and resulted in decreased proliferation of osteoblasts [150].

Interestingly, exogenous plasmin induced the expression of osteoprotegerin in plasminogen-deficient

mice. The bone mineral density (BMD) of the trabecular bone at four to six weeks of age was significantly

lower in the tibia of plasminogen-deficient mice compared to wild-type mice, but this difference

diminished over time. The cortical bone BMD was lower in plasminogen-deficient mice into adulthood (up

to 18 weeks old) [150].

Persistent inflammation of the periodontium induces osteoclast activation and resorption of alveolar

bone, which leads to flexibility of the teeth and eventual tooth loss. As we have shown, plasminogen-

deficient mice develop spontaneous periodontitis in which the alveolar bone is severely degraded.

Exogenous plasminogen was not only able to heal the periodontium in these mice, but it also induced

alveolar bone re-growth (Paper I, this thesis). Thus, a more thorough understanding of the role of the PA

system in bone remodeling might lead to novel therapeutic interventions for treating bone diseases.

1.6.3. Other tissue remodeling processes requiring the plasminogen activator system

The study of mice deficient in most members of the PA system has revealed the importance

of the PA system in many physiological and pathological events, including reproduction, angiogenesis, and

cancer. These are processes where tissue remodeling is crucial.

Reproduction involves complex biological processes, such as ovulation, fertilization,

embryo implantation, and embryogenesis, in which the PA system may play an important role. However,

20  

studies of gonadotropin-induced and physiological ovulation in plasminogen-deficient mice have

demonstrated only slightly reduced ovulation efficiency [151]. Plasminogen-deficient mice treated with a

broad-spectrum MMP inhibitor showed only a 14% reduction in ovulation efficiency [152]. However,

because plasminogen plays a major role in wound healing and angiogenesis, plasminogen-deficient mice

might recover poorly after litter delivery, which in turn could affect subsequent reproduction processes.

Interestingly, the serine protease inhibitor protease nexin-1 (PN-1), which modulates the proteolytic

activity of plasminogen, PAs, thrombin, and trypsin, was shown to play a crucial role in male fertility. Mice

lacking PN-1 are infertile due to enhanced proteolytic activity in the seminal fluid [153].

Angiogenesis, the formation of new blood vessels, is important during several

physiological and pathological processes, such as placental development, embryonic development, corpus

luteum formation, and wound healing [154]. Pathological angiogenesis occurs during the progress of

several disease conditions, such as tumor growth and metastasis, rheumatoid arthritis, and

atherosclerosis [154]. During angiogenesis, the endothelial cells of existing blood vessels degrade the

basement membranes and make space for the formation of new vessels [155]. This degradation process

involves the PA system and other systems such as the MMPs [156, 157].

Tumor growth and metastasis requires degradation of the ECM of the basement

membrane. The PA and MMP systems provide the proteolytic activity that is required for this process. The

PA system appears to be involved not only in tumor cell migration and invasion but also in other processes

in tumors such as angiogenesis and desmoplasia (stimulation of fibroblast proliferation and synthesis of

ECM proteins) [158]. Mice lacking plasminogen or PAs generally show a reduced rate of tumor growth

[159-161]. In humans, high levels of uPA and its receptor uPAR are associated with poor prognosis in

many types of cancer [49].

1.7. The plasminogen activator system in infection

An infection is caused by the invasion of a pathogenic bacteria, virus or parasite into a host

organism and depends on successful colonization of the host, avoidance of host defenses, and replication.

Infectious and parasitic diseases currently cause approximately one-third of all human deaths in the

world, which is more than all forms of cancer combined [162].

Some bacterial pathogens use host plasmin-mediated proteolysis to enhance their invasion

abilities and evade the innate immune system [163]. However, the PA system also participates in the

host’s defense against infection through its important roles in the inflammatory response. Numerous

bacteria, both gram-negative and gram-positive, carry receptors for plasmin/plasminogen or PAs [164,

165], and this provides them with the ability to invade the host by using the proteolytic activity of the host-

derived PA system. Several pathogens also produce pathogenic PAs, such as streptokinase (from various

species of streptococci), staphylokinase (from Staphylococcus aureus), and the Pla protease (from

Yersinia pestis) [163, 166]. Streptokinase, produced by group A streptococci, is very specific to human

plasminogen and has very low or no activity against other species, including mice. In transgenic mice

expressing human plasminogen, streptokinase caused increased mortality following a skin infection [167].

21  

The periodontal bacterium Fusobacterium nucleatum subsp. nucleatum binds

plasminogen in vitro, after which it becomes a proteolytic organism with the potential to invade

periodontal tissues [168]. Furthermore, the periodontal pathogen Porphyromonas gingivalis is able to

inactivate plasmin inhibitors α2-AP and α2-M [169, 170]. Although a number of bacterial species bind

plasminogen, the pathogenic properties of plasminogen activation have only been studied in a limited

group of tissue invasive bacteria. In vivo data that reveal the role of PA system in bacterial infections are

limited and have come from studies using plasminogen-deficient mice, even though plasminogen has been

shown to have a protective role in a Staphylococcus aureus-induced arthritis model [171]. Based on the

collective evidence, the PA system appears to play a dual role during infection.

1.8. The plasminogen activator system in inflammation

Inflammation is a protective process through which an organism removes injurious stimuli,

such as pathogens, damaged cells, or irritants, and initiates the healing process. Inflammation can be

classified as acute or chronic. Acute inflammation is the initial response to harmful stimuli in which

leukocytes migrate from the blood and into the injured tissues. A cascade of biochemical events leads to

the inflammatory response and involves the local vascular system, the immune system, and various cells

within the injured tissue. Chronic inflammation differs in the type of cells present at the site of

inflammation and is characterized by continuous destruction and healing of the tissue under the

stimulation of inflammatory processes [172, 173].

The PA system participates in all steps of the inflammation process, including

vasodilatation, exudation, release of pain mediators, and the clearance of the fibrinous exudates [174].

Plasmin is capable of inducing an inflammatory response by activating the complement system that

generates C3a and C5a. In turn, C3a and C5a induce mast cell degranulation, histamine-dependent

vasodilatation, and an increased permeability of the venules. uPAR-mediated generation of bradykinin, a

potent endothelium-dependent vasodilator, provides another functional role of the PA system in

vasodilation. Acute inflammation disturbs the equilibrium of fluid and protein exchange between the

microcirculation and tissues that provides cells and fluids that are needed at the site of injury. The PA

system plays a major role in leukocyte recruitment during inflammation. Plasmin triggers the aggregation

of neutrophils and the degranulation of platelets [175], and it also induces expression of cytokines and

tissue factor (TF) in human monocytes [136, 176, 177]. Plasmin binds to a variety of cell types, including

inflammatory cells, through its lysine binding sites. Such cell-surface binding leads to a full-scale pro-

inflammatory activation [136]. The lack of uPA is associated with an impaired influx of macrophages into

the injured area, which results in delayed or abolished cardiovascular wound healing [178, 179]. In vivo

studies also have shown a critical role for uPAR in the regulation of neutrophil recruitment to inflamed

sites [134].

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2. Periodontitis

The inherited or acquired disorders in the tissues surrounding and supporting the teeth are

assigned to eight major classes of periodontal disease: gingival diseases (I), chronic periodontitis (II),

aggressive periodontitis (III), periodontitis as a manifestation of systematic diseases (IV), necrotizing

periodontal diseases (V), abscesses of the periodontium (VI), periodontitis associated with endodontic

lesions (VII), and developmental and acquired deformities and conditions (VIII) [180, 181]. Most

periodontal diseases are inflammatory in nature and are caused by pathogenic oral microflora. Depending

on severity, periodontal diseases can be broadly divided into gingivitis and periodontitis. The mildest form

of periodontal disease, gingivitis is a highly prevalent inflammation of the gums caused by bacterial plaque

that accumulates on the teeth at the gingival margin. Gingivitis is usually reversible through effective

dental hygiene. In some patients gingivitis can develop into periodontitis – a severe and chronic

inflammatory disease affecting tissues that support and anchor teeth (the periodontium). Periodontitis

appears as progressive and irreversible loss of gingival tissue, the periodontal ligament, and alveolar bone

that eventually leads to loss of teeth [180]. Periodontal diseases are highly prevalent and affect up to 90%

of the worldwide population [182]. Until now, no treatment for periodontitis had been developed that

would be able to regenerate all of the damaged periodontal tissues, especially the alveolar bone.

Smoking and type 1 and type 2 diabetes are major risk factors for periodontitis, and

microorganisms such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and

Tannerella forsythia (formely Bacteroides forsythus) are considered to be main risk indicators [183].

Severe periodontal disease often occurs in individuals whose host response mechanisms or immune

systems have been impaired, for example, by defects in neutrophil function. Various systemic diseases

such as leukemia, thrombocytopenia, and leukocyte disorders are associated with the increased severity of

periodontal disease [180]. Periodontitis itself can be one of the risk factors for preterm delivery, diabetes,

cardiovascular disease, and pulmonary disease by spreading inflammatory factors as a result of persistent

inflammation in the oral cavity.

The PA system is closely associated to processes such as bacterial invasion, tissue

degradation, bone remodeling, and inflammation, all of which typically occur during the development of

periodontal disease. The PA system can also participate in the regeneration of periodontal tissues due to

its ability to activate growth factors and regulate tissue remodeling processes.

2.1. The periodontium in health and disease

The tissues surrounding the teeth, such as the gingiva, the periodontal ligament, the root

cementum, and the alveolar bone are called the periodontium (Figure 4). The main function of the

periodontium is to support and anchor teeth, but it also provides tissue integrity, defense against the

invasion of pathogens, and maintenance of local hemostasis; damage to any one of the periodontal tissues

can lead to periodontal disease. The protection against periodontal infection is provided by the extrinsic

and intrinsic defense systems. The extrinsic protection is derived from secretions of the salivary glands

that are rich in immunoglobulin A. The intrinsic protection is located within and beneath the oral gingival

epithelium. The gingivial epithelium contains a specific tissue architecture and leukocytes, which are the

most important for defense of periodontium [184].

23  

Figure 4. The periodontium comprises the following tissues: the gingiva, the periodontal ligament, the

root cementum, and the alveolar bone. The tooth consists of the crown and the roots, where the crown is

covered with enamel and root surface with the cementum layer. Dental pulp, surrounded by dentin,

contains nerves and blood vessels. Modified from J. Lindhe et al [185].

2.1.1. Gingiva

The gingiva is the part of the oral mucosa that covers the alveolar bone and surrounds the

teeth. The gingival epithelium provides a physical barrier between infection agents from the environment

and the host tissues. The major population of cells in the gingival epithelium consists of keratinocytes, but

this layer also contains melanocytes, Langerhans cells, and Merkel cells. These epithelial cells are

important for innate host defense by responding to attempted bacterial invasions and by transmitting

signals to other parts of the host organism.

The junctional epithelium is firmly attached to the tooth surface and forms an epithelial

barrier against bacterial plaque. It plays an important role by providing access for gingival fluid,

inflammatory cells, and components of the immune defense to the gingival margin. Junctional epithelial

cells exhibit rapid turnover, which contributes to the host-bacteria equilibrium and to the rapid repair of

damaged tissue. In the case of periodontitis, the junctional epithelium migrates down to the pocket, and

this lowers the level of the cemento-enamel junction (CEJ).

The gingival connective tissue (lamina propria) is the predominant tissue of the gingiva.

This gingival connective tissue has a high turnover rate, a good healing and regenerative capacity, and

leaves little evidence of scarring after surgical procedures. The major components of the connective tissue

are collagen fibers (60% by volume), fibroblasts (5%), blood vessels, nerves and matrix (35%) [185]. The

gingival connective tissue fibers are produced mostly by fibroblasts and consist of collagen, reticular

fibers, and elastic fibers. The collagen fibers hold the marginal gingiva firmly against the tooth and

24  

provide rigidity against the forces that develop during chewing. Collagen type I is most predominant in the

lamina propria and provides the tensile strength of the gingival tissue.

Different cell types, such as fibroblasts, mast cells, macrophages, and inflammatory cells,

are present in the connective tissue. The fibroblasts are the most predominant connective tissue cell type

(65% of the total cell population) and produce various types of fibers and ECM components. Fibroblasts

also participate in immune reactions and inflammation [186]. Cytokines released during inflammation

play a central role in the modulation of inflammatory systems. They are produced not only by

inflammatory/immune cells but also by epithelial, endothelial cells, and fibroblasts. Cells respond to

various inflammatory cytokines and growth factors and notably influence the balance between ECM

synthesis and degradation. Alterations in this balance can lead to pathological lesions, such as tissue

destruction in periodontitis.

2.1.2. Periodontal ligament and root cementum

The periodontal ligament is a highly vascularized connective tissue that surrounds the roots

of the teeth and joins the root cementum to the alveolar bone [185]. The functions of the periodontal

ligament can be grouped into physical functions, formative and remodeling functions, and nutritional and

sensory functions. The periodontal ligament anchors the tooth to the alveolar bone and distributes

masticatory forces. Its brush-like fibrils, mainly containing type I collagen, form a meshwork that

stretches out and connects the cementum of one tooth to that of the adjacent tooth (Figure 4) and to the

alveolar bone [187]. Type III collagen is present as mixed fibers, and type I collagen is present as Sharpey’s

fibers that insert from the periodontal ligament into the alveolar bone and provide a stable connection

with the tooth [188]. Type I, V and XII collagens are expressed by osteoblasts, and type III and some type

XII collagen fibers appear to be produced by fibroblasts during the formation of the periodontal ligament.

The collagen fibrils in the bone are stabilized by intermolecular cross-linking [189].

The periodontal ligament consists of fibroblasts, osteoblasts, cementoblasts, osteoclasts,

and epithelial and nerve cells. The periodontal ligament is also rich in mesenchymal stem cells, which are

important for regeneration. The fibroblasts are aligned along the principal fibers, the cementoblasts line

the surface of the cementum, and the osteoblasts line the alveolar bone surface. The periodontal ligament

undergoes constant remodeling in which old cells and fibers are broken down and replaced by new ones.

The cementum, bone, and gingiva are supplied with needed nutrients through a rich blood vessel network

that extends throughout the periodontal ligament.

The root cementum is a calcified tissue that covers the root of the tooth and provides an

attachment for the periodontal ligament fibers to the tooth (Figure 5). The cementum contains no blood or

lymph vessels, has no innervation, and does not undergo physiologic resorption or remodeling, but it is

continually deposited throughout the life of the organism. The cementum’s main function is to attach the

fibers of the periodontal ligament to the tooth, but the cementum also contributes to the process of repair

after damage to the root surface.

25  

Figure 5. A histological cross-section illustrating the periodontal ligament between two molars. The

periodontal ligament, containing brush-like fibrils of collagen, anchors the tooth in the pocket by

connecting cementum layers between teeth and the alveolar bone. The Safranin O-stained lower jaw of a

wild type mouse, magnification 400×.

In the case of periodontitis, persistent inflammation leads to severe tissue degradation that

includes the degradation of the periodontal ligament. The cementum layer is gradually resorbed, and the

fibers of the periodontal ligament loosen. Eventually this leads to the flexibility of the tooth and, in a worst

case scenario, to the loss of teeth accompanied by severe alveolar bone degradation.

2.1.3. Alveolar bone in health and disease

The alveolar bone, also called the alveolar process, is a specialized part of the mandible

(lower jaw) and maxilla (upper jaw) that forms the primary support for the teeth. The alveolar bone

consists of two components: cortical bone (also called the alveolar proper), which lines the walls of tooth

sockets, and the cancellous (trabecular) bone, which fills up space between the sockets and between the

compact jaw bone walls. Compared to other bone tissues in the body, the alveolar bone undergoes more

rapid remodeling due to tooth eruption and masticatory demands. Osteoblasts and osteoclasts are

involved in bone remodeling and form and resorb the mineralized connective tissues of bone, respectively.

The balance between the resorption by osteoclasts and the formative properties of osteoblasts must be

tightly regulated. The formation of bone involves the proliferation of stem cells and their differentiation

into osteoblasts. This differentiation is regulated by various hormones, cytokines, and growth factors. The

organic matrix that is produced by osteoblasts consists predominantly of type I collagen and various other

non-collageneous bone proteins. Following maturation, osteoblasts may undergo apoptosis, become

encased in matrix as osteocytes, or remain on the bone surface as bone lining cells [190]. Resorption of

mineralized bone requires osteoclasts, which are produced by the monocyte/macrophage lineage of

hematopoietic cells derived from bone marrow [190]. Osteoclasts are large, multinucleated cells with a

26  

ruffled border on one side of the cell. These cells are located in small cavities called Howship’s lacunae

that are formed during the digestion of the underlying bone. Resorption occurs through the release of

acidic substances that are able to dissolve the mineralized matrix of the bone. The remaining organic part

of the bone is eliminated by osteoclastic enzymes and phagocytosis [185].

Figure 6. The relationship between an inflammatory infiltration and periodontal bone loss. When

inflammatory infiltrate is at a distance from bone (left panel), it causes a mild, reversible form of oral

disease – gingivitis. If inflammation moves closer to the bone (right panel), osteoclastogenesis is induced

and irreversible bone resorption occurs that leads to a severe, irreversible form of oral disease –

periodontitis. Modified from D. T. Graves et al [191].

Inflammation and bone loss are the main symptoms of periodontitis (Figure 6). The

inflammation-mediated bone loss in periodontitis is enhanced by osteoclast activity without any

corresponding increase in bone formation [192]. Bone resorption during periodontitis depends on the

concentration of proinflammatory cytokines, such as IL-1, IL-6, IL-11, IL-17, and TNF-α, in gingival

tissues and how far these inflammatory mediators penetrate the alveolar bone [193, 194]. The combined

inhibition of IL-1 and TNF-α in an experimental model of periodontitis reduced alveolar bone loss by up

to 60% [195]. Receptor activator of nuclear factor kappa-B ligand (RANKL) is critical for bone metabolism

regulation because it is a key mediator in the process of osteoclast formation. RANKL is expressed mainly

on osteoblasts, and its increased expression can tip the balance towards osteoclastogenesis and an

increase in the bone loss observed during the development of periodontitis [192]. Thus, there is a great

need for a treatment to stimulate the formation of the alveolar bone that is lost during periodontal disease.

2.2. Pathogenesis of periodontitis

Bacteria and their pathogenic components are responsible for initiation of the tissue

destruction that occurs during the development of periodontitis. Pathogens that cause periodontitis have

classically been identified as gram-negative anaerobic bacteria that can survive in the gingival sulcus (the

27  

space between the tooth and the adjacent gingival epithelium) [191, 196]. Over 700 species of bacteria

have been identified in the human oral cavity, and one individual usually has approximately 100–200

different species of periodontal bacteria. Porphyromonas gingivalis, Tannerella forsythia, and

Aggregatibacter actinomycetemcomitans have been identified as key pathogens [197]. Interestingly, the

most pathogenic complex, comprised of Porphyromonas gingivalis, Tannerella forsythia, and

Treponema denticola, was shown to be dependent on prior colonization of the tooth pocket by a complex

of less pathogenic organisms [198]. Dental plaque is a biofilm containing several species of colonizing

bacteria [199]. Bacteria induce tissue destruction indirectly by activating host defense cells, which in turn

produce and release inflammatory mediators that stimulate connective tissue destruction [196]. The

components released by bacterial plaque induce the infiltration of inflammatory cells including

lymphocytes, macrophages, and polymorphonuclear leukocytes (PMNs) [200]. Bacterial LPS activates the

macrophages to synthesize and secrete the proinflammatory cytokines IL-1 and TNF-α, prostaglandins

(especially PGE2), and hydrolytic enzymes. The cytokines IL-1 and TNF-α are the main proinflammatory

mediators that lead to tissue and alveolar bone degradation in periodontitis [195, 201]. IL-1 and TNF-α

influence endothelial cells to recruit neutrophils and monocytes to the inflammation site. Moreover, IL-1

stimulates the macrophages and gingival fibroblasts to produce PGE2, a vasodilator and a cofactor

involved in increased vascular permeability and bone demineralization. Periodontal research has

traditionally focused on the pathogenesis of periodontitis by analyzing bacterial infection and risk factors,

but there have been a number of recent studies that highlight the host response factors that actually drive

the development of periodontal disease.

2.3. The plasminogen activator system in periodontitis

The PA system plays a central role in the degradation of the ECM by generating plasmin

that can degrade non-collagenous ECM proteins such as laminin and fibronectin. Plasmin can also act

indirectly by activating proMMPs into proteases that are specific for other connective tissue proteins such

as elastin and collagen [202]. The role of MMPs in gingival/periodontal tissues has been extensively

studied, while the PA system, which has a central regulatory role, has received less attention [203].

All members of the PA system are localized to periodontal tissues. Plasmin has been found

in the gingival fluid of both healthy and diseased patients, and the level of plasmin activity has been

observed to increase along with the severity of periodontitis and is related to the bone resorption rate

[204]. The concentrations of tPA and PAI-2 are approximately 300- and 10-times higher, respectively, in

the gingival cervical fluid compared to their plasma levels [205], and uPA and PAI-1 have also been

detected in gingival cervical fluid at concentrations above normal plasma levels. These factors are thought

to be produced locally [205]. In vitro studies have shown that gingival fibroblasts can alter their

production of tPA and PAI-2 in response to exposure to LPS from the periodontal pathogens A.

actinomycetemcomitans, F. nucleatum, and P. gingivalis [206]. Furthermore, the expression of uPA and

uPAR by gingival fibroblasts increases following exposure to LPS from P. gingivalis [207]. Bacteria induce

tissue destruction indirectly by activating host defense cells, which in turn produce and release mediators

that stimulate the effector molecules of connective tissue breakdown [199].

The direct involvement of plasminogen in oral health is demonstrated by human

plasminogen deficiency. Clinical plasminogen mutations have been reported in patients with ligneous

28  

gingivitis/periodontitis, which is a rare genetic periodontal disease with gingival enlargement and

periodontal breakdown [208-211]. In these patients, the disease re-occurs even after surgical treatments

or the application of heparin combined with antibiotics [212].

In Paper I of this thesis, we show that the lack of plasminogen in mice leads to

periodontitis. The intravenous restoration of plasminogen for 10 days in plasminogen-deficient mice

resolved the persistent inflammation of the gum tissue and induced alveolar bone regeneration.

Preventing or minimizing alveolar bone degradation is a major clinical aim in dentistry, and the

restoration of bone mass is extremely difficult. The regeneration of periodontal tissues involves complex

interactions between different cell types, the extracellular matrix, and local mediators such as growth

factors and cytokines. There are many conventional treatments that are currently used for periodontitis,

but few of them result in the complete regeneration of periodontal soft tissue [213]. The development of

therapeutic treatments to treat bone diseases are more focused on inhibition of the formation or activity of

osteoclasts, and far less attention has been paid to promoting bone formation [214]. Based on our data, we

suggest that plasminogen could be a candidate for the treatment of periodontitis by promoting the

clearance of persistent inflammation, stimulating the healing process, and, possibly, regenerating the

alveolar bone.

2.4. Animal models in periodontal research

Several animal models have been developed for studying the events associated with

inflammatory periodontal disease. Selection of the appropriate animal model depends on the similarity of

the periodontium and the nature of the disease to that of humans. In most mammalian species, the

inflammatory destruction of the periodontium can be spontaneous or experimentally induced.

Non-human primates have very similar dental formulas, root morphologies and

periodontium structure to humans, and they also have similarly occurring dental plaque, calculus, and

oral microbial pathogens (e.g., P. gingivalis). Late in life, these primates often acquire naturally occurring

periodontal disease. Although periodontitis in primates most closely resembles the human disease, the

expense and special requirements of these animals limit their use in periodontal studies [215]. In addition,

they are prone to infectious diseases such as tuberculosis, which makes them a less practical model for

studying periodontal diseases.

Mouse models make up the bulk of animal studies related to periodontal research [216].

The availability of different strains and transgenic mice allows for the study periodontal disease under

well-defined conditions [217, 218]. We have utilized mouse models in our studies, which has allowed us to

use mice deficient in plasminogen and PAs.

Rats and hamsters, like mice, have different dental formulas compared to humans, but

rats are often used as models of experimental periodontitis because the periodontal anatomy in the molar

region shares some similarities with that of humans. Furthermore, rats are easy to handle, can be obtained

with different genomes and microbial status, and are easily induced with infections. There is clear

evidence from the literature demonstrating horizontal bone loss in rats infected with A.

actinomycetemcomitans [219] and P. gingivalis [220].

29  

Beagle dogs are also widely used in periodontal research. They have two sets of teeth,

primary and permanent, with similar root morphology as humans. Gingivitis and periodontitis lesions in

dogs are more closely related to humans than other non-primate laboratory animals [221].

Each animal model for periodontal disease has advantages and disadvantages; finding an

animal model of periodontal disease that can reproduce all the aspects of human disease is difficult.

30  

3. Wound Healing

Wound healing is a very complex biological process that requires the interplay between

different tissue structures, a large number of resident and infiltrating cell types, and a variety of factors

released into the cellular milieu. The tissue damage must be repaired quickly to avoid complications, such

as blood loss and infection, and to reduce scarring. There are a number of wound types – incisions

(surgeries, accidents), excisions, burns, ulcers, and chronic wounds due to diabetes mellitus, infections, or

immunodeficiencies. The discovery of effective therapeutic treatments, that accelerate wound healing and

perhaps even result in non-scarring healing of the wound, would be of great benefit clinically. However,

due to the multiplicity and complexity of wound healing events, this is challenging.

3.1. Phases of wound repair

The healing of all wound types share the same ordered steps that can be divided into three

overlapping phases: the inflammatory phase, the proliferative phase (or the re-epithelialization phase),

and the remodeling phase [222]. Wound healing involves many cell types, as well as the ECM, and

chemical mediators. Each cell type has a specific function (or functions) in achieving normal repair of

damaged tissue, and disturbances of the healing process can lead to complications such as delayed and

improper healing, infection, and scarring.

3.1.1. The inflammatory phase

One of the first steps in wound healing is blood clotting, which is induced by platelets

exposed to collagen and the ECM at the site of the wound (Figure 7). The platelets release clotting factors,

growth factors, and cytokines [223], and this initiates hemostasis in the form of a fibrin clot and wound

contractions. The clot, formed from fibrin and fibronectin, also provides a matrix scaffold for the

recruitment of monocytes, fibroblasts, neutrophils, and endothelial cells to the injured site where the

inflammatory phase occurs [224].

Lymphocytes, monocytes and neutrophils are the main immune cells active in the

inflammatory process and subsequent tissue repair [225]. During the inflammatory phase, neutrophils are

first to infiltrate the wound site and play a key role in protecting the tissue against infection. These

neutrophils are responsible for phagocytosis, activation of the complement system, and producing

antibacterial oxygen radicals. However, the reactive oxygen species and enzymes (e.g., myeloperoxidase)

that are released by neutrophils may also cause damage to the host tissue. During the latter part of the

inflammatory phase, macrophages migrate into the wound site and phagocytize the neutrophils. At the

same time, the macrophages supply the wound with a continuous stream of cytokines and growth factors

that are necessary for the formation of new tissue in the wound [226].

31  

Figure 7. The inflammatory phase of wound repair three days after injury. When a blood clot is formed,

the platelets release growth factors and cytokines. Neutrophils are the first to arrive at the site of wound to

phagocytize debris, pathogens, and dead cells. Macrophages then migrate to the wound to clear the

neutrophils and to release cytokines and growth factors. The keratinocytes at the wound edges start to

migrate towards each other to close the open wound. Modified from R.A.F. Clark [222].

3.1.2. The proliferative phase

Within a few hours after a wound has been made, the epithelial cells at the edge of the

wound undergo a phenotypic transition in response to growth factors, such as platelet-derived growth

factor, transforming growth factors α and β, and cytokines [226]. These epithelial cells loosen from the

basement membrane and, with help of integrin receptors, migrate over the wound separating the

hemostatic scab from the underlying viable granulation tissue [227, 228]. The degradation of the ECM

and collagen through the activation of plasminogen and MMPs allows migrating epidermal cells to dissect

their way between the collagenous dermis and the fibrin clot [229]. After the first or second day post-

injury, epidermal cell proliferation is induced behind the actively migrating keratinocytes to maintain the

thickness of the epidermal cell layer [222, 226].

Fibroblasts that migrate to the wound site produce new ECM and collagen that are

deposited in the granulation tissue (Figure 8). The amount of collagen deposited during this phase is a

determining factor for the tensile strength of the tissue after healing. The collagen also serves as a scaffold

for the keratinocytes to migrate on and adhere to as soon as they separate the granulation tissue from the

fibrin clot [230]. The epidermal attachment to the basement membrane is re-established in a zipper-like

fashion [231, 232]. The formation of new blood vessels is a complex process, which relies on the ECM and

a number of growth factors [233]. Increased angiogenesis at the wound site is important in providing

essential nutrients during the wound repair process [234].

32  

Figure 8. The proliferative phase of wound repair five days after injury. During this phase, the wounded

area is divided into the hemostatic scab and granulation tissue. The granulation tissue consists of collagen

and the ECM that has been newly produced by fibroblasts. Angiogenesis occurs, and the vascular network

is re-established in the wounded area. Modified from R.A.F. Clark [222].

3.1.3. The remodeling phase

The matrix formation and the remodeling phase follow the re-epithelialization and

granulation formation phase. During this remodeling phase, the collagen formed in the granulation tissue

is collected into larger bundles that are held together by an increasing number of cross-linkages [235]. The

remodeling of collagen is crucial for the strength of the healed tissue and scarring. The collagen

remodeling process is dependent on the continuous synthesis and degradation of collagen that is

controlled by several of the proteolytic enzymes released by macrophages, fibroblasts, and epidermal and

endothelial cells [222]. Unfortunately, the tensile strength of a mature scar never exceeds 70–80% that of

undamaged skin [236, 237]. Depending on the severity of injury, this remodeling phase can take over a

year.

3.2. The plasminogen activator system in wound healing

The PA system is intricately involved in all of the processes that are important for wound

healing, including fibrinolysis, ECM degradation, activation of proMMPs, regulation of cell migration,

release and activation of growth factors, angiogenesis, and collagen remodeling [146, 229, 238-240].

Using PA- and plasminogen-deficient mice, different groups have shown the importance of plasminogen

in wound healing [144, 241, 242][Paper II and paper III in this thesis]. Initially it was reported that the

lack of plasminogen in mice leads to impaired wound repair. However, even in plasminogen-deficient

mice, cutaneous wounds will eventually achieve complete epithelial closure (144). Treatment with the

MMP inhibitor galardin, in combination with plasminogen deficiency, arrested wound healing completely

33  

in plasminogen-deficient mice [229]. Wound closure in mice that are doubly deficient in uPA and tPA is

not as severely impaired as it is in plasminogen-deficient mice [145]. Notably, treatment of doubly

deficient uPA and tPA mice with the MMP inhibitor galardin further delayed, but did not completely

block, wound closure, which occurred in plasminogen-deficient mice treated with galardin [145].

Furthermore, the impaired wound re-epithelization in plasminogen-deficient mice was fully restored by

an additional deficiency in fibrinogen [146]. In a different type of wound healing model – tympanic

membrane perforation – the healing process was shown to be completely arrested in the absence of

plasminogen [241]. This earlier study of tympanic membrane healing was the impetus for us to further

investigate the novel roles of plasminogen in wound repair. Our studies described in Paper II show that

burn and incision wounds successfully re-epithelialized with some delay in plasminogen-deficient mice.

However, neutrophils and fibrin deposits are present in the underlying tissues even after 60 days of

healing, and the healing is, therefore, not complete. In contrast, healing was restored in plasminogen-

deficient mice that were treated with human plasminogen, and the quality of healed burn wounds was

comparable to that of wild-type mice.

Plasminogen has been continuously observed in human wound biopsies analyzed at 10 days

after a wounding event in association with macrophages and fibroblasts. uPA was also detected in

macrophages and fibroblasts, whereas uPAR is only observed in macrophages of human wound

granulation tissue [243]. uPAR provides a mechanism to localize the production of the proteolytic activity

to the wounded area. The expression of uPA and uPAR, as well as PAI-1, is induced in the migrating

epithelial cell layer early during the re-epithelialization phase, when the proteolytic activity of plasmin

plays crucial role [144, 244, 245]. uPAR is also important for signal transduction in platelets and during

cell migration [246-249].

Increasing in vitro evidence also suggests that plasminogen is able to induce cell signaling

and cause the release and/or activation of growth factors, expanding the role the PA system in wound

repair [23, 250, 251]. Our data in Paper III demonstrate that plasminogen is a key regulatory molecule

with a pronounced pro-inflammatory effect that potentiates the early inflammatory response during

wound healing via the STAT3 pathway.

3.3. Cutaneous wound models in mice

Wound healing disorders are likely to increase, especially as the rates of diseases such as

diabetes, hypertension, and obesity increase. Animals have been used to experimentally determine the

molecular and cellular mechanisms underlying and controlling the healing process. A variety of

specialized wound models, such as those for incisions, excisions, burns, and chronic wounds, are needed

because each wound type has a unique healing pattern that depends on which tissues are wounded and

the manner in which they are wounded. The most common types of wounds established in mice models

are incision and excision wounds, burns and ulcers. The use of gene deficient or transgenic mice provides

an opportunity to study the role of specific genes during the wound healing processes.

The immediate reaction to an incisional or excisional injury is the formation of a fibrin-rich

hemostatic clot to stop the bleeding. Incision wounds are very abundant in a number of surgical

procedures and cause the least damage to epithelial and connective tissues. Incision wound models are the

most commonly used for studying the cellular mechanisms of wound healing with the aim of accelerating

34  

the healing process and diminishing scaring. Like all other wound types, the healing of excisional wounds

follows the inflammatory, proliferative and remodeling phases of wound healing but requires greater

amounts of granulation tissue formation, re-epithelialization, and cell proliferation. The excision model is

often used to monitor the effect of treatments on the rate of wound healing and to dissect the molecular

mechanisms of wound healing.

Burns cause cauterization of the blood vessels, and the top layer of the skin becomes

necrotic. Morphologically, severe burn wounds are divided into three layers. The top layer of the wound

consists of necrotic tissue that forms a burn scab. The underlying zone of stasis is still viable but risks

ischemic damage due to the decreased perfusion of oxygen to the area. The hyperemia zone sustains the

least amount of cellular damage and responds to the injury with vasodilatation and increased blood flow

[252].

Diabetes is one of the major causes of impaired wound healing [253, 254]. Patients tend to

develop wounds and have difficulty healing simple wounds, which often become chronic and infected.

Therefore, mouse strains that develop insulin resistance, for example, obese (ob/ob) and diabetic (db/db)

mice, are widely used as a chronic wound model [255].

35  

Results and Discussion

This chapter summarizes the three papers (Paper I-III) included in this thesis. The figures cited

correspond to those used in the original papers.

Plasmin is essential in preventing periodontitis in mice (Paper I)

Periodontitis involves infection, inflammation of the periodontium, degradation of gum

tissue, and alveolar bone resorption, all of which are processes in which the PA system is tightly involved.

We first monitored the oral health of plasminogen-deficient mice in comparison to wild-

type mice. Interestingly, all of the plasminogen-deficient mice developed periodontitis, while no

pathological changes were observed in the periodontal tissues of wild-type mice up to 20 weeks of age

(Figure 1). The alveolar bone in wild-type mice was intact (Figure 2A), but in plasminogen-deficient mice,

it was severely degraded (Figure 2B). The quantification of alveolar bone degradation by measuring the

distance between the CEJ and the alveolar bone crest revealed a significant age-associated increase in

bone degradation in plasminogen-deficient mice (Figure 2C). Furthermore, extensive neutrophil

accumulation was detected in the diseased gums of plasminogen-deficient mice, showing that recruitment

of inflammatory cells to the site of inflammation was not impaired (Figure 3D, F, and H). Fibrin deposits

were detected only in the damaged gum tissue (Figure 4D and 4F), suggesting that these deposits are a

consequence of disease, not the cause of it. Additionally, we demonstrated that mice doubly deficient in

uPA and tPA developed periodontitis in an identical manner to plasminogen-deficient mice (Figure 5),

leading to the conclusion that the proteolytic activity of plasmin is essential for normal oral health.

The supplementation of plasminogen-deficient mice with human plasminogen for 10 days

reestablished the oral health of two age groups of mice (8–12 and 17–20 weeks of age); the necrotic tissue

was significantly reduced (Figure 7A and 7B) and new epithelium and connective tissue fibers formed and

reattached (Figure 6C and 6D). Surprisingly, alveolar bone re-growth was observed along with soft gum

tissue regeneration (Figure 7C and 7D). The distance between the CEJ and alveolar bone crest in

plasminogen-treated plasminogen-deficient mice decreased by 17 to 25% and indicated a significant re-

growth of alveolar bone. Alkaline phosphatase staining confirmed osteoblast activity at the site of bone

formation (Figure 7E). As expected, there was no alkaline phosphatase activity detected at the alveolar

bone of PBS-treated plasminogen-deficient mice.

The data collected for this manuscript revealed the importance of plasmin for maintenance

of healthy periodontal tissues and that plasminogen has the potential for future development into a

therapeutic treatment for periodontal diseases.

36  

Plasminogen is a critical regulator of cutaneous wound healing (Paper II)

In a previous study, the lack of plasminogen caused a delayed healing of cutaneous incision wounds in

mice, which was caused by slower fibrin degradation in the wound and subsequent impairment of

keratinocyte migration over the wound [144]. Full re-epithelialization in some plasminogen-deficient mice

was not reached until 20–45 days, while in wild-type mice, full re-epithelialization occurred within 10

days [144]. However, our data on the role of plasminogen in tympanic membrane healing had revealed

that plasminogen is essential in healing of tympanic membrane perforations [242]. We therefore extended

our studies by monitoring cutaneous wound healing for up to 60 days in plasminogen-deficient mice. Both

incisional and burn wounds were used. Incision and burn wounds differ in the type of tissue damage.

Incision wounds have minimal damage to the surrounding tissue and form a fibrin-rich hemostatic clot,

whereas burn wounds caused by heat give, in addition to a clot, a layer of necrotic tissue closest to the heat

source and demand more effort to re-epithelialize the broader wound area.

We have confirmed that incision wounds in plasminogen-deficient mice eventually achieve

wound closure. However, morphologic Masson-Trichrome staining revealed a lower density of collagen in

re-epithelialized wound areas in plasminogen-deficient mice compared to wild-type mice (Figure 1F and

1H). Immunohistochemical staining allowed us to detect the accumulation of neutrophils, fibrin, and

fibrinogen in re-epithelialized incision wounds of plasminogen-deficient mice, even after 30 and 60 days

post-wounding (Figure 2E-H). Neutrophils, fibrin, and fibrinogen were absent in re-epithelialized incision

wounds of wild-type mice after 14 days (Figure 2A and 2B). Using a burn wound model, we showed that

plasminogen deficient mice also had delayed healing compared to wild-type mice (Figure 3). Neutrophils,

fibrin, and fibrinogen accumulated under the re-epithelialized area of the burn wound in plasminogen-

deficient mice after 30 and 60 days post-wounding (Figure 4E-H), and none of these were detected in

wild-type mice after 14 days of healing (Figure 4A and 4B). The persistent accumulation of neutrophils

around the edge of the wound in plasminogen-deficient mice suggests a nonresponsive inflammatory

system in these mice, and there remains the possibility that the presence of fibrinogen might play a role as

a chemoattractant for neutrophils [224, 256].

We also performed plasminogen reconstitution experiments to determine if daily

intravenous or subcutaneous injections of purified human plasminogen could improve or even restore the

wound healing process in burn wounds of plasminogen-deficient mice. Intravenous injections were

started at the time of the wounding and continued for two weeks. After four to five days of treatment of

plasminogen-deficient mice with human plasminogen, the rate of wound healing was similar to the

control group. However, at 7 days post-wounding, the plasminogen-treated group demonstrated increased

speed in wound closure and the acceleration of healing continued for the duration of the treatment period

(Figure 5A). After two weeks, the wounds of the plasminogen-treated mice had completely healed (Figure

5C), while approximately 40% of the wound area was not re-epithelialized in the PBS-treated mice (Figure

5A and 5E). The morphology and time of healing of the plasminogen-deficient mice that were treated with

plasminogen mimicked the wild-type mouse burn wound healing pattern (Figure 3C and 5C).

Subcutaneous injections around burn wounds were started five days after administration of

the burn in plg-deficient mice. During the first five to seven days of treatment, there was no obvious

37  

improvement in the healing of plasminogen-treated mice compared to PBS-treated plg-defient mice. By

two weeks after wounding, however, the mice treated locally with plasminogen showed an increased

wound healing rate compared to control mice, and only 25% of the wound area had yet to be re-

epithelialized (Figure 5A). Plasminogen-treated mice had completely healed wounds (Figure 5D), while

PBS-treated mice still had 10% of the open wound remaining on the last day of treatment (Figure 5B and

5F).

Improper and slow wound healing is a major concern in medical care. Delayed re-

epithelialization might lead to wound infection and, in the worst case scenario, to sepsis. In addition,

proper wound closure and maturation is very important to avoid scarring. Our results show that

plasminogen could be a possible therapeutic agent for improvement of wound healing by accelerating the

healing rate and resolving persistent inflammation around the wound area.

Plasminogen is a key pro-inflammatory regulator that accelerates the

healing of acute and diabetic wounds (Paper III)

The finding that plasminogen, in addition to its fibrinolytic properties, has the ability to

affect inflammatory processes and to induce cell signaling encouraged us to continue more detailed

studies on wound healing to dissect the molecular mechanisms behind plasminogen and to test whether

plasminogen can be used as a therapeutic agent. In the current study, we used a standard burn injury

mouse model to explore the healing potential of plasminogen and to reveal the molecular mechanism

underlying its effect in acute and diabetic wounds.

In the present study, we demonstrated that plasminogen supplementation improves and

speeds up skin wound healing in wild-type mice. Full-thickness standardized burn wounds were induced

in wild-type mice, and immediately after the injury, the mice were given 2 mg of plasminogen by

intravenous injection. The control group received 2 mg of bovine serum albumin (BSA) by intravenous

injection. Quantification of the wound area at different time points showed that from day 6 post-injury,

the healing rate of the plasminogen-treated group was significantly faster compared to that of the control

group (Figure 1A). A morphological analysis at 11 days post-injury revealed that the epithelium layer in the

control group remained open and was covered by a large scab (Figure 1C). However, in the plasminogen-

treated group, the re-epithelization was complete and only a small scab remained lightly attached above

the wound (Figure 1D). Furthermore, we also demonstrated that plasminogen levels specifically increase

at the inflammation site and in a limited area around the wound. As shown in Figure 2A, the plasminogen

levels were highest during the early inflammatory phase, became lower during the tissue formation phase,

and nearly returned to basal levels during the late tissue remodeling phase. Moreover, a spatial analysis

demonstrated that the accumulation of plasminogen was wound-specific (Figure 2B and 2C). The level of

plasminogen at the wound site was increased up to 20-fold when wild-type mice were supplemented

intravenously with plasminogen (Figure 2D). Taken together, these data suggest that the healing effect of

plasminogen is related to its wound-specific accumulation and that the extent of the plasminogen

accumulation can be further enhanced by supplementation with exogenous plasminogen.

38  

Inflammatory cell depletion experiments revealed that the accumulation of plasminogen at

the wound site is mainly due to migrating neutrophils and macrophages. Plasminogen seems to be

transported to the inflammation site bound to surface receptors present on these cells. As shown in Figure

3A, there was no significant reduction in plasminogen accumulation in the wounds of fibrinogen-depleted

mice compared to the control group. This indicates that the binding of plasminogen to fibrin is not a

major mechanism for the local accumulation of plasminogen in the wound. However, the level of

plasminogen in the wound was reduced by 52% following macrophage depletion, compared with the no

depletion control group (Figure 3B). Similarly, the plasminogen accumulation was reduced by 23%

following neutrophil depletion (Figure 3C). The inflammatory phase-specific accumulation of

plasminogen at the wound site suggests that plasminogen has a regulatory role as a pro-inflammatory

regulator in wound healing. Thus, we measured the level of IL-6, which is an important mediator of

inflammatory host responses to tissue injury. As shown in Figure 4A, IL-6 levels in unwounded skin were

low irrespective of genotype and treatment. However, the level of IL-6 in the wounded skin of wild-type

mice was significantly higher than in the wounded skin of plasminogen-deficient mice. After plasminogen

treatment, the levels of IL-6 were enhanced in plasminogen-deficient mice and were further elevated in

the wild-type mice. Moreover, the level of phosphorylated STAT3 (pSTAT3), which is an intracellular

inflammatory mediator, was higher in wounded skin than in unwounded skin, and the pSTAT3 level was

further increased following plasminogen treatment (Figure 4B). These data indicate that during wound

healing, plasminogen treatment enhances the expression of pro-inflammatory cytokines and activates

intracellular signaling events.

An imbalanced inflammatory response is one reason for wound healing defects in diabetic

patients. To explore the healing potential of plasminogen in diabetic wounds, we utilized db/db mice,

which suffer from a severe wound healing impairment. Immediately after the burn injury, the mice were

intravenously treated with plasminogen, whereas in the control group, the mice received BSA. As shown in

Figure 5, the healing rate in the plasminogen-treated group was significantly faster than that of the control

group. Furthermore, we determined the plasminogen level in wounded and unwounded skin in db/db and

non-diabetic control mice. As shown in Figure 6A, the plasminogen levels in the unwounded skin were low

irrespective of genotype or treatment. After burn induction, the plasminogen levels in the wounds of the

db/db mice were only slightly enhanced compared with the control mice, where the plasminogen level

after wounding increased approximately 4-fold. However, after receiving systemic plasminogen treatment,

the plasminogen levels in the wounds of the db/db mice reached approximately the same level as in the

control mice and the IL-6 levels in the wounds of the db/db mice were clearly increased (Figure 6B). The

levels of pSTAT3 in the wound sites increased after wounding and further increased after plasminogen

treatment (Figure 6C). These data indicate that the impaired wound healing in diabetic db/db mice may,

in part, be due to the dysregulation of plasminogen. Plasminogen supplementation appears to rebalance

the early inflammatory response in these mice, which leads to improved wound healing.

Taken together, our data show that plasminogen is a key regulatory molecule with a

pronounced pro-inflammatory effect that potentiates the early inflammatory response during wound

healing. Administration of additional plasminogen not only accelerates the healing of acute wounds but

also initiates and improves the healing of chronic diabetic wounds. Plasminogen treatment may, therefore,

become a novel therapeutic strategy for the treatment of cutaneous wounds.

39  

Concluding Remarks

Plasminogen-deficient mice spontaneously develop periodontitis, and the administration of plasminogen

to these mice leads to regeneration of the periodontium and partial re-growth of the alveolar bone.

Plasminogen accumulated at the area of wound and is essential for proper cutaneous wound healing.

In addition to its role in extracellular matrix degradation, plasminogen plays a pivotal role as a key pro-

inflammatory factor that potentiates the early inflammatory response during wound healing.

Delivery of plasminogen to the wounded area seems to be dysregulated in diabetic wound healing, and

plasminogen supplementation results in significantly improved healing of diabetic wounds.

40  

Acknowledgements

I am lucky to meet many good helpful people and I appreciate each of them for supporting and making me

happy during all those years here in Umeå.

For my PhD time I would like to thank my supervisor Tor Ny. Thank you for sharing with me your

experience in research and teaching me how to become well organized and thoughtful. Best wishes and

huge thanks to previous and current group members – Guo for fun conversations, your smile and help;

Shen for knowing and sharing all lab news; Peter for your calm attitude, Elin – for ‘surviving’ with me

introduction to the lab; Patrik for conceding your lab-bench for me – it was very inspirational; Nina for

making our lab so organized and all the fun we had sharing an office; Chun – it was great to have you

around to talk and laugh; Jinan for periodontitis project. Kui Liu and his group, especially Reddy,

Wenjing and Krishna. Malgorzata for friendship and laughs, chance to share all, work related and

personal – without you I wouldn’t make it. Nelson, I still can’t decide to curse you or to thank you, in any

case I am very happy I got a chance to meet you. Tomas Lindh for your endless enthusiasm and good

mood. Thomas Borén, I extremely your knowledge, advices and generosity. Anna Arnqvist for

Christmas gatherings at the most welcoming lab. Richard Lundmark for helping me out by involving a

bit in your projects. Erik Lundgren and Kristina Ruuth for introducing me to research world at Umeå

University. Ulf Ahlgren for my happiest period in Umeå – you have a golden heart! Ingrid, the most

elegant and gorgeous woman I have ever met, for the friendship, advices and warmth. Kenneth for your

irreplaceable personality and immediate help in any situation. Elisabeth for your smile and always good

mood. Animal house staff, especially Lena for your hard work and enthusiasm. Cecilia Elofsson, my

sunshine especially when it comes to paper work/applications-your good mood was very supportive.

My colleagues/friends: Katja – for supporting me and always overpricing me, for best ‘caipirinha’ talks –

I miss you a lot. Annelie – you are my star, your goodness and lack of selfishness are just endless! Love

you very much. Sara – I love you for being so honest, smart, and fun. I always have a great time with you.

Anna S, Zhanna, Olena, Melissa, and Jenny – you are all so charming in so different ways and I

always enjoy your lovely company. Pär, Miriam, Mantas, Anna E, Isabelle, Louise, Göran L,

Erika, Emma, Peter, Karin, Linus, Lisette, Göran B, Anna G, Björn, Verena and Sarantis for

sharing and enjoying ‘coffee tea vodka’ time with me. Malin for finally making teaching enjoyable.

Nasim and Nils – it’s so simple to love you for being so cute and friendly. Clas – for so many things…

caring so much and making me smile all the time.

My new colleagues at Molecular Periodontology – Ingrid, Ulf, Lillemor, Inger, Anita, Chrissie,

Anders, Cecilia, Ali and Fredrik – you are very welcoming and caring – I am very lucky to work with

you. Special thanks to my gorgeous boss Pernilla – your smile can move mountains.

Lelle (Lennart Carlsson), you do have one of the biggest hearts – thank you for helping me! Amir and

Tomas for all that fun we had working together – thank you for being there for me in good and hard

times.

41  

Patrycja – you are so brave and gorgeous friend of mine – I have missed you so badly. Tomek for

hangouts together with nice food and great mood. Lijana and Fredrik – už jūsų neeilinį svetingumą.

Jūratei ir Dariui už jaukius jūsų namus, kur iškart pasijunti savas, aukšto pilotažo kulinariją ir krepšinio

transliacijas. Arūnui ir Ramunei už tai, kad jūs tiesiog nepaprastai smagūs ir svetingi – visada smagu

jus aplankyt. Eglei ir Ernestui – už džiazą ir skanėstus. Kotryna, ačiū, kad priverti mane pasitempti bet

kuriuo klausimu and Andreas for making our queen of dresses so happy. Milžiniškas dėkui draugams ir

šeimai Lietuvoj. Rokai, Rasele, Loreta, Dalyte, Arūnai ir Gintai – ačiū, kad visada jaučiuos laukiama

ir atvykus jaučiuos lyg niekad nebuvau išvykus. Jūs man be galo brangūs! Evelina, mano ištikimiausia

vaikystės drauge, tu mane įkvėpi savo kūrybingumu ir paprastumu. Jovita ir Evaldai, nuo studijų laikų

mes kartu ir nesvarbu, kad mus dabar skiria jūra. Ačiū jum už neišsenkantį laukimą.

Mamyte, už man įdiegtą optimizmą ir paveldėtą smagų charakterį, dar tik tavo stiprybės pritrūkau.

Sesutei Elytei, už nepakeičiamumą – visada buvimą su manimi, kur aš bebūčiau. Algimantui didžiulis

ačiū už visus reikalų tvarkymus ir pagalbą.

Rimui už kavą ir smagius pasakojimus, o Juzefai už nepamainomą pagalbą prižiūrint mažylius tiek

rašant šias tezes, tiek apsiginant.

Karoliui, Gretai ir Ariui už tą neapsakomą laimės ir džiaugsmo jausmą patiriamą kiekvieną dieną.

Paskutinės eilutės skiriamos labai ypatingam žmogui, kurio jau deja nėra tarp mūsų... Mylimam Tėčiui.

Žinau, kad labai džiaugtumeis ir didžiuotumeis manim. Prisiminimai apie Tave visada priverčia

nusišypsoti.

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