wykład i mechanizmy angiogenezy...
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Wykład I Mechanizmy angiogenezy
25.04.2010 Prof. dr hab. Józef Dulak
Zakład Biotechnologii Medycznej Wydział Biochemii, Biofizyki i Biotechnologii UJ
Email: jozef. [email protected]
Web page: http://biotka.mol.uj.edu.pl/zbm
William Harvey (1578-1657) using a deer to demonstrate the circulatorysystem. His research laid the foundation for a scientific approach to medicine.
William Harvey - 1628 the first systematic description of circulatory system
chicken embryo
Marcello Malpighi – observed capillaries in 1661
Wilhelm His – 1865 – coined the term „endothelium”,to differentiate the inner lining of body cavities from „epithelium”
Die Häute und Höhlen des Körpers. Basel, Schwighauser, 1865.A new classification of tissues based on histogenesis. In the present work, His put forth the basic concepts of tissue embryology. Using serial sections and three-dimensional models to illustrate his theories, he showed that the serous spaces in the embryo are mesodermal in origin and that they are lined by the special layer which he was the first to term "endothelial"
End of XIX/begining XX century –description of lymphatic system –Henryk Hoyer – Jagiellonian University
Diagram of the organization of lymphatic and vein systems in chordate1, general schema; 2, lamprey; 3, dogfish; 4, fish; 5, newt and salamander; 6, frog and toad; 7, lizard; 8, crocodile; 9, bird; 10, mammals.
Distribution of the surface lymphatic veins on the head of trout (Salmo trutta).
The vascular wall
Structure of blood vessels and lymphatics Structure of blood vessels and lymphatics
Jain R, Nature Med. June 2003
large vessel
Circulatory system and blood vessels
Endothelial cells
The crucial player in blood vessel formation
Endothelial cells in culture – cobblestone appearance
1973 & 1974 – Jaffe et al. & Gimbrone et al. –isolation of human endothelial cells (EC) from the umbilical vein
Jaffe EA, Nachman RL, Becker CG, Minick CRCulture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745-56
Gimbrone MA Jr , Cotran RS, Folkman J.Endothelial regeneration: studies with human endothelial cells in culture.Ser Haematol. 1973;6(4):453-5.Links
Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolavers of closely opposed, polygonal large cellswhereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor'sblood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.
Ades EW, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC, Lawley TJ. HMEC-1: establishment of an immortalized human microvascular endothelial cell line.J Invest Dermatol. 1992 Dec;99(6):683-90.
Endothelial cells
- one of the most quiescent and genetically stable cells of the body –turnover time is usually hundred of days
- proliferation is inhibited due to the contact with the capillary basement membrane
Endothelial cell markers used to identify microvasculature in tissues
Marker Ligand
CD31 (PECAM-1) CD31 on endothelial cells, leukocytes; glycosaminoglycans
CD34 L-selectin
CD54 (ICAM-1) LFA-1 integrin
CD62E (E-selectin) Sialyl-Lewis-X antigen and other carbohydrates
CD105 (endoglin) TGF-β1 and –β3
CD106 (VCAM-1) VLA-4 integrin
CD141 (thrombomodulin) Thrombin
CD144 (VE-cadherin) CD144 homotypic interaction
Endothelin receptor B (ETBR) ET-1
Ephrin B2 EphB4
EphB4 Ephrin B2
Tie-2 Angiopoietins
VEGFR-2 VEGF
von Willebrand Factor (vWF) Factor VIII
Uptake of Ac-LDL Scavenger receptor
Morphological differentiation of endothelial cells Morphological differentiation of endothelial cells
Skeletal muscle, heart,lung, brain
Endocrine and exocrine organsintestinal villi
Cleaver O & Melton DA, Nature Med., June 2003
How the blood vessels are formed?
Blood vessel formationa.) vasculogenesis:
de novo blood vessel generation from vascular progenitor cells
b.) angiogenesis:
formation of new blood vessels via extension or remodeling of existing blood vessels
c.) arteriogenesis:
maturation of blood vessels via increasing
the lumen of vessels
Blood vessel formation
• Vasculogenesis: a.) during embryonic development;
• Angiogenesis:a.) embryonic development
b.) adulthood: wound healing, menstrual cycle, tumour-angiogenesis…
b.) during adulthood associated with circulating progenitor cells
angioblast capillary
bFGF
VEGF
VEGF
Ang1, bFGF
Vasculogenesiscapillaries are formed from
vascular progenitor cells
Angiogenesis formation of new blood vessels from pre-existing vessels
Arteriogenesisformation of mature blood vessels; differentiation into
veins and arteries
Three ways of formation of blood vessels Three ways of formation of blood vessels
MCP-1, PDGF
Ang-2
Major growth factors and receptors involvedin blood vessels formation
VEGF – vascular endothelial growth factorsVEGF-A – crucial mediator of angiogenesis
VEGF-R – receptors for vascular endothelial growth factors
Angiopoietins (Ang-1, 2) Tie- 2 – receptor for Ang-1, -2
FGFs – fibroblasts growth factors
PDGF – platelet-derived growth factor
EC – endothelial cellP – pericyteF – fibroblastVEGF – vascular endothelial
growth factorPDGF – platelet-derived
growth factorAng-1 – angiopoetin-1
Mechanisms of new blood vessels formation
Vasculogenesis
EC
P
EndothelialProgenitor
Cell
Capillaryblood vessel
Angiogenesis
EC
P
EC
EC
P
EC
P
Capillaryblood vessel Network of capillaries
VEGF
Arteriogenesis
EC
P
EC
SMCSMC
SMCSMC
F
F
Primaryblood vessel
Mature artery
increased blood flowVEGF + PDGFVEGF + Ang-1
Model organisms in vascular research
Vasculogenesis Vasculogenesis begins very early after the initiation of gastrulation in the mammalian embryo, with the formation of the blood islands in the yolk sac and angioblast precursors in the head mesenchyme
The human yolk sac is a membrane outside the embryo that is connected by a tube (the yolk stalk) though the umbilical opening to the embryo's midgut. The yolk sac serves as an early site for the formation of blood and in time is incorporated into the primitive gut of the embryo
capillaries are formed from vascular progenitor cells
Vasculogenesis
1. First phase• Initiated from the generation of hemangioblasts;
2. Second phase• Angioblastsproliferate and differentiate into
endothelial cells
3. Third phase
• Endothelial cells form primary capillary plexus
Formation of a vascular network
Hematopoiesis in zebrafish (Danio rerio)
Expression of the flk-1 represents the earliest marker of the developing endothelial lineage during vasculogenesis
SCL transcription factor is crucial for the developmentof blood cells and blood vessels
Common vascular progenitor cells Common vascular progenitor cells for endothelial cells and for endothelial cells and
vascular smooth muscle cells vascular smooth muscle cells
Hemangioblast
Angioblast
Venous Arterialendothelium endothelium
VEGF
Commonvascular
progenitor cells
(flk-1+)
VEGF
PDGF-BBPericytes/smooth muscles
progenitor cells of vascular smooth muscles
(various forms)
Blood cells Skeletal muscles
Origin of endothelial and mural cells from different progenitor cells
PDGF-BB
Vasculogenesis in adult
Vasculogenesis occurs also in adult organismVasculogenesis occurs also in adult organism
Previously, postnatal vascularization was thought to occur exclusively due to angiogenesis. However, recent investigations have shown that vasculogenesis is involved in blood vessel formation also during postnatal live and the cells responsible for that are EPCs.
JCI 1999;103:1231-1236
• Classic Paradigm: Angiogenesis– Mature ECs migrate and
proliferate to form new vessels
• New Model:Angiogenesis + Vasculogenesis– Bone Marrow derived EPCs
circulate to sites of neovascularization
Progenitor cells
Ancestor cells that can form mature cells to restore function in tissues
Endothelial progenitor cells
A primitive cell made in the bone marrow (or otherorgans) that can enter the bloodstream and go to areas
of blood-vessel injury to help repair the damage
Stem cells
Undifferentiated cells that can develop into any type of cell in the body
Adult stem cellsAdult stem cells
hematopoietic stem cells
mesenchymal stem cells
bone marrow
13
Differentiation patwhays for pluripotent bone marrow stromal cells
Plasticity of adult stem cells
the ability to form specialized cell types of other tissues
(also called transdifferentiation)
14
Endothelial Progenitor Cells
• 1997 Asahara et al. reported putative EPCs or angioblasts
– Cells display progenitor phenotype
– Differentiate into mature ECs in vitro– Participate in neovascularization when injected
into ischemic animal model
Science 1997;275:964-967
DiI AcLDL uptake
EPC
1:200 2h
Isolectin binding by EPC
control
isolectin
Lectin from Bandeira simplicifolia
Immunohistochemical characterisation of culture-expanded EPCs
Cytoplasmic factor VIII (von Willebrand factor)
Uptake of acetylated low density lipoproteins
2 weeks culture
tube formation on Matrigel
Dzau et al., Hypertension 2005,
Markers of endothelial progenitor cells
CD34VEGFR2CD133 (prominin, AC133)
CD133 is absent on mature endothelial cells and monocytic cells
Numerous origins of endothelial progenitor cells Numerous origins of endothelial progenitor cells
Urbich & Dimmeler, Trends Cardiovasc Med. 2004
Mechanisms of EPC homing and differentiation
Urbich & Dimmeler, Circ Res 2004
Vascular progenitors in the embryo and in the adults
Blood vessel formationa.) vasculogenesis:
de novo blood vessel generation from vascular progenitor cells
b.) angiogenesis:
formation of new blood vessels via extension or remodeling of existing blood vessels;
c.) arteriogenesis:
maturation of blood vessels via increasing
the lumen of vessels
Angiogenesis
• Majority of vascular development occurs via angiogenesis
• Growth of new blood vessels from existing vessels
• Two distinct mechanisms available
a.) sprouting angiogenesisb.) intussusceptive angiogenesis
Sprouting angiogenesis
• Sprouting: invasion of new capillaries into unvascularized tissue from existing mature vasculature
- degradation of matrix proteins
- detachment and migration of ECs
- proliferation
Sprouting angiogenesis
Intussusceptiveor non sprouting angiogenesis
- remodelling of existing vessels
- interendothelial contact is needed
- splits into two vessels
a: Remodeling: A pillar appears in close proximity of the branching angle
b,c: From the pillar a fold develops toward the tissue of the branching angle and finally the pillar tissue merges with the interstitium of the branching angle
d: As a result, the branching angle is relocated proximalad and the angle is altered
e: Pruning: A column of pillars arises close to the branching angleThe pillars can merge along a line indicated by the arrows. The eccentric location of the pillars can lead to alterations in the diameter of a vascular branch.
f: Ultimately, a branch can be completely pruned by repetition of the process (arrows along a theoretical line).
Scanning electron micrographs of chicken chorioallantoic membrane (CAM) vessels showing intussusceptive branching remodeling:
Burri, PH Dev Dyn. 2004
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Etapy angiogenezy Etapy angiogenezy
� zwiększenie przepuszczalności ściany i odkładanie włóknika�rozluźnienie warstwy komórek wspomagających (pericytów) � uwolnienie proteinaz przez komórki śródbłonka � trawienie błony podstawnej i macierzy pozakomórkowej otaczającej
naczynie krwionośne�migracja i proliferacja komórek śródbłonka� tworzenie struktur naczyniowych � łączenie (fuzja) nowych naczyń� inicjacja przepływu krwi
- zahamowanie proliferacji komórek śródbłonka - zahamowanie migracji komórek śródbłonka
� synteza błony podstawnej
Stages of angiogenesis Stages of angiogenesis
� increase in vessel permeability and thrombin deposition � loosening of pericyte contact � proteinase release from endothelial cells � digestion of basement membrane and extracellular matrix �migration and proliferation of endothelial cells � formation of vascular structures � fusion of new vessels � initiation of blood flow
- inhibition of endothelial cell proliferation - inhibition of the migration of endothelial cells
� formation of basement membrane
Endoglin is an auxiliary receptor for the transforming growth factor-beta family of cytokines and is required for angiogenesis and heart development.
Summary of the mechanisms of angiogenesis
arterio/venous
differentiation(ephrins/Eph)
Błona podstawna(basement membrane)
Specialised form of extracellular matrix
Basement membrane in various organs
Basement membrane of the blood vessels
- collagen IV - collagen XV- collagen XVIII- laminin - heparan-sulphate proteoglycans- perlecans - nidogen/entactin - SPARC/BM-40/osteopontin
Crucial role of metalloproteinases in angiogenesis
MMP-2 – gelatinase AMMP-9 – gelatinase B
MMP family MMP Name ECM Substrate
Collagenases Interstitial Collagenase (MMP-1) fibrillar collagens
Neutrophil Collagenase (MMP-8)
Collagenase-3 (MMP-13)
MMP-18 (Collagenase 4, xenopus collagenase)
Stromelysins Stromelysin-1 (MMP-3) laminin, fibronectin, non-fibrillar collagens
Stromelysin-2 (MMP-10)
Stromelysin-3 (MMP-11)
Matrilysins Matrilysin (MMP-7) PUMP laminin, fibronectin, non-fibrillar collagens
MMP-26 (Matrilysin-2, endometase)
The membrane-bound MMPs
MT1-MMP (MMP-14) gelatinase A, fibrillarcollagens, proteoglycans,ECM glycoproteins
MT2-MMP (MMP-15) gelatinase A
MT3-MMP (MMP-16) gelatinase A
MT4-MMP (MMP-17)
Gelatinases Gelatinase A (MMP-2) type I, IV, V and fibrillarcollagens; gelatin
Gelatinase B (MMP-9) type IV, V collagen,gelatin
Others MMP (examples) MMP-19 (RASI-1) gelatin
Enamelysin (MMP-20) type V collagen
metalloelastase (MMP-12) elastin
Metalloproteinases Metalloproteinases
Prodomain Catalytic domain Hemopexin
PRCGxPD
gelatinbinding
Zn2+
Zn2+
Zn2+
Zn2+
Zn2+
Collagenase family
Gelatinase family
MT-MMPs
MMP-7, MMP-23,MMP-26
Hingeregion
Stromelysin family
Transmembrane
Matrix metalloproteinases
Tissue inhibitors of metalloproteinases (TIMPs)
Proteolytic enzymes
Serine proteinase (plasminogen activators)
Matrix metalloproteinases (MMPs)zinc-dependent endopeptidases
Differentiation of endothelial cells on basement membrane Differentiation of endothelial cells on basement membrane
Physiological and pathological angiogenesis Physiological and pathological angiogenesis
Physiological angiogenesis in adults is restricted Physiological angiogenesis in adults is restricted
placenta uterus
Hair growthWound healing
New capillary formation in response to wounding
RheumathoidRheumathoidArthritis Arthritis
TumorsTumorsAIDS AIDS
complicationscomplications
Psoriasis Psoriasis
Infertility Infertility
SclerodermiaSclerodermia
Cardiovascular Cardiovascular diseasesdiseases
UlcersUlcers
StrokeStroke
Sight lossSight loss
angiogenesis
Exagerrated Exagerrated
InsufficientInsufficient
Take home messages
1. Three mechanisms of formation of blood vessels
2. Common origin of blood cells and endothelial cells
3. Endothelial progenitor cells play a role in blood vessel formation also in adults
4. Numerous mediators are involved in blood vessels formation
5. Physiological angiogenesis in adults is restricted, but it is a significant component of numerous diseases, such as cancer or atherosclerosis