osteo-progenitors in vascular calcification

551Journal of Atherosclerosis and Thrombosis　Vol.18, No.7

Review

Osteo-Progenitors in Vascular Calcification

Shripad Nagesh Pal and Jonathan Golledge

Vascular Biology Unit, Department of Surgery, School of Medicine and Dentistry, James Cook University, Townsville, QLD, Australia

Vascular calcification has been associated with the incidence of cardiovascular events and thus there has been interest in better understanding its pathogenesis. Early theories considered vascular calcification to be a passive process which occurred as a non-specific response to tissue injury or ne-crosis. More recent theories propose vascular calcification results from loss of molecular inhibitors or via an active cell mediated process. The origin of the cells responsible for vascular calcification is con-troversial and may vary in different sites and patients. Calcification has been reported as result of apoptosis or death of vascular smooth muscle cells for example. One novel source of cells controlling vascular calcification is from the bone marrow. A circulating immature bone marrow derived popula-tion has been identified and a small subset of this bone marrow population has been reported to pos-sess bone forming properties in vitro and hence termed osteo-progenitors. This article reviews evi-dence supporting the contribution of these naive bone marrow derived circulating osteo-progenitor cells in vascular calcification.

J Atheroscler Thromb, 2011; 18:551-559.

Key words; Aorta, Calcification, Atherosclerosis, Bone marrow, Osteo-progenitors

Introduction

Vascular calcification has been previously consid-ered as a complication of ageing and hence little sig-nificance has been given to its clinical consequences1); however, in the past two decades a large range of stud-ies have shown the association of vascular calcification, particularly coronary artery calcification, with inci-dent cardiovascular mortality2). These findings have fuelled a greater research focus on the aetiology, mech-anisms and consequences of vascular calcification2). Over the years, several molecular determinants have been reported to be associated with vascular calcifica-tion1, 2) (Table 1).

Previously, vascular calcification was considered to be a passive process in which elevated calcium and

phosphorous levels promoted apatite nucleation and crystal growth3); however, evidence over the past two decades suggests that it could be an actively regulated process that may arise via several different mechanisms3)(Table 2). More recently, a new mecha-nism that may contribute towards vascular calcifica-tion has been postulated3, 4). Ongoing studies suggest an active role for circulating cells arising from sources such as bone marrow (BM) in vascular calcification4-6). The focus of this review is the role of circulating cells originating from the bone marrow (BM) in vascular calcification (Fig.1).

Passive and active theories of vascular calcification

Vascular calcification has been considered to be a passive process which leads to uncontrolled precipita-tion of calcium phosphate associated with tissue ne-crosis and metabolic calcium and phosphate imbal-ance3, 7). Previously it was reported that calcification may be initiated at the sites of deposits of acellular material8, 9, 10). It was postulated that acellular calcifi-

Address for correspondence: Jonathan Golledge, The Vascular Biology Unit, Department of Surgery, School of Medicine and Dentistry, James Cook University Townsville, QLD, Australia 4811E-mail: [email protected]: January 18, 2011Accepted for publication: February 17, 2011

― A Circulating Cell Theory

552 Pal and Golledge 553Osteo-Progenitors in Vascular Calcification

Table 1. Molecular determinants of vascular calcification

Molecular protein Function

Collagen type Ⅰ may act as a nucleator3, 8, 18)

Elastin may act as a nucleator3, 8, 18)

Bone sialoprotein may act as a nucleator8, 18)

Osteopontin adhesion molecule which binds cells to apatite, thus inhibiting crystal growth7, 8, 17, 18)

Osteoprotegerin osteoclastogenesis inhibitory factor3, 8, 9, 18)

inhibitor of vascular calcification, knock-out mouse has medial calcification8, 9, 13)

Matrix Gla protein found in association with areas of calcification3, 8, 9, 15, 18)

Gla residues inhibitor of vascular calcification, knock-out mouse has medial calcification capable of binding to apatite8, 9)

Osteonectin high affinity for apatite and collagen3, 8, 17, 18)

Osteocalcin Gla residues capable of binding hydroxyapatite3, 8, 18)

Klotho inhibitor of vascular calcification, knock-out mouse has medial calcification3, 9, 18)

Examples of determinants of vascular calcification. Of the molecular determinants listed, the roles of OPG, OPN, MGP and Klotho have been studied in murine knockout models to determine their association with the progression of vascular calcifi-cation. (OPG: Osteoprotegerin; OPN: Osteopontin; MGP: Matrix Gla protein)

Table 2. Experimental evidence suggesting that vascular calcification is active

Investigations Summary of evidence

In vitro studies 1) hVSMC undergo calcification in vitro under the action of calcification-inducing factors such as β-glycerophosphate and dexamethasone.11, 12)

2) CVC of human origin undergo in vitro calcification, suggesting an osteogenic lineage for VSMC and CVC in atherosclerotic plaques.9, 16)

Cellular migration studies Researchers have documented VSMC migration across the internal elastic lamina from the tunica media into the sub-endothelial layer where they proliferate and contribute to atherosclerosis and calcification.10, 22)

Molecular studies 1) OPG levels were observed to decrease in cell culture studies of calcifying VSMC. When calcium levels were raised in the culture medium, there was a significantly decreased level of secreted OPG, suggesting its association with calcification.8, 9, 14)

2) Human osteoblastic cells have been cultured with TNF, IL-1β and ALP activity. Deposited calcium was measured in these cells. When tested at different concentrations, these cytokines resulted in significant decreases in ALP levels while the deposition of calcium within the cell layer decreased in cells treated with TNF-α and IL-1β suggesting that these cytokines are capable of influencing osteoclast activity within the vasculature.14, 17)

3) OPN is reported to be abundant at sites of ectopic calcification in human atherosclerotic lesions. In calcified arteries, OPN is highly localized to the surfaces of calcified deposits. OPN also inhibits calcium deposition in calcifying VSMC in vitro, which suggests a regulatory role for OPN in vascular calcification.8, 9, 10, 17)

Bone remodelling studies Bone remodelling, specifically osteoclastic resorptive activity, is associated with vascular calcification. These findings suggest that vascular calcification is linked to osteoclastic resorption. Studies have reported that soft tissue calcification is promoted by crystal nuclei generated at sites of bone resorption that circulate in the blood stream and lodge in soft tissue, thereby inducing tissue mineralization.9, 14, 18)

A summary of research investigations in the past decade suggesting that vascular calcification is an active cell-regulating process. These studies in-clude reports from in vitro studies, VSMC migration studies, genetic and molecular evidence and bone remodelling studies in animal models. (hVSMC: human vascular smooth muscle cells; CVC: Calcifying vascular cells; SMC: Smooth muscle cells; OPG: Osteoprotegerin; TNF-α: Tu-mor necrosis factor-alpha; IL-Interleukin; ALP: Alkaline phosphatase; OPN: Osteopontin)


cation arises because hydroxyapatite may persist long after cell apoptosis, thus suggesting that calcification may not be a cell-mediated process8, 9). Atherosclerotic thrombus was also thought to promote calcification8); however research findings from animal, in vitro and human studies provide considerable evidence that vas-cular calcification is an active, cell-mediated process (Table 2)2, 8, 10-12). These studies suggest that cells, such as macrophages, mast cells, and vascular smooth mus-cle cells (VSMC), are involved in the initiation and progression of vascular calcification8, 11, 12). Animal ex-periments also suggest that vascular calcification is a process that is contributed to by molecular proteins such as osteoprotegerin (OPG), osteopontin (OPG), osteonectin (OCN), matrix gla-protein (MGP) and bone matrix protein (BMP)13-17) (Table 2). Based on these cumulative findings, four different and non-mu-tually exclusive theories of vascular calcification have been postulated18, 19) (Fig.2).

Circulating cell theory

Circulating progenitor biology is evolving as one of the most promising fields under current investiga-tion, with huge potential in the field of regeneration. Circulating stem cells have been postulated to give rise

to VSMCs based on the identification of VSMC pro-genitors in circulating blood20). The origin of these cells is not clearly defined but it was postulated that the cells possibly emerge from the BM21-23). Further, this BM-derived cell population may possibly seed the peripheral vasculature and contribute to both artery disease and repair23). This novel mechanism was termed the ‘circulating cell theory’24). The contribu-tion of these circulating BM-derived naïve progenitor cells to vascular disease progression forms the basis of this circulating cell hypothesis. Over the past decade, several experiments in animal models (Table 3) and human investigations (Table 4) have supported this theory8, 11, 15, 21, 22, 24).

Fig.2. Schematic illustration summarizing four previous the-ories illustrating molecular mechanisms of vascular calcification

1. Loss of inhibition: A deficiency in certain proteins, such as MGP, OPG, OPN, results in osteoporosis and various degrees of mineralization at different areas in the vasculature, confirming the inhibitory roles of these genes in calcification. These proteins act as inducible inhibitors in the adaptive response of vascular injury by inhibiting crystal growth.2. Induction of bone formation: This theory suggests that vascular calcification is a similar process to bone formation and that a plu-ripotent population present in the artery wall is capable of osteo-genic differentiation that might be involved in vascular calcifica-tion under pathological conditions.3. Circulating nucleational complexes: This theory has associated bone remodelling, specifically osteoclastic resorptive activity, with vascular calcification.4. Apoptosis: Apoptosis has been regarded as a major mechanism for vascular calcification. This theory suggests that apoptotic bod-ies derived from VSMC can act as initiating and nucleating sites for calcium deposition.(MGP: matrix galactosidase protein; OPG: Osteoprotegerin; OPN: Osteopontin: VSMC: Vascular smooth muscle cells)

Fig.1. Circulating cell theory: A novel theory suggesting that BM-derived osteo-progenitors home to diseased arter-ies and contribute to vascular calcification

1. Osteo-progenitor cells are released from the BM environment under the influence of stem cell-mobilising cytokines.2. These osteo-progenitors home to the diseased vasculature.3. Under ideal osteogenic conditions these osteo-progenitors dif-ferentiate into osteoblasts.4. This differentiation results in the proliferation of osteoblasts within the vessel wall, resulting in calcification. (BM: Bone mar-row)


Osteo-progenitors and vascular calcification

Progenitor cells have only been studied in the last decade and understanding of their role in the vascula-ture is still evolving22, 24-27). BM-derived mesenchymal stem cells, in particular, have the ability to differenti-ate into many cell types, one of which is an osteogenic

lineage21, 28) (Fig.3). This lineage development occurs by a transdifferentiation process whereby a cell type committed to and progressing along a specific devel-opmental lineage switches to another cell type of a dif-ferent lineage through genetic reprogramming28, 29). Such BM-derived populations with osteogenic poten-tial are referred to as osteo-progenitors23). These osteo-

Table 3. Data highlighting the circulating cell theory in animal model experiments

Animal Investigation Summary of evidence

Transplant studies 1) BM transplantation was performed from LacZ mice to wild-type mice. Four weeks after transplantation, most of the neointimal cells were LacZ positive.21, 40)

2) Wild-type hearts were transplanted into wild-type mice whose BM had been reconstituted with that of transgenic mice that express GFP; the GFP-positive cells then accumulated on the luminal side of the transplanted coronary arteries, indicating that recipient BM cells can substantially contribute to neointimal formation in transplanted hearts.8, 9)

Cellular studies OCN＋ MNC from OPG－/－ mice have been demonstrated in the peripheral circulation and have been further identified to be deposited within the vasculature.42)

In vitro studies Haematopoietic stem cells derived from ROSA 26 mice differentiated into vascular cells that contributed to the pathogenesis of vascular diseases.8, 9, 22)

Histology studies Rats received intramyocardial injections of either DiI-labelled total BM cells or the same number of DiI-labelled clonally expanded BM-derived multipotent stem cells. Histological studies of these infarcted rat hearts identified spotty calcified areas. H & E staining demonstrated deep blue to purple regions of stained areas, suggesting the presence of calcium particles spread within regions of fibrosis. Von kossa staining demonstrated black metallic deposits of calcification.9)

A summary of recent research investigations in animal models that suggest a novel ‘circulating cell theory’ in the pathogenesis of vascular calcifica-tion. These reports include findings from BM transplant experiments, in vitro studies, cellular studies and histological observations. (BM: Bone marrow; GFP: Green fluorescent protein; OCN＋ MNC: Osteocalcin-positive mononuclear cells; OPG－/－: Osteoprotegerin-deficient; H & E: Hae-matoxylin and eosin)

Table 4. Data highlighting the circulating cell theory in human experiments

Human Investigation Summary of evidence

In vitro studies 1) hMSC when cultured in the presence of osteogenic factors differentiate into an osteogenic lineage producing bone-like nodules with a mineralized extracellular matrix containing hydroxyapatite. 27, 28)

2) Primary cultures of VSMC from normal vessels expressed MGP, collagen types Ⅰ and Ⅱ, OCN, ALP, OPN, and BMP-2, indicating that VSMC can express osteoblast-specific genes.11, 33)

3) CVC of human origin undergo in vitro calcification, suggesting an osteogenic lineage for VSMCs and CVC in atherosclerotic plaques.22, 24)

4) Human OCN＋ cells have been reported to stimulate mineralization both in vitro and when injected into mice.4, 23)

Cellular studies 1) Smooth muscle progenitor cells derived from the MNC of human blood have been demonstrated to differentiate into a VSMC lineage upon treatment with VEGF and PDGF.20, 22, 31)

2) OCN＋ MNC from patients diagnosed with PAD has been demonstrated in the peripheral circulation. Further, the severity of aortic calcification was found to correlate with this OCN＋ population.42)

A summary of recent research investigations based on human studies that suggest a novel ‘circulating cell theory’ in the pathogenesis of vascular cal-cification. These reports include findings from in vitro human cell lines and cellular studies. (hMSC: Human mesenchymal cells; VSMC: Vascular smooth muscle cells, MGP: Matrix Gla protein; OCN: Osteocalcin; OPN: Osteopontin; ALP: Alkaline phosphatase; BMP: Bone matrix protein; CVC: Calcifying vascular cells, OCN＋ MNC: Osteocalcin-positive mononuclear cells; PAD: Peripheral artery diseases)


progenitors mobilise out of the BM environment and enter the peripheral circulation24, 30, 31). Further, they home into the diseased vessel wall and contribute to disease complexity30-32).

Mobilization of osteo-progenitors

Recruitment of immature cells from the BM en-vironment to the blood under the influence of cyto-kines is a process termed mobilization30-32). This pro-cess involves enhancement of the physiological release of stem cells and progenitors from the BM reservoir in response to stress signals during injury and inflamma-tion33, 34) (Fig.4). The stem cell mobilization process is initiated by stimulation of marrow cells with cytokines such as granulocyte colony-stimulating factor (G-CSF), resulting in the release of progenitor cells30, 33). Cytokines involved in the increased release of these cells are G-CSF, granulocyte-macrophage colony-stim-ulating factor (GM-CSF) and stem cell factor (SCF)34-36). Chemokines such as stromal cell-derived

factor (SDF-1α) are also associated with the mobiliza-tion process32, 35). These molecules differ in their mode of administration, the time frame required to achieve mobilization, the lineage of cells mobilized, and their efficiency34).

Homing and proliferation in diseased vasculature

During the process of homing, circulating cells actively cross the endothelium and lodge transiently in the vessel wall by activation of adhesion interactions prior to their proliferation24, 32, 34)(Fig.4). The endo-thelium is the first anchoring site for homing cells. This endothelium layer expresses adhesion molecules and chemo-attractants24). Among these chemokines, SDF-1α signalling has been reported as an important factor involved with the migration and mobilization of haematopoietic stem cells during vessel wall inju-ry34, 37). Vascular damage involves initial endothelial denudation and platelet accumulation22, 24). Platelets,

Fig.3. Schematic diagram illustrating the multi-potential characteristics of MSC

MSC are multipotent stem cells that can differentiate into a variety of cell types. Their ability to differentiate into an osteogenic lineage suggests that they may be relevant to vascular calcifica-tion. (MSC: Mesenchymal stem cells)


along with VSMC, secrete growth factors and cyto-kines, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and SDF-1α24). Among these, PDGF is important for VSMC migration and proliferation while VEGF and SDF-1α are crucial for the mobilization of progenitor cells from the BM reservoir24, 38). After mobilization, these progenitor cells are believed to migrate to the site of damage where adhesion molecules and chemo-kines mediate their integration within the vascula-ture30, 32, 34). Soon after contact with these molecules the cells are activated, causing adhesion to the endo-thelium through interactions with vascular cell adhe-sion molecule (VCAM)-139). As soon as these imma-ture cells integrate into the endothelial layer the pro-

cess of proliferation and differentiation commences, which can potentially contribute to vascular repair or lesion formation22, 24, 31).

Current research investigations

Animal studiesBM transplant experiments conducted in animal

models suggested that BM-derived cells may contrib-ute to vascular disease progression40, 41). These BM transplant experiments also indicated that BM-derived progenitor cells could potentially achieve a VSMC lin-eage41). In vitro differentiation studies also suggest that these immature cells derived from various mouse models differentiate into osteoclast-like cells in the

Fig.4. Mobilization, homing and recruitment of progenitor cells

Vascular damage involves initial endothelial denudation and platelet accumulation. Cytokines, including PDGF, VEGF and SDF-1α, are crucial for the mobilization of progenitor cells from the BM reservoir. Once mobilized, these progenitor cells migrate to the site of damage where adhesion molecules mediate the integration of these cells within the vasculature. These imma-ture cells integrate into the endothelial layer and the process of proliferation and differentiation commences, which contributes to disease progression. (VSMC: Vascular smooth muscle cells; PDGF: Platelet-derived growth factor; VEGF: Vascular endothelial growth factor; SDF: Stro-mal cell-derived factor; BM: Bone marrow; kit L: kit ligand; MMP: matrix metalloproteinase)


presence of several chemo-attractants and growth fac-tors ideal for osteogenic lineage development4, 6, 21, 28). In previous studies, we assessed aortic calcification in an osteoprotegerin knockout (OPG－/－) mouse model. We also measured the number of circulating osteocal-cin-positive mononuclear cells (OCN＋ MNC) in the peripheral blood of the same mice42). This circulating OCN＋ MNC population was observed to be signifi-cantly increased in OPG－/－ mice and positively associ-ated with the aortic calcium concentration. To mea-sure the homing of these cells to the vasculature, we also quantified the number of OCN＋ cells within the aortas of OPG－/－ mice42). The results suggested a three-way association between the circulating BM-de-rived OCN＋ MNC, bone-forming cells within the vasculature and the severity of vascular calcification41). Protein cytokine studies performed in OPG－/－ mice also suggested a role for stem cell-mobilising cytokines in stimulating the release of immature osteo-progeni-tors from the BM environment43). Under the influ-ence of chemo-attractants, these bone marker-positive cells may home to diseased arteries. This cell popula-tion may further undergo osteogenic differentiation in the lesions, which could promote vessel mineraliza-tion.

Human studiesEvidence from human MSC studies also sup-

ports the hypothesis that BM cells play a role in the pathogenesis of vascular diseases28). Human osteogen-ic cells have been reported to stimulate mineralization both in vitro and when injected into mice44). The os-teogenic potential of adult vascular cells has been demonstrated in vitro and in vivo45). The lineage of these cells, which is similar to that of marrow stromal cells and their capacity for self-renewal, suggests that they are of MSC origin45, 46). Further, osteoclast and osteoblast-like cells have been demonstrated within ar-eas of vascular calcification in the human vascula-ture47). We also undertook a human investigation in patients suffering from peripheral arterial disease42). The aortic calcification severity in these patients was found to be associated with the percentage of circulat-ing OCN＋ MNCs. This cell population is thought to originate from the BM environment43). As observed in the mouse model studies, plasma concentrations of G-CSF and SCF were positively correlated with aortic calcification volumes and the size of the OCN＋ popu-lation44). Plasma concentrations of SDF-1α and G-CSF were approximately 2-3-fold higher in patients with aortic calcification than those reported in healthy subjects (normal G-CSF concentrations: ~27 pg/mL43, 48); normal SDF-1α concentrations: ~34 ng/mL47)). Plas-

ma SCF levels were only slightly higher than reported normal values (~2100 pg/mL48)). A positive associa-tion among the concentration of these cytokines, aor-tic calcification volumes and OCN＋ MNCs suggests a possible role for circulating osteo-progenitors in arte-rial mineralization in patients43).

Conclusions and future directions

The research reported in the current review sug-gests an association between cells previously identified as osteogenic progenitors and vascular calcification; however, further work is necessary to confirm the role of these BM-derived immature cells in vascular calcifi-cation. Possible further studies include tracing the cir-culatory path of these immature cells and determining their final lineage within the vasculature. BM cell pop-ulations could be extracted from the marrow environ-ment and labelled with green fluorescent protein (GFP), which has been reported to tag immature cells without influencing their lineage. This fluorescently labeled population could then be injected back in vivo. Further, these tagged cells could be traced in the peripheral circulation via in vivo imaging techniques. Another method of further assessing the circulating cell theory could be to block the effect of stem cell-mobilizing cytokines using anti-cytokine blocking agents. Controlling this mobilising activity would re-duce the release of osteo-progenitors from the marrow environment into the peripheral circulation and limit vascular calcification if the circulating theory is cor-rect. These and similar studies are required to better determine the contribution of circulating cells to vas-cular calcification.

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osteo-progenitors in vascular calcification

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