Mesenchymal stromal cells: inhibiting PDGF receptors or depleting fibronectin induces mesodermal progenitors with endothelial potential.

Realizing the full therapeutic potential of mesenchymal stromal/stem cells (MSCs) awaits improved understanding of mechanisms controlling their fate. Using MSCs cultured as spheroids to recapitulate a three-dimensional cellular environment, we show that perturbing the mesenchymal regulators, platelet-derived growth factor (PDGF) receptors or fibronectin, reverts MSCs toward mesodermal progenitors with endothelial potential that can potently induce neovascularization in vivo. MSCs within untreated spheroids retain their mesenchymal spindle shape with abundant smooth muscle α-actin filaments and fibronectin-rich matrix. Inhibiting PDGF receptors or depleting fibronectin induces rounding and depletes smooth muscle α-actin expression; these cells have characteristics of mesenchymoangioblasts, with enhanced expression of mesendoderm and endoderm transcription factors, prominent upregulation of E-cadherin, and Janus kinase signaling-dependent expression of Oct4A and Nanog. PDGF receptor-inhibited spheroids also upregulate endothelial markers platelet endothelial cell adhesion molecule 1 and vascular endothelial-cadherin and secrete many angiogenic factors, and in vivo they potently stimulate neovascularization, and their MSCs integrate within functional blood vessels that are perfused by the circulation. Thus, MSC potency and vascular induction are regulated by perturbing mesenchymal fate.

Chondrogenic ATDC5 cells: an optimised model for rapid and physiological matrix mineralisation.

The development of chondrogenic cell lines has led to major advances in the understanding of how chondrocyte differentiation is regulated, and has uncovered many signalling pathways and gene regulatory mechanisms required to maintain normal function. ATDC5 cells are a well established in vitro model of endochondral ossification; however, current methods are limited for mineralisation studies. In this study we demonstrate that culturing cells in the presence of ascorbic acid and 10 mM ß-glycerophosphate (ßGP) significantly increases the rate of extracellular matrix (ECM) synthesis and reduces the time required for mineral deposition to occur to 15 days of culture. Furthermore, the specific expression patterns of Col2a1 and Col10a1 are indicative of ATDC5 chondrogenic differentiation. Fourier transform-infrared spectroscopy analysis and transmission electron microscopy (TEM) showed that the mineral formed by ATDC5 cultures is similar to physiological hydroxyapatite. Additionally, we demonstrated that in cultures with ßGP, the presence of alkaline phosphatase (ALP) is required for this mineralisation to occur, further indicating that chondrogenic differentiation is required for ECM mineralisation. Together, these results demonstrate that when cultured in the presence of ascorbic acid and 10 mM ßGP, ATDC5 cells undergo chondrogenic differentiation and produce a physiological mineralised ECM from Day 15 of culture onwards. The rapid and novel method for ATDC5 culture described in this study is a major improvement compared with currently published methods and this will prove vital in the pursuit of underpinning the molecular mechanisms responsible for poor linear bone growth observed in a number of chronic diseases such as cystic fibrosis, chronic kidney disease, rheumatological conditions and inflammatory bowel disease.

HtrA1: a novel regulator of physiological and pathological matrix mineralization?

HtrA1 (high-temperature requirement protein A1) is a secreted multidomain protein with proven serine protease activity and the ability to regulate TGF-beta (transforming growth factor-beta)/BMP (bone morphogenetic protein) signalling. There is increasing evidence that HtrA1 regulates several pathological processes, including tumour development, Alzheimer's disease, age-related macular degeneration and osteoarthritis, although the mechanism(s) by which it regulates these processes have not been fully elucidated. Using overexpression and knock-down strategies, we have evidence demonstrating that HtrA1 is also a key regulator of physiological and pathological matrix mineralization in vitro. We propose that HtrA1 regulates mineralization by inhibiting TGF-beta/BMP signalling and/or by cleaving specific matrix proteins, including decorin and MGP (matrix Gla protein). Taken together, these studies suggest that HtrA1 may be a novel therapeutic target for several diseases.

Hepatocyte growth factor and c-Met expression in pericytes: implications for atherosclerotic plaque development.

Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c-Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31-positive endothelial cells. c-Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT-PCR, we demonstrated expression of HGF and c-Met in a rat pericyte cell-line, TR-PCT1, and in primary pericytes. HGF treatment of TR-PCT1 cells induced their migration, but not their proliferation, in a dose-dependent manner (10-100 ng/ml, p<0.01), an effect mediated by activation of the serine/threonine kinase Akt, shown by western blot analysis. Treating the cells with the PI3K inhibitors Wortmannin (0.1 microM) or LY294002 (10 microM) abolished these effects. This work demonstrates the expression of c-Met and HGF in human atherosclerotic arteries, in association with SM-actin-positive cells and CD-31-positive cells, respectively. HGF induces pericyte migration via PI3-kinase and Akt activation in vitro. HGF and c-Met may be involved in neovascularization during plaque development, and may recruit pericytes to neovessels. Since pericytes are thought to mechanically stabilize new blood vessels, these factors may function to protect against haemorrhage.

Contribution of VCAF-positive cells to neovascularization and calcification in atherosclerotic plaque development.

Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF-positive cells were detected within the intima and adventitia in areas where sporadic 3G5-positive pericytes were localized. Furthermore, VCAF-positive cells were identified in newly formed microvessels in association with CD34-positive/CD146-positive/c-kit-positive cells as well as in intact CD31-positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease.

Angiogenesis and pericytes in the initiation of ectopic calcification.

Ectopic calcification of blood vessels, heart valves, and skeletal muscle is a major clinical problem. There is now good evidence that angiogenesis is associated with ectopic calcification in these tissues and that it is necessary, but not sufficient, for calcification to occur. Angiogenesis may regulate ectopic calcification in several ways. First, many angiogenic factors are now known to exert both direct and indirect effects on bone and cartilage formation. Second, cytokines released by endothelial cells can induce the differentiation of osteoprogenitor cells. Third, the new blood vessels provide oxygen and nutrients to support the growing bone. Finally, the new blood vessels can serve as a conduit for osteoprogenitor cells. These osteoprogenitor cells may be derived from the circulation or from pericytes that are present in the neovessels themselves. Indeed, there is now compelling evidence that pericytes can differentiate into osteoblasts and chondrocytes both in vitro and in vivo. Other vascular cells, including adventitial myofibroblasts, calcifying vascular cells, smooth muscle cells, and valvular interstitial cells, have also been shown to exhibit multilineage potential in vitro. Although these cells share many properties with pericytes, the precise relationship between them is not known. Furthermore, it still remains to be determined whether all or some of these cells contribute to the ectopic calcification observed in vivo. A better understanding of the underlying mechanisms that link angiogenesis, pericytes, and ectopic calcification should provide a basis for development of therapeutic strategies to treat or arrest this clinically significant condition.

Chondrogenic and adipogenic potential of microvascular pericytes.

BACKGROUND: Previous studies have shown that pericytes can differentiate into osteoblasts and form bone. This study investigated whether pericytes can also differentiate into chondrocytes and adipocytes. METHODS AND RESULTS: Reverse transcription-polymerase chain reaction demonstrated that pericytes express mRNA for the chondrocyte markers Sox9, aggrecan, and type II collagen. Furthermore, when cultured at high density in the presence of a defined chondrogenic medium, pericytes formed well-defined pellets comprising cells embedded in an extracellular matrix rich in sulfated proteoglycans and type II collagen. In contrast, when endothelial cells were cultured under the same conditions, the pellets disintegrated after 48 hours. In the presence of adipogenic medium, pericytes but not endothelial cells expressed mRNA for peroxisome proliferator-activated receptor-gamma2 (an adipocyte-specific transcription factor) and incorporated lipid droplets that stained with oil red O. To confirm that pericytes can differentiate along the chondrocytic and adipocytic lineages in vivo, these cells were inoculated into diffusion chambers and implanted into athymic mice for 56 days. Accordingly, mineralized cartilage, fibrocartilage, and a nonmineralized cartilaginous matrix with lacunae containing chondrocytes were observed within these chambers. Small clusters of cells that morphologically resembled adipocytes were also identified. CONCLUSIONS: These data demonstrate that pericytes are multipotent cells that may contribute to growth, wound healing, repair, and/or the development and progression of various pathological states.

The involvement of matrix glycoproteins in vascular calcification and fibrosis: an immunohistochemical study.

Calcification and fibrointimal proliferation are associated with advanced complicated atherosclerosis in large arteries but may also occur in smaller vessels, resulting in ischaemic tissue necrosis. This study investigates whether the mechanisms of calcification and intimal fibrosis are similar in vessels of different sizes. The localization of osteopontin (OPN), matrix Gla protein (MGP), thrombospondin-1 (TSP-1), and cartilage oligomeric matrix protein (COMP) was investigated in three types of human vascular lesions: atherosclerosis, chronic vascular rejection (CVR) in renal allografts, and calcific uraemic arteriolopathy (calciphylaxis). These lesions were chosen as they affect different sized blood vessels and they exhibit a fibroproliferative intimal reaction, with or without calcification, resulting in luminal obliteration and ischaemic complications. OPN, MGP, TSP-1, and COMP were not detected in normal blood vessels. However, OPN and MGP were expressed at sites of calcification within atherosclerotic lesions and in microvessels in calciphylaxis, suggesting that calcification in different sized vessels may occur by a common mechanism. These proteins were not detected in areas of fibrointimal proliferation. In contrast, TSP-1 was localized primarily within the fibrous tissue of atherosclerotic lesions and was also expressed in the expanded fibrous intima of arteries showing CVR. COMP was localized primarily within the fibrous tissue under the lipid core of the majority of advanced atherosclerotic lesions. TSP-1 and COMP were also detected in areas of microcalcification in atherosclerotic lesions and TSP-1 was detected adjacent to areas of calcification in calciphylaxis. However, neither TSP-1 nor COMP was localized to calcific foci within these lesions. The localization of OPN, MGP, TSP-1, and COMP to pathological, but not normal arterial intima supports a pathogenetic role for these proteins in the development of vascular fibrosis and calcification. Modulation of their production and activity may offer a novel approach to the therapy of a number of vascular diseases.

Upregulation of collagen VIII following porcine coronary artery angioplasty is related to smooth muscle cell migration not angiogenesis.

Type VIII collagen is upregulated after vessel injury, and this collagen has been implicated in both smooth muscle cell migration and angiogenesis. This study examines the temporal and spatial pattern of expression of type VIII collagen in porcine coronary vessels at specific time points after balloon angioplasty. In situ hybridization studies demonstrated that collagen VIII messenger ribonucleic acid (mRNA) was markedly elevated in the neoadventitia at 3 days post-angioplasty. By 14 days, elevated collagen VIII message was seen mainly in the neointima and this expression decreased to background levels by 90 days. The distribution of collagen VIII protein, detected using immunohistochemistry, was similar but the up-regulation lagged behind the mRNA increase by a few days. Pre-treatment of sections with pepsin highlighted variations in the organization and appearance of extracellular collagen VIII containing structures in both injured and normal vessels. New vessel formation was evident in the neoadventitia after 3 days, but there was no colocalization of type VIII collagen immunostaining with that of von Willebrand factor (a marker of endothelial cells) in the neoadventitia. These data show that up-regulation of collagen VIII in the neoadventitia is an important early marker of the coronary arterial response to injury, and is not associated with new vessel formation.

1 alpha,25-dihydroxyvitamin D(3) inhibits angiogenesis in vitro and in vivo.

Modulation of angiogenesis is now a recognized strategy for the prevention and treatment of pathologies categorized by their reliance on a vascular supply. The purpose of this study was to evaluate the effect of 1 alpha,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)], the active metabolite of vitamin D(3), on angiogenesis by using well-characterized in vitro and in vivo model systems. 1,25(OH)(2)D(3) (1 x 10(-9) to 1 x 10(-7) mol/L) significantly inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting and elongation in vitro in a dose-dependent manner and had a small, but significant, inhibitory effect on VEGF-induced endothelial cell proliferation. 1, 25(OH)(2)D(3) also inhibited the formation of networks of elongated endothelial cells within 3D collagen gels. The addition of 1, 25(OH)(2)D(3) to endothelial cell cultures containing sprouting elongated cells induced the regression of these cells, in the absence of any effect on cells present in the cobblestone monolayer. Analysis of nuclear morphology, DNA integrity, and enzymatic in situ labeling of apoptosis-induced strand breaks demonstrated that this regression was due to the induction of apoptosis specifically within the sprouting cell population. The effect of 1,25(OH)(2)D(3) on angiogenesis in vivo was investigated by using a model in which MCF-7 breast carcinoma cells, which had been induced to overexpress VEGF, were xenografted subcutaneously together with MDA-435S breast carcinoma cells into nude mice. Treatment with 1,25(OH)(2)D(3) (12.5 pmol/d for 8 weeks) produced tumors that were less well vascularized than tumors formed in mice treated with vehicle alone. These results highlight the potential use of 1,25(OH)(2)D(3) in both the prevention and regression of conditions characterized by pathological angiogenesis.

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.

Role of pericytes in vascular calcification: a review.

Pericytes are defined by their location in vivo; the pericyte partially surrounds the endothelial cell of the microvessel and shares a common basement membrane with it. As an integral part of the microvasculature, pericytes play a fundamental role in maintaining local and tissue homeostasis. Current evidence also suggests that pericytes function as progenitor cells capable of differentiating into a variety of different cell types including osteoblasts, chondrocytes and adipocytes. It is now apparent that cells resembling microvascular pericytes, and termed 'pericyte-like' cells, have a widespread distribution in vivo. Pericyte-like cells have been identified in the inner intima, the outer media, and in the vasa vasora of the adventitia of large, medium and small human arteries (1, 2). Moreover, recent studies have suggested that these cells may be responsible, at least in part, for mediating the calcification commonly associated with atherosclerosis (1, 3, 4). In this review, we a) examine the evidence that microvascular pericytes deposit a bone-like mineralised matrix in vitro, b) compare the morphological and biochemical properties of microvascular pericytes, calcifying vascular cells (CVCs) and 'classical' smooth muscle cells (SMCs) isolated from bovine aorta, c) demonstrate that microvascular pericytes deposit a well-organised matrix of bone, cartilage and fibrous tissue in vivo, and d) discuss recent studies designed to gain a better understanding of how pericyte differentiation is regulated.

Matrix Gla protein is differentially expressed during the deposition of a calcified matrix by vascular pericytes.

PCR-based subtractive hybridisation was used to identify genes up-regulated when pericytes undergo osteogenic differentiation and deposit a calcified matrix. cDNA pools were generated from confluent pericytes and from pericyte cultures containing calcified nodules. A pericyte cDNA library was screened with the product of the subtraction procedure (calcified minus confluent cDNA) and the majority of the positive clones were identified as matrix Gla protein (MGP). Northern analysis and immunohistochemistry demonstrated that MGP was only expressed by pericytes in calcified nodules. Antibodies to MGP inhibited the deposition of a calcified matrix by pericytes, suggesting that MGP regulates both cell differentiation and calcification.

Gene expression during vascular pericyte differentiation.

Pericytes, an integral part of the microvasculature, are involved in a number of different processes, including angiogenesis. Many of the early studies on these cells are descriptive and concentrate on the location of pericytes in vivo, surrounding the endothelial cells in the microvessels. These studies led to the proposals that pericytes have a function in maintaining blood flow and contribute to the mechanical strength of the microvessels. However, with the advancement of tissue culture techniques and molecular technology it has been shown that these cells also have the ability to differentiate into a variety of different cell types, including osteoblasts, chondrocytes, adipocytes, fibroblasts, and smooth muscle cells. This review concentrates on the differentiation of pericytes along the osteogenic pathway. Pericytes behave like osteoblasts in vitro, by forming a mineralized matrix and expressing a number of genes that are also expressed by osteoblasts. These cells also form a well-defined matrix of bone, cartilage, and fibrous tissue in vivo, although it is not clear under what circumstances pericytes express osteogenic potential in situ. This review highlights the potential functional importance of pericytes in the growth, maintenance, and repair of the skeleton and in diseases involving ectopic ossification and calcification.

Vascular pericytes express osteogenic potential in vitro and in vivo.

At postconfluence, cultured bovine pericytes isolated from retinal capillaries form three-dimensional nodule-like structures that mineralize. Using a combination of Northern and Southern blotting, in situ hybridization, and immunofluorescence we have demonstrated that this process is associated with the stage-specific expression of markers of primitive clonogenic marrow stromal cells (STRO-1) and markers of cells of the osteoblast lineage (bone sialoprotein, osteocalcin, osteonectin, and osteopontin). To demonstrate that the formation of nodules and the expression of these proteins were indicative of true osteogenic potential, vascular pericytes were also inoculated into diffusion chambers and implanted into athymic mice. When recovered from the host, chambers containing pericytes were found reproducibly to contain a tissue comprised of cartilage and bone, as well as soft fibrous connective tissue and cells resembling adipocytes. This is the first study to provide direct evidence of the osteogenic potential of microvascular pericytes in vivo. Our results are also consistent with the possibility that the pericyte population in situ serves as a reservoir of primitive precursor cells capable of giving rise to cells of multiple lineages including osteoblasts, chondrocytes, adipocytes, and fibroblasts.

Association of thrombospondin-1 with osteogenic differentiation of retinal pericytes in vitro.

Vascular pericytes can differentiate into osteoblast-like cells in vitro, suggesting that these cells may represent a potential source of osteoprogenitor cells in the adult. Pericyte differentiation is associated with a characteristic pattern of nodule formation and mineralisation. Nodules are formed in post-confluent cultures by the retraction of multilayered areas. Crystals of hydroxyapatite are deposited on the extracellular matrix of these nodules which then becomes mineralised. We now demonstrate that thrombospondin-1 (TSP-1) gene expression is modulated during pericyte differentiation in vitro. That is, the relative levels of TSP-1 (protein and mRNA) increased markedly during nodule formation and then decreased when mineralisation of the nodules had taken place. TSP-1 was localised throughout non-mineralised nodules but it was largely excluded from the inner mass of mineralised nodules. The production of a mineralised matrix by vascular pericytes was promoted by the presence of antibodies to TSP-1 in the culture medium and was inhibited by exogenous TSP-1. These effects did not appear to be mediated through the activation of latent TGF-beta, since neither exogenous TGF-beta nor neutralising antibodies to TGF-beta had any effect on the rate or extent of mineralisation seen in the pericyte cultures. Taken together these results suggest that high levels of TSP-1 inhibit pericyte mineralisation, supporting the view that this protein plays a role in pericyte differentiation and bone formation.

Pericyte differentiation.

Pericytes are defined in vivo by their location: They are embedded within the basement membrane of microvessels. They form an integral part of the microvascular wall and are believed to participate in angiogenesis, although their precise role is not clear. Pericytes derived from the retinal microvasculature have been cultured and identified by a series of phenotypic characteristics that clearly distinguishes them from other stromal cells such as smooth muscle cells. Pericytes in vitro form multicellular nodules rich in extracellular matrix. This matrix becomes mineralized in the presence of growth medium containing serum, without exogenous beta-glycerophosphate. These results indicate that pericytes represent primitive mesenchymal cells able to differentiate into an osteogenic phenotype. Pericyte differentiation also is defined by alterations in their response to transforming growth factor beta 1 and changes in the synthesis and/or deposition of various extracellular matrix proteins such as laminin, Type IV collagen, tenascin, Type X collagen osteonectin, and thrombospondin-1. Angiogenesis is associated commonly with mineralization. These data suggest that pericytes may contribute to mineralization in vivo.

Evidence that tenascin and thrombospondin-1 modulate sprouting of endothelial cells.

Cultured endothelial cells undergo a reversible transition from a resting (cobblestone) phenotype to an angiogenic (sprouting) phenotype. This transition mimics the early events of angiogenesis. We have previously reported that the addition of exogenous xylosides inhibits endothelial cel sprouting and modifies the extracellular matrix (ECM) synthesised by the cells. We have now investigated whether endothelial sprouting is mediated by the nature of the extracellular matrix in contact with the cells. Accordingly, cell-free matrices deposited by bovine aortic endothelial cells (BAEC) were isolated. These matrices were produced under conditions in which the formation of the sprouting phenotype was permitted (controls) or inhibited (by the addition of exogenous xylosides). BAEC were then plated on these matrices and grown under conditions which promote sprouting. Sprouting proceeded normally on control matrices, whereas it was inhibited when the cells were grown on matrices deposited in the presence of xylosides. The composition of the permissive and inhibitory matrices was then analysed. Inhibitory matrices contained reduced levels of tenascin and increased levels of thrombospondin-1 by comparison to the permissive matrices. In contrast, no differences were detected in the relative levels of laminin. The roles of tenascin and thrombospondin-1 in endothelial sprouting were confirmed using specific antibodies. Immunolocalisation studies revealed the presence of both proteins in sprouting cells. Antibodies to tenascin inhibited the formation of sprouting cells on permissive matrices and on gelatin-coated dishes without affecting cell growth. Tenascin synthesis was increased when sprouting cells were present in the cultures. Antibodies to thrombospondin-1 stimulated sprouting on inhibitory matrices. These results suggest that the transition from a resting to a sprouting phenotype is promoted by tenascin and inhibited by thrombospondin-1.