Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.     

Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor

Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin receptor points to a role for the integrins in the interaction of endothelial cells with TSP. We also found that TSP modulates endothelial cell growth. When added to quiescent LE-II cells, it inhibited the mitogenic effects of serum and the angiogenic factor bFGF, in a dose-dependent manner. The inhibition of DNA synthesis detected in the mitogenic assay resulted in a true inhibition of BAEC and LE-II cell growth, as assessed by proliferation assay. This work indicates that TSP affects endothelial cell adhesion, spreading, motility and growth. TSP, therefore, has the potential to modulate the angiogenic process.     

Matrix-bound thrombospondin promotes angiogenesis in vitro

Thrombospondin (TSP) is a multidomain adhesive protein postulated to play an important role in the biological activity of the extracellular matrix. To test this hypothesis, TSP-containing fibrin and collagen matrices were evaluated for their capacity to support angiogenesis and cell growth from explants of rat aorta. This serum-free model allowed us to study the angiogenic effect of TSP without the interference of attachment and growth factors present in serum. TSP promoted dose- dependent growth of microvessels and fibroblast-like cells. The number of microvessels in TSP-containing collagen and fibrin gels increased by 136 and 94%, respectively. The TSP effect was due in part to cell proliferation since a 97% increase in [3H]thymidine incorporation by the aortic culture was observed. The effect was TSP-specific because TSP preparations adsorbed with anti-TSP antibody showed no activity. TSP did not promote angiogenesis directly since no TSP-dependent growth of isolated endothelial cells could be demonstrated. Rather TSP directly stimulated the growth of aortic culture-derived myofibroblasts which in turn promoted microvessel formation when cocultured with the aortic explants. Angiogenesis was also stimulated by myofibroblast- conditioned medium. Partial characterization of the conditioned medium suggests that the angiogenic activity is due to heparin-binding protein(s) with molecular weight > 30 kD. These results indicate that matrix-bound TSP can indirectly promote microvessel formation through growth-promoting effects on myofibroblasts and that TSP may be an important stimulator of angiogenesis and wound healing in vivo.     

A recombinant NH(2)-terminal heparin-binding domain of the adhesive glycoprotein, thrombospondin-1, promotes endothelial tube formation and cell survival: a possible role for syndecan-4 proteoglycan.

Thrombospondin-1 (TSP-1) is a multifunctional protein known to modulate angiogenesis, endothelial cell adhesion and apoptosis. In this study, we have demonstrated that TSP18, a recombinant 18 kDa protein encompassing the N-terminal residues 1-174 of human TSP-1, accelerated the process of tube-like structures formation by human umbilical vein endothelial cells (HUVECs) when included in fibrin matrices at 0.55-2.2 microM concentrations, for times ranging from 24 to 72 h. This effect was specifically inhibited by V58A4, a Mab raised against TSP18. Whole TSP-1 showed a dual effect, weakly enhancing tube formation at 22 nM (10 microg/ml), but causing inhibition at 45 and 90 nM (20 and 40 microg/ml, respectively). In order to investigate the possible effects of TSP18 on cell adhesion and viability, we performed adhesion assays on different protein supports. HUVECs adhered more weakly on TSP-1-coated surfaces, remaining round-shaped, as compared to the well-spread phenotype displayed on fibronectin and gelatin. Cells adhering on TSP18-coated surfaces displayed a well spread phenotype, with this adhesion strongly inhibited by heparin. The binding of TSP18 to endothelial membrane extracts was blocked by a monoclonal IgG directed against the cell surface proteoglycan syndecan-4. The DNA fragmentation patterns and the nuclear morphology were comparable for HUVECs adhering on all proteins, including TSP18, showing minimal cell apoptosis. Our results indicate that the N-terminal region of TSP-1 constitutes a suitable adhesive support for HUVECs, protecting them from apoptosis, possibly mediated by syndecan-4 proteoglycan.     

The effect of heparin on fibronectin and thrombospondin synthesis and mRNA levels in cultured human endothelial cells.

Studies to eludicate the effect of heparin on the synthesis of extracellular matrix components by cultured human umbilical vein endothelial cells (EC) were conducted. Using pulse-labeling and ELISA techniques, we found that EC grown in the presence of heparin (90 micrograms/ml) and endothelial cell growth factor (ECGF) synthesized 50% less fibronectin (FN) than did ECGF-treated control cultures. No change in the synthesis of thrombospondin (TSP) was induced by heparin. The effect of heparin on EC FN synthesis was independent of whether the cells were cultivated on plastic or gelatin substrates. However, ECGF modulates the effect of heparin on EC synthesis of FN. RNA slot-blot analysis demonstrated that heparin treatment specifically decreased the steady-state mRNA levels for both FN and TSP in the cells. Steady-state levels of mRNA for two intracellular proteins, actin and tubulin, were unchanged. These data suggest that heparin decreases EC expression of FN at least in part by decreasing the amount of FN mRNA available for translation. The failure of heparin to inhibit TSP expression, although it reduces TSP mRNA levels, points to the possibility that the rate of EC synthesis of TSP is translationally or post-translationally regulated.     

Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics.

Endothelial and other cell types synthesize thrombospondin (TSP), secrete it into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a Kd of 25 nM, and the number of binding sites is 2.6 X 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70-kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [125I]EC-TSP and of the 125I-labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

TSP50 promotes the Warburg effect and hepatocyte proliferation via regulating PKM2 acetylation

Metabolic reprogramming is a hallmark of malignancy. Testes-specific protease 50 (TSP50), a newly identified oncogene, has been shown to play an important role in tumorigenesis. However, its role in tumor cell metabolism remains unclear. To investigate this issue, LC–MS/MS was employed to identify TSP50-binding proteins and pyruvate kinase M2 isoform (PKM2), a known key enzyme of aerobic glycolysis, was identified as a novel binding partner of TSP50. Further studies suggested that TSP50 promoted aerobic glycolysis in HCC cells by maintaining low pyruvate kinase activity of the PKM2. Mechanistically, TSP50 promoted the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our findings indicate that TSP50-mediated low PKM2 pyruvate kinase activity is an important determinant for Warburg effect in HCC cells and provide a mechanistic link between TSP50 and tumor metabolism.Subject terms: Cancer metabolism, Oncogenes, Tumour biomarkers

Gao et al. shows that testes-specific protease 50 (TSP50) binds to PKM2 and promotes the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our study reveals a link between an oncogene and a key enzyme in HCC glycolysis, which provides new ideas for human HCCs treatment with TSP50 as the target.     

Syndecan-4 contributes to endothelial tubulogenesis through interactions with two motifs inside the pro-angiogenic N-terminal domain of thrombospondin-1

Thrombospondin-1 (TSP-1) is an extracellular matrix protein that modulates focal adhesion in mammalian cells and exhibits dual roles in angiogenesis. In a previous work, we showed that a recombinant 18 kDa protein encompassing the N-terminal residues 1-174 of human TSP-1 (TSP18) induced tubulogenesis of human umbilical vein endothelial cells and protected them from apoptosis. Our results indicated that these effects were possibly mediated by syndecan-4 proteoglycan, since binding of TSP18 to endothelial extracts was inhibited by anti-syndecan-4 antibody. Syndecan-4 is a heparan-sulfate proteoglycan that regulates cell-matrix interactions and is the only member of its family present in focal adhesions. In this report, we demonstrate that a monoclonal antibody against syndecan-4 blocks TSP18-induced tubulogenesis. Furthermore, through 2D adhesion and 3D angiogenic assays, we demonstrate that two sequences, TSP Hep I and II, retain the major pro-angiogenic activity of TSP18. These TSP-1 motifs also compete with the fibronectin Hep II domain for binding to syndecan-4 on endothelial cell surface, indicating that they may exert their effects by interfering with the recognition of fibronectin by syndecan-4. Additionally, TSP18 and its derived peptides activate the PKC-dependent Akt-PKB signaling pathway. Blockage of PKC activation prevented HUVEC spreading when seeded on TSP18 fragment, and on TSP Hep I and TSP Hep II peptides, but not on gelatin-coated substrates. Our results identify syndecan-4 as a novel receptor for the N-terminus of TSP-1 and suggest that TSP-1 N-terminal pro-angiogenic activity is linked to its capacity of interfering with syndecan-4 functions in the course of cell adhesion.     

BIM deficiency differentially impacts the function of kidney endothelial and epithelial cells through modulation of their local microenvironment

The extracellular matrix (ECM) acts as a scaffold for kidney cellular organization. Local secretion of the ECM allows kidney cells to readily adapt to changes occurring within the kidney. In addition to providing structural support for cells, the ECM also modulates cell survival, migration, proliferation, and differentiation. Although aberrant regulation of ECM proteins can play a causative role in many diseases, it is not known whether ECM production, cell adhesion, and migration are regulated in a similar manner in kidney epithelial and endothelial cells. Here, we demonstrate that lack of BIM expression differentially impacts kidney endothelial and epithelial cell ECM production, migration, and adhesion, further emphasizing the specialized role of these cell types in kidney function. Bim -/- kidney epithelial cells demonstrated decreased migration, increased adhesion, and sustained expression of osteopontin and thrombospondin-1 (TSP1). In contrast, bim -/- kidney endothelial cells demonstrated increased cell migration, and decreased expression of osteopontin and TSP1. We also observed a fivefold increase in VEGF expression in bim -/- kidney endothelial cells consistent with their increased migration and capillary morphogenesis. These cells also had decreased endothelial nitric oxide synthase activity and nitric oxide bioavailability. Thus kidney endothelial and epithelial cells make unique contributions to the regulation of their ECM composition, with specific impact on adhesive and migratory properties that are essential for their proper function.     

Type V collagen selectively inhibits human endothelial cell proliferation

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.     

Thrombospondin-1 inhibits VEGF receptor-2 signaling by disrupting its association with CD47

Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. We now describe a more potent mechanism by which TSP1 inhibits VEGF receptor-2 (VEGFR2) activation through engaging its receptor CD47. CD47 ligation is known to inhibit downstream signaling targets of VEGFR2, including endothelial nitric-oxide synthase and soluble guanylate cyclase, but direct effects on VEGFR2 have not been examined. Based on FRET and co-immunoprecipitation, CD47 constitutively associated with VEGFR2. Ligation of CD47 by TSP1 abolished resonance energy transfer with VEGFR2 and inhibited phosphorylation of VEGFR2 and its downstream target Akt without inhibiting VEGF binding to VEGFR2. The inhibitory activity of TSP1 in large vessel and microvascular endothelial cells was replicated by a recombinant domain of the protein containing its CD47-binding site and by a CD47-binding peptide derived from this domain but not by the CD36-binding domain of TSP1. Inhibition of VEGFR2 phosphorylation was lost when CD47 expression was suppressed in human endothelial cells and in murine CD47-null cells. These results reveal that anti-angiogenic signaling through CD47 is highly redundant and extends beyond inhibition of nitric oxide signaling to global inhibition of VEGFR2 signaling.     

Thrombin-Stimulated Calcium Mobilization Is Inhibited by Thrombospondin via CD36

Activation of the G-protein-linked thrombin receptor in endothelial cells normally leads to an increase in free intracellular calcium, [Ca²⁺]_i, which is the proximate stimulus for many important cell functions. We present evidence showing that signals from CD36, the thrombospondin (TSP) receptor, can inhibit this thrombin-mediated calcium response. Human endothelial cells preloaded with Indo-1 exhibited rapid calcium mobilization in response to thrombin. The presence of TSP inhibited the thrombin-stimulated calcium response in CD36-positive microvascular endothelial cells but not in CD36-negative umbilical vein endothelial cells. This TSP effect was mimicked by anti-CD36 antibodies and a TSP peptide (CSVTCG), but not by an alternative CD36 ligand (collagen IV) or an antibody to an alternative TSP receptor (α_vβ₃). TSP also inhibited the calcium response to the thrombin receptor-tethered ligand peptide, SFLLRN. In addition, TSP and anti-CD36 antibodies inhibited the calcium response of a closely related receptor, the trypsin/SLIGKVD-activated receptor PAR-2. TSP did not indiscriminately inhibit all calcium release pathways, since histamine- or VEGF-stimulated calcium responses were not inhibited by TSP. We conclude that cross-talk from the CD36 receptor influences the responsive state of the endothelial thrombin receptor family and/or its signaling pathway.     

Identification of an endothelial cell growth-inhibitory activity produced by human monocytes

The control of endothelial cell proliferation is important in a variety of processes including wound healing and tumor-induced angiogenesis. We have observed that normal unstimulated human monocytes isolated from the blood can inhibit human endothelial cell proliferation. Monocyte-conditioned medium was fractionated by gel filtration chromatography, yielding a 175-fold enrichment of a growth inhibitory activity, designated monocyte-derived endothelial cell inhibitory factor (MECIF). MECIF was found to be protease sensitive, resistant to acid treatment, and heat labile. When conditioned medium was subjected to HPLC gel filtration, the inhibitory activity was eluted as a single peak with a molecular weight of 50-70 kDa. Several characteristics distinguish MECIF from previously described monocyte/macrophage-derived inhibitory factors. Unlike TGF-beta, MECIF is heat labile and does not induce a mitogenic response in growth-arrested normal rat kidney cells. In addition, polyclonal antibodies specific for TGF-beta or INF-gamma do not inhibit MECIF activity. MECIF preparations show low levels of TNF-alpha, insufficient to promote the observed growth inhibitory effect. MECIF activity on human endothelial cells was found to be dose dependent and reversible. MECIF also appeared to be target cell selective in that it did not significantly alter the growth of human smooth muscle cells or skin fibroblasts. These data suggest that monocyte-derived factors may play a key role in inhibiting endothelial cell proliferation.     

Endothelial‐derived thrombospondin‐1 promotes macrophage recruitment and apoptotic cell clearance

Rapid apoptotic cell engulfment is crucial for prevention of inflammation and autoimmune diseases and is conducted by special immunocompetent cells like macrophages or immature dendritic cells. We recently demonstrated that endothelial cells (ECs) also participate in apoptotic cell clearance. However, in contrast to conventional phagocytes they respond with an inflammatory phenotype. To further confirm these pro‐inflammatory responses human ECs were exposed to apoptotic murine ECs and changes in thrombospondin‐1 (TSP‐1) expression and in activation of intracellular signalling cascades were determined by real‐time qPCR, immunoblotting and immunocytochemistry. Human primary macrophages or monocytic lymphoma cells (U937) were incubated with conditioned supernatant of human ECs exposed to apoptotic cells and changes in activation, migration and phagocytosis were monitored. Finally, plasma levels of TSP‐1 in patients with anti‐neutrophil cytoplasmic antibody(ANCA)‐associated vasculitis (AAV) were determined by ELISA. We provided evidence that apoptotic cells induce enhanced expression of TSP‐1 in human ECs and that this increase in TSP‐1 is mediated by the mitogen‐activated protein kinases (MAPK) ERK1 and 2 and their upstream regulators MEK and B‐Raf. We also showed that plasma TSP‐1 levels are increased in patients with AAV. Finally, we showed that conditioned supernatant of ECs exposed to apoptotic cells induces pro‐inflammatory responses in monocytes or U937 cells and demonstrated that increased TSP‐1 expression enhances migration and facilitates engulfment of apoptotic cells by monocyte‐derived macrophages or U937 cells. These findings suggest that under pathological conditions with high numbers of uncleared dying cells in the circulation endothelial‐derived elevated TSP‐1 level may serve as an attraction signal for phagocytes promoting enhanced recognition and clearance of apoptotic cells.     

Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1

Thrombospondin-1 (TSP1) has potent biological effects on vasculature smooth muscle cells (SMCs) and endothelial cells. The regulation of extracellular accumulation of TSP1 is mediated by a previously obscure process of endocytosis which leads to its lysosomal degradation. Since members of the low density lipoprotein receptor (LDLR) family have been found to mediate endocytosis which leads to degradation of a diverse array of ligands, we evaluated their possible role in the uptake and degradation of TSP1 by vascular SMCs, endothelial-cells and fibroblasts. 125I-TSP1 was found to be internalized and degraded lysosomally by all these cell types. Both the internalization and degradation of 125I-TSP1 could be inhibited by a specific antagonist of the LDLR family, the 39-kD receptor-associated protein (RAP). Antibodies to the LDLR-related protein (LRP) completely blocked the uptake and degradation of 125I-TSP1 in SMCs and fibroblasts but not endothelial cells. Solid-phase binding assays confirmed that LRP bound to TSP1 and that the interaction was of high affinity (Kd = 5 nM). Neither RAP nor LRP antibodies inhibited the binding of 125I-TSP1 to surfaces of SMCs. However, cell surface binding, as well as, endocytosis and degradation could be blocked by heparin or by pre- treatment of the cells with either heparitinase, chondroitinase or beta- D-xyloside. The data indicates that cell surface proteoglycans are involved in the LRP-mediated clearance of TSP1. A model for the clearance of TSP1 by these cells is that TSP1 bound to proteoglycans is presented to LRP for endocytosis. In endothelial cells, however, the internalization of TSP1 was not mediated by LRP but since RAP inhibited TSP1 uptake and degradation, we postulate that another member of the LDLR family is likely to be involved.     

Specific inhibition of endothelial cell proliferation by isolated endothelial plasma membranes

Cultures of human vascular endothelial cells were used to study the phenomenon of density-dependent inhibition of cell growth. Endothelial cells were disrupted by nitrogen cavitation, and a plasma membrane-enriched fraction was prepared by differential centrifugation followed in some cases by sucrose density gradient fractionation. Membrane suspension was added to low-density early-passage endothelial cultures grown in microwells. Hemocytometer cell counts and 6 hr 3H-thymidine pulses were performed in triplicate wells at varying intervals. Plasma membranes suppressed cell proliferation in a reversible, dose-dependent fashion. Increasing the ambient concentration of endothelial cell growth factor did not alter the inhibitory effect. The antiproliferative effect was sensitive to heat and trypsin and to incubation with 0.1 M sodium carbonate, pH 11.5. Membrane vesicles selectively derived from the apical cell surface also suppressed proliferation. This phenomenon showed at least some specificity for cell type and species in both human and bovine models. Therefore, cell-cell contact is capable of regulating endothelial cell proliferation in vitro despite the presence of available growth surfaces and of optimally supportive culture medium.