Role of extracellular signaling on endothelial cell proliferation and protein N-glycosylation

In vitro studies of angiogenic phenomenon have been limited due to nonavailability of a simple and biologically relevant model of the capillary wall. Recent development of a capillary endothelial cell line from the vascular bed of bovine adrenal medulla made us to study the effect of heparin, thrombin, thyroxine, glucagon, insulin, and phorbol myristate acetate (PMA) on the proliferative and metabolic activities such as glycosylation of asparagine-linked glycoproteins of these cells in culture. Out of six different agents studied here, only heparin, thrombin, and thyroxine reduced the doubling time of these cells by 24 hr with no observed morphological abnormality. Glucagon, showed marginal reduction in the cell doubling time. By contrast, insulin and PMA enhanced the doubling time. Insulin treatment though induced the S phase of cell cycle but it blocked the cells entry into the G2 + M phase. PMA arrested the cells in G0/G1 phase. The cellular response to protein N-glycosylation is increased in the presence of thyroxine, insulin, and thrombin and the effect is dose dependent. Further analysis on SDS-PAGE indicated that glycosylation of 80-120 kDa and 43 kDa glycoprotein species are enhanced when these cells are treated with insulin and thrombin. Glycopeptide generated from these glycoproteins suggested that they all carry "high mannose" and "complex" type oligosaccharide chains attached to their protein core.     

The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells

Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.     

Isolation and properties in culture of human adrenal capillary endothelial cells

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.     

Importance of size, sulfation, and anticoagulant activity in the potentiation of acidic fibroblast growth factor by heparin

Heparin was previously reported to potentiate the mitogenic activity of endothelial cell mitogens in a crude extract of bovine hypothalami (Thornton, S. C., Mueller, S. N., and Levine, E. M. (1983) Science 222, 623-625). We and others (Gospodarowicz, D., and Cheng, J. (1986) J. Cell. Physiol. 128, 475-484) have reported that the growth stimulatory effects of acidic fibroblast growth factor (aFGF) are potentiated in a similar manner. We have used these observations as the basis of an assay to characterize the importance of size, sulfation, and anticoagulant activity of heparin in mediating this effect. Partial nitrous acid depolymerization of heparin from porcine intestinal mucosa resulted in a mixture of heparin fragments, containing oligosaccharides ranging from disaccharides to polysaccharides of about 40 monosaccharides in length. This mixture was fractionated by ion exchange chromatography and gel permeation chromatography to obtain size-homogeneous oligosaccharides with different degrees of sulfation. Assay of these heparin-derived saccharides in the presence of a suboptimal concentration of aFGF revealed that a minimum chain length and a certain degree of sulfation is required in order to potentiate the action of aFGF. Low sulfate oligosaccharides (4-16 units) were unable to potentiate aFGF, whereas medium sulfate fractions of octadecasaccharides and larger were able to moderately potentiate aFGF. The potentiation of aFGF by the high sulfate fraction correlated with the saccharide size: 12 or more monosaccharide units were necessary to achieve potentiation equivalent to whole heparin, octa- and decasaccharides were mildly stimulatory, and hexasaccharides were without effect. In the absence of aFGF, intact heparin as well as all the oligosaccharides examined, inhibited the proliferation of capillary endothelial cells to approximately the same degree, between 20 and 50% inhibition. When a tetradecasaccharide was separated into a binding and a nonbinding fraction on matrix-bound antithrombin III, no difference was seen for these fractions in the endothelial cell proliferation assay. These results indicate that both size and sulfation of a heparin-derived oligosaccharide contribute to its ability to interact with aFGF and/or endothelial cells and that this interaction is independent of anticoagulant activity. In addition, our findings suggest that the inhibitory and potentiating effects of heparin on capillary endothelial cells have different structural requirements.     

Upregulation of the low density lipoprotein receptor at the blood-brain barrier: intercommunications between brain capillary endothelial cells and astrocytes

In contrast to the endothelial cells in large vessels where LDL receptors are downregulated, brain capillary endothelial cells in vivo express an LDL receptor. Using a cell culture model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes, we observed that the capacity of endothelial cells to bind LDL is enhanced threefold when cocultured with astrocytes. We next investigated the ability of astrocytes to modulate endothelial cell LDL receptor expression. We have shown that the lipid requirement of astrocytes increases the expression of endothelial cell LDL receptors. Experiments with dialysis membranes of different pore size showed that this effect is mediated by a soluble factor(s) with relative molecular mass somewhere between 3,500 and 14,000. Substituting astrocytes with smooth muscle cells or brain endothelium with endothelium from the aorta or the adrenal cortex did not enhance the luminal LDL receptor expression on endothelial cells, demonstrating the specificity of the interactions. This factor(s) is exclusively secreted by astrocytes cocultured with brain capillary endothelial cells, but it also upregulates the LDL receptor on other cell types. This study confirms the notion that the final fine tuning of cell differentiation is under local control.     

Uptake of rat plasma HDL subfractions labeled with [3H]cholesteryl linoleyl ether or with 125I by cultured rat hepatocytes and adrenal cells

Rat plasma low- and high-density lipoproteins were labeled with [3H]cholesteryl linoleyl ether and isolated by rate-zonal ultracentrifugation into apolipoprotein B-containing LDL, apolipoprotein E-containing HDL1 and apolipoprotein E-poor HDL2. These fractions were incubated with cultured rat hepatocytes and comparable amounts of all lipoproteins were taken up by the cells. Rat HDL was isolated at d 1.085-1.21 g/ml and apolipoprotein E-free HDL was prepared by heparin Sepharose chromatography. The original HDL and the apolipoprotein E-free HDL were labeled with 125I or with [3H]cholesteryl linoleyl ether and incubated with rat hepatocytes or adrenal cells in culture. The uptake of apolipoprotein E-free [3H]cholesterol linoleyl ether HDL by the cultured hepatocytes was 20-40% more than that of the original HDL. Comparison of uptake of cholesteryl ester moiety (represented by uptake of [3H]cholesteryl linoleyl ether) and of protein moiety (represented by metabolism of 125I-labeled protein) was carried out using both original and apolipoprotein E-free HDL. In experiments in which low concentrations of HDL were used, the ratio of 3H/125I exceeded 1.0. In cultured adrenal cells, the uptake of [3H]cholesteryl linoleyl ether-labeled HDL was stimulated 3-6-fold by 1 X 10(-7) M ACTH, while the uptake of 125I-labeled HDL increased about 2-fold. The ratio of 3H/125I representing cellular uptake was 2-3 and increased to 5 in ACTH-treated cells. The present results indicate that in cultured rat hepatocytes the uptake of homologous HDL does not depend on the presence of apolipoprotein E. Evidence was also presented for an uptake of cholesteryl ester independent of protein uptake in cultured rat adrenal cells and to a lesser extent in rat hepatocytes.     

GPIHBP1, an endothelial cell transporter for lipoprotein lipase

Interest in lipolysis and the metabolism of triglyceride-rich lipoproteins was recently reignited by the discovery of severe hypertriglyceridemia (chylomicronemia) in glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1)-deficient mice. GPIHBP1 is expressed exclusively in capillary endothelial cells and binds lipoprotein lipase (LPL) avidly. These findings prompted speculation that GPIHBP1 serves as a binding site for LPL in the capillary lumen, creating "a platform for lipolysis." More recent studies have identified a second and more intriguing role for GPIHBP1-picking up LPL in the subendothelial spaces and transporting it across endothelial cells to the capillary lumen. Here, we review the studies that revealed that GPIHBP1 is the LPL transporter and discuss which amino acid sequences are required for GPIHBP1-LPL interactions. We also discuss the human genetics of LPL transport, focusing on cases of chylomicronemia caused by GPIHBP1 mutations that abolish GPIHBP1's ability to bind LPL, and LPL mutations that prevent LPL binding to GPIHBP1.     

Exogenous and endogenous force regulation of endothelial cell behavior

Endothelial cells live in a dynamic environment where they are constantly exposed to external hemodynamic forces and generate cytoskeletal-based endogenous forces. These exogenous and endogenous forces are critical regulators of endothelial cell health and blood vessel maintenance at all generations of the vascular system, from large arteries to capillary beds. The first part of this review highlights the role of the primary exogenous hemodynamic forces of shear, cyclic strain, and pressure forces in mediating endothelial cell response. We then discuss the emergent role of the mechanical properties of the extracellular matrix and of cellular endogenous force generation on endothelial cell function, implicating substrate stiffness and cellular traction stresses as important mediators of endothelial cell health. The intersection of exogenous and endogenous forces on endothelial cell function is discussed, suggesting some of the many remaining questions in the field of endothelial mechanobiology.     

Heparin and acidic fibroblast growth factor interact to decrease prostacyclin synthesis in human endothelial cells by affecting both prostaglandin H synthase and prostacyclin synthase

Prostaglandin production by cultured human endothelial cells varies with growth conditions. We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI2) by human umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin-binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PGI2 production during culture in medium lacking these factors. Decreased PGI2 production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1-100 micrograms/ml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI2 production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF, mediated by alterations in two key enzymes in the arachidonate metabolic pathway.     

Modulation of endothelial cells growth induced by heparin

We have previously shown that heparin was bound and internalized by cultured human endothelial cells. In this study, we have investigated the effect of heparin on endothelial cells growth. We found that heparin inhibited 3H-thymidine uptake as well as actual cell growth in a dose-dependent manner in the presence of low concentrations of human serum. Inhibition was maximal at 1% serum concentration and was abolished at 10%. Chasing experiments supported the role of membrane-bound heparin in this inhibition. Low molecular weight heparin fractions, or pentosan polysulfate, were equally effective in inhibiting 3H-thymidine uptake. On the other hand, the simultaneous addition of heparin and ECGS was synergic in stimulating 3H-thymidine uptake. These results suggest a modulatory role of heparin in endothelial cells growth.     

Specific binding of vascular permeability factor to endothelial cells

Vascular permeability factor (VPF), also known as vascular endothelial cell growth factor, has recently been purified from guinea pig, human, and bovine sources. We show that various fetal or adult endothelial cell strains originating from either capillary or large vessels possess specific high affinity and saturable binding sites for guinea pig tumor-derived [125I]VPF. Two classes of sites with KDs of approximately 10 pM and 1 nM were detected for all endothelial cell types examined. Guinea pig [125I]VPF binding to endothelial cells was inhibited by human VPF (ID50 = 0.8 ng/ml) and by suramin (ID50 = 75 micrograms/ml) but not by heparin. Cross-linking experiments revealed specific [125I]VPF-receptor complexes of two types. Most of the complexes migrated very slowing in SDS-PAGE, indicating that they were of very high molecular weight and probably highly cross-linked. A portion of the molecules migrated as 270 kDa complexes, indicating that the molecular weight of the endothelial cell VPF receptor is about 230 kDa.     

Characterization of the receptor to vasculotropin on bovine adrenal cortex-derived capillary endothelial cells

Recently a new growth factor was purified to homogeneity, and its bioactivity seemed to be restricted to vascular endothelial derived cells. As it was also angiogenic in vivo, it was provisionally named vasculotropin (VAS). As an iodination procedure used to label VAS did not damage the molecule, it was possible to undertake binding studies. The binding of iodinated vasculotropin to bovine adrenal cortex-derived capillary endothelial cells was saturable at 250 pM, and half-maximal binding occurred at 47 pM. Scatchard's analysis of the data demonstrated two apparent classes of binding sites with apparent dissociation constants of 2 and 82 pM displaying 280 and 3400 binding sites, respectively. The binding was specific; half-displacement was observed with a 2-fold excess of unlabeled VAS. The structurally related platelet-derived growth factor did not compete in a radioreceptor assay. 125I-VAS was displaced by suramin and not by heparin. 125I-VAS was covalently cross-linked to its cell surface receptor on intact bovine adrenal cortex-derived capillary endothelial cells using the homobifunctional agents ethylene glycol bis(succinimidyl succinate) or disuccinimidyl tartarate. A major macromolecular species with an apparent molecular mass of 230,000 Da was labeled under reducing and nonreducing conditions. These data demonstrate the existence of a specific binding protein for VAS and an estimation of the size at 185,000 Da.     

Retinoids and TIMP1 prevent radiation-induced apoptosis of capillary endothelial cells

Radiation-induced changes in capillaries constitute a basic injury in the pathogenesis of chronic radiation damage to the heart, lung, liver, kidney and brain. It is important to identify new radioprotectors for capillary endothelial cells for use during radiotherapy to minimize normal tissue damage and possibly to increase the deliverable dose. Previously we demonstrated that exposure to ionizing radiation (10 Gy) results in death of bovine adrenal capillary endothelial cells in confluent monolayers by apoptosis. We also showed that retinoids inhibit the growth of endothelial cells, induce their differentiation, down-regulate matrix metalloproteinase (MMP) production, and up-regulate tissue inhibitors of matrix metalloproteinases (TIMPs). In the present studies, we demonstrated that radiation (10 Gy) induced an immediate increase in the amounts and activation of MMP1 and MMP2 in the cell fraction and medium of bovine capillary endothelial cells followed by an incidence of apoptosis. We also obtained data indicating that radiation-induced apoptosis can be inhibited by exposing bovine capillary endothelial cells to all-trans-retinol or all-trans-retinoic acid for 6 days before irradiation, even when the vitamins were removed 24 h before irradiation. Finally, we determined that inhibition of MMPs by TIMP was sufficient to block radiation-induced apoptosis, suggesting that the mechanism of protection by retinoids is through the alteration of levels of MMPs and TIMPs produced by the cells.     

Localization, transport, and uptake of D-aspartate in the rat adrenal and pituitary glands

Large amounts of D-aspartate (D-Asp) are present in the rat adrenal and pituitary glands. D-Asp is thought to be synthesized in the mammalian body and also accumulates in various tissues following intraperitoneal or intravenous administration. This report examines the origins of D-Asp in the adrenal and pituitary glands. We administered D-Asp to male rats intraperitoneally and immunolocalized this exogenous D-Asp in adrenal and pituitary tissue, using an anti-D-Asp antiserum which was previously developed in our laboratory. D-Asp levels in the rat adrenal gland have been shown to undergo a transient increase at 3 weeks of age and to decrease rapidly thereafter. We found that in the adrenal gland, exogenous D-Asp administered intraperitoneally was incorporated into the same region of the adrenal cortex in which endogenous D-Asp was present. By Northern and Western blot analysis and immunohistochemistry of glutamate (Glu) transporter, we also found that expression of the Glu transporter (GLAST), which has an affinity for D-Asp, transiently increased at 3 weeks of age and that localization patterns of the Glu transporter within the tissue were almost coincident with those of endogenous D-Asp. These observations suggest that D-Asp in the adrenal cortex of 3-week-old male rats is primarily acquired by uptake from the vascular system. We have previously shown that D-Asp is specifically localized in prolactin (PRL)-containing cells in the anterior lobe of the adult rat pituitary gland. Here we report that in the pituitary gland, exogenous D-Asp accumulated in endothelial cells, but not in PRL-containing cells. Northern and Western blot analysis and immunohistochemistry of Glu transporter revealed that developmental changes in the Glu transporter (GLAST) expression did not correlate with tissue levels of D-Asp and that the Glu transporter was not expressed in PRL-containing cells. These observations suggest that, in contrast to the adrenal gland, most of the D-Asp in the pituitary gland of adult male rats originates inside the gland itself.     

Increased expression of β-catenin in brain microvessels of a segmentally trisomic (Ts65Dn) mouse model of Down syndrome

We examined the distribution of β-catenin and endogenous blood serum albumin at the ultrastructural level in blood microvessels (capillaries) from brains of control and trisomic Ts65Dn mice. Morphological examination revealed an increased immunolabeling for β-catenin in endothelial substructures of the capillary network, such as intercellular junctions, cytoplasm, and nuclei. These immunosignals were significantly increased in all endothelial substructures from trisomic mice. These changes, however, did not affect the blood–brain barrier function of the entire microvascular network, because the increased uptake of albumin by endothelial cells and the eventual escape of this protein (microleakage) into the perivascular neuropil were noted only in a few capillary profiles. Nevertheless, these findings suggest the involvement of some segments of the microvascular network in the brain pathology associated with DS.     

Phagocytosis of hepatocyte mitochondria by rat Kupffer cells in vitro

Kupffer cells in primary culture bind and endocytose rapidly added rat liver mitochondria. Using phase contrast microscopy various stages of the uptake and digestion of these organelles were documented. Activities of mitochondrial enzymes within the Kupffer cells increased during the early phase of phagocytosis; they later declined, reaching the endogenous level of the Kupffer cell mitochondria after 3 to 4 h. The uptake was enhanced in the presence of heparin or rat serum, while iodoacetate, cytochalasin B or anti-fibronectin antisera were inhibitory. The transient presence of enzymatically active hepatocyte mitochondria renders Kupffer cells capable of producing urea. This mechanism partially explains earlier observations of urea formation in non-parenchymal rat liver cells.     

Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein

Acetylated-low density lipoprotein (Ac-LDL) is taken up by macrophages and endothelial cells via the "scavenger cell pathway" of LDL metabolism. In this report, aortic and microvascular endothelial cells internalized and degraded 7-15 times more [125I]-Ac-LDL than did smooth muscle cells or pericytes. Bound [125I]-Ac-LDL was displaced by unlabeled Ac-LDL, but not unmodified LDL. The ability to identify endothelial cells based on their increased metabolism of Ac-LDL was examined using Ac-LDL labeled with the fluorescent probe 1,1'- dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-Ac- LDL). When cells were incubated with 10 micrograms/ml Dil-Ac-LDL for 4 h at 37 degrees C and subsequently examined by fluorescence microscopy, capillary and aortic endothelial cells were brilliantly fluorescent whereas the fluorescent intensity of retinal pericytes and smooth muscle cells was only slightly above background levels. Dil-Ac-LDL at the concentration used for labeling cells had no effect on endothelial cell growth rate. When primary cultures of bovine adrenal capillary cells were labeled with 10 micrograms/ml of Dil-Ac-LDL for 4 h at 37 degrees C, then trypsinized and subjected to fluorescence-activated cell sorting, pure cultures of capillary endothelial cells could be obtained. Utilizing this method, large numbers of early passage microvascular endothelial cells can be obtained in significantly less time than with conventional methods.     

Angiotensin II stimulates receptor-mediated uptake of LDL by bovine adrenal cortical cells in primary culture

Bovine adrenal cells were isolated from the subcapsular region of the gland to obtain cultures enriched in cells of the zona glomerulosa. The cells kept in primary cultures were shown to respond to angiotensin II and adrenocorticorticotropin (ACTH) by a significant increase in aldosterone production. These primary adrenal cultures were used to study the effect of angiotensin II on LDL metabolism. Addition of angiotensin II for 48 h to the culture medium resulted in a 200-300% increase in LDL metabolism, and the lowest effective concentration was 10(-8) -10(-9) M. The angiotensin II effect became evident after 12-16 h of incubation. To compare the metabolism of the 125I-labeled protein moiety to that of cholesteryl ester of LDL, the lipoprotein was labeled also with cholesteryl linoleyl ether, a nonhydrolyzable analog of cholesteryl ester. Under basal conditions and in the presence of angiotensin II or ACTH the ratio of [3H]cholesteryl linoleyl ether to 125I indicate some preferential uptake of the cholesteryl ester moiety. Stimulation of specific LDL binding at 4 degrees C and LDL metabolism at 37 degrees C by 10(-7) M angiotensin II occurred at all concentrations of LDL studied. Linearization of the kinetic data showed that angiotensin II increased the LDL receptor number significantly but not the affinity of the LDL receptor for its ligand. The present findings indicate that in analogy to ACTH, angiotensin II can influence receptor-mediated uptake of LDL by adrenal cortical cells. It remains to be shown whether the angiotensin II effect on LDL metabolism is limited to adrenal cells or will affect other cells which express the angiotensin II receptor.     

Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules

Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10(12) binding sites/mm2 ECM with an apparent kD of 610nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 micrograms/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 micrograms/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.     

Both actin and polyproline interactions of profilin-1 are required for migration, invasion and capillary morphogenesis of vascular endothelial cells

The objective of the present study was to evaluate how different ligand interactions of profilin-1 (Pfn1), an actin-binding protein that is upregulated during capillary morphogenesis of vascular endothelial cells (VEC), contribute to migration and capillary forming ability of VEC. We adopted a knockdown-knockin experimental system to stably express either fully functional form or mutants of Pfn1 that are impaired in binding to two of its major ligands, actin (H119E mutant) and proteins containing polyproline domains (H133S mutant), in a human dermal microvascular cell line (HmVEC) against near-null endogenous Pfn1 background. We found that silencing endogenous Pfn1 expression in HmVEC leads to slower random migration, reduced velocity of membrane protrusion and a significant impairment in matrigel-induced cord formation. Only re-expression of fully functional but not any of the two ligand-binding deficient mutants of Pfn1 rescues the above defects. We further show that loss of Pfn1 expression in VEC inhibits three-dimensional capillary morphogenesis, MMP2 secretion and ECM invasion. VEC invasion through ECM is also inhibited when actin and polyproline interactions of Pfn1 are disrupted. Together, these experimental data demonstrate that Pfn1 regulates VEC migration, invasion and capillary morphogenesis through its interaction with both actin and proline-rich ligands.