Cell adhesion molecule T-cadherin regulates vascular cell adhesion, phenotype and motility

T-cadherin (T-cad), an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion molecules, is widely expressed in the cardiovascular system. The expression profile of T-cad within diseased (atherosclerotic and restenotic) vessels indicates some relationship between expression of T-cad and the phenotypic status of resident cells. Using cultures of human aortic smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) we investigate the hypothesis that T-cad may function in modulating adhesive properties of vascular cells. Coating of culture plates with recombinant T-cad protein or with antibody against the first amino-terminal domain of T-cad (anti-EC1) significantly decreased adhesion and spreading of SMC and HUVEC. HUVECs adherent on T-cad or anti-EC1 substratum exhibited an elongated morphology and associated redistribution of the cytoskeleton and focal adhesions to a distinctly peripheral location. These changes are characteristic of the less-adhesive, motile or pro-migratory, pro-angiogenic phenotype. Boyden chamber migration assay demonstrated that the deadhesion induced by T-cad facilitates cell migration towards a serum gradient. Overexpression of T-cad in vascular cells using adenoviral vectors does not influence cell adhesion or motility per se, but increases the detachment and migratory responses induced by T-cad substratum. The data suggest that T-cad acts as an anti-adhesive signal for vascular cells, thus modulating vascular cell phenotype and migration properties.     

Induction of cell adhesion molecules, E-selectin, VCAM-1 and ICAM-1, in a co-culture of human endothelial and smooth muscle cells

Using immunofluorescence and flow cytometry, we studied the surface expression of cell adhesion molecules, E-selectin, VCAM-1 and ICAM-1, in human umbilical vein endothelial cells (HUVEC) co-cultured with human aortic intimal smooth muscle cells (SMC). It was found that inactivated HUVEC constitutively expressed only ICAM-1. After 3-4 h of co-culturing with SMC in the Transwell system we observed the appearance of E-selectin and VCAM-1, and the increase of ICAM-1 content on the cell surface. In all the cases, the maximum expression of these molecules (85-100% of positively stained cells) was detected within 18-24 h after co-culturing. Similar effect was exerted by SMC-conditioned culture medium, whose action well compared with that of a direct addition of interleukin-1 to EC at a concentration of 5-10 u/ml. The obtained data suggest that the cytokines secreted by SMC may participate in the regulation of endothelial cell adhesion molecule expression, and influence cell accumulation in sites of inflammation, immune disorders, etc.     

Flow effects on cultured vascular endothelial and smooth muscle cell functions.

Cultured vascular endothelial cells were exposed to fluid shear stress by means of a rotary-disc shear-loading device, and the physiological effects of the conditioned medium (CM) and the homogenate (HM) of the cells on migration, adhesion and growth of endothelial cells (EC) or smooth muscle cells (SMC) were studied. Effects of shear stress on the production and secretion of collagen, one of the extracellular matrices of EC, were also studied. CM stimulated the adhesion and growth of SMC, but not of EC themselves. The ability to stimulate SMC adhesion and growth was similar in CM obtained from the static and shear-loaded cells. HM of the shear-loaded EC stimulated SMC migration. Further, HM of the shear-loaded EC contained increased amounts of collagen compared with the static EC. These results suggest that: 1) EC produce and secrete accelerators for the adhesion and growth of SMC, 2) EC react to the physical stimulus of fluid shear stress to produce stimulators of SMC migration, and 3) EC produce collagen, the production of which is enhanced by fluid shear stress.     

Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin alphavbeta3 on vascular endothelial cells

Vascular smooth muscle cell growth-promoting factor (VSGP) was originally isolated from bovine ovarian follicular fluid as a stimulator of vascular smooth muscle cell proliferation. Homology searches indicate that bovine and human VSGPs are orthologs of rat F-spondin. Here, we examined whether recombinant human VSGP/F-spondin affected the biological activities of endothelial cells. VSGP/F-spondin did not affect the proliferation of human umbilical vein endothelial cells (HUVECs); however, it did inhibit VEGF- or bFGF-stimulated HUVEC migration. To clarify the mechanism of this inhibitory effect, we examined the adhesion of HUVECs to extracellular matrix proteins. VSGP/F-spondin specifically inhibited the spreading of HUVECs on vitronectin via the functional blockade of integrin alphavbeta3. As a result, VSGP/F-spondin inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) when HUVECs were plated on vitronectin. Moreover, VSGP/F-spondin inhibited the activation of Akt when HUVECs on vitronectin were stimulated with VEGF. VSGP/F-spondin inhibited tube formation by HUVECs in vitro and neovascularization in the rat cornea in vivo. These results indicate that VSGP/F-spondin inhibits angiogenesis at least in part by the blockade of endothelial integrin alphavbeta3.     

Maspin Regulates Endothelial Cell Adhesion and Migration through an Integrin Signaling Pathway

Maspin has been identified as a potent angiogenesis inhibitor. However, the molecular mechanism responsible for its anti-angiogenic property is unclear. In this study, we examined the effect of maspin on endothelial cell (EC) adhesion and migration in a cell culture system. We found that maspin was expressed in blood vessels ECs and human umbilical vein endothelial cells (HUVECs). Maspin significantly enhanced HUVEC cell adhesion to various matrix proteins. This effect was dependent on the activation of integrin β₁, which subsequently led to distribution pattern changes of vinculin and F-actin. These results indicated that maspin affects cell adhesion and cytoskeleton reorganization through an integrin signal transduction pathway. Analysis of HUVECs following maspin treatment revealed increased integrin-linked kinase activities and phosphorylated FAK levels, consistent with increased cell adhesion. Interestingly, when HUVECs were induced to migrate by migration stimulatory factor bFGF, active Rac1 and cdc42 small GTPase levels were decreased dramatically at 30 min following maspin treatment. Using phosphorylated FAK at Tyr³⁹⁷ as an indicator of focal adhesion disassembly, maspin-treated HUVECs had elevated FAK phosphorylation compared with the mock treated control. The results were a reduction in focal adhesion disassembly and the retardation in EC migration. This study uncovers a mechanism by which maspin exerts its effect on EC adhesion and migration through an integrin signal transduction pathway.     

Mesenchymal stem cell modification of endothelial matrix regulates their vascular differentiation

Mesenchymal stem cells (MSCs) respond to a variety of differentiation signal provided by their local environments. A large portion of these signals originate from the extracellular matrix (ECM). At the same time, MSCs secrete various matrix‐altering agents, including proteases, that alter ECM‐encoded differentiation signals. Here we investigated the interactions between MSC and ECM produced by endothelial cells (EC‐matrix), focusing not only on the differentiation signals provided by EC‐matrix, but also on MSC‐alteration of these signals and the resultant affects on MSC differentiation. MSCs were cultured on EC‐matrix modified in one of three distinct ways. First, MSCs cultured on native EC‐matrix underwent endothelial cell (EC) differentiation early during the culture period and smooth muscle cell (SMC) differentiation at later time points. Second, MSCs cultured on crosslinked EC‐matrix, which is resistant to MSC modification, differentiated towards an EC lineage only. Third, MSCs cultured on EC‐matrix pre‐modified by MSCs underwent SMC‐differentiation only. These MSC‐induced matrix alterations were found to deplete the factors responsible for EC‐differentiation, yet activate the SMC‐differentiation factors. In conclusion, our results demonstrate that the EC‐matrix contains factors that support MSC differentiation into both ECs and SMCs, and that these factors are modified by MSC‐secreted agents. By analyzing the framework by which EC‐matrix regulates differentiation in MSCs, we have uncovered evidence of a feedback system in which MSCs are able to alter the very matrix signals acting upon them. J. Cell. Biochem. 107: 706–713, 2009. Published 2009 Wiley‐Liss, Inc.     

Effect of different cell sheet ECM microenvironment on the formation of vascular network

The repair and reconstruction of large bone defects remains as a significant clinical challenge mainly due to the insufficient vascularization. The prefabrication of vascular network based on cell sheet technique brings a promising potential for sufficient vascularization due to rich extracellular matrix (ECM) of cell sheets. However, the effect of different cell sheet ECM micro-environment on the formation of a vascular network has not been well understood. Here our goal is to study the effect of different cell sheets on the formation of a vascular network. First we cultured human bone marrow mesenchymal stem cells (hBMSCs) under two culture conditions to obtain osteogenic differentiated cell sheet (ODCS) and undifferentiated cell sheet (UDCS), respectively. Then the human umbilical vein endothelial cells (HUVECs) were seeded onto the surface of the two sheets at different seeding densities to fabricate pre-vascularized cell sheets. Our results indicated that the two sheets facilitated the alignment of HUVECs and promoted the formation of vascular networks. Quantitative analysis showed that the number of networks in ODCS was higher than that in the UDCS. The ECM of the two sheets was remodeled and rearranged during the tubulogenesis process. Furthermore, results showed that the optimal seeding density of HUVECs was 5 × 10⁴ cell/cm². In summary, these results suggest that the vascularized ODCS has a promising potential to construct pre-vascularized tissue for bone repair.     

Influence of low density lipoproteins on vascular smooth muscle cell growth and motility: modulation by extracellular matrix.

Low density lipoproteins (LDL) are thought to play a major role in cardiovascular diseases such as atherosclerosis. Much remains to be done to understand the cellular effects of LDL and how the extracellular matrix (ECM) influences these effects. We found that LDL produced a dose dependent increase in vascular smooth muscle cell (SMC) proliferation. The ECM altered the proliferative response of SMC to LDL: on collagen I there was a 66% inhibition, endothelial cell derived-ECM a 2-fold increase, and collagen IV no difference in proliferation compared to paired controls. LDL affected SMC motility (cell area and shape factor) but the extent and direction of the effect depended on whether the cells were cultured on uncoated or coated dishes. LDL treated cultures had a 5-fold lower migration rate but net movement was not different, suggesting that LDL decreased SMC random movement. There was a dose-dependent accumulation of lipid by SMC incubated with LDL and, subsequently, cytoplasmic lipid droplets were observed. Cells cultured on uncoated plates showed an increased cholesterol content as a function of LDL concentration. In contrast, cells cultured on a collagen IV matrix showed no net change in cholesterol content over the range of LDL concentrations studied. Hence, the uptake of LDL cholesterol appears to be completely inhibited by this matrix. These studies indicate that the influence of LDL on several SMC parameters is modulated by ECM components.     

The majority of type 1 plasminogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissue-type plasminogen activator

The interactions between exogenously added tissue-type plasminogen activator (t-PA) and the active form of type 1 plasminogen activator inhibitor (PAI-1) produced by and present in cultured human umbilical vein endothelial cells (HUVECs) were investigated. Immunoblotting analysis of the conditioned media obtained from monolayers of HUVECs treated with increasing concentrations of t-PA (less than or equal to 10 micrograms/ml) revealed a dose-dependent formation of both t-PA/PAI-1 complexes, and of a 42,000-Mr cleaved or modified form of the inhibitor. Immunoradiometric assays indicated that t-PA treatment resulted in a fourfold increase in PAI-1 antigen present in the conditioned media. This increase did not result from the release of PAI-1 from intracellular stores, but rather reflected a t-PA-dependent decrease in the PAI-1 content of the Triton X-100 insoluble extracellular matrix (ECM). Although the rate of t-PA-mediated release of PAI-1 was increased by the removal of the monolayer, similar quantities of PAI-1 were removed in the presence or absence of the cells. These results suggest that the cells only represent a semipermeable barrier between ECM-associated PAI-1 and exogenous t-PA. Treatment of HUVECs with t-PA (1 microgram/ml, 2 h) to deplete the ECM of PAI-1 did not affect the subsequent rate of PAI-1 production and deposition into the ECM. Immunogold electron microscopy of HUVECs not only confirmed the location of PAI-1 primarily in the region between the culture substratum and ventral cell surface but failed to demonstrate significant (less than 1%) PAI-1 on the cell surface. Thus, the majority of PAI-1 associated with cultured HUVEC monolayers is present under the cells in the ECM and is accessible to solution-phase t-PA.     

Microvascular pericytes contain muscle and nonmuscle actins

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.     

Inducible nitric oxide and prostacyclin productions are differently controlled by extracellular matrix and cell density in human vascular endothelial cells

Both cell-matrix and cell-cell interactions are important regulators of the function of most human cells. In this study we investigated how these interactions controlled the production of vasodilators nitric oxide (NO), and prostacyclin (PGI₂), in freshly isolated human umbilical vein endothelial cells (HUVECs). On the reconstituted extracellular matrix (ECM) Matrigel freshly isolated HUVECs treated with interleukin-1β, lipopolysaccharide, and interferon-γ, produced more NO, but less PGI₂, than on gelatin substratum. High cell density was essential for inducibility of NO production in cells plated on gelatin substratum, but not on ECM. In cells plated on gelatin substratum at low cell density, which mimicked conventional HUVEC culturing conditions, both inducible NO production and the inducible NO synthase (iNOS) mRNA levels, detected by competitive RT-PCR, were low. However, inducible PGI₂ production remained high in these cells. Highest inducible NO productions were observed in HUVECs that presumably had best maintained their original differentiated phenotype. Thus our data imply that the inducible NO and PGI₂ productions of freshly isolated HUVECs were differently controlled by the extracellular matrix and cell density. Our data suggest that both cell-matrix and cell-cell interactions may have a strong influence on the proinflammatory cytokine responses of human vascular endothelial cells. J. Cell. Biochem. 64:538–546. © 1997 Wiley-Liss, Inc.     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.     

Human macrovascular endothelial cells: Optimization of culture conditions

Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage.     

Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells

Tissue engineering may offer patients new options when replacement or repair of an organ is needed. However, most tissues will require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network would be promotion of vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, we isolated CD34(+)/CD133(+) endothelial progenitor cells (EPC) from human umbilical cord blood and expanded the cells ex vivo as EPC-derived endothelial cells (EC). The EPC lost expression of the stem cell marker CD133 but continued to express the endothelial markers KDR/VEGF-R2, VE-cadherin, CD31, von Willebrand factor, and E-selectin. The cells were also shown to mediate calcium-dependent adhesion of HL-60 cells, a human promyelocytic leukemia cell line, providing evidence for a proinflammatory endothelial phenotype. The EPC-derived EC maintained this endothelial phenotype when expanded in roller bottles and subsequently seeded on polyglycolic acid-poly-l-lactic acid (PGA-PLLA) scaffolds, but microvessel formation was not observed. In contrast, EPC-derived EC seeded with human smooth muscle cells formed capillary-like structures throughout the scaffold (76.5 +/- 35 microvessels/mm(2)). These results indicate that 1) EPC-derived EC can be expanded in vitro and seeded on biodegradable scaffolds with preservation of endothelial phenotype and 2) EPC-derived EC seeded with human smooth muscle cells form microvessels on porous PGA-PLLA scaffolds. These properties indicate that EPC may be well suited for creating microvascular networks within tissue-engineered constructs.     

Long-term effect of losartan administration on blood pressure, heart and structure of coronary artery of young spontaneously hypertensive rats

Alterations in geometry and structure of coronary arteries have marked consequences on blood flow to the respective area. We evaluated long-term effect of losartan on blood pressure (BP), heart weight/body weight (HW/BW), geometry and structure of septal branch of coronary artery (RS) of young SHR and Wistar rats. Four-week-old Wistar rats and SHR were used. Losartan was administered (20 mg/kg/day) in drinking water by gavage for 5 weeks. BP was measured by plethysmographic method. Cardiovascular system was perfused with a fixative (120 mm Hg). RS was processed for electron microscopy. Wall thickness of intima + media (WT), inner diameter (ID), cross-sectional area of intima + media (CSA), volume densities (VD) of endothelial cells (EC), extracellular matrix (ECM) of intima, smooth muscle cells (SMC) and ECM of media were evaluated. BP of 4-week-old SHR did not differ from that of Wistar rats. BP, HW/BW, WT, CSA, WT/ID, CSAs of SMC, ECM of media were increased in 9-week-old SHR, whereas their VD and CSA of EC were decreased. Losartan administration decreased BP and HW/BW in both groups. Geometry of RS was affected only in SHR (reduction of WT, CSA, WT/ID and increased of ID, circumferential tension, VD and CSA of EC). Losartan administration reduced BP and myocardial mass in both groups and beneficially affected geometry and structure of coronary artery in SHR.     

Extracellular matrix is a source of mitogenically active platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF A_s (short splice version) is secreted, PDGF A_L (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and η-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions. © 1996 Wiley-Liss, Inc.