Vascular Endothelial Cell Behavior Research Articles

Pharmacological control of angiogenesis by regulating phosphorylation of myosin light chain 2

BackgroundControl of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis. Methods and resultsWe exposed transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs. ConclusionThe present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis.

Dominant geometrical factors of collective cell migration in flexible 3D gelatin tube structures

Collective cell migration is a dynamic and interactive behavior of cell cohorts essential for diverse physiological developments in living organisms. Recent studies have revealed the importance of three-dimensional (3D) topographical confinements to regulate the migration modes of cell cohorts in tubular confinement. However, conventional in vitro assays fail to observe cells’ behavior in response to 3D structural changes, which is necessary for examining the geometric regulation factors of collective migration. Here, we introduce a newly developed assay for fabricating flexible 3D structures of capillary microtunnels to examine the behavior of vascular endothelial cells (ECs) as they progress through the successive transition across wide or narrow tube structures. The microtunnels with altered diameters were formed inside gelatin-gel blocks by photo-thermal etching with micrometer-sized spot heating of the focused infrared laser absorption. The ECs migrated and spread two-dimensionally on the inner surface of gelatin capillary microtunnels as a monolayer instead of filling the entire capillary. In the straight cylindrical topographical constraint, leading ECs exhibited no apparent diameter dependence for the maximum peak migration velocity. However, widening the diameter in the narrow-wide structures caused a decrease in migration velocity following in direct proportion to the diameter increase ratio, whereas narrowing the diameter in wide-narrow microtunnels increased the speed without obvious correlation between velocity change and diameter change. The results demonstrated the ability of the newly developed flexible 3D gelatin tube structures for collective cell migration, and the findings provide insights into the dominant geometric factor of the emerging migratory modes for endothelial migration as asymmetric fluid flow-like behavior in the borderless cylindrical cell sheets.

Vascularization and neuralization of bioactive calcium magnesium phosphate/hydrogels for wound healing

Reconstruction of the microenvironment at the site of injury plays an important role in tissue regeneration and functional recovery. With calcium magnesium phosphate (MCPC) as a model, the effects of MCPC on the vascularization and neurotization in the process of wound regeneration were investigated. Herein, a series of MCPC/hydrogels with different concentrations (0–4%) of MCPC were synthesized. The results showed that MCPC could stimulate the migration behavior of vascular endothelial cells (HUVECs) and up-regulate the expression of VEGF and HIF-1α genes. Further in vivo results revealed that MCPC/hydrogels significantly improved wound healing and blood vessel formation. In addition, we demonstrated that MCPC/hydrogels could also largely accelerate the regeneration of nerves, which would finally contribute to wound healing due to the proper release of magnesium and calcium ions. This research also provides an insight toward the positive role of MCPC in vascular and nerve reconstruction during wound healing.

Reelin Signaling in Vascular Endothelial Cell Biology

IntroductionEstablishment of the coronary circulation is a critical step in heart development. Formation of functional coronary vessels is required for the heart to undergo successful compaction of the ventricular myocardium. Oxygen and nutrients are delivered to the contractile myocardium through the coronary vasculature. Molecular signaling pathways that control formation of the coronary vessels are being identified. Immunofluorescent studies from our lab indicate that the Reelin extracellular matrix glycoprotein is expressed within the vascular endothelial cells of developing and mature coronary vessels of the mouse heart. The Reelin signaling pathway is known to contribute to brain and lymphatic system development by influencing neuronal and endothelial cell behaviors such as adhesion and migration. However, its role in coronary vascular development is as yet unidentified.Study ObjectiveThe goal of this study was to elucidate the potential roles of Reelin in formation of the coronary vasculature by identifying how it regulates vascular endothelial cell behaviors. We hypothesized that Reelin signaling regulates establishment of coronary vessel integrity during cardiogenesis by influencing vascular endothelial cell adhesion as well as cell membrane permeability and resistance.MethodsWe utilized the primary human dermal microvascular endothelial cell line (HDMEC) as an in vitro system to examine contributions of Reelin to vascular endothelial cell behavior. A small interfering RNA‐mediated approach was used to silence the endogenous RELN gene in HDMECs and reduce Reelin protein expression. Transfected HDMECs were subjected to cell‐based assays to assess changes in vascular endothelial cell behavior in response to RELN silencing. We assessed vascular endothelial cell adhesion on extracellular matrices that are required during coronary vessel development. Vessel integrity was examined by assaying endothelial cell membrane permeability and resistance in RELN‐silenced cells. Also, we examined the effect of Reelin expression on cell viability.ResultsRELN‐silenced HDMECs exhibited a >90% reduction in Reelin expression in comparison to non‐targeting, negative control HDMECs. This significant Reelin reduction altered vascular endothelial cell adhesion to certain extracellular matrices. These RELN‐silenced HDMECs exhibited a significant decrease in baseline cell membrane permeability and concomitant changes in cell membrane resistance. Furthermore, we observed changes in cell morphology in RELN‐silenced cells versus control cells. Preliminary data suggests that loss of RELN expression affects vascular endothelial cell morphology and expression of components within the Reelin signaling pathway.ConclusionsBased on this evidence, we conclude that Reelin influences adhesion, morphology, membrane permeability and membrane resistance of vascular endothelial cells in order to regulate vessel wall integrity. These findings may provide key evidence as to how Reelin expression in nascent vascular endothelial cells impacts formation of the coronary vessels during mammalian cardiovascular development.Support or Funding InformationAHA 17AIREA3360773 and PCOM Center for Chronic Disorders of AgingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vascular Endothelial Cell Behavior in Complex Mechanical Microenvironments

The vascular mechanical microenvironment consists of a mixture of spatially and temporally changing mechanical forces. This exposes vascular endothelial cells to both hemodynamic forces (fluid flow, cyclic stretching, lateral pressure) and vessel forces (basement membrane mechanical and topographical properties). The vascular mechanical microenvironment is "complex" because these forces are dynamic and interrelated. Endothelial cells sense these forces through mechanosensory structures and transduce them into functional responses via mechanotransduction pathways, culminating in behavior directly affecting vascular health. Recent in vitro studies have shown that endothelial cells respond in nuanced and unique ways to combinations of hemodynamic and vessel forces as compared to any single mechanical force. Understanding the interactive effects of the complex mechanical microenvironment on vascular endothelial behavior offers the opportunity to design future biomaterials and biomedical devices from the bottom-up by engineering for the cellular response. This review describes and defines (1) the blood vessel structure, (2) the complex mechanical microenvironment of the vascular endothelium, (3) the process in which vascular endothelial cells sense mechanical forces, and (4) the effect of mechanical forces on vascular endothelial cells with specific attention to recent works investigating the influence of combinations of mechanical forces. We conclude this review by providing our perspective on how the field can move forward to elucidate the effects of the complex mechanical microenvironment on vascular endothelial cell behavior.

TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1.

Endothelial cell adhesion and migration are critical steps of the angiogenic process, whose dysfunction is associated with tumor growth and metastasis. The TRPM8 channel has recently been proposed to play a protective role in prostate cancer by impairing cell motility. However, the mechanisms by which it could influence vascular behavior are unknown. Here, we reveal a novel non-channel function for TRPM8 that unexpectedly acts as a Rap1 GTPase inhibitor, thereby inhibiting endothelial cell motility, independently of pore function. TRPM8 retains Rap1 intracellularly through direct protein-protein interaction, thus preventing its cytoplasm-plasma membrane trafficking. In turn, this mechanism impairs the activation of a major inside-out signaling pathway that triggers the conformational activation of integrin and, consequently, cell adhesion, migration, in vitro endothelial tube formation, and spheroid sprouting. Our results bring to light a novel, pore-independent molecular mechanism by which endogenous TRPM8 expression inhibits Rap1 GTPase and thus plays a critical role in the behavior of vascular endothelial cells by inhibiting migration.

Interleukin-32α Inhibits Endothelial Inflammation, Vascular Smooth Muscle Cell Activation, and Atherosclerosis by Upregulating Timp3 and Reck through suppressing microRNA-205 Biogenesis.

Interleukin-32 (IL-32) is a multifaceted cytokine that promotes inflammation and regulates vascular endothelial cell behavior. Although some IL-32 isoforms have been reported to contribute to vascular inflammation and atherosclerosis, the functional role of IL-32α in vascular inflammation and atherogenesis has not been studied.Methods: IL-32α function was assessed in cells with transient IL-32α overexpression or treated with recombinant human IL-32α by western blotting and mRNA expression analysis. Vascular smooth muscle cell (VSMC) proliferation and migration was examined by BrdU incorporation and wound healing assays, respectively. In addition, the participation of IL-32α on vascular inflammation, arterial wall thickening, and atherosclerosis in vivo was monitored in human IL-32α transgenic (hIL-32α-Tg) mice with or without ApoE knockout (ApoE-/-/hIL-32α-Tg).Results: Our analyses showed that IL-32α suppresses genes involved in the inflammatory and immune responses and cell proliferation, and by limiting matrix metalloproteinase (MMP) function. In vivo, administration of hIL-32α inhibited vascular inflammation and atherosclerosis in hIL-32α-Tg and ApoE-/-/hIL-32α-Tg mice. Subsequent microarray and in silico analysis also revealed a marked decreased in inflammatory gene expression in hIL-32α-Tg mice. Collectively, our studies demonstrated that IL-32α upregulates the atheroprotective genes Timp3 and Reck by downregulating microRNA-205 through regulation of the Rprd2-Dgcr8/Ddx5-Dicer1 biogenesis pathway.Conclusion: Our findings provide the first direct evidence that IL-32α is an anti-inflammatory and anti-atherogenic cytokine that may be useful as a diagnostic and therapeutic protein in atherosclerosis.

Cervical Carcinoma Cells Stimulate the Angiogenesis through TSLP Promoting Growth and Activation of Vascular Endothelial Cells

To explore whether cervical carcinomas cells-derived thymic stromal lymphopoietin (TSLP) modulates the biologic behavior of vascular endothelial cells and herein participates in the angiogenesis in the cervical cancer pathogenesis. We analyzed expression of TSLP and its receptor (TSLPR) in cervical cancer cells by immunohistochemistry, ELISA, and flow cytometry, respectively. We further investigated the effects of TSLP on the proliferation, apoptosis, activation, and angiogenesis in vitro of human umbilical vein endothelial cells (HUVECs). It has been found that the cervical cancer cells translate TSLP and endothelial cells express TSLPR in cervical cancer tissues. Both HeLa and CaSki cells secret TSLP in a time-dependent manner, and the ratio of TSLPR-positive HUVECs is about 30%. It has been showed that recombinant human TSLP (rhTSLP) can significantly increase Ki67 and CD62E expression in HUVECs and interleukin-6 (IL-6) levels from HeLa and CaSki cells; on the contrary, anti-human TSLP or TSLPR neutralizing antibody down-regulates the expression of Ki67, angiogenesis-relative molecules CD62E, and CD105 in HUVECs cocultured with HeLa or CasKi cells and inhibits IL-6 secretion from HeLa and CaSki cells. Moreover, both rhTSLP and endogenous TSLP from HeLa or CaSki cells obviously stimulate the proliferation, activation, and angiogenesis, but not influence the apoptosis of HUVECs in vitro. This study has demonstrated that TSLP secreted by cervical carcinomas cells is involved in the angiogenesis of cervical cancer in a paracrine manner.

Low Concentration of S100A8/9 Promotes Angiogenesis-Related Activity of Vascular Endothelial Cells: Bridges among Inflammation, Angiogenesis, and Tumorigenesis?

Previous studies showed that several members of the S100A family are involved in neovascularization and tumor development. This study checked whether low concentrations of S100A8 or S100A9 has any effect on the behaviour of vascular endothelial cells. A human umbilical vascular endothelial cell (HUVEC) line was used to measure vascular endothelial cell bioactivity related to angiogenesis, such as cell proliferation, migration, and vessel formation. In the low concentration range up to 10 μg/mL, either each alone or in combination, S100A8 and S100A9 proteins promoted proliferation of HUVEC cells in a dose-dependent manner. The presence of both proteins in culture showed additive effects over each single protein. Both proteins enhanced HUVEC cells to migrate across the transwell membrane and to form tube-like structures on the Matrigel surface. When mixed in Matrigel and injected subcutaneously in Balb/c mice, both proteins increased vessel development in the gel plugs. Microarray assay of HUVEC cells treated with 10 μg/mL S100A8 revealed that ribosome pathway, pathogenic Escherichia coli infection pathway, apoptosis, and stress response genes were modulated by S100A8 treatment. We propose that S100A8 and S100A9 proteins from either infiltrating inflammatory cells or tumor cells play an important role in the interplay among inflammation, angiogenesis, and tumorigenesis.

Enhanced Cellular Function of Human Vascular Endothelial Cell on Poly (ε-caprolactone)/Gelatin Coaxial-Electrospun Scaffold

An extracellular matrix-like poly (ε-caprolactone) (PCL)/gelatin core-shell nanofibrous scaffold with high hydrophilicity was developed by coaxial-elecrospinning. However, the behavior of vascular endothelial cells (ECs) on the modified scaffold remains limited. In this study, human umbilical vein ECs (HUVECs) were seeded onto PCL scaffolds with or without gelatin. Morphological changes of HUVECs were observed under confocal laser scanning microscopy (LSCM). HUVECs’ adhesion, proliferation and apoptosis were detected by MTT assay and flow cytometry (FCM). Our results showed that HUVECs on PCL/gelatin scaffolds with identical polygonal and cobblestone-like characteristics reached confluence after 7 days. Modification of PCL nanofibers significantly promoted the attachment of HUVECs onto scaffolds within 1 hour. Compared to pristine PCL, a two-fold increase in proliferation of HUVECs was also observed after 7 days, whereas the apoptosis of HUVECs was obviously reduced by 40% on the modified scaffolds. In summary, these results indicated modified PCL/gelatin scaffold developed by coaxial-elecrospinning can increase the adhesion, proliferation, and suppress apoptosis of HUVECs, suggesting it has a great potential and promising vascular graft in tissue engineering applications.

The role of substratum compliance of hydrogels on vascular endothelial cell behavior

Cardiovascular disease (CVD) remains a leading cause of death both within the United States (US) as well as globally. In 2006 alone, over one-third of all deaths in the US were attributable to CVD. The high prevalence, mortality, morbidity, and socioeconomic impact of CVD has motivated a significant research effort; however, there remain significant knowledge gaps regarding disease onset and progression as well as pressing needs for improved therapeutic approaches. One critical area of research that has received limited attention is the role of biophysical cues on the modulation of endothelial cell behaviors; specifically, the impact of local compliance, or the stiffness, of the surrounding vascular endothelial extracellular matrix. In this study, the impact of substratum compliance on the modulation of cell behaviors of several human primary endothelial cell types, representing different anatomic sites and differentiation states in vivo, were investigated. Substrates used within our studies span the range of compliance that has been reported for the vascular endothelial basement membrane. Differences in substratum compliance had a profound impact on cell attachment, spreading, elongation, proliferation, and migration. In addition, each cell population responded differentially to changes in substratum compliance, documenting endothelial heterogeneity in the response to biophysical cues. These results demonstrate the importance of incorporating substratum compliance in the design of in vitro experiments as well as future prosthetic design. Alterations in vascular substratum compliance directly influence endothelial cell behavior and may participate in the onset and/or progression of CVDs.

Modulation of human vascular endothelial cell behaviors by nanotopographic cues

Basement membranes possess a complex three-dimensional topography in the nanoscale and submicron range which have been shown to profoundly modulate a large menu of fundamental cell behaviors. Using the topographic features found in native vascular endothelial basement membranes as a guide, polyurethane substrates were fabricated containing anisotropically ordered ridge and groove structures and isotropically ordered pores from 200 nm to 2000 nm in size. We investigated the impact of biomimetic length-scale topographic cues on orientation/elongation, proliferation and migration on four human vascular endothelial cell-types from large and small diameter vessels. We found that all cell-types exhibited orientation and alignment with the most pronounced response on anisotropically ordered ridges ≥800 nm. HUVEC cells were the only cell-type examined to demonstrate a decrease in proliferation in response to the smallest topographic features regardless of surface order. On anisotropically ordered surfaces all cell-types migrated preferentially parallel to the long axis of the ridges, with the greatest increase in cell migration being observed on the 1200 nm pitch. In contrast, cells did not exhibit any preference in direction or increase in migration speed on isotropically ordered surfaces. Overall, our data demonstrate that surface topographic features impact vascular endothelial cell behavior and that the impact of features varies with the cell behavior being considered, topographic feature scale, surface order, and the anatomic origin of the cell being investigated.

Biophysical Cueing and Vascular Endothelial Cell Behavior

Human vascular endothelial cells (VEC) line the vessels of the body and are critical for the maintenance of vessel integrity and trafficking of biochemical cues. They are fundamental structural elements and are central to the signaling environment. Alterations in the normal functioning of the VEC population are associated with a number of vascular disorders among which are some of the leading causes of death in both the United States and abroad. VECs attach to their underlying stromal elements through a specialization of the extracellular matrix, the basement membrane. The basement membrane provides signaling cues to the VEC through its chemical constituents, by serving as a reservoir for cytoactive factors and through its intrinsic biophysical properties. This specialized matrix is composed of a topographically rich 3D felt-like network of fibers and pores on the nano (1–100 nm) and submicron (100–1,000 nm) size scale. The basement membrane provides biophysical cues to the overlying VECs through its intrinsic topography as well as through its local compliance (relative stiffness). These biophysical cues modulate VEC adhesion, migration, proliferation, differentiation, and the cytoskeletal signaling network of the individual cells. This review focuses on the impact of biophysical cues on VEC behaviors and demonstrates the need for their consideration in future vascular studies and the design of improved prosthetics.

Endothelial Mechanisms of Flow-Mediated Athero-Protection and Susceptibility

See related article, pages 97–105 The arterial endothelium survives remarkably well as the interface between blood and vessel wall in an environment of constantly changing biomechanical stresses as well as acute and chronic exposure to inflammatory stimulants (eg, cytokines and hypercholesterolemia respectively).1 Cell turnover, which tends to occur in regional clusters,2 is otherwise very low in this monolayer. The endothelium also plays an important regulatory role in the pathogenesis of vascular disease. The cells readily respond to diverse stimuli through a repertoire of mechanisms to enhance their own survival even as they facilitate inflammatory, proatherogenic responses in the subendothelial tissue. The necessity to be a responsive cellular interface probably accounts for much of the endothelial phenotype heterogeneity that exists between vascular beds as well as within discrete regions of the arterial circulation.3,4 Hemodynamic characteristics that vary with blood vessel geometry predict the location of arterial sites that are susceptible to atherosclerosis.5 Curved and branching vessel geometries create sites of flow separation that contain transient flow reversals, lower average shear stresses, and occasional turbulence, (collectively, disturbed flow) and that are predictive of lesion formation. In contrast, pulsatile unidirectional laminar flow (and higher average shear stresses) is associated with regions where atherosclerosis rarely occurs, despite there being equivalent exposure to plasma risk factors such as hypercholesterolemia throughout the circulation. Although the signatures of endothelial phenotype in such regions in vivo are varied and complex, data are emerging from genomic5–7 and protein8 analyses of endothelium at such sites that identify molecular differences. Some of these are accessible for study in vitro to investigate detailed mechanisms under more controlled conditions. …

Effect of Pore Size of Self-Organized Honeycomb-Patterned Polymer Films on Spreading, Focal Adhesion, Proliferation, and Function of Endothelial Cells

The design of nano- and microstructures based on self-organization is a key area of research in the search for new materials, and it has a variety of potential applications in tissue engineering scaffolds. We have reported a honeycomb-patterned polymer film (honeycomb film) with highly regular pores that is formed by self-organization. This study describes the behavior of vascular endothelial cells (ECs) on honeycomb films with four different pore sizes (5, 9, 12, and 16 microm) as well as on a flat film. We examined the influence of the honeycomb pattern and pore size on cell behavior. The changes in cell morphologies, actin filaments, vinculin clusters, cell proliferation, and secreted extracellular matrix (ECM) (fibronectin, laminin, type IV collagen, and elastin) production profiles were observed by using optical, fluorescence, and scanning electron microscopy. The ECs that adhered to the flat film showed an elongated morphology with random orientation; the actin filaments and focal adhesions were not conspicuous. On the other hand, the ECs on the honeycomb films exhibited greater spreading and flattening; the degree of spreading of the ECs increased with an increase in the pore size. The actin filaments and focal adhesions appeared conspicuous, and the focal adhesions localized along the edge of the honeycomb pores were distributed over the entire projected cell area. The honeycomb film with a pore size of 5 microm showed the highest cell proliferation and ECM production profiles. These results suggest that the honeycomb film is a suitable material for designing a new vascular device.

Regulation of adrenomedullin release from human endothelial cells by sex steroids and angiotensin-II

It is well documented that there are gender differences in the incidence and patterns of cardiovascular diseases but the reasons are unclear. Sex steroids may modulate the behaviour of vascular endothelial cells, which in turn act by paracrine processes to alter adjacent vascular smooth muscle activity. We hypothesised that the sex steroids alter the percentage of vascular endothelial cells that secrete the vasodilator peptide, adrenomedullin and modify the adrenomedullin-stimulating action of angiotensin-II. The percentage of adrenomedullin-secreting human aortic endothelial cells was determined using the cell immunoblot method. Cells were incubated with selected concentrations of angiotensin-II, oestradiol and testosterone alone and sex steroids in combination with angiotensin-II. Adrenomedullin mRNA expression in endothelial cells was quantified by real-time PCR. It was observed that at 4 h, angiotensin-II increased the percentage of adrenomedullin-secreting cells in a concentration-dependent manner. Testosterone at physiological concentrations was observed to increase the number of adrenomedullin-secreting cells whilst oestradiol had no effect. Addition of testosterone to angiotensin-II resulted in less than additive increases in the number of cells secreting adrenomedullin. Oestradiol reduced the angiotensin-II-induced increase in adrenomedullin-secreting cells. Adrenomedullin mRNA expression was significantly increased by testosterone and there was also a trend for an increase in adrenomedullin mRNA expression, which occurred when cells were incubated with angiotensin-II. Our results point to a complex interplay between the sex steroids and angiotensin-II in regulating adrenomedullin production by human endothelial cells, which may contribute to gender-related differences in vascular disease in humans.

Normal Force Exerted on Vascular Endothelial Cells

Hemodynamic forces play an important role in the normal and pathological behavior of vascular endothelial cells as recent studies on the shear stress over the endothelium have shown. Based on computational investigation and scaling analysis, our study shows that the normal force contributes significantly to the total force on the endothelial cells even in large vessels. Therefore, our study suggests that the functions of endothelial cells are also affected by the normal forces exerted on them. The effects of the normal force are more pronounced for smaller vessels and/or less spread cells.

Cell Density-dependent Regulation of Proteoglycan Synthesis by Transforming Growth Factor-β1 in Cultured Bovine Aortic Endothelial Cells

The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-beta(1) (TGF-beta(1)) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-beta(1) on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-beta(1) also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(3)H]glucosamine, [(35)S]sulfate, or (35)S-labeled amino acids in the presence of TGF-beta(1). The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-beta(1) on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-beta(1) induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of approximately 50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of approximately 400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-beta(1) cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-beta(1) promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.

Establishment and characterization of a novel in vitro angiogenesis model using a microvascular endothelial cell line, F-2C, cultured in chemically defined medium.

The behavior of vascular endothelial cells (EC) is an important factor in the processes involved in angiogenesis, but the regulatory mechanisms of angiogenesis, especially underlying the tubulogenesis by EC are not yet clear. Although a number of in vitro experimental models of tubulogenesis have been developed by use of cultured EC, most of those models are too complex to be easily handled and further, the culture media are usually supplemented with serum, creating problems in interpretation of experimental results. To generate a simple in vitro angiogenesis study model under serum-free culture conditions, we adapted a murine microvascular endothelial cell line, F-2, to a chemically defined medium, Cos Medium 001, and successfully established a subline of F-2, designated F-2C, which revealed a unique growth pattern. In Cos Medium 001, F-2C proliferates in a cobblestone pattern at an early growth stage, but, at a late growth stage, spontaneously differentiates to form three-dimensional honeycomblike tubular structures without the supplementation of any specific factors. The cell aggregation activity of F-2C in the presence of Ca2+ was much greater than that of F-2. The amount of subendothelial matrix deposited by F-2C was significantly higher than that by F-2, and increased prominently after the F-2C cells reached the differentiating stage of tubulogenesis. These findings indicate that F-2C is a new EC line in which tubulogenesis is spontaneously induced by the marked deposition of basement membrane analog to the subendothelial matrix and by the enhancement of presumable cadherin activity. We suggest that this cell line, F-2C, represents a simple and useful in vitro angiogenesis model.