Release of prostacyclin,endothelium-derived relaxing factor and endothelin by freshly harvested cells attached to microcarrier beads

Summary Cultured endothelial cells have been used in the past as a source of endothelium-derived relaxing factor (EDRF) and of prostacyclin (PGI₂). Although cell cultures are essential for observation of prolonged exposure to media or when there is delayed response, they are time consuming and sterile conditions are essential. In the present study, we report that endothelial cells, freshly harvested from bovine aortas, readily attached themselves to cytodex-3 microcarrier beads and released an endothelium-derived relaxing factor (EDRF), prostacyclin (PGI₂) and increased the amount of cyclic GMP in vascular smooth muscle. Attachment to microcarrier beads was essential since it increased the surface area and the number of attached cells and permited collection of cell free filtrates because of the formation of dense networks of cells and beads. As a result superfusion of cells and beads on the filter did not dislodge bound cells which remain on the filter. Conditioned filtrates from freshly harvested endothelial cells attached to microcarrier beads caused marked relaxation of endothelium-deprived bovine pulmonary artery strips. The degree of relaxation depended on the number of cells; maximal relaxation occurred with 50 million cells at ED₅₀ of 14 million. High values of cyclic GMP were found in vascular smooth muscle exposed to conditioned filtrate. The calcium ionophore A23187 further increased the amount of cyclic GMP. Large amounts of PGI₂ were released by freshly harvested endothelial cells particularly after stimulation with the calcium ionophore. In contrast, endothelin production by freshly harvested cells attached to microcarrier beads was barely detectable after 30 min incubation and was beyond the limit of detection by bioassay procedures. Freshly harvested endothelial cells attached to microcarrier beads appear to be a useful adjunct to tissue cultures under specific experimental conditions.Abbreviations EDRF Endothelium-Derived Relaxing Factor - PGI₂ Prostacyclin - K-H Krebs-Henseleit solution - cyclic GMP cyclic Guanosine Monophosphate - fmoles femtomoles - IB Ibuprofen     

Role of endothelial cells in the proliferative response of cultured pulmonary vascular smooth muscle cells to reduced oxygen tension

Summary The development of pulmonary hypertension in a wide variety of human disease states and experimental animal models characterized by chronic alveolar hypoxia is mediated by two pathologic vascular processes, a) vasoconstriction and b) vasoconstruction (structural remodeling). The anatomic changes seen within the pulmonary circulation include a) increased deposition of collagen and elastin in the adventitial layer and b) aberrant pulmonary vascular smooth muscle cell proliferation and maturation in the medial segments. Despite the demonstrated ability of pharmacologic manipulation in the experimental animal to ameliorate both the structural and hemodynamic changes, the actual etiologic mechanisms are only beginning to be explored. Using the cell culture technique of co-cultivation, we have investigated the potential role of bovine pulmonary arterial endothelial cell-derived factors in mediating abnormal bovine smooth muscle cell growth under conditions of reduced oxygen tension. We have demonstrated that these cultured endothelial cells exposed in vitro to reduced levels of atmospheric oxygen concentrations of 5.0% and 2.5% O₂ for durations of 24 to 72 h produce and secrete soluble growth factor(s) which stimulate smooth muscle cell proliferation when compared to cells maintained under standard tissue culture oxygen conditions of 95% room air. This growth-stimulatory effect required the concomitant presence of serum factors (0.5% fetal bovine serum), was inhibited by heparin, was distinct from platelet-derived growth factor, and seemed to have a molecular weight greater than 14 000 Da. We conclude that reduced levels of oxygen tension in vitro can selectively induce pulmonary arterial endothelial cells to release mitogen(s) which can stimulate vascular smooth muscle replication. Furthermore, we speculate that this in vitro finding may be of importance as an etiologic mechanism to explain the accelerated smooth muscle cell growth characteristic of hypoxic pulmonary arteriopathy.     

αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-α_vβ₃ antibodies prevent cell adhesion to FGF-2. Also, purified human α_vβ₃ binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-α_vβ₃ monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with α_vβ₃ integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.     

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.     

Microparticle formation after co-culture of human whole blood and umbilical artery in a novel in vitro model of flow

Cardiovascular disease (CVD) is now the largest killer in western society, and the importance of interactions between vascular endothelium and circulating blood components in disease pathogenesis is well established. Microparticles are a heterogeneous population of <1 μm blood borne particles that arise from blebbing or shedding of cell membranes. The microparticle population includes several classes of apoptotic bodies; however, increased numbers of procoagulant microparticles have been described in plasma from people with CVD. We have previously demonstrated that interactions of monocytes and platelets with isolated inflamed endothelial cells lead to production of pro-coagulant tissue factor bearing microparticles under laminar flow conditions. Here we have investigated microparticle production after perfusion of human whole blood through intact inflamed human umbilical artery. When blood was perfused through umbilical arteries which had been pre-stimulated with tumour necrosis factor (TNFα) for 18 h under flow conditions, there was significantly increased production of microparticles from both platelet and non-platelet sources, in particular from erythrocytes. To determine whether microparticles generated during interactions with inflamed endothelium could induce a pro-inflammatory response in trans, we isolated microparticles by centrifugation after co-culture and incubated with isolated quiescent endothelial cells followed by measurement of reactive oxygen species formation. Microparticles derived from co-culture with inflamed endothelium induced significantly enhanced levels of reactive oxygen species (ROS). These data suggest that presence of an inflamed endothelium causes release of pro-inflammatory microparticles from circulating blood cells, which could contribute to prolonged endothelial activation and subsequent atherosclerotic changes in blood vessels subjected to inflammatory insult.     

Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterization of vascular endothelial growth factor (VEGF) as the mitogen.

BACKGROUND: New vessel growth is often associated with ischemia, and hypoxic tissue has been identified as a potential source of angiogenic factors. In particular, ischemia is associated with the development of neovascularization in a number of ocular pathologies. For this reason, we have studied the induction of endothelial cell mitogens by hypoxia in retinal cells. MATERIALS AND METHODS: Human retinal pigment epithelium (hRPE) were grown under normoxic and hypoxic conditions and examined for the production of endothelial mitogens. Northern analysis, biosynthetic labeling and immunoprecipitation, and ELISA were used to assess the levels of vascular endothelial growth factor/vascular permeability factor (VEGF) and basic fibroblast growth factor (bFGF), two endothelial cell mitogens and potent angiogenic factors. Soluble receptors for VEGF were employed as competitive inhibitors to determine the contribution of the growth factor to the hypoxia-stimulated mitogen production. RESULTS: Following 6-24 hr of hypoxia, confluent and growing cultures of hRPE increase their levels of VEGF mRNA and protein synthesis. Biosynthetic labeling studies and RT-PCR analysis indicate that the cells secrete VEGF121 and VEGF165, the soluble forms of the angiogenic factor. In contrast, hRPE cultured under hypoxic conditions show reduced steady-state levels of basic fibroblast growth factor (bFGF) mRNA and decreased bFGF protein synthesis. Unlike VEGF, bFGF is not found in conditioned media of hRPE following 24 hr of hypoxia. Using a soluble high-affinity VEGF receptor as a competitive inhibitor of VEGF, we demonstrate that a VEGF-like activity is the sole hypoxia-inducible endothelial mitogen produced by cultured hRPE. CONCLUSIONS: From this comparison we conclude that hRPE do not respond to hypoxia with a general, nonspecific increase in the overall levels of growth factors, as is seen during cell wounding responses or serum stimulation. The physiological relevance of data from this in vitro model are affirmed by separate studies in an animal model of retinal ischemia-induced ocular neovascularization (1) in which retina-derived VEGF levels have been shown to correlate spatio-temporally with the onset of angiogenesis. Taken together, these data support the hypothesis that the induction of VEGF by hypoxia mediates the rapid, initial angiogenic response to retinal ischemia.     

The correlation between leaf-surface and leaf-tissue secondary metabolites: a case study with pyrrolizidine alkaloids in <Emphasis Type="Italic">Jacobaea</Emphasis> hybrid plants

Introduction

Usually whole plant or whole leaf extracts are analyzed to study the chemical ecology of insect-plant interactions. For herbivore species the contact with the leaf surface enables them to estimate the quality of the plant. The relationship between the leaf-surface and leaf-tissue secondary metabolites (SMs) could offer important new insights in insect-plant interactions mediated by SMs. Pyrrolizidine alkaloids (PAs), typical defense chemicals in Jacobaea species, are repellent for generalist herbivores but are attractive to specialists.

Objectives

Explore whether the PAs on the leaf surface are a reliable representation of the PAs in the leaf tissue in PA-containing plants.

Method

The concentration of individual PAs present on the leaf surface and in the corresponding leaf tissue from 37 genotypes (one plant from each genotype) of an F₂ generation of a cross between Jacobaea vulgaris and Jacobaea aquatica was measured by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). PAs were removed from the leaf surface by extraction with a slightly acidic aqueous solution.

Results

The total amount of PAs present on the surface of the leaves was only 0.015% (range 0.001–0.163%) of the total amount present in the leaf tissue. Most PAs present in the leaf tissue were also found on the surface, except for jaconine, dehydrojaconine, dehydrojacoline and usaramine N-oxide. Positive correlations between leaf-surface and leaf-tissue concentrations were found for most of the jacobine-like and otosenine-like PAs, but correlations for total PA, senecionine- and erucifoline-like PAs were not significant.

Conclusion

These results indicate that PA variation on the leaf surface only partially reflects the PA variation in the leaf tissue. Because most herbivores are affected in a different manner by individual PAs, this result means that the leaf surface does not give a reliable estimate of plant quality to herbivores.

     

Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases

We have previously shown that capillary endothelial cells grown on the surface of three-dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary-like tubular structures in response to tumor-promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine-specific phosphatases, to determine whether this agent could induce the expression of an angiogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasminogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase-type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.     

Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells

Proper bone remodeling requires an active process of angiogenesis which in turn supplies the necessary growth factors and stem cells. This tissue cooperation suggests a cross‐talk between osteoblasts and endothelial cells. This work aims to identify the role of paracrine communication through vascular endothelial growth factor (VEGF) in co‐culture between osteoblastic and endothelial cells. Through a well defined direct contact co‐culture model between human osteoprogenitors (HOPs) and human umbilical vein endothelial cells (HUVECs), we observed that HUVECs were able to migrate along HOPs, inducing the formation of specific tubular‐like structures. VEGF₁₆₅ gene expression was detected in the HOPs, was up‐regulated in the co‐cultured HOPs and both Flt‐1 and KDR gene expression increased in co‐cultured HUVECs. However, the cell rearrangement observed in co‐culture was promoted by a combination of soluble chemoattractive factors and not by VEGF₁₆₅ alone. Despite having no observable effect on endothelial cell tubular‐like formation, VEGF appeared to have a crucial role in osteoblastic differentiation since the inhibition of its receptors reduced the co‐culture‐stimulated osteoblastic phenotype. This co‐culture system appears to enhance both primary angiogenesis events and osteoblastic differentiation, thus allowing for the development of new strategies in vascularized bone tissue engineering. J. Cell. Biochem. 106: 390–398, 2009. © 2009 Wiley‐Liss, Inc.     

Apolipoprotein E: A potent inhibitor of endothelial and tumor cell proliferation

Recombinant human apolipoprotein E3 (apoE), purified from E. coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose- and time-dependent manner. ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number. Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin-binding growth factors. Thus, at low serum concentrations (0–2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold. The bFGF-dependent proliferation was dramatically inhibited by apoE with an IC₅₀ ≈ 50 nM. Under conditions where cell proliferation was mainly serum-dependent, apoE also suppressed growth but required higher concentrations to be effective (IC₅₀ ≈ 500 nM). ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells. The IC₅₀ values obtained with these cells were generally 3–5 times higher than with BAE cells. Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture. In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF. Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE. The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growth, and metastasis.     

Factor Xa and thrombin evoke additive calcium and proinflammatory responses in endothelial cells subjected to coagulation

Endothelial cells react to factor Xa and thrombin by proinflammatory responses. It is unclear how these cells respond under physiological conditions, where the serine proteases factor VIIa, factor Xa and thrombin are all simultaneously generated, as in tissue factor-driven blood coagulation. We studied the Ca(2+) signaling and downstream release of interleukins (ILs), induced by these proteases in monolayers of human umbilical vein endothelial cells. In single cells, factor Xa, but not factor VIIa, complexed with tissue factor, evoked a greatly delayed, oscillatory Ca(2+) response, which relied on its catalytic activity and resembled that of SLIGRL, a peptide specifically activating the protease-activated receptor 2 (PAR2). Thrombin even at low concentrations evoked a rapid, mostly non-oscillating Ca(2+) response through activation of PAR1, which reinforced the factor Xa response. The additive Ca(2+) signals persisted, when factor X and prothrombin were activated in situ, or in the presence of plasma that was triggered to coagulate with tissue factor. Further, thrombin reinforced the factor Xa-induced production of IL-8, but not of IL-6. Both interleukins were produced in the presence of coagulating plasma. In conclusion, under coagulant conditions, factor Xa and thrombin appear to contribute in different and additive ways to the Ca(2+)-mobilizing and proinflammatory reactions of endothelial cells. These data provide first evidence that these serine proteases trigger distinct signaling modules in endothelium that is activated by plasma coagulation.     

β3-Adrenergic Receptor Stimulation Induces E-Selectin-mediated Adipose Tissue Inflammation

Inflammation induced by wound healing or infection activates local vascular endothelial cells to mediate leukocyte rolling, adhesion, and extravasation by up-regulation of leukocyte adhesion molecules such as E-selectin and P-selectin. Obesity-associated adipose tissue inflammation has been suggested to cause insulin resistance, but weight loss and lipolysis also promote adipose tissue immune responses. While leukocyte-endothelial interactions are required for obesity-induced inflammation of adipose tissue, it is not known whether lipolysis-induced inflammation requires activation of endothelial cells. Here, we show that β₃-adrenergic receptor stimulation by CL 316,243 promotes adipose tissue neutrophil infiltration in wild type and P-selectin-null mice but not in E-selectin-null mice. Increased expression of adipose tissue cytokines IL-1β, CCL2, and TNF-α in response to CL 316,243 administration is also dependent upon E-selectin but not P-selectin. In contrast, fasting increases adipose-resident macrophages but not neutrophils, and does not activate adipose-resident endothelium. Thus, two models of lipolysis-induced inflammation induce distinct immune cell populations within adipose tissue and exhibit distinct dependences on endothelial activation. Importantly, our results indicate that β₃-adrenergic stimulation acts through up-regulation of E-selectin in adipose tissue endothelial cells to induce neutrophil infiltration.     

Interactions of adipocytes and their precursor cells with endothelial cells in culture

Factors involved in the growth of adipose tissue were examined by testing interactions under cell culture conditions between cellular components of this tissue and plasma from overfed rats. The cellular factors were capillary fragments, endothelial cells during growth and after confluence, fibroblasts, adipocytes and adipose precursor cells before determination (adipoblasts) and after determination (preadipocytes). Multiplying adipose precursor cells stimulated markedly the multiplication of endothelial cells, while their own multiplication was inhibited. The stimulatory effect was partially transferred into the culture medium but not remaining in culture dishes conditioned by preceding cultures of adipose precursor cells, removed by Tris-EDTA buffer or mechanically. The activity was apparently not dependent on feeding conditions. Plasma from overfed rats did not affect endothelial or adipose precursor cell multiplication, but caused more rapid lipid filling of the latter. Endothelial cells facilitated lipid accumulation of preadipocytes. These results indicate that when adipose tissue is expanding by adipocyte multiplication capillarization is stimulated secondarily, being then capable of facilitating triglyceride accumulation in adipocytes.     

Integrin-mediated Signaling Events in Human Endothelial Cells

Vascular endothelial cells are important in a variety of physiological and pathophysiological processes. The growth and functions of vascular endothelial cells are regulated both by soluble mitogenic and differentiation factors and by interactions with the extracellular matrix; however, relatively little is known about the role of the matrix. In the present study, we investigate whether integrin-mediated anchorage to a substratum coated with the extracellular matrix protein fibronectin regulates growth factor signaling events in human endothelial cells. We show that cell adhesion to fibronectin and growth factor stimulation trigger distinct initial tyrosine phosphorylation events in endothelial cells. Thus, integrin-dependent adhesion of endothelial cells leads to tyrosine phosphorylation of both focal adhesion kinase and paxillin, but not of several growth factor receptors. Conversely, EGF stimulation causes receptor autophosphorylation, with no effect on focal adhesion kinase or paxillin tyrosine phosphorylation. Adhesion to fibronectin, in the absence of growth factors, leads to activation of MAPK. In addition, adhesion to fibronectin also potentiates growth factor signaling to MAPK. Thus, polypeptide growth factor activation of MAPK in anchored cells is far more effective than in cells maintained in suspension. Other agonists known to activate MAPK were also examined for their ability to activate MAPK in an anchorage-dependent manner. The neuropeptide bombesin, the bioactive lipid lysophosphatidic acid (LPA), and the cytokine tumor necrosis factor α, which signal through diverse mechanisms, were all able to activate MAPK to a much greater degree in fibronectin-adherent cells than in suspended cells. In addition, tumor necrosis factor α activation of c-Jun kinase (JNK) was also much more robust in anchored cells. Together, these data suggest a cooperation between integrins and soluble mitogens in efficient propagation of signals to downstream kinases. This cooperation may contribute to anchorage dependence of mitogenic cell cycle progression.     

Role of polyamines in regulating silymarin production in Silybum marianum (L.) Gaertn (Asteraceae) cell cultures under conditions of calcium deficiency

As part of our efforts to identify the possible role of polyamines (PAs) in silymarin (Sm) production, the effects of calcium deprivation on cell growth and on endogenous PAs levels and Sm production by milk thistle (Silybum marianum (L.) Gaertn) grown in cell cultures were examined. Young cultured cells of the H2 line of S. marianum were transferred to a medium without calcium and with ethylene glycol-bis-(β-aminoethyl) ether-N,N,N′,N′-tetraacetic acid present to chelate any free calcium in order to analyze the effects of this medium on the levels of PAs and Sm produced by the cells. During the 17 days of exposure to this calcium-free medium most of the cell populations were in the G0/G1 phase (from day 7 to day 14 of culture) while PA levels underwent a progressive decline up to day 17, after which they were no longer detectable. We observed that putrescine (Put) accumulation was always lower than that observed under normal conditions. The lack of calcium in the MS medium advances the onset of the stationary phase, whose beginning is marked by an increase in the Put/spermidine (Spd) index, raising the production of Sm; the suspensions were productive for a longer time and hence produced more of the substance. Our results indicate that under stress conditions the production of Sm in young-cell suspensions of S. marianum is not associated with high levels of PAs in the medium – contrary to what one would expect – allowing us to conclude that growth inhibition appears to be the factor responsible for the maximum Sm accumulation while PAs are not directly involved in the Sm synthesis pathway by milk thistle grown in culture.     

The role of vascular actors in two dimensional dialogue of human bone marrow stromal cell and endothelial cell for inducing self-assembled network

Angiogenesis is very important for vascularized tissue engineering. In this study, we found that a two-dimensional co-culture of human bone marrow stromal cell (HBMSC) and human umbical vein endothelial cell (HUVEC) is able to stimulate the migration of co-cultured HUVEC and induce self-assembled network formation. During this process, expression of vascular endothelial growth factor (VEGF₁₆₅) was upregulated in co-cultured HBMSC. Meanwhile, VEGF₁₆₅-receptor2 (KDR) and urokinase-type plasminogen activator (uPA) were upregulated in co-cultured HUVEC. Functional studies show that neutralization of VEGF₁₆₅ blocked the migration and the rearrangement of the cells and downregulated the expression of uPA and its receptor. Blocking of vascular endothelial-cadherin (VE-cad) did not affect the migration of co-cultured HUVEC but suppressed the self-assembled network formation. In conclusion, co-cultures upregulated the expression of VEGF₁₆₅ in co-cultured HBMSC; VEGF₁₆₅ then activated uPA in co-cultured HUVEC, which might be responsible for initiating the migration and the self-assembled network formation with the participation of VE-cad. All of these results indicated that only the direct contact of HBMSC and HUVEC and their respective dialogue are sufficient to stimulate secretion of soluble factors and to activate molecules that are critical for self-assembled network formation which show a great application potential for vascularization in tissue engineering.     

Cellular mechanisms and intracellular signaling pathways for the modulation of eNOS in pulmonary arteries by 15-HETE

The 15-hydroxyeicosatetraenoic acid (15-HETE), a lipid metabolite and vasoconstrictor, plays an important role in hypoxic contraction of pulmonary arteries (PAs) through working on smooth muscle cells (SMCs). Previous studies have shown that vascular endothelium is also involved in PAs tone regulation. However, little is known as to how the pulmonary artery endothelial cells (PAECs) are related to the 15-HETE-induced vasoconstriction and that which intracellular signaling systems are critical. To test this hypothesis, we examined PAs constriction in isolated rat PAs rings, the expression and activity of endothelial nitric oxide synthase (eNOS) with western blot, and nitric oxide (NO) production using the DAF-FM DA fluorescent indicator. The results showed that the 15-HETE-induced PAs constriction was diminished in endothelium-intact rings. In the presence of the eNOS inhibitor L-NAME, vasoconstrictor responses to KCl were greater than the control. The activation of eNOS was activated by Ca²⁺ released from intracellular stores and the PI3K/Akt pathway. Phosphorylations of the eNOS at Ser-1177 and Akt at Ser-473 were necessary for their activity. A prolonged 15-HETE treatment (30?min) led to a decrease in NO production by phosphorylation of eNOS at Thr-495, leading to augmentation of PAs constriction. Therefore, 15-HETE initially inhibited the PAs constriction through the endothelial NO system, and both Ca²⁺ and the PI3K/Akt signaling systems are required for the effects of 15-HETE on PAs tone regulation.     

Vascular Permeability Factor/Vascular Endothelial Growth Factor Inhibits Anchorage-Disruption-Induced Apoptosis in Microvessel Endothelial Cells by Inducing Scaffold Formation

Survival and proliferation of endothelial cells requires both growth factors and an appropriate extracellular matrix to which cells can attach. In the absence of either, endothelial cells rapidly undergo apoptosis. Thus, when human microvascular endothelial cells (HDMEC) are plated on a hydrophobic surface such as untreated polystyrene, they rapidly undergo apoptosis and die. The present study demonstrates that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-selective cytokine, inhibits apoptosis of HDMEC cultured on untreated polystyrene and induces these cells to adhere, spread, and proliferate. VPF/VEGF-induced HDMEC adhesion was time-dependent, requiredde novoprotein synthesis, and was inhibited by a soluble RGD peptide but not by an inhibitor of collagen synthesis. Under the conditions of these experiments, VPF/VEGF downregulated expression of collagen IV and fibronectin but did not change collagen I mRNA levels. VPF/VEGF-induced HDMEC adhesion was inhibited by antibodies to αvβ5 and vitronectin but not by antibodies to αvβ3. Other endothelial growth factors and cytokines such as bFGF, HGF, and TGFβ did not reproduce the VPF/VEGF effect. We suggest that VPF/VEGF induces endothelial cells to deposit a scaffolding (likely involving vitronectin) that allows them to attach to and proliferate on an otherwise nonsupportive surface (hydrophobic polystyrene) and in this manner serves as both a survival factor and a growth factor.     

pH regulates vascular endothelial growth factor binding to fibronectin: a mechanism for control of extracellular matrix storage and release

Hypoxia is one of the major signals that induces angiogenesis. Hypoxic conditions lead to reduced extracellular pH. Vascular endothelial growth factor (VEGF) binding to endothelial cells and the extracellular matrix (ECM) increases at acidic pH (7.0-5.5). These interactions are dependent on heparan sulfate proteoglycans, but do not depend on the presence of VEGF receptors. Here we report that VEGF(165) and VEGF(121) binding to fibronectin also increased at acidic pH, and that these interactions are further enhanced by the addition of heparin. These results reveal that the accepted non-heparin-binding isoform of VEGF (VEGF(121)) is converted into a heparin-binding growth factor under acidic conditions. Interestingly, we did not observe increased binding of VEGF to collagen type I at acidic pH in the presence or absence of heparin, indicating that this effect is not a general property of all heparin-binding ECM proteins. The high level of VEGF binding at acidic pH was also rapidly reversed as demonstrated by increased rates of VEGF dissociation from fibronectin and fibronectin-heparin matrices as the pH was raised. The VEGF released from fibronectin retained its ability to stimulate the activation of extracellular-regulated kinase 1/2 in endothelial cells. These results suggest that VEGF may be stored in the extracellular matrix via interactions with fibronectin and heparan sulfate in tissues that are in need of vascularization so that it can aid in directing the dynamic process of growth and migration of new blood vessels.