Expression and localization of recombinant human EDG-1 receptors in Pichia pastoris

The receptor for human endothelial differentiation gene-1 protein (EDG-1) was C-terminally tagged with green fluorescent protein and expressed in the methylotrophic yeast, Pichia pastoris. EDG-1 expression was driven by the highly inducible alcohol oxidase 1 promoter. Expression of EDG-1 recombinant protein was detected by Western blot analysis and confocal microscopy. The recombinant EDG-1 receptor protein was located in the plasma membrane. Radioligand binding assays demonstrated that the␣EDG-1 receptors expressed in Pichia pastoris␣have specific and saturation binding of ³²P-labeled sphingosine 1-phosphate.     

Internalization of CD40 regulates its signal transduction in vascular endothelial cells

The CD40 ligand (CD40L)-CD40 dyad can ignite proinflammatory and procoagulatory activities of the vascular endothelium in the pathogenesis and progression of atherosclerosis. Besides being expressed on the activated CD4(+) T cell surface (mCD40L), the majority of circulating CD40L reservoir (sCD40L) in plasma is released from stimulated platelets. It remains debatable which form of CD40L triggers endothelial inflammation. Here, we demonstrate that the agonistic antibody of CD40 (G28.5), which mimics the action of sCD40L, induces rapid endocytosis of CD40 independent of TRAF2/3/6 binding while CD40L expressed on the surface of HEK293A cells captures CD40 at the cell conjunction. Forced internalization of CD40 by constitutively active mutant of Rab5 preemptively activates NF-kappaB pathway, suggesting that CD40 was able to form an intracellular signal complex in the early endosomes. Internalized CD40 exhibits different patterns of TRAF2/3/6 recruitment and Akt phosphorylation from the membrane anchored CD40 complex. Finally, mCD40L but not sCD40L induces the upregulation of proinflammatory cytokines and cell adhesion factors in the primary human vascular endothelial cells in vitro, although both forms of CD40L activate NF-kappaB pathway. These results therefore may help understand the molecular mechanism of CD40L signaling that contributes to the pathophysiology of atherosclerosis.     

High glucose increases B1-kinin receptor expression and signaling in endothelial cells

The loss of endothelial function is the initiating factor in the development of diabetic vascular disease. Kinins control endothelial function by the activation of two receptors: the B2 which is constitutively expressed, and the B1 which is highly induced in pathological conditions. In the present study, we observed that the levels of B1-receptor mRNA and protein are induced in endothelial cells incubated in high glucose. An increase in B1-receptor was also observed in the endothelial layer of aortas, from 4-week diabetic rats. When cells were grown in high glucose, the B1 agonist des-Arg9-BK increased nitrite levels, whereas in normal glucose nitrite levels were unchanged. Nitrite increase was blocked by L-NAME and 1400W indicating the participation of the inducible Nitric Oxide Synthase (iNOS). iNOS protein levels were also increased in high glucose. These results demonstrate the participation of the B1 receptor in the signaling pathways mediated by kinins in high glucose.     

Human endothelial progenitors constitute targets for environmental atherogenic polycyclic aromatic hydrocarbons

Cigarette smoking, a well-known cardiovascular risk factor, has been recently demonstrated to decrease circulating endothelial progenitor cell (EPC) number. Owing to the fact that polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) constitute major components of tobacco smoke, the present study was designed to analyze the effects of these chemicals on the development of human EPC cultures from peripheral blood mononuclear cells. Treatment by BP markedly impaired EPC number and EPC colonies in a dose-dependent manner. Such deleterious effects were abrogated using 3'-methoxy-4'-nitroflavone, a pure antagonist of the aryl hydrocarbon receptor, highlighting the involvement of this receptor in PAH toxicity towards EPCs. Additional events such as cytochrome P-450-dependent PAH metabolism and formation of PAH-related adducts to cellular macromolecules were also required. Overall, these data established EPCs as new cellular targets of PAHs, which may contribute to the deleterious cardiovascular effects of environmental substances containing these chemicals, especially tobacco smoke.     

Modulation of angiogenesis by tissue inhibitor of metalloproteinase-4

Despite the importance of MMP activity in the regulation of angiogenesis, relatively little is known about the role of TIMP-4, the most recently discovered endogenous MMP inhibitor, in modulating neovascularization. It has largely been assumed that all TIMPs are capable of inhibiting angiogenesis in vivo. However, it is now widely appreciated that TIMPs-1, -2, and -3 differ significantly in their ability to modulate angiogenic processes in vitro and angiogenesis in vivo. In order to study the effect of TIMP-4 in controlling angiogenesis, we have cloned and expressed TIMP-4 in a Pichia pastoris expression system, purified it to homogeneity, and tested its ability to regulate angiogenesis in vivo and in vitro. Our studies demonstrate that TIMP-4 is an inhibitor of capillary endothelial cell migration, but not of proliferation or of angiogenesis in vivo.     

Cryopreservation does not alter the allogenicity and development of vasculopathy in post-transplant rat aortas

BACKGROUND: Cryopreservation is a valuable technique for storing heart valve and vascular allografts. However, the biological ramifications of cryopreservation are still unclear; therefore, using animal experiments we assessed how 'cryopreservation' influences graft allogenicity and cell viability. METHODS: Thoracic aortas of Lewis rats were prepared as fresh (F) or cryopreserved (CP) grafts, and implanted into the infrarenal aorta of Lewis or Brown Norway rats (BNs). The grafts and spleens were harvested at post-operative day 7 and 28 (POD7, POD28) for analyses. RESULTS: First, the systemic immune response to transplantation was estimated by mixed lymphocyte reaction analyses using spleen cells from na?ve or recipient BNs. The alloreactivity of the recipients increased to 1.5 times that of the na?ve BNs at POD7 and POD28, when stimulated by mitomycin C-treated Lewis spleen cells. Second, local immune response was estimated by TNFalpha, IFNgamma, and iNOS mRNA expression in the grafts by quantitative PCR, which revealed 20- to 40-fold increases at POD28 after allotransplantation. Third, endothelial cell viability was estimated by endothelial NOS mRNA expression level: it was similar and highest in F and CP grafts before transplantation then significantly decreased after both syngeneic and allogeneic transplantation. Finally, intimal hyperplasia, expressed by I/M ratio, developed over time after allotransplantation, reaching 2.5 times the thickness of F grafts before transplantation. The results of these experiments revealed no difference between F and CP grafts before and after transplantation. CONCLUSION: Cryopreservation did not modify the allogenicity of vascular allografts and had minimal adverse impacts on graft cell viability.     

Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions

Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.     

Coculture with endothelial cells reduces the population of cycling LeX neural precursors but increases that of quiescent cells with a side population phenotype

Neural stem cell proliferation and differentiation are regulated by external cues from their microenvironment. As endothelial cells are closely associated with neural stem cell in brain germinal zones, we investigated whether endothelial cells may interfere with neurogenesis. Neural precursor cells (NPC) from telencephalon of EGFP mouse embryos were cocultured in direct contact with endothelial cells. Endothelial cells did not modify the overall proliferation and apoptosis of neural cells, albeit they transiently delayed spontaneous apoptosis. These effects appeared to be specific to endothelial cells since a decrease in proliferation and a raise in apoptosis were observed in cocultures with fibroblasts. Endothelial cells stimulated the differentiation of NPC into astrocytes and into neurons, whereas they reduced differentiation into oligodendrocytes in comparison to adherent cultures on polyornithine. Determination of NPC clonogenicity and quantification of LeX expression, a marker for NPC, showed that endothelial cells decreased the number of cycling NPC. On the other hand, the presence of endothelial cells increased the number of neural cells having "side population" phenotype, another marker reported on NPC, which we have shown to contain quiescent cells. Thus, we show that endothelial cells may regulate neurogenesis by acting at different level of NPC differentiation, proliferation and quiescence.     

Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Deltap85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.