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We recently demonstrated that the 3-kb 5'-flanking region of the human ROBO4 gene directs endothelial cell-specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)-binding motif at -119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the -119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.

Mammalian endothelial cells (ECs) display marked phenotypic heterogeneity. Little is known about the evolutionary mechanisms underlying EC heterogeneity. The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates, which lived some time more than 500 million years ago. Features of ECs that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. The goal of this study was to determine whether the hagfish endothelium displays phenotypic heterogeneity. Electron microscopy of the aorta, dermis, heart, and liver revealed ultrastructural heterogeneity of the endothelium. Immunofluorescent studies demonstrated marked differences in lectin binding between vascular beds. Intravital microscopy of the dermis revealed histamine-induced adhesion of leukocytes in capillaries and postcapillary venules, but no such adhesion in arterioles. Together, these data suggest that structural, molecular, and functional heterogeneity of the endothelium evolved as an early feature of this cell lineage.

Background/Aims: Skin biopsies allow for direct phenotyping of the endothelium in clinical settings. Objectives: We hypothesize that in murine sepsis endothelial activation is manifested by changes in protein and mRNA expression in skin biopsies, and that such alterations differ from other organs. Methods: In two mouse models of sepsis [endotoxemia and cecal ligation puncture (CLP)], we measured circulating levels of endothelial biomarkers, quantitated mRNA expression of activation markers and assayed for protein expression using immunohistochemistry. Results: Endotoxemic mice demonstrated increased circulating levels of sE-selectin, sICAM-1, sVCAM-1 and sP-selectin at 24 h, while CLP was associated with increased levels of sE-selectin alone. In real-time PCR, mRNA levels for P-selectin, ICAM-1 and PAI-1 were increased in skin from endotoxemic mice. In CLP, mRNA levels for P-selectin, ICAM-1, E-selectin and PAI-1 were elevated, while VCAM-1 expression was reduced in skin. Most, but not all of these changes correlated with alterations in immunohistochemical staining. Expression patterns in skin differed from those in brain, heart, and lung. Conclusions: Skin biopsies demonstrated endothelial cell activation during sepsis. The expression patterns differed by type of sepsis model and between vascular beds of skin, brain, heart, and lung, providing a foundation for identifying skin microvascular-bed-specific molecule signatures. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The last common ancestor of hagfish and gnathostomes was also the last common ancestor of all extant vertebrates that lived some time more than 500 million years ago. Features that are shared between hagfish and gnathostomes can be inferred to have already been present in this ancestral vertebrate. We recently reported that hagfish endothelium displays phenotypic heterogeneity in ultrastructure, lectin binding, and mechanisms of leukocyte adhesion. Thus, phenotypic cell heterogeneity evolved as an early feature of the endothelium. In the present study, we wanted to extend these observations by determining whether hagfish endothelium plays a role in mediating vasomotor tone. Response of mesenteric and skeletal muscle arteries to a variety of mediators was assayed by videomicroscopy. Phenylephrine and acetylcholine induced vasoconstriction of mesenteric and skeletal muscle arteries. Bradykinin (BK) and ADP promoted vasorelaxation in precontracted mesenteric arteries but not those from skeletal muscle. BK- and ADP-mediated vasorelaxation of the mesenteric artery was abrogated by mechanical denudation of the endothelium but was unaffected by Nω-nitro-L-arginine methyl ester. Indomethacin significantly inhibited the vasodilatory response to ADP but not BK. The nitric oxide donor sodium nitroprusside resulted in endothelium-independent relaxation of both mesenteric and skeletal muscle arteries. Together, these data suggest that site-specific endothelium-dependent vasorelaxation is an evolutionarily conserved property of this cell lineage. [ABSTRACT FROM AUTHOR]

Chronic ultraviolet-B irradiation of the skin results in epidermal hyperplasia, degradation of extracellular matrix molecules, and formation of wrinkles. To characterize the biologic role of the vascular system in the mediation of ultraviolet-B-induced skin damage, we performed quantitative analyses of cutaneous blood vessels of mice after 10 wk of ultraviolet-B irradiation. Skin vascularization was greatly increased after chronic ultraviolet-B exposure with a significant increase of both the number and the size of dermal blood vessels, associated with upregulation of vascular endothelial growth factor expression in the hyperplastic epidermis. To directly study whether inhibition of angiogenesis may diminish ultraviolet-B-induced cutaneous damage, wild-type and transgenic mice with skin-specific overexpression of the endogenous angiogenesis inhibitor thrombospondin-1 were subjected to the same ultraviolet-B irradiation regimen. Ultraviolet-B-irradiated thrombospondin-1 transgenic mice showed a significantly reduced skin vascularization, decreased endothelial cell proliferation, and increased endothelial cell apoptosis rates, compared with wild-type mice. Moreover, dermal photo-damage and wrinkle formation were greatly reduced in thrombospondin-1 transgenic mice. These results reveal an important role of the cutaneous vascular system in mediating ultraviolet-B-induced skin damage and suggest inhibition of angiogenesis as a potential new approach for the prevention of chronic cutaneous photo-damage. [ABSTRACT FROM AUTHOR]

Abstract: Endothelial cell phenotypes are differentially regulated between different sites of the vascular tree. We tested the hypothesis that endocan, a novel soluble dermatan sulfate proteoglycan, is differentially expressed in the intact endothelium and that site-specific expression is mediated by signals in the local microenvironment. Using a combination of Northern blot analyses, Taqman RT-PCR, and in situ hybridizations, endocan was shown to be preferentially expressed in the endothelial lining of tumor xenografts, including human non-small cell lung cancer, rat glioma, and human renal cell carcinoma. In contrast, endocan mRNA was expressed at low levels in embryos between E4.5 and E18.5. Under in vitro conditions, endocan expression in human umbilical vein endothelial cells (HUVEC) was upregulated by tumor cell-conditioned medium, an effect that was inhibited by the addition of neutralizing antibody to vascular endothelial growth factor (VEGF). Moreover, treatment of HUVEC with VEGF resulted in a dose- and time-dependent increase in endocan mRNA. The results suggest that endocan is preferentially expressed in tumor endothelium in vivo and that its expression is regulated by tumor-derived factors. [Copyright &y& Elsevier]

The endothelium, as the interface between blood and all tissues, plays a critical role in inflammation. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, highly abundant in plasma, that potently regulates endothelial responses through interaction with its receptors (S1 PRs). Here, we studied the role of S1PR2 in the regulation of the proadhesion and proinflammatory phenotype of the endothelium. By using genetic approaches and a S1PR2-specific antagonist (JTE013), we found that S1PR2 plays a key role in the permeability and inflammatory responses of the vascular endothelium during endotox-emia. Experiments with bone marrow chimeras (S1pr2+/+ →S1pr2+/+, S1pr2+/+ → S1pr2-/-, and S1pr2-/- → S1pr2+/+) indicate the critical role of S1PR2 in the stromal compartment, in the regulation of vascular permeability and vascular inflammation. In vitro, JTE013 potently inhibited tumor necrosis factor α-induced endothelial inflammation. Finally, we provide detailed mechanisms on the downstream signaling of S1PR2 in vascular inflammation that include the activation of the stress-activated protein kinase pathway that, together with the Rho-kinase nuclear factor kappa B pathway (NF-kB), are required for S1 PR2-mediated endothelial inflammatory responses. Taken together, our data indicate that S1PR2 is a key regulator of the proinflammatory phenotype of the endothelium and identify S1PR2 as a novel therapeutic target for vascular disorders. [ABSTRACT FROM AUTHOR]