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Sepsis, the systemic inflammatory response to infection, is a leading cause of morbidity and mortality. The mechanisms of sepsis pathophysiology remain obscure but are likely to involve a complex interplay between mediators of the inflammatory and coagulation pathways. An improved understanding of these mechanisms should provide an important foundation for developing novel therapies. In this study, we show that sepsis is associated with a time-dependent increase in circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in animal and human models of sepsis. Adenovirus-mediated overexpression of soluble Flt-1 (sFlt-1) in a mouse model of endotoxemia attenuated the rise in VEGF and PlGF levels and blocked the effect of endotoxemia on cardiac function, vascular permeability, and mortality. Similarly, in a cecal ligation puncture (CLP) model, adenovirus–sFlt-1 protected against cardiac dysfunction and mortality. When administered in a therapeutic regimen beginning 1 h after the onset of endotoxemia or CLP, sFlt peptide resulted in marked improvement in cardiac physiology and survival. Systemic administration of antibodies against the transmembrane receptor Flk-1 but not Flt-1 protected against sepsis mortality. Adenovirus-mediated overexpression of VEGF but not PlGF exacerbated the lipopolysaccharide-mediated toxic effects. Together, these data support a pathophysiological role for VEGF in mediating the sepsis phenotype.

IntroductionPrevious reports suggest that endothelial activation is an important process in sepsis pathogenesis. We investigated the association between biomarkers of endothelial cell activation and sepsis severity, organ dysfunction sequential organ failure assessment (SOFA) score, and death.MethodsThis is a prospective, observational study including adult patients (age 18 years or older) presenting with clinical suspicion of infection to the emergency department (ED) of an urban, academic medical center between February 2005 and November 2008. Blood was sampled during the ED visit and biomarkers of endothelial cell activation, namely soluble fms-like tyrosine kinase-1 (sFlt-1), plasminogen activator inhibitors -1 (PAI-1), sE-selectin, soluble intercellular adhesion molecule (sICAM-1), and soluble vascular cell adhesion molecule (sVCAM-1), were assayed. The association between biomarkers and the outcomes of sepsis severity, organ dysfunction, and in-hospital mortality were analyzed.ResultsA total of 221 patients were included: sepsis without organ dysfunction was present in 32%, severe sepsis without shock in 30%, septic shock in 32%, and 6% were non-infected control ED patients. There was a relationship between all target biomarkers (sFlt-1, PAI-1, sE-selectin, sICAM-1, and sVCAM-1) and sepsis severity, P < 0.05. We found a significant inter-correlation between all biomarkers, including the strongest correlations between sFlt-1 and sE-selectin (r = 0.55, P < 0.001), and between sFlt-1 and PAI-1 (0.56, P < 0.001). Among the endothelial cell activation biomarkers, sFlt-1 had the strongest association with SOFA score (r = 0.66, P < 0.001), the highest area under the receiver operator characteristic curve for severe sepsis of 0.82, and for mortality of 0.91.ConclusionsMarkers of endothelial cell activation are associated with sepsis severity, organ dysfunction and mortality. An improved understanding of endothelial response and associated biomarkers may lead to strategies to more accurately predict outcome and develop novel endothelium-directed therapies in sepsis.

M.Streit and P.Velasco contributed equally to this workThe function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP-1 in the skin, using a keratin 14 expression cassette. TSP-1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full-thickness skin wounds was greatly delayed in TSP-1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP-1 potently inhibited ®broblast migration in vivo and in vitro. These ®ndings demonstrate that TSP-1 preferentially interfered with wound healing-associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair. Keywords: blood vessels/skin/transgenic mice/wound healing IntroductionWound healing is characterized by the formation of a richly vascularized, hyperpermeable granulation tissue, supporting the increased nutritional needs of rapidly proliferating and migrating epidermal keratinocytes, ®broblasts and leukocytes (Dvorak, 1986). Several angiogenesis factors have been found to be upregulated in healing wounds, including vascular endothelial growth factor (VEGF) expressed by epidermal keratinocytes (Brown et al., 1992). Impaired wound healing in genetically diabetic mice is associated with diminished expression of VEGF by epidermal keratinocytes (Frank et al., 1995), as well as reduced expression of platelet-derived growth factor-A (PDGF-A), PDGF-B and of the B-type PDGF receptor (Beer et al., 1997). Moreover, mice de®cient for basic ®broblast growth factor were characterized by delayed wound healing (Ortega et al., 1998). While these data strongly suggest that impaired wound healing may be partially caused by insuf®cient stimulation of blood vessels, due to decreased activity of angiogenesis factors, the biological role of endogenous inhibitors of angiogenesis during tissue repair has remained controversial.Several naturally occurring angiogenesis inhibitors have been identi®ed, including thrombospondin-1 (TSP-1) (Iruela-Arispe et al., 1991), TSP-2 (Volpert et al., 1995), angiostatin (O'Reilly et al., 1994), endostatin (O'Reilly et al., 1997) and vasostatin (Pike et al., 1998). TSP-1 is a 450 kDa homotrimeric matricellular glycoprotein that regulates attachment, proliferation, migration and differentiation of various cell types (for review see Bornstein, 1995). TSP-1 interacts with several cell surface receptors, including the integrins a v b 3 , a 3 b 1 , a 4 b 1 , a 5 b 1 , the integrin-associated protein CD47, heparan sulfate proteoglycans and CD36 (Adams, 1997). TSP-1 inhibits proliferation and migration of vascular endotheli...

• Endothelial S1PR2 plays a critical role in the induction of vascular permeability and vascular inflammation during endotoxemia.• S1PR2 could be a novel therapeutic target to promote vascular integrity in inflammatory vascular disorders.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 (S1PRs). 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 endotoxemia. Experiments with bone marrow chimeras (S1pr2 1/1 → S1pr2, and S1pr2 2/2 → 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 a-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 S1PR2-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. (Blood. 2013;122(3):443-455)

ERG (Ets Related Gene) is an ETS transcription factor that has recently been shown to regulate a number of endothelial cell (EC) restricted genes including VE-cadherin, von Willebrand Factor (vWF), endoglin, and intercellular adhesion molecule-2 (ICAM-2). Our preliminary data demonstrate that unlike other ETS factors, ERG exhibits a highly EC-restricted pattern of expression in cultured primary cells and several adult mouse tissues including the heart, lung, and brain. In response to inflammatory stimuli, such as TNF-α, we observed a marked reduction of ERG expression in EC. To further define the role of ERG in the regulation of normal EC function we used RNA interference to knockdown ERG. Microarray analysis of RNA derived from ERG siRNA- or TNF-α–treated HUVEC revealed significant overlap (P value <0.01) in the genes that are up- or downregulated. Of particular interest to us was a significant change in expression of interleukin-8 (IL-8) at both protein and RNA levels. Exposure of EC to TNF-α is known to be associated with increased neutrophil attachment. We observed that knockdown of ERG in HUVEC is similarly associated with increased neutrophil attachment compared to control siRNA-treated cells. This enhanced adhesion could be blocked with IL-8 neutralizing or IL-8 receptor blocking antibodies. ERG can inhibit the activity of the IL-8 promoter in a dose dependent manner. Direct binding of ERG to the IL-8 promoter in EC was confirmed by chromatin immunoprecipitation. In summary, our findings support a role for ERG in promoting anti-inflammatory effects in EC through repression of inflammatory genes such as IL-8.

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.

A multidrug resistance gene, YDR1, of Saccharomyces cerevisiae, which encodes a 170-kDa protein of a member of the ABC superfamily, was identified. Disruption of YDR1 resulted in hypersensitivity to cycloheximide, cerulenin, compactin, staurosporine and fluphenazine, indicating that YDR1 is an important determinant of cross resistance to apparently-unrelated drugs. The Ydr1 protein bears the highest similarity to the S. cerevisiae Snq2 protein required for resistance to the mutagen 4-NQO. The drug-specificity analysis of YDR1 and SNQ2 by gene disruption, and its phenotypic suppression by the overexpressed genes, revealed overlapping, yet distinct, specificities. YDR1 was responsible for cycloheximide, cerulenin and compactin resistance, whereas, SNQ2 was responsible for 4-NQO resistance. The two genes had overlapping specificities toward staurosporine and fluphenazine. The transcription of YDR1 and SNQ2 was induced by various drugs, both relevant and irrelevant to the resistance caused by the gene, suggesting that drug specificity can be mainly attributed to the functional difference of the putative transporters. The transcription of these genes was also increased by heat shock. The yeast drug-resistance system provides a novel model for mammalian multidrug resistance.