Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased ␤1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of ␤1-integrins. Rather, we demonstrate that Notch4-expressing cells display ␤1-integrin in an active, high-affinity conformation. Furthermore, using function-activating ␤1-integrin antibodies, we demonstrate that activation of ␤1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting ␤1-integrinmediated adhesion to the underlying matrix.

No abstract

The effect of lipopolysaccharide (LPS) on endothelial cells is a key component of the inflammatory response seen in Gram-negative sepsis. LPS does not cause death of cultured human endothelial cells. However, when the expression of new proteins is inhibited by cycloheximide, microvascular endothelial cells in culture undergo apoptosis. This finding suggests that LPS induces apoptotic and antiapoptotic pathways, with the antiapoptotic response being dependent on the synthesis of new proteins. Concurrent activation of apoptotic and antiapoptotic pathways has previously been documented for tumor necrosis factor (TNF). In the case of TNF, the antiapoptotic signal has been attributed to at least two cytoprotective proteins: the Bcl-2 homologue, A1, and the zinc-finger protein, A20. In this study, we demonstrate that both these molecules are induced in microvascular endothelial cells by LPS. Enforced overexpression of either A1 or A20 inhibits LPS and cycloheximide-initiated apoptosis. Induction of A1 and A20 does not require synthesis of intermediary proteins, but is dependent on the presence of soluble CD14. In addition, we show that inhibition of signaling by the transcription factor, NF-κB, blocks accumulation of A1 and A20 mRNA. Taken together, our findings suggest that LPS directly induces expression of the cytoprotective proteins, A1 and A20, via a CD14-dependent pathway requiring activation of NF-κB. © 1998 by The American Society of Hematology.

Candida albicans, as an opportunistic pathogen, can cause superficial and life-threatening candidiasis in immunocompromised individuals. The formation of surface-associated biofilms and the appearance of drug resistance pose a significant challenge for clinical intervention. In this study, a total of 104 hospital-acquired C. alibcans clinical isolates were collected from sterile sites and mucosal lesions of 92 infectious disease patients in the Shanghai Public Health Clinical Center and analysed. The resistance rates to fluconazole, itraconazole and voriconazole were 12.5 %, 15.4 % and 11.5 % respectively. Multilocus sequence typing (MLST) analysis identified 63 diploid sequence types (DSTs) with a decentralized phylogeny, of which 37 DSTs (58.7 %) had not been reported in the online MLST database. Loss of heterozygosity was observed in ACC1 and ADP1 sequences obtained from six sequential isolates from a patient receiving antifungal treatment, which exemplified the effect of microevolution on C. albicans genetic alterations. Biofilm formation capability, an important virulence trait of C. albicans, was variable among strains isolated from different anatomical sites (P50.0302) and affected by genotypes (P50.0185). The mRNA levels of the azole antifungal target ERG11 gene and efflux pump genes (CDR1, CDR2 and MDR1) were detected in 9-18.1 % of azole-resistant and susceptible-dose dependent (S-DD) isolates. Twelve mutations encoding distinct amino acid substitutions in ERG11 were found in azoleresistant and S-DD isolates. Among them, A114S, Y132H and Y257H substitution in the ERG11 gene may be primarily related to azole resistance. Taken together, we observed a high level of diversity within C. albicans isolates. Multiple inter-related underlying mechanisms, including genetic and environmental factors, may account for high surface adhesion or azole resistance in clinical C. albicans infections.Abbreviations: AAT1, aspartate aminotransferase; ACC1, Acetyl-coenzyme A carboxylase; ACT1, actin; ADP1, ATP-dependent permease; ALS, hyphaspecific surface protein; BSI, nosocomial bloodstream infection; CDR1, CDR2, ATP-dependent efflux pumps; CLSI, Clinical and Laboratory Standard Institute; DST, diploid sequence type; ERG11, cytochrome P450 14a-demethylase; HWP1, hypha-specific cell wall protein; MDR1, major facilitator pump; MLST, multilocus sequence typing; MPI, mannose phosphate isomerase; SAP, secreted aspartyl protease; S-DD, susceptible-dose dependent; SYA1, alanyl-RNA synthetase; VPS13, vacuolar protein sorting protein; XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-2H-tetrazolium-5-carboxanilide; YPD, yeast extract, peptone, dextrose medium; ZWF1b, glucose-6-phosphate dehydrogenase.

No abstract

The effect of lipopolysaccharide (LPS) on endothelial cells is a key component of the inflammatory response seen in Gram-negative sepsis. LPS does not cause death of cultured human endothelial cells. However, when the expression of new proteins is inhibited by cycloheximide, microvascular endothelial cells in culture undergo apoptosis. This finding suggests that LPS induces apoptotic and antiapoptotic pathways, with the antiapoptotic response being dependent on the synthesis of new proteins. Concurrent activation of apoptotic and antiapoptotic pathways has previously been documented for tumor necrosis factor (TNF). In the case of TNF, the antiapoptotic signal has been attributed to at least two cytoprotective proteins: the Bcl-2 homologue, A1, and the zinc-finger protein, A20. In this study, we demonstrate that both these molecules are induced in microvascular endothelial cells by LPS. Enforced overexpression of either A1 or A20 inhibits LPS and cycloheximide-initiated apoptosis. Induction of A1 and A20 does not require synthesis of intermediary proteins, but is dependent on the presence of soluble CD14. In addition, we show that inhibition of signaling by the transcription factor, NF-κB, blocks accumulation of A1 and A20 mRNA. Taken together, our findings suggest that LPS directly induces expression of the cytoprotective proteins, A1 and A20, via a CD14-dependent pathway requiring activation of NF-κB. © 1998 by The American Society of Hematology.

Burn wound-related sepsis is associated with the development of systemic inflammatory response syndrome and multiple organ dysfunction syndrome (MODS). This study is aimed at investigating the development and progression of SIS and MODS in a mouse model of skin burn sepsis. C57BL/6J mice were randomly divided into the sham, burn, Pseudomonas, and burn/Pseudomonas groups. The back skin of the sham, burn, and burn/Pseudomonas groups was burned about 10% of total area with using 37°C or 98°C water for 8 s, respectively, followed by inoculating with Pseudomonas aeruginosa. The Pseudomonas group was infected with P. aeruginosa without burn injury. Their body weights, mortality, organ histology, and function as well as systemic inflammation were measured longitudinally. The burn/Pseudomonas mice lost more body weights than did mice from the other groups and had a significantly higher mortality rate (P < 0.05). The burn/Pseudomonas mice exhibited significantly higher levels of bacterial loads in different organs and serum endotoxin, interleukin 1β, interleukin 6, tumor necrosis factor α, and C-reactive protein than those in mice from the other groups (P < 0.05). The burn/Pseudomonas mice also displayed more severe liver, lung, and kidney tissue damage and impaired organ functions, particularly at 72 h after inoculation than did the burn and Pseudomonas groups of mice. Our data indicate that burn and P. aeruginosa infection induced severe sepsis and rapidly progressed into systemic inflammatory response syndrome and MODS in mice.

Avanafil, from 50 to 200 mg, is effective and well tolerated for the treatment of ED, and an increase in dosage is associated with a significant rise in effectiveness but not with significantly more adverse events.