Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2-to 3-day pulse stimulates angiogenesis by inducing an endothelial "tip cell" phenotype. TNF induces the known tip cell genes plateletderived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reversetranscription-polymerase chain reaction IntroductionNeovascularization, or the formation of new blood vessels, is a critical component of many physiologic as well as pathologic conditions, including development, reproduction, wound healing, diabetic retinopathy, and tumor growth. During wound healing, new vessel growth by angiogenesis is a necessary early step in rebuilding tissue, however the coordination of angiogenesis with the resolution of the acute inflammatory stage is not well understood. The earliest events after tissue damage include the generation of a fibrin clot and the bursting of platelets to release numerous growth factors. Fibrin provides a provisional matrix that promotes the accumulation of blood-derived monocytes that then differentiate into tissue macrophages. Activated macrophages synthesize several cytokines, including tumor necrosis factor (TNF), which activate local endothelial cells (ECs) and promote leukocyte recruitment. After 3 to 4 days, when the initial infection has been cleared, there is a switch toward tissue repair and concomitant with this is the acceleration of angiogenesis. 1,2 TNF is a major inflammatory mediator that induces multiple changes in EC gene expression including induction of adhesion molecules, integrins, and matrix metalloproteinases (MMPs). Its effects on angiogenesis have been the subject of some controversy. For example, TNF blocks EC proliferation and migration in vitro [3][4][5] and has been reported to down-regulate activity 6 and expression 7,8 of vascular endothelial cell growth factor receptor-2 (VEGFR2). On the other hand, TNF has also been shown to up-regulate VEGFR2 expression 9 and promote EC migration. 10 In vivo the situation is no clearer: TNF promotes angiogenesis in the cornea, 3,11 whereas loss of TNFR1 (p55 receptor) leads to enhanced angiogenesis in both retina 12 and wounded skin. 13 Further studies with TNF receptor-deficient mice have demonstrated enhanced hind limb angiogenesis after temporary ischemia in T...

Candida albicans must penetrate the endothelial cell lining of the vasculature to invade the deep tissues during a hematogenously disseminated infection. We compared 27 C. albicans mutants with their wild-type parent for their capacity to damage endothelial cells in vitro and cause a lethal infection in mice following tail vein inoculation. Of 10 mutants with significantly impaired capacity to damage endothelial cells, all had attenuated virulence. Therefore, the endothelial cell damage assay can be used as a screen to identify some virulence factors relevant to hematogenously disseminated candidiasis.During the initiation of hematogenously disseminated candidiasis, blood-borne organisms must adhere to and penetrate the endothelial cell lining of the blood vessels to invade the deep tissues. One mechanism by which Candida albicans can penetrate the vascular endothelium is by damaging and eventually killing the endothelial cells. Damaged endothelial cells detach from the basement membrane, leaving gaps through which the organism can invade. Also, the exposed basement membrane can be avidly bound by additional organisms (18).C. albicans damages human vascular endothelial cells in vivo and in vitro (5,10,17,25). Maximal endothelial cell damage (ECD) occurs in vitro when C. albicans adheres to and invades the endothelial cells and then secretes lytic enzymes (2,10,11,13,16). Moreover, some C. albicans mutants with filamentation defects cause significantly less ECD than the wild-type parent strain (24). These filamentation mutants also have attenuated virulence in various experimental models of infection (reviewed in reference 23). We hypothesized that the in vitro assay for C. albicans-induced ECD (ECD assay) can serve as a model for certain aspects of host-pathogen interactions in vivo, such as the ability of the organism to adhere to, invade, and injure host cells. This hypothesis predicts that some mutants with virulence defects will be defective in the ECD assay. The goal of the present study was to investigate this prediction.C. albicans strains. The genotypes and sources of the C. albicans strains used here are listed in Tables 1 and 2. Each strain was grown overnight in yeast nitrogen base broth (Difco, Detroit, Mich.) supplemented with 2% glucose (wt/vol) at 20°C on a rotating drum. The blastospores were harvested by centrifugation, washed with phosphate-buffered saline, enumerated with a hemacytometer, and suspended in RPMI 1640 medium (Irvine Scientific, Santa Ana, Calif.).ECD assay. We used our standard 51 Cr release ECD assay to determine the abilities of mutants of C. albicans to damage endothelial cells in vitro (24). The ECD assay was performed in 96-well tissue culture plates (Corning Inc., Acton, Mass.) with endothelial cells isolated from human umbilical cord veins, as described previously (24). The inoculum was 4 ϫ 10 4 organisms per well, and the organisms were incubated with the endothelial cells for 3 h in 5% CO 2 at 37°C. At the end of the incubation period, the wells were examined with an inv...

We examined the relationship between host survival and renal and splenic immune responses in a murine model of hematogenously disseminated candidiasis. Male BALB/c mice were infected via tail vein injection with wild-type C. albicans or with an isogenic, ⌬efg1/⌬efg1 hypha-deficient mutant. Host survival, organ fungal burden, intracellular cytokine content of splenic and kidney lymphocytes, and whole-organ cytokine profiles were determined. Wild-type C. albicans induced type 2 splenocyte responses with both nonfatal and fatal inocula. In the kidney, conversely, wild-type inocula causing no or low mortality induced type 1 responses and 100% fatal inocula induced type 2 or interleukin-10 (IL-10)-dominant responses. Hypha-deficient mutant C. albicans caused no or low mortality while inducing type 1 responses in both the spleen and kidney. To our knowledge, this is the first demonstration that host survival during systemic infection correlates with the type of immune response engendered in a nonlymphoid, parenchymal organ and not with the response in the spleen. Furthermore, the results provide in vivo confirmation that hyphal formation by C. albicans induces type 2 or IL-10-dominant host responses in tissues.Candida is the fourth most common bloodstream isolate in the nosocomial setting (31), and the cost associated with nosocomial candidemia in the United States approaches one billion dollars per year (18). Furthermore, disseminated candidiasis has an attributable mortality of nearly 40% overall (and Ͼ50% in myeloablated patients) even in the face of modern antifungal therapy (14,21,35). Strategies to potentiate host defense against the fungus are likely to be efficacious for preventing and/or treating this infection; hence, there has been intense interest in elucidating the precise nature of protective host defense mechanisms against Candida.The paradigm of type 1 and/or type 2 immunity integrates cell-mediated and humoral host defense (32). T-helper 1 (Th1) cells, which secrete gamma interferon (IFN-␥) but not interleukin-4 (IL-4), stimulate type 1 immunity characterized by intense phagocytic activity. Conversely, Th2 cells, which secrete IL-4 but not IFN-␥, stimulate type 2 immunity characterized by induction of high antibody titers and suppression of phagocytic activity. It has been suggested that a type 1 immune response is protective against both disseminated and mucocutaneous candidiasis, while a dominant type 2 immune response results in increased susceptibility to disseminated disease (6,10,17,28,30). Furthermore, during Candida sepsis there is a down-regulation of the host response to the fungus (33), suggesting that T-regulatory (Treg) or Th3 cells, which predominantly secrete the immunosuppressive cytokines IL-10 and transforming growth factor beta (TGF-␤) (8, 9, 11), regulate host immunity to high inoculum levels of Candida. This notion is supported by data from Romani's group, who recently reported that C. albicans induces IL-10 ϩ /TGF-␤ ϩ Treg cells that mediate oral tolerance following intragastric ...

The vacuole has crucial roles in stress resistance and adaptation of the fungal cell. Furthermore, in Candida albicans it has been observed to undergo dramatic expansion during the initiation of hyphal growth, to produce highly "vacuolated" subapical compartments. We hypothesized that these functions may be crucial for survival within the host and tissue-invasive hyphal growth. We also considered the role of the late endosome or prevacuole compartment (PVC), a distinct organelle involved in vacuolar and endocytic trafficking. We identified two Rab GTPases, encoded by VPS21 and YPT72, required for trafficking through the PVC and vacuole biogenesis, respectively. Deletion of VPS21 or YPT72 led to mild sensitivities to some cellular stresses. However, deletion of both genes resulted in a synthetic phenotype with severe sensitivity to cellular stress and impaired growth. Both the vps21⌬ and ypt72⌬ mutants had defects in filamentous growth, while the double mutant was completely deficient in polarized growth. The defects in hyphal growth were not suppressed by an "active" RIM101 allele or loss of the hyphal repressor encoded by TUP1. In addition, both single mutants had significant attenuation in a mouse model of hematogenously disseminated candidiasis, while the double mutant was rapidly cleared. Histological examination confirmed that the vps21⌬ and ypt72⌬ mutants are deficient in hyphal growth in vivo. We suggest that the PVC and vacuole are required on two levels during C. albicans infection: (i) stress resistance functions required for survival within tissue and (ii) a role in filamentous growth which may aid host tissue invasion.

TNF-α is a potent proinflammatory cytokine that induces endothelial cell (EC) adhesion molecules. In addition, TNF promotes angiogenesis by inducing an EC tip cell phenotype and the expression of jagged-1, a ligand for the notch pathway. Notch signaling is critical for vascular patterning and helps to restrict the proliferation of tip cells. Here we demonstrate that TNF induction of jagged-1 in human EC is rapid and dependent upon signaling through TNFR1, but not TNFR2. A luciferase reporter construct carrying 3.7 kb of 5′ promoter sequence from the human gene was responsive to both TNF and overexpression of NFκB pathway components. TNF-induced promoter activation was blocked by treatment with an NFκB inhibitor or co-expression of dominant-negative IKKβ. Mutations in a putative NFκB-binding site at −3.0 kb, which is conserved across multiple species, resulted in a loss of responsiveness to TNF and NFκB. Electromobility shift and chromatin immunoprecipitation assays revealed binding of both p50 and p65 to the promoter in response to TNF treatment. Full promoter activity also depends on an AP-1 site at −2.0 kb. These results indicate that canonical NFκB signaling is required for TNF induction of the notch ligand jagged-1 in EC.