Epidermal sensing of oxygen is essential for systemic hypoxic response.

Skin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1alpha inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.

Alport syndrome and autosomal dominant tubulointerstitial kidney disease frequently underlie end-stage renal disease of unknown origin-a single-center analysis.

BACKGROUND: The prevalence of end-stage renal disease of unknown etiology in adult patients is globally high and accounts for almost 20% of all dialysis patients. Recent studies have suggested that the percentage of adult patients with a causal genetic variant has been underestimated so far. Despite severe prognostic and therapeutic implications, awareness about prevalence and manifestations of genetic kidney diseases in adult renal patients is still limited. METHODS: We recruited 58 individuals from 39 families at our transplantation center, fulfilling at least one of the following criteria: (i) unclear etiology of kidney disease, (ii) clinically suspected genetic kidney disease and (iii) positive family history for nephropathies. The cohort consisted of patients waitlisted for kidney transplantation and patients in the follow-up after transplantation. Detailed documentation of family history and phenotype was obtained before initiating gene panel sequencing of 479 nephropathy-associated genes. RESULTS: With this study design, a molecular genetic diagnosis was established in one-third of all patients. Mutations in the collagen COL4A genes, and mutations in MUC1 and UMOD were the most frequent among all detected causal variants. Overall, rare genetic variants were detected in more than half of all cases. CONCLUSION: The combination of detailed phenotyping prior to next-generation sequencing diagnostics was highly efficient. Elucidating the underlying genetic causes in a cohort of adult renal patients has considerable clinical impact on medical management.

Renal transplantation after recovery from COVID-19 - a case report with implications for transplant programs in the face of the ongoing corona-pandemic.

BACKGROUND: The ongoing coronavirus pandemic has major impacts on both patients and healthcare systems worldwide, thus creating new realities. Patients on maintenance dialysis listed for renal transplantation are a vulnerable subgroup with many comorbidities and recurring contacts with the healthcare system. Due to the COVID-19 pandemic transplant numbers have dropped considerably, further increasing waiting times in this high-risk population. On the other hand, knowledge of the severity of SARS-CoV-2 infection in immunocompromised patients, development and persistence of neutralising antibodies in such patients is just emerging. It is unclear how best to address the dilemma of postponing the life-saving transplantation. CASE PRESENTATION: We present a case report of a successful kidney transplantation only 65 days after the recipient was hospitalized for treatment of COVID-19 pneumonia. In a follow up of 9 months, we observed no signs of recurrent disease and transplant function is excellent. Monitoring SARS-CoV-2 antibody response demonstrates stable IgG levels. CONCLUSION: This reassuring case provides guidance to transplant centers how to proceed with kidney transplantation safely during the pandemic. Careful consideration of risks and benefits of the organ offer, full recovery from COVID-19 symptoms and the presence of a positive SARS-CoV-2 IgG antibody test, qualifies for kidney transplantation.

Mononuclear phagocytes orchestrate prolyl hydroxylase inhibition-mediated renoprotection in chronic tubulointerstitial nephritis.

Prolyl hydroxylase domain enzyme inhibitors (PHDIs) stabilize hypoxia-inducible factors (HIFs), and are protective in models of acute ischemic and inflammatory kidney disease. Whether PHDIs also confer protection in chronic inflammatory kidney disease models remains unknown. Here we investigated long-term effects of PHDI treatment in adenine-induced nephropathy as a model for chronic tubulointerstitial nephritis. After three weeks, renal dysfunction and tubulointerstitial damage, including proximal and distal tubular injury, tubular dilation and renal crystal deposition were significantly attenuated in PHDI-treated (the isoquinoline derivative ICA and Roxadustat) compared to vehicle-treated mice with adenine-induced nephropathy. Crystal-induced renal fibrosis was only partially diminished by treatment with ICA. Renoprotective effects of ICA treatment could not be attributed to changes in adenine metabolism or urinary excretion of the metabolite 2,8-dihydroxyadenine. ICA treatment reduced inflammatory infiltrates of F4/80+ mononuclear phagocytes in the kidneys and supported a regulatory, anti-inflammatory immune response. Furthermore, interstitial deposition of complement C1q was decreased in ICA-treated mice fed an adenine-enriched diet. Tubular cell-specific HIF-1α and myeloid cell-specific HIF-1α and HIF-2α expression were not required for the renoprotective effects of ICA. In contrast, depletion of mononuclear phagocytes with clodronate largely abolished the nephroprotective effects of PHD inhibition. Thus, our findings indicate novel and potent systemic anti-inflammatory properties of PHDIs that confer preservation of kidney function and structure in chronic tubulointerstitial inflammation and might counteract kidney disease progression.

Association of donor hypertension and recipient renal function in living donor kidney transplantation: A single-center retrospective study.

Transplant centers now accept living donors with well-controlled hypertension. Little is known whether hypertension in living donors affects recipient's kidney function. We aimed to examine potential differences in kidneys from hypertensive donors compared to normotensive donors with respect to renal function over 36 months and histologic findings at transplantation (T0) and 12 months after transplantation (T1). Retrospective single-center analysis of 174 living donor-recipient pairs (age > 18; transplantation date 1/2008-3/2016). Hypertension in donors was defined as being on antihypertensive medication. All biopsies were assessed by the same blinded, experienced renal pathologist. Biopsies were scored for glomerulosclerosis, IFTA, and arteriosclerosis. Regression models were used to examine the relationship of donor hypertension with renal function and histologic changes. Hypertensive donors were significantly older than normotensive donors. Chronic changes such as tubular atrophy and atherosclerosis were more evident in kidneys from hypertensive donors at T0 as well as T1. Donor hypertension was independently associated with histologic changes at T0 and T1 but not with renal function over the follow period. Despite more pronounced histologic changes in kidneys from hypertensive living donors, these grafts exhibited a similar functional outcome. However, they subsequently might be at a greater risk and warrant thorough follow-up care.

HIF-1α promotes cyst progression in a mouse model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations of the PKD1 gene and characterized by growth of bilateral renal cysts. Cyst growth is accompanied by regional hypoxia and induction of hypoxia-inducible factor (HIF)-1α in cyst-lining epithelial cells. To determine the relevance of HIF-1α for cyst growth in vivo we used an inducible kidney epithelium-specific knockout mouse to delete Pkd1 at postnatal day 20 or 35 to induce polycystic kidney disease of different severity and analyzed the effects of Hif-1α co-deletion and HIF-1α stabilization using a prolyl-hydroxylase inhibitor. HIF-1α expression was enhanced in kidneys with progressive cyst growth induced by early Pkd1 deletion, but unchanged in the milder phenotype induced by later Pkd1 deletion. Hif-1α co-deletion significantly attenuated cyst growth in the severe, but not in the mild, phenotype. Application of a prolyl-hydroxylase inhibitor resulted in severe aggravation of the mild phenotype with rapid loss of renal function. HIF-1α expression was associated with induction of genes that mediate calcium-activated chloride secretion. Thus, HIF-1α does not seem to play a role in early cyst formation, but accelerates cyst growth during progressive polycystic kidney disease. This novel mechanism of cyst growth may qualify as a therapeutic target.

A randomized, phase 2 study of ASP0113, a DNA-based vaccine, for the prevention of CMV in CMV-seronegative kidney transplant recipients receiving a kidney from a CMV-seropositive donor.

Cytomegalovirus (CMV) is a latent infection in most infected individuals, but can be pathogenic in immunocompromised kidney transplant recipients. ASP0113 is a DNA-based vaccine for the prevention of CMV-related mortality and end-organ disease in transplant recipients. The efficacy, safety, and immunogenicity of ASP0113 was assessed in a phase 2, double-blind, placebo-controlled study in CMV-seronegative kidney transplant recipients receiving a kidney from a CMV-seropositive donor. Transplant recipients were randomized (1:1) to receive 5 doses of ASP0113 (5 mg; n = 75) or placebo (n = 74) on Days 30/60/90/120/180 posttransplant, and they received prophylactic valganciclovir/ganciclovir 10-100 days posttransplant. The primary endpoint was the proportion of transplant recipients with CMV viremia ≥1000 IU/mL from Day 100 through to 1 year after the first study vaccine injection. There was no statistically significant difference in the primary endpoint between the ASP0113 and placebo groups (odds ratio 0.79, 95% confidence interval 0.43-1.47; P = .307). There were similar numbers of transplant recipients with treatment-emergent adverse events between groups; however, more transplant recipients reported injection site pain in the ASP0113 group compared with placebo. ASP0113 did not demonstrate efficacy in the prevention of CMV viremia in this CMV-seronegative kidney transplant population, but demonstrated a safety profile similar to placebo. ClinicalTrials.gov registration number: NCT01974206.

Hypoxia-inducible protein 2 Hig2/Hilpda mediates neutral lipid accumulation in macrophages and contributes to atherosclerosis in apolipoprotein E-deficient mice.

Recently we identified hypoxia-inducible protein 2 (HIG2)/hypoxia-inducible lipid droplet-associated (HILPDA) as lipid droplet (LD) protein. Because HILPDA is highly expressed in atherosclerotic plaques, we examined its regulation and function in murine macrophages, compared it to the LD adipose differentiation-related protein (Adrp)/perilipin 2 (Plin2), and investigated its effects on atherogenesis in apolipoprotein E-deficient (ApoE-/-) mice. Tie2-Cre-driven Hilpda conditional knockout (cKO) did not affect viability, proliferation, and ATP levels in macrophages. Hilpda proved to be a target of hypoxia-inducible factor 1 (Hif-1) and peroxisome proliferator-activated receptors. In contrast, Adrp/Plin2 was not induced by Hif-1. Hilpda localized to the endoplasmic reticulum-LD interface, the site of LD formation. Hypoxic lipid accumulation and storage of oxidized LDL, cholesteryl esters and triglycerides were abolished in Hilpda cKO macrophages, independent of the glycolytic switch, fatty acid or lipoprotein uptake. Hilpda depletion reduced resistance against lipid overload and increased production of reactive oxygen species after reoxygenation. LPS-stimulated prostaglandin-E2 production was dysregulated in macrophages, demonstrating the substrate buffer and reservoir function of LDs for eicosanoid production. In ApoE-/- Hilpda cKO mice, total aortic plaque area, plaque macrophages and vascular Vegf expression were reduced. Thus, macrophage Hilpda is crucial to foam-cell formation and lipid deposition, and to controlled prostaglandin-E2 production. By these means Hilpda promotes lesion formation and progression of atherosclerosis.-Maier, A., Wu, H., Cordasic, N., Oefner, P., Dietel, B., Thiele, C., Weidemann, A., Eckardt, K.-U., Warnecke, C. Hypoxia-inducible protein 2 Hig2/Hilpda mediates neutral lipid accumulation in macrophages and contributes to atherosclerosis in apolipoprotein E-deficient mice.

Differential activation and antagonistic function of HIF-{alpha} isoforms in macrophages are essential for NO homeostasis.

Hypoxic response and inflammation both involve the action of the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha. Previous studies have revealed that both HIF-alpha proteins are in a number of aspects similarly regulated post-translationally. However, the functional interrelationship of these two isoforms remains largely unclear. The polarization of macrophages controls functionally divergent processes; one of these is nitric oxide (NO) production, which in turn is controlled in part by HIF factors. We show here that the HIF-alpha isoforms can be differentially activated: HIF-1alpha is induced by Th1 cytokines in M1 macrophage polarization, whereas HIF-2alpha is induced by Th2 cytokines during an M2 response. This differential response was most evident in polarized macrophages through HIF-alpha isoform-specific regulation of the inducible NO synthase gene by HIF-1alpha, and the arginase1 gene by HIF-2alpha. In silico modeling predicted that regulation of overall NO availability is due to differential regulation of HIF-1alpha versus HIF-2alpha, acting to, respectively, either increase or suppress NO synthesis. An in vivo model of endotoxin challenge confirmed this; thus, these studies reveal that the two homologous transcription factors, HIF-1alpha and HIF-2alpha, can have physiologically antagonistic functions, but that their antiphase regulation allows them to coordinately regulate NO production in a cytokine-induced and transcription-dependent fashion.

HIF-1α is a protective factor in conditional PHD2-deficient mice suffering from severe HIF-2α-induced excessive erythropoiesis.

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2α. In contrast, HIF-1α served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1α resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2α-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2α-dependent erythrocytosis, whereas HIF-1α protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia.

Inactivation of evenness interrupted (EVI) reduces experimental fibrosis by combined inhibition of canonical and non-canonical Wnt signalling.

OBJECTIVES: Canonical as well as non-canonical Wnt signalling pathways have emerged as core pathways of fibrosis. Their profibrotic effects are mediated via distinct intracellular cascades independently of each other. Thus, inhibition of both pathways may have additive antifibrotic effects. Here, we knocked down evenness interrupted (EVI) to simultaneously target for the first time canonical and non-canonical Wnt signalling in experimental fibrosis. METHODS: The antifibrotic effects of siRNA-mediated knockdown of EVI were evaluated in the mouse models of bleomycin-induced skin fibrosis and in fibrosis induced by adenoviral overexpression of a constitutively active TGF-ß receptor I (AdTBRI). RESULTS: Knockdown of EVI decreased the release of canonical and non-canonical Wnt ligands by fibroblasts and reduced the activation of canonical and non-canonical Wnt cascades in experimental fibrosis with decreased accumulation of ß-catenin and phosphorylated JNK and cJun. Inactivation of EVI exerted potent antifibrotic effects and reduced dermal thickening, myofibroblast differentiation and accumulation of collagen in the mouse models of bleomycin-induced and AdTBR-induced fibrosis. CONCLUSIONS: Inhibition of Wnt secretion by knockdown of EVI inhibits canonical and non-canonical Wnt signalling and effectively reduces experimental fibrosis in different preclinical models. Inhibition of Wnt secretion may thus be an interesting approach for the treatment of fibrosis.

Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis.

Angiogenesis and the development of a vascular network are required for tumour progression, and they involve the release of angiogenic factors, including vascular endothelial growth factor (VEGF-A), from both malignant and stromal cell types. Infiltration by cells of the myeloid lineage is a hallmark of many tumours, and in many cases the macrophages in these infiltrates express VEGF-A. Here we show that the deletion of inflammatory-cell-derived VEGF-A attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumours in mice. Vasculature in tumours lacking myeloid-cell-derived VEGF-A was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF-A decreases the phosphorylation of VEGF receptor 2 (VEGFR2) in tumours, even though overall VEGF-A levels in the tumours are unaffected. However, deletion of myeloid-cell VEGF-A resulted in an accelerated tumour progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumour cell death and decreased tumour hypoxia. Furthermore, loss of myeloid-cell VEGF-A increased the susceptibility of tumours to chemotherapeutic cytotoxicity. This shows that myeloid-derived VEGF-A is essential for the tumorigenic alteration of vasculature and signalling to VEGFR2, and that these changes act to retard, not promote, tumour progression.

Inactivation of tankyrases reduces experimental fibrosis by inhibiting canonical Wnt signalling.

OBJECTIVES: Canonical Wnt signalling has recently emerged as a key mediator of fibroblast activation and tissue fibrosis in systemic sclerosis. Here, we investigated tankyrases as novel molecular targets for inhibition of canonical Wnt signalling in fibrotic diseases. METHODS: The antifibrotic effects of the tankyrase inhibitor XAV-939 or of siRNA-mediated knockdown of tankyrases were evaluated in the mouse models of bleomycin-induced dermal fibrosis and in experimental fibrosis induced by adenoviral overexpression of a constitutively active TGF-ß receptor I (Ad-TBRI). RESULTS: Inactivation of tankyrases prevented the activation of canonical Wnt signalling in experimental fibrosis and reduced the nuclear accumulation of ß-catenin and the mRNA levels of the target gene c-myc. Treatment with XAV-939 or siRNA-mediated knockdown of tankyrases in the skin effectively reduced bleomycin-induced dermal thickening, differentiation of resting fibroblasts into myofibroblasts and accumulation of collagen. Potent antifibrotic effects were also observed in Ad-TBRI driven skin fibrosis. Inhibition of tankyrases was not limited by local or systemic toxicity. CONCLUSIONS: Inactivation of tankyrases effectively abrogated the activation of canonical Wnt signalling and demonstrated potent antifibrotic effects in well-tolerated doses. Thus, tankyrases might be candidates for targeted therapies in fibrotic diseases.

HIF-1α activation results in actin cytoskeleton reorganization and modulation of Rac-1 signaling in endothelial cells.

BACKGROUND: Hypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model. RESULTS: Microvascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1α and not HIF-2α as shown in cells with stable knockdown of HIF-α isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1α-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells. CONCLUSIONS: Our data demonstrates that PHD inhibition induces HIF-1α-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.

BK viremia and polyomavirus nephropathy in 352 kidney transplants; risk factors and potential role of mTOR inhibition.

BACKGROUND: Polyomavirus BK nephropathy (PyVAN) remains an important cause of early graft dysfunction and graft loss in kidney transplantation. METHODS: In this retrospective, single centre cohort study we studied the incidence and outcome of BK viral infection in 352 patients transplanted in 2008-2011. RESULTS: During follow-up viral replication was detected in 48 patients (13.6%); 22 patients (6.2%) had biopsy proven PyVAN.In multivariate logistic regression analyses risk factors for BK-viremia were lack of enrolment into randomized controlled trials (RCTs), biopsy proven acute rejections, cytomegaly virus (CMV) serostatus of both donor and recipient and previous transplantation.In patients without PyVAN reduction or switch of immunosuppression was associated with rapid viral clearance and stable graft function. In contrast, in most patients with PyVAN graft function deteriorated and 5 patients prematurely lost their allograft. Switch of immunosuppression to a low dose cyclosporine plus mTOR inhibitor based regimen in patients with PyVAN was safe, well tolerated and tended to be associated with a better short-term outcome in terms of graft function compared to reduction of existing immunosuppression alone. CONCLUSIONS: With the lack of licensed anti-polyoma viral drugs reduction or conversion of immunosuppression remains the mainstay of therapy in patients with PyVAN. The combination of low dose cyclosporine plus mTOR inhibition appears to be safe and warrants further investigation.

Loss of myeloid cell-derived vascular endothelial growth factor accelerates fibrosis.

Tissue injury initiates a complex series of events that act to restore structure and physiological homeostasis. Infiltration of inflammatory cells and vascular remodeling are both keystones of this process. However, the role of inflammation and angiogenesis in general and, more specifically, the significance of inflammatory cell-derived VEGF in this context are unclear. To determine the role of inflammatory cell-derived VEGF in a clinically relevant and chronically inflamed injury, pulmonary fibrosis, we deleted the VEGF-A gene in myeloid cells. In a model of pulmonary fibrosis in mice, deletion of VEGF in myeloid cells resulted in significantly reduced formation of blood vessels; however, it causes aggravated fibrotic tissue damage. This was accompanied by a pronounced decrease in epithelial cell survival and a striking increase in myofibroblast invasion. The drastic increase in fibrosis following loss of myeloid VEGF in the damaged lungs was also marked by increased levels of hypoxia-inducible factor (HIF) expression and Wnt/beta-catenin signaling. This demonstrates that the process of angiogenesis, driven by myeloid cell-derived VEGF, is essential for the prevention of fibrotic damage.

High Macrophage Densities in Native Kidney Biopsies Correlate With Renal Dysfunction and Promote ESRD.

Introduction: Macrophages and monocytes are main players in innate immunity. The relevance of mononuclear phagocyte infiltrates on clinical outcomes remains to be determined in native kidney diseases. Methods: Our cross-sectional study included 324 patients with diagnostic renal biopsies comprising 17 disease entities and normal renal tissues for comparison. All samples were stained for CD68+ macrophages. Selected groups were further subtyped for CD14+ monocytes and CD163+ alternatively activated macrophages. Using precise pixel-based digital measurements, we quantified cell densities as positively stained areas in renal cortex and medulla as well as whole renal tissue. Laboratory and clinical data of all cases at the time of biopsy and additional follow-up data in 158 cases were accessible. Results: Biopsies with renal disease consistently revealed higher CD68+-macrophage densities and CD163+-macrophage densities in cortex and medulla compared to controls. High macrophage densities correlated with impaired renal function at biopsy and at follow-up in all diseases and in diseases analyzed separately. High cortical CD68+-macrophage densities preceded shorter renal survival, defined as requirement of permanent dialysis. CD14+ monocyte densities showed no difference compared to controls and did not correlate with renal function. Conclusion: Precise quantification of macrophage densities in renal biopsies may contribute to risk stratification to identify patients with high risk for end-stage renal disease (ESRD) and might be a promising therapeutic target in renal disease.

Astrocyte hypoxic response is essential for pathological but not developmental angiogenesis of the retina.

Vascular/parenchymal crosstalk is increasingly recognized as important in the development and maintenance of healthy vascularized tissues. The retina is an excellent model in which to study the role of cell type-specific contributions to the process of blood vessel and neuronal growth. During retinal vascular development, glial cells such as astrocytes provide the template over which endothelial cells migrate to form the retinal vascular network, and hypoxia-regulated vascular endothelial growth factor (VEGF) has been demonstrated to play a critical role in this process as well as pathological neovascularization. To investigate the nature of cell-specific contributions to this process, we deleted VEGF and its upstream regulators, the hypoxia-inducible transcription factors HIF-1 alpha and HIF-2 alpha, and the negative regulator of HIF alpha, von Hippel-Lindau protein (VHL), in astrocytes. We found that loss of hypoxic response and VEGF production in astrocytes does not impair normal development of retinal vasculature, indicating that astrocyte-derived VEGF is not essential for this process. In contrast, using a model of oxygen-induced ischemic retinopathy, we show that astrocyte-derived VEGF is essential for hypoxia-induced neovascularization. Thus, we demonstrate that astrocytes in the retina have highly divergent roles during developmental, physiological angiogenesis, and ischemia-driven, pathological neovascularization.

Mutual regulation of hypoxia-inducible factor and mammalian target of rapamycin as a function of oxygen availability.

The mammalian target of rapamycin (mTOR) regulates cellular growth and proliferation, mainly by controlling cellular translation. Most tumors show constitutive activation of the mTOR pathway. In hypoxia, mTOR is inactivated, which is believed to be part of the program of the cell to maintain energy homeostasis. However, certain proteins are believed to be preferentially translated during hypoxia via 5' terminal oligopyrimidine tract mechanisms with controversial discussion about the involvement of the mTOR-dependent ribosomal protein S6 (rpS6). The hypoxia-inducible transcription factor (HIF) is the master regulator of hypoxic adaptation and itself strongly implicated in tumor growth. HIF is translationally regulated by mTOR. The regulatory features and the involvement of molecular oxygen itself in this regulation of HIF by mTOR are poorly understood. mTOR inhibition leads to profound attenuation of HIFalpha protein in the majority of primary and cancer cells studied. Under severe hypoxia, no influence of mTOR inhibitors was observed; thus, stimulation of HIFalpha by mTOR may only be relevant under mild hypoxia or even normoxia. HIF expression and phosphorylated rpS6 negatively correlate in experimental tumors. In cell culture, prolonged hypoxia abolishes rpS6 phosphorylation, which seems to be partly independent of the upstream p70S6 kinase. We show that hypoxic repression of rpS6 is largely dependent on HIF, implicating a negative feedback loop, which may influence cellular translational rates and metabolic homeostasis. These data implicate that the hypoxic microenvironment renders tumor cells resistant to mTOR inhibition, at least concerning hypoxic gene activation, which would add to the difficulties of other established therapeutic strategies in hypoxic cancer tissues.