PHD1, PHD2, and PHD3 are prolyl hydroxylase domain proteins that regulate the stability of hypoxiainducible factor ␣ subunits (HIF-␣). To determine the roles of individual PHDs during mouse development, we disrupted all three Phd genes and found that Phd2 ؊/؊ embryos died between embryonic days 12.5 and 14.5 whereas Phd1؊/؊ or Phd3 ؊/؊ mice were apparently normal. In Phd2 ؊/؊ mice, severe placental and heart defects preceded embryonic death. Placental defects included significantly reduced labyrinthine branching morphogenesis, widespread penetration of the labyrinth by spongiotrophoblasts, and abnormal distribution of trophoblast giant cells. The expression of several trophoblast markers was also altered, including an increase in the spongiotrophoblast marker Mash2 and decreases in the labyrinthine markers Tfeb and Gcm1. In the heart, trabeculae were poorly developed, the myocardium was remarkably thinner, and interventricular septum was incompletely formed. Surprisingly, while there were significant global increases in HIF-␣ protein levels in the placenta and the embryo proper, there was no specific HIF-␣ increase in the heart. Taken together, these data indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.

Polycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins, which tag hypoxia-inducible factor (HIF) ␣ subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-␣ stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1 ؊/؊ or Phd3 ؊/؊ mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2␣, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1␣ accumulated in both the kidney and liver. Elevated HIF-1␣ levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2␣/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway. (Blood. 2008; 111:3229-3235)

Background Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. While germline Vegfr-1−/− embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain are apparently healthy. Methods and Results We carried out Cre-loxP mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice, and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell (EC) proliferation, and facilitated angiogenesis of blood vessels which matured and perfused properly. Vascular permeability was normal at the basal level, but elevated in response to high doses of exogenous VEGF-A. In the post-infarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2+/− heterozygosity into Vegfr-1 somatic knockout mice. Conclusions Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1 deficient mice.

Here we investigate the role of hypoxia inducible factor (HIF)-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26CreERT2, Tie2Cre, and GFAPCre. Global Hif-2α disruption by Rosa26CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAPCre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP+ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2+ and PDGFRα+ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other's development, mediated at least in part by HIF-2α.

Background-The development of the vascular system is a complex process that involves communications among multiple cell types. As such, it is important to understand whether a specific gene regulates vascular development directly from within the vascular system or indirectly from nonvascular cells. Hypoxia-inducible factor-2␣ (Hif-2␣, or endothelial PAS protein-1 [EPAS-1]) is required for vascular development in mice, but it is not clear whether its requirement resides directly in endothelial cells. Methods and Results-To address this issue, we expressed Hif-2␣ cDNA in the vascular endothelium of Hif-2␣

Hypoxia promotes angiogenesis, proliferation, invasion and metastasis of pancreatic cancer. Essentially all studies of the hypoxia pathway in pancreatic cancer research to date have focused on fully malignant tumors or cancer cell lines, but the potential role of HIFs in the progression of pre-malignant lesions has not been critically examined. Here, we show that HIF2α is expressed early in pancreatic lesions both in human and in a mouse model of pancreatic cancer. HIF2α is a potent oncogenic stimulus but its role in Kras-induced pancreatic neoplasia has not been discerned. We used the Ptf1aCre transgene to activate KrasG12D and delete Hif2α solely within the pancreas. Surprisingly, loss of Hif2α in this model led to not reduced but rather markedly higher number of mPanIN lesions. These low-grade mPanIN lesions, however, failed to progress to high-grade mPanINs, associated with exclusive loss of β-catenin and SMAD4. The concomitant loss of HIF2α as well as β-catenin and Smad4 was further confirmed in vitro, whereby silencing of Hif2α resulted in reduced β-catenin and Smad4 transcription. Thus, with oncogenic Ras expressed in the pancreas, HIF2α modulates Wnt-signaling during mPanIN progression, by maintaining appropriate levels of both Smad4 and β-catenin.

In mice, retinal vascular and astrocyte networks begin to develop at birth, expanding radially from the optic nerve head (ONH) towards the retinal periphery. The retinal vasculature grows towards the periphery ahead of differentiated astrocytes, but behind astrocytic progenitor cells (APCs) and immature astrocytes. Endothelial cell specific Vegfr-2 disruption in newborn mice not only blocked retinal vascular development but also suppressed astrocytic differentiation, reducing the abundance of differentiated astrocytes while causing the accumulation of precursors. By contrast, retinal astrocytic differentiation was accelerated by the exposure of wild-type newborn mice to hyperoxia for 24 hours, or by APC specific deficiency in hypoxia inducible factor (HIF)−2α, an oxygen labile transcription factor. These findings reveal a novel function of the retinal vasculature, and imply that in normal neonatal mice, oxygen from the retinal circulation may promote astrocytic differentiation, in part by triggering oxygen dependent HIF-2α degradation in astrocytic precursors.

Phosphoinositide 3-kinase (PI3K) is activated by transmembrane tyrosine kinases such as vascular endothelial growth factor (VEGF) receptors and Tie2 (tunica intima endothelial kinase 2), both of which are key regulators of vascular development. However, the in vivo role of PI3K during developmental vascularization remains to be defined. Here we demonstrate that mice deficient in the p110␣ catalytic subunit of PI3K display multiple vascular defects, including dilated vessels in the head, reduced branching morphogenesis in the endocardium, lack of hierarchical order of large and small branches in the yolk sac, and impaired development of anterior cardinal veins. These vascular defects are strikingly similar to those in mice defective in the Tie2 signaling pathway. Indeed, Tie2 protein levels were significantly lower in p110␣-deficient mice.