Prolyl hydroxylase 3 controls the intestine goblet cell generation through stabilizing ATOH1.

Intestinal epithelia self-renew constantly and generate differentiated cells such as secretary goblet cells. The intestine goblet cells secrete gel-forming mucins that form mucus to create a barrier of defense. We reported previously that loss of prolyl hydroxylase (PHD) 3 led to disruption of the intestinal epithelial barrier function. However, the underlying mechanism remains elusive. Here, we demonstrate that PHD3 controls the generation of intestine goblet cell. We found that genetic ablation of Phd3 in mice intestine epithelial cells reduced the amount of goblet cells. Mechanistically, PHD3 bounds the E3 ubiquitin ligase HUWE1 and prevented HUWE1 from mediating ubiquitination and degradation of ATOH1, an essential driver for goblet cell differentiation. The prolyl hydroxylase activity-deficient variant PHD3(H196A) also prevented ATOH1 destruction. A genetic intestine epithelial PHD3(H196A)-knockin had no effect on ATOH1 expression or goblet cell amount in mice, suggesting that the PHD3 prolyl hydroxylase activity is dispensable for its ability to control ATOH1 expression and goblet cell generation. In dextran sulfate sodium (DSS)-induced experimental colitis, PHD3-knockout rather than PHD3(H196A)-knockin sensitized the mice to DSS treatment. Our results reveal an additional critical mechanism underlying the regulation of ATOH1 expression and goblet cell generation and highlight that PHD3 plays a role in controlling intestine goblet cell generation in a hydroxylase-independent manner.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1ß production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Inhibition of HIF-prolyl-4-hydroxylases prevents mitochondrial impairment and cell death in a model of neuronal oxytosis.

Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal damage. Here, we show that pharmacological inhibition of hypoxia-inducible factor prolyl-4-hydroxylases (HIF-PHDs) by adaptaquin inhibits lipid peroxidation and fully maintains mitochondrial function as indicated by restored mitochondrial membrane potential and ATP production, reduced formation of mitochondrial reactive oxygen species (ROS) and preserved mitochondrial respiration, thereby protecting neuronal HT-22 cells in a model of glutamate-induced oxytosis. Selective reduction of PHD1 protein using CRISPR/Cas9 technology also reduced both lipid peroxidation and mitochondrial impairment, and attenuated glutamate toxicity in the HT-22 cells. Regulation of activating transcription factor 4 (ATF4) expression levels and related target genes may mediate these beneficial effects. Overall, these results expose HIF-PHDs as promising targets to protect mitochondria and, thereby, neurons from oxidative cell death.

Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2 (phd2) deficiency during vertebrate development.

In the adult, new vessels and red blood cells form in response to hypoxia. Here, the oxygen-sensing system (PHD-HIF) has recently been put into focus, since the prolyl-hydroxylase domain proteins (PHD) and hypoxia-inducible factors (HIF) are considered as potential therapeutic targets to treat ischemia, cancers or age-related macula degeneration. While the oxygen-sensing system (PHD-HIF) has been studied intensively in this respect, only little is known from developing vertebrate embryos since mutations within this pathway led to an early decease of embryos due to placental defects. During vertebrate embryogenesis, a progenitor cell called hemangioblast is assumed to give rise to blood cells and blood vessels in a process called hematopoiesis and vasculogenesis, respectively. Xenopus provides an ideal experimental system to address these processes in vivo, as its development does not depend on a functional placenta and thus allows analyzing the role of oxygen directly. To this end, we adopted a computer-controlled four-channel system, which allowed us to culture Xenopus embryos under defined oxygen concentrations. Our data show that the development of vascular structures and blood cells is strongly impaired under hypoxia, while general development is less compromised. Interestingly, suppression of Phd2 function using specific antisense morpholinos or a chemical inhibitor resulted in mostly overlapping vascular defects; nevertheless, blood cell was formed almost normally. Our results provide the first evidence that oxygen via Phd2 has a decisive influence on the formation of the vascular network during vertebrate embryogenesis. These findings may be considered in certain potential treatment concepts.

PHD2/3-dependent hydroxylation tunes cardiac response to ß-adrenergic stress via phospholamban.

Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and PHD3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of Phd2 and Phd3 dramatically decreased expression of phospholamban (PLN), resulted in sustained activation of calcium/calmodulin-activated kinase II (CaMKII), and sensitized mice to chronic ß-adrenergic stress-induced myocardial injury. We have provided evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and PHD3 and is hydroxylated at 2 proline residues. Inhibition of PHDs increased the interaction between TR-α and nuclear receptor corepressor 2 (NCOR2) and suppressed Pln transcription. Together, these observations provide mechanistic insight into how oxygen directly modulates cardiac contractility and suggest that cardiac function could be modulated therapeutically by tuning PHD enzymatic activity.

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I.

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2ß2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.

Deletion of prolyl hydroxylase domain proteins (PHD1, PHD3) stabilizes hypoxia inducible factor-1 alpha, promotes neovascularization, and improves perfusion in a murine model of hind-limb ischemia.

BACKGROUND: There is an emerging focus on investigating innovative therapeutic molecules that can potentially augment neovascularization in order to treat peripheral arterial disease (PAD). Although prolyl hydroxylase domain proteins 1 and 3 (PHD1 and PHD3) may modulate angiogenesis via regulation of hypoxia inducible factor-1α (HIF-1α), there has been no study directly addressing their roles in ischemia-induced vascular growth. We hypothesize that PHD1(-/-) or PHD3(-/-) deficiency might promote angiogenesis in the murine hind-limb ischemia (HLI) model. STUDY DESIGN: Wild type (WT), PHD1(-/-) and PHD3(-/-) male mice aged 8-12weeks underwent right femoral artery ligation. Post-procedurally, motor function assessment and laser Doppler imaging were periodically performed. The mice were euthanized after 28days and muscles were harvested. Immunohistochemical analysis was performed to determine the extent of angiogenesis by measuring capillary and arteriolar density. VEGF expression was quantified by enzyme-linked immunosorbent assay (ELISA). Bcl-2 and HIF-1α were analyzed by immunofluorescence. Fibrosis was measured by picrosirius red staining. RESULTS: PHD1(-/-) and PHD3(-/-) mice showed significantly improved recovery of perfusion and motor function score when compared to WT after femoral artery ligation. These mice also exhibited increased capillary and arteriolar density, capillary/myocyte ratio along with decreased fibrosis compared to WT. VEGF, Bcl-2 and HIF-1α expression increased in PHD1(-/-) and PHD3(-/-) mice compared to WT. CONCLUSIONS: Taken together these results suggest that PHD1 and PHD3 deletions promote angiogenesis in ischemia-injured tissue, and may present a promising therapeutic strategy in treating PAD.

Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR.

Solid tumours are exposed to microenvironmental factors such as hypoxia that normally inhibit cell growth. However, tumour cells are capable of counteracting these signals through mechanisms that are largely unknown. Here we show that the prolyl hydroxylase PHD3 restrains tumour growth in response to microenvironmental cues through the control of EGFR. PHD3 silencing in human gliomas or genetic deletion in a murine high-grade astrocytoma model markedly promotes tumour growth and the ability of tumours to continue growing under unfavourable conditions. The growth-suppressive function of PHD3 is independent of the established PHD3 targets HIF and NF-κB and its hydroxylase activity. Instead, loss of PHD3 results in hyperphosphorylation of epidermal growth factor receptor (EGFR). Importantly, epigenetic/genetic silencing of PHD3 preferentially occurs in gliomas without EGFR amplification. Our findings reveal that PHD3 inactivation provides an alternative route of EGFR activation through which tumour cells sustain proliferative signalling even under conditions of limited oxygen availability.

Prolyl-hydroxylase PHD3 interacts with pyruvate dehydrogenase (PDH)-E1ß and regulates the cellular PDH activity.

Cells are frequently exposed to hypoxia in physiological and pathophysiological conditions in organisms. Control of energy metabolism is one of the critical functions of the hypoxic response. Hypoxia-Inducible Factor (HIF) is a central transcription factor that regulates the hypoxic response. HIF prolyl-hydroxylase PHDs are the enzymes that hydroxylate the α subunit of HIF and negatively regulate its expression. To further understand the physiological role of PHD3, proteomics were used to identify PHD3-interacting proteins, and pyruvate dehydrogenase (PDH)-E1ß was identified as such a protein. PDH catalyzes the conversion of pyruvate to acetyl-coA, thus playing a key role in cellular energy metabolism. PDH activity was significantly decreased in PHD3-depleted MCF7 breast cancer cells and PHD3(-/-) MEFs. PHD3 depletion did not affect the expression of the PDH-E1α, E1ß, and E2 subunits, or the phosphorylation status of E1α, but destabilized the PDH complex (PDC), resulting in less functional PDC. Finally, PHD3(-/-) cells were resistant to cell death in prolonged hypoxia with decreased production of ROS. Taken together, the study reveals that PHD3 regulates PDH activity in cells by physically interacting with PDC.

Prolyl-4-hydroxylase domain 3 (PHD3) is a critical terminator for cell survival of macrophages under stress conditions.

On a molecular level, cells sense changes in oxygen availability through the PHDs, which regulate the protein stability of the α-subunit of the transcription factor HIF. Especially, PHD3 has been additionally associated with apoptotic cell death. We hypothesized that PHD3 plays a role in cell-fate decisions in macrophages. Therefore, myeloid-specific PHD3(-/-) mice were created and analyzed. PHD3(-/-) BMDM showed no altered HIF-1α or HIF-2α stabilization or increased HIF target gene expression in normoxia or hypoxia. Macrophage M1 and M2 polarization was unchanged likewise. Compared with macrophages from WT littermates, PHD3(-/-) BMDM exhibited a significant reduction in TUNEL-positive cells after serum withdrawal or treatment with stauro and SNAP. Under the same conditions, PHD3(-/-) BMDM also showed less Annexin V staining, which is representative for membrane disruption, and indicated a reduced early apoptosis. In an unbiased transcriptome screen, we found that Angptl2 expression was reduced in PHD3(-/-) BMDM under stress conditions. Addition of rAngptl2 rescued the antiapoptotic phenotype, demonstrating that it is involved in the PHD3-mediated response toward apoptotic stimuli in macrophages.

Prolyl hydroxylase domain protein 2 plays a critical role in diet-induced obesity and glucose intolerance.

BACKGROUND: Recent studies suggest that the oxygen-sensing pathway consisting of transcription factor hypoxia-inducible factor and prolyl hydroxylase domain proteins (PHDs) plays a critical role in glucose metabolism. However, the role of adipocyte PHD in the development of obesity has not been clarified. We examined whether deletion of PHD2, the main oxygen sensor, in adipocytes affects diet-induced obesity and associated metabolic abnormalities. METHODS AND RESULTS: To delete PHD2 in adipocyte, PHD2-floxed mice were crossed with aP2-Cre transgenic mice (Phd2(f/f)/aP2-Cre). Phd2(f/f)/aP2-Cre mice were resistant to high-fat diet-induced obesity (36.7±1.7 versus 44.3±2.0 g in control; P<0.01) and showed better glucose tolerance and homeostasis model assessment-insulin resistance index than control mice (3.6±1.0 versus 11.1±2.1; P<0.01). The weight of white adipose tissue was lighter (epididymal fat, 758±35 versus 1208±507 mg in control; P<0.01) with a reduction in adipocyte size. Macrophage infiltration into white adipose tissue was also alleviated in Phd2(f/f)/aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2(f/f)/aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2(f/f)/aP2-Cre mice. Knockdown of PHD2 in 3T3L1 adipocytes induced a decrease in the glucose level and an increase in the lactate level in the supernatant with upregulation of glycolytic enzymes and reduced lipid accumulation. CONCLUSIONS: PHD2 in adipose tissue plays a critical role in the development of diet-induced obesity and glucose intolerance. PHD2 might be a novel target molecule for the treatment of obesity and associated metabolic abnormalities.

Hypoxia promotes the production and inhibits the destruction of human articular cartilage.

OBJECTIVE: To determine the effects of hypoxia on both anabolic and catabolic pathways of metabolism in human articular cartilage and to elucidate the roles played by hypoxia-inducible factors (HIFs) in these responses. METHODS: Normal human articular cartilage from a range of donors was obtained at the time of above-the-knee amputations due to sarcomas not involving the joint space. Fresh cartilage tissue explants and isolated cells were subjected to hypoxia and treatment with interleukin-1α. Cell transfections were performed on isolated human chondrocytes. RESULTS: Using chromatin immunoprecipitation, we found that hypoxia induced cartilage production in human tissue explants through direct binding of HIF-2α to a specific site in the master-regulator gene SOX9. Importantly, hypoxia also suppressed spontaneous and induced destruction of human cartilage in explant culture. We found that anticatabolic responses were predominantly mediated by HIF-1α. Manipulation of the hypoxia-sensing pathway through depletion of HIF-targeting prolyl hydroxylase-containing protein 2 (PHD-2) further enhanced cartilage responses as compared to hypoxia alone. Hypoxic regulation of tissue-specific metabolism similar to that in human cartilage was observed in pig, but not mouse, cartilage. CONCLUSION: We found that resident chondrocytes in human cartilage are exquisitely adapted to hypoxia and use it to regulate tissue-specific metabolism. Our data revealed that while fundamental regulators, such as SOX9, are key molecules both in mice and humans, the way in which they are controlled can differ. This is all the more important since it is upstream regulators such as this that need to be directly targeted for therapeutic benefit. HIF-specific hydroxylase PHD-2 may represent a relevant target for cartilage repair.

Deficiency of the oxygen sensor PHD1 augments liver regeneration after partial hepatectomy.

PURPOSE: Liver regeneration after partial hepatectomy (PH) occurs in conditions of reduced oxygen supply. HIF prolyl hydroxylase enzymes (PHD1, PHD2, and PHD3) are oxygen sensors involved in adaptive response to hypoxia. Specific functions of these PHD enzymes in liver regeneration have, however, remained enigmatic. Here, we investigated the significance of PHD1 in liver regeneration following hepatectomy. METHODS: Liver regeneration was studied in PHD1-deficient (PHD1(-/-)) and wild type (WT) mice subjected to 80% hepatectomy. For in vitro analyses, hepatocytes were isolated from PHD1(-/-) and WT livers. Cell cycle progression was studied via FACS-based analysis of nuclear DNA profile. Transcription factor binding assays, qRT-PCR, and immunoblotting were applied to study the relevance of PHD1 downstream effectors during liver regeneration. RESULTS: Liver regeneration was significantly enhanced in PHD1(-/-) mice compared to WT littermates. This effect was due to enhanced proliferation rather than to hypertrophy of liver cells. Cell cycle progression was significantly enhanced, and transcriptional activity of the cell cycle regulator c-Myc was increased in PHD1-deficient hepatocytes. These changes coincided with increased expression of cyclin D2, a cell cycle-promoting c-Myc target, and decreased expression of the cell cycle-delaying c-Myc target p21. CONCLUSIONS: Loss of PHD1 enhances liver regeneration by boosting hepatocyte proliferation in a c-Myc-dependent fashion. PHD1 might, therefore, represent a potential target to facilitate liver regeneration after surgical resection.

Gene-targeting of Phd2 improves tumor response to chemotherapy and prevents side-toxicity.

The success of chemotherapy in cancer treatment is limited by scarce drug delivery to the tumor and severe side-toxicity. Prolyl hydroxylase domain protein 2 (PHD2) is an oxygen/redox-sensitive enzyme that induces cellular adaptations to stress conditions. Reduced activity of PHD2 in endothelial cells normalizes tumor vessels and enhances perfusion. Here, we show that tumor vessel normalization by genetic inactivation of Phd2 increases the delivery of chemotherapeutics to the tumor and, hence, their antitumor and antimetastatic effect, regardless of combined inhibition of Phd2 in cancer cells. In response to chemotherapy-induced oxidative stress, pharmacological inhibition or genetic inactivation of Phd2 enhances a hypoxia-inducible transcription factor (HIF)-mediated detoxification program in healthy organs, which prevents oxidative damage, organ failure, and tissue demise. Altogether, our study discloses alternative strategies for chemotherapy optimization.

PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response.

The DNA damage response (DDR) is a complex regulatory network that is critical for maintaining genome integrity. Posttranslational modifications are widely used to ensure strict spatiotemporal control of signal flow, but how the DDR responds to environmental cues, such as changes in ambient oxygen tension, remains poorly understood. We found that an essential component of the ATR/CHK1 signaling pathway, the human homolog of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2), associated with and was hydroxylated by prolyl hydroxylase domain protein 3 (PHD3). HCLK2 hydroxylation was necessary for its interaction with ATR and the subsequent activation of ATR/CHK1/p53. Inhibiting PHD3, either with the pan-hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or through hypoxia, prevented activation of the ATR/CHK1/p53 pathway and decreased apoptosis induced by DNA damage. Consistent with these observations, we found that mice lacking PHD3 were resistant to the effects of ionizing radiation and had decreased thymic apoptosis, a biomarker of genomic integrity. Our identification of HCLK2 as a substrate of PHD3 reveals the mechanism through which hypoxia inhibits the DDR, suggesting hydroxylation of HCLK2 is a potential therapeutic target for regulating the ATR/CHK1/p53 pathway.

Effect of prolyl hydroxylase domain-2 haplodeficiency on the hepatocarcinogenesis in mice.

BACKGROUND & AIMS: The two major primary liver cancers in adults are hepatocellular carcinoma and cholangiocarcinoma. These tumors rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors. Recently, interest has grown in the regulators of these factors. Several reports have been published describing the role of prolyl hydroxylase domains--the key oxygen sensor responsible for the degradation of hypoxia inducible factors--tumor progression and vascularisation. The effect of prolyl hydroxylase domain 2 on the pathogenesis of liver cancer has never been studied. METHODS: A diethylnitrosamine-induced mouse model was used in this study, allowing primary hepatic tumors to occur as a result of chronic liver damage. Several parameters of prolyl hydroxylase domain 2-haplodeficient mice were compared to those of wild type mice, thereby focussing on the expression of angiogenic factors and on the hepatic progenitor cell activation and differentiation. RESULTS: This study shows that inhibiting prolyl hydroxylase domain 2 increases the hepatocarcinogenesis and stimulates the development of cholangiocarcinoma. Furthermore, PHD2 deficiency and the accompanying continuous HIF activation, selected for a more metastatic tumor phenotype. CONCLUSIONS: The effect of prolyl hydroxylase domain 2 deficiency on hepatocarcinogenesis hold a great potential for therapeutic intervention, since hypoxia and the selection for a more aggressive cholangiocarcinoma phenotype might also have a repercussion on patients receiving long-term treatment with anti-angiogenic compounds.

HIF prolyl hydroxylase-2 inhibition diminishes tumor growth through matrix metalloproteinase-induced TGFß activation.

A right amount of oxygen and nutrients is essential for a tumor to develop. The role of oxygen dependent pathways and their regulators is therefore of utmost importance although little is known about the detailed impact they can have. Recently we have shown that inhibition of the oxygen sensor PHD2 in tumor cells blocks tumor growth due to the anti-proliferative activity of TGFß. In this study, we refined these results by comparing different shPHD2 sequences in depth in the early phase of tumor growth. Our findings also reveal an intriguing role for MMP2 and MT1MMP in these settings, as these activated proteases display an anti-proliferative characteristic through the activation of downstream TGFß targets. In conclusion, PHD2 inhibition is essential for the regulation of the anti-tumoral activity in mouse tumor cells and might bring some new insight in our understanding of tumor growth inhibition.

Macrophage skewing by Phd2 haplodeficiency prevents ischaemia by inducing arteriogenesis.

PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion. We show that Phd2 (also known as Egln1) haplodeficient (Phd2(+/-)) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2(+/-) mice was due to an expansion of tissue-resident, M2-like macrophages and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2(+/-) macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

Reduced retinal neovascularization, vascular permeability, and apoptosis in ischemic retinopathy in the absence of prolyl hydroxylase-1 due to the prevention of hyperoxia-induced vascular obliteration.

PURPOSE: Prolyl hydroxylases (PHDs) are oxygen sensors that stabilize hypoxia-inducible factors (HIFs) to induce proinflammatory, vasopermeability, and proapoptotic factors. These may be potential targets to reduce the complications of ischemic retinopathies. METHODS: Oxygen-induced ischemic retinopathy (OIR) was generated as a model for retinopathy of prematurity (ROP) by placing 7-day-old mice in 75% oxygen for 5 days and returning them to the relative hypoxia of room air for 5 days. Neovascularization (NV) and avascular areas were assessed on retinal flat-mounts by image analysis. Blood-retinal barrier breakdown was assessed using ³H-mannitol as a tracer. Apoptosis was detected with TUNEL staining. HIF-1α and VEGF were quantified using Western blot analysis and ELISA. RESULTS: PHD1-deficient mice demonstrated reduced hyperoxia-associated vascular obliteration during oxygen-induced ischemic retinopathy. This was associated with subsequent reduced avascularity, vascular leakage, and pathologic NV during the hypoxic phase, which could be accounted for by a reduced expression of HIF-1α and VEGF. Apoptosis in the retina was also reduced in PHD1-depleted mice after 2 days in hyperoxia. CONCLUSIONS: PHD1 deficiency is associated with a reduction of ischemia-induced retinal NV. The regulatory mechanism in this model appears to be: PHD1 depletion prevents HIF-1α degradation in hyperoxia, which induces VEGF, thus preventing hyperoxia-related vessel loss. Without a vessel deficiency, there would not be relative hypoxia when the mice are returned to room air and there would be no need to initiate angiogenesis signaling. Blocking PHD1 may be beneficial for ischemic retinopathies and inflammatory and neurodegenerative disorders.

A feedback loop involving the Phd3 prolyl hydroxylase tunes the mammalian hypoxic response in vivo.

Hypoxia-inducible factor (HIF), consisting of a labile alpha subunit and a stable beta subunit, is a master regulator of hypoxia-responsive mRNAs. HIF alpha undergoes oxygen-dependent prolyl hydroxylation, which marks it for polyubiquitination by a complex containing the von Hippel-Lindau protein (pVHL). Among the three Phd family members, Phd2 appears to be the primary HIF prolyl hydroxylase. Phd3 is induced by HIF and, based on findings from in vitro studies, may participate in a HIF-regulatory feedback loop. Here, we report that Phd3 loss exacerbates the HIF activation, hepatic steatosis, dilated cardiomyopathy, and premature mortality observed in mice lacking Phd2 alone and produces a closer phenocopy of the changes seen in mice lacking pVHL than the loss of Phd2 alone. Importantly, the degree to which Phd3 can compensate for Phd2 loss and the degree to which the combined loss of Phd2 and Phd3 resembles pVHL loss appear to differ for different HIF-responsive genes and in different tissues. These findings highlight that the responses of different HIF target genes to changes in prolyl hydroxylase activity differ, quantitatively and qualitatively, in vivo and have implications for the development of paralog-specific prolyl hydroxylase inhibitors as therapeutic agents.