Myeloid PHD2 deficiency accelerates neointima formation via Hif-1α.

The key players of the hypoxic response are the hypoxia-inducible factors (Hif), whose α-subunits are tightly regulated by Prolyl-4-hydroxylases (PHD), predominantly by PHD2. Monocytes/Macrophages are involved in atherosclerosis but also restenosis and were found at hypoxic and sites of the lesion. Little is known about the role of the myeloid PHD2 in atherosclerosis and neointima formation. The study aimed to investigate the consequences of a myeloid deficiency of PHD2 in the process of neointima formation using an arterial denudation model. LysM-cre mice were crossed with PHD2fl/fl, PHD2fl/fl/Hif1αfl/fl and PHD2fl/fl/Hif2αfl/fl to get myeloid specific knockout of PHD2 and the Hif-α subunits. Denudation of the femoral artery was performed and animals were fed a western type diet afterwards with analysis of neointima formation 5 and 35 days after denudation. Increased neointima formation in myeloid PHD2 knockouts was observed, which was blunted by double-knockout of PHD2 and Hif1α whereas double knockout of PHD2 and Hif-2α showed comparable lesions to the PHD2 knockouts. Macrophage infiltration was comparable to the neointima formation, suggesting a more inflammatory reaction, and was accompanied by increased intimal VEGF-A expression. Collagen-content inversely correlated to the extent of neointima formation suggesting a destabilization of the plaque. This effect might be triggered by macrophage polarization. Therefore, in vitro results showed a distinct expression pattern in differentially polarized macrophages with high expression of Hif-1α, VEGF and MMP-1 in proinflammatory M1 macrophages. In conclusion, the results show that myeloid Hif-1α is involved in neointima hyperplasia. Our in vivo and in vitro data reveal a central role for this transcription factor in driving plaque-vascularization accompanied by matrix-degradation leading to plaque destabilization.

Prolyl hydroxylase domain 2 reduction enhances skeletal muscle tissue regeneration after soft tissue trauma in mice.

The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tissue regeneration. HIF-1 is negatively controlled by O2-dependent prolyl hydroxylases with a predominant role of prolyl hydroxylase 2 isoform (Phd2). Transgenic mice, hypomorphic for this isoform, accumulate more HIF-1 under normoxic conditions. Using these mice, we investigated the influence of Phd2 and HIF-1 on the regenerative capability of skeletal muscle tissue after myotrauma. Phd2-hypomorphic and wild type mice (on C57Bl/6 background) were grouped with regeneration times from 6 to 168 hours after closed mechanic muscle trauma to the hind limb. Tissue samples were analysed by immuno-staining and real-time PCR. Bone marrow derived macrophages of wild type and Phd2-hypomorphic mice were isolated and analysed via flow cytometry and quantitative real-time PCR. Phd2 reduction led to a higher regenerative capability due to enhanced activation of myogenic factors accompanied by induction of genes responsible for glucose and lactate metabolism in Phd2-hypomorphic mice. Macrophage infiltration into the trauma areas in hypomorphic mice started earlier and was more pronounced compared to wild type mice. Phd2-hypomorphic mice also showed higher numbers of macrophages in areas with sustained trauma 72 hours after myotrauma application. In conclusion, we postulate that the HIF-1 pathway is activated secondary to a Phd2 reduction which may lead to i) higher activation of myogenic factors, ii) increased number of positive stem cell proliferation markers, and iii) accelerated macrophage recruitment to areas of trauma, resulting in faster muscle tissue regeneration after myotrauma. With the current development of prolyl hydroxylase domain inhibitors, our findings point towards a potential clinical benefit after myotrauma.

Hypoxia-inducible factor (HIF)-prolyl hydroxylase 3 (PHD3) maintains high HIF2A mRNA levels in clear cell renal cell carcinoma.

Most clear cell renal cell carcinomas (ccRCCs) have inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL), resulting in the accumulation of hypoxia-inducible factor α-subunits (HIF-α) and their downstream targets. HIF-2α expression is particularly high in ccRCC and is associated with increased ccRCC growth and aggressiveness. In the canonical HIF signaling pathway, HIF-prolyl hydroxylase 3 (PHD3) suppresses HIF-2α protein by post-translational hydroxylation under sufficient oxygen availability. Here, using immunoblotting and immunofluorescence staining, qRT-PCR, and siRNA-mediated gene silencing, we show that unlike in the canonical pathway, PHD3 silencing in ccRCC cells leads to down-regulation of HIF-2α protein and mRNA. Depletion of other PHD family members had no effect on HIF-2α expression, and PHD3 knockdown in non-RCC cells resulted in the expected increase in HIF-2α protein expression. Accordingly, PHD3 knockdown decreased HIF-2α target gene expression in ccRCC cells and expression was restored upon forced HIF-2α expression. The effect of PHD3 depletion was pinpointed to HIF2A mRNA stability. In line with these in vitro results, a strong positive correlation of PHD3 and HIF2A mRNA expression in ccRCC tumors was detected. Our results suggest that in contrast to the known negative regulation of HIF-2α in most cell types, high PHD3 expression in ccRCC cells maintains elevated HIF-2α expression and that of its target genes, which may enhance kidney cancer aggressiveness.

HIF-1α metabolically controls collagen synthesis and modification in chondrocytes.

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.

Loss of Phd2 cooperates with BRAFV600E to drive melanomagenesis.

Prolyl hydroxylase domain protein 2 (PHD2) is a well-known master oxygen sensor. However, the role of PHD2 in tumor initiation remains controversial. We find that during the transition of human nevi to melanoma, the expression of PHD2 protein is significantly decreased and lower expression PHD2 in melanoma is associated with worse clinical outcome. Knockdown of PHD2 leads to elevated Akt phosphorylation in human melanocytes. Mice with conditional melanocyte-specific expression of Phd2lox/lox (Tyr::CreER;Phd2lox/lox) fail to develop pigmented lesions. However, deletion of Phd2 in combination with expression of BRafV600E in melanocytes (Tyr::CreER;Phd2lox/lox;BRafCA) leads to the development of melanoma with 100% penetrance and frequent lymph node metastasis. Analysis of tumor tissues using reverse phase protein arrays demonstrates that Phd2 deletion activates the AKT-mTOR-S6 signaling axis in the recovered tumors. These data indicate that PHD2 is capable of suppressing tumor initiation largely mediated through inhibiting of the Akt-mTOR signaling pathway in the melanocyte lineage.

Regulatory Role of Endothelial PHD2 in the Hepatic Steatosis.

BACKGROUND/AIMS: Liver disease is a leading cause of high mortality and morbidity worldwide. The aim of the present study is to investigate the regulatory role of prolyl hydroxylase-2 (PHD2)-hypoxia-inducible factor-2a (HIF-2α) axis on nonalcoholic fatty liver disease (NAFLD) and to explore the potential mechanisms by which endothelial (EC)-specific PHD2 deficiency regulates hepatic steatosis and fibrosis. METHODS: In the endothelial-specific PHD2 knockout (PHD2ECKO) mouse fed with normal diet or high fat diet (HFD), liver lipid accumulation and fibrosis were measured by Oil Red O and Masson trichrome staining. The fat and body weight (FW/BW) ratio and glucose tolerance were measured. The expression of HIF-2α, atrial natriuretic peptide (ANP), angiopoietin-2 (Ang-2), and transforming growth factor-b (TGF-ß) were analyzed by western blot analysis. RESULTS: The steatosis and fibrosis were significantly increased in the PHD2ECKO mice. FW/BW ratio was significantly increased in the PHD2ECKO mice. Moreover, knockout of endothelial PHD2 resulted in an impairment of glucose tolerance in mice. Western blot analysis showed that the expression of HIF-2α in liver tissues was not significantly increased. Interestingly, the expression of ANP was decreased, and Ang-2 and TGF-ß levels were significantly increased in the liver of PHD2ECKO mice. The FW/BW ratio was also significantly increased in the PHD2ECKO mice fed with HFD for 16 weeks. Feeding HFD resulted in a significant increase in hepatic steatosis in the control PHD2f/f mice, but did not further enhance hepatic steatosis in the PHD2ECKO mice. CONCLUSIONS: We concluded that the endothelial PHD2 plays a critical role in hepatic steatosis and fibrosis, which may be involved in the regulation of ANP and Ang-2/TGF-ß signaling pathway, but not the HIF-2α expression.

Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways.

Hypoxia-inducible factor 1α (HIF1α) induces the expression of several hundred genes in hypoxia aiming at restoration of oxygen homeostasis. HIF prolyl-4-hydroxylases (HIF-P4Hs) regulate the stability of HIF1α in an oxygen-dependent manner. Hypoxia is a common feature in inflammation and cancer and the HIF pathway is closely linked with the inflammatory NF-κB and tumor suppressor p53 pathways. Here we show that genetic inactivation or chemical inhibition of HIF-P4H-1 leads to downregulation of proinflammatory genes, while proapoptotic genes are upregulated. HIF-P4H-1 inactivation reduces the inflammatory response under LPS stimulus in vitro and in an acute skin inflammation model in vivo. Furthermore, HIF-P4H-1 inactivation increases p53 activity and stability and hydroxylation of proline 142 in p53 has an important role in this regulation. Altogether, our data suggest that HIF-P4H-1 inhibition may be a promising therapeutic candidate for inflammatory diseases and cancer, enhancing the reciprocal negative regulation of the NF-κB and p53 pathways.

Increased EPO Levels Are Associated With Bone Loss in Mice Lacking PHD2 in EPO-Producing Cells.

The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts, and hematopoietic cells (CD68:cre-PHD2f/f ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast-specific (Osx:cre-PHD2f/f ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2f/f ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research.

Neuronal deficiency of HIF prolyl 4-hydroxylase 2 in mice improves ischemic stroke recovery in an HIF dependent manner.

Hypoxia inducible factors (HIFs) mediate the endogenous adaptive responses to hypoxia. HIF prolyl 4-hydroxylase domain proteins (PHD) are important suppressors of the HIF pathway. Recently, we demonstrated that neuron-specific deletion of Phd2 reduces cerebral tissue damage in the very acute phase of ischemic stroke. In the present study, we investigated whether neuronal Phd2 ablation is likewise beneficial for stroke recovery, and aimed to identify underlying cellular mechanisms. Mice underwent permanent occlusion of the distal middle cerebral artery (pdMCAO) for either 7days (sub-acute stage) or 30days (chronic stage). One week after pdMCAO the infarct size of Phd2-deficient mice was significantly reduced as compared to wild-type (WT) mice. Accordingly, Phd2-deficient animals showed less impaired sensorimotor function. Neuronal loss of Phd2 upregulated vascular endothelial growth factor (VEGF) and significantly increased microvascular density along the infarct border in the sub-acute stage of stroke. Phd2-deficient mice showed reduced expression of pro-inflammatory cytokines and increased numbers of resting microglia/macrophages and reactive astrocytes within peri-infarct regions in comparison to WT littermates. Finally, brain tissue protection and increased angiogenesis upon sub-acute ischemic stroke was completely absent in Phd2 knockout mice that were additionally deficient for both Hif1a and Hif2a. Our findings suggest that lack of PHD2 in neurons improves histological and functional long-term outcome from ischemic stroke at least partly by amplifying endogenous adaptive neovascularization through activation of the HIF-VEGF axis.

Prolyl hydroxylase domain 2 deficiency promotes skeletal muscle fiber-type transition via a calcineurin/NFATc1-dependent pathway.

BACKGROUND: Hypoxia exposure is known to induce an alteration in skeletal muscle fiber-type distribution mediated by hypoxia-inducible factor (HIF)-α. The downstream pathway of HIF-α leading to fiber-type shift, however, has not been elucidated. The calcineurin pathway is one of the pathways responsible for slow muscle fiber transition. Because calcineurin pathway is activated by vascular endothelial growth factor (VEGF), one of the factors induced by HIF-1α, we hypothesized that the stabilization of HIF-1α may lead to slow muscle fiber transition via the activation of calcineurin pathway in skeletal muscles. To induce HIF-1α stabilization, we used a loss of function strategy to abrogate Prolyl hydroxylase domain protein (PHD) 2 responsible for HIF-1α hydroxylation making HIF-1α susceptible to ubiquitin dependent degradation by proteasome. The purpose of this study was therefore to examine the effect of HIF-1α stabilization in PHD2 conditional knockout mouse on skeletal muscle fiber-type transition and to elucidate the involvement of calcineurin pathway on muscle fiber-type transition. RESULTS: PHD2 deficiency resulted in an increased capillary density in skeletal muscles due to the induction of vascular endothelial growth factor. It also elicited an alteration of skeletal muscle phenotype toward the type I fibers in both of the soleus (35.8 % in the control mice vs. 46.7 % in the PHD2-deficient mice, p < 0.01) and the gastrocnemius muscle (0.94 vs. 1.89 %, p < 0.01), and the increased proportion of type I fibers appeared to correspond to the area of increased capillary density. In addition, calcineurin and nuclear factor of activated T cell (NFATc1) protein levels were increased in both the gastrocnemius and soleus muscles, suggesting that the calcineurin/NFATc1 pathway was responsible for the type I fiber transition regardless of PGC-1α, which responded minimally to PHD2 deficiency. Indeed, we found that tacrolimus (FK-506), a calcineurin inhibitor, successfully suppressed slow fiber-type formation in PHD2-deficient mice. CONCLUSIONS: Taken together, stabilized HIF-1α induced by PHD2 conditional knockout resulted in the transition of muscle fibers toward a slow fiber type via a calcineurin/NFATc1 signaling pathway. PHD2 conditional knockout mice may serve as a model for chronic HIF-1α stabilization as in mice exposed to low oxygen concentration.

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis.

Cancer-associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF-mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia-inducible factor (HIF)-1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF-1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co-injected with tumour cells similarly prevents CAF-induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.

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.

HIF hydroxylase pathways in cardiovascular physiology and medicine.

Hypoxia inducible factors (HIFs) are α/ß heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate-dependent dioxygenases that catalyze post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFα subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here, we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities, and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease.

Conditional knockout of prolyl hydroxylase domain protein 2 attenuates high fat-diet-induced cardiac dysfunction in mice.

Oxygen sensor prolyl hydroxylases (PHDs) play important roles in the regulation of HIF-α and cell metabolisms. This study was designed to investigate the direct role of PHD2 in high fat-diet (HFD)-induced cardiac dysfunction. In HFD fed mice, PHD2 expression was increased without significant changes in PHD1 and PHD3 levels in the heart. This was accompanied by a significant upregulation of myeloid differentiation factor 88 (MYD88) and NF-κB. To explore the role of PHD2 in HFD-induced cardiac dysfunction, PHD2 conditional knockout mice were fed a HFD for 16 weeks. Intriguingly, knockout of PHD2 significantly reduced MYD88 and NF-κb expression in HFD mouse hearts. Moreover, knockout of PHD2 inhibited TNFα and ICAM-1 expression, and reduced cell apoptosis and macrophage infiltration in HFD mice. This was accompanied by a significant improvement of cardiac function. Most importantly, conditional knockout of PHD2 at late stage in HFD mice significantly improved glucose tolerance and reversed cardiac dysfunction. Our studies demonstrate that PHD2 activity is a critical contributor to the HFD-induced cardiac dysfunction. Inhibition of PHD2 attenuates HFD-induced cardiac dysfunction by a mechanism involving suppression of MYD88/NF-κb pathway and inflammation.

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.

Wound healing improvement with PHD-2 silenced fibroblasts in diabetic mice.

BACKGROUND: Hypoxia-inducible factor 1α is the central regulator of the hypoxia-induced response which results in the up-regulation of angiogenic factors. Its activity is under precise regulation of prolyl-hydroxylase domain 2. We hypothesized that PHD2 silenced fibroblasts would increase the expression of angiogenic factors, which might contribute to the improvement of the diabetic wound healing. MATERIALS AND METHODS: 50 dB/db mice were employed and randomly assigned into five groups with 10 mice in each: group 1 (untreated cell), group 2 (PHD2 silenced cell), group 3 (L-mimosine treated cells), group 4 (nontargeting siRNA treated cells) and group 5 (sham control). Fibroblasts were cultivated from the dermis of mice in each group and treated with PHD2 targeting siRNA, L-mimosine and non-targeting siRNA respectively. A fraction of the fibroblasts were employed to verify the silencing rate of PHD2 after 48 hours. The autologous fibroblasts (treated and untreated) labeled with adenovirus-GFP were implanted around the wound (Φ6mm), which was created on the dorsum of each mouse. The status of wounds was recorded periodically. Ten days postoperatively, 3 mice from each group were sacrificed and wound tissues were harvested. Molecular biological examinations were performed to evaluate the expressions of cytokines. 28 days postoperatively, the remaining mice were sacrificed. Histological examinations were performed to evaluate the densities of GFP+ cells and capillaries. RESULTS: The expression of PHD2 reduced to 12.5%, and the expressions of HIF-1α and VEGFa increased significantly after PHD2 siRNA treatment. With the increasing expressions of HIF-1α and VEGFa, the time to wound closure in group 2 was less than 2 weeks. Increased numbers of GFP+ cells and capillaries were observed in group 2. CONCLUSION: PHD2 siRNA treatment not only increased the expression of HIF1α and VEGFa, but also improved the fibroblast proliferation. These effects might contribute to the improvement of the diabetic wound healing.