Prolyl Hydroxylase Domain Protein Research Articles

Local Delivery of PHD2 siRNA from ROS-Degradable Scaffolds to Promote Diabetic Wound Healing.

Small interfering RNA (siRNA) delivered from reactive oxygen species-degradable tissue engineering scaffolds promotes diabetic wound healing in rats. Porous poly(thioketal-urethane) scaffolds implanted in diabetic wounds locally deliver siRNA that inhibits the expression of prolyl hydroxylase domain protein 2, thereby increasing the expression of progrowth genes and increasing vasculature, proliferating cells, and tissue development in diabetic wounds.

Prolyl hydroxylase domain proteins regulate bone mass through their expression in osteoblasts

The roles of prolyl hydroxylase domain proteins (PHDs) in bone are incompletely understood. Here we deleted the expression of genes encoding PHD1, PHD2, and PHD3 in osteoblasts in mice by breeding the floxed Phd1–3 mice with Col1a1-Cre transgenic mice. Results showed that mice lacking PHD1-3 in osteoblasts (Phd1–3ob−/−) had increased bone mass. Bone parameters such as bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were increased, while trabecular spacing (Tb.Sp) was decreased in Phd1–3ob−/− relative to wild-type (WT) femurs. In contrast, loss of PHD1–3 in osteoblasts did not alter cortical thickness (Cort.Th). The mineralization apposition rate (MAR) was increased in Phd1–3ob−/− bone compared to that of wild-type (WT) bone, demonstrating an enhancement of osteoblast function. Loss of PHD1-3 increased the number of osteoblast progenitors (CFU-OBs) in bone marrow cultures. Interestingly, deleting Phd1–3 genes in osteoblasts increased osteoclast formation in vitro and in bone.

The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α.

Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude.

PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

Disruption of Prolyl hydroxylase domain protein-2 (PHD2) activity impairs TGFβ-dependent sphingolipid metabolism in murine placenta.

Disruption of Prolyl hydroxylase domain protein-2 (PHD2) activity impairs TGFβ-dependent sphingolipid metabolism in murine placenta.

Loss of prolyl hydroxylase domain protein 2 in vascular endothelium increases pericyte coverage and promotes pulmonary arterial remodeling.

Pulmonary arterial hypertension (PAH) is a leading cause of heart failure. Although pulmonary endothelial dysfunction plays a crucial role in the progression of the PAH, the underlying mechanisms are poorly understood. The HIF-α hydroxylase system is a key player in the regulation of vascular remodeling. Knockout of HIF-2α has been reported to cause pulmonary hypertension. The present study examined the role of endothelial cell specific prolyl hydroxylase-2 (PHD2) in the development of PAH and pulmonary vascular remodeling. The PHD2f/f mouse was crossbred with VE-Cadherin-Cre promoter mouse to generate an endothelial specific PHD2 knockout (Cdh5-Cre-PHD2ECKO) mouse. Pulmonary arterial pressure and the size of the right ventricle was significantly elevated in the PHD2ECKO mice relative to the PHD2f/f controls. Knockout of PHD2 in EC was associated with vascular remodeling, as evidenced by an increase in pulmonary arterial media to lumen ratio and number of muscularized arterioles. The pericyte coverage and vascular smooth muscle cells were also significantly increased in the PA. The increase in vascular pericytes was associated with elevated expression of fibroblast specific protein-1 (FSP-1). Moreover, perivascular interstitial fibrosis of pulmonary arteries was significantly increased in the PHD2ECKO mice. Mechanistically, knockout of PHD2 in EC increased the expression of Notch3 and transforming growth factor (TGF-β) in the lung tissue. We conclude that the expression of PHD2 in endothelial cells plays a critical role in preventing pulmonary arterial remodeling in mice. Increased Notch3/TGF-β signaling and excessive pericyte coverage may be contributing to the development of PAH following deletion of endothelial PHD2.

Prolyl hydroxylase 2 (PHD2) inhibition protects human renal epithelial cells and mice kidney from hypoxia injury.

Prolyl hydroxylase domain protein 2 (PHD2) is a key oxygen sensor, setting low steady-state level of hypoxia-inducible factor-α (HIF-α). Here, we showed that treatment of cobalt chloride (CoCl2), a hypoxia mimic, in HK-2 tubular epithelial cells induced PHD2 and HIF-1/2α expression as well as cell apoptosis and autophagy activation. Three methyladenine (3-MA), the autophagy inhibitor, blocked autophagy and protected HK-2 cells from CoCl2. Significantly, siRNA knockdown of PHD2 also protected HK-2 cells from CoCl2, possibly via increasing HIF-1α expression. Reversely, HIF-1α siRNA knockdown almost abolished cytoprotection by PHD2 siRNA in CoCl2-treated HK-2 cells. In vivo, pretreatment with a PHD inhibitor L-mimosine remarkably attenuated mice renal ischemia-reperfusion injuries. Molecularly, L-mimosine inhibited apoptosis and inflammatory responses in injured mice kidneys. Together, our results suggest that PHD2 silence or inhibition protects human renal epithelial cells and mice kidney from hypoxia injuries.

WITHDRAWN: Prolyl hydroxylase domain proteins regulate bone mass through their expression in osteoblasts

WITHDRAWN: Prolyl hydroxylase domain proteins regulate bone mass through their expression in osteoblasts

Tumour suppressor death-associated protein kinase targets cytoplasmic HIF-1α for Th17 suppression.

Death-associated protein kinase (DAPK) is a tumour suppressor. Here we show that DAPK also inhibits T helper 17 (Th17) and prevents Th17-mediated pathology in a mouse model of autoimmunity. We demonstrate that DAPK specifically downregulates hypoxia-inducible factor 1α (HIF-1α). In contrast to the predominant nuclear localization of HIF-1α in many cell types, HIF-1α is located in both the cytoplasm and nucleus in T cells, allowing for a cytosolic DAPK–HIF-1α interaction. DAPK also binds prolyl hydroxylase domain protein 2 (PHD2) and increases HIF-1α-PHD2 association. DAPK thereby promotes the proline hydroxylation and proteasome degradation of HIF-1α. Consequently, DAPK deficiency leads to excess HIF-1α accumulation, enhanced IL-17 expression and exacerbated experimental autoimmune encephalomyelitis. Additional knockout of HIF-1α restores the normal differentiation of Dapk−/− Th17 cells and prevents experimental autoimmune encephalomyelitis development. Our results reveal a mechanism involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that raising DAPK levels could be used for treatment of Th17-associated inflammatory diseases.

Activation of the hypoxia‐inducible factor pathway induced by PHD2 deficiency enhances the effect of running training in mice

BackgroundRecently, altitude training has widely been accepted by elite athletes as an efficient way to improve the sea‐level performance. The adaptation to the deprivation of O2, which is called hypoxic response and mediated by hypoxia‐inducible factors (HIFs), seems to contribute to the benefit of altitude training. However, the veracity of this benefit is still controversial, and the effect of biological processes mediated by the HIF pathway on the improvement in exercise performance after exercise training, if any, needs to be demonstrated. The main regulator of HIFs activity is prolyl hydroxylase domain protein 2 (PHD2). Previous reports showed that Phd2 deficiency induced HIFs activation and subsequent hematopoietic and angiogenic adaptations (Minamishima et al., 2008, Takeda et al., 2007). Overall, we believe that the Phd2‐deficient mouse model enables us to elucidate the influence of the activated HIF pathway on the exercise training effect. We therefore hypothesised that the stabilization of HIFs and the subsequent hypoxic response induced by Phd2 deficiency contribute to the improvement in exercise training. To examine this hypothesis, we investigated the maximal exercise capacity of Phd2‐deficient mice before and after a 4‐week regimen of exercise training.MethodsBecause Phd2 knockout is embryonic lethal, estrogen receptor (ER) agonist‐induced Phd2 cKO mouse using Cre‐loxP system was used. Two weeks after tamoxifen, an estrogen‐agonist, maximal running test using a rodent treadmill was performed. Control mice (n = 16) and Phd2 cKO mice (n = 15) were then divided into two groups: trained and non‐trained. The mice in the training group underwent 4‐week treadmill training on a rodent treadmill. After 4‐week period, all mice of both groups were tested again for their maximal running capacity and were sacrificed for blood and tissue examination.ResultsBecause of Phd2 deficiency, HIF‐α proteins accumulated. In line with increases in plasma erythropoietin levels (P < 0.01), the Phd2 cKO mice exhibited marked increases in haematocrit values (P < 0.01) and haemoglobin concentrations (P < 0.01). Increased expression of plasma vascular endothelial growth factor (P < 0.05) induced angiogenesis in the skeletal muscle of the Phd2 cKO mice (P < 0.05). Importantly, trained Phd2 cKO mice showed a significantly higher gain in running time by a factor of approximately 1.53 compared with the trained control mice (P < 0.05, Figure). On the other hand, we did not find any difference between the non‐trained control and non‐trained Phd2 cKO mice (P = 0.21). Collectively, these data suggest that the combination of training and the activation of the HIF pathway is important for maximizing the effect of running training.ConclusionsWe concluded that the activation of the HIF pathway induced by PHD2 deficiency enhanced the effect of running training.Support or Funding InformationThis work was supported by a JSPS Grant‐in‐Aid for JSPS fellows (26‐3209).

Role of prolyl hydroxylase domain proteins in the regulation of insulin secretion.

Type 2 diabetes is associated with impaired nutrient‐regulated anaplerosis and insulin secretion in pancreatic β‐cells. One key anaplerotic substrate that may be involved in regulating insulin release is α‐ketoglutarate (αKG). Since prolyl hydroxylase domain proteins (PHDs) can metabolize cytosolic αKG, we sought to explore the role of this enzyme in the regulation of β‐cell function. The oxygen‐sensing PHDs regulate the stability of hypoxia‐inducible factor 1α (HIF1α) as well as other proline‐containing proteins by catalyzing the hydroxylation of proline residues. This reaction is dependent on sufficient levels of oxygen, iron, and αKG. In the present study, we utilized both pharmacological and genetic approaches to assess the impact of inhibiting PHD activity on β‐cell function. We demonstrate that ethyl‐3,4‐dihydroxybenzoate (EDHB), a PHD inhibitor, significantly blunted glucose‐stimulated insulin secretion (GSIS) from 832/13 clonal cells, rat, and human islets. EDHB reduced glucose utilization, ATP/ADP ratio, and key TCA cycle intermediates such as pyruvate, citrate, fumarate, and malate. siRNA‐mediated knockdown of PHD1 and PHD3 inhibited GSIS, whereas siRNA‐mediated knockdown of PHD2 had no effect on GSIS. Taken together, the current results demonstrate an important role for PHDs as mediators of islet insulin secretion.

The Oxygen Sensor PHD2 Controls Dendritic Spines and Synapses via Modification of Filamin A.

SummaryNeuronal function is highly sensitive to changes in oxygen levels, but how hypoxia affects dendritic spine formation and synaptogenesis is unknown. Here we report that hypoxia, chemical inhibition of the oxygen-sensing prolyl hydroxylase domain proteins (PHDs), and silencing of Phd2 induce immature filopodium-like dendritic protrusions, promote spine regression, reduce synaptic density, and decrease the frequency of spontaneous action potentials independently of HIF signaling. We identified the actin cross-linker filamin A (FLNA) as a target of PHD2 mediating these effects. In normoxia, PHD2 hydroxylates the proline residues P2309 and P2316 in FLNA, leading to von Hippel-Lindau (VHL)-mediated ubiquitination and proteasomal degradation. In hypoxia, PHD2 inactivation rapidly upregulates FLNA protein levels because of blockage of its proteasomal degradation. FLNA upregulation induces more immature spines, whereas Flna silencing rescues the immature spine phenotype induced by PHD2 inhibition.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Oxidative Dimerization of PHD2 is Responsible for its Inactivation and Contributes to Metabolic Reprogramming via HIF-1α Activation.

Prolyl hydroxylase domain protein 2 (PHD2) belongs to an evolutionarily conserved superfamily of 2-oxoglutarate and Fe(II)-dependent dioxygenases that mediates homeostatic responses to oxygen deprivation by mediating hypoxia-inducible factor-1α (HIF-1α) hydroxylation and degradation. Although oxidative stress contributes to the inactivation of PHD2, the precise molecular mechanism of PHD2 inactivation independent of the levels of co-factors is not understood. Here, we identified disulfide bond-mediated PHD2 homo-dimer formation in response to oxidative stress caused by oxidizing agents and oncogenic H-rasV12 signalling. Cysteine residues in the double-stranded β-helix fold that constitutes the catalytic site of PHD isoforms appeared responsible for the oxidative dimerization. Furthermore, we demonstrated that disulfide bond-mediated PHD2 dimerization is associated with the stabilization and activation of HIF-1α under oxidative stress. Oncogenic H-rasV12 signalling facilitates the accumulation of HIF-1α in the nucleus and promotes aerobic glycolysis and lactate production. Moreover, oncogenic H-rasV12 does not trigger aerobic glycolysis in antioxidant-treated or PHD2 knocked-down cells, suggesting the participation of the ROS-mediated PHD2 inactivation in the oncogenic H-rasV12-mediated metabolic reprogramming. We provide here a better understanding of the mechanism by which disulfide bond-mediated PHD2 dimerization and inactivation result in the activation of HIF-1α and aerobic glycolysis in response to oxidative stress.

Deletion or Inhibition of the Oxygen Sensor PHD1 Protects against Ischemic Stroke via Reprogramming of Neuronal Metabolism

The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network. Instead, PHD1(-/-) neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1(-/-) neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose away from glycolysis. As a result, PHD1(-/-) neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke.

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.

Activation of HIF-2a-EPO Axis in Kidney or Liver Is Sufficient to Drive Erythrocytosis in a Novel Inducible HIF-2a Transgenic Mouse Model

Erythropoietin (Epo) plays a critical role in the regulation of red cell mass (RCM) according to tissue oxygen levels. Hypoxia inducible factor-2a (HIF-2a) is known to be a master regulator for Epo expression. The liver is the major source of Epo in mice during embryonic and early postnatal development. In adult erythropoiesis, the kidneys become the major source of Epo and only a small portion of systemic Epo is produced by non-renal tissue, such as hepatocytes. The hepatocytes retain the capacity for Epo production in adult hematopoiesis and can increase Epo production in certain situations, such as severe hypoxia, post-nephrectomy, or certain liver cancer conditions. Erythrocytosis is defined as an absolute increase in RCM, and can be described as either primary or secondary, depending on whether the defect is intrinsic or extrinsic to the erythrocyte progenitors. The main causes of the secondary erythrocytosis include the defects in the oxygen-sensing pathway (VHL-PHD2-HIF2a signaling pathway) resident in the kidneys and liver, that transduces changes in oxygen delivery to changes in Epo gene transcription. Human mutations which cause erythrocytosis are usually genetic or somatic mutations; thus, both intrinsic and extrinsic effects to the erythrocyte progenitors could contribute to erythrocytosis. It is unclear whether kidney or liver specific upregulation of HIF-2a and downstream target Epo production will be sufficient to develop secondary erythrocytosis. It is also unclear whether any other cell type(s) in/out of the bone marrow (BM) also contribute to erythrocytosis phenotypes.To model the gain-of-function (GOF) mutations of HIF-2a and excess Epo production in erythrocytosis, we generated an inducible HIF-2a transgenic allele with human HIF-2a mutant (P531A and N847A, double point mutant; DPM) under control of tetracycline response elements and CMV mini promoter. Using kidney and liver specific Cre (Pax3-Cre and Alb-Cre), Rosa26-loxP-Stop-loxP (LSL)-rtTA-IRES-GFP driver, and administering doxycycline, we could induce a stable HIF-2a protein (DPM) in the kidneys and liver. HIF-2a induced cells can also be detected by their GFP expression. We used Pax3-Cre/LSL-rtTA-GFP and Alb-Cre/LSL-rtTA-GFP mice without DPM transgenic allele as our control. After doxycycline administration, both Pax3-Cre/LSL-rtTA-GFP/DPM (Pax3-Cre/DPM) and Alb-Cre/LSL-rtTA-GFP/DPM (Alb-Cre/DPM) mice quickly developed typical erythrocytosis, while control mice did not show any abnormal phenotypes. We subsequently measured the peripheral blood (PB) Epo level with ELISA. The expression levels of Epo in the PB of Pax3-Cre/DPM and Alb-Cre/DPM mice were significantly higher than that from the control mice (control, 107 pg/ml; Alb-Cre/DPM, 2976 pg/ml; Pax3-Cre/DPM, 1810 pg/ml). The values of hematocrit (Ht, %: control, 39.9±1.2; Alb-Cre/DPM, 51.3±10.0; Pax3-Cre/DPM, 48.6±1.0) and hemoglobin (Hb, g/dl: control, 15.2±0.67; Alb-Cre/DPM, 20.8±2.84; Pax3-Cre/DPM, 17.3±1.04) in Alb-Cre/DPM and Pax3-Cre/DPM mice were significantly elevated compared to those in the control mice (control, n=10; Alb-Cre/DPM, n=11; Pax3-Cre/DPM, n=3, p<0.05 for both). White blood cell counts and platelet counts were comparable among the groups. Interestingly, PB monocytes from Alb-Cre/DPM mice were significantly increased compared to those from Pax3-Cre/DPM mice and control mice. The impact of these elevated monocytes on erythrocytosis phenotype is unclear. Prolyl hydroxylase domain protein (PHD) is critical for the degrading process of the HIF protein. Previously, it has been reported that PHD knockout in hematopoietic cells with Vav1-Cre caused erythrocytosis. However, when we induced HIF-2a DPM in hematopoietic cells using Vav1-Cre/LSL-rtTA-GFP/DPM, these mice did not show obvious erythrocytosis phenotype despite DPM and GFP expression in all the blood lineages.In conclusion, we have generated a novel inducible HIF-2a transgenic allele and established both kidney- and liver-driven erythrocytosis mouse models. We found that kidney or liver specific Epo overexpression resulting from stable HIF-2a DPM mutant is sufficient to develop erythrocytosis, while HIF-2a DPM mutant induced in hematopoietic cells cannot develop erythrocytosis. Our study suggests that targeting VHL-PHD2-HIF2a-EPO pathway in kidney/liver will benefit patients with erythrocytosis. DisclosuresNo relevant conflicts of interest to declare.

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

BackgroundHypoxia 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.ResultsPHD2 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.ConclusionsTaken 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0079-5) contains supplementary material, which is available to authorized users.

Abstract 19126: Endothelial-specific Deletion of Prolyl Hydroxylase Domain Protein 2 (PHD2) Results in Severe Pulmonary Hypertension in Mice by Enhancing the HIF2alpha Signaling Pathway

Rationale: Sustained pulmonary vasoconstriction and excessive vascular remodeling are major causes of elevated pulmonary vascular resistance which leads to increased pulmonary arterial pressure in patients with pulmonary hypertension. Hypoxic-inducible factor (HIF) and its upstream regulators have been linked to the hypoxia response in vascular remodeling and the development of pulmonary hypertension. In this study, we aimed at defining whether increased HIF1α and/or HIF2α, due to endothelial cell specific deletion of prolyl hydroxylase domain protein 2 (PHD2) under normoxic condition are involved in or required for the initiation and progression of pulmonary hypertension. Methods: PHD2, HIF1α and HIF2α conditional knockout mice were created. Right ventricle systolic pressures (RVSP), right ventricular hypertrophy by RV/(LV+S) ratios, and small pulmonary artery smooth muscle layer thickness were measured. Pulmonary arterial smooth muscle cells (PASMCs) and pulmonary arterial endothelial cells (PAECs) were isolated from wild type (WT) or knockout (KO) mice, followed with cell-based assays. Results: We report here that mice with targeted deletion of PHD2 developed severe pulmonary hypertension under normoxic condition. Conditional and inducible deletion of HIF2α in endothelial cells, but not smooth muscle cells, dramatically protected mice from hypoxia-induced pulmonary hypertension. HIF2α KO mice had significantly lower RVSP, RV/(LV+S) ratios, and displayed less pulmonary vascular remodeling when exposed to hypoxia compared to their WT mice. Conclusion: This work shows that the endothelium is responsible for the development of pulmonary hypertension and it demonstrates a crucial role of PHD2/HIF signaling for hypoxic response in pulmonary hypertension. These findings unveil temporally and spatially distinct functions for HIFs in the development of pulmonary hypertension.

Abstract 9781: Deletion of Prolyl Hydroxylase Domain Protein 2 in Myeloid Lineage suppressed Experimental Abdominal Aortic Aneurysm via NF-κB Inactivation

Background: Macrophage migration to the vessels is important for vascular inflammation and induces vascular degenerative diseases. Macrophages secrete matrix metalloprotainases (MMPs), which activate many cytokines and digest extracellular matrix of aorta. MMPs play an important role in the progression of aortic aneurysm. We previously reported that non-specific prolyl hydroxylase domain protein (PHD) inhibitor, Cobalt chloride (CoCl2), suppressed MMP-2 and -9 expression and attenuated experimental abdominal aortic aneurysm (AAA) formation in mice. In this report we investigated the myeloid specific PHD2 knockout effect on MMPs expression and aneurysm formation. Methods: Myeloid specific PHD2 conditional knockout (MyPHD2KO) mice were generated. Experimental AAA was induced by periaortic application of Calcium chloride (CaCl2) for 6 weeks. In vitro Lipopolysaccharide (LPS, 100ng/ml) was used to induce MMP-2 and MMP-9 expression with peritoneal macrophages. MMPs mRNA, protein expression and protein activity were analyzed by qRT-PCR, Western blot analysis and Zymography, respectively. ELISA-based NF-κB p65 Transcription Factor Assay was used to examine NF-κB p65 binding activity with consensus DNA binding site. Results: CaCl2-induced AAA was suppressed with MyPHD2KO mice (max diameter of aneurysm: 1.03mm±0.14mm in MyPHD2KO group, 1.63mm±0.34mm in Control AAA group, p&lt;0.01). In peritoneal macrophages CoCl2 reduced LPS-induced MMP-2 and MMP-9 mRNA and protein expression. PHD2 deletion in peritoneal macrophages from MyPHD2KO mice also suppressed LPS-induced MMP-2 and MMP-9 mRNA, protein expression and activity. PHD2 deletion suppressed LPS-induced NF-κB p65 phosphorylation via IκBα stabilization by Western blot analysis. NF-κB p65 binding activity was suppressed in MyPHD2KO macrophages (p&lt;0.01 vs control). Conclusion: Deletion of PHD2 in myeloid lineage attenuated MMPs expression by NF-κB inactivation and suppressed AAA formation. PHD2 in macrophage may be a novel target for cardiovascular disease treatment.