Prolyl Hydroxylase Domain-containing Protein Research Articles

Gingerol prevents prion protein-mediated neuronal toxicity by regulating HIF prolyl hydroxylase 2 and prion protein.

Prion diseases are a family of progressive neurodegenerative disorders, which are fatal in the majority of cases and affect both humans and domestic animals. Prion protein (PrP) (106–126) retains the neurotoxic properties of the entire pathological PrPsc and it is generally used as a reasonable model to study the mechanisms responsible for prion diseases. In our previous studies, we demonstrated that hypoxia-inducible factor (HIF)-1α is involved in the gingerol-mediated protection of neuronal cells. HIF mediates cellular adaptations to low oxygen. Prolyl hydroxylase domain-containing protein 2 (PHD2) is an oxygen sensor that hydroxylates the HIF-α-subunit, promoting its proteasomal degradation under normoxic conditions. Thus, in the present study we wished to determine whether gingerol inhibits the catalytic activity of PHD2 and prevents HIF-1α protein proteasomal degradation, thereby preventing the occurrence of PrP (106–126)-induced neuronal apoptosis. We used the pharmacological inhibition of PHD2 by dimethyloxalylglycine (DMOG) or deferoxamine (DFO) and the genetic inhibition of HIF-1α by HIF-1α small interfering RNA (siRNA) to block the effects of gingerol against PrP (106–126)-induced neurotoxicity. Our results demonstrated that gingerol prevented PrP (106–126)-induced neuronal apoptosis by upregulating HIF-1α and inhibiting the catalytic activity of PHD2 under normoxic conditions. Moreover, the protective effects of gingerol against PrP (106–126)-induced neuronal apoptosis were associated with the upregulation of the expression of cellular prion protein (PrPc). In conclusion, our results indicate that gingerol has therapeutic potential for use in the treatment or prevention of prion diseases, and its inhibitory effects on the catalytic activity of PHD2 may be of clinical benefit.

Conditional disruption of the prolyl hydroxylase domain-containing protein 2 (Phd2) gene defines its key role in skeletal development.

We have previously shown that the increase in osterix (Osx) expression during osteoblast maturation is dependent on the activity of the prolyl hydroxylase domain-containing protein 2 (Phd2), a key regulator of protein levels of the hypoxia-inducible factor family proteins in many tissues. In this study, we generated conditional Phd2 knockout mice (cKO) in osteoblast lineage cells by crossing floxed Phd2 mice with a Col1α2-iCre line to investigate the function of Phd2 in vivo. The cKO mice developed short stature and premature death at 12 to 14 weeks of age. Bone mineral content, bone area, and bone mineral density were decreased in femurs and tibias, but not vertebrae of the cKO mice compared to WT mice. The total volume (TV), bone volume (BV), and bone volume fraction (BV/TV) in the femoral trabecular bones of cKO mice were significantly decreased. Cross-sectional area of the femoral mid-diaphysis was also reduced in the cKO mice. The reduced bone size and trabecular bone volume in the cKO mice were a result of impaired bone formation but not bone resorption as revealed by dynamic histomorphometric analyses. Bone marrow stromal cells derived from cKO mice formed fewer and smaller nodules when cultured with mineralization medium. Quantitative RT-PCR and immunohistochemistry detected reduced expression of Osx, osteocalcin, and bone sialoprotein in cKO bone cells. These data indicate that Phd2 plays an important role in regulating bone formation in part by modulating expression of Osx and bone formation marker genes.

Neovascularization induced by hypoxia inducible transcription factor is associated with the improvement of cardiac dysfunction in experimental autoimmune myocarditis

Objective: Mechanisms of cardiac dysfunction in myocarditis have not been fully elucidated. Though it remains controversial whether angiogenesis is beneficial or harmful in inflammatory disease, significant vascular destruction might possibly impair cardiac function in myocarditis. The prolyl hydroxylase domain-containing protein (PHD) inhibitor is a potential drug for promoting angiogenesis as it stabilizes hypoxia inducible transcription factor (HIF). The authors examine whether the PHD inhibitor TM6008 could affect cardiac function by promoting angiogenesis in experimental autoimmune myocarditis (EAM).Methods: EAM was induced on BALB/c mice by immunizing them with a synthesized α myosin heavy-chain peptide. Every day, 200 mg/kg of TM6008 or vehicle was administered orally.Results: TM6008 improved left ventricular ejection fraction significantly on the 21st day of EAM. Though TM6008 did not affect the severity of myocardial cell infiltration, it tended to reduce cardiac fibrosis. Immunohistochemistry showed that CD31-positive blood vessels were preserved in the TM6008 group compared to the control group. Immunoblotting revealed that TM6008 increased the expression of HIF-1α, HIF-2α and vascular endothelial growth factor in myocarditis.Conclusion: Inhibition of PHD could ameliorate cardiac dysfunction in EAM, partially through promoting neovascularization. Relief of tissue hypoxia via neovascularization could improve cardiac function in myocarditis.

Inhibition of prolyl hydroxylase domain-containing protein on hypertension/renal injury induced by high salt diet and nitric oxide withdrawal

Nitric oxide just as prolyl hydroxylase domain-containing protein (PHD) is a regulator of hypoxia inducible factor-1 α (HIF-1α), a transcription factor complex that controls the expression of most genes involved in hypoxia and cardiovascular diseases. In the absence of nitric oxide, it is not clear how HIF-1α and PHD are regulated and to what extent they contribute to the ensuing disorder. Using the nitric oxide withdrawal/high salt diet model of hypertensive renal injury, this study tested the hypothesis that removal of the inhibition by nitric oxide on PHD predisposes to increased PHD but reduced HIF-1α expression, hypertension and renal injury. In animals treated with N-nitro-L-arginine (L-NNA; 250 mg/l in drinking water for 14 days) and high salt diet (4% NaCl), there was hypertension (41±5%, P<0.05), proteinuria (three-fold, P<0.05), kidney (22±3%, P<0.05) and heart enlargement (24±3%, P<0.05), as well as increased renal osteopontin (21±3%, P<0.05) and collagen IV (24±4%, P<0.05) expression. Accompanying these effects were increased expression of PHD1 (24±4%, P<0.05) and PHD2 (36±4%, P<0.05) but reduced HIF-1α (35±6%, P < 0.05) expression. Dimethyloxallyl glycine (5mg/kg), a PHD inhibitor, paradoxically exacerbated hypertension (46±7%, P<0.05), proteinuria (two-fold, P <0.05), and increased osteopontin (15±2%, P<0.05) and HIF-1α (31±5%, P<0.05) expression with no change in PHD1/2 expression or kidney and heart enlargement. These data suggest that the protective effect of physiological levels of nitric oxide may be by virtue of inhibition of PHD or increased HIF-1α expression, hence, the pathological changes produced following its withdrawal was accompanied by increased PHD or decreased HIF-1α expression. Exacerbation of hypertension and renal injury following PHD inhibition suggests a deleterious effect in the chronic setting and challenges the dogma that inhibition of PHD is useful in cardiovascular diseases.

Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment

To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (ΔO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (ΔΦm) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, ΔΦm was abolished in cells >500 μm from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined ΔO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500-2,000 μm from the source. In these cells, tissue ΔO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of ΔO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain ΔΦm, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue ΔO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining ΔΦm in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain ΔΦm in an otherwise anoxic microenvironment by decreasing tissue ΔO2 while activating oxygen-independent electron transport in mitochondria.

Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes

Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain-containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes. Although Hif regulates many metabolic pathways, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.

Novel complex crystal structure of prolyl hydroxylase domain-containing protein 2 (PHD2): 2,8-Diazaspiro[4.5]decan-1-ones as potent, orally bioavailable PHD2 inhibitors

We have discovered a novel complex crystal structure of the PHD2 enzyme with its inhibitor, the 2,8-diazaspiro[4.5]decan-1-one analogue 4b. The widely reported salt bridge between Arg383 of the enzyme and its inhibitors in all complex structures published thus far was not observed in our case. In our complex structure compound 4b forms several novel interactions with the enzyme, which include a hydrogen bond with Arg322, a π-cation interaction with Arg322, a π–π stacking with Trp389, and a π–π stacking with His313. Guided by the structural information, SAR studies were performed on the 2,8-diazaspiro[4.5]decan-1-one series leading to the discovery of compound 9p with high potency and good oral pharmacokinetic profile in mice.

Hypoxia-inducible factor prolyl-hydroxylase-2 mediates transforming growth factor beta 1-induced epithelial–mesenchymal transition in renal tubular cells

Transforming growth factor beta 1 (TGF-β1)-induced epithelial–mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGF-β1-induced signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGF-β1-induced EMT using cultured renal tubular cells. Our results showed that TGF-β1 stimulated EMT in renal tubular cells as indicated by the significant decrease in epithelial marker P-cadherin, and the increase in mesenchymal markers α-smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP-1). Meanwhile, we found that TGF-β1 time-dependently increased HIF-1α and that HIF-1α siRNA significantly inhibited TGF-β1-induced EMT, suggesting that HIF-1α mediated TGF-β1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1:PHD2=0.21:1.0). Importantly, PHD2 mRNA and protein, but not PHD1, were decreased by TGF-β1. Furthermore, over-expression of PHD2 transgene almost fully prevented TGF-β1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGF-β1-induced EMT. Finally, Smad2/3 inhibitor SB431542 prevented TGF-β1-induced PHD2 decrease, suggesting that Smad2/3 may mediate TGF-β1-induced EMT through PHD2/HIF-1α pathway. It is concluded that TGF-β1 decreased PHD2 expression via an Smad-dependent signaling pathway, thereby leading to HIF-1α accumulation and then EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGF-β1, and may be a new therapeutic target in chronic kidney diseases.

Overexpression of HIF Prolyl‐Hydoxylase‐2 transgene in the renal medulla induced a salt sensitive hypertension

Renal medullary hypoxia-inducible factor (HIF)-1α and its target genes, such as haem oxygenase and nitric oxide synthase, have been indicated to play an important role in the regulation of sodium excretion and blood pressure. HIF prolyl hydroxylase domain-containing proteins (PHDs) are major enzymes to promote the degradation of HIF-1α. We recently reported that high salt intake suppressed the renal medullary PHD2 expression and thereby activated HIF-1α-mediated gene regulation in the renal medulla in response to high salt. To further define the functional role of renal medullary PHD2 in the regulation of renal adaptation to high salt intake and the longer term control of blood pressure, we transfected PHD2 expression plasmids into the renal medulla in uninephrectomized rats and determined its effects on pressure natriuresis, sodium excretion after salt overloading and the long-term control of arterial pressure after high salt challenge. It was shown that overexpression of PHD2 transgene increased PHD2 levels and decreased HIF-1α levels in the renal medulla, which blunted pressure natriuresis, attenuated sodium excretion, promoted sodium retention and produced salt sensitive hypertension after high salt challenge compared with rats treated with control plasmids. There was no blood pressure change in PHD2-treated rats that were maintained in low salt diet. These results suggested that renal medullary PHD2 is an important regulator in renal adaptation to high salt intake and a deficiency in PHD2-mediated molecular adaptation in response to high salt intake in the renal medulla may represent a pathogenic mechanism producing salt sensitive hypertension.

Abstract 396: Inhibition of Prolyl Hydroxylase Domain-containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor

Background: Prolyl hydroxylase domain-containing protein (PHD) mediates hydroxylation of hypoxia-inducible factor (HIF)-1α and thereby induces proteasomal degradation of HIF-1α. Inhibition of PHD by hypoxia or hypoxia mimetics such as cobalt chloride (CoCl2) stabilizes HIF-1 and increases the expression of target genes such as vascular endothelial growth factor (VEGF). Although hypoxia activates the systemic renin angiotensin system (RAS), the role of PHD in regulating RAS remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin (Ang) II type 1 receptor (AT1R) and its signaling. Methods and Results: Hypoxia (1% O2), CoCl2 (100-300 μmol/L), and dimethyloxalylglycine (0.25-1.0 mmol/L), all known to inhibit PHD, reduced AT1R expression by 37.7±7.6, 39.6±8.4-69.7±9.9, and 13.4±6.1-25.2±7.0%, respectively (p&lt;0.01) in cultured vascular smooth muscle cell. The same stimuli increased the expression of nuclear HIF-1α and VEGF (p&lt;0.05), suggesting that PHD activity is inhibited. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT1R expression by 55.3±6.0% (p&lt;0.01). CoCl2 decreased AT1R mRNA through transcriptional and posttranscriptional mechanisms (p&lt;0.01 and &lt;0.05, respectively). CoCl2 and PHD2 knockdown diminished Ang II-induced ERK phosphorylation (P&lt;0.01). Over-expression of the constitutively active HIF-1α did not impact the AT1R gene promoter activity. Oral administration of CoCl2 (14 mg/kg/day) to C57BL/6J mice receiving Ang II infusion (490 ng/kg/min) for 4 weeks significantly reduced the expression of AT1R in the aorta by 60.9±11.3% (p&lt;0.05) and attenuated coronary perivascular fibrosis by 85% (p&lt;0.01) without affecting blood pressure. However, CoCl2 did not affect Ang II-induced renal interstitial fibrosis. Conclusion: PHD inhibition downregulates AT1R expression independently of HIF-1α, reduces the cellular response to Ang II, and attenuates profibrotic effect of Ang II on the coronary arteries. PHD inhibition may be beneficial for the treatment of cardiovascular diseases, in which activation of RAS plays a critical role.

Abstract 132: Hypoxia-inducible Factor Prolyl-hydroxylase-2 Mediates Transforming Growth Factor Beta 1-induced Epithelial-mesenchymal Transition In Renal Tubular Cells

Transforming growth factor beta 1 (TGFβ1)-induced epithelial-mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGFβ1 signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGFβ1-induced EMT in renal tubular cells. Our results showed that TGFβ1 treatment for 48 h stimulated EMT in cultured renal tubular cells as indicated by the decrease in epithelial marker P-cadherin from 1.0 ± 0.02 to 0.40 ± 0.05 ( P &lt; 0.05), and the increase in mesenchymal markers α-smooth muscle actin (2.14 ± 0.32 fold, P &lt; 0.05) and fibroblast-specific protein (2.0 ± 0.17 fold, P &lt; 0.05) as shown in Western blot assay. Meanwhile, TGFβ1 time-dependently increased HIF-1α, which reached its maximum value (2.36 ± 0.2 fold, P &lt; 0.05) at 24 h, and that HIF-1α siRNA significantly inhibited TGFβ1-induced EMT, suggesting that HIF-1α mediated TGFβ1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1 : PHD2 = 0.21:1.0). Importantly, TGFβ1 time-dependently decreased PHD2 mRNA and protein level, which reached their maximum value from 1.0 ± 0.15 to 0.45 ± 0.08 ( P &lt; 0.05) for mRNA at 16 h and from 1.0 ± 0.08 to 0.26 ± 0.08 ( P &lt; 0.05) for protein at 24 h, respectively. In contrast, TGFβ1 had no effect on PHD1 mRNA and protein levels. Furthermore, over-expression of PHD2 transgene almost fully prevented TGFβ1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGFβ1-induced EMT. Finally, Smad2 inhibitor SB431542 prevented TGFβ1-induced PHD2 decrease, suggesting that Smad2 may mediate TGFβ1-induced EMT through PHD2/HIF-1α. It is concluded that TGFβ1 decreased PHD2 expression via a Smad2-dependent signaling pathway, thereby leading to HIF-1α accumulation and EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGFβ1 and that manipulating PHD2/HIF-1α pathway may be used as a therapeutic strategy in chronic kidney diseases. (support: NIH grant HL89563 and HL106042)

GNAS1 and PHD2 Short-interfering RNA Support Bone Regeneration in Vitro and in an in Vivo Sheep Model

BackgroundOur ability to guide cells in biomaterials for in vivo bone repair is limited and requires novel strategies. Short-interfering RNA (siRNA) allows the regulation of multiple cellular pathways. Core binding factor alpha 1 (Cbfa1) and hypoxia-inducible factor 1 (HIF-1) pathways can be modulated to direct bone formation via siRNA against guanine nucleotide-binding protein alpha-stimulating activity polypeptide 1 (siGNAS1) and prolyl hydroxylase domain-containing protein 2 (siPHD2), respectively.Questions/PurposesWe determined whether the administration of siGNAS1 and siPHD2 in mesenchymal stem cells (MSCs) promotes osteogenic phenotype, the dose-dependent effects of siGNAS1 on MSC differentiation to osteogenic phenotype, and whether the two siRNAs promote bone formation in vivo.MethodssiRNAs were administered to MSCs at Day 0, and protein expression of bone-specific markers was assessed at Days 1, 2, and 4 (n = 3/group/time point). In an in vivo model using seven sheep, chambers containing silk fibroin-chitosan (SFCS) scaffolds with siRNA were implanted over the periosteum and harvested at Days 7, 21, 36, and 70 (n = 4/group/time point, except at Day 70 [n = 2]) to assess bone formation.ResultssiGNAS1 promoted collagen I and osteopontin expression, whereas siPHD2 had no effect in vitro. Dose-dependent effects of siGNAS1 on ALP expression were maximal at Day 1 for 10 μg/mL and Day 4 for 100 μg/mL. In vivo, by Day 70, mean bone volume increased compared to Day 7 for siGNAS1-SFCS (47.8 versus 1.8 mg/mL) and siPHD2-SFCS (61.3 versus 1.5 mg/mL).ConclusionsBoth siPHD2 and siGNAS1 support bone regeneration in vivo, whereas only siGNAS1 regulates bone phenotype in MSCs in vitro.Clinical RelevanceWhile the use of autologous tissue is limited for reconstructing critical-sized defects, the development of biomaterial-based approaches to promote bone formation may abrogate some of those limitations.

Roles for Peroxisome Proliferator-activated Receptor γ (PPARγ) and PPARγ Coactivators 1α and 1β in Regulating Response of White and Brown Adipocytes to Hypoxia

Obese white adipose tissue is hypoxic but is incapable of inducing compensatory angiogenesis. Brown adipose tissue is highly vascularized, facilitating delivery of nutrients to brown adipocytes for heat production. In this study, we investigated the mechanisms by which white and brown adipocytes respond to hypoxia. Brown adipocytes produced lower amounts of hypoxia-inducible factor 1α (HIF-1α) than white adipocytes in response to low O(2) but induced higher levels of hypoxia-associated genes. The response of white adipocytes to hypoxia required HIF-1α, but its presence alone was incapable of inducing target gene expression under normoxic conditions. In addition to the HIF-1α targets, hypoxia also induced many inflammatory genes. Exposure of white adipocytes to a peroxisome proliferator-activated receptor γ (PPARγ) ligand (troglitazone) attenuated induction of these genes but enhanced expression of the HIF-1α targets. Knockdown of PPARγ in mature white adipocytes prevented the usual robust induction of HIF-1α targets in response to hypoxia. Similarly, knockdown of PPARγ coactivator (PGC) 1β in PGC-1α-deficient brown adipocytes eliminated their response to hypoxia. These data demonstrate that the response of white adipocytes requires HIF-1α but also depends on PPARγ in white cells and the PPARγ cofactors PGC-1α and PGC-1β in brown cells.

Inhibition of Prolyl Hydroxylase Domain-Containing Protein

See related article, pp 386–393 Intermittent hypoxia, such as that observed in patients with sleep apnea, is frequently associated with hypertension and other cardiovascular diseases. Hypoxia also participates in pathophysiological remodeling of cardiac myocytes, as well as remodeling of the vasculature. Overreactivity of the renin-angiotensin system (RAS) has long been implicated in the etiology of hypertension associated with hypoxia,1 and a recent human study confirms the importance of the angiotensin II type I (AT1) receptor activation in this process.2 However, current knowledge regarding the molecular mechanism by which RAS is activated and mediates cardiovascular diseases under hypoxia remains quite limited. The cellular response to hypoxia involves the induction of the hypoxia-inducible factor 1α (HIF1α), considered to be the major transcription factor involved in gene regulation by hypoxia. In normoxia, HIF1α is hydroxylated by the cellular oxygen “sensor” prolyl-hydroxylase domain (PHD) proteins (PHD1, PHD2, and PHD3) and degraded by proteasomes. The PHDs are inhibited in hypoxia, resulting in stabilization and activation of transcription by the nonhydroxylated and stabilized form of HIF1α. PHDs are an evolutionarily conserved subfamily of dioxygenases that use oxygen and 2-oxoglutarate as cosubstrates …

Inhibition of Prolyl Hydroxylase Domain-Containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor

Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT(1)R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT(1)R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT(1)R expression. Cobalt chloride diminished angiotensin II-induced extracellular signal-regulated kinase phosphorylation. Cobalt chloride decreased AT(1)R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT(1)R downregulation.

Hypoxia-inducible factor-1α contributes to the profibrotic action of angiotensin II in renal medullary interstitial cells

To examine whether hypoxia-inducible factor (HIF)-1α mediates the profibrotic effects of angiotensin II, we treated cultured renal medullary interstitial cells with angiotensin II and found that it increased HIF-1α levels. This was accompanied by a significant upregulation of collagen I/III, the tissue inhibitor of metalloproteinase-1, elevation of the proliferation marker proliferating cell nuclear antigen, and a transdifferentiation marker vimentin. All these effects of angiotensin II were completely blocked by siRNA for HIF-1α but not HIF-2α. Overexpression of a prolyl-hydroxylase domain-containing protein 2 (PHD2) transgene, the predominant renal HIF prolyl-hydroxylase, attenuated the effects of angiotensin II and its gene silencing enhanced the effects of angiotensin II. Removal of hydrogen peroxide eliminated angiotensin II-induced profibrotic effects. A 2-week infusion of rats with angiotensin II increased the expression of HIF-1α and α-smooth muscle actin, another marker of transdifferentiation, in renal medullary interstitial cells in vivo. Thus, our study suggests that HIF-1α mediates angiotensin II-induced profibrotic effects through activation of cell transdifferentiation. We propose that redox regulation of prolyl-PHD2 plays a critical role in angiotensin II-induced activation of HIF-1α in renal cells.

Loss of Hypoxia-Inducible Factor Prolyl Hydroxylase Activity in Cardiomyocytes Phenocopies Ischemic Cardiomyopathy

Ischemic cardiomyopathy is the major cause of heart failure and a significant cause of morbidity and mortality. The degree of left ventricular dysfunction in this setting is often out of proportion to the amount of overtly infarcted tissue, and how decreased delivery of oxygen and nutrients leads to impaired contractility remains incompletely understood. The Prolyl Hydroxylase Domain-Containing Protein (PHD) prolyl hydroxylases are oxygen-sensitive enzymes that transduce changes in oxygen availability into changes in the stability of the hypoxia-inducible factor transcription factor, a master regulator of genes that promote survival in a low-oxygen environment. We found that cardiac-specific PHD inactivation causes ultrastructural, histological, and functional changes reminiscent of ischemic cardiomyopathy over time. Moreover, long-term expression of a stabilized hypoxia-inducible factor alpha variant in cardiomyocytes also led to dilated cardiomyopathy. Sustained loss of PHD activity and subsequent hypoxia-inducible factor activation, as would occur in the setting of chronic ischemia, are sufficient to account for many of the changes in the hearts of individuals with chronic coronary artery disease.

Prolyl hydroxylase domain-containing protein inhibitors as stabilizers of hypoxia-inducible factor: small molecule-based therapeutics for anemia

Importance of the field: Anemia caused by chronic kidney disease and other chronic diseases or conditions can be managed by the treatment of biologic-based erythropoiesis stimulating agents (ESAs). Although these ESAs are successful in treating these anemic conditions, a small molecule-based anti-anemia medicine can potentially revolutionize the treatment of anemia by bringing convenience to patients and being cost effective. Prolyl hydroxylase domain-containing protein (PHD) inhibitors may provide an opportunity for the development of small molecule anti-anemia medicines.Areas covered in this review: This review covers efforts to target PHD enzymes for stabilization of hypoxia-inducible factor (HIF)-α subunits under normal oxygen levels as an attractive strategy to upregulate the expression of erythropoietin and genes involved in iron metabolism for the treatment of anemia.What the reader will gain: The reader will gain a brief summary of recent advances in HIF and PHD biology and a review of patents/patent applications on the subject of PHD inhibitors as HIF stabilizers for the treatment of anemia.Take home message: Several classes of PHD enzyme inhibitors have been disclosed and several are currently in clinical trials for the development of small molecule-based therapeutics for the treatment of anemia.

Inhibition of Hypoxia-inducible Factor-targeting Prolyl Hydroxylase Domain-containing Protein 2 (PHD2) Enhances Matrix Synthesis by Human Chondrocytes

Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-alpha isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2alpha stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interference-mediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2alpha levels and therefore enhanced SOX9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.

Hearts of Hypoxia-inducible Factor Prolyl 4-Hydroxylase-2 Hypomorphic Mice Show Protection against Acute Ischemia-Reperfusion Injury

Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2(gt/gt)) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34-47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hif-p4h-2(gt/gt) hearts. When isolated Hif-p4h-2(gt/gt) hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2(gt/gt) hearts, indicating enhanced glycolysis and an improved cellular energy state. Our data suggest that chronic stabilization of Hif-1alpha and Hif-2alpha by genetic knockdown of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function, and blood pressure.