Matsuura et al Role of PHD2 in Adipocytes 2079Several cell culture studies have revealed that hypoxia and HIF convert cell metabolism that is dependent on aerobic glucose oxidation and fatty acid synthesis into that which is dependent on anaerobic glycolysis. HIF not only upregulates a series of glycolytic enzymes 15,16 but also actively inhibits oxidative phosphorylation in mitochondria by inducing pyruvate dehydrogenase kinase 1 (PDK1).17,18 PDK1 inhibits pyruvate dehydrogenase activity and consequently reduces the conversion of pyruvate to acetyl CoA, an essential substrate for oxidative phosphorylation. 17,18 In addition, HIF inhibits adipogenesis by inducing DEC1/Stra13. 13 These HIF-induced metabolic alterations such as increased glucose consumption and less fatty acid synthesis might be beneficial for nutrient excess in obese or diabetic subjects. Although HIF could be a potential therapeutic target, direct manipulation of HIF is often difficult in vivo. In contrast, PHD is an ideal target to manipulate HIF levels, and several chemical inhibitors of PHD have been developed. 19 However, the role of adipocyte PHD in the development of obesity-induced glucose intolerance has not been determined. In the present study, we generated mice lacking PHD2, also known as Egl 9 homolog1 (EglN1) in adipocytes, because PHD2 is the most crucial isoform to regulate HIF level in vitro 20 and in vivo 21 among 3 PHD isoforms (PHD1, PHD2, and PHD3). We found that PHD2 deletion in adipocyte attenuates weight gain and alleviates glucose intolerance induced by a high-fat diet (HFD). MethodsAdditional details of the experimental procedures are included in the online-only Data Supplement.All animal procedures were approved by the Animal Care and Use Committee of Kyushu University and conducted in accordance with the institutional guidelines. Previously generated Phd2-floxed mice f/f mice. Phd2 f/f mice served as controls. These mice were fed an HFD containing 60% kcal fat (High Fat Diet 32, Clea Japan, Inc) from 12 to 18 weeks of age. Mice 12 and 18 weeks of age were analyzed. Preparation of cell lysate and total RNA, Western blot analysis, quantitative reverse transcription-polymerase chain reaction, luciferase assay, and histological/immunohistochemical analysis were performed using conventional methods. The primer sequences for quantitative reverse transcription-polymerase chain reaction are shown in Table I Figure 1A and Figure IA in the online-only Data Supplement). Expression of PHD2 in heart and bone marrowderived macrophages was slightly reduced. We did not find any apparent abnormalities in the appearance in Phd2 Figure 2B and 2D). However, the extent of HFD-induced adipocyte hypertrophy was significantly reduced in Phd2 f/f /aP2-Cre mice compared with control mice (Figure 2C and 2D). A detailed analysis of the size distribution of the adipocytes revealed that WAT from controls contained a greater number of larger adipocytes (>10 000 μm 2 ) than that Enhanced Angiogenesis in WAT From HFD-Fed Phd2f/f /aP2-Cre Mice B...

Objective-Prolyl hydroxylase domain-containing proteins (PHDs) play pivotal roles in oxygen-sensing system through the regulation of ␣-subunit of hypoxia-inducible factor (HIF), a key transcription factor governing a large set of gene expression to adapt hypoxia. Although tissue hypoxia plays an essential role in maintaining inflammation, the role of PHDs in the inflammatory responses has not been clearly determined. Here, we investigated the role of PHDs in lipopolysaccharide (LPS)-induced tumor necrosis factor ␣ (TNF-␣) induction in macrophages. Methods and Results-Northern blot analysis and ELISA revealed that LPS-induced TNF-␣ upregulation was strongly suppressed by PHD inhibitors, dimethyloxallyl glycine (DMOG), and TM6008 in RAW264.7 macrophages. DMOG suppressed LPS-induced TNF-␣ upregulation in HIF-1␣-depleted cells and HIF-1␣ overexpression failed to suppress the induction of TNF-␣. DMOG rather suppressed LPS-induced NF-B transcriptional activity. Downregulation of Phd1 or Phd2 mRNA by RNA interference partially attenuated LPS-induced TNF-␣ induction. DMOG also inhibited LPS-induced TNF-␣ production in peritoneal macrophages as well as human macrophages. Conclusions-PHD inhibition by DMOG or RNA interference inhibited LPS-induced TNF-␣ upregulation in macrophages possibly through NF-B inhibition, which is independent of HIF-1␣ accumulation. This study suggests that PHDs are positive regulators of LPS-induced inflammatory process, and therefore inhibition of PHD may be a novel strategy for the treatment of inflammatory diseases. Key Words: tumor necrosis factor -alpha Ⅲ prolyl hydroxylase domain-containing protein Ⅲ hypoxia-inducible factor Ⅲ inflammation Ⅲ hypoxia I nflammation is a fundamental process for the protection of our body against outside pathogen. Tissues with inflammation are characterized by several features including the accumulation of inflammatory cells such as macrophages, lymphocytes, and neutrophils, limited blood supply attributable to impaired local microcirculation, and abnormal angiogenesis. 1 Inflammatory cells are metabolically active and consume a large amount of oxygen and nutrient. These cells are, therefore, eventually exposed to hypoxic and nutrient-deprived condition. 2 Thus, the inflammatory cells need to adapt these hypoxic conditions to perpetuate inflammatory reaction. 3 The reduced oxygen concentration is directly sensed by an innate oxygen-sensing system. 4 -6 The hypoxia-inducible factor (HIF) is a key transcription factor that mediates cellular adaptive responses to hypoxia. 7 HIF is a heterodimer consisting of an oxygen-labile ␣-subunit and a stable ␤-subunit. The stability of the ␣-subunit of HIF-1 and HIF-2 (HIF-1␣ and HIF-2␣) is regulated through the hydroxylation at the 4-position of specific proline residues in HIF-1␣ and HIF-2␣ by prolyl hydroxylase domain-containing proteins (PHDs). 8,9 Because PHD activity depends on the availability of molecular oxygen, PHDs are able to serve as a sensor for oxygen concentration. Under normal oxygen concentration, HIF-␣ i...

OBJECTIVE: LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and Results: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)—a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)—a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis. Conclusions: Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.

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BackgroundHypertension induces cardiovascular hypertrophy and fibrosis. Infiltrated macrophages are critically involved in this process. We recently reported that inhibition of prolyl hydroxylase domain protein 2 (PHD2), which hydroxylates the proline residues of hypoxia‐inducible factor‐α (HIF‐α) and thereby induces HIF‐α degradation, suppressed inflammatory responses in macrophages. We examined whether myeloid‐specific Phd2 deletion affects hypertension‐induced cardiovascular remodeling.Methods and ResultsMyeloid‐specific PHD2‐deficient mice (MyPHD2KO) were generated by crossing Phd2‐floxed mice with LysM‐Cre transgenic mice, resulting in the accumulation of HIF‐1α and HIF‐2α in macrophage. Eight‐ to ten‐week‐old mice were given NG‐nitro‐L‐arginine methyl ester (L‐NAME), a nitric oxide synthase inhibitor, and Angiotensin II (Ang II) infusion. L‐NAME/Ang II comparably increased systolic blood pressure in control and MyPHD2KO mice. However, MyPHD2KO mice showed less aortic medial and adventitial thickening, and macrophage infiltration. Cardiac interstitial fibrosis and myocyte hypertrophy were also significantly ameliorated in MyPHD2KO mice. Transforming growth factor‐β and collagen expression were decreased in the aorta and heart from MyPHD2KO mice. Echocardiographic analysis showed that left ventricular hypertrophy and reduced ejection fraction induced by L‐NAME/Ang II treatment in control mice were not observed in MyPHD2KO mice. Administration of digoxin that inhibits HIF‐α synthesis to L‐NAME/Ang II‐treated MyPHD2KO mice reversed these beneficial features.ConclusionsPhd2 deletion in myeloid lineage attenuates hypertensive cardiovascular hypertrophy and fibrosis, which may be mediated by decreased inflammation‐ and fibrosis‐associated gene expression in macrophages. PHD2 in myeloid lineage plays a critical role in hypertensive cardiovascular remodeling.

BackgroundClinical trials have shown that treatment of patients with type 2 diabetes with pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, reduces cardiovascular events. However, the effect of PPARγ agonists on endoplasmic reticulum (ER) stress that plays an important role in the progression of atherosclerosis has not been determined. We sought to determine the effect of PPARγ agonists on ER stress induced by palmitate, the most abundant saturated fatty acid in the serum.Methods and ResultsProtein expression of ER stress marker was evaluated by Western blot analysis and stearoyl-CoA desaturase1 (SCD-1) mRNA expression was evaluated by qRT-PCR. Macrophage apoptosis was detected by flowcytometry. Pioglitazone and rosiglitazone reduced palmitate-induced phosphorylation of PERK, a marker of ER stress, in RAW264.7, a murine macrophage cell line. Pioglitazone also suppressed palmitate-induced apoptosis in association with inhibition of CHOP expression, JNK phosphorylation and cleavage of caspase-3. These effects of pioglitazone were reversed by GW9662, a PPARγ antagonist, indicating that PPARγ is involved in this process. PPARγ agonists increased expression of SCD-1 that introduces a double bond on the acyl chain of long-chain fatty acid. 4-(2-Chlorophenoxy)-N-(3-(3-methylcarbamoyl)phenyl)piperidine-1-carboxamide, an inhibitor of SCD-1, abolished the anti-ER stress and anti-apoptotic effects of pioglitazone. These results suggest that PPARγ agonists attenuate palmitate-induced ER stress and apoptosis through SCD-1 induction. Up-regulation of SCD-1 may contribute to the reduction of cardiovascular events by treatment with PPARγ agonists.

Abstract-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. (Hypertension. 2011;58:386-393.) • Online Data Supplement Key Words: angiotensin II type 1 receptor Ⅲ renin angiotensin system Ⅲ prolyl hydroxylase domain-containing protein Ⅲ vascular remodeling R enin-angiotensin system (RAS) physiologically and pathophysiologically plays a pivotal role in the cardiovascular system. RAS modulates blood pressure, fluid and electrolyte homeostasis, and neuronal function. 1 RAS is also critical for the pathogenesis of cardiovascular diseases, such as hypertension, atherosclerosis, ischemic heart disease, and congestive heart failure. 2 Angiotensin II (Ang II), the primary active circulating component of the RAS, is a multifunctional hormone responsible for many cellular processes, such as inflammation, fibrosis, migration, proliferation, hypertrophy, and apoptosis, resulting in the cardiovascular remodeling. 3 The effects of Ang II are mediated by Ang II receptors, and 2 distinct isoforms of 7-transmembrane, G protein-coupled receptors have ever been cloned, Ang II type 1 receptor (AT 1 R) 4 and Ang II type 2 receptor. 5 It is generally accepted that AT 1 R mainly contributes to the progression of cardiovascular diseases. Indeed, many large-scale randomized clinical trials showed the beneficial effects of AT 1 R antagonists in the treatment of cardiovascular diseases. 6 Cardiovascular diseases are intimately related to the reduced oxygen concentration state (hypoxia). Cardiomyocytes in ischemic heart disease, peripheral organs in heart failure, ischemic limb in arteriosclerosis obliterans, and the brain in cerebral infarction are subject to hypoxia. Recently, it was reported that hypoxia activates both circulating and local RAS. 7,8 Hypoxia-inducible factor 1 (HIF-1) ...

In the present study we sought to determine the effect of CoCl2, an inhibitor of PHD (prolyl hydroxylase domain protein), on the development of AAA (abdominal aortic aneurysm). AAA was induced in C57BL/6 mice by periaortic application of CaCl2 (AAA group). NaCl (0.9%)-treated mice were used as a sham control (SHAM group). Mice were treated with 0.05% CoCl2 in the drinking water (AAA/CoCl2 group). At 1 and 6 weeks after the operation, aortic tissue was excised for further examination. After 6 weeks of CaCl2 treatment, aortic diameter and macrophage infiltration into the aortic adventitia were increased in the AAA group compared with the SHAM group. Treatment with CoCl2 reduced the aneurysmal size and macrophage infiltration compared with the AAA group. Aortic expression of inflammatory cytokines and MCP-1 (monocyte chemoattractant protein-1) and the activities of MMP-9 (matrix metalloproteinase-9) and MMP-2 were enhanced in the AAA group and attenuated in the AAA/CoCl2 group. Expression of cytokines and the activities of MMPs were already increased after 1 week of CaCl2 treatment, but were suppressed by CoCl2 treatment in association with reduced NF-κB (nuclear factor κB) phosphorylation. Treatment with CoCl2 in mice prevented the development of CaCl2-induced AAA in association with reduced inflammation and ECM (extracellular matrix) disruption. The results of the present study suggest that PHD plays a critical role in the development of AAA and that there is a therapeutic potential for PHD inhibitors in the prevention of AAA development.