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...

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) ...

The MDM2 antagonist nutlin-3 inhibits VSMC proliferation, migration, and NF-κB activation, and also attenuates neointimal hyperplasia after vascular injury in mice, which is associated with suppression of vascular cell proliferation and an inflammatory response. Targeting MDM2 might be a potential therapeutic strategy for the treatment of vascular proliferative diseases.

Donepezil {(RS)-2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one} is a reversible acetylcholinesterase inhibitor and used for treatment of patients with AD (Alzheimer's disease). Recent studies showed that treatment with donepezil reduced production of inflammatory cytokines in PBMCs (peripheral blood mononuclear cells). It was also reported that muscle-derived inflammatory cytokines play a critical role in neovascularization in a hindlimb ischaemia model. We sought to determine whether donepezil affects angiogenesis. A hindlimb ischaemia model was created by unilateral femoral artery ligation. Blood flow recovery examined by laser Doppler perfusion imaging and capillary density by immunohistochemical staining of CD31-positive cells in the ischaemic hindlimb were significantly decreased in donepezil- and physostigmine-treated mice compared with control mice after 2 weeks. Donepezil reduced expression of IL (interleukin)-1β and VEGF (vascular endothelial growth factor) in the ischaemic hindlimb. Intramuscular injections of IL-1β to the ischaemic hindlimb reversed the donepezil-induced VEGF down-regulation and the anti-angiogenic effect. Hypoxia induced IL-1β expression in C2C12 myoblast cells, which was inhibited by pre-incubation with ACh (acetylcholine) or LY294002, a PI3K (phosphoinositide 3-kinase) inhibitor. Donepezil inhibited phosphorylation of Akt [also known as PKB (protein kinase B)], a downstream kinase of PI3K, in the ischaemic hindlimb. These findings suggest that cholinergic stimulation by acetylcholinesterase inhibitors suppresses angiogenesis through inhibition of PI3K-mediated IL-1β induction, which is followed by reduction of VEGF expression. Acetylcholinesterase inhibitor may be a novel anti-angiogenic therapy.