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Age-related macular degeneration (AMD) is the leading cause of low vision and blindness for which there is currently no cure. Increased matrix metalloproteinase-9 (MMP-9) was found in AMD and potently contributes to its pathogenesis. Resident microglia also promote the processes of chronic neuroinflammation, accelerating the progression of AMD. The present study investigates the effects and mechanisms of the natural compound theissenolactone B (LB53), isolated from Theissenia cinerea, on the effects of RPE dysregulation and microglia hyperactivation and its retinal protective ability in a sodium iodate (NaIO

Intracerebral hemorrhage (ICH) is a devastating neurological disorder characterized by an exacerbation of neuroinflammation and neuronal injury, for which few effective therapies are available at present. Inhibition of excessive neuroglial activation has been reported to alleviate ICH-related brain injuries. In the present study, the anti-ICH activity and microglial mechanism of ergosta-7,9(11),22-trien-3β-ol (EK100), a bioactive ingredient from Asian medicinal herb Antrodia camphorate, were evaluated. Post-treatment of EK100 significantly attenuated neurobehavioral deficit and MRI-related brain lesion in the mice model of collagenase-induced ICH. Additionally, EK100 alleviated the inducible expression of cyclooxygenase (COX)-2 and the activity of matrix metalloproteinase (MMP)-9 in the ipsilateral brain regions. Consistently, it was shown that EK100 concentration-dependently inhibited the expression of COX-2 protein in Toll-like receptor (TLR)-4 activator lipopolysaccharide (LPS)-activated microglial BV-2 and primary microglial cells. Furthermore, the production of microglial prostaglandin E2 and reactive oxygen species were attenuated by EK100. EK100 also attenuated the induction of astrocytic MMP-9 activation. Among several signaling pathways, EK100 significantly and concentration-dependently inhibited activation of c-Jun N-terminal kinase (JNK) MAPK in LPS-activated microglial BV-2 cells. Consistently, ipsilateral JNK activation was markedly inhibited by post-ICH-treated EK100 in vivo. In conclusion, EK100 exerted the inhibitory actions on microglial JNK activation, and attenuated brain COX-2 expression, MMP-9 activation, and brain injuries in the mice ICH model. Thus, EK100 may be proposed and employed as a potential therapeutic agent for ICH.

Oxidative stress is an important factor to cause the pathogenesis of diabetic retinopathy (DR) because the retina has high vascularization and long-time light exposition. Cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes can convert arachidonic acid (AA) into eicosanoids, which are important lipid mediators to regulate DR development. COX-derived metabolites appear to be significant factors causative to oxidative stress and retinal microvascular dysfunction. Several elegant studies have unraveled the importance of LOX-derived eicosanoids, including LTs and HETEs, to oxidative stress and retinal microvascular dysfunction. The role of CYP eicosanoids in DR is yet to be explored. There is clear evidence that CYP-derived epoxyeicosatrienoic acids (EETs) have detrimental effects on the retina. Our recent study showed that the renin-angiotensin system (RAS) activation augments retinal soluble epoxide hydrolase (sEH), a crucial enzyme degrading EETs. Our findings suggest that EETs blockade can enhance the ability of RAS blockade to prevent or mitigate microvascular damage in DR. This review will focus on the critical information related the function of these eicosanoids in the retina, the interaction between eicosanoids and reactive oxygen species (ROS), and the involvement of eicosanoids in DR. We also identify potential targets for the treatment of DR.

Aims Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. Materials & methods We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC–MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. Results Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. Conclusion Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.

Nearly all patients with type 1 diabetes and 60% of those with type 2 diabetes develop diabetic retinopathy (DR). Thus, there is an urgent need to identify new therapeutic targets to prevent or at least to minimize microvascular dysfunction associated with DR. Activation of the renin-angiotensin system (RAS) contributes to breakdown of blood-retinal barrier (BRB) and neovascularization in DR. Clinical studies underscored RAS as a promising therapeutic target to treat DR. Although, blockade of RAS has reduced the incidence of DR in clinical studies, it did not reduce the progression of DR. Cytochrome-P450 metabolizes arachidonic acid to the pro-angiogenic and -permeability mediators, epoxyeicosatrienoic acids (EETs) which are metabolized by the soluble epoxide hydrolase (sEH) to inactive products. Our experiments demonstrated that: 1.) Angiotensin II (Ang II) increases retinal expression of sEH by 2.8 fold (2.84 ± 1.1 vs. 1 ± 0.08, P < 0.05, n = 4), which is blunted by angiotensin receptor-1 (AT1) inhibition; 2.) both sEH blockade and knockout (KO), which accumulate EETs, enhance Ang II-induced BRB injury as shown by 1.58 fold increase in retinal albumin leakage (1.58 ± 0.4 vs. 1 ± 0.13, P < 0.05, n = 4) and 2.8 fold (2.8 ± 1.7 vs. 1 ± 0.09, P < 0.05, n = 4), respectively. These results were confirmed by fluorescein angiogram analysis; 3.) Administration of synthetic 11,12-EET enhances intravitreal Ang II-induced retinal albumin leakage by 2.2 fold (2.2 ± 0.5 vs. 1 ± 0.18, P < 0.05, n = 4); and 4.) sEH KO potentiates diabetes (STZ)-induced retinal damage. This was associated with upregulation of retinal vascular endothelial growth factor (VEGF) and glucose transporter-1 (GLUT-1). In conclusion, our data demonstrate that in DR, activation of retinal AT1 triggers a compensatory mechanism by increasing retinal sEH that decreases EETs production. Thus, the use of combined EETs blockade with AT1 blocker holds the promise, as a new therapeutic strategy to prevent or reduce DR. Disclosure M. Al-Shabrawey: None. A. Ibrahim: None. A.M. Tawfik: None. M.H. Wang: None. Funding American Heart Association (SE00144); National Institutes of Health (R01EY023315 to M.A-S.)

// Weijing Feng 1, 2, * , Kun Zhang 1, 2, * , Yu Liu 3, * , Jie Chen 1, 4, * , Qingqing Cai 5 , Yinyin Zhang 1, 2 , Mongheng Wang 6 , Jingfeng Wang 1, 2 , Hui Huang 1, 2 1 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Department of Cardiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 2 Laboratory of RNA and Major Diseases of Brain and Heart, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 3 Department of Cardiology, the People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, China 4 Department of Radiation Oncology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, China 5 Department of Medical Oncology, Sun Yat-sen University Cancer Center, Sun Yat-sen University, Guangzhou, China 6 Department of Physiology, Georgia Regents University, Augusta, GA, USA * These authors have contributed equally to this work Correspondence to: Hui Huang, email: huanghui765@hotmail.com Keywords: vascular smooth muscle cells, osteogenic switching, vascular calcification, angiotensin II, apocynin Received: January 27, 2016 Accepted: October 19, 2016 Published: November 08, 2016 ABSTRACT Vascular calcification (VC) is a significant risk factor for cardiovascular morbidity and mortality. We recently reported that apocynin had benefits for preventing cardiovascular diseases. However, whether apocynin could attenuate VC is unknown. Here, we investigated the role of apocynin in VC and its underlying mechanisms. 163 participants with high or normal ankle–brachial index (ABI) were enrolled in this study for analyzing the demographic and biochemical data. In vitro, vascular smooth muscle cells (VSMCs) were exposed to calcification medium containing b-glycerophosphate and angiotensin II (Ang II) for 24 hours. The results showed that serum level of Ang II was significantly increased in patients with high ABI (P

Both clinical and experimental studies have demonstrated that vascular calcification (VC) is a common pathology shared in many chronic diseases such as chronic kidney disease (CKD) and diabetes. It's an independent risk factor for cardiovascular events. Since the pathogenesis of VC is complicated, current therapies have limited effects on the regression of VC. Therefore, it is urgent to investigate the potential mechanisms and find new targets for the treatment of VC. Aldosterone (Aldo), a mineralocorticoid hormone, is the metabolite of renin-angiotensin-aldosterone system (RAAS) activation, which can exert genomic and non-genomic effects on the cardiovascular system. Recent data suggests that Aldo can promote VC. Here, we summarized the roles of Aldo in the process of VC and a series of findings indicated that Aldo could act as a potentially therapeutic target for treating VC.

Despite the optimization of blood glucose control and the therapeutic management of risk factors, obesity- and diabetes-induced cardiovascular diseases are still major health problems in the United States. Arachidonic acid (AA), an endogenous 20-carbon polyunsaturated fatty acid, is metabolized by cytochrome P450 (CYP) epoxygenases into epoxyeicosatrienoic acids (EETs), which are important lipid mediators with many beneficial effects in type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and obesity- and diabetes-induced cardiovascular diseases. EETs can be further metabolized to less active dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). It has been demonstrated that the use of sEH blockers, which prevent EET degradation, is a promising pharmacological approach to promoting insulin secretion, preventing endothelial dysfunction, decreasing blood pressure, and protecting against target organ damage in obesity and metabolic diseases. This review will focus on biochemistry of CYP monooxygenase system as well as the pharmacology and physiological significance of EETs and sEH. We will also discuss the role of EETs/sEH in T1DM, T2DM, and obesity- and diabetes-induced cardiovascular diseases.

Clinical studies have suggested that the renin-angiotensin system (RAS) may be a promising therapeutic target in treating diabetic retinopathy (DR). While AT1 receptor blockade decreased the incidence of DR in the DIRECT trial, it did not reduce the DR progression. Lack of understanding of the molecular mechanism of retinal microvascular damage induced by RAS is a critical barrier to the use of RAS blockade in preventing or treating DR. The purpose of this study is to investigate the interaction between soluble epoxide hydrolase (sEH) and the AT1 receptor in Angiotensin II (Ang II)- and diabetes-induced retinal microvascular damage. We demonstrate that Ang II increases retinal sEH levels, which is blunted by an AT1 blocker; administration of 11,12-epoxyeicosatrienoic acid (EET) exacerbates intravitreal Ang II-induced retinal albumin leakage; while sEH knockout (KO) and blockade reduce Ang II-induced retinal vascular remodeling, sEH KO causes retinal vascular leakage in Ang II-sEH KO mice; and sEH KO potentiates diabetes-induced retinal damage via promoting retinal vascular endothelial growth factor (VEGF) but reducing expression of tight junction proteins (ZO-1 and occludin). Our studies hold the promise of providing a new strategy, the use of combined EETs blockade with AT1 blocker, to prevent or reduce DR.

Arachidonic acid (AA) is metabolized by cyclooxygenase (COX) and cytochrome P450 (CYP) enzymes into eicosanoids, which are involved in cardiovascular diseases and stroke. Evidence has demonstrated the important functions of these eicosanoids in regulating cerebral vascular tone, cerebral blood flow, and autoregulation of cerebral circulation. Although COX-2 inhibitors have been suggested as potential treatments for stroke, adverse events, including an increased risk of stroke, occur following long-term use of coxibs. It is important to note that prolonged treatment with rofecoxib increased circulating levels of 20-hydroxyeicosatetraenoic acid (20-HETE), and 20-HETE blockade is a possible strategy to prevent coxib-induced stroke events. It appears that 20-HETE has detrimental effects in the brain, and that its blockade exerts cerebroprotection against ischemic stroke and subarachnoid hemorrhage (SAH). There is clear evidence that activation of EP2 and EP4 receptors exerts cerebroprotection against ischemic stroke. Several elegant studies have contributed to defining the importance of stabilizing the levels of epoxyeicosatrienoic acids (EETs), by inhibiting or deleting soluble epoxide hydrolase (sEH), in stroke research. These reports support the notion that sEH blockade is cerebroprotective against ischemic stroke and SAH. Here, we summarize recent findings implicating these eicosanoid pathways in cerebral vascular function and stroke. We also discuss the development of animal models with targeted gene deletion and specific enzymatic inhibitors in each pathway to identify potential targets for the treatment of ischemic stroke and SAH.

Advanced oxidation protein products (AOPPs) are independent risk factor for various cardiovascular diseases. Cardiomyocyte apoptosis has been implicated as an important mechanism in cardiac remodeling in chronic kidney disease (CKD). However, whether AOPPs affect cardiomyocyte apoptosis and subsequent cardiac remodeling in CKD is still not very clear. Here, we assessed the role of AOPPs in cardiomyocyte apoptosis in CKD. H9C2 rat cardiomyoblast cells were exposed to AOPPs. Apoptotic cells were determined by TUNEL assay. The expression of apoptotic markers (cleaved caspase-3 and Bax), JNK signaling, and endoplasmic reticulum stress were explored. Serum AOPPs were measured in male Sprague-Dawley rats that underwent sham surgery and 5/6 nephrectomy, respectively. In vitro, our findings showed that AOPPs activated JNK signaling and endoplasmic reticulum stress and significantly aggravated H9C2 rat cardiomyoblast cells apoptosis. These effects were partially ameliorated by apocynin with inhibition of oxidative stress. In vivo, serum levels of AOPPs were progressively elevated with the increasing time course in CKD rats compared with sham-operated rats ( P0.05). Serum AOPP levels were positively associated with cardiomyocyte apoptosis ( R

In traditional Chinese medicine, Nitraria sibirica Pall. (Nitrariaceae) is used to treat hypertension. This study determined the effects of the total alkaloids of the leaves of Nitraria sibirica (NSTA) on blood pressure and albuminuria in mice treated with angiotensin II and a high-salt diet (ANG/HS). Adult mice were divided into three groups: control; infused with angiotensin II and fed a diet containing 4% NaCl (ANG/HS; and ANG/HS plus injection of NSTA (1 mg·kg(-1)·d(-1), i.p.). After treatment of these regimens, daily water and food intake, kidney weight, blood pressure, urinary albumin excretion, renal concentrations of inflammatory markers, including soluble intercellular adhesion molecule-1 (sICAM-1) and monocyte chemoattractant protein-1 (MCP-1), and the expression of renal fibrosis markers were determined. Compared to the control group, the ANG/HS group had higher blood pressure and urinary albumin excretion. Treatment with NSTA in ANG/HS mice for three weeks significantly reduced blood pressure and urinary albumin excretion. ANG/HS treatment caused elevated levels of sICAM-1 and MCP-1, as well as increased fibrosis markers. Concurrent treatment with ANG/HS and NSTA attenuated the levels and expression of renal inflammatory and fibrosis markers. Treatment with NSTA effectively reduces hypertension-induced albuminuria through the reduction of renal inflammatory and fibrosis markers.

Background and aims Therapeutic angiogenesis is a pivotal strategy for ischemic heart disease. The aim of the present study was to determine the effect and molecular mechanism of Shexiang Baoxin pills, a widely-used traditional Chinese medicine for ischemic heart disease, on angiogenesis in a rat model of myocardial infarction (MI). Methods We used the occlusion of left anterior descending coronary artery of Sprague-Dawley rats as a model of MI. The MI rats were treated with distilled water, Shexiang Baoxin pills, or Shexiang Baoxin pills + HET0016 (a selective blocker of the biosynthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) at 10 mg/kg/day), respectively. Sham-operated rats were used as controls. Results Treatment with Shexiang Baoxin pills increases the level of serum 20-HETE in MI rats, which can be suppressed by HET0016 treatment. Shexiang Baoxin pills shows cardio-protective effects on MI rats, including improving cardiac function, decreasing infarction area, and promoting angiogenesis in peri-infarct area. The protective effects of Shexiang Baoxin pills are partly inhibited by HET0016. Furthermore, Shexiang Baoxin pills enhances the number of circulating endothelial progenitor cells (EPCs) and the expression of the vascular endothelial growth factor (VEGF), based on immunohistochemical analysis, in peri-infarct area of MI rats, which is partly suppressed by HET0016. Conclusions Shexiang Baoxin pills may partially participate in angiogenesis in MI rats. The protective mechanism of Shexiang Baoxin pills may be mediated via up-regulation of 20-HETE, which promotes EPCs mobilization and VEGF expression.

Background: Coxibs, selective cyclooxygenase-2 (COX-2) inhibitors, significantly improved the quality of life of millions of individuals affected by pain and inflammation. Unfortunately, increased risk of stroke with the use of coxibs limited their clinical usefulness. The goal of this study is to investigate the role of 20-hydroxyeicosatetraenoic acid (20-HETE) metabolism by COX-2 in rofecoxib-induced cerebrovascular damage. We treated MC38 mice (1.4 x 10 6 MC38 cells/mouse) with rofecoxib (50 mg/L) + HET0016 (20-HETE blocker; 5 mg/kg/day, i.p.), rofecoxib, or vehicle for 3 weeks. We then subjected these MC38 and control mice to thromboembolic stroke. Finally, we treated B6 mice with 20-OH PGE 2 (250 ng/h; osmotic pump) and vehicle for 7 days, and then subjected them to ischemic stroke. Results: 1 ). Rofecoxib significantly reduced MC38 tumor size (from 693 ± 106 to 329 ± 40 mm 3 , P < 0.05). Within its therapeutic dose, rofecoxib selectively increased circulating 20-HETE levels (100 ± 18 vs. 150 ± 11%, P < 0.05), which was reversed by HET0016 (150 ± 11 vs. 100 ± 16%, P < 0.05). We did not find significant change on cyp4a (20-HETE synthesizing enzymes) expression in the brain microvessels after rofecoxib treatment. 2 ). A major prostaglandin (PG) metabolite, which is 20-OH PGE 2 , was generated when we incubated purified COX-2 with 20-HETE (5 uM). Strikingly, rofecoxib (1 uM) inhibited 20-OH PGE 2 synthesis by 71% (from 100 ± 16 to 29 ± 8%, P < 0.05). 3 ). Hemorrhagic transformation (HT) was greater in rofecoxib group (4/5 mice showing bleeding), a vascular injury that was prevented by co-treatment with HET0016 (0/6 mice showing bleeding). 4 ). 20-OH PGE 2 supplementation reduced both infarct size (45 ± 10 vs. 17 ± 8% contralateral hemisphere, P < 0.05) and neurological deficits (3.6 ± 0.5 vs. 2 ± 1, P < 0.05). Conclusion: 1 ) Rofecoxib increases circulating levels of 20-HETE, which exacerbates stroke damage. 2 ) COX-2 is the key player in regulating 20-HETE metabolism. 3 ) 20-HETE blockade prevents HT induced by rofecoxib. 4 ) 20-OH PGE 2 exerts cerebroprotective after ischemic stroke. In summary , these results suggest that both 20-HETE blockade and 20-OH PGE 2 supplementation are new approaches to ameliorate coxib-induced cerebrovascular damage and adverse stroke outcomes.

PGE2 has an established role in renal water handling. The present study was undertaken to examine the role of microsomal prostaglandin E synthase-1 (mPGES-1) in the diuretic response to acute and chronic water loading. Compared with wild-type (+/+) controls, mPGES-1 −/− mice exhibited impaired ability to excrete an acute, but not chronic water load. In response to acute water loading, urinary PGE2 excretion in the +/+ mice increased at 2 h, in parallel with increased urine flow. In contrast, the −/− mice exhibited a delayed increase in urinary PGE2 excretion, coinciding with the stimulation of renal medullary mRNA expression of cytosolic prostaglandin E synthase but not mPGES-2. At baseline, renal aquaporin-2 (AQP2) expression in mPGES-1 −/− mice was enhanced compared with the +/+ control. In response to acute water loading, renal AQP2 expression in the +/+ mice was significantly reduced, and this reduction was blunted in the −/− mice. Despite striking changes in AQP2 protein expression, renal AQP2 mRNA in both genotypes largely remained unchanged. Overall, these data support an important role of mPGES-1 in provoking the diuretic response to acute water loading.

Prostaglandin (PG) E 2 has an established role in the regulation of vascular tone and reactivity. The present study examined the role and mechanism of microsomal PG synthase-1 (mPGES-1) in vascular response to angiotensin II (Ang II) infusion. A 7-day Ang II infusion at 0.35 mg/kg per day via osmotic minipump had no obvious effect on mean arterial blood pressure in mPGES-1 +/+ mice but induced a marked hypertensive response in mPGES-1 −/− mice, associated with a parallel increase in urinary 8-isoprostane excretion and aortic NADPH oxidase activity and mRNA expression of p47 phox , gp91 phox , and Nox1. The hypertension in mPGES-1 −/− mice was completely prevented by Tempol treatment and was fully restored on termination of the antioxidant. Apocynin induced a similar blood pressure–lowering effect as Tempol. The Ang II infusion induced mRNA expression of mPGES-1, as well as mPGES-2 and cytosolic PGE synthase in the aortas as assessed by real-time RT-PCR. Immunohistochemistry revealed remarkably enhanced immunoreactivity of mPGES-1 mostly in vascular smooth muscle cells. In cultured vascular smooth muscle cells, Ang II exerted a direct stimulatory effect on reactive oxygen species production, NADPH oxidase activity, and expression of p47 phox , gp91 phox , and Nox1 that were all inhibited by PGE 2 . The −/− mice also exhibited enhanced renal hemodynamic response to acute Ang II infusion at 150 nmol/kg per minute via a jugular vein over a period of 40 minutes. These results suggest that mPGES-1–derived PGE 2 buffers Ang II–induced vasoconstriction via inhibition of NADPH oxidase–dependent reactive oxygen species production.

Type 2 diabetes and dyslipidemia oftentimes present in combination. However, the relative roles of diabetes and diet-induced dyslipidemia in mediating changes in vascular structure, mechanics, and function are poorly understood. Our hypothesis was that addition of a high-fat diet would exacerbate small artery remodeling, compliance, and vascular dysfunction in type 2 diabetes. Vascular remodeling indices [media/lumen (M/L) ratio, collagen abundance and turnover, and matrix metalloproteinase dynamics], mechanical properties (vessel stiffness), and reactivity to pressure and vasoactive factors were measured in third-order mesenteric arteries in control Wistar and type 2 diabetic Goto-Kakizaki (GK) rats fed either a regular or high-fat diet. M/L ratios, total collagen, and myogenic tone were increased in diabetes. Addition of the high-fat diet altered collagen patterns (mature versus new collagen) in favor of matrix accumulation. Addition of a high-fat diet caused increased constriction to endothelin-1 (0.1–100 nM), showed impaired vasorelaxation to both acetylcholine (0.1 nM–1 μM) and sodium nitroprusside (0.1 nM–1 μM), and increased cardiovascular risk factors in diabetes. These results suggest that moderate elevations in blood glucose, as seen in our lean GK model of type 2 diabetes, promote resistance artery remodeling resulting in increased medial thickness, whereas addition of a high-fat diet contributes to diabetic vascular disease predominantly by impairing vascular reactivity in the time frame used for this study. Although differential in their vascular effects, both hyperglycemia and diet-induced dyslipidemia need to be targeted for effective prevention and treatment of diabetic vascular disease.

Clofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR alpha) agonist, increases renal tubular cytochrome P450 4a (Cyp4a) expression thereby increasing 20-hydroxyeicosatetraenoic acid (20-HETE) production. To determine if clofibrate affects blood pressure regulation we studied mice with DOCA-salt induced hypertension in wild-type and PPAR alpha knockout mice. Wild-type mice treated with DOCA-salt had higher mean arterial pressures and higher cumulative sodium balance, but lower renal 20-HETE production than did vehicle-treated mice. Treating DOCA-salt mice with clofibrate attenuated the increase in mean arterial pressure and cumulative sodium balance while increasing 20-HETE production and renal Cyp4a expression. In contrast the PPAR alpha knockout mice treated with clofibrate and DOCA-salt showed no attenuation in the increase of blood pressure, cumulative sodium balance, renal 20-HETE production or Cyp4a protein expression. Expression of the PPAR alpha protein was greater in proximal tubules than in renal microvessels. Our results show that PPAR alpha pathway induces renal tubular 20-HETE production which affects sodium retention and blood pressure regulation in DOCA-salt-treated mice.

OBJECTIVE—The implication of innate immunity in type 1 diabetes development has long been proposed. High-mobility group box 1 (HMGB1), an evolutionarily conserved chromosomal protein, was recently recognized to be a potent innate inflammatory mediator when released extracellularly. We sought to test the hypothesis that HMGB1 acts as an innate immune mediator implicated in type 1 diabetes pathogenesis.RESEARCH DESIGN AND METHODS—Eight- and 12-week-old NOD mice were treated with an HMGB1 neutralizing antibody once a week until 25 weeks of age and monitored for insulitis progression and diabetes onset. The underlying mechanisms of HMGB1 regulation of autoimmune response were further explored.RESULTS—During autoimmunity, HMGB1 can be passively released from damaged pancreatic β-cells and actively secreted by islet infiltrated immune cells. Extracellular HMGB1 is potent in inducing NOD dendritic cell maturation and stimulating macrophage activation. Blockade of HMGB1 significantly inhibited insulitis progression and diabetes development in both 8- and 12-week-old NOD mice. HMGB1 antibody treatment decreased the number and maturation of pancreatic lymph node (PLN) CD11c++CD11b+ dendritic cells, a subset of dendritic cells probably associated with autoantigen presentation to naïve T-cells, but increased the number for PLN CD4+Foxp3+ regulatory T-cells. Blockade of HMGB1 also decreased splenic dendritic cell allo-stimulatory capability associated with increased tolergenic CD11c+CD8a+ dendritic cells. Interestingly, the number of CD8+interferon-γ+ (Tc1) T-cells was increased in the PLNs and spleen after blockade of HMGB1, which could be associated with retarded migration of activated autoreactive T-cells into the pancreatic islets.CONCLUSIONS—Extracellular HMGB1 functions as a potent innate immune mediator contributing to insulitis progression and diabetes onset.

Epithelial-to-mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of tubulointerstitial fibrosis. EMT is typically induced by transforming growth factor-β1(TGF-β1) and inhibited by hepatocyte growth factor (HGF). The present study was undertaken to evaluate the potential role of cyclooxygenase (COX)-2-derived PGE2in regulation of EMT in cultured Madin-Darby canine kidney (MDCK) cells, in the setting of HGF treatment. Exposure to 50 ng/ml HGF significantly induced COX-2 protein expression and PGE2release, whereas other growth factors, including epidermal growth factor, the insulin-like growth factor I protein, platelet-derived growth factor-BB, and TGF-β1, had no effects on COX-2 expression or PGE2release. COX-2 induction by HGF was preceded by activation of ERK1/2, and an ERK1/2-specific inhibitor, U-0126 (10 μM), completely abolished HGF-induced COX-2 expression. Exposure of MDCK cells to 10 ng/ml TGF-β1for 72 h induced EMT as evidenced by conversion to the spindle-like morphology, loss of E-cadherin, and activation of α-smooth muscle actin. In contrast, treatment with 1 μM PGE2completely blocked EMT, associated with a significant elevation of intracellular cAMP and complete blockade of TGF-β1-induced oxidant production. cAMP-elevating agents, including 8-Br-cAMP, forskolin, and IBMX, inhibited EMT and associated oxidative stress induced by TGF-β1, but inhibition of cAMP pathway with Rp-cAMP, the cAMP analog, and H89, the protein kinase A (PKA) inhibitor, did not block the effect of PGE2. The effect of HGF on EMT was inhibited by ∼50% in the presence of a COX-2 inhibitor SC-58635 (10 μM). Therefore, our data suggest that PGE2inhibits EMT via inhibition of oxidant production and COX-2-derived PGE2partially accounts for the antifibrotic effect of HGF.

Previous studies have shown that the synthesis of renal cytochrome P-450 (CYP)-derived eicosanoids is downregulated in genetic or high-fat diet-induced obese rats. Experiments were designed to determine whether fenofibrate, a peroxisome proliferator-activated receptor (PPAR)-α agonist, would induce renal eicosanoid synthesis and improve endothelial function in obese Zucker rats. Administration of fenofibrate (150 mg·kg−1·day−1for 4 wk) significantly reduced plasma insulin, triglyceride, and total cholesterol levels in obese Zucker rats. CYP2C11 and CYP2C23 proteins were downregulated in renal vessels of obese Zucker rats. Consequently, renal vascular epoxygenase activity decreased by 15% in obese Zucker rats compared with lean controls. Chronic fenofibrate treatment significantly increased renal cortical and vascular CYP2C11 and CYP2C23 protein levels in obese Zucker rats, whereas it had no effect on epoxygenase protein and activity in lean Zucker rats. Renal cortical and vascular epoxygenase activities were consequently increased by 54% and 18%, respectively, in fenofibrate-treated obese rats. In addition, acetylcholine (1 μM)-induced vasodilation was significantly reduced in obese Zucker kidneys (37% ± 11%) compared with lean controls (67% ± 9%). Chronic fenofibrate administration increased afferent arteriolar responses to 1 μM of acetylcholine in obese Zucker rats (69% ± 4%). Inhibition of the epoxygenase pathway with 6-(2-propargyloxyphenyl)hexanoic acid attenuated afferent arteriolar diameter responses to acetylcholine to a greater extent in lean compared with obese Zucker rats. These results demonstrate that the PPAR-α agonist fenofibrate increased renal CYP-derived eicosanoids and restored endothelial dilator function in obese Zucker rats.

A correlation exists between obesity and hypertension. In the currently available models of diet-induced obesity, the treatment of rats with a high fat (HF) diet does not begin until adulthood. Our aim was to develop and characterize a model of pre-pubescent obesity-induced hypertension. Male Sprague-Dawley rats were fed a HF diet (35% fat) for 10 weeks, beginning at age 3 weeks. Blood pressure was measured by tail-cuff, and a terminal blood sample was obtained to measure fasting blood glucose, insulin, plasma renin, aldosterone, thiobarbitutic acid reactive substances (TBARS), and free 8-isoprostanes levels. The vascular reactivity in the aorta was assessed using a myograph. Blood pressure was increased in rats fed the HF diet (HF, 161 ± 2 mm Hg vs. control, 137 ± 2 mm Hg, P < 0.05). Blood glucose (HF, 155 ± 4 mg/dL vs. control, 123 ± 5 mg/dL, P < 0.05), insulin (HF, 232 ± 63 pM vs. control, 60 ± 11 pM, P < 0.05), TBARS (expressed as nM of malondialdehyde [MDA]/ml [HF, 1.8 ± 0.37 nM MDA/ml vs. control 1.05 ± 0.09 nM MDA/ml, P < 0.05]), and free 8-isoprostanes (HF, 229 ± 68 pg/ml vs. control, 112 ± 9 pg/ml, P < 0.05) levels were elevated in the HF diet group. Interestingly, plasma renin and aldosterone levels were not different between the groups. The maximum vasoconstriction to phenylephrine (10−4 M) was increased in the HF diet group (HF, 26.1 ± 1.5 mN vs. control 22.3 ± 1.2 mN, P < 0.05). In conclusion, pre-pubescent rats become hypertensive and have increased oxidative stress and enhanced vasoconstriction when fed a HF diet. Surprisingly, this occurs without the increase in renin or aldosterone levels seen in the adult models of diet-induced obesity.

We previously reported that inhibition of nitric oxide (NO) synthesis by N-nitro-l-arginine methyl ester (l-NAME) during late pregnancy leads to increased production of renal vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 (CYP) 4A-derived vasoconstrictor, in pregnant rats. However, the effect of upregulation of vascular 20-HETE production on renal function after NO inhibition is not known. To test the hypothesis that increased gestational vascular 20-HETE synthesis after NO inhibition is involved in mediating blood pressure and renal functional changes, we first determined the IC50value of the effect of nitroprusside (SNP), a NO donor, on renal 20-HETE production in cortical microsomes. We then divided pregnant rats and age-matched virgin rats into a vehicle control group, an l-NAME treatment group (0.25 mg/ml in drinking water), and a group treated with l-NAME plus N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; CYP4A-selective inhibitor, 10 mg·kg−1·day−1iv). After 4 days of treatment, we measured blood pressure, renal blood flow (RBF), renal vascular resistance (RVR), and glomerular filtration rate (GFR) in each group. The addition of SNP (IC50= 22 μM) decreased renal cortical 20-HETE production. In pregnant rats, l-NAME treatment led to significantly higher mean arterial pressure (MAP) and RVR, and lower RBF and GFR. Combined treatment with DDMS and l-NAME significantly attenuated the increases in MAP and RVR and the decrease in GFR, but not the reduction in RBF induced by l-NAME treatment. l-NAME and l-NAME plus DDMS had no significant impact on renal hemodynamics in virgin rats. In addition, chronic treatment with DDMS selectively inhibited cortical 20-HETE production without a significant effect on CYP4A expression in l-NAME-treated pregnant rats. In conclusion, NO effectively inhibits renal cortical microsomal 20-HETE production in female rats. In pregnant rats, the augmentation of renal 20-HETE production after NO inhibition is associated with increased MAP and RVR, whereas decreased GFR is negated by treatment of a selective and competitive CYP4A inhibitor. These results demonstrate that the interaction between renal 20-HETE and NO is important in the regulation of renal function and blood pressure in pregnant rats.

Background Renal cytochrome P450 (CYP)-derived eicosanoids, hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs), have been shown to affect renal function and blood pressure (BP). We recently reported that high fat (HF) diet treatment in male rats increases BP and decreases production of these eicosanoids in the kidneys. However, at what level the downregulation of renal eicosanoid synthesis occurs and whether the HF diet has any effects on the regulation of renal eicosanoid synthesis in female rats are not known. The purpose of this study was to determine renal CYP-derived eicosanoid synthesis and its association with BP regulation in HF male and female rats. Methods In the first set of experiments, male and female rats were fed the HF or control diet for 10 weeks. In the second set of experiments, male and female rats were fed the HF diet for 10 days. In the third set of experiments, HF-fed and control female rats were treated with 5α-dihydrotestosterone for 4 weeks. After treatment, BP, urinary sodium, sodium balance, eicosanoid production, and CYP enzyme expression were determined. Results An elevation of BP and a decrease of renal cortical eicosanoid production were found in HF male rats, but no BP and eicosanoid production changes were observed in HF female rats. The HF treatment also caused a significant decrease of eicosanoid production and a decrease of CYP4A and 2C23 expression in the proximal tubules of HF male rats. Moreover, the HF diet treatment in male rats caused an increase in cumulative sodium balance and an elevation of BP, whereas no change in cumulative sodium balance and BP was observed in female rats. The treatment of 5α-dihydrotestosterone increased BP and 20-HETE production in the renal microvessels, but had no effect on urinary sodium excretion and renal microvessel EET production in both control and HF-fed female rats. Conclusions These results demonstrate that there are gender-specific differences in regulation of renal eicosanoid synthesis, sodium balance, and BP caused by HF treatment, and it appears that androgens play some role in upregulation of renal microvessel 20-HETE production in both HF and control female rats.

We examined the effect of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (HET0016), an inhibitor of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthesis on the omega-hydroxylation and epoxidation of arachidonic acid (AA) catalyzed by recombinant cytochrome P450 4A1 (CYP4A1), CYP4A2 and CYP4A3, and characterized the enzyme inhibitory profile of HET0016. The IC50 values of HET0016 for recombinant CYP4A1-, CYP4A2- and CYP4A3-catalyzed 20-HETE synthesis averaged 17.7 nM, 12.1 nM and 20.6 nM, respectively. The IC50 value for production of 11,12-epoxy-5,8,14-eicosatrienoic acid (11,12-EET) by CYP4A2 and 4A3 averaged 12.7 nM and 22.0 nM, respectively. The IC50 value for CYP2C11 activity was 611 nM which was much greater than that for CYP4As. The initial velocity study showed the Ki value of HET0016 for CYP4A1 was 19.5 nM and a plot of Vmax versus amount of recombinant CYP4A1 added shows HET0016 is an irreversible non-competitive inhibitor. These results indicate that HET0016 is a selective, non-competitive and irreversible inhibitor of CYP4A.

Divalent metal ions such as copper, manganese, and cobalt are essential for cell development, differentiation, function and survival. These essential metal ions are delivered into intracellular domains as cofactors for enzymes involved in neuropeptide and neurotransmitter synthesis, superoxide metabolism, and other biological functions in a target specific fashion. Altering the homeostasis of these essential metal ions is known to connect to a number of human diseases including Alzheimer disease, amyotrophic lateral sclerosis, and pain. It remains unclear how these essential metal ions are delivered to intracellular targets in mammalian cells. Here we report that rat spinal cord dorsal horn neurons express ACDP4, a member of Ancient Conserved Domain Protein family. By screening a pretransformed human fetal brain cDNA library in a yeast two-hybrid system, we have identified that ACDP4 specifically interacts with COX11, an intracellular metal ion chaperone. Ectopic expression of ACDP4 in HEK293 cells resulted in enhanced toxicity to metal ions including copper, manganese, and cobalt. The metal ion toxicity became more pronounced when ACDP4 and COX11 were co-expressed ectopically in HEK293 cells, suggesting a functional coupling between them. Our results indicate a role of ACDP4 in metal ion homeostasis and toxicity. This is the first report revealing a functional aspect of this ancient conserved domain protein family. We propose that ACDP is a family of transporter protein or chaperone proteins for delivering essential metal ions in different mammalian tissues. The expression of ACDP4 on spinal cord dorsal horn neurons may have implications in sensory neuron functions under physiological and pathological conditions.

Epoxyeicosatrienoic acids (EETs), which belong to cytochrome P-450 (CYP)-derived eicosanoids, have been implicated to vasodilate renal arterioles, inhibit sodium transport in the nephron, and regulate blood pressure in several animal models. Because pregnancy is associated with changes of blood pressure, the aim of this study was to examine whether renal EET synthesis is altered and whether EETs are involved in blood pressure regulation during pregnancy in rats. Renal microsomal epoxygenase activity increased by 47, 97, and 63% on days 6, 12, and 19 of gestation, respectively. The elevation of epoxygenase activity during pregnancy was associated with an increase in CYP2C11, CYP2C23, and CYP2J2 protein expression on days 6, 12, and 19 of gestation. Moreover, immunohistochemical analysis showed that renal tubular CYP2C11, CYP2C23, and CYP2J2 expression was significantly increased in pregnant rats on days 6, 12, and 19 of gestation. Administration of 6-(2-propargyloxyphenyl)hexanoic acid (PPOH), a selective epoxygenase inhibitor, caused a dose-dependent inhibition of microsomal expoxygenase activity without a significant effect on ω-hydroxylase activity in female rats. Interestingly, administration of PPOH (20 mg·kg−1·day−1for 4 days starting on day 15 of pregnancy) increased blood pressure by 21 mmHg and caused a significant decrease in the body weight of fetal pups (1.3 ± 0.08 g in control vs. 1.1 ± 0.06 g in PPOH). Moreover, PPOH treatment significantly decreased renal microsomal epoxygenase activity and the expression of CYP2C11, CYP2C23, and CYP2J in pregnant rats. This study demonstrates that EET synthesis in the kidney is elevated during pregnancy, and CYP2C11, 2C23, and CYP2J2 are responsible for the change of renal EET synthesis. The inhibition results demonstrate that the downregulation of renal epoxygenase activity by PPOH causes hypertension in pregnant rats. This study suggests that EETs may contribute to the control of blood pressure during pregnancy.

Background/Aims:Recent work has shown that female animals are more resistant to ischemic acute renal failure (ARF) than male animals. The mechanism underlying the gender difference is unclear. Moreover, whether the gender difference holds true for ARF induced by other insults is unknown. This study sought to determine the gender differences in ischemic and nephrotoxic ARF. Methods: Gender differences were tested in two experimental models of ARF. For ischemic ARF, bilateral clamping of renal pedicles was conducted in C57BL/6 and129/Sv mice followed by reperfusion. For nephrotoxic ARF, cisplatin was administered to the animals. Renal function, tissue damage, animal survival, and renal cell apoptosis were examined. Results: Ischemic ARF was significantly ameliorated in female mice, as shown by lower serum creatinine and blood urea nitrogen (BUN). Female mice also showed better renal histology, less apoptosis and caspase activation, and a much better survival rate than male mice following ischemic insult. On the contrary, female mice were more sensitive to cisplatin-induced ARF. In these animals, BUN increased at day 1 following cisplatin injection, while in males BUN increases were not shown until day 3. Higher levels of serum creatinine were also recorded in female mice. Renal histology showed severer necrotic tubular damage in females, although apoptosis and caspase activation appeared similar in both genders. Consistently, male mice survived better than females in the nephrotoxic model. Conclusion: While female mice were resistant to ischemic ARF, they appeared more sensitive to cisplatin-induced ARF. Investigation of the gender differences at the cellular and molecular levels might provide a new area for mechanistic study of ARF.

Apoptosis has been implicated in ischemic renal injury. Thus one strategy of renal protection is to antagonize apoptosis. However, apoptosis inhibitory approaches remain to be fully explored. Zn2+has long been implicated in apoptosis inhibition; but systematic analysis of the inhibitory effects of Zn2+is lacking. Moreover, whether Zn2+blocks renal cell apoptosis following ischemia is unknown. Here, we demonstrate that Zn2+is a potent apoptosis inhibitor in an in vitro model of renal cell ischemia. ATP depletion induced apoptosis in cultured renal tubular cells, which was accompanied by caspase activation. Zn2+at 10 μM inhibited both apoptosis and caspase activation, whereas Co2+was without effect. In ATP-depleted cells, Zn2+partially prevented Bax activation and cytochrome c release from mitochondria. In isolated cell cytosol, Zn2+blocked cytochrome c-stimulated caspase activation at low-micromolar concentrations. In addition, Zn2+could directly antagonize the enzymatic activity of purified recombinant caspases. We conclude that Zn2+is a potent inhibitor of apoptosis in renal tubular cells following ATP depletion. Zn2+blocks apoptosis at multiple steps including Bax activation, cytochrome c release, apoptosome function, and caspase activation.

The incidence of essential hypertension increases with obesity; however, the mechanisms that link obesity with hypertension are unclear. Renal cytochrome P450 (CYP)-derived eicosanoids—hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs)—have been shown to play an important role in the regulation of renal function, vascular tone, and blood pressure. The objective of this study was to examine CYP-derived eicosanoid synthesis in the different renal zones (cortex, medulla, and papilla) of rats fed a high fat diet (HF). Male Sprague-Dawley rats were fed a HF diet or regular rat chow for 10 weeks. After 10 weeks, HF rats showed significantly higher systolic blood pressure, body weight, and fat:body weight ratio. The renal ω-hydroxylase activity was decreased by 46% in cortex, 43% in medulla, and 46% in papilla of HF rats. The renal epoxygenase activity was decreased by 46% in cortex, 31% in medulla, and 56% in papilla of HF rats. Interestingly, the changes in the rate of 20-HETE and EET formation in different renal zones were consistent with the levels of expression of CYP4A and CYP2C23 proteins, respectively. Furthermore, there were no significant changes in the synthesis of these metabolites in the renal microvessels. These results demonstrate that HF diet causes the downregulation of CYP4A and CYP2C23 in renal tubules, and these proteins are responsible for renal 20-HETE and EET formation. The reduction in the synthesis of these eicosanoids may play an important role in the regulation of renal function and blood pressure in obesity-induced hypertension.

Background— Twenty-hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450 metabolite of arachidonic acid that produces vasoconstriction and inhibition of renal tubular sodium transport. In Dahl rats, a 20-HETE deficiency plays a role in salt-sensitive (SS) hypertension. In humans, there are no data on regulation of 20-HETE by salt intake or on a role for this compound in SS hypertension. Methods and Results— Thirteen salt-resistant (SR) and 13 SS hypertensive subjects had urine 20-HETE excretion measured during salt-loading and depletion. In all patients, 20-HETE was 66.6% higher in the salt-replete (1.75±0.25 μg/h) than in the salt-depleted state (1.05±0.16, P r =0.61, P r =0.66, P r =−0.79, P Conclusions— We demonstrate for the first time that 20-HETE excretion is regulated by salt intake in hypertension. We find a disrupted relationship between sodium excretion and 20-HETE in SS patients, which results in dependence of their salt excretion on blood pressure and may be related to the magnitude of their obesity. We conclude that salt-sensitivity of blood pressure in essential hypertension may result from impairment of a natriuretic mechanism dependent on 20-HETE.

20-HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca2+-activated K+ channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low- K m arachidonic acid ω-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC50 from 0.37 ± 0.04 μM in plasmid-transfected arteries to 0.07 ± 0.01 μM in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine.

We examined whether renal 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis is altered during gestation. Renal microsomal arachidonic acid ω-hydroxylase activity increased by 50 and 48% in rats on days 12 and 19 of gestation, respectively. Renal microvessel 20-HETE synthesis increased by 50 and 82% in rats on days 6 and 12 of gestation, respectively, and returned to control levels at day 19 of gestation. In contrast, 20-HETE synthesis in isolated medullary thick ascending limb was unchanged from control levels on days 6and 12 of gestation, but it increased twofold on day 19 of gestation. This increase on day 19 of gestation was associated with a twofold increase in urinary 20-HETE excretion, and it coincided with a 23-mmHg fall in blood pressure. Moreover, change in the rate of 20-HETE synthesis in microvessels was consistent with the level of expression of cytochrome P450 (CYP)4A proteins. Administration of the CYP4A inhibitor 1-aminobenzotriazole (ABT) for 2 days on day 12 of pregnancy or for 5 days starting on day 15 of pregnancy caused a transient but significant reduction in systolic blood pressure. ABT treatment also decreased urinary sodium, urinary 20-HETE, and renal and microvessel 20-HETE synthesis. This study, to our knowledge, is the first to demonstrate that 20-HETE synthesis in the kidney is altered in time- and site-specific manners during pregnancy. The localized pattern of changes suggests that there are distinct regulatory mechanisms for 20-HETE synthesis in the kidney during pregnancy.

20-Hydroxyeicosatetraenoic acid (20-HETE), Cyp4a-derived eicosanoid, is a lipid mediator that promotes tumor growth, as well as causing detrimental effects in cerebral circulation. We determined whether concurrent inhibition of cyclooxygenase-2 (COX-2) and 20-HETE affects colon tumor growth and ischemic stroke outcomes. The expression of Cyp4a and COXs and production of 20-HETE and PGE2were determined in murine colon carcinoma (MC38) cells. We then examined the effects of combined treatment with rofecoxib, a potent COX-2 inhibitor, and HET0016, a potent Cyp4a inhibitor, on the growth and proliferation of MC38 cells. Subsequently, we tested the effects of HET0016 plus rofecoxib in MC38 tumor and ischemic stroke models. Cyp4a and COXs are highly expressed in MC38 cells. Respectively, HET0016 and rofecoxib inhibited 20-HETE and PGE2formation in MC38 cells. Moreover, rofecoxib combined with HET0016 had greater inhibitory effects on the growth and proliferation of MC38 cells than did rofecoxib alone. Importantly, rofecoxib combined with HET0016 provided greater inhibition on tumor growth than did rofecoxib alone in MC38 tumor-bearing mice. Prolonged treatment with rofecoxib selectively induced circulating 20-HETE levels and caused cerebrovascular damage after ischemic stroke, whereas therapy with rofecoxib and HET0016 attenuated 20-HETE levels and reduced rofecoxib-induced cerebrovascular damage and stroke outcomes during anti-tumor therapy. Thus these results demonstrate that combination therapy with rofecoxib and HET0016 provides a new treatment of colon tumor, which can not only enhance the anti-tumor efficacy of rofecoxib, but also reduce rofecoxib-induced cerebrovascular damage and stroke outcomes.

The cytochrome P-450 4A (CYP4A)-derived arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) affects renal tubular and vascular functions and has been implicated in the control of arterial pressure. We examined the effect of antisense oligonucleotide (ODN) to CYP4A1, the low K m arachidonic acid ω-hydroxylating isoform, on vascular 20-HETE synthesis, vascular reactivity, and blood pressure in the spontaneously hypertensive rat (SHR). Administration of CYP4A1 antisense ODN decreased mean arterial blood pressure from 137 ± 3 to 121 ± 4 mmHg ( P < 0.05) after 5 days of treatment, whereas treatment with scrambled antisense ODN had no effect. Treatment with CYP4A1 antisense ODN reduced the level of CYP4A-immunoreactive proteins along with 20-HETE synthesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine (EC50 0.69 ± 0.17 vs. 1.77 ± 0.40 μM). Likewise, mesenteric arterioles from antisense ODN-treated rats revealed attenuation of myogenic constrictor responses to increases of transmural pressure. The decreased vascular reactivity and myogenic responses were reversible with the addition of 20-HETE. These data suggest that CYP4A1-derived 20-HETE facilitates myogenic constrictor responses in the mesenteric microcirculation and contributes to pressor mechanisms in SHR.

20-Hydroxyeicosatetraenoic acid (HETE), the cytochrome P-450 (CYP) 4A ω-hydroxylation product of arachidonic acid, has potent biological effects on renal tubular and vascular functions and on the control of arterial pressure. We have expressed high levels of the rat CYP4A1, -4A2, -4A3, and -4A8 cDNAs, using baculovirus and Sf 9 insect cells. Arachidonic acid ω- and ω-1-hydroxylations were catalyzed by three of the CYP4A isoforms; the highest catalytic efficiency of 947 nM−1⋅ min−1for CYP4A1 was followed by 72 and 22 nM−1⋅ min−1for CYP4A2 and CYP4A3, respectively. CYP4A2 and CYP4A3 exhibited an additional arachidonate 11,12-epoxidation activity, whereas CYP4A1 operated solely as an ω-hydroxylase. CYP4A8 did not catalyze arachidonic or linoleic acid but did have a detectable lauric acid ω-hydroxylation activity. The inhibitory activity of various acetylenic and olefinic fatty acid analogs revealed differences and indicated isoform-specific inhibition. These studies suggest that CYP4A1, despite its low expression in extrahepatic tissues, may constitute the major source of 20-HETE synthesis. Moreover, the ability of CYP4A2 and -4A3 to catalyze the formation of two opposing biologically active metabolites, 20-HETE and 11,12-epoxyeicosatrienoic acid, may be of great significance to the regulation of vascular tone.

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol ⋅ kg body wt−1⋅ day−1iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.

Erectile dysfunction (ED) is estimated to affect more than 30 million American men and 152 million men worldwide. Therapeutic agents targeting the nitric oxide/cyclic GMP signaling pathway have successfully treated patients with ED; however, the efficacies of these treatments are significantly lower in specific populations such as patients with diabetes. The goal of this study was to discover and identify new endothelium-derived relaxing factors involved in the regulation of erectile function, providing alternative therapeutic targets for treatment of ED. Immunoblotting results showed that protein expressions of epoxygenases from cytochrome P450 (CYP)2B, 2C and 2J subfamilies, as well as NADPH CYP reductase were present in rat corpora cavernosa, which was confirmed by immunohistochemical analysis. Furthermore, CYP2C was localized in cavernosal endothelial cells using double immunolabeling. CYP epoxygenase activity was analyzed by reverse-phase high-pressure liquid chromatography; and the results showed that 11,12- epoxyeicosatrienoic acid (EET) was the major product metabolized by CYP epoxygenases in rat corpora cavernosa. Inhibition of EETs function by injection of an EETs antagonist into rat penis significantly decreased intracavernosal pressure-induced by electrical stimulation of the major pelvic ganglion in vivo. In conclusion, our results suggest that EETs, produced by CYP epoxygenases, in penile endothelial cells serve as vasodilators. Inhibition of this pathway attenuated erectile function, suggesting that EETs are required for normal erection.

Objective Inhibition of soluble epoxide hydrolase (Ephx2) has been shown to play a protective role in cardiac hypertrophy, but the mechanism is not fully understood. We tested the hypothesis that deletion of soluble epoxide hydrolase attenuates cardiac hypertrophy via down-regulation of cardiac fibroblasts-derived fibroblast growth factor-2. Design Prospective, controlled, and randomized animal study. Setting University laboratory. Subjects Male wild-type C57BL/6 mice and Ephx2 (-/-) mice. Interventions Male wild-type or Ephx2 (-/-) mice were subjected to transverse aorta constriction surgery. Measurements and main results Four weeks after transverse aorta constriction, Ephx2 (-/-) mice did not develop significant cardiac hypertrophy as that of wild-type mice, indicated by no changes in the ratio of heart weight/body weight and ventricular wall thickness after transverse aorta constriction. Cardiac fibroblast growth factor-2 increased in wild-type-transverse aorta constriction group but this did not change in Ephx2 (-/-)-transverse aorta constriction group, and the serum level of fibroblast growth factor-2 did not change in both groups. In vitro, cardiac fibroblasts were stimulated by angiotensin II to analyze the expression of fibroblast growth factor-2. The effect of increased fibroblast growth factor-2 from cardiac fibroblasts induced by angiotensin II was attenuated by soluble epoxide hydrolase deletion. ERK1/2, p38, and AKT kinase were involved in fibroblast growth factor-2 expression regulated by angiotensin II, and soluble epoxide hydrolase deletion lowered the phosphorylation of ERK1/2 not p38 or AKT to mediate fibroblast growth factor-2 expression. In addition, soluble epoxide hydrolase deletion did not attenuate cardiomyocytes hypertrophy induced by exogenous fibroblast growth factor-2. Conclusions Our present data demonstrated that deletion of soluble epoxide hydrolase prevented cardiac hypertrophy not only directly to cardiomyocytes but also to cardiac fibroblasts by reducing expression of fibroblast growth factor-2.

Inner medulla collecting duct (IMCD) cells are the key part for urinary concentration. Hypotonic stress may trigger apoptosis of IMCD cells and induce renal injury. Epoxyeicosatrienoic acids (EETs) play an important role in anti-apoptosis, but their roles in hypotonic-induced apoptosis of IMCD cells are still unclear. Here we found increasing exogenous 11, 12-EET or endogenous EETs with Ad-CMV-CYP2C23-EGFP transfection decreased apoptosis of IMCD cells induced by hypotonic stress. Moreover, up-regulation of γ-ENaC induced by hypotonic stress was abolished by elevation of exogenous or endogenous EETs. Collectively, this study illustrated that EETs attenuated hypotonic-induced apoptosis of IMCD cells, and that regulation of γ-ENAC may be a possible mechanism contributing to the anti-apoptotic effect of EETs in response to hypotonic stress.

In order to study the interaction between human cytochrome P450 2E1 (h2E1) and NADPH–P450 oxidoreductase (hOR) in a native membrane environment, we used two approaches to express both h2E1 and hOR in a baculovirus expression system. For a dual-expression system, h2E1 and hOR were coexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus carrying both h2E1 and hOR cDNAs (v-h2E1-hOR). The h2E1 cDNA was expressed under the control of the polyhedrin promoter P Polh , whereas hOR cDNA was expressed under the control of the P 10 promoter. The expressed enzymes were catalytically active in the cell membrane preparations. The estimated molar expression ratio of h2E1 to hOR in the membranes was 1:5. The apparent K m and k cat for N -nitrosodimethylamine (NDMA) demethylase activity were 145 μ M and 2.4 min −1 , respectively. When Sf9 cells were co-infected with the dual-expression virus (v-h2E1-hOR) and human cytochrome b 5 recombinant virus (v-hb 5 ), a 9-fold decrease in the K m of NDMA demethylase activity (16 μ M ) was observed in the membrane preparations, whereas the k cat was increased to 4 min −1 . In the second approach, recombinant viruses of h2E1 and hOR (v-h2E1 and v-hOR) were used to co-infect the Sf9 cells. In this double-expression system, with a fixed amount of v-h2E1, the expression of h2E1 in the Sf9 cells decreased as the amount of v-hOR increased. Western blot analysis of the membrane preparations showed that the level of hOR increased, but the level of h2E1 decreased with increasing amounts of v-hOR. A corresponding decrease in h2E1 mRNA, however, was not observed. In the presence of human cytochrome b 5 (hb 5 ), the optimal h2E1:hOR molar ratio for h2E1 catalytic activity was 1:1. In order to further investigate the hb 5 effect on h2E1-catalyzed reactions in the native membranes, we co-infected Sf9 cells with v-h2E1, v-hOR, and v-hb 5 and obtained a membrane preparation containing h2E1, hOR, and hb 5 . Stoichiometric analysis with membrane preparations from double-infection and triple-infection systems revealed that the presence of hb 5 decreased NADPH oxidation and H 2 O 2 formation by 72 and 80%, respectively, but increased product formation from NDMA 13-fold. These results suggest that hb 5 enhances the coupling between h2E1 and hOR for product formation. These studies also demonstrate that the baculovirus–insect cell system can produce high levels of expression of functional h2E1, hOR, and hb 5 for mechanistic studies.

The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the alpha-methyl and alpha-methylene groups, leading to the formation of reactive species which can pyridyloxobutylate and methylate DNA, respectively. The present study examined the kinetic parameters of NNK-derived keto alcohol (alpha-methyl hydroxylation), and keto aldehyde (alpha-methylene hydroxylation) formation catalyzed by human P450s heterologously expressed by either the baculovirus-insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane preparations of the expressed P450s catalyzed the alpha-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol. Human P450 2A6 showed the lowest KM (118 microM) for the formation of keto aldehyde. A similar KM was observed for keto alcohol formation by expressed P450 2A6, but the kcat was lower than the value obtained for keto aldehyde formation. The addition of exogenous b5 increased the expressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both alpha-hydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capacity for keto alcohol formation; however, their KM values for this reaction were in the millimolar range. Expressed human P450 3A4 oxidized NNK to keto aldehyde also with a high KM. Ten human liver microsomal samples were each shown to activate NNK to keto aldehyde and keto alcohol. A positive correlation coefficient of 0.74 was found between keto aldehyde formation and both coumarin 7-hydroxylation (P450 2A6) and 6 beta-testosterone hydroxylation (3A4) activity in characterized human liver microsomes. Keto alcohol formation showed a significant correlation with ethoxyresorufin O-dealkylation (P450 1A2) in human liver microsomes. Both coumarin and troleandomycin, specific inhibitors of P450 2A6 and 3A4, respectively, inhibited the formation of keto aldehyde, but inhibited the formation of keto alcohol only slightly in human liver microsomes. Both furafylline, a P450 1A2 inhibitor, and N-nitrosodimethylamine, a P450 2E1 substrate, inhibited the formation of keto alcohol but not keto aldehyde in human liver microsomes. Quinidine, a specific inhibitor of P450 2D6, was not an effective inhibitor of NNK metabolism. These results demonstrate that P450s 2A6 and 3A4 may be important P450s for the activation of NNK to a DNA-methylating agent and keto aldehyde via the alpha-methylene hydroxylation pathway. P450s 1A2, 2E1, and 2D6 are shown to be selective for alpha-methyl hydroxylation of NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent.