Frank Fan Zhang

D-4F Induces Heme Oxygenase-1 and Extracellular Superoxide Dismutase, Decreases Endothelial Cell Sloughing, and Improves Vascular Reactivity in Rat Model of Diabetes

Background—Apolipoprotein A1 mimetic peptide, synthesized from D-amino acid (D-4F), enhances the ability of HDL to protect LDL against oxidation in atherosclerotic animals. Methods and Results—We investigated the mechanisms by which D-4F provides antioxidant effects in a diabetic model. Sprague-Dawley rats developed diabetes with administration of streptozotocin (STZ). We examined the effects of daily D-4F (100 &mgr;g/100 g of body weight, intraperitoneal injection) on superoxide (O2−), extracellular superoxide dismutase (EC-SOD), vascular heme oxygenase (HO-1 and HO-2) levels, and circulating endothelial cells in diabetic rats. In response to D-4F, both the quantity and activity of HO-1 were increased. O2− levels were elevated in diabetic rats (74.8±8×103 cpm/10 mg protein) compared with controls (38.1±8×103 cpm/10 mg protein; P<0.01). D-4F decreased O2− levels to 13.23±1×103 (P<0.05 compared with untreated diabetics). The average number of circulating endothelial cells was higher in diabetics (50±6 cells/mL) than in controls (5±1 cells/mL) and was significantly decreased in diabetics treated with D-4F (20±3 cells/mL; P<0.005). D-4F also decreased endothelial cell fragmentation in diabetic rats. The impaired relaxation typical of blood vessels in diabetic rats was prevented by administration of D-4F (85.0±2.0% relaxation). Western blot analysis showed decreased EC-SOD in the diabetic rats, whereas D-4F restored the EC-SOD level. Conclusions—We conclude that an increase in circulating endothelial cell sloughing, superoxide anion, and vasoconstriction in diabetic rats can be prevented by administration of D-4F, which is associated with an increase in 2 antioxidant proteins, HO-1 and EC-SOD.

Endothelial Dysfunction and Hypertension in Rats Transduced With CYP4A2 Adenovirus

Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114±1 to 133±1 mm Hg, P<0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2–transduced rats produced more 20-HETE (129±10 versus 97±7 pmol/mg protein, P<0.01) and less nitric oxide (NO; 4.2±1.6 versus 8.4±1 nmol nitrite+nitrate/mg; P<0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation.

20-Hydroxy-5,8,11,14-eicosatetraenoic Acid Mediates Endothelial Dysfunction via IκB Kinase-Dependent Endothelial Nitric-Oxide Synthase Uncoupling

Endothelial dysfunction and activation occur in the vasculature and are believed to contribute to the pathogenesis of cardiovascular diseases. We have shown that 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a cytochrome P450 4A-derived eicosanoid that promotes vasoconstriction in the microcirculation, uncouples endothelial nitric-oxide synthase (eNOS) and reduces nitric oxide (NO) levels via the dissociation of the 90-kDa heat shock protein (HSP90) from eNOS. It also causes endothelial activation by stimulating nuclear factor-κB (NF-κB) and increasing levels of pro-inflammatory cytokines. In this study, we examined signaling mechanisms that may link 20-HETE-induced endothelial dysfunction and activation. Under conditions in which 20-HETE inhibited NO production, it also stimulated inhibitor of NF-κB (IκB) phosphorylation. Both effects were prevented by inhibition of tyrosine kinases and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). It is noteworthy that inhibitor of IκB kinase (IKK) activity negated the 20-HETE-mediated inhibition of NO production. Immunoprecipitation experiments revealed that treatment of ionophore-stimulated cells with 20-HETE brings about a decrease in HSP90-eNOS association and an increase in HSP90-IKKβ association, suggesting that the activation by 20-HETE of NF-κB is linked to its action on eNOS. Furthermore, addition of inhibitors of tyrosine kinase MAPK and IKK restored the 20-HETE-mediated impairment of acetylcholine-induced relaxation in rat renal interlobar arteries. The results indicate that 20-HETE mediates eNOS uncoupling and endothelial dysfunction via the activation of tyrosine kinase, MAPK, and IKK, and these effects are linked to 20-HETE-mediated endothelial activation.

Endothelial Sirtuin 1 Deficiency Perpetrates Nephrosclerosis through Downregulation of Matrix Metalloproteinase-14: Relevance to Fibrosis of Vascular Senescence

Sirtuin 1 (SIRT1) depletion in vascular endothelial cells mediates endothelial dysfunction and premature senescence in diverse cardiovascular and renal diseases. However, the molecular mechanisms underlying these pathologic effects remain unclear. Here, we examined the phenotype of a mouse model of vascular senescence created by genetically ablating exon 4 of Sirt1 in endothelial cells (Sirt1(endo-/-)). Under basal conditions, Sirt1(endo-/-) mice showed impaired endothelium-dependent vasorelaxation and angiogenesis, and fibrosis occurred spontaneously at low levels at an early age. In contrast, induction of nephrotoxic stress (acute and chronic folic acid-induced nephropathy) in Sirt1(endo-/-) mice resulted in robust acute renal functional deterioration followed by an exaggerated fibrotic response compared with control animals. Additional studies identified matrix metalloproteinase-14 (MMP-14) as a target of SIRT1. In the kidneys of Sirt1(endo-/-) mice, impaired angiogenesis, reduced matrilytic activity, and retention of the profibrotic cleavage substrates tissue transglutaminase and endoglin accompanied MMP-14 suppression. Furthermore, restoration of MMP-14 expression in SIRT1-depeleted mice improved angiogenic and matrilytic functions of the endothelium, prevented renal dysfunction, and attenuated nephrosclerosis. Our findings establish a novel mechanistic molecular link between endothelial SIRT1 depletion, downregulation of MMP-14, and the development of nephrosclerosis.

Androgen-Dependent Hypertension Is Mediated by 20-Hydroxy-5,8,11,14-Eicosatetraenoic Acid–Induced Vascular Dysfunction: Role of Inhibitor of κB Kinase

Increased vascular synthesis of 20-HETE is associated with increased vascular contraction, endothelial dysfunction and endothelial activation; all are believed to account for 20-HETE pro-hypertensive properties. We previously demonstrated that the 20-HETE-dependent inhibition of NO production is mediated through IκB kinase (IKK) suggesting a cross talk between 20-HETE-mediated endothelial dysfunction and activation. In this study, we examined the temporal relationship among blood pressure, endothelial dysfunction and endothelial activation and the role of IKK in the rat model of androgen-driven 20-HETE-mediated hypertension. In Sprague-Dawley rats treated with 5α-dihydrotestosterone (DHT), renal vascular 20-HETE levels increased by day 2 of treatment from 17.7±2.4 to 57.7±9.7 ng/mg, while blood pressure elevation reached significance by day 3 (132.7±1.7 vs 117.2±0.8 mmHg). In renal interlobar arteries, when compared to vehicle, DHT treatment increased the sensitivity to phenylephrine-induced vasoconstriction by 3.5-fold, decreased acetylcholine-induced vasorelaxation and increased NF-kB activity, all of which were attenuated by treatment with the 20-HETE antagonist, 20-HEDE. Co-treatment with parthenolide, an IKK inhibitor, attenuated the androgen-dependent 20-HETE-mediated elevation in blood pressure (from 133.7±3.1 to 109.8±3.0 mmHg). In addition, parthenolide treatment negated 20-HETE-mediated inhibition of the relaxing response to acetylcholine and 20-HETE-mediated increase in vascular NF-kB activity. These findings suggest that inhibition of IKK attenuates the androgen-dependent 20-HETE-mediated increase in blood pressure by inhibiting both 20-HETE-dependent endothelial activation and dysfunction.Increased vascular synthesis of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is associated with increased vascular contraction, endothelial dysfunction, and endothelial activation; all are believed to account for 20-HETE prohypertensive properties. We demonstrated previously that the 20-HETE–dependent inhibition of NO production is mediated through inhibitor of &kgr;B kinase (IKK), suggesting a cross-talk between 20-HETE–mediated endothelial dysfunction and activation. In this study, we examined the temporal relationship among blood pressure, endothelial dysfunction, and endothelial activation and the role of IKK in the rat model of androgen-driven 20-HETE–mediated hypertension. In Sprague-Dawley rats treated with 5&agr;-dihydrotestosterone, renal vascular 20-HETE levels increased by day 2 of treatment from 17.7±2.4 to 57.7±9.7 ng/mg, whereas blood pressure elevation reached significance by day 3 (132.7±1.7 versus 117.2±0.8 mm Hg). In renal interlobar arteries, when compared with vehicle, 5&agr;-dihydrotestosterone treatment increased the sensitivity to phenylephrine-induced vasoconstriction by 3.5-fold, decreased acetylcholine-induced vasorelaxation, and increased nuclear factor &kgr;B activity, all of which were attenuated by treatment with the 20-HETE antagonist, 20 hydroxyeicosa-6(Z),15(Z)-dienoic acid, (20-6,15-HEDE). Cotreatment with parthenolide, an IKK inhibitor, attenuated the androgen-dependent 20-HETE–mediated elevation in blood pressure (from 133.7±3.1 to 109.8±3.0 mm Hg). In addition, parthenolide treatment negated 20-HETE–mediated inhibition of the relaxing response to acetylcholine and 20-HETE–mediated increase in vascular nuclear factor &kgr;B activity. These findings suggest that inhibition of IKK attenuates the androgen-dependent 20-HETE–mediated increase in blood pressure by inhibiting both 20-HETE–dependent endothelial activation and dysfunction.

Dual Pathways of Carbon Monoxide–Mediated Vasoregulation. Modulation by Redox Mechanisms

Rationale: Vascular tissues produce carbon monoxide (CO) via HO-dependent and HO-independent mechanisms; the former in tandem with biliverdin and iron and the latter as a lone product. CO has been shown to function as both a vasoconstrictor and vasodilator; however, factors that dictate the vasoregulatory phenotype of this gas are unknown. Objective: We investigated whether CO-mediated vasoconstriction is mechanistically linked to enhanced reactive oxygen species production that masks vasodilatory pathways. Methods and Results: Sprague–Dawley rat interlobar and interlobular arteries were examined in terms of superoxide (O2·−) generation and vascular reactivity in the absence and presence of antioxidants. Both authentic CO and the CO-releasing molecule (CORM)-3 constricted renal arteries and increased O2·− production in a dose-dependent manner. The antioxidants tempol, ebselen, and deferoxamine inhibited CO-induced O2·− production and converted CO from constrictor to dilator. CO-induced O2·− generation was found to involve the activity of multiple oxidases including nitric oxide synthase, NADPH oxidase, xanthine oxidase, and complex IV of the mitochondrial electron chain. Furthermore, inhibition of these enzymes converted CO from constrictor to dilator. Similarly, biliverdin and bilirubin inhibited CO-induced O2·− production and vasoconstriction, allowing for a vasodilatory response to CO to be expressed. CO-induced vasoconstriction was dependent on a non-thromboxane agonist of the thromboxane receptor, whereas vasodilatory mechanisms of CO relied on the activation of soluble guanylate cyclase and calcium-gated potassium channels. Conclusions: CO-induced vasoconstriction involves the generation of reactive oxygen species, which, when negated, allows for the expression of vasodilatory pathways which are masked by the primary oxidative stress response to this gas.

20-HETE Signals Through G Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension

Rationale: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction, and vascular diseases. Objective: To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo. Methods and Results: Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified G-protein receptor 75 (GPR75), currently an orphan G-protein–coupled receptor (GPCR), as a specific target of 20-HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated G&agr;q/11 protein dissociation and increased inositol phosphate accumulation and GPCR-kinase interacting protein-1-GPR75 binding, which further facilitated the c-Src–mediated transactivation of epidermal growth factor receptor. This results in downstream signaling pathways that induce angiotensin-converting enzyme expression and endothelial dysfunction. Knockdown of GPR75 or GPCR-kinase interacting protein-1 prevented 20-HETE–mediated endothelial growth factor receptor phosphorylation and angiotensin-converting enzyme induction. In vascular smooth muscle cells, GPR75–20-HETE pairing is associated with G&agr;q/11- and GPCR-kinase interacting protein-1–mediated protein kinase C–stimulated phosphorylation of MaxiK&bgr;, linking GPR75 activation to 20-HETE–mediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE–dependent hypertension prevented blood pressure elevation and 20-HETE–mediated increases in angiotensin-converting enzyme expression, endothelial dysfunction, smooth muscle contractility, and vascular remodeling. Conclusions: This is the first report to identify a GPCR target for an eicosanoid of this class. The discovery of 20-HETE–GPR75 pairing presented here provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases.

20-HETE induces remodeling of renal resistance arteries independent of blood pressure elevation in hypertension

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 (Cyp)-derived arachidonic acid metabolite that has been shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation, and promote hypertension. We examined if 20-HETE contributes to microvascular remodeling in hypertension. In Sprague-Dawley rats, administration of the 20-HETE biosynthesis inhibitor HET0016 or the 20-HETE antagonist N-20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented 5α-dihydrotestosterone (DHT)-induced increases in blood pressure as well as abrogated DHT-induced increases in the media-to-lumen ratio (M/L), media thickness, and collagen IV deposition in renal interlobar arteries. Reserpine prevented blood pressure elevation in DHT-treated rats but did not affect microvascular remodeling (M/L, media thickness, and collagen deposition); under these conditions, treatment with the 20-HETE antagonist attenuated microvascular remodeling, suggesting that 20-HETE contributes to DHT-induced vascular remodeling independent of blood pressure elevation. In Cyp4a14(-/-) mice, which display androgen-driven and 20-HETE-dependent hypertension, treatment with the 20-HETE antagonist abolished remodeling of renal resistance arteries measured as media thickness (24 ± 1 vs. 15 ± 1 μm) and M/L (0.29 ± 0.03 vs. 0.17 ± 0.01). Moreover, in Cyp4a12 transgenic mice in which the expression of Cyp4a12-20-HETE synthase is driven by a tetracycline-sensitive promoter, treatment with doxycycline resulted in blood pressure elevation (140 ± 4 vs. 92 ± 5 mmHg) and a significant increase in remodeling of renal resistance arteries (media thickness: 23 ± 1 vs. 16 ± 1 μm; M/L: 0.39 ± 0.04 vs. 0.23 ± 0.02); these increases were abrogated by cotreatment with 20-HEDE. This study demonstrated that 20-HETE is a key regulator of microvascular remodeling in hypertension; its effect is independent of blood pressure elevation and androgen levels.

Androgen-induced hypertension in angiotensinogen deficient mice: role of 20-HETE and EETS.

20-HETE is a potent inducer of endothelial ACE in vitro and administration of lisinopril or losartan attenuates blood pressure in models of 20-HETE-dependent hypertension. The present study was undertaken to further define the relationship between 20-HETE and the renin-angiotensin system in hypertension using an angiotensinogen-deficient mouse (Agt+/-). Treatment of male AGT+/- with 5α-dihydrotestosterone (DHT) increased systolic BP from 102±2 to 125±3mmHg; in comparison, the same treatment raised BP in wild type (WT) from 110±2 to 138±2mmHg. DHT increased vascular 20-HETE levels in AGT+/- and WT from 1.5±0.7 and 2.1±0.6 to 13.0±2.0 and 15.8±4.0ng/mg, respectively. Concurrent treatment with the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented the increases in BP in both AGT+/- and WT mice. Administration of 20-HEDE at the peak of the DHT-induced BP increase (12 days) reduced BP to basal levels after 48h. Interestingly, basal levels of renal microvascular EETs were higher in AGT+/- compared to WT (55.2±9.7 vs 20.0±4.1ng/mg) and treatment of AGT+/- with DHT decreased the levels of EETs (28.4±5.1ng/mg). DHT-mediated changes in vascular EET level were not observed in WT mice. Vascular Cyp4a12 and ACE protein levels were increased in both AGT+/- and WT by 30-40% and decreased with concomitant administration of 20-HEDE. Lisinopril was as effective as 20-HEDE in preventing DHT-mediated increases in BP in both AGT+/- and WT mice. This study substantiates our previous findings that the RAS plays an important role in 20-HETE-mediated hypertension. It also proposes a novel interaction between 20-HETE and EETs.

Angiotensin II receptor blockade or deletion of vascular endothelial ACE does not prevent vascular dysfunction and remodeling in 20-HETE-dependent hypertension

Increased vascular 20-HETE is associated with hypertension and activation of the renin-angiotensin system (RAS) through induction of vascular angiotensin-converting enzyme (ACE) expression. Cyp4a12tg mice, whose Cyp4a12-20-HETE synthase expression is under the control of a tetracycline (doxycycline, DOX) promoter, were used to assess the contribution of ACE/RAS to microvascular remodeling in 20-HETE-dependent hypertension. Treatment of Cyp4a12tg mice with DOX increased systolic blood pressure (SBP; 136 ± 2 vs. 102 ± 1 mmHg; P < 0.05), and this increase was prevented by administration of 20-HEDGE, lisinopril, or losartan. DOX-induced hypertension was associated with microvascular dysfunction and remodeling of preglomerular microvessels, which was prevented by 20-HEDGE, a 20-HETE antagonist, yet only lessened, but not prevented, by lisinopril or losartan. In ACE 3/3 mice, which lack vascular endothelial ACE, administration of 5α-dihydrotestosterone (DHT), a known inducer of 20-HETE production, increased SBP; however, the increase was about 50% of that in wild-type (WT) mice (151 ± 1 vs. 126 ± 1 mmHg). Losartan and 20-HEDGE prevented the DHT-induced increase in SBP in WT and ACE 3/3 mice. DHT treatment increased 20-HETE production and microvascular remodeling in WT and ACE 3/3 mice; however, remodeling was attenuated in the ACE 3/3 mice as opposed to WT mice (15.83 ± 1.11 vs. 22.17 ± 0.92 μm; P < 0.05). 20-HEDGE prevented microvascular remodeling in WT and ACE 3/3 mice, while losartan had no effect on microvascular remodeling in ACE 3/3. Taken together, these results suggest that RAS contributes to 20-HETE-mediated microvascular remodeling in hypertension and that 20-HETE-driven microvascular remodeling independent of blood pressure elevation does not fully rely on ACE activity in the vascular endothelium.