Volkmar Gross

Increased Vascular Smooth Muscle Contractility in TRPC6−/− Mice

ABSTRACT Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6 − / − smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6 −/− smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.

Impaired Endothelium-Derived Hyperpolarizing Factor-Mediated Dilations and Increased Blood Pressure in Mice Deficient of the Intermediate-Conductance Ca2+-Activated K+ Channel

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca2+-activated K+ channels (KCa), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance KCa (IKCa/KCa3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of KCa3.1. KCa3.1-deficient mice (KCa3.1−/−) were generated by conventional gene-targeting strategies. Endothelial KCa currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the KCa3.1 gene abolished endothelial KCa3.1 currents and significantly diminished overall current through KCa channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in KCa3.1−/− mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in KCa3.1−/− mice. Moreover, the loss of KCa3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial KCa3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Hypertension-Induced End-Organ Damage: A New Transgenic Approach to an Old Problem

Angiotensin (Ang) II-induced organ damage has fascinated students of hypertension since the work of Wilson and Byrom. We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the vascular wall accompanies PAI-1, MCP-1, and VEGF expression. The expression of TGF-beta and deposition of extracellular matrix proteins follows, which is accompanied by fibrinoid vasculitis in small vessels of the heart and kidneys. Angiotensin-converting enzyme inhibitors and AT1 receptor blockers each lowered blood pressure and shifted pressure natriuresis partially leftward by different mechanisms. When combined, they normalized blood pressure, pressure natriuresis, and protected from vasculopathy completely. Renin inhibition lowered blood pressure partially, but protected from vasculopathy completely. Endothelin receptor blockade had no influence on blood pressure but protected from vasculopathy and improved survival. We show evidence that Ang II stimulates oxidative stress directly or indirectly via endothelin 1 and that NFkappaB is upregulated in this model. We speculate that the transcription factors NFkappaB and AP-1 are involved with initiating chemokine and cytokine expression, leading to the above cascade. The unique model and our pharmacological probes will enable us to test these hypotheses.

Endothelial dysfunction and elevated blood pressure in MAS gene-deleted mice.

Mas codes for a G protein–coupled receptor that is implicated in angiotensin-(1-7) signaling. We studied the cardiovascular phenotype of Mas-deficient mice backcrossed onto the FVB/N genetic background using telemetry and found that they exhibit higher blood pressures compared with controls. These Mas−/− mice also had impaired endothelial function, decreased NO production, and lower endothelial NO synthase expression. Reduced nicotinamide-adenine dinucleotide phosphate oxidase catalytic subunit gp91phox protein content determined by Western blotting was higher in Mas−/− mice than in controls, whereas superoxide dismutase and catalase activities were reduced. The superoxide dismutase mimetic, Tempol, decreased blood pressure in Mas−/− mice but had a minimal effect in control mice. Our results show a major cardiovascular phenotype in Mas−/− mice. Mas-deletion results in increased blood pressure, endothelial dysfunction, and an imbalance between NO and reactive oxygen species. Our animals represent a promising model to study angiotensin-(1-7)–mediated cardiovascular effects and to evaluate Mas agonistic compounds as novel cardioprotective and antihypertensive agents based on their beneficial effects on endothelial function.

Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease

We aimed to identify genetic variants associated with heart failure by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F2 crosses between spontaneously hypertensive heart failure (SHHF) rats and reference strains. We combined linkage analyses with genome-wide expression profiling and identified Ephx2 as a heart failure susceptibility gene in SHHF rats. Specifically, we found that cis variation at Ephx2 segregated with heart failure and with increased transcript expression, protein expression and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we tested the role of Ephx2 in heart failure using knockout mice. Ephx2 gene ablation protected from pressure overload–induced heart failure and cardiac arrhythmias. We further demonstrated differential regulation of EPHX2 in human heart failure, suggesting a cross-species role for Ephx2 in this complex disease.

Blood Pressure–Independent Effects in Rats With Human Renin and Angiotensinogen Genes

The blood pressure-independent effects of angiotensin II (Ang II) were examined in double transgenic rats (dTGR) harboring human renin and human angiotensinogen genes, in which the end-organ damage is due to the human components of the renin angiotensin system. Triple-drug therapy (hydralazine 80 mg/L, reserpine 5 mg/L, and hydrochlorothiazide 25 mg/L in drinking water) was started immediately after weaning. Triple-drug therapy normalized blood pressure and coronary resistance, only partially prevented cardiac hypertrophy, and had no effect on ratio of renal weight to body weight. Although triple-drug therapy delayed the onset of renal damage, severe albuminuria nevertheless occurred. Semiquantitative scoring of ED-1-positive and MIB-5-positive (nuclear cell proliferation-associated antigen Ki-67) cells showed profound perivascular monocyte/macrophage infiltration and cell proliferation in kidneys and hearts of untreated dTGR. Triple-drug therapy had only a minimal effect on local inflammatory response or vascular cell proliferation. In contrast, a novel orally active human renin inhibitor (HRI), 30 mg/kg by gavage for 4 weeks, normalized blood pressure and coronary resistance and also prevented cardiac hypertrophy and albuminuria. ED-1-positive cells and MIB-5-positive cells were decreased by HRI in hearts and kidneys almost to levels observed in normotensive Sprague-Dawley rats. The renoprotective effects of HRI were at least in part due to improved renal hemodynamics and distal tubular function, since HRI shifted renal pressure-diuresis/natriuresis curves leftward by approximately 35 mm Hg, increased glomerular filtration rate and renal blood flow, and shifted the fractional water and sodium excretion curves leftward. In untreated dTGR, plasma Ang II was increased by 400% and renal Ang II level was increased by 300% compared with Sprague-Dawley rats. HRI decreased plasma human renin activity by 95% and normalized Ang II levels in both plasma and kidney compared with triple-drug therapy. Our findings indicate that in dTGR harboring human renin and angiotensinogen genes, Ang II causes end-organ damage and promotes inflammatory response and cellular growth largely independent of blood pressure.

Mouse Cyp4a isoforms: enzymatic properties, gender- and strain-specific expression, and role in renal 20-hydroxyeicosatetraenoic acid formation

AA (arachidonic acid) hydroxylation to 20-HETE (20-hydroxyeicosatetraenoic acid) influences renal vascular and tubular function. To identify the CYP (cytochrome P450) isoforms catalysing this reaction in the mouse kidney, we analysed the substrate specificity of Cyp4a10, 4a12a, 4a12b and 4a14 and determined sex- and strain-specific expressions. All recombinant enzymes showed high lauric acid hydroxylase activities. Cyp4a12a and Cyp4a12b efficiently hydroxylated AA to 20-HETE with V(max) values of approx. 10 nmol x nmol(-1) x min(-1) and K(m) values of 20-40 microM. 20-Carboxyeicosatetraenoic acid occurred as a secondary metabolite. AA hydroxylase activities were approx. 25-75-fold lower with Cyp4a10 and not detectable with Cyp4a14. Cyp4a12a and Cyp4a12b also efficiently converted EPA (eicosapentaenoic acid) into 19/20-OH- and 17,18-epoxy-EPA. In male mice, renal microsomal AA hydroxylase activities ranged between approx. 100 (NMRI), 45-55 (FVB/N, 129 Sv/J and Balb/c) and 25 pmol x min(-1) x mg(-1) (C57BL/6). The activities correlated with differences in Cyp4a12a protein and mRNA levels. Treatment with 5alpha-dihydrotestosterone induced both 20-HETE production and Cyp4a12a expression more than 4-fold in male C57BL/6 mice. All female mice showed low AA hydroxylase activities (15-25 pmol x min(-1) x mg(-1)) and very low Cyp4a12a mRNA and protein levels, but high Cyp4a10 and Cyp4a14 expression. Renal Cyp4a12b mRNA expression was almost undetectable in both sexes of all strains. Thus Cyp4a12a is the predominant 20-HETE synthase in the mouse kidney. Cyp4a12a expression determines the sex- and strain-specific differences in 20-HETE generation and may explain sex and strain differences in the susceptibility to hypertension and target organ damage.

Reduction of circulating soluble Flt-1 alleviates preeclampsia-like symptoms in a mouse model

Preeclampsia (PE) is characterized by widespread endothelial damage with hypertension, proteinuria, glomeruloendotheliosis and elevated soluble Flt‐1 (sFlt‐1), a natural occurring antagonist of vascular endothelial growth factor (VEGF). Cancer patients receiving anti‐VEGF therapy exhibit similar symptoms. We suggested that a decrease in circulating sFlt‐1 would alleviate the symptoms associated with PE. Adenoviral (Adv) overexpression of sFlt‐1 induced proteinuria, caused glomerular damage and increase in blood pressure in female Balb/c mice. Circulating level of sFlt‐1 above 50 ng/ml plasma induced severe vascular damage and glomerular endotheliosis. Albumin concentration in urine was elevated up to 30‐fold, compared to control AdvGFP‐treated animals. The threshold of kidney damage was in the range of 20–30 ng/ml sFlt‐1 in plasma (8–15 ng/ml in urine). Co‐administration of AdvsFlt‐1 with AdvVEGF to neutralize circulating sFlt‐1 resulted in more than a 70% reduction in free sFlt‐1 in plasma, more than 80% reduction in urine and rescued the damaging effect of sFlt‐1 on the kidneys. This demonstrates that below a critical threshold sFlt‐1 fails to elicit damage to the fenestrated endothelium and that co‐expression of VEGF is able to rescue effects mediated by sFlt‐1 overexpression.

Interaction Between P450 Eicosanoids and Nitric Oxide in the Control of Arterial Tone in Mice

Objective—Epoxyeicosatrienoic acids (EETs) serve as endothelial-derived hyperpolarizing factors (EDHF), but may also affect vascular function by other mechanisms. We identified a novel interaction between EETs and endothelial NO release using soluble epoxide hydrolase (sEH) −/− and +/+ mice. Methods and Results—EDHF responses to acetylcholine in pressurized isolated mesenteric arteries were neither affected by the sEH inhibitor, N-adamantyl-N′-dodecylurea (ADU), nor by sEH gene deletion. However, the EDHF responses were abolished by catalase and by apamin/charybdotoxin (ChTx), but not by iberiotoxin, nor by the cytochrome P450 inhibitor PPOH. All four EETs (order of potency: 8,9-EET >14,15-EET≈5,6-EET >11,12-EET) and all 4 dihydroxy derivatives (14,15-DHET≈8,9-DHET≈11,12-DHET >5,6-DHET) produced dose-dependent vasodilation. Endothelial removal or L-NAME blocked 8,9-EET and 14,15-DHET-dependent dilations. The effects of apamin/ChTx were minimal. 8,9-EET and 14,15-DHET induced NO production in endothelial cells. ADU (100 &mgr;g/mL in drinking water) lowered blood pressure in angiotensin II–infused hypertension, but not in L-NAME–induced hypertension. Blood pressure and EDHF responses were similar in L-NAME–treated sEH +/+ and −/− mice. Conclusions—Our data indicate that the EDHF response in mice is caused by hydrogen peroxide, but not by P450 eicosanoids. Moreover, P450 eicosanoids are vasodilatory, largely through their ability to activate endothelial NO synthase (eNOS) and NO release.

Vascular Endothelial Cell-Specific NF-κB Suppression Attenuates Hypertension-Induced Renal Damage

Nuclear factor kappa B (NF-&kgr;B) participates in hypertension-induced vascular and target-organ damage. We tested whether or not endothelial cell–specific NF-&kgr;B suppression would be ameliorative. We generated Cre/lox transgenic mice with endothelial cell–restricted NF-&kgr;B super-repressor I&kgr;B&agr;&Dgr;N (Tie-1-&Dgr;N mice) overexpression. We confirmed cell-specific I&kgr;B&agr;&Dgr;N expression and reduced NF-&kgr;B activity after TNF-&agr; stimulation in primary endothelial cell culture. To induce hypertension with target-organ damage, we fed mice a high-salt diet and N(omega)-nitro-l-arginine-methyl-ester (L-NAME) and infused angiotensin (Ang) II. This treatment caused a 40-mm Hg blood pressure increase in both Tie-1-&Dgr;N and control mice. In contrast to control mice, Tie-1-&Dgr;N mice developed a milder renal injury, reduced inflammation, and less albuminuria. RT-PCR showed significantly reduced expression of the NF-&kgr;B targets VCAM-1 and ICAM-1, compared with control mice. Thus, the data demonstrate a causal link between endothelial NF-&kgr;B activation and hypertension-induced renal damage. We conclude that in vivo NF-&kgr;B suppression in endothelial cells stops a signaling cascade leading to reduced hypertension-induced renal damage despite high blood pressure.