Meng Luo

EETs Elicit Direct Increases in Pulmonary Arterial Pressure in Mice.

OBJECTIVE The biological role of epoxyeicosatrienoic acids (EETs) in the regulation of pulmonary circulation is currently under debate. We hypothesized that EETs initiate increases in right ventricular systolic pressure (RVSP) via perhaps, pulmonary vasoconstriction. METHODS Mice were anesthetized with isoflurane. Three catheters, inserted into the left jugular vein, the left carotid artery, and the right jugular vein, were used for infusing EETs, monitoring blood pressure (BP), and RVSP respectively. BP and RVSP were continuously recorded at basal conditions, in response to administration of 4 regioisomeric EETs (5,6-EET; 8,9-EET; 11,12-EET, and 14,15-EET; 1, 2, 5 and 10 ng/g body weight (BW) for each EET), and during exposure of mice to hypoxia. RESULTS All 4 EETs initiated dose-dependent increases in RVSP, though reduced BP. 11,12-EET elicited the greatest increment in RVSP among all EET isoforms. To clarify the direct elevation of RVSP in a systemic BP-independent manner, equivalent amounts of 14,15-EET were injected over 1 and 2 minutes respectively. One-minute injection of 14,15-EET elicited significantly faster and greater increases in RVSP than the 2-minute injection, whereas their BP changes were comparable. Additionally, direct injection of low doses of 14,15-EET (0.1, 0.2, 0.5, and 1 ng/g BW) into the right ventricle caused significant increases in RVSP without effects on BP, confirming that systemic vasodilation-induced increases in venous return are not the main cause for the increased RVSP. Acute exposure of mice to hypoxia significantly elevated RVSP, as well as 14,15-EET-induced increases in RVSP. CONCLUSIONS EETs directly elevate RVSP, a response that may play an important role in the development of hypoxia-induced pulmonary hypertension (PH).

EET-dependent potentiation of pulmonary arterial pressure: sex-different regulation of soluble epoxide hydrolase.

We tested the hypothesis that suppression of epoxyeicosatrienoic acid (EET) metabolism via genetic knockout of the gene for soluble epoxide hydrolase (sEH-KO), or female-specific downregulation of sEH expression, plays a role in the potentiation of pulmonary hypertension. We used male (M) and female (F) wild-type (WT) and sEH-KO mice; the latter have high pulmonary EETs. Right ventricular systolic pressure (RVSP) and mean arterial blood pressure (MABP) in control and in response to in vivo administration of U46619 (thromboxane analog), 14,15-EET, and 14,15-EEZE [14,15-epoxyeicosa-5(z)-enoic acid; antagonist of EETs] were recorded. Basal RVSP was comparable among all groups of mice, whereas MABP was significantly lower in F-WT than M-WT mice and further reduced predominantly in F-KO compared with M-KO mice. U46619 dose dependently increased RVSP and MABP in all groups of mice. The increase in RVSP was significantly greater and coincided with smaller increases in MABP in M-KO and F-WT mice compared with M-WT mice. In F-KO mice, the elevation of RVSP by U46619 was even higher than in M-KO and F-WT mice, associated with the least increase in MABP. 14,15-EEZE prevented the augmentation of U46619-induced elevation of RVSP in sEH-KO mice, whereas 14,15-EET-induced pulmonary vasoconstriction was comparable in all groups of mice. sEH expression in the lungs was reduced, paralleled with higher levels of EETs in F-WT compared with M-WT mice. In summary, EETs initiate pulmonary vasoconstriction but act as vasodilators systemically. High pulmonary EETs, as a function of downregulation or deletion of sEH, potentiate U46619-induced increases in RVSP in a female-susceptible manner.

Sexually dimorphic phenotype of arteriolar responsiveness to shear stress in soluble epoxide hydrolase-knockout mice

We hypothesized that potentiating the bioavailability of endothelial epoxyeicosatrienoic acids (EETs) via deletion of the gene for soluble epoxide hydrolase (sEH), or downregulation of sEH expression, enhances flow/shear stress-induced dilator responses (FID) of arterioles. With the use of male (M) and female (F) wild-type (WT) and sEH-knockout (KO) mice, isolated gracilis muscle arterioles were cannulated and pressurized at 80 mmHg. Basal tone and increases in diameter of arterioles as a function of perfusate flow (5, 10, 15, 20, and 25 μl/min) were recorded. The magnitude of FID was significantly smaller and associated with a greater arteriolar tone in M-WT than F-WT mice, revealing a sex difference in FID. This sex difference was abolished by deletion of the sEH gene, as evidenced by an enhanced FID in M-KO mice to a level comparable with those observed in F-KO and F-WT mice. These three groups of mice coincidentally exhibited an increased endothelial sensitivity to shear stress (smaller WSS50) and were hypotensive. Endothelial EETs participated in the mediation of enhanced FID in M-KO, F-KO, and F-WT mice, without effects on FID of M-WT mice. Protein expression of sEH was downregulated by approximately fourfold in vessels of F-WT compared with M-WT mice, paralleled with greater vascular EET levels that were statistically comparable with those observed in both male and female sEH-KO mice. In conclusion, sex-different regulation of sEH accounts for sex differences in flow-mediated dilation of microvessels in gonadally intact mice.

Female-favorable attenuation of coronary myogenic constriction via reciprocal activations of epoxyeicosatrienoic acids and nitric oxide

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid via CYP/epoxygenases, which are catabolized by soluble epoxide hydrolase (sEH) and known to possess cardioprotective properties. To date, the role of sEH in the modulation of pressure-induced myogenic response/constriction in coronary arteries, an important regulatory mechanism in the coronary circulation, and the issue as to whether the disruption of the sEH gene affects the myogenic response sex differentially have never been addressed. To this end, experiments were conducted on male (M) and female (F) wild-type (WT) and sEH-knockout (KO) mice. Pressure-diameter relationships were assessed in isolated and cannulated coronary arteries. All vessels constricted in response to increases in intraluminal pressure from 60 to 120 mmHg. Myogenic vasoconstriction was significantly attenuated, expressed as an upward shift in the pressure-diameter curve of vessels, associated with higher cardiac EETs in M-KO, F-WT, and F-KO mice compared with M-WT controls. Blockade of EETs via exposure of vessels to 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) prevented the attenuated myogenic constriction in sEH-KO mice. In the presence of 14,15-EEZE, pressure-diameter curves of females presented an upward shift from those of males, exhibiting a sex-different phenotype. Additional administration of N(ω)-nitro-l-arginine methyl ester eliminated the sex difference in myogenic responses, leading to four overlapped pressure-diameter curves. Cardiac sEH was downregulated in F-WT compared with M-WT mice, whereas expression of endothelial nitric oxide synthase and CYP4A (20-HETE synthase) was comparable among all groups. In summary, in combination with NO, the increased EET bioavailability as a function of genetic deletion and/or downregulation of sEH accounts for the female-favorable attenuation of pressure-induced vasoconstriction.

Sexually Dimorphic Adaptation of Cardiac Function: Roles of Epoxyeicosatrienoic Acid and Peroxisome Proliferator-Activated Receptors

Epoxyeicosatrienoic acids (EETs) are cardioprotective mediators metabolized by soluble epoxide hydrolase (sEH) to form corresponding diols (DHETs). As a sex‐susceptible target, sEH is involved in the sexually dimorphic regulation of cardiovascular function. Thus, we hypothesized that the female sex favors EET‐mediated potentiation of cardiac function via downregulation of sEH expression, followed by upregulation of peroxisome proliferator‐activated receptors (PPARs). Hearts were isolated from male (M) and female (F) wild‐type (WT) and sEH‐KO mice, and perfused with constant flow at different preloads. Basal coronary flow required to maintain the perfusion pressure at 100 mmHg was significantly greater in females than males, and sEH‐KO than WT mice. All hearts displayed a dose‐dependent decrease in coronary resistance and increase in cardiac contractility, represented as developed tension in response to increases in preload. These responses were also significantly greater in females than males, and sEH‐KO than WT. 14,15‐EEZE abolished the sex‐induced (F vs. M) and transgenic model‐dependent (KO vs. WT) differences in the cardiac contractility, confirming an EET‐driven response. Compared with M‐WT controls, F‐WT hearts expressed downregulation of sEH, associated with increased EETs and reduced DHETs, a pattern comparable to that observed in sEH‐KO hearts. Coincidentally, F‐WT and sEH‐KO hearts exhibited increased PPARα expression, but comparable expression of eNOS, PPARβ, and EET synthases. In conclusion, female‐specific downregulation of sEH initiates an EET‐dependent adaptation of cardiac function, characterized by increased coronary flow via reduction in vascular resistance, and promotion of cardiac contractility, a response that could be further intensified by PPARα.

Inhibition of soluble epoxide hydrolase reduces portal pressure by protecting mesenteric artery myogenic responses in cirrhotic rats

Hyperdynamic circulation contributes to the progress of portal hypertension in liver cirrhosis. We investigated the effects of soluble epoxide hydrolase (sEH) inhibition on portal pressure and the myogenic response of mesenteric arteries isolated from cirrhotic rats using the sEH inhibitor t-TUCB (trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]cyclohexyloxy}benzoic acid). Cirrhotic tissues had a higher ratio of epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs) following increased CYP2C11 expression, which may be a protective response. In comparison with controls, myogenic responses of mesenteric arteries from cirrhotic rats were attenuated at 80-140mmHg, while inhibition of sEH partly reversed the impaired myogenic constriction at 100-140mmHg and exhibited better feedback of vascular smooth muscle to pressure variation. Inhibition of sEH reduced portal pressure by decreasing endothelial synthesis of nitric oxide. An imbalance between EETs and nitric oxide may account for hyperdynamic circulation. sEH inhibitors may provide a novel approach for treating cirrhosis of the liver.

Intrahepatic upregulation of MRTF-A signaling contributes to increased hepatic vascular resistance in cirrhotic rats with portal hypertension.

BACKGROUND Portal hypertension in cirrhosis is mediated, in part, by increased intrahepatic resistance, reflecting massive structural changes associated with fibrosis and intrahepatic vasoconstriction. Activation of the Rho/MRTF/SRF signaling pathway is essential for the cellular regulatory network of fibrogenesis. The aim of this study was to investigate MRTF-A-mediated regulation of intrahepatic fibrogenesis in cirrhotic rats. METHODS Portal hypertension was induced in rats via an injection of CCl4 oil. Hemodynamic measurements were obtained using a polyethylene PE-50 catheter and pressure transducers. Expression of hepatic fibrogenesis was measured using histological staining. Expression of protein was measured using western blotting. RESULTS Upregulation of MRTF-A protein expression in the livers of rats with CCl4-induced cirrhosis was relevant to intrahepatic resistance and hepatic fibrogenesis in portal hypertensive rats with increased modeling time. Inhibition of MRTF-A by CCG-1423 decelerated hepatic fibrosis, decreased intrahepatic resistance and portal pressure, and alleviated portal hypertension. CONCLUSION Increased intrahepatic resistance in rats with CCl4-induced portal hypertension is associated with an upregulation of MRTF-A signaling. Inhibition of this pathway in the liver can decrease hepatic fibrosis and intrahepatic resistance, as well as reduce portal pressure in cirrhotic rats with CCl4-induced portal hypertension.

Effects of NLRP6 on the proliferation and activation of human hepatic stellate cells

&NA; Nod‐like receptor pyrin domain‐containing proteins (NLRPs) are known to take part in the pathogenesis of chronic liver diseases, including liver fibrosis. However, no known direct role of NLRP6, a member of NLRPs, has been reported in liver fibrosis. Here, we found that NLRP6 expression was decreased in fibrotic and cirrhotic livers. In a human hepatic stellate cell line, LX‐2, overexpression of NLRP6 suppressed cell proliferation, hydroxyproline accumulation, as well as the expression of type I and type III collagens (Col‐I and Col‐III), &agr;‐smooth muscle actin (&agr;‐SMA) and matrix metalloproteinases (MMP2 and MMP9), whereas NLRP6 knockdown displayed reverse effects. Furthermore, NLRP6 significantly suppressed the phosphorylation of Smad2/3 (p‐Smad2/3) and enhanced the expression of protein phosphatase magnesium dependent 1 A (PPM1A), the only phosphatase for Smad2/3. NLRP6 overexpression abrogated TGF‐&bgr;1‐stimulated hydroxyproline accumulation and p‐Smad2/3. Co‐immunoprecipitation assay demonstrated that NLRP6 was able to form a complex with PPM1A. NLRP6 overexpression did not change the level of p‐Smad2/3 in LX‐2 cells with PPM1A knockdown. These data indicated that PPM1A was required for the inhibitory effects of NLRP6 on TGF‐&bgr;1/Smad2/3 signaling. In conclusion, our results suggest that NLRP6 exerts anti‐fibrotic effects in LX‐2 cells via regulating PPM1A/Smad2/3 and that NLRP6 may be an effective target in the treatment of liver fibrosis.

Comparison of physical parameter measurements between peripheral and portal blood samples in patients with portal hypertension

BACKGROUND Measuring portal venous pressure is necessary to examine, diagnose, and treat portal hypertension, but current methods are invasive. OBJECTIVE This study aimed to determine whether a noninvasive peripheral blood measurement could be used to estimate portal venous pressure by investigating correlations between certain physical parameter measurements in the peripheral blood with those obtained in portal blood samples. METHODS A total of 128 peripheral and portal blood samples from patients (n= 128) were analyzed for blood rheology and routine blood parameters. RESULTS The mean peripheral and portal whole blood viscosities under the shear rates of 200 s-1 (BV 200 s-1) were 2.97 ± 0.50 mPa.s and 3.06 ± 0.39 mPa.s. The mean peripheral and portal BV 30 s-1 values were 3.96 ± 0.79 mPa.s and 4.16 ± 0.64 mPa.s. We observed strong correlations between peripheral and portal blood measurements of BV 200 s-1 (r2= 0.9649), BV 30 s-1 (r2= 0.9622), BV 5 s-1 (r2= 0.9610), and BV 1 s-1 (r2= 0.9623). CONCLUSIONS Our results indicate that peripheral blood can be used to evaluate certain parameters in portal blood for use in biofluid mechanics studies, and to provide noninvasive measurement of portal venous pressure.

Soluble epoxide hydrolase inhibition with t-TUCB alleviates liver fibrosis and portal pressure in carbon tetrachloride-induced cirrhosis in rats

BACKGROUND/AIMS Fibrosis and increased intrahepatic vascular resistance are the hallmarks of chronic inflammatory disorders of the liver and cirrhosis. Inhibitors of the enzyme soluble epoxide hydrolase reduce fibrosis in several disease models. The present study aimed at investigating the effects of soluble epoxyhydrolase inhibition with t-TUCB in tetrachloride-induced cirrhosis in rats. METHODS The models were established by CCl4 (2ml/kg) given subcutaneously for 14 weeks. t-TUCB was concomitantly administered from the tenth week of modelling time. After the models were successfully built, the rats were anesthetized with sodium phenobarbital and portal pressure was determined in the groups. After that, the rats were killed and part of liver tissues were taken for histological analysis. In addition, the levels of intrahepatic inflammatory message factors were measured using real-time polymerase chain reaction (PCR) analysis. The remaining liver samples were processed for assessment of oxidative stress. RESULTS t-TUCB administration significantly attenuated portal pressure relative to CCl4-only rats. This improvement was associated with decreased deposition of collagen in liver, which was supported by reduced mRNA expression of α-smooth muscle actin (α-SMA), Collagen I, Collagen III, transforming growth factor (TGF)-β and tissue inhibitor of metalloproteinase-1 (TIMP-1) and increased matrix metalloproteinase-1, -13 (MMP-1, -13) mRNA expression. In addition, t-TUCB decreased the levels of proinflammatory cytokines, including IL-1β, IL-6, IL-10, tumor necrosis factor-α (TNF-α) and NF-κB, within cirrhotic hepatic tissue. Meanwhile, oxidative stress was also alleviated following inhibition of sEH in CCl4-induced models, as evidenced by down-regulated levels of malondialdehyde (MDA) and up-regulated levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). CONCLUSION The soluble epoxyhydrolase inhibitor, t-TUCB alleviates liver fibrosis and portal hypertension through attenuation of inflammatory response and oxidative stress in tetrachloride induced cirrhosis.