Blockade of Mas Receptor or Mas-Related G-Protein Coupled Receptor Type D Reduces Portal Pressure in Cirrhotic but Not in Non-cirrhotic Portal Hypertensive Rats

Figures

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  Figure 1. Schematic representation of the renin angiotensin system (RAS) showing the pathways responsible for the generation of angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)]. Ang II acts via its type 1 receptor (AT1R) to exert vasoconstrictive effects. Ang II is degraded to Ang-(1-7) by angiotensin converting enzyme 2 (ACE2). Ang-(1-7) opposes Ang II effects through its receptors, MasR and MrgD.

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  Figure 2. Schematic representation of the experimental plan adopted in the pressure measurement study. Thirty minutes after the cannulation of portal vein and femoral artery, the angiotensin receptor blockers were administered as a bolus injection via the femoral vein catheter. In the experiments using a single receptor blocker injection of either A779, D-Pro, or PD123319, pressure measurement was continued for 30 min after the injection (A). In the experiments using combined treatment, losartan was injected first, followed by either A779 or D-Pro 20 min later. The pressure measurement was continued for a total of 35 min after losartan injection (B).

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  Figure 3. Changes in portal pressure (PP) after intravenous bolus injections of either the MasR blocker A779 (10 μg/kg), MrgD blocker D-Pro (10 μg/kg), or AT2R blocker PD123319 (1 mg/kg) in CCl4 (A), BDL (B), and PPVL (C) models. Saline injection served as the control. Pressure measurement was commenced 5 min prior to injection and continued for 30 min after the injection. Each time point represents the mean ± SEM profile from six to seven rats per treatment group. ****p < 0.05, **p < 0.01, *p < 0.05 baseline vs. A779, ####p < 0.05, ###p < 0.005, #p < 0.05 baseline vs. D-Pro, θθθθp < 0.05, θθθp < 0.01, θp < 0.05 baseline vs. PD123319.

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  Figure 4. Changes in mean arterial pressure (MAP) with intravenous bolus injections of either MasR blocker A779 (10 μg/kg), MrgD blocker D-Pro (10 μg/kg) or AT2R blocker PD123319 (1 mg/kg) in CCl4 (A) BDL (B), and PPVL (C) models. Saline injection served as the control. Each time point represents the mean ± SEM profile from six to seven rats per treatment group. *p < 0.05 baseline vs. A779; ##p < 0.01, #p < 0.05, baseline vs. D-Pro.

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  Figure 5. Changes in mean arterial pressure after the treatment with AT1R blocker losartan (1 mg/kg) in the CCl4 (A) and BDL (B) models. Note that the two groups that received losartan injections in each model were pooled for t-test analysis. Bottom panels show mean arterial pressure of the two losartan groups after receiving either MasR blocker A779 (10 μg/kg) or MrgD blocker D-Pro (10 μg/kg) in the CCl4 (C) and BDL (D) models. Losartan significantly reduced MAP in all groups; however, A779 or D-Pro which was given 20 min after losartan failed to counteract the hypotensive effect of losartan. Each time point represents the mean ± SEM profile from 12 to 14 (A,B) or 6 to 7 (C,D) rats per treatment group. Data in (C) and (D) were analyzed by repeated-measures ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0005 baseline vs. post-losartan injection.

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  Figure 6. Changes in portal pressure after the treatment with AT1R blocker losartan (1 mg/kg) in the CCl4 (A) and BDL (B) models. Note that the two groups that received losartan injections in each model were pooled for t-test analysis. Bottom panels show portal pressure of the two losartan groups after receiving either MasR blocker A779 (10 μg/kg) or MrgD blocker D-Pro (10 μg/kg) in the CCl4 (C) and BDL (D) models. Losartan significantly reduced portal pressure in all groups; however, A779 or D-Pro which was given 20 min after losartan failed to affect portal pressure. Each time point represents the mean ± SEM profile from 12 to 14 (A,B) or 6 to 7 (C,D) rats per treatment group. Data in (C) and (D) were analyzed by repeated-measures ANOVA. **p < 0.01, ***p < 0.001, ****p < 0.0005 baseline vs. post-losartan injection.

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  Figure 7. Plasma concentrations of Ang-(1-7) peptide (10 μg/kg) and peptide receptor blockers A779 (10 μg/kg) and D-Pro (10 μg/kg) after a bolus injection of intravenous administration. Each time point represents the mean ± SEM profile from 4 rats per treatment group. ****p < 0.005 concentration of each peptide at 2 min vs. baseline.

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  Figure 8. Receptor gene expression of MasR (A), MrgD (B), and AT1R (C) analyzed by qPCR in mesenteric vascular bed of the CCl4, BDL and PPVL rats compared with sham-operated and healthy control rats. Data have been normalized to endogenous control gene 18S, and healthy control group was given an arbitrary value of 1. Each time point represents the mean ± SEM profile from six to seven rats per treatment group.

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  Figure 9. Receptor gene expression of MasR (A), MrgD (B), and AT1R (C) analyzed by qPCR in the livers of the CCl4, BDL, and PPVL rats compared with sham-operated and healthy control rats. Data have been normalized to endogenous control gene 18S, and healthy control group was given an arbitrary value of 1. Each time point represents the mean ± SEM profile from six to seven rats per treatment group.

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  Figure 10. Immunohistochemical localization of MasR (left column) and MrgD (right column) in the liver. MasR (C) and MrgD (D) staining of CCl4 livers were compared with those of healthy control livers (A,B, respectively). Note that in cirrhotic livers (C), there was strong positive staining for MasR in liver sinusoids (arrow) which is consistent with the localization of hepatic stellate cells and/or liver sinusoidal endothelial cells. Also note that positive staining for MasR in bile duct epithelial cells (arrow head-large) and hepatic arterioles (arrow head-small) in the cirrhotic liver. However, there was no positive staining for MrgD in the cirrhotic liver (D). Livers from PPVL rats showed no positive staining for both receptors (G,H) compared to the controls (E,F). Original images were captured at the X20 magnification.