Mas‐related G protein‐coupled receptor type D antagonism improves portal hypertension in cirrhotic rats

Figures

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  FIGURE 1. In vivo hemodynamic changes in rats with cirrhotic portal hypertension induced by carbon tetrachloride intoxication or bile duct ligation surgery and noncirrhotic portal hypertension induced by partial portal vein ligation surgery compared with respective controls. Rats were given 2 weeks (CCl4 and BDL) or 1 week (PPVL) continuous infusion of MrgD blocker D‐pro or MasR blocker A779. (A) Splanchnic vascular resistance, (B) mesenteric blood flow, (C) hepatic vascular resistance, and (D) portal pressure in three models are shown. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, diseased (CCl4 or BDL or PPVL) versus olive oil‐injected/sham‐operated healthy controls. # p < 0.05, ## p < 0.01, ### p < 0.005, #### p < 0.001, saline‐infused diseased controls versus diseased rats treated with D‐pro or A779. θ p < 0.05, D‐pro versus A779‐treated diseased rats. Each bar represents the mean ± SEM profile from 10 to 15 rats per treatment or control group. A779, D‐Ala7‐Ang‐(1–7); BDL, bile duct ligation; CCl4, carbon tetrachloride; D‐Pro, D‐Pro7‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D; PPVL, partial portal vein ligation

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  FIGURE 2. Effects of MrgD blocker D‐pro and MasR blocker A779 on acetylcholine‐induced vasodilatory responses. (A,B) Healthy and (C,D) carbon tetrachloride‐intoxicated cirrhotic rats were analyzed for the effects of MrgD blocker D‐pro and MasR blocker A779 on acetylcholine‐induced vasodilatory responses in (A,C) proximal and (B,D) distal mesenteric resistance vessels. The A779 response curve in panel B is not shown due to a technical reason. Each data point represents mean ± SEM profile of vascular responses from n = 4–6 rats per group. *p < 0.05, D‐pro versus saline or A779 groups. A779, D‐Ala7‐Ang‐(1–7); D‐Pro, D‐Pro7‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D

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  FIGURE 3. Gene expression of MrgD and MasR. (A,B) MrgD and MasR were analyzed in cirrhotic mesenteric arteries isolated from carbon tetrachloride‐intoxicated and bile duct‐ligated rats, cirrhotic rats treated with the MrgD blocker D‐pro or MasR blocker A779, and healthy controls. (C–H) Protein expression of MrgD and MasR quantified by western blot and immunohistochemistry (magnification ×200), respectively, is shown. Each bar represents the mean ± SEM profile from 10 to 15 rats per group. Arrowheads show positive endothelial staining. *p < 0.05, ***p < 0.005, ****p < 0.001, diseased (CCl4 or BDL) versus olive oil‐injected or sham‐operated healthy controls. # p < 0.05, ## p < 0.01, #### p < 0.001, diseased saline versus D‐pro or A779‐treated diseased rats. θ p < 0.05, θθ p < 0.01, D‐pro versus A779‐treated diseased rats. A779, D‐Ala7‐Ang‐(1–7); BDL, bile duct ligation; CCl4, carbon tetrachloride; D‐Pro, D‐Pro7‐ Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D

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  FIGURE 4. Gene expression of MrgD and MasR. (A,B) MrgD and MasR were analyzed in cirrhotic livers isolated from carbon tetrachloride‐intoxicated and bile duct‐ligated rats, cirrhotic rats treated with the MrgD blocker D‐pro or MasR blocker A779, and healthy controls. Up‐regulation of MasR protein expression as quantified by western blot (C,D) and immunohistochemistry (F,G) (magnification ×200) is shown. Positive staining of MasR in endothelium (large arrowhead), bile duct epithelial cells (arrow), and hepatic arterioles (small arrowhead) is shown. MrgD protein was not detectable by western blot or by immunohistochemistry (E) (magnification ×200) in cirrhotic or control livers. Each bar represents the mean ± SEM profile from 10 to 15 rats per group. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, diseased (CCl4 or BDL) versus olive oil‐injected/sham‐operated healthy controls. A779, D‐Ala7‐Ang‐(1–7); BDL, bile duct ligation; CCl4, carbon tetrachloride; D‐Pro, D‐Pro7‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D

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  FIGURE 5. Gene expression in omental vessels of patients with cirrhosis with PSC (n = 6) and subjects without cirrhosis (n = 3). (A) MrgD. (B) MasR. (C) Immunohistochemical staining (magnification ×200) of MasR and MrgD protein in omental vessels are indicated by arrowheads. (D) Liver gene and (E,F) protein expression of MasR by real‐time quantitative polymerase chain reaction and western blot, respectively, in PSC (n = 6) and ALC (n = 8) and subjects without cirrhosis (n = 5). (G,H) Positive immunostaining of MasR protein was detected in endothelium (large arrowhead), bile duct epithelial cells (arrow), and hepatic arterioles (small arrowhead) (magnification ×200). However, liver MrgD protein was not detected by western blot (data not shown) or (G) immunohistochemistry (magnification ×200). Each bar represents the mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.001. ALC, alcoholic cirrhosis; MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D; PSC, primary sclerosing cholangitis

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  FIGURE 6. Gene expression of MrgD and MasR in mesenteric arteries isolated from PPVL rats with noncirrhotic portal hypertension, PPVL rats treated with the MrgD blocker D‐pro or MasR blocker A779, and sham‐operated controls. (A) MrgD. (B) MasR. (C,D) Western blot analysis of mesenteric arterial protein expression of (C) MrgD and (D) MasR showed no difference between the four groups. (E) MrgD and MasR protein was not detected by immunohistochemical staining, likely due to very low expression levels. Each bar represents the mean ± SEM profile from 10 to 15 rats per group. A779, D‐Ala7‐Ang‐(1–7); D‐Pro, D‐Pro7‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D; PPVL, partial portal vein ligation

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  FIGURE 7. Relationship between hemodynamic changes and the expression of MasR and MrgD in different vascular beds of healthy and cirrhotic carbon tetrachloride‐intoxicated rats. Each bar represents the mean ± SEM profile of 10–15 rats. Fold changes are shown on top of each bar of control (open bars) and cirrhotic (closed bars) rats. Thickness of receptor blocking lines indicates relative contribution by the receptor. Broken line denoting a receptor‐blocking step represents a possible pathway. A779, D‐Ala7‐Ang‐(1–7); D‐Pro, D‐Pro7‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D; ns, nonsignificant