| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 103 === Introduction: Adipokines, released from perivascular adipose tissue (PVAT), regulating vascular tone in a paracrine role have been established for several years. In some previous studies, PVAT inhibits endothelial nitric oxide synthase
(eNOS)-derived nitric oxide (NO) production. Endothelial dysfunction is one of the characteristics of hypertension. Comparing to normotensive rats, the genetic model of spontaneously hypertensive rats (SHR) exhibits downregulated caveolin-1 (Cav-1), a eNOS modulator, expression in aortas. Furthermore, the release of endothelium-derived contracting factors (EDCF), like prostacyclin (PGI2), is thought to be the main attribution of endothelial dysfunction in SHR via thromboxane A2 receptor (TP receptor). The activation of TP receptor recruits Ras homolog gene family member A (RhoA)/Rho-associated kinase (ROK) to the plasma membrane of smooth muscle and induces calcium sensitization. Cav-1 is probably implicated in RhoA/ROK pathway, because of colocalization of Cav-1 with RhoA in mice aortas. Cav-1 not only plays a main role in regulation of vascular tone, but also modulates eNOS function as PVAT does. Therefore, effects of PVAT in vascular tone regulation in hypertension were examined.
Materials and methods: Isometric tension studies in isolated thoracic aortas from Wistar (200 to 300 g) rats in the absence or presence of PVAT incubated solution were carried out to examine the vascular reactivity and endothelial function. After administration of several inhibitors, the effects of PVAT incubated solution were observed in the absence or presence of the PVAT. The protein expression of NO regulators, including eNOS, phosphorylated-eNOSThr495 (p-eNOSThr495), p-eNOSSer1177, AMPK, and Cav-1 and the NO production in aortic homogenates were also examined. Furthermore, thoracic aortas from animal model of hypertension, SHR (12-week old), and its comparable normotensive rats, Wistar-Kyoto (WKY, 12-week old), were used to study the effects of PVAT releasing factors. Systolic blood pressure was determined in conscious restrained rats by tail-cuff system in these two species. For NO, 6-Keto prostaglandin F1α (6-Keto PGF1α, a stable hydrolyzed product of unstable PGI2), and thromboxane B2 (TxB2, a stable metabolites of thromboxane A2 (TxA2)) measurements, aortas were incubated with or without ACh or PVAT incubation for 5 to 15 min, and then the incubation solutions were collected. In addition, the remaining aortic rings were stored at -80°C for eNOS, Cav-1, and RhoA protein detection. Moreover, co-localization of RhoA with Cav-1 and eNOS with Cav-1 were confirmed by immunofluorescence microscopy analysis.
Results: Results demonstrated that PVAT incubated solution enhanced contractile response to phenylephrine and reduced the endothelium-dependent dilatory response to acetylcholine (ACh). After the endothelium was denuded, the administration of endothelium-independent vasodilator sodium nitroprusside or NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride, the inhibitory effect of PVAT incubated solution was diminished. According to the results, we suggest that PVAT releasing factors plays an inhibitory role in endothelial NO production. We also observed eNOS and its activity by measuring the protein expression of eNOS, p-eNOSThr495, p-eNOSSer1177, AMPK, and Cav-1. We found that PVAT releasing factors significantly increased the protein expression of aortic Cav-1; Meanwhile, the aortic NO production was inhibited significantly. We further performed the PVAT effects on aortic rings from WKY and SHR. The results from WKY were similar to those from Wistar. Interestingly, the vasoconstriction induced by high concentration of ACh (10 μM) in SHR aortas was disappeared after PVAT treatment. Because activation of TP receptor is known to be involved in the high concentration of ACh (10 μM)-induced vasoconstriction in SHR aortas, we further investigated the effects of PVAT on TP receptor signal pathway. The co-localization of RhoA (the downstream factor of TP receptor signal pathway) with Cav-1 indicated that Cav-1 is interacted with RhoA. As shown in our previous study, PVAT releasing factors also enhanced aortic Cav-1 and reduced ROK-2 (C-20) protein expressions in SHR. The enhancement of Cav-1 protein expression by PVAT releasing factors could inhibit the high concentration of ACh (10 μM) induced vasoconstriction via regulation of TP receptor RhoA/ROK signal pathway.
Conclusions: The paracrine roles of PVAT involve in regulation of blood pressure through Cav-1, which modulates eNOS or TP receptor RhoA/ROK signal pathway, in normal and hypertensive rats.
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