Leukemia-associated RhoGEF在高血壓大鼠血管平滑肌細胞鈣離子敏感化的重要性

Abstract

Human hypertension is associated with physiological and biochemical changes in vascular walls. These changes include excessive contractions of vascular smooth muscles that lead to an increase in peripheral resistance. The extent of vascular smooth muscle contraction depends on the phosphorylated level of myosin light chain (MLC), regulated by the balance between the activity of MLC kinase (MLCK) and MLC phosphatase (MLCP). MLCP activity is independent of Ca2+, the MLCP-mediated regulation of vascular tone is known as Ca2+ sensitization. This dissertation contains two parts. The first part studied the cell physiology of VSMCs including the effect of Ang II on calcium sensitization pathway. And the second part studied the comparison of RhoGEFs properties difference between hypertensive (SHR) and normotensive (WKY) subject. In the first part, we identify a specific molecule(s) that binds monomeric G protein Rho A and activates the Rho A/Rho kinase/MYPT1 axis, by stimulation of Angiotensin II (Ang II). Primary cultured vascular smooth muscle cells (VSMCs) from Sprague-Dawley rats were treated with or without siRNAs against leukemia-associated RhoGEF (LARG) and then treated with Ang II plus PD123319, Ang II plus losartan, or Val5-Ang II. Subsequently, mRNA and protein levels were determined by quantitative real-time PCR and Western blot analysis. An aortic ring contractile tension experiment was carried out using an isometric force recording method. The results indicated that after Ang II stimulation, LARG mRNA was significantly increased at 0.5hours. The amounts of LARG protein, Rho A activity, and phosphorylation of myosin phosphatase target subunit 1 (MYPT1), were increased at 3, 6, and 9 hours after treatment with Ang II plus PD123319 or Val5-Ang II. Moreover, knockdown of LARG by siRNA reduced the effects of AT1 receptors activation in VSMCs. The ex vivo contractile force study using aortic rings, confirmed the LARG siRNA diminishing effect on Rho A/MYPT1 activity in response to Ang II. Our results provide direct evidence that Ang II upregulates LARG gene expression and activates the LARG/Rho A/MYPT1 axis via AT1, thereby maintaining vascular tone. In the second part, the different properties of the three RhoGEF including leukemia-associated RhoGEF (LARG), p115-RhoGEF, and PDZ-RhoGEF expression in vascular smooth muscle between hypertensive and normotensive rats were compared. Primary cultured VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were treated with angiotensin II (Ang II) plus Ang II type 2 receptor antagonists PD123319, or Ang II type 1 receptors (AT1) agonist Val5-Ang II. The mRNA levels of RhoGEFs were measured by qPCR. The protein levels of RhoGEFs were measured by Western blot analysis. Aortic rings were pretreated with LARG siRNA or scrambled siRNA for 48 h; and then, the contractile force was recorded. The results show that the baseline levels of p115-RhoGEF, PSD-95/Disc-large/ZO-1 homology (PDZ)-RhoGEF, and LARG mRNA in VSMCs of WKY does not increase with age. However, there is a significant elevation of these RhoGEFs’ mRNA in the 12-week old SHR (SHR-12W) compared to the 5-week old SHR (SHR-5W). After AT1 activation by agonists, only LARG mRNA of WKY-5W, WKY-12W, and SHR-5W significantly increased at 0.5 and 6 h. The LARG protein increases at 6 h after AT1 activation in WKY-12W, but it remains unchanged in SHR-12W. Moreover, the ex vivo LARG-knockdown can correct excessive contraction of the aortic ring of SHR-12W. We show the differences of age-dependent RhoGEF expression and in response to Ang II stimulation between SHR and WKY rats. The LARG is differentially regulated and knockdown of LARG reduces more contraction of VSMCs in SHR. The findings support that LARG gene expression maybe related to the genesis of hypertension in SHR. In summary, Ang II activate LARG protein, the upstream molecule of Rho A, by AT1 and activate Rho A/Rho kinase/MYPT1 axis to maintain the vascular basal tone. The spontaneously hypertensive subject may be because of the large amount of AT1 on VSMC, leading to the increased expression of LARG regulated by Ang II. These excessively enhance the activity of the Rho A/Rho kinase/MYPT1 axis and lead to excessive vasoconstriction and hypertension via increased peripheral resistance.