Differential baseline expression and angiotensin II-stimulation of leukemia-associated RhoGEF in vascular smooth muscle cells of spontaneously hypertensive rats.

PURPOSE: Studies to explore angiotensin II (Ang II) and its downstream signaling pathways via Rho guanine nucleotide exchange factors (RhoGEFs) and RhoA signaling are crucial to understanding the mechanisms of smooth muscle contraction leading to hypertension. This study aimed to investigate the Ang II-induced expression of RhoGEFs in vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) and to identify the possible regulator associated with hypertension. METHODS: Cultured VSMCs of the aorta from SHRs and Wistar-Kyoto (WKY) rats were treated with or without Ang II or Ang II plus Ang II type 2 receptor antagonists. The expression levels of RhoGEF messenger RNA (mRNA) and protein were determined. To evaluate the changes of aortic ring contractile force in response to Ang II, a nonviral carrier system was adopted to deliver the leukemia-associated RhoGEF (LARG) small interfering RNA via nanoparticles into aortic rings. RESULTS: The baseline mRNA levels of three RhoGEFs in cultured VSMCs of WKY rats did not increase with age, but they were significantly higher in 12-week-old SHRs than in 5-week-old SHRs. Expression levels of LARG mRNA were higher in SHRs than in age-matched WKY rats. The baseline LAGR protein of 12-week-old SHRs was about four times higher than that of WKY rats of the same age. After Ang II-stimulation, LAGR protein expression was significantly increased in 12-week-old WKY rats but remained unchanged in 12-week-old SHRs. LARG small interfering RNA was successfully delivered into aortic rings using nanoparticles. LARG knockdown resulted in 12-week-old SHRs showing the greatest reduction in aortic ring contraction. CONCLUSION: There were differences in age-related RhoGEF expression at baseline and in response to Ang II-stimulation between SHRs and WKY rats in this study. Nanotechnology can assist in studying the silencing of LARG in tissue culture. The findings of this study indicate that LARG gene expression may be associated with the genesis of hypertension in SHRs.

Angiotensin II regulates the LARG/RhoA/MYPT1 axis in rat vascular smooth muscle in vitro.

AIM: To identify a key protein that binds monomeric G protein RhoA and activates the RhoA/Rho kinase/MYPT1 axis in vascular smooth muscle cells (VSMCs) upon angiotensin II (Ang II) stimulation. METHODS: Primary cultured VSMCs from Sprague-Dawley rats were transfected with siRNAs against leukemia-associated RhoGEF (LARG), and then treated with Ang II, losartan, PD123319, or Val(5)-Ang II. The target mRNA and protein levels were determined using qPCR and Western blot analysis, respectively. Rat aortic rings were isolated, and the isometric contraction was measured with a force transducer and recorder. RESULTS: Stimulation with Ang II (0.1 µmol/L) for 0.5 h significantly increased the level of LARG mRNA in VSMCs. At 3, 6, and 9 h after the treatment with Ang II (0.1 µmol/L) plus AT(2) antagonist PD123319 (1 µmol/L) or with AT(1) agonist Val(5)-Ang II (1 µmol/L), the LARG protein, RhoA activity, and phosphorylation level of myosin phosphatase target subunit 1 (MYPT1) in VSMCs were significantly increased. Knockdown of LARG with siRNA reduced these effects caused by AT(1) receptor activation. In rat aortic rings pretreated with LARG siRNA, Ang II-induced contraction was diminished. CONCLUSION: Ang II upregulates LARG gene expression and activates the LARG/RhoA/MYPT1 axis via AT(1), thereby maintaining vascular tone.