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

人類高血壓的形成與血管壁之生理學上及生化學上的改變有關。這些改變中包括了血管平滑肌的過度收縮所導致的周邊阻力的增加。血管平滑肌收縮的程度取決於肌凝蛋白輕鏈(myosin light chain, MLC)磷酸化的程度，而其磷酸化程度又受MLC激酶(myosin light chain kinase, MLCK)與MLC磷酸酶(myosin light chain phosphatase, MLCP)兩者活性的調控。MLCP的活性不屬於鈣離子從屬性(calcium dependent manner)，其所調節的血管張力稱為鈣離子敏感化(calcium sensitization)。本論文包含兩個部分，第一部分有關血管平滑肌細胞(vascular smooth muscle cells, VSMCs)內的細胞生理, 探討血管張力素II (angiotensin II, Ang II)對於鈣離子敏感化訊息路徑的作用。而第二部分比較了高血壓個體和正常血壓個體, 其VSMCs中此鈣離子敏感化訊息路徑的差異。在第一部分中，我們找尋確認在VSMCs中，Ang II刺激之後，會接合到單體G蛋白(小G蛋白)(monomeric G protein, small G protein) Rho A以及活化Rho A/Rho kinase/MYPT1訊息軸線的專一蛋白分子。將Sprague-Dawley大鼠的初級培養之血管平滑肌細胞處理以Leukemia-associated RhoGEF (LARG)的小分子干擾核糖核酸(Small Interfering RNA, siRNA)，接著處理以Ang II 加上 PD123319(Ang II第二型受體拮抗劑，AT2 antagonist), Ang II加上losartan (Ang II第一型受體拮抗劑，AT1 antagonist), 或者 Val5-Ang II (Ang II第一型受體促進劑，AT1 agonist)。隨後以定量及時聚合酶連鎖反應(quantitative real-time PCR, qPCR)來測定mRNA含量，以西方墨點法(Western blot analysis)測定蛋白含量。另外使用等長收縮力量紀錄法(an isometric force recording method)來測量主動脈環收縮力。結果顯示，在Ang II刺激之後的0.5小時，leukemia-associated RhoGEF (LARG) mRNA增加達顯著差異。以Ang II加上PD123319刺激，或以Val5-Ang II刺激後的3、6、和9小時，其LARG蛋白量、Rho A蛋白活性、以及myosin phosphatase target subunit 1 (MYPT1)的磷酸化程度皆有顯著增加。此外，用siRNA將LARG基因減弱(knock down)之後，降低了VSMCs對於AT1受體活化後所造成的收縮效應。主動脈環的收縮力研究也證實了LARG siRNA會降低Rho A/MYPT1對Ang II刺激的活化效應。這部分的結果提供了一個直接證據，就是Ang II透過AT1受體, 向上調節LARG的基因表達，並且會活化LARG/Rho A/MYPT1訊息軸線，因而維持血管張力。在第二部分，我們比較高血壓和正常血壓大鼠其血管平滑肌中，三個RhoGEF包括LARG，p115-RGEF，以及PSD-95/Disc-large/ZO-1 homology (PDZ)-RhoGEF表現的不同特性。自發性高血壓鼠(spontaneously hypertensive rats, SHR)和Wistar-Kyoto rats (WKY)的初級培養血管平滑肌細胞(primary cultured VSMCs)處理以Ang II加上PD123319或者Val5-Ang II。之後，三種RhoGEFs的mRNA含量以qPCR測量。以西方墨點法測定三種RhoGEFs蛋白的含量。主動脈環則先以LARG siRNA或者scramble siRNA處理48小時，之後進行收縮力紀錄實驗。實驗結果發現，WKY的VSMCs中，p115-RhoGEF, PDZ-RhoGEF，和LARG其mRNA的基礎表現量並未隨年齡而增加。然而，在12週大的成年SHR，這三種RhoGEF的mRNA表現量，相較於5週大的未成年SHR有顯著增加。而在AT1活化之後，只有WKY(包括5週和12週)、5週大SHR的LARG的mRNA分別在0.5小時和6小時有顯著增加。蛋白方面, 12週大WKY，在AT1活化之後6小時，觀察到LARG蛋白增加，但在12週大的SHR其LARG蛋白表現在AT1活化之後仍然沒有改變。再者，在12週大的SHR，主動脈環LARG knockdown可以修正其主動脈環過度的收縮。我們這部分的研究結果顯示了，這三種RhoGEF基因的基礎表現，以及其對於AT1促進劑刺激的反應，而這些表現在SHR和WKY之間也有年齡從屬性(age-dependent manner)的差異。這些結果顯示LARG的基因表現情形與或許與SHR高血壓的發生有某些關聯。綜合言之，Ang II透過AT1來活化Rho A的上游分子LARG，因此活化Rho A/Rho kinase/MYPT1訊息軸線，而維持了血管基本張力；自發性高血壓個體可能因為其VSMCs的AT1數量較多，導致Ang II透過AT1調控LARG的基因表現增多，因而過度加強了此軸線的活性，而導致血管過度收縮，周邊阻力增加，而引起高血壓。

關鍵字

血管張力素II ； Ang II第一型受體；血管平滑肌細胞；鈣離子敏感化；自發性高血壓鼠；年齡從屬性； LARG ； SHR ； WKY

並列摘要

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.

並列關鍵字

Angiotensin II ； AT1 ； LARG ； Rho A ； MYPT1 ； vascular smooth muscle ； RhoGEF ； spontaneously hypertensive rat (SHR) ； Wistar-Kyoto rats (WKY)

參考文獻

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Banerjee J, Wedegaertner PB (2004) Identification of a novel sequence in PDZ-RhoGEF that mediates interaction with the actin cytoskeleton. Mol Biol Cell 15:1760-1775.

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國際替代計量

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

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