Role Of Abl-knockout In Mediating Smooth Muscle Tone

Essential hypertension, stroke, ischemic heart disease, pulmonary hypertension, and other cardiovascular pathologies are the primary factors of the mortality and morbidity in the fully industrialized societies. Despite billons of dollars spent on the clinical studies, the problem has not been solved yet. Keeping in mind that all above-mentioned pathologies involve abnormalities of vascular tone, the fundamental studies regarding regulation of the smooth muscle contractility remain an urgent task of nowadays science. Smooth muscle contraction is interpreted in terms of the sliding of thick filaments and thin filaments. In this process, myosin (forms thick filaments) experiences light chain phophorylation, which initiate sliding of thick and thin filaments. The actin (forms thin filaments) cytoskeleton in smooth muscle has long been thought to be a fixed structure. Recent studies have documented that the actin architecture of smooth muscle is in a dynamic state. Actin polymerization and depolymerization plays an fundamental role in force development in smooth muscle. There is a wealth of evidence that a non-receptor tyrosine kinase Abl regulates the actin cytoskeleton system. Abl has been implicated to function in a range of cellular processes including regulation of the actin cytoskeleton that mediates cell migration and spreading, membrane ruffling and cell adhesion, etc. However, the role of Abl in regulating force development and actin cytoskeleton in smooth muscle has no been investigated.【Objective】Based on previous theory, we used Abl-knockout and wildtype mice to investigate blood pressure of tail arteriy, media smooth muscle wall thickness of artery, actin cytoskeleton change after contractile stimulation, myosin light chain phosphorylation and Abl substrate and contractile protein expression.Part 1. Blood pressure of tail artery and media wall thickness of artery.1. Method:The blood pressure was measured with XBP1000 noninvasive Tail Blood Pressure System (Kent Scientific Corporation). Result: Blood pressure of tail artery of Abl knockout mice is lower than those of wildtype mice.2. Method: After euthanized in CO2 chamber, carotid arteries was gathered from mice. After cryosectioned, phase contraction photos were taken under microscope. Media wall thickness was measured by softwere.Result: There is no significant difference between media wall thickness of carotid artery of Abl-knockout mice and wildtype mice.Part 2. Myosin light chain phosphorylation assay and actin polymerization assay after contractile stimulation1. Myosin light chain phosphorylation assay Groups: a. Widtype control b. Wildtype phenylephrine stimulated c. Abl-knockout control d. Abl-knockout phenylephrine stimulatedMethod: After euthanized in CO2 chamber, carotid arteries, mesenteric arteries, femoral arteries, aorta were gathered from mice. After stimulation with phenylephrine, western blot was performed to test myosin light chain phosphorylation.Result: After phenylephrine stimulation, there are no difference between myosin light chain phosphorylation level of Abl-knockout mice and wildtype mice. 2. Actin polymerization assay Groups: a. Widtype control b. Wildtype phenylephrine stimulated c. Wildtype angiotensin II stimulated d. Abl-knockout control e. Abl-knockout phenylephrine stimulated f. Abl-knockout angiotesin II stimulatedMethod: After euthanized in CO2 chamber, carotid arteries, mesenteric arteries, femoral arteries, aorta were gathered from mice. After stimulation with phenylephrine and angiontesin II, immunoflurescent test was performed to test actin polymerization level.Result: After phenylephrine and angiotensin 2 stimulation, actin polymerization level of arteries from Abl-knockout mice was significant lower than those from wildtype mice.Part 3. Crk-associated substrate phosphorylation after contractile stimulation and contractile associated protein expression assay.1. Crk-associated substrate (CAS) phosphorylation test Groups: a. Widtype control b. Wildtype phenylephrine stimulated c. Wildtype angiotensin II stimulated d. Abl-knockout control e. Abl-knockout phenylephrine stimulated f. Abl-knockout angiotesin II stimulatedMethod: After euthanized in CO2 chamber, aorta, mesenteric arteries and femoral arteries were gathered from mice. After stimulation with phenylephrine and angiontesin II, western blotting was performed to test CAS phosphorylation level. Result: After stimulation with phenylephrine and angiontesin II, CAS phosphorylation level was increased in arteries from wildtype mice. This increment was significant lower in arteries from Abl-knockout mice.2. Contractile associated protein expression levelMethod: After euthanized in CO2 chamber, aorta, mesenteric arteries, femoral arteries and lung tissues were gathered from mice. Western blot was performed to test paxillin, vinculin andα-actin protein level in arteries. Q-RT-PCR test was performed to test paxillin, vinculin and GAPDH mRNA level in lung tissue.Result: In arteries from Abl-knockout mice, paxillin and vinculin protein level was significantly lower than arteries from wildtype mice. In lung tissues from Abl-knockout mice, paxillin and vinculin mRNA level was significantly lower than lung tissues from wildtype mice.【Conclusion】1. Blood pressure of tail artery of Abl-knockout mice is lower than of wildtype mice.2. In arteries from Abl-knockout mice, increment of actin polymerization after contractile stimulation was attenuated. Whereas myosin light chain phosphorylation level was similar in arteries from Abl-knockout and wildtype mice. Suggesting that Abl might regulate smooth muscle tone through affecting actin cytoskeleton.3. In arteries from Abl-knockout mice, increment of CAS phosphorylation level after contractile stimulation was inhibited. Suggesting that Abl might regulate cytoskeleton structure through mediating CAS phosphorylation level.4. In arteries and non-muslce organs from Abl-knockout mice, protein level of paxillin and vinculin is lower than wildtype mice. As well as mRNA level is lower in Abl-knockout mice than wildtype mice. Suggesting that Abl might regulate actin cytoskeleton structure through mediating actin cytoskeleton associated protein expression level.