Probucol Protects Vasodilation And Its Effect On Smooth Muscle Cell Phenotypes

Part Ⅰ-Probucol Protects Endothelium-Dependent Vasodilation from the Oxidative Damage of Hydrogen Peroxide through activation of eNOSBackgroundProbucol is a synthetic anti-oxidative agent. As a diphenolic compound, it also has the lipid-lowering, anti-inflammatory and anti-atherosclerotic property. However, the clinical application of probucol is almost centered in lipid-lowering effect. In vitro and in vivo studies found out that probucol had direct effect on vascular contraction and dilation, which was independent of its lipid-lowering property.As we know, oxidative stress induced by ROS is the important initial part of the onset and progression of many vascular diseases, such as hypertension and atherosclerosis. Vasomotor can be influenced by oxidative stress through oxidative injuries on vascular cells. While probucol was found to not only significantly inhibit the oxidation of lipids (such as low density lipoprotein, LDL), but also clear the free radicals in bloodstream and tissues. This prompts that probucol can inhibit oxidation throughout vascular diseases, and finally attenuates the oxidative stress to promote vascular functions.Endothelium can maintain the vascular hemostasis via composing the mechanic and biological barrier and participating in many bioactivities. Endothelial dysfunction can lead to imbalance of vasoconstriction and vasodilation, enhanced cell adhesion, increased infiltration of inflammatory cells and anti-coagulation disorder. In humans, endothelial dysfunction is believed to be an important risk factor of cardiovascular events. Oxidative stress promotes vascular diseases through endothelium damage, leading to endothelial dysfunction. Therefore, endothelial dysfunction caused by oxidative injury may serve as a potent reason for atherosclerosis, hypertension and diabetesObjectiveBesides the lipid-lowering effect, we plan to investigate how the other properties of probucol work on vascular functions. We aim to explore the effect of probucol on vasomotor and its possible mechanisms. Accoding to its anti-oxidatiove property, we also try to figure out the effect of probucol on vasomotor under the condition of H2O2-induced oxidative stress.MethodsAn in-vitro model of H2O2-induced selective damage to vasodilation was established with thoracic arterial rings of rats.(1) The vascular activity of thoracic arterial rings of rats was studied by myograph, which measured the KC1or PE-induced vascular contraction and ACh or SNP-induced relaxation (the endothelium-dependent or independent relaxation). Compared with the basal vasomotor, the effect of H2O2on vascular activity was also showed.(2) Upon the intervention of H2O2, the alteration of vascular activity by probucol was recorded by myograph. Western-blot was applied to analyse the protein level of eNOS in order to figure out if probucol changes the effect of H2O2through eNOS pathway.(3) Organ culture technology was used to indicate the vascular damage by a different environment (without FBS) and the influence of probucol on vasodilation.The atherosclerotic model of apoE-/-mice was built and probucol was applied to see the effect of probucol on vascular dilation in atherosclerosis.Primary HUVECs were extracted from human umbilical veins and identified by immunofluorescence technique. We also established a cellular model of H2O2-induced selective damage to HUVECs, for application in the following parts:(1) the mRNA expression of eNOS was analyzed by Real-time qPCR. The protein level of eNOS and p-eNOS/eNOS extent was showed by Western-blot, indicating the activation of eNOS by probucol.(2) With H2O2intervention, the effect of probucol was also showed in mRNA and protein level of eNOS.(3) NO production was detected by immunofluenscence microscopy and microplate reader system.Results1. H2O2can cause oxidative damage to arteries, and selectively inhibit ACh-induced endothelium-dependent vasodilation. No effect of H2O2was observed in vascular contractility. But H2O2can cause irreversable loss of all vascular activity in high concentration or long intervention.2. Under H2O2-induced oxidative stress, probucol can protect endothelium-dependent vasodilatation from the oxidative damage of H2O2in thoracic arterial rings of rats.3. Probucol protects endothelium-dependent vasodilatation of thoracic aorta of atherosclerotic apoE-/-mice.3. Probucol protects endothelial function through preserve eNOS expression in thoracic arterial rings of rats, with no effects on contraction.5. During organ culture of thoracic arterial rings of rats, the decrease of ACh-induced vasodilation can be inhibited by probucol.6. In HUVECs, probucol enhanced eNOS phosphorylation at Ser-1177and promote its activation. H2O2can inhibit the expression level of eNOS and probucol preserves the phosphorylation of eNOS.7. In HUVECs, probucol promote the production of NO as the consequence of eNOS activation. But with H2O2induced stress, such effect of probucol was absent.ConclusionsProbucol protects endothelium from H2O2-induced damage and improve the endothelium-dependent vasodilation, probably through upregulating the phosphorylation of eNOS and activating its oxidase activity, increasing NO production and finally improving vascular relaxation. Part II-Effects of Probucol on Smooth Muscle Cell Phenotypes with association with SMC Proliferation and MigrationBackgroundVascular smooth muscle cells (VSMCs), as the important components of arteral walls, are capable of regulating vascular functions. Its dysfunction is related to many vascular diseases, including atherosclerosis, hypertension, diabetes and restenosis after ballon angioplasty. VSMCs also participate the atherogenesis, in forms of invasive phenotype migrating into subendothelium or synthetic type producing lots of cytokines and matrix metallproteases. Traditionally, SMC was devided into two phenotypes-contractile and proliferatiye types. The latter type was almost found in vascular diseases and was thought to be the dediffererntiation from contractile SMCs in tunica media. While new researches found that the proliferative SMCs in damaged arteries were derived from another cell type, which was stem-cell like and can differenciate into synthetic SMCs. Highly differenciated SMCs are marked by some contractile proteins, includinga-actin, SM22, calponin and SM-MHC (SM1、SM2), which can help confirm the phenotypes of VSMCs.It was reported already that probucol can inhibit the arota restenosis after ballon angioplasty of rabbits through inhibiting the proliferation and migration of VSMCs. But the effect of PB on VSMCs has not been explained in phenotype way before.ObjectiveConsidering the relationship between SMC phenotype and vascular diseases, this study is aimed to explore how PB works on VSMCs phenotype switch and if PB inhibits the pathogenic property of VSMCs due to influence on VSMCs phenotypes. Methods(1) Primary culture of rat arota tissues to get the primary vascular smooth muscle cells, to detect the contractile SMC markers by immunofluorescence technique.(2) Using cell counting methods to explore the effect of PB on primary VSMCs migration from arota tissues and the proliferation of VSMCs.(3) Using Transwell cell migration technique to study the influence of PB on vascular cell motion(4) With RT-qPCR and western-blot technique, the expression changes of phenotypic proteinsa-actin, SM22, Sox-10and activating transcription factor, myocardin, were measured in VSMCs by PB and oxLDL.Results(1) Highly differenciated VSMCs are marked with a-actin, SM22and MHC.(2) PB inhibits the expression of Sox-10, myocardin, and contractile proteins a-actin and SM22. H2O2-induced oxidative stress can promote the expression of Sox-10and downregulate myocardin.(3) PB inhibits the migration of VSMCs from arota tissues and from the transwell membrane. PB inhibits the proliferation of VSMCs in a time-related pattern.