Changes Of Focal Adhesion Kinase Induced By Ethanol And The Related Cytotoxicity To Endothelial Cells

Drinking has a long history in China, and with the development of socialactivities, drinking is becoming more and more common. The related socialproblems have been serious. In recent years, injury cases after drinking havebeen increasing, and the related cases are always complicated to judge. In1963, some researchers indicated that traumatic subarachnoid hemorrhage(tSAH) was related with heavy drinking. In forensic medicine cases, drinkingleads to high incidence and mortality of intracranial hemorrhage with a slightexternal force. Although some mechanisms have been studied, such as chagesof heart rate and blood flow, vascular permeability，vasomotor function, andthe imbalance between coagulation and anticoagulation, it is still difficult toexplain the reasons. There are some challenges in forensic practice: how toevaluate the contribute degree of external force and alcohol and how toevaluate the case nature.Ethanol (EtOH) is the most important component of alcoholic beverages.Aftering drinking, the ethanol is taken into blood and the blood alcoholconcentration (BAC) increases rapidly. With blood stream, ethanol is taken toorgans in the body. Vascular endothelial cells (EC) lining the blood vesselsform the interface between the bloodstream and the vessel wall and as suchthey are continuously subjected to ethanol and metabolites from the flowingblood after drinking. And EC take part in ethanol metabolism. As result, ECare susceptible to ethanol totoxicity.Endothelial cells (EC) are the substaintial constitution of vessels, whotake responsibility to material exchange, angiotasis regulation, bloodcoagulation, angiogenesis and leukocyte migration. In addition, EC ‘sense’and transmit the biosignals from shear stress, cycle tension and bloodcomponents and coordinate diverse cells to make biological response. In summary, the toxicity of ethanol to EC will lead injury of vascular disorder.Cerebral vessels are charactered with autonomic regulation, so they aresensitive to EC injury. The current study was designed to study toxicity ofethanol to cerebral vessels, which will be helpful to futher study aboutmechanisms of tSAH with slight blow after drinking.EC line the wall of all blood vessels with monolayer of cells, so thedelicate balance between cell-cell interaction and EC-matrix adhesion areimportant to endothelial function. Focal adhesions (FAs) are complexstructures that assemble at the plasma membrane in discrete regions ofintegrin-mediated recognition of extracellular matrix (ECM) components.They connect the extracellular filamentous meshwork to the intracellularcytoskeleton controlling or mediating bidirectional signal transductionthrough endothelial cell.Focal adhesion kinase (FAK) is an important member in FA pathwaytransducting in-outside or outside-in signals. FAK is a non receptor proteintyrosine kinase (PTK), taking part in composition of focal adhesion (FA)complex and regulation of cytoskeleton. In the one hand, several factors, suchas integrin clustering, growth factor receptors and shear stress, regulate FAK.In another hand, FAK is a scaffolding protein binding with other proteins. SoFAK is involved in multiple pathways. There is evidence that FAK is relatedwith adhesion, migration, proliferation and mechanotransduction. Inpathological conditions, FAK related signal pathways become disorder. Thecurrent study was designed to observe the changes of FAK induced byexcessive ethanol, and to explore the mechanisms.Part I: The cytotoxicity of ethanol to endothelial cellsObjective: To observe the cytotoxicity of excessive ethanol to vessels invivo and in vitro and to study systematically the effects of ethanol on structureand functions of endothelial cells.Methods:1The rats were given alcoholic beverage with a blunt tipped needle, andBAC was detected after drinking. Masson’s trichrome staining and transmission electron microscope (TEM) were used to observe themorphological changes of capillaries and basilar arteries (BA) from alcoholicrats with different duration of drinking;2Human umbilical vein endothelial cells were given differentconcentrations or different duration of ethanol treatment, and the viability wasdetected with MTT, representing the cytotoxicity of ethanol;3Apoptotic cells were determined by Annexin V/PI apoptosis detectionkit with flow cytometry;4The morphological changes of the endothelial cell after ethanol treatedwere observed with light microscope, scanning electron microscope (SEM)and transmission electron microscope (TEM);5After the ethanol treatment, the cells were harvested by TrypLE Express, and allowed to adhere in plates coated with fibronectin (FN) for1-2h.The adhesion was detected with MTT and the average areas were measured byimage soft; the role of integrin pathway in adhesion was observed by blockingintegrin with specific blocking antibody;6Wound healing assay was used to observe the migration of HUVECafter ethanol treatment.Results:1The rats with short-time drinking appeared changes in capillaries:obvious vasogenic edema, enlarged perivascular space, microvillus decreasedand vacuolus in mitochondria;2The rats with long-time drinking appeared vascular remodeling: SMChyperplasia in tunica media and collagen deposition in tunica adventitia, thewall: lumen ratio increasing (p<0.05), and endothelial desquamation;3Excessive ethanol treatment decreased the HUVEC viability in a dose-and time-dependent manner. Compared to the control group, there were moreearly apoptotic cells (p<0.05) showing unregular nucleus, increased electrondensity of chromatin with half moon shape, and decreased cytoplasm;4HUVEC after excessive treatment lost normal shape, even withorganelle injury. 5Excessive ethanol treatment decreased adhesion rate (p<0.05) andspreading areas of HUVEC (p<0.05). The antibody to block integrin β1reduced adhesion rate of control EC, without affects on ethanol treated EC.6Excessive ethanol treatment decreased migration of HUVEC in woundhealing assay.Summary: Excessive ethanol caused endothelial injury, includingmorphological changes, viability decreasing, and interference of adhesion,spreading, and migration of EC. The adhesion injury induced by ethanol mightbe related with integrin pathway.Part Ⅱ: Changes of FAK and the related mechanisms in EC caused byexcessive ethanolObjective: This part was designed to study the distribution, proteinexpression and phosphorylation of FAK after excessive ethanol treatment, andto explore the related metablisms.Methods:1Distribution of pFAK Y397in spreading and migarating EC wasobserved with immunofluorenscence.2Total protein level and phosphorylation level of FAK were detectedwith western blot.3To study the relationship between the metabolism of ethanol and theFAK changes, specific ADH inhibitor4MP and ethanol metaboliteacetaldehyde (Ach) were given to HUVEC.4The oxidative stress from ethanol or Ach was detected with specificprobe DCFH-DA.5To study the relationship between oxidative stress caused by themetabolism of ethanol and the FAK changes, exogenous H2O2and nonspecific ROS scanvenger N-acetyl-L-cysteine (NAC) were given to HUVEC.Results:1In control group, pFAK397appeared as large pelltes, especially at edgeof cells during spreading and in parapodia during migrating. In ethanol group,pFAK397appeared as small dots; 2Ethanol increased pFAK in a dose-and time-dependent manner(p<0.05，p<0.01), without affect on total FAK protein;3Increased pFAK induced by ethanol was attenuated by4MP (p<0.05);Ach increased pFAK397significantly (p<0.05);4Both ethanol and Ach increased ROS level of HUVEC;5Exogenous H2O2incresased FAK phosphorylation (p<0.01); IncreasedpFAK induced by ethanol or Ach was attenuated by NAC (p<0.05).Summary：Ethanol disordered the distribution of pFAK, and increasedthe level of pFAK; disordered distribution of pFAK was involved in FA mature,leading to injury in EC survival, adhesion and migration; Ach and oxidativestress induced by ethanol metabolism was one of important factors of FAKphosphorylation.Part Ⅲ: The role of FAK phosphorylation on cytotoxicity of excessiveethanol to ECObjective：This part was designed to explore the relationship betweenincrease of pFAK and NO-mediated homeostasis, and to evaluate whetherPI3K/AKT pathway was involved in this process.Methods:1NO production was detected with specific probe DAF-FM; eNOSinhibitor L-NAME, iNOS inhibitor L-NAME L-canavanine and inhibitor ofFAK autophosphorylation PF228were employed to assess the origin of NO;2NT production was dectected by western blot;3The pathways involving in eNOS activation were vertified withPI3K/AKT inhibitor wortmannin and PF228.Results:1Ethanol increased NO production (p<0.05)，which was attenuated bypretreatment of L-NAME or PF228(p<0.05), but not by L-canavanine.2NT production was significantly increased after treated with200mMethanol for24h (p<0.05).3Increase of AKT and eNOS phosphorylation induced by ethanol wasattenuated by both wortmannin and PF228(p<0.05). Summary: Excessive ethanol increased NO generation and toxic NTproduction; FAK phosphorylation induced by ethanol promote the activationof eNOS via PI3K/AKT, which broke the NO-mediated endothelialhomeostasis.Conclusions:1Short-time drinking leads to microvasular morphological and structuralchanges, while long-time drinking causes vascular remodeling of cerebralarteries. Endothelial cells are the injury target of ethanol cytotoxicity.2Ethanol disorders the distribution of FA, inducing decrease of survivalrate, and down-regulation of adhesion and migration ability.3Metabolites and oxidative stress from ethanol increase FAKphosphorylation, which up-regulates FAK/PI3K/AKT/eNOS pathway.Overproduction of NO and ROS generates NT, taking part in endothelialinjury.