The Effect Of Lysophosphatidylcholine And GRP78 On The Cardiac Ischemia/reperfusion Injury

Coronary heart disease(CHD)is a major cause of death and disability worldwide.Thanks to myocardial reperfusion strategy including the use of thrombolytic therapy or primary percutaneous coronary intervention,the CHD mortality rates have declined over the past four decades.However the process of restoring of blood flow to the ischemia myocardium provokes myocardial reperfusion injury.Several abrupt biochemical and metabolic changes happens during myocardial reperfusion including Ca2+ overload,mitochondrial reenergization and the accumulation of reactive oxygen species.Recent studies have established that ischemia/reperfusion(I/R)generates endoplasmic-reticulum stress that greatly protects the ischemia injury.The understanding of mechanism of cardiac protection during I/R injury is greatly in need.The cardiac injury during myocardial reperfusion causes four types of cardiac dysfunction:The first type is myocardial stunning,a term denoting the "mechanical dysfunction that persists after reperfusion.The myocardium usually recovers from injury after several days or weeks.The second type of cardiac dysfunction,the no-reflow phenomenon,was originally defined as the "inability to reperfuse the previously ischemic region".It refers to the impedance of microvascular blood flow.The third type of cardiac dysfunction is reperfusion arrhythmias and the last type is lethal reperfusion injury.Lysophosphatidylcholine(LPC,lysoPC)is a class of chemical compounds containing phosphorus and is derived from polar surface phosphatidylcholine of lipoproteins or from cell membrane-derived phosphatidylcholine as a result of phospholipase A2 hydrolysis.And phospholipase A1(PLA1)is able to cleave the sn-1 ester bond of phosphatidylcholine.The activation of PLA2 is known to lead to an accumulation of arachidonic acid(AA)within the membrane phospholipid pool of the ischemic myocardium.Some metabolites of AA produce detrimental effects in the ischemia heart and proinflammatory effects during reperfusion,whereas others have been recently shown to reduce I/R injury in the heart.Besides,LPC is a known arrhythmogenic agent causing the metabolic and mechanical dysfunction of cardiomyocytes by inducing calcium overload.Many disturbances,including those of cellular redox regulation,cause accumulation of unfolded proteins in the ER.Previous study has showed that unfolded protein response is activated during cardiac I/R injury by using a murine model.The expression of GRP78 is highly induced in ischemia zone compared with the border zone,remote region.Overexpression Xbp1s induces the expression of GRP78 and therefore protects cardiomyocytes from I/R injury.Recent study has established the activity of iPLA2? increases ER stress cause apoptosis.Here we test the hypothesis that I/R injury increases the level of LPC and thus activates the ER stress.GRP78 overexpression in vivo protects ischemia/reperfusion(I/R)injury by reducing infarct size and activating PI3K/Akt pathway.On the other hand,the upregulation of LPC induces the inflammation of endothelial cells,and may play a critical role in I/R related no-reflow phenomenon.Part 1:The level of LPC and the regulation of ER stress under stimulated I/R conditionAim:To investigate the level of cell death and alteration of LPC content under sI/R condition.To access the activation of ER stress and the expression of chemical chaperones in different time point under sI/R.Method and Result:We performed a time course study of simulated I/R using NRVM.The level of cell death reached nearly 40%within 3 hours ischemia followed by overnight reperfusion.By 6 hours ischemia and reperfusion overnight the cell death increased to 78%and caused apoptosis with clearly changes of cell size and complexity.The intracellur level of LPC was upregulated under sI/R condition and the supernatant LPC level was also increased.Under sI/R condition,the expression of GRP78 was increased more than 2-fold.The other important chaperone GRP94 was also upregulated at protein level.The intensity of band indicating cell surface GRP78 was increased by nearly 8-fold upon sI/R compared to the normoxia.In contrast,the total intracellular amount of GRP78 increased by 3-fold.This result suggests that sI/R promotes the expression of GRP78 at cell surface.Conclusion:The intracellur and supernatant level of LPC were highly increased and the ER stress chaperones GRP78 and GRP94 were also induced under stimulated I/R condition.Part 2:The effect of LPC and ER stress chaperone GRP78 in cardiac I/R injuryAim:To identify the effect of LPC on the expression of GRP78 and investigate the role of GRP78 in regulation of cardiac I/R injury.Method and Result:A dose course study of LPC was conducted to access the expression of GRP78.We found the expression of GRP78 was highly induced by 60?M?80?M LPC.By using a cardiomyocyte inducible GRP78-overexpressing mouse model,we found that GRP78 overexpression in vivo reduced infarct size and fibrosis upon I/R injury.Besides,the PI3K/Akt pathway was activated after overexpression of GRP78.Whereas the cardiomyocyte specific GRP78-knockout caused the death of mice and cardiac dysfunction.Conclusion:The expression of GRP78 was induced after the stimulation of high dose LPC.GRP78 induction reduces infarct size and fibrosis upon I/R injury by activating PI3K/Akt pathway.GRP78 is essential for maintaining the heart function.Part 3:The effect and mechanism of LPC on the inflammation of endothelial cellsAim:To determine the role of LPC on the endothelial inflammation and further access the underlying mechanism.Method and Result:To assess the effect of LPC on IL-8 production,HUVECs were stimulated with LPC at concentrations of 40,60 and 80?M.After 3 hours treatment,the production of IL-8 increased in a dose-dependent manner.As the NF-?B pathway plays a central role in inflammation and chemokine expression,experiments were performed to address whether NF-?B was involved in the action of the LPC treatment.Immunofluorescence staining showed that p65 subunit of NF-?B translocated into the nucleus after LPC treatment.More importantly,mutation of NF-?B and AP-1 sites in the IL-8 promoter led to a strong diminishment of LPC-mediated induction by using Dual luciferase assay.Small Ubiquitin-like Modifier(SUMO)protein are a family of small proteins that detached from other proteins to modify their function.Human SUMO E2 ligase is Ubc9 and the SUMO E3 ligase is HDAC4.Silencing of Ubc9 and HDAC4 inhibited LPC-induced IL-8 expression.Compared with the control siRNA group,LPC-induced degradation of I?B? was delayed at early stage of stimulation when Ubc9 was knockdown.Morever,treatment of LPC enhanced the adhesion of THP-1 cells to HUVECs and IL-8 neutralization attenuated the THP-1 adhesion induced by LPC.Conclusion:LPC induced the expression of IL-8 by activating the SUMOylation modified NF-?B pathway,and therefore promoted the adhesion of THP-1 cells to HUVECs.