The Role Of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 (LOX-1) In Mechanical Stress-Induced Cardiac Hypertrophy

It has been proved that, without the involvement of angiotensin Ⅱ (AngⅡ), mechanical stress-activated AngⅡ type 1 receptor (AT1R) is profoundly relevant to pathogenesis of cardiac hypertrophy. Inverse agonist in AT1R blockers (ARBs), but not neutral antagonist, is effective in blocking this kind of agonist-independent activity of AT1R and consequent cardiac hypertrophy. Based on this finding, an enormous effort has been focused on exploring new ARBs. However, looking for a new therapeutic target may yield twice the result with half the effort. Here we demonstrate that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays a critical role in the pathological process of mechanical stress-induced cardiac hypertrophy.We firstly investigated whether LOX-1 participated in pressure overload-or mechanical stretch-induced cardiac hypertrophy in angiotensinogen-knockout (ATG-/-) mice or the primary cultured cardiomyocytes harvested from neonatal ATG-/- mice respectively. Treatment with LOX-1 neutralizing antibody (NAB) in vivo or LOX-1-shRNA in vitro significantly reduced pressure overload-or mechanical stretch-induced hypertrophic response respectively.Next we found that LOX-1 might directly participated in mechanical stretch-induced cardiac hypertrophy through JNK signaling by transfecting the plasmids of LOX-1 or AT1R into COS7 cells cultured in silicon-based plates pre-coated with collagen for 24 h before mechanically stretched.Finally, we found that Src, but not Gαq11 and Jak2, was involved in LOX-1-JNK signaling activated by mechanical stretch without the participation of AngⅡ.Collectively, we found that LOX-1-Src-JNK signaling was directly activated by mechanical stress without the involvement of AngⅡ, indicating LOX-1 as a promising therapeutic target for pressure overload-induced cardiac hypertrophy.Part ⅠLOX-1 participated in pressure overload-induced cardiac hypertrophy without the involvement of AngⅡObjective:To investigate whether LOX-1 participates in pressure overload-induced cardiac hypertrophy.Methods:Transverse aorta constriction (TAC) was carried out on 10 week-old ATG-/-mice to establish the cardiac hypertrophy model. Olmesartan (OLM,3 mg·kg-1-day-1, p.o.) or LOX-1 neutralizing antibody (NAB,0.06 mg·kg-1·day-1, i.p.) was administered from the day of TAC. Two weeks later, mice were subjected to echocardiography, heart catheterization, cardiac histology and examination of relative genes and proteins expression in myocardium.Results:Echocardiographic results showed increased left ventricular wall thickness and decreased left ventricular cavity in TAC group, compared with those in Sham group. Besides, an increased heart weight/body weight (HW/BW) and a raised cross-sectional area (CSA) of cardiomyocytes were observed. Additionally, the reprogramming of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and skeletal α-actin (SAA) was seen in TAC group. All the above alterations suggested a significant cardiac hypertrophy at that time in TAC group. However, all these maladaptive responses were ameliorated significantly by treatment with OLM or NAB.All of the increased phosphorylation of ERKs, P38 and JNKs was significantly reduced by OLM. Nevertheless, only the phosphorylation of ERKs and JNKs was obviously abolished by NAB.Conclusion:These evidences suggest that LOX-1 participates in pressure overload-induced cardiac hypertrophy.Part ⅡLOX-1 participated in mechanical stretch-induced cardiomyocyte hypertrophy without the involvement of AngⅡObjective:To investigate whether LOX-1 participates in mechanical stretch-induced cardiomyocyte hypertrophy in vitro.Methods:Cardiomyocytes from the hearts of neonatal ATG-/- mice were cultured in silicon-based plates pre-coated with collagen. Cardiomyocytes were pretreated with control-shRNA or LOX-1-shRNA for 4 d before mechanical stretched to 120% for 8/15/30 min or 24 h for the extraction of total protein and RNA respectively.Results:Compared with control-shRNA, pretreatment with LOX-1-shRNA significantly reduced mechanical stretch-induced increases in cardiomyocyte size, ERKs and JNKs phosphorylation and the mRNA expression of the fetal genes ANP and SAA without affecting mechanical stretch-induced phosphorylation of P38.Conclusion:These evidences suggest that LOX-1 participates in mechanical stretch-induced cardiomyocyte hypertrophy.Part Ⅲ LOX-1 mediated mechanical stretch-induced JNKs phosphorylationObjective:To investigate whether LOX-1 can directly mediate mechanical stretch-induced MAPK phosphorylation.Methods:COS7 cells, which lack expression of endogenous Angll, AT1R and LOX-1, were used in this part of the study. We transiently transfected the empty plasmid or the plasmid of LOX-1 or AT1R into COS7 cells cultured in silicon-based plates pre-coated with collagen for 24 h and then mechanically stretched the cells to 120% for 8 min,15 min or 30 min for the measurement of pERKs, pP38 or pJNKs respectively.Results:Compared with empty plasmid-transfected group, the phosphorylation of ERKs, P38 and JNKs was significantly upregulated by mechanical stretch in ATlR-transfected group, which was in agreement with previous studies. Not the phosphorylation of ERKs and P38, but the phosphorylation of JNKs, was directly induced by mechanical stretch in LOX-1-transfected COS7 cells.Conclusion:These results indicated that LOX-1 could directly mediate mechanical stretch-induced JNKs phosphorylation.Part Ⅳ Src was involved in LOX-1-JNK signaling pathway activated by mechanical stretchObjective:To examine whether G proteins or non-receptor-type tyrosine kinases such as the Janus kinase (Jak) family and the Src family are activated in LOX-1-JNK signaling activated by mechanical stretch.Methods:COS7 cells cultured in silicon-based plates pre-coated with collagen were transiently transfected with the empty plasmid, LOX-1 plasmid or AT1R plasmid for 24 h and then mechanically stretched to 120% for 5 min,5 min or 10 min for the examination of cytosol Gaql 1, lysate pJak2, and lysate pSrc, respectively. PP2 (a Src tyrosine kinase inhibitor,5μM) was used to examine whether LOX-1-dependent JNKs phosphorylation in the condition of mechanical stretch requires Src.Results:Compared with empty plasmid-transfected group, in AT1R-transfected group, stimulation with mechanical stretch significantly redistributed Gaq11 subunits into the cytosol of COS7 cells, significantly upregulated the phosphorylation of Jak2 and Src, which was in agreement with previous studies. In LOX-1-transfected group, no obvious redistribution of Gaqll subunits and no significant phosphorylation of Jak2 was observed in the condition of mechanical stretch; however, the phosphorylation of Src increased significantly after mechanical stretch. Further experiments revealed that PP2 abolished mechanical stretch-induced JNKs activation in LOX-1-transfected COS7 cells.Conclusion:These results indicated that Src, but not Gaqll protein and Jak2, was involved in LOX-1-JNK signaling activated by mechanical stretch without the involvement of AngⅡ.Conclusion1. LOX-1 directly mediates mechanical stress-induced cardiac hypertrophy without the involvement of AngⅡ.2. Activation of LOX-1-Src-JNK signaling is one of the mechanisms by which mechanical stress induce cardiac hypertrophy without the involvement of AngⅡ.The potential application and novelty of this project1.We explored the mechanism for mechanical stress-induced cardiac hypertrophy independent of AngⅡ.2. We found that mechanical stretch could directly activate the LOX-1-Src-JNK signaling, indicating LOX-1-Src-JNK signaling as one of the mechanisms by which mechanical stress induce cardiac hypertrophy.3. Our findings indicated that LOX-1 could be a new therapeutic target for pressure overload-induced cardiac hypertrophy without the involvement of AngⅡ.4. Developing a LOX-1 antagonist for the treatment of myocardial hypertrophy would be promising.