Heme Oxygenase-1-Endogenous Carbon Monoxide System On Prevention Of Atherosclerosis And Restenosis Of Balloon Injuried Carotid Artery In Rabbit

Background Coronary heart disease is the major cause of morbidity and mortality in the West, its major manifestation is atherosclerosis (AS) . Percutaneous transluminal coronary angioplasty (PTCA) and percutaneous intracoronary stent implantation are effective interventional therapy means for coronary heart disease,but the pathobiologic process of blood vessel restenosis (RS ) following balloon angioplasty continues to be an enigmatic problem in clinical settings. Although the exact mechanisms responsible for atherosclerosis and restenosis are not yet fully resolved,it is believed that oxidation,inflammation and vascular smooth muscle cells (VSMCs) proliferation are 3 crucial events involoved in the development of atherosclerotic lesions and restenosis.Some recent studies demonstrated that heme oxygenase-1-endogenous carbon monoxide-cycle guanosine monophosphate (HO-1-CO-cGMP) cell signaling system involve in many pathogphsiological processes and the regulation of cardiovascular system.Heme oxygenase is the primary and rate-limiting enzyme of heme degradation,which converts the cellular heme to carbon monoxide (CO) ,biliverdin and free iron. Recently it is suggested that endogenous CO play an important role in regulating vascular tone under both physiological and pathological conditions,and CO is the cell messenger of cardiovascular system. CO,like the similar diatomic gas nitric oxide (NO) ,inhibits VSMCs proliferation by activating soluble guanylate cyclase (sGC) and elevating intracellular sGMP levels.This,in turn, relaxes blood vessels and inhibits platelet aggregation.Biliverdin is subsequently and rapidly converted to bilirubin via biliverdin reductase.Bilirubin,which is characterized as a chain-breaking reactive oxygen species (ROS) ,is potent antioxidants and have been shown to inhibit the lipid peroxidation,while free iron,although itself an oxidant,stimulates ferritin synthesis which exerts an additional antioxidant effect. Several lines of evidence strongly suggest that HO-1 plays an important physiological and pathophysiological role in cardiovascular diseases such as atherosclerosis,intimal hyperplasia,and pulmonaryhypertension. However,how does change the expression of HO-1and the HO-1/CO system in various development stages of atherosclerotic lesions and restenosis? Do those changes associate with the extent of atherosclerotic lesions and restenosis? There are very fewer reports about these questions. Insulin-like growth factor-I (IGF-I), which is an important mitoge, promotes and regulates VSMCs proliferation and migration in an autocrine/paracrine manner.Some recent studies showed higher IGF-I mRNA expression in the atherosclerotic lesions, the neointima after balloon injury and restenotic lesions after angioplasty. The findings suggest the importance of the IGF-I in atherosclerosis and restenosis.But whether or not heme oxygenase-1-endogenous carbon monoxide system exerts an effect on VSMCs proliferation induced by IGF-I? Which will be investigated in the present study.Objective 1 .To investigate the change of endogenous CO and the expression of heme oxygenase-1 (HO-1 )in rabbit aorta with atherosclerosis induced by high cholesterol diet and inffluence of HO-1 system of heme catabolism on the atherosclerotic process.2. To investigate the change of endogenous CO and the expression of HO-1 in carotid artery after balloon injury and the inffluence of HO-1 system of heme catabolism on vascular remodeling following arterial injury in atherosclerotic rabbit.3.To determine the effect of hemin on the expression of HO-1 in rabbit aortic VSMCs and the inffluence of endogenous carbon monoxide on VSMCs proliferation induced by insulin-like growth factor-I (IGF-I) .4.To study the molecular mechasisms of heme oxygenase-1-endogenous carbon monoxide system on prevention of atherosclerosis and restenosis development after angioplasty and suppression of VSMCs proliferation in order to provide experimental information and theortic bases for preventing thes diseases.Methods-Section I the rabbits received 1.5% cholesterol diet (Ch group) or 1.5% cholesterol diet plus HO-1 inducer hemin (Hm,15mg · kg-1· d-1,ip.,Hm group) or HO-1 inhibitor zincprotoporphyrin IX(Znpp-IX,45μmol·kg-1 · d-1,ip.,Zn group) .After 4 weeks,8 weeks and12 weeks, animals were sacrificed and tissues obtained,serum lipids (TC TG LDL-C HDL-C) and serum total bilirubin (TBil) were determined, aortic CO protduction were assessed by enzyme-linked immunosorbent assay (ELISA) ,the area of atherosclerotic plaques in aorta ware measured by oil red O-staining and image analyse softwere, the extant of atherosclerotic lesions was examined by by light microscopy and transmission electronmicroscopy ( TEM ) , neointimal expression of HO-1 protein was detected by immunohistochemistry techniques (IHC) , intramural expression of HO-1 mRNA and protein were examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively.Methods-Section II Set up atherosclerotic and balloon injury restenosis model in rabbits. Rabbits were randomly divided into 5 groups, including control group (C group ) ,cholesterol group (Ch group ) ,Znpp-IX group (Zn group ) ,hemin group ( Hm group ) and sham-operation group (SH group) . 2 weeks,6 weeks and 10 weeks after the operation,animals were sacrificed and tissues obtained,the thickness of intima and media ware measured by image analyse system,serum lipids (TC TG LDL-C HDL-C) and serum TBil were assessed, arterial CO protdction were determined by ELISA, neointimal expression of HO-1 protein was detected by immunohistochemistry techniques, intramural expression of HO-1 mRNA and protein were examined by RT-PCR and Western blot analysis, respectively.Methods-Section III Rabbit aortic VSMCs were cultured in vitro and induced to proliferate by IGF-I, hemin (a substrate and inducer of HO-1) orZnpp-IX (an inhibitor of HO-1) were added to induce and inhibit the expression of HO-1,in turn,induce and inhibit CO production, respectively. The expression of HO-1 mRNA and protein were detected by RT-PCR and Western blot analysis, respectively.The relative amount of CO released into the media was quantitated as carbon monoxide hemoglobin (COHb) by ELISA,the proliferation of VSMCs was determined by 3H-TdR incorporation experiment and MTT test ,and cell proliferation cycle was analyzed by flow cytometry.Results I 1 .High cholesterol diet markedly enhanced the levels of serum TC, TG and LDL-C, but both hemin and Znpp-IX did not alter serum lipids concentration.2.Compared with control group (C group) at various time points,CO production and the expression of HO-1 mRNA and protein in aorta were higher significantly in Ch group;the aortic lesion area was( 10.54±1.30)%, (34.13 ± 3.26)% and(54.00 ± 4.16)% respectively;monocytes, macrophage and foam cells in the intima of aortas were observed;the necrosis of aortic endothelial cell and the disruption of elastic layer were obvious;medial smooth muscle cells proliferated and migrated to intima in Ch group.Immunostaining clearly demonstrated that the expression of HO-1 was prominent in endothelium and foam cells/macrophages ofthickened intima in lesions in Ch group.3.Compared with control group at various time points,Znpp-IX injection decreased markedly CO production and the expression of HO-lmRNA and protein in aorta;Compared with Ch group, the aortic lesions that developed in Zn group were increased byl8.69%,25.90% and 13.20% respectivefywhich were predominantly atheromatous plaques. There were more monocytes, macrophage and foam cells in the intima of aortas;The necrosis of aortic endothelial cell, the disruption of elastic layer and the proliferation and migration of medial smooth muscle cells were more obvious in Zn group than Ch group;Immunostaining demonstrated that the expression of HO-1 was devoid of in endothelium and foam cells/macrophages of thickened intima in lesions in Zn group.4.Compared with Ch group at various time points,hemin injection increased markedly not only serum TBil concentration,but also CO production and the expression of HO-1mRNA and protein in aorta;The aortic lesion areas that developed in Hm group ware reduced by -66.22%,-62.09% and -66.89% respectively, at various time points.Furthermore, the aortic lesions that developed in hemin-treated rabbits were not as advanced as thos observed in Ch group,which were predominantly fibrous-atheromatous plaques.There were very few foam cells in Hm group.The expression of HO-1 was prominent in endothelium and also detected in medial VSMCs in Hm group.Results II 1.High cholesterol diet markedly enhanced the levels of serum TC,TG,and LDL-C,but both hemin and Znpp-IX did not alter serum lipids concentration.2.In Ch group, intramural CO production and expression of HO-lmRNA significantly enhanced, compared with control group at various time points; neointimal thickness (I) and neointimal thickness (I) / medial thickness (M) ratio markedly attenuated, compared with Zn group at various time points. Immunostaining clearly demonstrated that the expression of HO-1 was prominent in VSMCs of neointima and about internal elastic lamina in Ch group.3.Administration of HO-1 inducer hemin markedly increased not only serum TBil concentration (about 5-fold, 6-fold and 7-fold,respectively) ,but also intramural CO production and expression of HO-1mRNA and protein,compared with control group, at various time points.Treatment of hemin significantly attenuated I (-37%,-35% and -36% respectively),and 1/ M ratio(-38%,-36% and -35% respectively), compared with cholesterol group at various time points. The expression of HO-1 was prominent in VSMCs of neointima and about internal elastic lamina and also detected in medial VSMCs in HmgroupACompared with control group at various time points,HO-1 inhibitor zin-cprotoporphyrin IX (Znpp-IX) treatment, however, effectively inhibited vascular HO-1 induction and CO production, Administration of Znpp-IX significantly enhanced I (+57%,+59% and +58% respectively) , and I/M ratio (+55%,+76% and +91% respectively) , compared with cholesterol group at various time points.Immunostaining demonstrated that the expression of HO-1 was devoid of in VSMCs of neointima and about internal elastic lamina in Zn group.Results III 1.IGF-I increased VSMCs 3H-TdR incorporation and A value, and accelerated cell cycle progression ,and resulted in an increase in the S and G2/M phase and a decrease in the G0/G1 phase (P<0.01) .2.With hemin added in gradient concentration, A values were reduced, the IGF-I -induced 3H-TdR incorporations of VSMCs were reduced by29.6%, 45.0% and 54. 1%,respectively (P<0.01) .The reduction was dependent on the amount of added hemin and the concentration of COHb in the media. Opposite results were observed in VSMCs treated with Znpp-IX (P<0.01,P<0.05) . 3.The addition of hemin in gradient concentration, resulted in an increase in the G0/G1 phase and a decrease in the S and G2/M phase (P<0.01) decelerated cell cycle progression. The change was dependent on the amount of added hemin and the concentration of COHb in the media.Opposite results were observed in VSMCs treated with Znpp-IX (P<0.01). 4.The expression of HO-1mRNA and protein in VSMCs and the amount of COHb in the media were increased significantly by hemin (P<0.01) .The increase was dependent on the amount of added hemin in the media.Meanwhile the proliferation of VSMCs was suppressed markedly. Opposite results were observed in VSMCs treated with Znpp-IX (P<0.01 ) .Conclusion This study demonstrated that 1.The HO-1/CO system can inhibit atherosclerotic formation and attenuate development of atherosclerosis by suppressing inflammatory reaction and VSMCs proliferation and protecting endothelial cells.2.HO-1 and the products of heme catabolism can attenuate the arterial response to injury and ensuing vascular wall remodeling and the extent of restenosis by reducing vascular oxidative damage and inhibiting VSMCs proliferation and vascular inflammatory reaction 3.Endogenous HO-1/CO system may be directly involoved in the regulation of VSMCs proliferation by inhibiting VSMCs DNA synthesis,energy metabolism and decelerateing cell cycle progression.4.Up-regulation of VSMC endogenous HO-1 represents a cellularprotective response to oxidative stress or injury.5.The enhancement of HO-1mRNA and protein expression is the molecular base of antiatherosclerosis, antirestenosis and antiproliferation of endogenous CO. The HO-1/CO system thus provides a potential target for development of new therapeutic approach to prevent atherosclerosis and restenosis.