張宇紅　鄧麗玉　林　彬　涂　勝　顧新元　唐利龍

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缺氧誘導因子對急性心肌梗死后心肌細胞及細胞外基質的影響

張宇紅鄧麗玉林彬涂勝顧新元唐利龍

【摘要】急性心肌梗死是指冠狀動脈閉塞、血流中斷，使部分心肌因嚴重的缺血缺氧而發(fā)生局部壞死。而缺氧誘導因子作為機體應對缺氧應激的適應性反應的調控因子，參與了急性心肌梗死時心臟的適應性保護。該文主要介紹缺氧誘導因子的結構、功能及其對急性心肌梗死后心肌細胞及細胞外基質的影響。

【關鍵詞】急性心肌梗死；缺氧誘導因子；心肌細胞；細胞外基質；心臟保護

作者單位：350108福建醫(yī)科大學附屬協(xié)和醫(yī)院心內科(張宇紅，鄧麗玉，林彬，涂勝)；512000廣東省韶關市粵北人民醫(yī)院心內科(顧新元)；411199湖南省湘潭市中心醫(yī)院心內科(唐利龍)

1概述

缺氧誘導因子(hypoxia-inducible factor, HIF)是由α亞基和β亞基組成的異二聚體。哺乳動物有3種HIFα亞基的亞型,包括HIF1α、2α和3α,其蛋白穩(wěn)定性受氧濃度影響；而HIF的β亞基則可穩(wěn)定地在核內表達且不受氧濃度調節(jié)。α亞基和β亞基都是螺旋-環(huán)-螺旋肽蛋白家族的成員。

缺氧時,脯氨酸羥化酶的活性受抑制,HIF1α通過HLH和PAS區(qū)域與β亞基異源二聚體化,并與轉錄的共激活劑CBP/P300相結合,形成活性轉錄復合體,轉位至細胞核,介導靶基因缺氧應答部分的反向激活。HIF異二聚體可以識別并結合靶基因上的缺氧應答序列,這些序列擁有共同的堿基片段5-(A/G)CGTG-3。

HIF1α在多細胞生物的各種類型細胞中均表達；而HIF2α僅在脊椎動物的特定細胞如角質形成細胞表達,參與缺氧條件下新生血管形成；HIF3α也僅在某些特定細胞通過與HIF1α、HIF2α、HIF1β結合，抑制負性調節(jié)HIF1α、HIF2α的轉錄活性。迄今，大量實驗證明HIF可以調節(jié)超過200多種基因的轉錄活性，在應對缺氧應激損傷的適應性變化中發(fā)揮著重要作用。

2HIF對急性心肌梗死后心肌的影響

HIF是急性心肌梗死(AMI)后心臟適應性變化過程中的主要調節(jié)因子之一。在人和鼠都觀察到，提高HIF活性后心肌梗死面積縮小,左室收縮功能提高，存活率提高[8-9]。HIF的心肌保護作用是由多因素介導的，包括一系列HIF靶基因的轉錄及其相應信號轉導通路的活化。

2.1調節(jié)心肌收縮

心血管活性肽是心血管系統(tǒng)穩(wěn)態(tài)的重要調節(jié)者,也是HIF1的靶基因之一，在AMI早期有維持心肌收縮功能的作用[10]。心肌梗死后24 h內，機體通過激活HIF通路使心血管活性肽水平急劇升高[11],心血管活性肽再通過磷酸肌醇信號途徑PKCε和細胞外信號調節(jié)激酶途徑ERK1/2,激活下游肌球蛋白輕鏈激酶。肌球蛋白輕鏈被磷酸化后，Ca2+敏感性增強,促進心肌肌絲交聯(lián),使心肌收縮力增強[12],從而實現(xiàn)心臟保護作用。

2.2調節(jié)心肌細胞能量代謝

HIF1α通過多種靶基因共同作用,減少AMI后基礎氧耗,提高葡萄糖利用效率。HIF介導AMI后心肌有氧代謝受抑,無氧酵解增強的過程。實驗發(fā)現(xiàn)，HL-1心肌細胞在缺氧后24 h，葡萄糖轉運體-1和乳酸水平分別增高5和15倍；線粒體電子傳遞鏈上的各種酶類包括復合體Ⅰ、Ⅳ和順烏頭酸酶的活性也不同程度地降低[13]。HIF1α促進正葡萄糖轉運蛋白-1(GLUT-1)、正葡萄糖轉運蛋白-3(GLUT-3)和己糖激酶Ⅰ基因(HK-1)、己糖激酶Ⅱ基因(HK-2)基因表達,使葡萄糖形成脫氧葡萄糖而不能進一步代謝[14]; HIF1α促進蛋白激酶(PDK)基因表達,磷酸化丙酮酸脫氫酶(PDH)的催化區(qū)域使之失活,使丙酮酸進入糖酵解途徑[15-16]；HIF1α促進乳酸脫氫酶(LDH)和MOT4基因的表達,使丙酮酸轉化為乳酸排出體外,同時減少線粒體源性自由基形成以減少心肌細胞死亡[17];另外正丙酮酸激酶PKM2既參與糖酵解過程，也與HIF1α結合促進其反向激活功能，以正反饋機制實現(xiàn)缺氧狀態(tài)下心肌細胞代謝轉化[18]。

2.3調節(jié)線粒體功能

HIF通過綜合作用使線粒體產生的自由基減少,起到心肌保護作用。線粒體源性自由基增加會對細胞造成氧化應激損傷[19]，HIF1有助于維持AMI后自由基的穩(wěn)態(tài)。HIF上調線粒體中線粒體蛋白BNIP3的水平,與自噬基因Beclin1競爭結合癌基因Bcl2后，游離的Beclin1協(xié)同磷脂酰肌醇激酶3激活缺氧誘導性線粒體自溶[20]; HIF抑制線粒體電子傳遞鏈中復合體1和4的活性[21]; HIF激活微小核糖核酸miRNA-210，抑制鐵硫蛋白ISCU1/2，抑制三羧酸循環(huán)順烏頭酶和電子傳遞鏈復合體1的活性[22]。

2.4調節(jié)炎癥反應

炎癥趨化因子(如基質細胞衍生因子-12和單核細胞趨化蛋白-5)以及血管黏附分子(如細胞間黏附素和血管源性細胞黏附素)的mRNA表達在心肌梗死區(qū)域升高[23]。而HIF1可控制炎癥反應，起心臟保護作用。有實驗表明，在缺血再灌注前激活HIF活性,可減少心肌細胞表達趨化因子如角質細胞源性趨化因子、巨噬細胞炎癥蛋白-2、單核細胞趨化因子-1、中性粒細胞趨化因子和細胞間黏附分子-1的表達,從而抑制中性粒細胞的聚集，明顯降低了梗死面積[24]。

3心臟細胞外基質組成和功能

正常情況下,細胞外基質(ECM)由結構蛋白、非結構蛋白和由基質金屬蛋白酶(MMP)、金屬蛋白酶組織抑制物(TIMP)組成的蛋白水解系統(tǒng)構成。AMI后，左室細胞外基質發(fā)生了一系列形態(tài)和功能的變化,稱為心室重構。異常的心室重構會導致心臟纖維化或心室過度擴張[25-26]。

心臟細胞外基質主要由纖維母細胞活化、增殖分化形成的心肌成纖維細胞合成[27]。基質結構蛋白包括膠原纖維、層黏連蛋白和纖連蛋白,基質非結構蛋白包括結締組織生長因子、血栓調節(jié)蛋白、骨橋蛋白、骨黏連蛋白等[28]。其中,結構蛋白參與維持心室正常結構和功能;非結構蛋白通過細胞表面受體、生長因子、蛋白酶等參與調節(jié)結構蛋白。

心臟細胞外基質的MMP，主要包括MMP-1、MMP-2、MMP-3、MMP-9、MMP-14。MMP可降解心臟所有基質成分,受促炎因子如白細胞介素-1、腫瘤壞死因子-α和促纖維化因子如轉化生長因子-β、血管緊張素Ⅱ的調節(jié)[29-30]。MMP也受心肌成纖維細胞來源的TIMP調節(jié)，主要包括TIMP-1和TIMP-2[31]。TIMP是內源性MMP活性抑制劑,在維持成纖維細胞和MMP對細胞外基質的合成降解平衡中起重要作用[32]。

4HIF對AMI后細胞外基質的影響

AMI時心肌細胞不可逆性死亡,梗死區(qū)域炎癥細胞浸潤,使心肌成纖維細胞迅速被激活、增殖,釋放炎癥介質以及MMP，降解細胞外基質并吞噬組織碎片[33-34]。HIF在該過程中通過促基質細胞遷移、低氧條件下細胞存活、細胞分化、生長因子釋放和基質合成等發(fā)揮重要作用[35]。組織缺氧時,HIF1α上調P4HA2、P4HA2和PLOD2的基因表達,從而促進膠原沉積[36]；HIF1α直接誘導TIMP-1、纖溶酶原激活物抑制劑-1、結締組織生長因子的表達[37],促進損傷部位膠原沉積；HIF1α激活心臟成纖維細胞內的DNA甲基轉移酶-1、DNA甲基轉移酶-3β,引起細胞內廣泛的DNA甲基化,激活轉化生長因子-β信號通路,使細胞表達促纖維化基因產物如Ⅰ型、Ⅲ型膠原和α平滑肌肌動蛋白[38]。HIF1α過度表達則可使成纖維細胞過度增殖、分化，導致過多的基質形成及心臟舒張功能障礙。

5HIF促進AMI后新生血管合成

AMI后HIF1通過刺激各種來源的促血管生成基因的轉錄和表達,如刺激血管內皮生長因子、胎盤生長因子、血管生成素Ⅰ和Ⅱ、血小板源性生長因子和基質細胞因子1以促進缺血部位新生血管與側支循環(huán)形成,從而減少梗死面積、保存心功能及降低患者死亡率[39]。在冠狀動脈疾病患者中，HIF1α水平上調與側支循環(huán)形成密切相關[40]。在大鼠心肌實驗觀察到HIF還可通過激活絲氨酸/蘇氨酸激酶-AMP依賴的蛋白激酶信號途徑，參與微血管內皮細胞的血管形成[41]。HIF1α可通過各種信號通路啟動組織缺氧損傷后的組織修復進程,但其特異性調節(jié)機制尚未了解。

參考文獻

[1]Semenza GL. Hypoxia. Cross talk between oxygen sensing and the cell cycle machinery. Am J Physiol Cell Physiol, 2011, 301(3):C550-552.

[2]Lee JW, Bae SH, Jeong JW, et al. Hypoxia- inducible factor (HIF-1) alpha: its protein stability and biological functions. Exp Mol Med, 2004, 36(1):1-12.

[3]Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-alpha to the von Hippel- Lindau ubiquitylation complex by O2-regulated prolylhydroxylation. Science, 2001, 292(5516):468-472.

[4]Cowburn AS, Alexander LE, Southwood M, et al.Epidermal deletion of HIF-2α stimulates wound closure. J Invest Dermatol, 2014, 134(3):801-808.

[5]Nauta TD, van Hinsbergh VW, Koolwijk P. Hypoxic signaling during tissue repair and regenerative medicine. Int J Mol Sci, 2014, 15(11):19791-19815.

[6]Ong SG, Hausenloy DJ. Hypoxia-inducible factor as a therapeutic target for cardioprotection. Pharmacol Ther, 2012, 136(1):69-81.

[7]Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell, 2012,148(3):399-408.

[8]Lee SH, Wolf PL, Escudero R, et al. Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med, 2000, 342(9):626-633.

[9]Kerkel? R, Karsikas S, Szabo Z, et al. Activation of hypoxia response in endothelial cells contributes to ischemic cardioprotection. Mol Cell Biol, 2013, 33(16):3321-3329.

[10]Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem, 1997, 272(36):22642-22647.

[11]Ronkainen VP, Ronkainen JJ, et al. Hypoxia inducible factor regulates the cardiac expression and secretion of apelin. FASEB J. 2007, 21(8):1821-1830.

[12]Perjés á, Skoumal R, Tenhunen O, et al. Apelin increases cardiac contractility via protein kinase Cε- and extracellular signal-regulated kinase-dependent mechanisms. PLoS One, 2014, 9(4):e93473.

[13]Kleinz MJ, Davenport AP. Emerging roles of apelin in biology and medicine. Pharmacol Ther, 2005, 107(2):198-211.

[14]Colson BA, Locher MR, Bekyarova T, et al. Differential roles of regulatory light chain and myosin binding protein-c phosphorylations in the modulation of cardiac force development.J Physiol, 2010, 588(Pt 6):981-993.

[15]Ambrose LJ, Abd-Jamil AH, Gomes RS, et al. Investigating mitochondrial metabolism in contracting HL-1 cardiomyocytes following hypoxia and pharmacological HIF activation identifies HIF-dependent and independent mechanisms of regulation. J Cardiovasc Pharmacol Ther, 2014, 19(6):574-585.

[16]Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1α. Genes Dev, 1998, 12(2):149-162.

[17]Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism. J Biol Chem, 2006, 281(14):9030-9037.

[18]Luo W, Hu H, Chang R, et al. Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell, 2011, 145(5):732-744.

[19]Goswami SK, Maulik N, Das DK. Ischemia-reperfusion and cardioprotection: a delicate balance between reactive oxygen species generation and redox homeostasis. Ann Med, 2007, 39(4):275-289.

[20]Zhang H, Bosch-Marce M, Shimoda LA, et al. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J Biol Chem, 2008, 283(16):10892-10903.

[21]Cockman ME, Masson N, Mole DR, et al. Hypoxia inducible factor-alpha binding and ubiquitylation by the von hippel-lindau tumor suppressor protein. J Biol Chem, 2000, 275 (33):25733-25741.

[22]Chan SY, Zhang YY, Hemann C, et al. MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2. Cell Metab, 2009, 10(4):273-284.

[23]Everaert BR, Nijenhuis VJ, Reith FC, et al. Adiponectin deficiency blunts hypoxia-induced mobilization and homing of circulating angiogenic cells. Stem Cells Int, 2013, 2013:260156.

[24]Natarajan R, Salloum FN, Fisher BJ, et al. Activation of hypoxia-inducible factor-1 via prolyl-4 hydoxylase-2 gene silencing attenuates acute inflammatory responses in postischemic myocardium. Am J Physiol Heart Circ Physiol, 2007, 293(3):H1571-H1580.

[25]Ling L, Cheng Y, Ding L, et al. Association of serum periostin with cardiac function and short-term prognosis in acute myocardial infarction patients. PLoS One, 2014, 9(2):e88755.

[26]Go AS, Mozaffarian D, Roger VL, et al. Heart disease and stroke statistics: 2013 update: a report from the American Heart Association. Circulation,2013, 127(1):e6-e245.

[27]李秀，劉巍.肌成纖維細胞在心肌梗死后重構中的作用及機制.國際心血管病雜志，2014,41(2):88-90.

[28]Frangogiannis NG. Matricellular proteins in cardiac adaptation and disease. Physiol Rev, 2012, 92(2):635-688.

[29]Li YY, McTiernan CF, Feldman AM. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res, 2000, 46(2):214-224.

[30]Turner NA, Porter KE. Regulation of myocardial matrix metalloproteinase expression and activity by cardiac fibroblasts. IUBMB Life, 2012, 64(2):143-150.

[31]Turner NA, Warburton P, O′Regan DJ, et al. Modulatory effect of interleukin-1α on expression of structural matrix proteins, MMPs and TIMPs in human cardiac myofibroblasts: role of p38 MAP kinase. Matrix Biol, 2010, 29(7):613-620.

[32]Ma Y, Halade GV, Lindsey ML. Extracellular matrix and fibroblast communication following myocardial infarction. J Cardiovasc Transl Res, 2012, 5(6):848-857.

[33]Frey H, Schroeder N, Manon-Jensen T, et al. Biological interplay between proteoglycans and their innate immune receptors in inflammation. Febs J, 2013, 280(10):2165-2179.

[34]Watson CJ, Collier P, Tea I, et al. Hypoxia-induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype. Hum Mol Genet, 2014, 23(8):2176-2188.

[35]Hong WX, Hu MS, Esquivel M, et al. The Role of hypoxia-inducible factor in wound healing. Adv Wound Care (New Rochelle), 2014, 3(5):390-399.

[36]Ruthenborg RJ, Ban JJ, Wazir A, et al. Regulation of wound healing and fibrosis by hypoxia and hypoxia-inducible factor-1. Mol Cells, 2014, 37(9):637-643.

[37]Gilkes DM, Bajpai S, Chaturvedi P, et al. Hypoxia-inducible factor 1 (HIF-1) fibroblasts P4HA1, P4HA2, and PLOD2 expression in under hypoxic conditions by inducing promotes extracellular matrix remodeling. J Biol Chem, 2013, 288(15):10819-10829.

[38]Watson CJ, Collier P, Tea I, et al. Hypoxia-induced epigenetic modifications are associated with cardiac tissue fibrosis and the development of a myofibroblast-like phenotype. Hum Mol Genet, 2014, 23(8):2176-2188.

[39]Ahluwalia A, Tarnawski AS. Critical role of hypoxia sensor--HIF-1alpha in VEGF gene activation. Implications for angiogenesis and tissue injury healing. Curr Med Chem, 2012, 19(1):90-97.

[40]Chen SM, Li YG, Zhang HX, et al. Hypoxia-inducible factor-1alpha induces the coronary collaterals for coronary artery disease. Coron Artery Dis, 2008, 19(3):173-179.

[41]張普,劉銘雅,朱偉,等.Apelin經Akt/AMPK信號通路促進心肌微血管內皮細胞血管生成.國際心血管病雜志,2013,40(1):44-48.

(收稿：2015-06-08修回：2015-08-03)

(本文編輯：丁媛媛)

通信作者：顧新元，Email：guxinyuanbob@sina.com

基金項目：國家自然科學基金項目資助(81170241)

doi:10.3969/j.issn.1673-6583.2016.01.013