突变型人低氧诱导因子1α对大鼠后肢缺血模型的促血管新生作用
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摘要
研究背景:
冠心病是一种常见的心脏病,是我国城乡居民的主要死亡原因,并将成为全世界首要的死亡原因和最大的疾病负担。目前的治疗手段主要有药物治疗和包括冠状动脉旁路移植术(coronary artery bypass graft,CABG)及经皮冠状动脉介入术(percutaneous coronary intervention,PCI)在内的血运重建技术(revascularization)。终末期冠心病患者约占冠心病患者中的10%,因冠状动脉病变弥漫,或病变血管细小等无条件进行CABG及PTCA治疗,药物治疗又不能满意地控制其心绞痛症状,需要寻求一种新的更加适合于他们的治疗方法。90年代以来,人们发现,一些在机体生理和病理性的血管生成中起重要作用的生长因子能够促进缺血组织的血管新生,加速侧支循环的建立,有可能通过直接提高缺血区的血供来改善组织缺血,并设想导入外源性生长因子以促进血管新生,即所谓治疗性血管生成(therapeutic angiogenesis)。这一方法为冠心病的治疗提供了新思路。
血管新生(neovascularization)包括vasculogenesis和angiogenesis两种情况。Vascularogenesis主要是指由胚胎发育时的angioblast形成的原始血管网络,继而出现血管重塑(remodeling),包括血管内皮、平滑肌细胞、支持细胞和细胞外基质相互作用,形成完整的血管的过程。Angiogenesis是指从已存在的血管上,通过成熟血管内皮细胞增殖、游走,形成新的毛细血管网的过程。血管新生的目的是侧支血管形成并形成血流,以保障所有细胞的充足氧供。在成人缺血性心脏病和肿瘤的缺血缺氧组织中都会有血管数量的增多,低氧诱导的HIF-1α的激活是主要的分子机制。血管再生(Angiogenesis)、血运重建是一个复杂的病理生理过程,需要有许多血管生长因子的参与,一些大样本量、随机、安慰剂对照试验的结果提示单一生长因子的上调作用对于血管生成可能是不足的,成熟的血管形成需要大量不同的细胞类型的相互作用以及后继的大量生长因子及它们的受体的共同表达。所以,联合基因治疗成为新的研究热点。
人低氧诱导因子-1(hypoxia inducible factor 1,HIF-1)被公认是一个在应答细胞氧张力变化的基因表达方面的重要调节因子,由两个亚单位组成,氧调节亚单位HIF-1α和构成性表达的亚单位HIF-1β。HIF-1α可调控下游60多种与血管生长、血管张力、糖代谢、红细胞生成及干细胞诱导分化等相关的基因。低氧刺激可诱导HIF-1α产生,而HIF-1α可通过促进下游一系列靶基因(如VEGF、VEGFR1、VEGFR2、NOS、IGF-Ⅱ等)的转录,触发机体血管新生的反应,促进侧支循环的建立,有望诱导生理功能完整的血管新生。
但由于其独特的结构,HIF-1α只有在低氧条件下才能发挥功能,其蛋白形式在常氧(21%O_2)下5分钟即发生降解。主要与氧依赖降解区(oxygen dependentdegradation domain,ODDD)564位的脯氨酸(Prolyl,Pro)和402位的脯氨酸(Prolyl,Pro)发生羟基化有关,在低氧的状态下,脯氨酰羟化酶的活性受到抑制,Pro564和Pro402羟基化反应受阻,导致HIF-1α降解途径中断,细胞内HIF-1α水平增加,与HIF-1β二聚化形成具有生理活性的HIF-1,通过与靶基因如VEGF的低氧反应元件(hypoxia response element,HRE)结合,进而激活靶基因的转录。而HIF-1α转录激活区(transactivation domain,TAD)的活性则主要受C-TAD端803位天门冬氨酰(Asparageine,Asn)的羟化调节,在常氧下,Asn 803可在HIF抑制因子(factor inhibiting HIF-1α,FIH)作用下发生羟基化,使得C-TAD与转录协同激活因子CBP/p300的结合受抑制,从而抑制其转录活性。以上发现阐明了HIF-1α两个重要结构区的功能,为此,《Sincese》曾发表评论称“这些发现将为缺血性心脏病、中风等许多疾病启开一种新的治疗的可能性”。我们先期分别对HIF-1α的关键位点Pro564、Pro402和Asn803单一或联合定点突变为Ala564、Ala402和Ala803,获得了人突变体HIF-1α基因。
为提高外源性基因的转染效率,必须借助于载体。腺病毒因可转染静止细胞,转染率高,对于非复制状态的心肌细胞来说较为适宜;且不整合进染色体,避免了插入突变的危险。虽维持时间较短,但这对于只需短时间表达基因产物的心血管病基因治疗来说,更为适合。故我们将人突变体HIF-1α基因成功包装了腺病毒,获得了三种不同位点突变的重组腺病毒人突变体HIF-1α基因。
目的:
探讨不同位点突变的重组腺病毒人突变体HIF-1α基因(Ad-HIF-1α-Ala564,简称Ad-H_(564);Ad-HIF-1α-Ala 564-Ala 402,简称Ad-H_(564/402);Ad-HIF-1α-Ala564-Ala 803,简称Ad-H_(564/803))在大鼠急性下肢缺血模型内的表达及对血管新生的影响。
方法:
①在NEK 293A细胞中大量扩增腺病毒、氯化铯浓度梯度离心,透析纯化,以获得高纯度、高活性的重组腺病毒人突变体HIF-1α基因。应用电子显微镜、PCR、测序等方法鉴定腺病毒基因信息,用分光光度计法测定病毒滴度。②构建急性大鼠下肢缺血模型,随机分为6组,分别以Ad-LacZ、Ad-H_0、Ad-H_(564)、Ad-H_(564/402)、Ad-H_(564/803)和生理盐水(NS)转染术侧下肢骨骼肌,③于转染后1、3、5、7天,RT-PCR测定骨骼肌中HIF-1αmRNA的表达量;④于转染后第28天,用免疫组化的方法检测骨骼肌中HIF-1α的蛋白表达及CD31蛋白表达;⑤基因转染后28天,选择性下肢动脉造影及动脉铸型观察血管密度。
结果:
①经过在HEK 293A细胞中大量扩增、氯化铯密度梯度离心、透析纯化后,各种重组腺病毒人突变体HIF-1α基因纯度提高,滴度达到10~(11) OPU/ml,所携带的目的基因信息无丢失或变异,体外实验显示转染效率高,未发现野生型腺病毒产生。②三种重组腺病毒人突变体HIF-1α基因转染大鼠急性下肢缺血模型后,均可促进骨骼肌内HIF-1αmRNA的表达,且以第7天的表达量最高(F=24.942,P=0.000);各突变体HIF-1α基因组的表达量均高于野生型HIF-1α基因组(F=99.380,P=0.000);三种突变体基因组间相比,表达量最高的是Ad-HIF-1α_(564/402)组(0.7952±0.1563,P=0.000),其次为Ad-HIF-1α_(564/803)组(0.5815±0.0994,P=0.000),再次为Ad-HIF-1α_(564)组(0.5447±0.1413)。③三种重组腺病毒人突变体HIF-1α基因转染大鼠急性下肢缺血模型后第28天,各突变体HIF-1α基因组大鼠的骨骼肌组织间及间质组织中均可见明显的HIF-1α蛋白表达阳性细胞和CD31蛋白表达阳性细胞,且在血管周围比较密集,微小血管着色更深,与野生型HIF-1α基因组及对照组的差异显著。;各突变体HIF-1α基因相比,以Ad-H_(564/803)组的最为密集。④三种重组腺病毒人突变体HIF-1α基因转染大鼠急性下肢缺血模型后第28天,血管造影及动脉铸型显示均可触发机体血管新生,促进侧支循环的建立。虽侧支血管不及野生型HIF-1α基因组的粗大,但末梢可见更多的绒毛样微细血管;各突变体基因组间相比,以Ad-HIF-1α_(564/803)组的促血管新生作用最强,其次为Ad-HIF-1α_(564/402)组,再次为Ad-HIF-1α_(564)组。
结论:
①外源性重组腺病毒人突变体基因Ad-H_(564)、Ad-H_(564/402)、Ad-H_(564/803)均可促进骨骼肌组织中的HIF-1αmRNA的表达和HIF-1α蛋白的表达,且较野生型HIF-1α基因的作用更强。②外源性重组腺病毒人突变体基因Ad-H_(564)、Ad-H_(564/402)、Ad-H_(564/803)转染大鼠急性下肢缺血模型后第28天,既可促进双侧下肢的侧支血管建立,又可促进局部小血管形成(angiogenesis)。且促进局部绒毛样微小血管形成的作用强于野生型HIF-1α基因。③各突变体基因组间相比较,以Ad-HIF-1α_(564/803)组的促血管新生作用最强,其次为Ad-HIF-1α_(564/402)组。
Introduction
Coronary artery disease(CAD) is the most common type of heart disease.It is the leading cause of death in urban and rural residents in China and will become the most disease burden and primary death causes in the world in 2020.Now,the main treatment of CAD are medicine and revascularization,including percutaneous coronary intervention(PCI) and coronary artery bypass graft(CABG) surgery.A significant proportion of patients with coronary artery disease,were called end stage patients,have symptoms refractory to medical treatment,yet are unsuitable for conventional revascularization techniques,like PCI and CABG.Such patients are potential candidates for alternative forms of coronary revascularization,like therapeutic angiogenesis.Two major avenues for achieving therapeutic angiogenesis are currently under intense investigation:gene therapy(the introduction of new genetic material into somatic cells to synthesize proteins that are missing,defective, or desired for specific therapeutic purposes) and protein-based therapy (administration of the growth factors,instead of the genes encoding for the growth factors responsible for angiogenesis.) From 90's,it was found that angiogenic growth factors,which plays an important role in physiologic and pathologic angiogenesis,could contribute to neovascularization and promote collateral artery development in animal models of myocardial and hindlimb ischemia.Stimulation of angiogenesis represents an important new therapeutic approach for CAD.
Vascular development involves vasculogenesis,in which endothelial cells form a primary tubular network,as well as angiogenesis,in which vessel size and structure are modified based upon flow and branching occurs to insure that all cells receive adequate O_2 delivery.In adults,angiogenesis occurs in response to tissue hypoxia/ ischemia and plays an important role in determining the progression of ischemic heart disease and cancer.A critical molecular pathway induced by hypoxia/ischemia is the activation of hypoxia-inducible factor 1,a transcriptional activator of genes encoding vascular endothelial growth factor and other important mediators of angiogenesis. Angiogenesis is a highly complex,orchestrated process that plays a critical role in normal development and in the pathophysiology of common diseases.It requires a balance of multiple angiogenic factors.Results of several randomized,double-blind. placebo-controlled trial of therapeutic angiogenesis by gene transfer have been reported that therapeutic angiogenesis strategies that involve administration of a single angiogenic factor was insufficient.Different angiogenic factors appear to play complementary roles in the process of vascular development.So combined gene therapy become the research hotspots of therapeutic angiogenesis.
Hypoxia-inducible factor 1(HIF-1) is a heterodimeric transcription factor that functions as a master regulator of mammalian oxygen homeostasis.At a molecular level.HIF-1 is composed of an Q_2-regulated HIF-1αsubunit and a constitutively expressed HIF-1βsubunit.The HIF-1αsubunit is 826-amino-acid protein that is regulatory and is unique to the hypoxic response.In response to low level of oxygen, subunit HIF-1αexpression is up-regulated and transactivates its target genes essential (＞60) for glucose uptake,energy metabolism,erythropoiesis and vascular development.
There are two key domains in HIF-1α,which named oxygen dependent degradation domain(ODDD) and transactivation domains(TAD).When cells maintained at low oxygen tension are returned to a nonhypoxic environment,HIF-1αprotein levels decay rapidly,as the protein is degraded via the ubiquitin-proteosome pathway.It was related to the prolyl hydroxylation of the two proline residus Pro564 and Pro402 in ODD.Under normoxic conditions,HIF-1αis hydroxylated on proline residues 402 and 564,and this modification functions as a molecular signal for ubiquitination and degradation of the protein.As a result,HIF-1αhas an extremely short half-life under normoxic conditions.Since O_2 is rate-limiting for the prolyl hydroxylases that perform this reaction,prolyl hydroxylation of HIF-1αis inhibited in hypoxic/ischemic tissues.This causes accumulation of the protein,which dimerizes with HIF-1β,generating a functional heterodimer that can bind to specific nucleotide sequences in target genes such as VEGF and activate their transcription.The activaty of transcription of HIF-1 is related to the hydroxylation of Asn803 in TAD.These dates identified the function of two primary domains in HIF-1α."Discovery of these could open up new therapeutic possibilities for the many diseases such as ischemic heart disease and strock"(Science).
We have constructed the adenovirus mutant HIF-1α-Ala564(Ad-H_(564)), adenovirus mutant HIF-1α-Ala564-Ala402(Ad-H_(564/402)) and adenovirus mutant HIF-1α-Ala564-Ala803(Ad-H_(564/803)) in last 3-4 years.In subsequent investigations, we establish the model of rat hindlimb ischemia,intramuscular transfect mutant HIF-1 a genes respectively,and evaluate the function of these mutant HIF-1αgenes.
The transfection efficiency of genes could be promoted by vector.Adenoviruses is the vectors most often studied likely because of the ease of production,reasonable transfection efficiency,and expression in nonproliferating cells.The life cycle does not normally involve integration into the host genome,rather they replicate as episomal elements in the nucleus of the host cell and consequently there is no risk of insertional mutagenesis.It is more suitable for cardiomyocyte because of its specific features.
Object
To investigate the gene expression and compare the function of mutant HIF-1αadenoviral-mediated gene in various sites(Ad-H_(564),Ad-H_(564/402) and Ad-H_(564/803)) in the rat model of hindlimb ischemia.
Methods
(1) In the first experiment,mutant HIF-1αgene adenovirus vector was amplified in HEK293A cells and purified by ultracentrifugation in CsC1 step gradient solutions and transfect into the hela cells.Then the adenovirus was determined by electron microscope,PCR and sequencing,and the amount of viral particles was determined by spectrophotometry.
(2) In the second experiment,the acute rat ischemic hindlimb models were produced and the Ad-LacZ,Ad-H_0,Ad-H_(564),Ad-H_(564/402),Ad-H_(564/803) and normal saline (NS) 500μl were administered respectively intramuscularly into the ischemic limb in 5 sites.The gene expression was evaluated by reverse transcriptase polymerase chain reaction(RT-PCR) after 1,3,5,7 days of intramuscular gene transfer in skeletal muscles of rat in vivo.
(3) The protein expression of HIF-1αand CD31 were observed by immunohistochemistry stain after 28 days of intramuscular gene transfer in skeletal muscles of rat in vivo.
(4) Selective internal iliac angiograghy was performed by the same angiogragher to illustrate the microvessel density at the 28~(th) day after intramuscular gene transfer.
Results
(1) The correct mutant adenovirus HIF-1αwas obtained after being amplified and purified.The last titre of adenovirus was about 10~(11) OPU/ml.Not found the wild type adenovirus.
(2) The results of RT-PCR showed that mutant adenovirus HIF-1αimprove the gene expression in ischemic muscle in vivo,and the relative expression was the highest at the 7~(th) day after intramuscular gene transfer(F=24.942,P=0.000).It is higher in all groups of mutant adenovirus HIF-1αthan that of control groups (F=99.380,P=0.000).The relative expression in group Ad-HIF-1α_(564/402) was the highest(0.7952±0.1563,P=0.000) in three groups of mutant adenovirus HIF-1α, then in group Ad-HIF-1α_(564/803)(0.5815±0.0994,P=0.000),then the Ad-HIF-1α_(564) (0.5447±0.1413)。
(3) Many positive cells of HIF-1αand CD31 by immunohistochemistry stain were found in skeletal muscles and interstitial tissues in groups of mutant HIF-1αgene at the 28~(th) day after intramuscular gene transfer,and the positive cells was more densely around the blood vessels.The positive cells in all groups of mutant HIF-1αgene was more densely than that of nature HIF-1αgene and was the most densely in group of Ad-H_(564/803).The more minimal the vessel is,the darker the positive cells was been stained.
(4) The results of angiograghy and vascular cast showed that mutant adenovirus HIF-1αcould induce angiogenesis and promote collateral artery development in vivo after 28 days of gene transfer,and the function of mutant adenovirus HIF-1αis more powerful than that of the nature HIF-1α.The microvessel density in group Ad-H_(564/803) was the greatest in three groups of mutant adenovirus HIF-1α.
Conclusion
The mutant adenovirus-mediated gene transfer of HIF-1αimproves the gene expression of HIF-1αmRNA and HIF-1αprotein in vivo and contributes to neovascularization and promotes collateral artery development in rat models of hindlimb ischemia.The function of the Ad-HIF-1α_(564/803) is more powerful than that of Ad-HIF-1α_(564) and Ad-HIF-1α_(564/402).
冠心病是一种常见的心脏病,是我国城乡居民的主要死亡原因,并将成为全世界首要的死亡原因和最大的疾病负担。目前的治疗手段主要有药物治疗和包括冠状动脉旁路移植术(coronary artery bypass graft,CABG)及经皮冠状动脉介入术(percutaneous coronary intervention,PCI)在内的血运重建技术(revascularization)。终末期冠心病患者约占冠心病患者中的10%,因冠状动脉病变弥漫,或病变血管细小等无条件进行CABG及PTCA治疗,药物治疗又不能满意地控制其心绞痛症状,需要寻求一种新的更加适合于他们的治疗方法。90年代以来,人们发现,一些在机体生理和病理性的血管生成中起重要作用的生长因子能够促进缺血组织的血管新生,加速侧支循环的建立,有可能通过直接提高缺血区的血供来改善组织缺血,并设想导入外源性生长因子以促进血管新生,即所谓治疗性血管生成(therapeutic angiogenesis)。这一方法为冠心病的治疗提供了新思路。
血管新生(neovascularization)包括vasculogenesis和angiogenesis两种情况。Vascularogenesis主要是指由胚胎发育时的angioblast形成的原始血管网络,继而出现血管重塑(remodeling),包括血管内皮、平滑肌细胞、支持细胞和细胞外基质相互作用,形成完整的血管的过程。Angiogenesis是指从已存在的血管上,通过成熟血管内皮细胞增殖、游走,形成新的毛细血管网的过程。血管新生的目的是侧支血管形成并形成血流,以保障所有细胞的充足氧供。在成人缺血性心脏病和肿瘤的缺血缺氧组织中都会有血管数量的增多,低氧诱导的HIF-1α的激活是主要的分子机制。血管再生(Angiogenesis)、血运重建是一个复杂的病理生理过程,需要有许多血管生长因子的参与,一些大样本量、随机、安慰剂对照试验的结果提示单一生长因子的上调作用对于血管生成可能是不足的,成熟的血管形成需要大量不同的细胞类型的相互作用以及后继的大量生长因子及它们的受体的共同表达。所以,联合基因治疗成为新的研究热点。
人低氧诱导因子-1(hypoxia inducible factor 1,HIF-1)被公认是一个在应答细胞氧张力变化的基因表达方面的重要调节因子,由两个亚单位组成,氧调节亚单位HIF-1α和构成性表达的亚单位HIF-1β。HIF-1α可调控下游60多种与血管生长、血管张力、糖代谢、红细胞生成及干细胞诱导分化等相关的基因。低氧刺激可诱导HIF-1α产生,而HIF-1α可通过促进下游一系列靶基因(如VEGF、VEGFR1、VEGFR2、NOS、IGF-Ⅱ等)的转录,触发机体血管新生的反应,促进侧支循环的建立,有望诱导生理功能完整的血管新生。
但由于其独特的结构,HIF-1α只有在低氧条件下才能发挥功能,其蛋白形式在常氧(21%O_2)下5分钟即发生降解。主要与氧依赖降解区(oxygen dependentdegradation domain,ODDD)564位的脯氨酸(Prolyl,Pro)和402位的脯氨酸(Prolyl,Pro)发生羟基化有关,在低氧的状态下,脯氨酰羟化酶的活性受到抑制,Pro564和Pro402羟基化反应受阻,导致HIF-1α降解途径中断,细胞内HIF-1α水平增加,与HIF-1β二聚化形成具有生理活性的HIF-1,通过与靶基因如VEGF的低氧反应元件(hypoxia response element,HRE)结合,进而激活靶基因的转录。而HIF-1α转录激活区(transactivation domain,TAD)的活性则主要受C-TAD端803位天门冬氨酰(Asparageine,Asn)的羟化调节,在常氧下,Asn 803可在HIF抑制因子(factor inhibiting HIF-1α,FIH)作用下发生羟基化,使得C-TAD与转录协同激活因子CBP/p300的结合受抑制,从而抑制其转录活性。以上发现阐明了HIF-1α两个重要结构区的功能,为此,《Sincese》曾发表评论称“这些发现将为缺血性心脏病、中风等许多疾病启开一种新的治疗的可能性”。我们先期分别对HIF-1α的关键位点Pro564、Pro402和Asn803单一或联合定点突变为Ala564、Ala402和Ala803,获得了人突变体HIF-1α基因。
为提高外源性基因的转染效率,必须借助于载体。腺病毒因可转染静止细胞,转染率高,对于非复制状态的心肌细胞来说较为适宜;且不整合进染色体,避免了插入突变的危险。虽维持时间较短,但这对于只需短时间表达基因产物的心血管病基因治疗来说,更为适合。故我们将人突变体HIF-1α基因成功包装了腺病毒,获得了三种不同位点突变的重组腺病毒人突变体HIF-1α基因。
目的:
探讨不同位点突变的重组腺病毒人突变体HIF-1α基因(Ad-HIF-1α-Ala564,简称Ad-H_(564);Ad-HIF-1α-Ala 564-Ala 402,简称Ad-H_(564/402);Ad-HIF-1α-Ala564-Ala 803,简称Ad-H_(564/803))在大鼠急性下肢缺血模型内的表达及对血管新生的影响。
方法:
①在NEK 293A细胞中大量扩增腺病毒、氯化铯浓度梯度离心,透析纯化,以获得高纯度、高活性的重组腺病毒人突变体HIF-1α基因。应用电子显微镜、PCR、测序等方法鉴定腺病毒基因信息,用分光光度计法测定病毒滴度。②构建急性大鼠下肢缺血模型,随机分为6组,分别以Ad-LacZ、Ad-H_0、Ad-H_(564)、Ad-H_(564/402)、Ad-H_(564/803)和生理盐水(NS)转染术侧下肢骨骼肌,③于转染后1、3、5、7天,RT-PCR测定骨骼肌中HIF-1αmRNA的表达量;④于转染后第28天,用免疫组化的方法检测骨骼肌中HIF-1α的蛋白表达及CD31蛋白表达;⑤基因转染后28天,选择性下肢动脉造影及动脉铸型观察血管密度。
结果:
①经过在HEK 293A细胞中大量扩增、氯化铯密度梯度离心、透析纯化后,各种重组腺病毒人突变体HIF-1α基因纯度提高,滴度达到10~(11) OPU/ml,所携带的目的基因信息无丢失或变异,体外实验显示转染效率高,未发现野生型腺病毒产生。②三种重组腺病毒人突变体HIF-1α基因转染大鼠急性下肢缺血模型后,均可促进骨骼肌内HIF-1αmRNA的表达,且以第7天的表达量最高(F=24.942,P=0.000);各突变体HIF-1α基因组的表达量均高于野生型HIF-1α基因组(F=99.380,P=0.000);三种突变体基因组间相比,表达量最高的是Ad-HIF-1α_(564/402)组(0.7952±0.1563,P=0.000),其次为Ad-HIF-1α_(564/803)组(0.5815±0.0994,P=0.000),再次为Ad-HIF-1α_(564)组(0.5447±0.1413)。③三种重组腺病毒人突变体HIF-1α基因转染大鼠急性下肢缺血模型后第28天,各突变体HIF-1α基因组大鼠的骨骼肌组织间及间质组织中均可见明显的HIF-1α蛋白表达阳性细胞和CD31蛋白表达阳性细胞,且在血管周围比较密集,微小血管着色更深,与野生型HIF-1α基因组及对照组的差异显著。;各突变体HIF-1α基因相比,以Ad-H_(564/803)组的最为密集。④三种重组腺病毒人突变体HIF-1α基因转染大鼠急性下肢缺血模型后第28天,血管造影及动脉铸型显示均可触发机体血管新生,促进侧支循环的建立。虽侧支血管不及野生型HIF-1α基因组的粗大,但末梢可见更多的绒毛样微细血管;各突变体基因组间相比,以Ad-HIF-1α_(564/803)组的促血管新生作用最强,其次为Ad-HIF-1α_(564/402)组,再次为Ad-HIF-1α_(564)组。
结论:
①外源性重组腺病毒人突变体基因Ad-H_(564)、Ad-H_(564/402)、Ad-H_(564/803)均可促进骨骼肌组织中的HIF-1αmRNA的表达和HIF-1α蛋白的表达,且较野生型HIF-1α基因的作用更强。②外源性重组腺病毒人突变体基因Ad-H_(564)、Ad-H_(564/402)、Ad-H_(564/803)转染大鼠急性下肢缺血模型后第28天,既可促进双侧下肢的侧支血管建立,又可促进局部小血管形成(angiogenesis)。且促进局部绒毛样微小血管形成的作用强于野生型HIF-1α基因。③各突变体基因组间相比较,以Ad-HIF-1α_(564/803)组的促血管新生作用最强,其次为Ad-HIF-1α_(564/402)组。
Introduction
Coronary artery disease(CAD) is the most common type of heart disease.It is the leading cause of death in urban and rural residents in China and will become the most disease burden and primary death causes in the world in 2020.Now,the main treatment of CAD are medicine and revascularization,including percutaneous coronary intervention(PCI) and coronary artery bypass graft(CABG) surgery.A significant proportion of patients with coronary artery disease,were called end stage patients,have symptoms refractory to medical treatment,yet are unsuitable for conventional revascularization techniques,like PCI and CABG.Such patients are potential candidates for alternative forms of coronary revascularization,like therapeutic angiogenesis.Two major avenues for achieving therapeutic angiogenesis are currently under intense investigation:gene therapy(the introduction of new genetic material into somatic cells to synthesize proteins that are missing,defective, or desired for specific therapeutic purposes) and protein-based therapy (administration of the growth factors,instead of the genes encoding for the growth factors responsible for angiogenesis.) From 90's,it was found that angiogenic growth factors,which plays an important role in physiologic and pathologic angiogenesis,could contribute to neovascularization and promote collateral artery development in animal models of myocardial and hindlimb ischemia.Stimulation of angiogenesis represents an important new therapeutic approach for CAD.
Vascular development involves vasculogenesis,in which endothelial cells form a primary tubular network,as well as angiogenesis,in which vessel size and structure are modified based upon flow and branching occurs to insure that all cells receive adequate O_2 delivery.In adults,angiogenesis occurs in response to tissue hypoxia/ ischemia and plays an important role in determining the progression of ischemic heart disease and cancer.A critical molecular pathway induced by hypoxia/ischemia is the activation of hypoxia-inducible factor 1,a transcriptional activator of genes encoding vascular endothelial growth factor and other important mediators of angiogenesis. Angiogenesis is a highly complex,orchestrated process that plays a critical role in normal development and in the pathophysiology of common diseases.It requires a balance of multiple angiogenic factors.Results of several randomized,double-blind. placebo-controlled trial of therapeutic angiogenesis by gene transfer have been reported that therapeutic angiogenesis strategies that involve administration of a single angiogenic factor was insufficient.Different angiogenic factors appear to play complementary roles in the process of vascular development.So combined gene therapy become the research hotspots of therapeutic angiogenesis.
Hypoxia-inducible factor 1(HIF-1) is a heterodimeric transcription factor that functions as a master regulator of mammalian oxygen homeostasis.At a molecular level.HIF-1 is composed of an Q_2-regulated HIF-1αsubunit and a constitutively expressed HIF-1βsubunit.The HIF-1αsubunit is 826-amino-acid protein that is regulatory and is unique to the hypoxic response.In response to low level of oxygen, subunit HIF-1αexpression is up-regulated and transactivates its target genes essential (＞60) for glucose uptake,energy metabolism,erythropoiesis and vascular development.
There are two key domains in HIF-1α,which named oxygen dependent degradation domain(ODDD) and transactivation domains(TAD).When cells maintained at low oxygen tension are returned to a nonhypoxic environment,HIF-1αprotein levels decay rapidly,as the protein is degraded via the ubiquitin-proteosome pathway.It was related to the prolyl hydroxylation of the two proline residus Pro564 and Pro402 in ODD.Under normoxic conditions,HIF-1αis hydroxylated on proline residues 402 and 564,and this modification functions as a molecular signal for ubiquitination and degradation of the protein.As a result,HIF-1αhas an extremely short half-life under normoxic conditions.Since O_2 is rate-limiting for the prolyl hydroxylases that perform this reaction,prolyl hydroxylation of HIF-1αis inhibited in hypoxic/ischemic tissues.This causes accumulation of the protein,which dimerizes with HIF-1β,generating a functional heterodimer that can bind to specific nucleotide sequences in target genes such as VEGF and activate their transcription.The activaty of transcription of HIF-1 is related to the hydroxylation of Asn803 in TAD.These dates identified the function of two primary domains in HIF-1α."Discovery of these could open up new therapeutic possibilities for the many diseases such as ischemic heart disease and strock"(Science).
We have constructed the adenovirus mutant HIF-1α-Ala564(Ad-H_(564)), adenovirus mutant HIF-1α-Ala564-Ala402(Ad-H_(564/402)) and adenovirus mutant HIF-1α-Ala564-Ala803(Ad-H_(564/803)) in last 3-4 years.In subsequent investigations, we establish the model of rat hindlimb ischemia,intramuscular transfect mutant HIF-1 a genes respectively,and evaluate the function of these mutant HIF-1αgenes.
The transfection efficiency of genes could be promoted by vector.Adenoviruses is the vectors most often studied likely because of the ease of production,reasonable transfection efficiency,and expression in nonproliferating cells.The life cycle does not normally involve integration into the host genome,rather they replicate as episomal elements in the nucleus of the host cell and consequently there is no risk of insertional mutagenesis.It is more suitable for cardiomyocyte because of its specific features.
Object
To investigate the gene expression and compare the function of mutant HIF-1αadenoviral-mediated gene in various sites(Ad-H_(564),Ad-H_(564/402) and Ad-H_(564/803)) in the rat model of hindlimb ischemia.
Methods
(1) In the first experiment,mutant HIF-1αgene adenovirus vector was amplified in HEK293A cells and purified by ultracentrifugation in CsC1 step gradient solutions and transfect into the hela cells.Then the adenovirus was determined by electron microscope,PCR and sequencing,and the amount of viral particles was determined by spectrophotometry.
(2) In the second experiment,the acute rat ischemic hindlimb models were produced and the Ad-LacZ,Ad-H_0,Ad-H_(564),Ad-H_(564/402),Ad-H_(564/803) and normal saline (NS) 500μl were administered respectively intramuscularly into the ischemic limb in 5 sites.The gene expression was evaluated by reverse transcriptase polymerase chain reaction(RT-PCR) after 1,3,5,7 days of intramuscular gene transfer in skeletal muscles of rat in vivo.
(3) The protein expression of HIF-1αand CD31 were observed by immunohistochemistry stain after 28 days of intramuscular gene transfer in skeletal muscles of rat in vivo.
(4) Selective internal iliac angiograghy was performed by the same angiogragher to illustrate the microvessel density at the 28~(th) day after intramuscular gene transfer.
Results
(1) The correct mutant adenovirus HIF-1αwas obtained after being amplified and purified.The last titre of adenovirus was about 10~(11) OPU/ml.Not found the wild type adenovirus.
(2) The results of RT-PCR showed that mutant adenovirus HIF-1αimprove the gene expression in ischemic muscle in vivo,and the relative expression was the highest at the 7~(th) day after intramuscular gene transfer(F=24.942,P=0.000).It is higher in all groups of mutant adenovirus HIF-1αthan that of control groups (F=99.380,P=0.000).The relative expression in group Ad-HIF-1α_(564/402) was the highest(0.7952±0.1563,P=0.000) in three groups of mutant adenovirus HIF-1α, then in group Ad-HIF-1α_(564/803)(0.5815±0.0994,P=0.000),then the Ad-HIF-1α_(564) (0.5447±0.1413)。
(3) Many positive cells of HIF-1αand CD31 by immunohistochemistry stain were found in skeletal muscles and interstitial tissues in groups of mutant HIF-1αgene at the 28~(th) day after intramuscular gene transfer,and the positive cells was more densely around the blood vessels.The positive cells in all groups of mutant HIF-1αgene was more densely than that of nature HIF-1αgene and was the most densely in group of Ad-H_(564/803).The more minimal the vessel is,the darker the positive cells was been stained.
(4) The results of angiograghy and vascular cast showed that mutant adenovirus HIF-1αcould induce angiogenesis and promote collateral artery development in vivo after 28 days of gene transfer,and the function of mutant adenovirus HIF-1αis more powerful than that of the nature HIF-1α.The microvessel density in group Ad-H_(564/803) was the greatest in three groups of mutant adenovirus HIF-1α.
Conclusion
The mutant adenovirus-mediated gene transfer of HIF-1αimproves the gene expression of HIF-1αmRNA and HIF-1αprotein in vivo and contributes to neovascularization and promotes collateral artery development in rat models of hindlimb ischemia.The function of the Ad-HIF-1α_(564/803) is more powerful than that of Ad-HIF-1α_(564) and Ad-HIF-1α_(564/402).
引文
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2.Murray CJ,Lopez AD.Alternative projection of mortality and disarbility by cause 1990-2020:Globle Burden of Disease Study.Lancet 1997,349:1498-1504
3.霍勇,王日胜.冠心病介入治疗的现状与进展.实用老年医学;2005;19(3):118-120
4.Henry TD,Annex BH,McKendall GR et al.The VIVA trial:vascular endothelial growth factor in ischemia for vascular angiogenesis.Circulation 2003;107:1359-1365
5.Zheng Y,Murakami M,Takahashi H,et al.Chimeric VEGF-ENZ7/PlGF Promotes Angiogenesis Via VEGFR-2 Without Significant Enhancement of Vascular Permeability and Inflammation.Arterioscler Thromb Vase Biol.2006 Jun 22;[Epub ahead of print].
6.Darland DC,D'Amore PA.Blood vessel maturation:vascular development comes of age.J Clin Invest 1999;103:157-158.
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