CN110167564A - 调节tjp1表达以调节心脏细胞的再生 - Google Patents
调节tjp1表达以调节心脏细胞的再生 Download PDFInfo
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Abstract
本发明涉及治疗受试者中的心脏病、特别是急性心肌梗死(AMI)的方法,包括向受试者施用Tjp1抑制剂的步骤,其中所述Tjp1抑制剂的施用促进心肌细胞增殖。本发明还包括Tjp1抑制剂在制造用于心脏病的药物中的用途、贴剂和编码Tjp1抑制剂的核酸。在具体实施方案中,Tjp1抑制剂是核酸,即Tjp1的siRNA或shRAN。本发明还包括施用所述Tjp1抑制剂与神经调节蛋白1(NRG1)、成纤维细胞生长因子(FGF)、血管内皮生长因子(VEGF)或滤泡稳定蛋白样1(Fstl1)的组合,并且其中以血清型9的腺伴随病毒(AAV 9)中递送所述抑制剂。
Description
对相关申请的交叉引用
本申请要求2016年9月14日提交的新加坡临时申请第10201607673U的优先权权益,通过引用将其内容整体全部并入本文用于所有目的。
技术领域
本发明一般性地涉及生物化学。特别是,本发明涉及通过靶向Tjp1治疗心脏病的方法。
背景技术
现代的久坐生活方式、饮食、预期寿命增加和如糖尿病等疾病的增加导致患有心血管疾病(包括急性心肌梗死(AMI))的患者爆发。当代治疗,包括血管成形术、支架置入术和辅助药物治疗降低了AMI的早期死亡率。然而,尽管特别遵守指南推荐的疗法,但2010年当地28天AMI病例死亡率明显超过2009年OECD平均水平(12.7对7.9%)。这些相对更差的结果的一个原因可能是AMI后的心力衰竭(HF)。在AMI期间,数十亿心肌细胞(muscle cell)或心肌细胞(cardiomyocytes,CM)丧失并被成纤维细胞取代形成富含胶原蛋白的瘢痕组织。这种瘢痕不会收缩,并会通过代偿机制和额外的CM丧失进一步削弱心脏,最终导致HF和死亡。成体心脏在受伤后无法充分再生,使全世界数百万人承受着AMI的后果,相关的死亡率特别令人担忧。因此,在MI之后允许存活或替换丧失的CM的治疗方法将具有巨大的经济和社会影响。鉴于上述问题,需要提供治疗受试者的心脏病的替代方法。
发明概述
在一个方面,提供了治疗受试者中的心脏病的方法,包括向所述受试者施用治疗有效量的Tjp1抑制剂的步骤。在一个实施方案中,Tjp1抑制剂是核酸。在一个实施方案中,核酸选自由siRNA、shRNA、反义寡核苷酸、gapmer和短发夹反义寡核苷酸(hairpin AntisenseOligonucleotide,shAON)组成的组。在一个实施方案中,核酸与编码Tjp1的mRNA结合并形成核酸-mRNA复合体。在一个实施方案中,核酸-mRNA复合体中的mRNA被切割和/或不被翻译。在一个实施例中,编码抑制剂的核酸与选自以下组成的组的序列具有至少90%的同一性:SEQ ID NO:1(CCGGGCCTGCATACAATAAAGCAAACTCGAGTTTGCTTTATTGTATGCAGGCTTTTTG)、SEQ ID NO:2(CCGGGGAACCACTCTATCAAGTATTCTCGAGAATACTTGATAGAGTGGTTCCTTTTTG)、SEQID NO:3(CCGGCGTGGATTGAACTTACTAAATCTCGAGATTTAGTAAGTTCAATCCACGTTTTTG)和SEQ IDNO:4(CCGGCCGCGAAGTTATGAGCAAGTTCTCGAGAACTTGCTCATAACTTCGCGGTTTTTG)。
在一个实施方案中,所述方法还包括与Tjp1抑制剂一起或单独施用选自由多肽和核酸组成的组的其他因子。在一个实施方案中,所述核酸编码细胞周期蛋白A2。在一个实施方案中,所述多肽与ErbB4受体结合。在一个实施方案中,结合ErbB4受体的多肽选自由神经调节蛋白-1、神经调节蛋白-2(NRG2)、神经调节蛋白-3(NRG3)、乙胞素(BTC)、上皮调节蛋白(EPR)、肝素结合EGF样生长因子(HB-EGF)、表皮生长因子(EGF)、β-纤维素(cellulin)、转化生长因子α(TGFα)和双调蛋白(AR)组成的组。在一个实施方案中,结合ErbB4受体的多肽是神经调节蛋白-1(NRG1)。在一个实施方案中,所述多肽激活Wnt信号传导。在一个实施方案中,所述激活Wnt信号传导的多肽选自由Wnt、Norrin和R-spondin组成的组。在一个实施方案中,所述多肽是生长因子或分泌因子。在一个实施方案中,生长因子是成纤维细胞生长因子(FGF)或血管内皮生长因子(VEGF)。在一个实施方案中,分泌因子是滤泡稳定蛋白样1(Fstl1)。
在一个实施方案中,其中当抑制剂是核酸抑制剂时,所述方法包括病毒介导的递送系统。在一个实施方案中,所述病毒选自由逆转录病毒、腺病毒、腺伴随病毒和单纯疱疹病毒组成的组。在一个实施方案中,病毒是腺伴随病毒。在一个实施方案中,腺伴随病毒是AAV血清型9。
在另一个方面,提供了Tjp1抑制剂在制造用于治疗心脏病的药物中的用途。在一个实施方案中,心脏病选自由以下组成的组:心肌梗死、急性心肌梗死(AMI)、心力衰竭、心肌症、先天性心脏病、获得性心血管疾病、心肌细胞缺陷、心脏缺血再灌注损伤、心脏创伤和在心脏细胞再生有益的患者中的其他心脏损伤。
还在另一个方面,提供了贴剂,其中贴剂包含如本文定义的Tjp1抑制剂。
还在另一个方面,提供了编码Tjp1抑制剂的核酸,其中所述核酸与选自由以下组成的组的序列具有至少90%的同一性:SEQ ID NO:1(CCGGGCCTGCATACAATAAAGCAAACTCGAGTTTGCTTTATTGTATGCAGGCTTTTTG),SEQ ID NO:2(CCGGGGAACCACTCTATCAAGTATTCTCGAGAATACTTGATAGAGTGGTTCCTTTTTG),SEQ ID NO:3(CCGGCGTGGATTGAACTTACTAAATCTCGAGATTTAGTAAGTTCAATCCACGTTTTTG)和SEQ ID NO:4(CCGGCCGCGAAGTTATGAGCAAGTTCTCGAGAACTTGCTCATAACTTCGCGGTTTTTG)。
附图的简要描述
当结合非限制性示例和附图考虑时,参考详细描述会更好地理解本发明,其中:
图1所示的一组实验结果说明了有效诱导心肌细胞(CM)中的Tjp1缺失。图1A显示了在对照(Ctrl;Tjp1F/F Myh6-CreERT2)和两个独立的来自三苯氧胺诱导的小鼠Tjp1cKO心脏的裂解物中Tjp1表达的蛋白质印迹分析;GAPDH用作加样对照。分析了3只对照和6只Tjp1cKO小鼠,并显示了来自代表性实验的印迹。图1B显示的一对照片描绘了对照和Tjp1cKO心脏的免疫组织化学结果。对照和Tjp1cKO心脏的切片用Tjp1抗体(箭状物所示)和DAPI(箭头所示)染色。观察到Tjp1定位于对照心脏中的闰盘及其从Tjp1cKO心脏的有效缺失。分析了3只对照和3只Tjp1cKO小鼠,并显示了代表性实验。图1C显示的照片描绘了检测LacZ表达的组织化学结果。使Tjp1cKO(Tjp1F/F Myh6-CreERT2)小鼠与lacZ报告系杂交,并每日给予腹膜内三苯氧胺(他莫昔芬)注射,持续5天,以诱导Cre表达。检测到LacZ表达作为Cre表达激活的间接证据。显示了三个经分析的Tjp1cKO小鼠之一的代表性实验。图1D所示的照片描绘了苏木精和曙红(H&E)染色的结果。三苯氧胺诱导后一天或者七天收获对照和Tjp1cKO心脏,切片,并用H&E染色。Tjp1的缺失不导致明显的组织学变化。每个时间点检查3只对照和Tjp1cKO小鼠的每只,并显示了来自代表性实验的图像。图1E所示的一对照片描绘了凋亡细胞检测的组织化学结果。三苯氧胺诱导后1天、3天、7天和30天收获对照和Tjp1cKO心脏,切片,并进行Tunel染色处理以检测凋亡细胞。细胞核用DAPI染色。在三苯氧胺诱导后1天未检测到凋亡,在三苯氧胺诱导后3天、7天和30天收集的心脏中获得相似结果(数据未显示)。每个时间点检查3只对照和3只Tjp1cKO小鼠。显示了来自代表性实验的图像。图1F所示的一对照片比较了对照和Tjp1cKO小鼠的心脏大小。用三苯氧胺将对照和Tjp1cKO小鼠诱导5天。在三苯氧胺诱导后7天解剖心脏并拍照。图1G所示的柱状图描绘了对照和Tjp1cKO小鼠的心脏与体重之比。测定体重(克)和心脏重量(克),计算比例并绘图。因此图1说明了在CM中的Tjp1失活之后,Tjp1cKO心脏未显示明显异常或凋亡。然而,与对照相比,Tjp1cKO心脏的心脏大小和心脏与体重之比略高。
图2所示的一组实验结果说明Tjp1的缺失诱导成体心脏中的细胞增殖。图2A所示一对照片描绘了Edu掺入的组织化学结果。用三苯氧胺诱导对照和Tjp1cKO小鼠5天,并在处死前1小时腹膜内注射Edu。收获心脏并将切片染色以显示具有Edu掺入的细胞核(如箭状物所示),表示增殖细胞。细胞核用DAPI染色(如箭头所示)。图2B所示的柱状图描绘了心脏切片中Edu和DAPI阳性细胞核的定量。计数Edu-和DAPI-阳性细胞核,计算Edu阳性细胞核的百分比并作图。使用6只对照和6只Tjp1cKO小鼠。每个心脏都取10个视野(view)。图2C所示的一组蛋白质印迹描绘了增殖标志物和细胞周期标志物的表达。在最后一次三苯氧胺诱导后1天收集对照和两个Tjp1cKO心脏的裂解物,并通过蛋白质印迹分析所示细胞周期调节蛋白的表达。GAPDH用作加样对照。分析了2只对照和4只Tjp1cKO小鼠,并显示了来自代表性实验的印迹。图2D所示的一对照片描绘了纤维化检测的组织化学结果。在三苯氧胺诱导后3、7和30天收获对照和Tjp1cKO心脏,切片并进行Masson's Trichrome染色处理以检测纤维化。尽管大量细胞增殖,但没有检测到纤维化,并且在诱导后7天和30天收集的心脏中获得了类似的结果(数据未显示)。检查了各3只对照和Tjp1cKO小鼠,并显示了来自代表性实验的数据。图2E所示的一对照片描绘了检测Edu阳性和MHC阳性心肌细胞(CM)的免疫组织化学结果。用三苯氧胺诱导对照和Tjp1cKO小鼠5天。在三苯氧胺诱导后,通过腹膜内注射Edu 3天。收获心脏并进行切片共标记以检测Edu掺入和CM制造者MHC。箭状物表明MHC阳性CM明显与Edu阳性细胞核相关,这与增殖一致。细胞核用DAPI染色。图2F所示的柱状图描绘了Edu阳性MHC阳性细胞的定量。计数与MHC阳性心肌细胞相关的Edu阳性细胞核和总Edu阳性细胞核,计算Edu阳性MHC阳性细胞的百分比并作图。使用5只对照和5只Tjp1cKO小鼠。每个心脏都取10个视野。只对明确与MHC阳性细胞相关的Edu阳性细胞核评分,因此CM增殖的程度很可能被低估。图2G所示的照片描绘了成体心肌细胞(CM)的解离。用三苯氧胺诱导对照和Tjp1cKO小鼠5天,然后在1天后处死。解离心脏细胞并通过相差显微镜成像。图2H所示的一组照片描绘了检测单核和多核Edu阳性心肌细胞(CM)的组织化学结果。用三苯氧胺诱导对照和Tjp1cKO小鼠5天,然后每天3次Edu注射,并在一天后处死。解离心脏细胞并标记掺入的Edu(如箭状物所示)和MHC(如星号所示)。显示了单核和双核MHC阳性CM的实例。细胞核用DAPI标记(如箭头所示)。图2I所示的柱状图描绘了单核或多核Edu阳性MHC阳性细胞的定量。计数单核或多核Edu阳性MHC阳性细胞和总MHC阳性细胞,测定单核或双核Edu阳性CM的分数并作图。图2J所示的柱状图描绘了在多个时间点对Edu阳性心肌细胞(CM)进行定量。发现Edu阳性CM的分数不随时间而改变。用三苯氧胺诱导对照和Tjp1cKO小鼠5天,然后每天3次Edu注射。30天后处死小鼠。解离心脏细胞并标记掺入的Edu和MHC。计数单核或多核Edu阳性MHC阳性细胞和总MHC阳性细胞,测定单核或二核Edu阳性CM的分数并作图。图2K所示的柱状图描绘了在多个时间点对总Edu阳性心脏细胞进行定量。发现总Edu阳性心脏细胞的分数随时间而降低。用三苯氧胺诱导对照和Tjp1cKO小鼠5天,然后用Edu腹膜内注射3天。在3天Edu脉冲后1天、25天和137天,收集心脏并处理以检测掺入的Edu。观察到Edu阳性细胞逐渐下降。图2L所示的柱状图描绘了137天后测量的心脏与体重之比。发现心脏与体重之比随时间正常化。测定体重(克)和心脏重量(克),计算比例并绘图。因此图2阐明Tjp1缺失诱导细胞增殖,因为Tjp1cKO心脏中Edu阳性细胞核丰富,观察到更高水平的针对有丝分裂和增强的细胞增殖的标志物,并且未观察到纤维化。
图3所示的一组实验结果说明了Tjp1缺失后的心肌细胞(CM)去分化。图3A所示的一组照片描绘了肌节结构观察的组织化学结果。用三苯氧胺诱导对照和Tjp1cKO小鼠5天,并在1或7天后收获心脏。将心脏切片,MHC染色并以640x或1600x放大率成像。观察诱导后1天和第7天恢复时的无组织肌节结构。细胞核用DAPI标记。分析每个时间点的3只对照和3只Tjp1cKO小鼠,并显示了来自代表性实验的数据。图3B和3C所示的一组照片描绘了免疫组织化学结果,其表明一些Edu阳性细胞表达心肌细胞(CM)祖细胞标志物Gata-4。用三苯氧胺诱导对照和Tjp1cKO小鼠5天。在三苯氧胺诱导1天后、于处死前以Edu脉冲1小时。将对照和Tjp1cKO心脏切片并染色,以检测掺入的Edu和Gata-4(图3B)或Nkx2.5(图3C)。箭状物指向Edu阳性细胞核和Gata-4-或Nkx2.5阳性细胞核。细胞核用DAPI标记。图3D和3E所示的一对柱状图描绘了Edu阳性Gata-4或Nkx2.5阳性细胞核的定量。计数Edu阳性Gata-4阳性或Edu阳性Nkx2.5阳性细胞核和Edu阳性细胞核,计算Edu阳性Gata-4阳性细胞(图3D)或Edu阳性Nkx2.5阳性细胞(图3E)的百分比并作图。使用3只对照和3只Tjp1cKO小鼠。图3F所示的一组蛋白质印迹描绘了早期心脏祖细胞标志物和去分化标志物的表达。将在三苯氧胺诱导后1天收集的对照和2个Tjp1cKO心脏裂解并通过蛋白质印迹分析针对去分化的不同标志物的表达。GAPDH用作加样对照。分析了2只对照和4只Tjp1cKO小鼠,并显示了来自代表性实验的印迹。尽管通过免疫组织化学观察到Gata-4的细胞核表达增强(参见图3C),但是观察到蛋白质水平并没有增加,这表明细胞溶质的核重定位增强。因此,图3说明了与心脏发育相关的蛋白质水平的增加,意味着CM在Tjp1缺失后经历部分去分化。
图4所示的一组实验结果说明响应Tjp1缺失的Erk、Stat3、Akt和Wnt通路的激活有助于成体心脏中的细胞增殖。图4A所示的一组蛋白质印迹描绘了表明不同信号传导通路的激活的各种标志物的表达。在最后一次三苯氧胺注射后一天收集对照裂解物和来自三苯氧胺诱导的小鼠的两个Tjp1cKO心脏裂解物,并处理以分析用于激活不同的信号传导通路的所示标志物的表达。GAPDH用作加样对照。分析了2只对照和4只Tjp1cKO小鼠,并且呈现了来自代表性实验的数据。图4B所示的一组蛋白质印迹描绘了Mek抑制剂PD0325901对信号传导的影响。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,同时共注射或不共注射PD0325901,持续5天,并在最后一次注射之后一天收集心脏并处理用于蛋白质印迹分析。分析了三对对照和Tjp1cKO动物。显示了来自代表性实验的图版。图4C所示的一组照片描绘了免疫组织化学结果,所述结果说明了Mek抑制剂PD0325901对细胞核pErk和pStat3的影响。如图4B所述获得心脏,并进行处理用于免疫组织化学以检测pErk或pStat3。图4D所示的一组照片描述了免疫组织化学结果,所述结果说明抑制Mek-Erk通路阻断细胞增殖。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,持续5天,同时共注射或不共注射PD0325901。在最后一次诱导之后一天,在处死小鼠前一小时注射Edu。标记心脏以检测掺入的Edu(如箭状物所示)和细胞核(DAPI,如箭头所示)。图4E所示的柱状图描绘了在暴露于PD0325901或不暴露于PD0325901的情况下心脏中Edu阳性细胞核的定量。测定在暴露或不暴露于PD0325901的心脏中Edu阳性细胞核的百分比并作图。使用经Mek处理或未经Mek处理的6只Tjp1cKO小鼠。每个心脏都取10个视野。图4F、4G和4H所示的一组柱状图描绘了Stat3抑制剂S31-301、Akt抑制剂API-2和Wnt抑制剂LGK974对Tjp1cKO心脏细胞增殖影响的定量。测定暴露于或不暴露于S31-301(图4F)、API-2(图4G)或LGK974(图4H)的心脏中Edu阳性细胞核的百分比(参见图7),并作图。使用经或未经不同抑制剂处理的5只(API-2)或6只(S31-201、LGK974)Tjp1cKO小鼠。每个心脏都取10个视野。因此,图4说明了参与各种信号传导通路的组分的抑制证实了在Tjp1缺失时那些信号传导通路的激活驱动细胞增殖。
图5所示的一组实验结果说明在缺乏Tjp1情况下ErbB4有助于增殖增强,并且Tjp1结合并抑制ErbB4信号传导。图5A所示的一组蛋白质印迹说明了EGF/ErbB受体抑制剂Ast1306对信号传导的影响。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,同时共注射Ast1306或不共注射Ast1306,持续5天。在最后一次注射之后一天收集心脏并处理用于蛋白质印迹分析。用Mek抑制剂注射三对对照和Tjp1cKO小鼠并检查。显示了来自代表性实验的印迹。图5B所示的一对照片描述了免疫组织化学结果,所述结果说明抑制EGF/ErbB受体阻断细胞增殖。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,持续5天,同时共注射或不共注射Ast1306。在最后一次诱导之后一天,在处死小鼠前一小时注射Edu。标记心脏以检测掺入的Edu(如箭状物所示)和细胞核(DAPI,如箭头所示)。图5C所示柱状图描绘了在暴露于或不暴露于Ast1306C的情况下心脏中Edu阳性细胞核的定量。测定了在暴露于或不暴露于Ast1306的心脏中Edu阳性细胞核的百分比并作图。使用经Ast1306处理或未经Ast1306处理的6只Tjp1cKO小鼠。每个心脏都取10个视野。图5D所示的一组蛋白质印迹说明Tjp1的缺失增加了总ErbB4蛋白水平并激活了心脏中的ErbB4蛋白水平。在最后一次三苯氧胺注射之后一天收集对照裂解物和来自三苯氧胺诱导的小鼠的Tjp1cKO心脏裂解物,并处理以分析ErbB4或激化的pErbB4的表达。GAPDH用作加样对照。分析了2只对照和4只Tjp1cKO小鼠,并显示了来自代表性实验的数据。图5E所示的一组照片描绘了免疫组织化学结果,所述结果说明缺乏Tjp1增强了Nrg1对成体心脏中的细胞增殖的刺激作用。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,持续5天。三周后,每天给小鼠注射Edu和媒介(vehicle)或Nrg1,持续5天。在最后一次注射之后一天,收获心脏并切片染色以检测掺入的Edu(如箭状物所示)和细胞核(DAPI,如箭头所示)。图5F所示的柱状图描绘了暴露于或未暴露于Nrg1的对照或Tjp1cKO心脏中edu阳性细胞核定量。测定暴露于或未暴露于Nrg1的对照或Tjp1cKO心脏中Edu阳性细胞核的百分比并作图。每组使用6只对照和6只Tjp1cKO小鼠。每个心脏都取10个视野。图5G所示的一组蛋白质印迹说明了沉默Tjp1增强和维持Nrg1介导的Erk活化并稳定ErbB4蛋白水平。将对照或Tjp1shRNA处理的MCF-7细胞与Nrg1一起孵育所示的时间段。然后裂解细胞并处理用于蛋白质印迹分析。GAPDH用作加样对照。图5H所示的一组蛋白质印迹说明了沉默Tjp1使细胞对Nrg1刺激敏感。将对照或Tjp1shRNA处理的MCF-7细胞与所示的各种Nrg1浓度一起孵育,然后裂解并处理用于蛋白质印迹分析。GAPDH用作加样对照。配体刺激和剂量反应实验重复至少三次。显示了来自代表性实验的数据。图5I所示的一组氨基酸序列比较了ErbB2和Erb4的C末端中的PDZ结合基序。图5J所示的一组蛋白质印迹说明了ZO-1(Tjp1)PDZ结构域结合ErbB4但不结合缺乏C-末端PDZ结合基序的突变体。将GST或携带Tjp1的3个PDZ结构域的GST融合蛋白(GST-Tjp1PDZ1-3)与来自过表达ErbB4或缺乏PDZ结合基序的突变体(ErbB4ΔPBM)的细胞的裂解物一起孵育。洗脱结合的蛋白质并使用抗ErbB4抗体通过蛋白质印迹检测。运行细胞裂解物的等分式试样(10μg)作为对照(输入),并通过用丽春红(Ponceau)染色对膜进行染色使GST或GST-Tjp1PDZ1-3融合蛋白可视化。图5K所示的一组蛋白质印迹说明了ZO-1(Tjp1)PDZ结构域结合ErbB2。将GST或携带Tjp1的3个PDZ结构域的GST融合蛋白(GST-Tjp1PDZ1-3)或携带密切相关的Tjp2的3个PDZ结构域的GST融合蛋白(GST-Tjp2PDZ1-3)或携带Tjp3的3个PDZ结构域的GST融合蛋白(GST-Tjp3PDZ1-3)与过表达ErbB2的细胞的裂解物一起温育。洗脱结合的蛋白质并使用抗ErbB2抗体通过蛋白质印迹检测。运行细胞裂解物的等分试样(10μg)作为对照(输入),并通过用丽春红染色对膜进行染色使GST或GST-Tjp1PDZ1-3融合蛋白可视化。GST结合实验至少进行3次,并显示来自代表性实验的数据。因此,图5表明Tjp1对心脏中Nrg1介导的ErbB4信号传导具有抑制作用。因此,Tjp1沉默(例如shRNA介导的Tjp1沉默)维持Nrg1诱导的ErbB4信号传导,从而促进CM增殖。
图6所示的一组实验结果说明,在实验性心肌梗死(MI)和Tjp1缺失后观察到心肌细胞(CM)增殖增强和持续的心脏功能。图6A所示一对照片描绘了免疫组织化学结果,所述结果表明了对照和Tjp1cKO心脏中MI诱导的纤维化。通过左冠状动脉前降支的永久性结扎诱导MI。手术后,通过每日三苯氧胺注射,持续5天,诱导Tjp1缺失。一周后收集心脏,切片并用Masson's Trichrome染色以使纤维化可视。基于Masson's Trichrome染色结果,对照和Tjp1cKO心脏显示了相似的染色强度,从而表明诱导MI的手术对对照和Tjp1cKO心脏导致了相似的初始损伤。图6B所示的一组照片描绘了免疫组织化学结果,所述结果说明了MI边界区域的CM增殖。在如上所述诱导MI和三苯氧胺诱导后,每天施用Edu,持续三天,并处死小鼠。标记对照或Tjp1cKO心脏以检测掺入的Edu和MHC以鉴定CM。细胞核用DAPI标记。箭状物指向Edu阳性MHC阳性细胞,表明在Tjp1cKO梗死边界区域中更多的增殖性CM。观察到梗死组织中的大量MHC阴性细胞核,推测成纤维细胞造成纤维化瘢痕形成。图6C和6D所示的柱状图描绘了对照和Tjp1cKO心脏的Edu阳性和MHC阳性细胞的定量。计数对照和Tjp1cKO心脏的MI边界区域中的Edu阳性和MHC阳性细胞,并归一化至MI边界区域(图6C)。每个心脏都取超过10个视野。或者,计数MI边界区域和远程非梗死区域中Edu阳性和MHC阳性细胞和总Edu阳性细胞,测定Edu阳性CM的分数并绘图(图6D)。每个心脏都取10个视野。图6E、6F、6G和6H所示的柱状图和几个散点图说明了心脏功能分析的结果。在MI和三苯氧胺诱导后,记录超声心动图以估计初始梗死面积(图6E)。用所示时间间隔的记录数据计算舒张期末容积(EDV;图6F)、左心室射血分数(LVEF,图6G)和梗死边界区壁厚度(图6H)。监测7只对照和8只Tjp1cKO小鼠。使用黑色方块指示对照小鼠的数据点,并使用具有白色星号的灰色方块指示Tjp1cKO小鼠的数据点。图6I显示了用于说明Tjp1在调节CM增殖中的作用的模型的图解说明。在成体心脏中,Tjp1抑制ErbB2/ErbB4信号传导和下游效应物Erk、Akt、Stat3和Wnt信号传导的激活。Tjp1缺失导致部分CM去分化和旁分泌因子/自分泌因子释放。Tjp1缺乏还使ErbB2/ErbB4对Nrg1或其他配体敏感,所述配体可能存在于组织中或响应于Tjp1缺失和/或MI而释放,以通过激活Erk、Akt、Stat3和Wnt信号传导来驱动CM增殖。因此,图6说明在MI后Tjp1缺失导致CM增殖增强,特别是在瘢痕边界区域,从而限制不利的重塑和保持心脏功能。
图7所示的一组实验结果说明Stat3、Akt和Wnt的抑制剂在成体心脏中不同程度地抑制由Tjp1缺失诱导的信号传导和增殖。图7A所示的一组蛋白质印迹说明了Stat抑制剂S31-20对信号传导的影响。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,同时共注射或不共注射S31-20,持续5天。在最后一次注射后一天收集心脏并处理用于蛋白质印迹分析。图7B所示的一组照片描绘了免疫组织化学结果,其说明了Stat3抑制剂S31-20对细胞核pStat3和pErk水平的影响。处理如图7A所述获得的心脏用于免疫组织化学,以检测pErk或pStat3。图7C所示的一对照片描绘了免疫组织化学结果,其说明Stat3途径的抑制阻断细胞增殖。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,持续5天,同时共注射或不共注射S31-20。在最后一次诱导后一天,在处死小鼠前一小时注射Edu。标记心脏以检测掺入的Edu(如箭状物所示)和细胞核(DAPI,如箭头所示)。量化结果如图4F所示。图7D所显示的一组蛋白质印迹说明了Akt抑制剂API-2对信号传导的影响。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,同时共注射或不共注射API-2,持续5天。在最后一次注射后一天收集心脏并处理用于蛋白质印迹分析。图7E所示的一对照片描绘了免疫组织化学结果,其说明Akt途径的抑制部分地阻断细胞增殖。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,持续5天,同时共注射或不共注射API-2。在最后一次诱导后一天,在处死小鼠前一小时注射Edu。标记心脏以检测掺入的Edu(如箭状物所示)和细胞核(DAPI,如箭头所示)。量化的结果如图4G所示。图7F所示的一组蛋白质印迹说明了Wnt抑制剂LGK974对信号传导的影响。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,同时共注射或不共注射LGK974,持续5天。在最后一次注射后一天收集心脏并处理用于蛋白质印迹分析。图7G所示的一对照片描绘了免疫组织化学结果,其说明Wnt途径的抑制部分地阻断细胞增殖。每天用三苯氧胺诱导对照和Tjp1cKO小鼠,持续5天,同时共注射或不共注射LGK974。在最后一次诱导后一天,在处死小鼠前一小时注射Edu。标记心脏以检测掺入的Edu(如箭状物所示)和细胞核(DAPI,如箭头所示)。量化结果如图4H所示。因此,图7类似于图4,说明Stat3、Akt和Wnt的抑制抑制了Tjp1cKO心脏中的细胞增殖。
图8所示的一组实验结果说明用shRNAs沉默小鼠Tjp1和Tjp2。图8A所示的一组蛋白质印迹说明了靶向Tjp1的shRNA在Tjp1/ZO-1表达中的作用。用靶向Tjp1的不同shRNA(TJP1#1,TJP1#2)或非靶向对照shRNA(NT)转染小鼠Hepa1-6细胞,并通过蛋白质印迹分析测定对Tjp1/ZO-1表达的影响。图8B显示了一组shRNA序列。显示了TJP1#1(mTJP1shRNA#1;SEQ IDNO:3)和TJP1#2(mTJP1shRNA#2;SEQ ID NO:4)的序列。因此,图8显示可以使用靶向Tjp1的shRNA沉默小鼠Tjp1。
发明详述
心脏病是全世界成年人住院和死亡的主要原因。例如,在美国,心力衰竭导致每4例死亡中的1例死亡。先天性或获得性心血管疾病导致心肌细胞(CM)缺乏,这通常导致心力衰竭和死亡。当心肌细胞(CM)不可恢复地丧失并被纤维化取代时,发生心肌梗死(MI),然后也会发生心力衰竭(HF)。尽管受益于阻断肾素-血管紧张素-醛固酮和交感神经系统的影响,但HF的结果仍然令人沮丧,5年生存率仅为50%。与人类不同,在一些其他脊椎动物中,心脏具有固有的再生能力。切除高达20%的心室后,斑马鱼可在2个月内再生丧失的组织。通过预先存在的CM的去分化和增殖来实现再生。在哺乳动物中,新生小鼠的心脏具有在出生后7天内丧失的再生能力。然而,在成体小鼠和其他哺乳动物中,心脏损伤导致瘢痕组织形成以及CM和心脏功能的丧失。
心脏再生的主要挑战是人成体心脏细胞(例如人成体心肌细胞)的增殖能力有限。MI后心脏细胞增殖能力的缺乏是有问题的,因为它可以影响MI后心功能的恢复和HF的预防。鉴于上述情况,需要提供治疗受试者的心脏病并诱导心脏细胞增殖的替代方法。
本公开的发明人已经发现了治疗受试者的心脏病的替代方法。因此,一方面,本发明提供了用于治疗受试者中的心脏病的方法,包括向受试者施用(治疗有效量的)Tjp1抑制剂的步骤。令人惊讶地发现,施用Tjp1抑制剂抑制Tjp1表达。这种抑制导致Tjp1活性的抑制,从而诱导心脏细胞增殖。因此,另一方面,本发明提供了在有需要的受试者中增殖心脏细胞的方法,包括向受试者施用(治疗有效量的)Tjp1抑制剂的步骤。在一些实例中,受试者已患有心脏病。在不受理论束缚的情况下,心肌梗死后心脏细胞的这种增殖通过刺激心脏细胞的再生、特别是心肌细胞的再生来防止或减少瘢痕组织的形成。本文所述的抑制Tjp1的方法可用于诱导患有心脏病的人的心肌细胞增殖。
如本文所用,术语Tjp1(紧密连接蛋白1)和ZO-1(Zonula Occludens-1)可互换使用。术语“Tjp1”和“ZO-1”是指在心肌细胞中定位于闰盘的肌动蛋白结合支架蛋白。如本文所用,术语“心脏细胞”是指形成心脏结构的细胞类型。心脏细胞的实例包括但不限于心肌细胞、成纤维细胞、心外膜细胞、内皮细胞、平滑肌细胞等。术语“心脏细胞”不是指不形成心脏结构的细胞类型,例如血细胞。因此,在一个实例中,本文所述的Tjp1抑制剂还诱导包括但不限于心肌细胞、成纤维细胞、心外膜细胞、内皮细胞、平滑肌细胞等细胞的增殖。在一个实例中,由本文所述的Tjp1抑制剂诱导增殖的细胞是心肌细胞。在一个实例中,心肌细胞是成熟或分化的心肌细胞。在一个实例中,本发明人惊奇地观察到,在施用Tjp1抑制剂后,心肌细胞去分化成不成熟的心肌细胞并表现出再生表型。
本领域技术人员应当理解,本文包括的再生表型列表并非穷举。再生表型的实例包括但不限于无组织肌节结构、Dab2(Disabled-2)的上调、Myh7(肌球蛋白重链7)的上调、解聚蛋白(Destrin)的上调、Runx1(Runt相关转录因子1)的上调、转录调节剂(如GATA4(GATA结合蛋白4)或Nkx2.5(NK2同源框5))从细胞质到细胞核的上调和/或再分布、DNA复制(如Edu(5-乙炔基-2'-脱氧尿苷)阳性染色所示)和/或Myh6(肌球蛋白重链6)的下调。如本文所用,术语“上调”或“下调”是指与Tjp1抑制之前的水平相比增加或减少。在例如Myh7、Dab2、解聚蛋白、Runx1的mRNA和/或蛋白质水平上的表达增加表明去分化,因为它们是胚胎CM标志物。Myh6是成熟的CM标志物,因此Myh6的表达降低也可以作为去分化的标志。
在一个实例中,当心肌细胞去分化成不成熟的心肌细胞时表现出的再生表型包括但不限于本文所述再生表型中的至少一种、或至少两种、或至少三种或更多种。例如如图3所示,与心脏发育相关的再生表型水平的变化表明CM在Tjp1缺失后经历部分去分化。在一个实例中,所述再生表型包括但不限于无组织肌节结构、Dab2的上调、Myh7的上调、解聚蛋白的上调、Runx1的上调、转录调节剂(例如GATA4或Nkx2.5)从细胞质到细胞核的重新分布、DNA复制(例如Edu阳性染色所示)、Myh6的下调等。
在一个实例中,本文所述的Tjp1抑制剂抑制Tjp1表达。本文所用的术语“抑制”通常是指与未治疗的受试者(或待治疗的患者)或对照相比量减少。然而,为避免疑义,“抑制”是指足以刺激心脏细胞、特别是心肌细胞再生的减少。在另一个实例中,“抑制”是指与未治疗的受试者(或待治疗的患者)相比,Tjp1表达减少至少10%、或至少约20%、或至少约30%、或至少约40%相比、或至少约50%、或至少约60%、或至少约70%、或至少约80%、或至少约90%、或高达并包括100%,或与未治疗的受试者(或待治疗的患者)相比介于10-100%之间的Tjp1表达的任何减少。如本文所使用的,术语“对照”是指在统计上与被测试组相似的群体,对所述群体不实施任何改变。本文所用的对照的非限制性实例是野生型小鼠(即没有Tjp1敲除的小鼠)。
据信Tjp1对心肌细胞的特异性抑制足以刺激心脏中各种细胞类型的增殖。在一些实例中,除了预期的自分泌或分泌因子对心肌细胞的影响(例如心肌细胞本身的增殖)之外,Tjp1的缺失还令人惊讶地诱导旁分泌或分泌因子影响非心肌细胞(例如但不限于成纤维细胞)。因此,在一个实例中,由本文所述的Tjp1抑制剂诱导增殖的细胞是成纤维细胞。
本发明人惊奇地发现Tjp1表达的抑制激活了多种受体。在一个实例中,如例如图5所示,Tjp1对心脏中Nrg1介导的ErbB4信号传导具有抑制作用。因此,在一个实例中,Tjp1表达的抑制增强了一种或至少一种受体的表面表达,所述受体包括但不限于ErbB1受体(EGF受体)、ErbB2受体、ErbB4受体等。
除了抑制Tjp1表达外,在一个实例中,Tjp1抑制剂抑制Tjp1生物学活性。本发明人惊奇地发现,抑制Tjp1生物活性激活多种信号传导通路。如例如图4所示,Erk、Stat3、Akt和Wnt途径响应于Tjp1缺失而被激活。因此,在一个实例中,Tjp1生物活性的抑制激活了至少一种信号传导通路,所述信号传导通路包括但不限于促分裂原激活的蛋白激酶(MAPK)途径、Stat3信号传导通路、Akt信号传导通路、Wnt信号传导通路、Hippo途径等。
如上文所述,Tjp1表达的抑制诱导心脏细胞增殖。因此,控制Tjp1抑制的持续时间是重要的。因此,在一个实例中,Tjp1抑制剂是瞬时(可逆或非永久)抑制剂或永久(不可逆)抑制剂。如本文所用,术语“瞬时或可逆或非永久性抑制剂”是指不改变心脏细胞的基因和/或可以容易地从抑制剂结合的靶标中去除的Tjp1抑制剂。如本文所用,术语“永久性或不可逆抑制剂”是指改变心脏细胞的基因和/或不能容易地从抑制剂结合的靶标中去除的Tjp1抑制剂。永久性或不可逆抑制剂的非限制性实例是靶向和遗传修饰心脏细胞基因的抑制剂。基因的靶向和修饰可以使用本领域已知的任何方法实施,包括但不限于CRISPR/Cas9、TALEN、锌指核酸酶等。
Tjp1永久性失活的非限制性实例(例如在Tjp1缺失后)是本文所述的Tjp1cKO小鼠。然而,还观察到缺失后的Edu掺入随时间而降低,从而表明Tjp1也能被瞬时失活。在一个实例中,Tjp1抑制剂是在足以获得效果(例如心脏细胞再生)的一段时间内使Tjp1表达或活性失活的抑制剂。在获得所需效果后,不再需要抑制剂来抑制Tjp1的表达或活性。在足以获得效果的时间内使Tjp1表达或活性失活的抑制剂的非限制性实例是使用AAV(腺伴随病毒)引入的shRNA。shRNA将作为游离质粒保留在心肌细胞中,并在细胞增殖期间被稀释或进行自然更新。已知通过AAV引入的序列在数周至超过一年内保持活跃,这是稳定心脏功能所需的时间范围的实例。
应当理解,本领域已知能够减少或失活Tjp1表达或活性的任何方法都适用于本公开的方法。本领域技术人员应当理解,本文包括的Tjp1抑制剂类型的列表并非穷举。Tjp1抑制剂类型的实例包括但不限于小分子、抗体、多肽、核酸和能够抑制Tjp1表达、功能或活性的任何其他生物或化学实体。在一个实例中,Tjp1抑制剂包括但不限于小分子、抗体、多肽、核酸等。本领域技术人员还应当理解,在一些实施方案中,可用作Tjp1抑制剂的抗体是单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、嵌合抗体、多特异性抗体或其抗体片段。在一些实例中,抗体包括但不限于Fab片段、Fab'片段、F(ab')2片段、Fv片段、双抗体或单链抗体分子。在一些实例中,抗体是人抗体。
如本领域技术人员所理解的,诸如载体、DNA插入物、shRNA等核酸序列可经设计以靶向Tjp1。在一个实例中,用作Tjp1抑制剂的核酸包括但不限于siRNA、shRNA、反义寡核苷酸、gapmer、短发夹反义寡核苷酸(shAON)等。在一个实例中,当用核酸作为Tjp1抑制剂时,核酸可与编码Tjp1的mRNA结合或相互作用,并形成核酸-mRNA复合体。在一个实例中,当用作Tjp1抑制剂的核酸与编码Tjp1的mRNA结合或相互作用并形成核酸-mRNA复合体时,核酸-mRNA复合体中的mRNA被切割和/或不翻译。
使用核酸作为与编码Tjp1的mRNA结合或相互作用的Tjp1抑制剂的方法也称为“反义寡核苷酸疗法”。如本文所用,术语“反义寡核苷酸疗法”是指合成会与Tjp1产生的信使RNA(mRNA)结合并使其失活的核酸(例如DNA、RNA、化学类似物等)链,从而将该基因有效地“关闭”的方法。这是因为mRNA必须是单链的才能被翻译。“反义寡核苷酸疗法”还指合成经靶向而与前mRNA上的剪接位点结合、修饰mRNA外显子内容、并因此mRNA不会被翻译的核酸(例如DNA、RNA、化学类似物等)链的方法。本领域技术人员应当理解,用作反义寡核苷酸疗法的Tjp1抑制剂的核酸包括但不限于shRNA、siRNA、吗啉代等。用于反义寡核苷酸疗法的核酸可以用本领域已知的任何方式递送。用于反义寡核苷酸疗法的核酸递送的方法包括但不限于使用腺伴随病毒的递送方法、使用其他病毒的递送方法、使用脂质体的递送方法、通过结合用于靶向的适体的递送方法等。
本领域技术人员应当理解,可以使用任何核苷酸序列编码用作Tjp1抑制剂的核酸。在一个实例中,如例如图5G所示,蛋白质印迹分析表明,可以使用靶向Tjp1的shRNA沉默Tjp1。本领域技术人员认识到,靶向Tjp1的shRNA是具有与编码Tjp1的cDNA和/或mRNA的序列互补和/或结合的序列的shRNA。靶向Tjp1的shRNA可以根据本领域已知的各种方法设计。因此,靶向Tjp1的shRNA或shRNA抑制剂可以具有任何序列,并且不限于本文所列的序列。在一个实例中,编码Tjp1抑制剂的核酸与下述序列具有至少70%的同一性、或至少75%的同一性、或至少80%的同一性、或至少85%的同一性、或至少90%的同一性、或至少95%的同一性、或至少98%的同一性、或至少99%的同一性、或至少99.5%的同一性:SEQ ID NO:1(CCGGGCCTGCATACAATAAAGCAAACTCGAGTTTGCTTTATTGTATGCAGGCTTTTTG)和/或SEQ ID NO:2(CCGGGGAACCACTCTATCAAGTATTCTCGAGAATACTTGATAGAGTGGTTCCTTTTTG),并且其中所述序列仍保留编码Tjp1抑制剂的能力。在一个实例中,编码Tjp1抑制剂的核酸与下述序列具有至少70%的同一性、或至少75%的同一性、或至少80%的同一性、或至少85%的同一性、或至少90%的同一性、或至少95%的同一性、或至少98%的同一性、或至少99%的同一性、或至少99.5%同一性:SEQ ID NO:3(CCGGCGTGGATTGAACTTACTAAATCTCGAGATTTAGTAAGTTCAATCCACGTTTTTG)和/或SEQ ID NO:4(CCGGCCGCGAAGTTATGAGCAAGTTCTCGAGAACTTGCTCATAACTTCGCGGTTTTTG),并且其中所述序列仍保留编码Tjp1抑制剂的能力。在一个实例中,编码Tjp1抑制剂的核酸具有序列SEQ ID NO:1(CCGGGCCTGCATACAATAAAGCAAACTCGAGTTTGCTTTATTGTATGCAGGCTTTTTG)和/或SEQ ID NO:2(CCGGGGAACCACTCTATCAAGTATTCTCGAGAATACTTGATAGAGTGGTTCCTTTTTG)。在一个实例中,编码Tjp1抑制剂的核酸具有序列SEQ ID NO:3(CCGGCGTGGATTGAACTTACTAAATCTCGAGATTTAGTAAGTTCAA TCCACGTTTTTG)和/或SEQ ID NO:4(CCGGCCGCGAAGTTATGAGCAAGTTCTCGAGAACTTGCTCATAACTTCGCGGTTTTTG)。
还在另一个方面,本发明提供了编码Tjp1抑制剂的核酸,其中所述核酸与选自以下的序列具有至少70%的同一性、或至少75%的同一性、或至少80%的同一性、或至少85%的同一性、或至少90%的同一性、或至少95%的同一性、或至少98%的同一性、或至少99%的同一性、或至少99.5%的同一性:SEQ ID NO:1(CCGGGCCTGCATACAATAAAGCAAACTCGAGTTTGCTTTATTGTATGCAGGCTTTTTG)、SEQ ID NO:2(CCGGGGAACCACTCTATCAAGTATTCTCGAGAATACTTGATAGAGTGGTTCCTTTTTG)、SEQ ID NO:3(CCGGCGTGGATTGAACTTACTAAATCTCGAGATTTAGTAAGTTCAATCCACGTTTTTG)和SEQ ID NO:4(CCGGCCGCGAAGTTATGAGCAAGTTCTCGAGAACTTGCTCATAACTTCGCGGTTTTTG),并且其中所述序列仍保留编码Tjp1抑制剂的能力。在一个实例中,本发明提供了编码Tjp1抑制剂的核酸,其中所述核酸与选自以下的序列具有至少70%的同一性、或至少75%的同一性、或至少80%的同一性、或至少85%的同一性、或至少90%的同一性、或至少95%的同一性、或至少98%的同一性、或至少99%的同一性、或至少99.5%的同一性:SEQID NO:3(CCGGCGTGGATTGAACTTACTAAATCTCGAGATTTAGTAAGTTCAATCCACGTTTTTG)和SEQ IDNO:4(CCGGCCGCGAAGTTATGAGCAAGTTCTCGAGAACTTGCTCATAACTTCGCGGTTTTTG),并且其中所述序列仍保留编码Tjp1抑制剂的能力。
如本领域技术人员所理解的,本文公开的核酸序列(例如SEQ ID NO:1和/或SEQ IDNO:2)是DNA插入物。在一些实例中,将核酸(即DNA插入物)插入的载体(例如质粒载体)中,从其产生shRNA。本领域技术人员理解,由载体产生的shRNA结合靶标的mRNA(例如可翻译为Tjp1的mRNA)中与所产生的shRNA序列互补的区域。在一个实例中,可以将SEQ ID NO:1和SEQ ID NO:2公开的核酸序列二者都插入单个载体中,从而单个载体会表达一种以上的shRNA。在不希望受理论束缚的情况下,多种shRNA的组合可以增加shRNA对Tjp1表达或活性的抑制效果。本领域技术人员应当理解,用作Tjp1抑制剂的核酸可以从衍生自MHC或Tttn2的启动子表达,其会允许抑制性核酸在心肌细胞中的特异性表达。在一个实例中,用于表达Tjp1抑制剂的载体是腺伴随病毒(AAV)载体。基于AAV血清型9(AAV9)的载体可以有效地转导心肌细胞。在另一个实例中,称为自身互补腺伴随病毒(scAAV)的改变形式的AAV可用于转导抑制性核酸。尽管AAV包装单链DNA并且必须等待其第二链合成,但scAAV包装两条彼此互补的较短链。通过避免第二链合成,scAAV能够更快地表达。
本发明人还发现,当Tjp1抑制剂与其他因子一起施用时,Tjp1抑制剂的功效得到改善。本发明人已观察到Tjp1缺失本身激活Wnt信号传导。因此,在不希望受理论束缚的情况下,并且如标题为“Tjp1缺失激活驱动CM增殖的信号传导通路”的实施例中所示,添加Wnt、Norrin或R-Spondin似乎与Tjp1缺失协同作用。因此,在一个实例中,Tjp1抑制剂与至少一种或至少两种或至少三种或至少四种或至少五种或更多种其他因子一起施用,所述其他因子包括但不限于小分子(例如Wnt信号传导通路的激活剂)、抗体或其片段、多肽(例如DARPin(设计的锚蛋白重复蛋白))、核酸(例如适体、单链RNA和单链DNA)等。在一个实例中,Tjp1抑制剂与Tjp1抑制剂一起施用或分别施用。
本领域技术人员应当理解,因子列表可包括这些因子、抗体、肽、适体、darpins的可与ErbB受体结合的片段,并且与Tjp1失活组合,增强ErbB受体信号传导。如本领域技术人员还应当理解的,ErbB4可以与ErbB/Her2同源二聚化以及异源二聚化,并且Tjp1和/或Tjp2也可以结合ErbB1/EgfR(其可以与ErbB2/Her2同源二聚化和异源二聚化)和ErbB2/Her2(其可以与Her1EgfR、ErbB3和ErbB4同源二聚化和异源二聚化),并且Tjp1也可以通过与这些受体组合结合的配体来调节信号传导。
在一个实例中,与Tjp1抑制剂一起施用的核酸是编码细胞周期蛋白A2的核酸。在一个实例中,当Tjp1抑制剂与多肽一起施用时,所述多肽包括但不限于生长因子、细胞因子、趋化因子、分泌因子、刺激因子等。在一个实例中,当Tjp1抑制剂与多肽一起施用时,所述多肽是生长因子和/或分泌因子(或分泌蛋白)。如本文所用,术语“刺激因子”是指能够刺激相邻细胞的配体/受体的细胞外结构域(例如,Notch途径)。
在一个实例中,与Tjp1抑制剂一起施用的多肽与ErbB4受体结合。在一个实例中,与ErbB4受体结合并与Tjp1抑制剂一起施用的多肽包括但不限于成纤维细胞生长因子(FGF)、血管内皮生长因子(VEGF)、神经调节蛋白-1、神经调节蛋白-2(NRG2)、神经调节蛋白-3(NRG3)、乙胞素(BTC)、上皮调节蛋白(EPR)、肝素结合EGF样生长因子(HB-EGF)、表皮生长因子(EGF)、β-纤维素、转化生长因子α(TGFα)、双调蛋白(AR)等。在一个实例中,与ErbB4受体结合并与Tjp1抑制剂一起施用的多肽是神经调节蛋白-1(NRG1)。在一个实例中,与Tjp1抑制剂一起施用的多肽是分泌因子。如本文所用,术语“分泌因子”是指分泌而离开细胞的因子。在一个实例中,分泌因子是卵泡抑素样1(Fstl1)。在一个实例中,与Tjp1抑制剂一起施用的多肽激活Wnt信号传导。在一个实例中,与Tjp1抑制剂一起施用并激活Wnt信号传导的多肽包括但不限于Wnt、Norrin、R-spondin等。
本领域技术人员应当理解,Tjp1抑制剂可以使用本领域已知的任何递送系统施用,并且用于施用本文列出的Tjp1抑制剂的递送系统列表并非穷举。这种递送系统的实例包括但不限于病毒介导的递送系统、基于聚合物的递送系统、可植入的贴剂、位点特异性递送系统(例如通过外膜导管进入血管周围空间和外膜)、可注射系统(任选地与支架递送系统组合)、可植入释放递送系统等。在Tjp1抑制剂是核酸抑制剂的一个实例中,使用包括但不限于以下的系统施用所述核酸:病毒介导的递送系统、基于聚合物的递送系统、可植入的贴剂、位点特异性递送系统(例如通过外膜导管进入血管周围空间和外膜)等。
本领域技术人员应当理解,通过本文列出的病毒介导的递送系统施用Tjp1抑制剂的病毒列表并非穷举。这些病毒的实例包括但不限于逆转录病毒、腺病毒、腺伴随病毒、单纯疱疹病毒等。在Tjp1抑制剂的递送系统是病毒介导的递送系统的一个实例中,病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、单纯疱疹病毒等。在Tjp1抑制剂的递送系统是病毒介导的递送系统的一个实例中,病毒是腺伴随病毒。可用于递送Tjp1抑制剂的腺伴随病毒具有多种血清型。在一个实例中,用于递送Tjp1抑制剂的腺伴随病毒包括但不限于AAV血清型1、AAV血清型2、AAV血清型3、AAV血清型4、AAV血清型5、AAV血清型6、AAV血清型7、AAV血清型8、AAV血清型9、AAV血清型10、AAV血清型11等。在一个实例中,用于递送Tjp1抑制剂的腺伴随病毒是AAV血清型9。在Tjp1抑制剂的递送系统是病毒介导的递送系统的一个实例中,抑制剂的剂量为4.00x107vg/kg至4.00x1015vg/kg、或4.00x108vg/kg至4.00x1014vg/kg、或4.00x109vg/kg至4.00x1013vg/kg、或4.00x1010vg/kg至4.00x1012vg/kg、或约4.00x107vg/kg、或约4.00x108vg/kg、或约4.00x109vg/kg、或约4.00x1010vg/kg、或约4.00x1011vg/kg、或约4.00x1012vg/kg、或约4.00x1013vg/kg、或约4.00x1014vg/kg、或约4.00x1015vg/kg。如本文所用术语“vg/kg”是指每公斤受试者体重的病毒(载体)基因组。
在Tjp1抑制剂是小分子、抗体或多肽的一个实例中,递送系统包括但不限于局部递送系统、全身递送系统等。在一个实例中,Tjp1抑制剂的局部递送系统包括但不限于基于聚合物的递送系统、可注射系统(任选地与支架递送系统组合)、外膜导管、可植入释放递送系统等。在一个实例中,Tjp1抑制剂的全身递送系统包括但不限于口服递送系统(即口服制剂,例如胶囊、溶液、片剂等)、栓剂、静脉内递送系统、腹膜内递送系统、肌内递送系统、皮内递送系统、皮下注射等。在Tjp1抑制剂是小分子、抗体或多肽的一个实例中,抑制剂的剂量为1μg/kg至1000mg/kg、或10mg/kg至100mg/kg、或约10mg/kg、或约20mg/kg、或约30mg/kg、或约40mg/kg、或约50mg/kg、或约60mg/kg、或约70mg/kg、或约80mg/kg、或约90mg/kg、或约100mg/kg。
为了使本发明人能够提供通过施用Tjp1抑制剂和促进心脏细胞增殖来治疗心脏病的替代方法,需要提供鉴定活性化合物的方法。因此,另一方面,本发明提供了鉴定能够促进心肌细胞增殖的化合物的方法,其中所述方法包括以下步骤:(a)使化合物与Tjp1敲除宿主或其中Tjp1已经受到抑制的宿主接触,和(b)观察宿主中是否存在心肌细胞增殖,其中心肌细胞增殖的存在表明该化合物能够促进心肌细胞增殖。本文所用术语“敲除”是指部分或完全抑制细胞中由内源DNA序列编码的至少一部分Tjp1的表达。
还在另一个方面,本发明提供了鉴定响应Tjp1抑制而分泌的因子(有或没有心肌梗死)的方法,其中所述方法包括:(a)向宿主施用Tjp1抑制剂或使用Tjp1敲除宿主(有或没有心肌梗死诱导),(b)在步骤(a)前后从宿主收集样品,和(c)确定在步骤(a)之后分泌的因子,从而鉴定响应Tjp1抑制而分泌的因子。
在一个实例中,在鉴定能够促进心肌细胞增殖的化合物或响应Tjp1抑制而分泌的因子的方法中使用的宿主是非人哺乳动物宿主。在一个实例中,非人哺乳动物宿主包括但不限于小鼠、猪、大鼠、豚鼠、仓鼠、兔、非人灵长类动物、猫、狗等。在一个实例中,非人哺乳动物宿主是小鼠或猪。在一个实例中,鉴定能够促进心肌细胞增殖的化合物或响应Tjp1抑制而分泌的因子的方法使用包括但不限于抗体阵列、鉴定和定量细胞因子和信号转导蛋白的测定(例如Luminex多重测定)等测定进行。
还在另一方面,本发明提供用于疗法中的Tjp1抑制剂。还在另一方面,本发明提供了Tjp1抑制剂在制造用于治疗心脏病的药物中的用途。Tjp1抑制剂抑制Tjp1表达。Tjp1表达的抑制能够导致Tjp1活性的抑制。因此,Tjp1的抑制最终会诱导心脏细胞(例如心肌细胞)的增殖。在一个实例中,心脏病包括但不限于心肌梗死、急性心肌梗死(AMI)、心力衰竭、心肌症、先天性心脏病、获得性心血管疾病、心肌细胞缺乏、心脏缺血再灌注损伤、心脏创伤、心肌梗死后能通过心脏细胞(例如心肌细胞)的再生来治疗的心脏疾病等。在一个示例中,心脏病还包括在心脏细胞再生有益的患者中的其他心脏损伤。
还在另一方面,本发明提供了包含本文所述的Tjp1抑制剂的药物组合物。在一个实例中,将包含Tjp1抑制剂的组合物与至少一种本文所述的其他因子一起施用。还在另一方面,本发明提供了贴剂,其中贴剂包含本文所述的Tjp1抑制剂。在一个实例中,贴剂包含Tjp1抑制剂是可植入的。本文所用术语“贴剂”是指与心脏接触以向心脏提供本文所述的Tjp1抑制剂的装置或植入物。可以使用本领域已知的任何方法制备贴剂。
如在本申请中所使用的,单数形式“一个/种(a)”,“一个/种(an)”和“所述(the)”包括复数指代,除非上下文另有明确说明。例如,术语“一个/种Tjp1抑制剂”包括多种Tjp1抑制剂,包括其混合物和组合。
如本文所用,在物质浓度、物质大小、时间长度或其他所述值的上下文中,术语“约”是指所述值的+/-5%,或所述值的+/-4%,或所述值的+/-3%,或所述值的+/-2%,或所述值的+/-1%,或所述值的+/-0.5%。
在整个本公开中,某些实施方案可以以范围形式公开。应当理解,范围形式的描述仅仅是为了方便和简洁,并且不应该被解释为对所公开范围的死板限制。因此,范围的描述应当视为具体公开了所有可能的子范围以及该范围内的各个数值。例如,应当认为对诸如1-6范围的描述已具有特别公开了子范围,例如1-3、1-4、1-5、2-4、2-6、3-6等,以及该范围内的单独数字,例如1、2、3、4、5和6。无论范围的广度如何,这都适用。
本文例证性地描述的本发明可以在缺少本文未具体公开的任何一种或多种要素、任何一种或多种限制的情况下适当地实施。因此,例如,术语“包含(comprising)”、“包括(including)”、“含有(containing)”等应当被广泛地解读而不受限制。另外,本文所用的术语和表达已被用作描述的术语而非限制,并且无意使用这些术语和表达来排除所示和所述特征的任何等同物或其部分,但应认识到在所要求保护的本发明的范围内可以进行各种修改。因此,应该理解,尽管已经通过优选实施方案和可选特征具体公开了本发明,但是本领域技术人员可以采用本文公开的本发明的修改和变化,并且这些修改和变化是被认为在本发明的范围内。
本文已广泛和一般性地描述了本发明。落入上位公开内容中的每个较窄类别和下位群组也构成本发明的一部分。这包括本发明的上位描述,带有从所述上位概念中去除任何主题的附带条件或否定限制,而不管是否在本文中具体叙述了被去除的材料。
其他实施方案在所附权利要求和非限制性实施例内。另外,在根据马库什群组描述本发明的特征或方面的情况下,本领域技术人员应当认识到,也因此按照马库什群组的任何单个成员或成员子群的形式描述了本发明。
实施例
紧密连接蛋白1(Tjp1),也称为Zonula Occludens-1(ZO-1),是在CM中定位于闰盘的肌动蛋白结合支架蛋白。这种质膜亚结构域含有基于N-钙粘蛋白的粘附连接、桥粒和间隙连接,并提供CM之间的机械偶联和电偶联。Tjp1结合与N-钙粘蛋白结合的α-连环蛋白以及Cx-43。Tjp1调节小鼠ES细胞的自我更新和分化,并且Tjp1缺陷小鼠显示了早期胚胎致死性。对Tjp1在CM中的功能角色知之甚少。在MI诱导的心脏重塑期间,Tjp1在间隙连接内化后与Cx43结合。编码Tjp1的前两个PDZ结构域的肽的表达导致大鼠心室CM中粘附物和间隙连接的异常亚细胞结构。用人HF研究的Tjp1水平在衰竭心脏中显示出较低的Tjp1水平,但在扩张和缺血性心肌症中上调。
这里,显示与生长因子提供的针对CM增殖的刺激提示相反,结构蛋白Tjp1抑制MI后的CM增殖和再生。成体CM中Tjp1的诱导性失活足以通过激活ErbB受体和下游Mek-Erk、Stat3、Akt和Wnt信号传导来驱动心脏细胞(包括丰富的CM)的增殖。Tjp1直接结合ErbB4,从而抑制Nrg1介导的信号传导。MI后,在没有Tjp1的情况下,梗死区域中CM增殖的增强限制了病理性重塑,并防止心脏功能恶化。因此,MI后沉默Tjp1可提供新的治疗机会。
材料和方法
小鼠CM中可诱导的Tjp1缺失
已经描述了敲入(floxed)的Tjp1(Tjp1F/F)小鼠的制备。为了特地使CM中的Tjp1缺失,使Tjp1F/F C57BL/6N小鼠与Myh6-CreERT2(MerCreMer)C57BL/6小鼠(由KristinB.Andersson(Institute for Experimental Medical Research,Oslo UniversityHospital,Norway with permission from Jeff Molkentin,Howard Hughes MedicalInstitute,Cincinnati Children's Hospital Medical Center)惠赠)杂交,后者在CM特异性α-肌球蛋白重链(Myh6,MHC)启动子的控制下以三苯氧胺可诱导的方式表达Cre。将小鼠与C57BL/6N背景回交超过5代。将三苯氧胺(Sigma)溶解在乙醇中并在高压灭菌的向日葵油(Sigma)中稀释。给6-8周龄的小鼠腹膜内注射20mg/ml三苯氧胺5天,每日剂量为100mg/kg体重。通过免疫染色和蛋白质印迹分析验证ZO-1蛋白的缺失。在实验中使用以三苯氧胺诱导的对照(Tjp1+/+Myh6-CreERT2)和Tjp1cKO(Tjp1F/F Myh6-CreERT2)小鼠。所有动物实验方案都获得相关IACUC委员会的批准。
基因分型
从剪切的尾部分离基因组DNA,并在PCR基因分型中使用两种引物。引物1(5′-CTT CTCTGA CCC TAC ACA GCT ACC-3′;SEQ ID NO:5)和引物2(5′-ATC GTG TGG GAA AGA CAA GC-3′;SEQ ID NO:6)扩增针对野生型等位基因的279bp片段和条件突变等位基因的471bp片段。
组织学分析
将新鲜解剖的心脏在4%多聚甲醛中固定过夜,包埋在石蜡中,切成5μm厚,并用苏木精和曙红染色。为了检测纤维化,心脏切片用Masson's Trichrome染色试剂盒(Polyscience)染色。
蛋白质印迹分析
用研钵将新鲜解剖的心脏匀浆,并在冰上的裂解缓冲液(50mM Tris-HCl,pH 7.5,100mM NaCl,1mM MgCl2和0.5%Triton X-100)中裂解15min(分钟)。将裂解物进行超声处理并在4℃通过离心(13,000×g,15min)澄清。通过SDS-聚丙烯酰胺凝胶电泳使上清液(等量的蛋白质)分级,并进行蛋白质印迹分析。使用针对以下的抗体:ZO-1(兔;InvitrogenCat.#617300或ThermoFisher Scientific Cat.#61-7300)、Dab2(小鼠;BD BiosciencesCat.#610464)、β-连环蛋白(小鼠;BD Biosciences,Cat.#610154)、LEF1(兔;CellSignalling,Cat.#2230)、Slug(兔;Cell Signalling,Cat.#9585)、Snail(兔;CellSignalling,cat.#3895)、Sox2(兔;Cell Signalling,Cat.#2748)、Gsk3β(兔;CellSignalling,Cat.#9315)、pGsk3β(兔;Cell Signalling,Cat.#9323;)、Erk1/2(兔;CellSignalling,Cat.#9102或#4695)、pErk1/2(兔;Cell Signalling,Cat.#9101或#4695)、JNK(兔;Cell Signalling,Cat.#9258)、pJnk(兔;Cell Signalling,Cat.#9255)、p38兔8690;Cell Signalling)、磷-p38(兔;Cell Signalling,Cat.#4511)、Akt(兔;Cell Signalling,Cat.#2920)、pAkt(S473)(兔;Cell Signalling,Cat.#4060)、Stat3(兔;Cell Signalling,Cat.#4904)、pStat3(兔;Cell Signalling,Cat.#9145)、cMyc(兔;Cell Signalling,Cat.#13987)、pH3(S10)(兔;Cell Signalling,Cat.#3377)、细胞周期蛋白D1(兔;CellSignalling,Cat.#2922)、细胞周期蛋白E(小鼠;Cell Signalling,Cat.#4129)、细胞周期蛋白A2(兔;Cell Signalling,Cat.#4656)、Yap(兔;Cell Signalling,Cat.#8418)、Runx1(兔;Cell Signalling,Cat.#4336)、MEF2c(兔;Cell Signalling,Cat.#5030)、GP130(兔;Santa Cruz,Cat.#SC-656)、细胞周期蛋白B1(兔;Santa Cruz,Cat.#SC-752)、GATA4(兔;Santa Cruz,Cat.#SC-9053)、Axin2(兔;Abcam,Cat.#Ab332197)、p21(兔;Abcam,Cat.#Ab7960)、SMA(兔;Abcam,Cat.#Ab5964)、PCNA(小鼠;Abnova,Cat.#H00005111)、MF20(小鼠;The Developmental Studies Hybridoma Bank at the University of Iowa,Cat.#MF20-C)、ErbB4(兔;Cell Signalling Cat.#4795)、pErbB4(兔;Cell Signalling,Cat.#4757)、EGFR(兔;Cell Signalling,Cat.#4267)和pEGFR(兔;Cell Signalling,Cat.#3777)。
免疫染色
将石蜡切片脱蜡,并通过在2100Retriever(Pick Cell Laboratories)中将载玻片蒸汽处理20分钟来回收抗原。将切片与抗ZO-1的一抗(兔;Invitorgen,Cat.#617300)和MF20(小鼠;The Developmental Studies Hybridoma Bank at the University of Iowa,Cat.#MF20-C)一起孵育,然后基本上如前所述,与Invitrogen的适当标记的二抗一起孵育。细胞核用4,6-二脒基-2-苯基吲哚(DAPI)标记。
细胞凋亡测定
将石蜡切片脱蜡,并根据制造商的方案,用TMR Red In Situ Cell Death Detection试剂盒(Roche Diagnostics)进行末端脱氧核苷酸转移酶介导的dUTP-生物素切口末端标记(TUNEL)测定。
增殖分析
在处死小鼠前1小时,将每克体重50μg的5-乙炔基-2'-脱氧尿苷(Edu;Invitrogen)腹膜内注射到小鼠体内。如上所述解剖出心脏并进行处理。将石蜡切片脱蜡并根据制造商的方案用Click-iTTMEdU Imaging Kits(成像试剂盒)(Invitrogen)染色。
用药理学抑制剂进行体内处理
为了阻断不同的信号传导通路,通过每日腹膜内注射给动物施用Mek抑制剂PD0325901(25mg/kg小鼠体重;Sigma)、Stat3抑制剂S31-201(5mg/kg小鼠体重;Santa CruzBiotechnology)、Wnt抑制剂LGK974(3mg/kg小鼠体重;Biovision),或EGFR/ErbB抑制剂Ast1306(25mg/kg小鼠体重;Selleckchem),持续5天。使用相同的给药途径和方案施用Nrg1(2.5μg/小鼠;溶于PBS,0.1%BSA;ProsPec,Cat.#cyt-407c)。
针对MI的外科手术程序和心脏功能评估
如前所述,在小鼠中诱导心肌梗死。简而言之,雄性小鼠(10-12周龄;20-25g)用0.5mg/kg美托咪定(Pfizer Animal Health,Exton,PA,USA)、5.0mg/kg多美康(Dormicum)(Sciencelab.com,Inc.,Texas,USA)和0.05mg/kg芬太尼(Pfizer PharmaceuticalsGroup,New York,USA)的混合物麻醉,然后进行左动脉前降支的永久性结扎。通过皮下注射0.5mg/kg阿替美唑(Pfizer Animal Health,Exton,PA,USA)和5mg/kg氟马西尼(SagentPharmaceuticals,Illinois,USA)使小鼠恢复,然后皮下注射0.1mg/kg Temgesic(HospiraInc.,Illinois,USA)用于镇痛。手术后,每天通过腹膜内途径给小鼠注射100mg/kg三苯氧胺,持续5天。外科医生不知道小鼠基因型。
如上所述,由高频超声系统Vevo 2100(Visualsonics)评估心脏功能,并由经验丰富的不知道小鼠基因型的研究人员用版本1.7.0的2100软件进行分析。简而言之,使用自动3D电机(Visualsonics)并使用集成呼吸和ECG门控以减少伪迹来获取B模式3D数据集。在基线,MI后第1、4、8和12周,在全身麻醉下(1-1.5%异氟醚,Baxter,新加坡)对小鼠进行超声心动图检查。用直肠探针监测体温并保持在36-37℃。
统计分析
使用具有Bonferroni事后分析的双向ANOVA或用于重复测量的GLM进行组间随时间的比较。值报告为平均值±SEM。P值≤0.05视为统计学上显著的。用SPSS(Statistics,版本22.0)和Prism(版本6,GraphPad Software)软件分析数据。
cDNA和shRNA质粒
未标记的pcDNA3.1-ERBB4由Yardena Samuels(Addgene plasmid#29527)馈赠。为了产生ErbB4突变体,根据制造商(Agilent Technologies)的说明书,使用Quik Change定点诱变试剂盒将ErbB4的最后三个C-末端氨基酸突变为丙氨酸以消除PDZ结合基序。使用标准克隆方案将人ZO-1的PDZ 1-3结构域克隆到pGEX 4T-1载体中。ZO1或TJP1和对照shRNA质粒获自Mission shRNA(Sigma)。
GSTl拉下实验(Pull down assay)
根据制造商的说明书,使用JetPrimePolyPlus转染试剂盒(PolyPlus Transfections)将ErbB4和突变体转染到HEK293细胞中。在裂解缓冲液(20mM Tris-HCl(pH7.4)、150mMNaCl、1mM EDTA、0.25%脱氧胆酸盐、1%NP-40和蛋白酶抑制剂)中制备细胞裂解物。使用标准方案产生、纯化GST和GST ZO-1PDZ1-3结构域融合蛋白,并与谷胱甘肽Sepharose-4B(GEHealthcare Life Sciences)结合。通过使用已知量的BSA作为标准物,通过SDS-PAGE定量结合的蛋白质。将18μl GST珠与1μg GST融合蛋白一起孵育。通过用冰冷的PBS洗涤除去未结合的蛋白质。然后加入300μg转染的细胞,并在4℃孵育2小时,然后用冰冷的裂解缓冲液洗涤4次。用4x SDS-PAGE样品缓冲液洗脱结合的级分,通过SDS PAGE分离,免疫印迹到PVDF膜上并用兔抗ErbB4抗体探测。
MCF7细胞中ZO-1shRNA敲减和NRG-1刺激
根据制造商(Life Technologies)的方案,使用293FT细胞和Virapower Lentiviral包装混合物和Lipofectamine 2000试剂生成慢病毒颗粒。然后将慢病毒颗粒加入MCF7细胞中,并用嘌呤霉素进行稳定选择。然后将细胞接种在60mm的培养皿上,次日用OPTI-MEM培养基洗涤,并孵育过夜。第二天,在不同时间点用人NRG-1(Cell Signalling Technology,Cat.#5218)刺激血清饥饿细胞。将细胞在RIPA缓冲液(20mM Tris-HCl(pH7.4)、150mMNaCl、1mM EDTA、0.25%脱氧胆酸盐、1%TritonX-100、0.1%SDS和蛋白酶抑制剂)中裂解,定量并在SDS-PAGE和蛋白质印迹上分析。对于剂量反应实验,如上所述,将细胞接种在60mm的培养皿,并血清饥饿过夜。第二天,用不同浓度的NRG-1刺激细胞15分钟,随后在冰冷的PBS中洗涤,用RIPA缓冲液裂解并处理用于SDS-PAGE和蛋白质印迹。
实验结果
CM中Tjp1的失活导致成体小鼠心脏细胞增殖
在Cre介导的CM中Tjp1失活后,心脏中的Tjp1蛋白水平降低(图1A),并且在大多数CM中检测不到蛋白质(图1B)。这与在三苯氧胺诱导后Tjp1F/F Myh6-CreERT2/Rosa26-LacZ报告子小鼠细胞系的大多数CM中广泛分布的LacZ染色相关,表明Cre-重组酶表达(图1C)。在三苯氧胺诱导后高达一年内,LacZ染色模式保持相似(数据未显示),表明野生型细胞未替代Tjp1缺失的CM。最后一次三苯氧胺注射后1天或7天Tjp1cKO心脏的组织学分析未显示明显异常(图1D)或细胞凋亡(图E)。
尽管组织学正常,但与对照相比,Tjp1cKO小鼠的心脏大小(图1F)和心脏与体重之比(图1G)略高。为了分析较大的Tjp1cKO心脏大小是否反映细胞增殖,在最后一次施用三苯氧胺后一天注射Edu。在对照切片中很少发现的Edu阳性细胞核在Tjp1cKO心脏中很丰富(图2A、B)。与有丝分裂和增强的细胞增殖一致,在成体Tjp1cKO心脏中检测到更高的PCNA、pS10-H3、细胞周期蛋白D1、细胞周期蛋白E、细胞周期蛋白A2和细胞周期蛋白B1蛋白水平(图2C)。Masson’s Trichrome染色未显示Tjp1cKO心脏纤维化(图2D)。
CM标志物心脏α-肌球蛋白重链(MHC;Myh6)的共标记显示至少5%的Edu阳性细胞是MHC阳性CM(图2E、F)。在解离的CM中(图2G),在最后一次三苯氧胺注射后3天,约50%的Edu-和MHC-阳性细胞含有单个细胞核(图2I),并且该分数在一个月后没有变化(图2J)。相反,总Edu阳性细胞的分数随时间下降(图2K),可能解释了137天后心脏与体重之比的正常化(图2L)。
Tjp1缺失后CM经历部分去分化
预先存在的CM的去分化随后增殖和再分化是可以生成新CM的一种机制。在最后一次三苯氧胺注射后一天制备的MHC染色的心脏切片的高放大倍数可视化揭示了Tjp1cKO心脏中肌节组织的变化(图3A),这表示去分化。一周后,肌节结构恢复。超过2%的Edu阳性细胞用GATA4(图3B、C)或Nkx2.5(图3D、E)标记,它们是CM分化的两个早期标志物。Dab2在胚胎心脏中高度表达并在出生后被抑制并被认为是CM去分化的标志物,在Tjp1缺陷心脏中被上调(图3F)。也与心脏发育有关的Runx1、MHC、α-SMA或Nkx2.5蛋白质水平的变化不太明显。
Tjp1缺失激活驱动CM增殖的信号传导通路
接下来,评估在Tjp1cKO心脏中激活的信号传导通路,其集中于与CM增殖相关的通路。促分裂原激化蛋白激酶(MAPK)通路被与CM增殖有关的受体激活,包括ErbB2和ErbB4。斑马鱼中Stat3的抑制,虽然不影响生长相关的CM增殖,但限制了损伤引起的心脏再生。通过PI3K激活Akt刺激CM增殖,Wnt信号传导也是如此。
与增加的细胞增殖一致,MAPK信号传导响应于Tjp1的缺失而被激活(例如更高的pThr202/Tyr204-Erk水平)(图4A)。相反,p38和JNK,其在被抑制时促进CM的细胞周期再进入和胞质分裂,在Tjp1cKO心脏中被抑制(例如,较低的pThr180/Tyr182-p38和pThr183/Tyr185-JNK水平)。在缺乏Tjp1的心脏中,总Stat3以及pY705-Stat3蛋白水平升高(图4A)。与Stat3激活一致,下游靶基因c-Myc被上调。在Stat3上游的Gp130没有变化。在Tjp1cKO心脏中Akt1、特别是pS473-Akt蛋白水平增加。尽管三苯氧胺诱导后总β-连环蛋白水平略有降低,但其共激活剂LEF1和若干下游靶基因,包括Axin2、Slug、Snail和Sox2的表达高度升高(图4A),表明Wnt信号传导的激活。
为了探讨不同信号传导通路的贡献并确认它们的激活在Tjp1缺失时驱动细胞增殖,使用在短期全身暴露期间小鼠良好耐受的化学抑制剂。在5天内将所述抑制剂与每日三苯氧胺注射同时施用以诱导Tjp1的缺失。在最后一次注射后,收集心脏并分析。Mek磷酸化并激活其直接下游靶标Erk。PD0325901,是特异性Mek抑制剂,抑制不同效应物的激活(图4B、C),并因此细胞增殖由于Tjp1缺失而激活(图4D、E)。Stat3抑制剂S31-201防止Stat3和Erk激活,适度抑制Akt和Wnt信号传导并抑制Tjp1cKO心脏中的细胞增殖(图4F、图7)。Akt抑制剂API-2显著抑制Akt磷酸化,部分减少Wnt信号传导,但不影响pErk或pStat3水平。LGK974,是Porcupine(其活性是Wnt分泌所必需的)的小分子抑制剂,抑制Wnt信号传导,适度降低Erk和Akt,但不降低Stat3磷酸化。与它们的部分效应一致,API-2和LGK974将Tjp1缺失诱导的细胞增殖减少到更适中的程度(约50%;图4G、H)。
Tjp1缺失后的CM增殖需要ErbB受体激活并且被Nrg1增强
Erk、Stat3和Akt是ErbB生长因子受体家族的常见下游效应物,表明这些受体可在Tjp1缺失后被激活。因此确定AST1306是否抑制下游效应物的激活,AST1306是阻断配体诱导的自身磷酸化和激活的ErbB受体的激酶活性的不可逆抑制剂。AST1306强烈消除Tjp1cKO心脏中磷酸化的Erk、Stat3和Akt的增加,c-Myc水平也是如此(图5A)。较低的Axin-2蛋白水平也表明Wnt信号传导的抑制。所述化合物强烈降低Tjp1cKO心脏中的pS10-H3和PCNA表达(图5A)。在经处理的Tjp1cKO心脏中存在少于1%的Edu阳性细胞,而在未处理的Tjp1cKO心脏中则存在7%的Edu阳性细胞(图5B、C)。
有趣的是,EGFR蛋白水平和磷酸化在Tjp1缺失时升高,并且这被AST1306消除(图5A)。由于Nrg1/ErbB4轴与CM增殖有关,而AST1306是pan-ErbB抑制剂,因此分析了Tjp1cKO心脏中的ErbB4激活。与EGFR类似,在Tjp1cKO心脏中ErbB4蛋白和磷酸化水平增加(图5D)。为了评估Nrg1对细胞增殖的可能作用,在Tjp1缺失后3周,此时Edu阳性细胞的数量下降(图2K;5E、F),每天注射5次Nrg1和Edu。与未处理或处理的对照心脏相比,Nrg1显著增加Tjp1cKO心脏中的细胞增殖(图5E、F)。Nrg1应用在Tjp1cKO心脏中产生丰富的Edu和MHC阳性细胞(576中的10个),与对照心脏中主要是MHC阴性增殖细胞相反。
Tjp1与ErbB4结合以抑制Nrg1介导的信号传导
上述数据表明了Tjp1对心脏中Nrg1介导的ErbB4信号传导的抑制作用。为了证实这一点并进一步分析其机制,使用了比成体CM更容易培养和分析的MCF7细胞。shRNA介导的Tjp1沉默维持Nrg1诱导的ErbB4信号传导(图5G)。在对照细胞中,Erk磷酸化在配体添加后约15分钟达到峰值,随后在60-120分钟之间下降至基础水平。相反,在Tjp1已经沉默的细胞中,Erk在240分钟后仍然保持磷酸化。伴随着Nrg1诱导的信号传导增强和如在心脏中观察到的,当Tjp1沉默时,ErbB4蛋白水平增加,表明对受体运输和/或加工的影响。剂量-反应实验表明,Tjp1的沉默使细胞对Nrg1高度敏感(图5H)。尽管对照细胞中最大的Erk激活需要50-100ng/ml的Nrg1,但是在耗尽Tjp1后仅用2.5ng/ml就可实现。
ErbB4含有C末端PDZ结合基序(PBM)(图5I)。为了测试该PBM是否可以与Tjp1的PDZ结构域相互作用,使用携带Tjp1的三个PDZ结构域的GST融合蛋白(Tjp1PDZ1-3)和来自表达野生型ErbB4或具有破坏的PBM的突变体(ErbB4ΔPBM)的细胞的裂解物进行拉下实验。Tjp1PDZ结构域能够下拉ErbB4,并且这种结合需要完整的PBM(图5J)。ErbB4辅助受体ErbB2还含有推定的PBM,并且同样与Tjp1PDZ结构域相互作用(图5I和K)。
MI后Tjp1的失活促进CM增殖并保持心脏功能
接下来研究Tjp1缺失对MI后心脏再生和功能的影响。永久结扎左动脉前降支手术后第一天,给小鼠注射5天三苯氧胺以使Tjp1缺失。用Edu注射3天后,在1周或1个月后处死小鼠。Masson’s Trichrome染色显示对照和Tjp1cKO心脏的左心室(LV)的梗死区域形成瘢痕(图6A)。在Tjp1cKO心脏的梗死边界区域发现显著更多的Edu和MHC阳性CM(图6B和C)。有趣的是,梗死边界区域中增殖性细胞为MHC阳性CM的增殖性细胞是未损伤的心脏组织中的2倍(图6D),表明梗死小生境和Tjp1缺乏对CM增殖的协同效应。
用超声心动图评估心脏功能。MI后7天,对照和Tjp1cKO小鼠的初始梗死面积相似(43.7%对43.1%;p=0.0573;图6E)。在MI后,对照和Tjp1cKO LV的左心室舒张期末容积(EDV)最初增加(图6F),如同针对类似梗死面积所预期的那样。然而,随着时间的推移,与Tjp1cKO心脏相比,EDV在对照中持续增加到显著更大的程度,与Tjp1cKO心脏中较少的不利重构一致。对照和Tjp1cKO心脏的LV射血分数(EF)下降至约30%,与可比较的初始梗死面积一致。然而,虽然LVEF在对照中持续下降,但在没有Tjp1的情况下其得以保留(第8周p=0.033,第12周p=0.006;GLM用于重复测量,基因型之间p=0.024;图6G),表明MI后心脏功能障碍因Tjp1缺乏而减弱。通过超声心动图估计的梗死边界区壁在第1周时在Tjp1cKO心脏中明显变厚(对照为1.00±0.04mm,而Tjp1cKO为1.15±0.04mm;p=0.029;图6H),可能反映了CM和非CM两者的增殖。这种差异在MI后第12周变得更少(WT为0.86±0.09mm,KO为1.08±0.06mm;p=0.081),可能是由于非CM丧失和不利的LV重构减少,如EDV指示的LV扩张减少所示。总之,MI后Tjp1缺失导致CM增殖增强,特别是在瘢痕边缘区域,从而限制不利的重构和保持心脏功能至少12周。
讨论
几十年来,克服终末分化的成体哺乳动物CM中缺乏再生能力一直备受关注。成体CM去分化和增殖的能力有限阻碍了急性心脏损伤和细胞丧失后功能的恢复,导致HF。迄今为止,HF的药物疗法虽然有效,但仍会使患者预后不良。鉴定成体心脏再生的细胞机制提供了靶向用于优化对损伤的心脏适应性反应的关键通路的可能性,从而预防或逆转HF。在人体临床试验中使用基于Nrg1的疗法,Nrg1/ErbB4轴已作为有前景的治疗靶点出现。提供了结构蛋白Tjp1充当成体小鼠心脏中Nrg1/ErbB4信号传导和CM增殖的抑制剂的第一个证据。在实验性MI后,耗尽Tjp1增强特别是在梗死边界区域中的CM再生,从而保留心脏功能。
在去分化之后,可以由心脏前体或预先存在的CM生成新的CM。对闰盘的N-钙粘蛋白复合体中Tjp1丧失的即时反应可能是CM的部分和瞬时解偶联和去分化,如肌节结构的变化和早期心脏谱系标志物(例如Nkx2.5、Gata4、Dab-2)的表达所示。抑制细胞增殖的药理学抑制剂也降低Dab-2蛋白水平,表明去分化和增殖是相互关联的。在组成型活性ErbB2表达后也观察到CM去分化和增殖。由于大多数鼠CM是双核或多核的,因此在Tjp1cKO心脏中存在相似数量的单核和双核Edu阳性CM进一步支持它们来自先前存在的CM。新生成的CM持续存在于心脏中,但Edu阳性非CM细胞随时间而下降(在5个月内从约20%降至5%)。虽然尚不清楚如何去除过量的成纤维细胞,但它可能解释了心脏大小的适度瞬时增加和缺乏纤维化。然而,Tjp1cKO心脏中的细胞仍然接受增殖性刺激,例如Nrg1给药。
最近证实Tjp1结合并抑制EGFR的活性,EGFR是ErbB受体家族的另一成员。CM和MCF-7细胞中Tjp1的耗尽导致ErbB4受体激活,可能通过对较低的配体浓度敏感和/或维持激活状态。后者可能包括Tjp1对ErbB4受体运输或加工的影响,这是鉴于在Tjp1耗尽后心脏细胞和MCF-7细胞中的EGFR和ErbB4蛋白水平增加。Nrg1/ErbB4和ErbB2,其也结合Tjp1,通过激活Erk、Stat3、Akt和Wnt信号传导刺激成体心肌细胞增殖。这些效应物和通路在Tjp1cKO心脏中也被激活,并且如使用特定药理学抑制剂所证明的,是细胞增殖所需的。有趣的是,CM中Tjp1的缺失不仅刺激CM的增殖,还刺激心脏中其他细胞的增殖。假设了旁分泌/自分泌机制,据此Tjp1缺陷型CM分泌的生长因子刺激非CM细胞的增殖。由激活的非CM分泌的这些或其他因子可能进一步分别以自分泌或旁分泌方式对CM发挥作用。例如,内皮细胞分泌多种心脏活动因子,包括Nrg1。
在急性MI的小鼠模型中,如通过临床相关参数评估的,在梗死后去除Tjp1改善了总体结果。Tjp1缺失导致瘢痕边缘区域中增殖的CM显著增加。与未受损的Tjp1cKO心脏组织相比,在梗死边界中检测到两倍的增殖性CM。MI梗死后,受损的心脏和浸润免疫细胞释放生长因子和细胞因子,它们可刺激缺乏Tjp1的CM增殖。推测梗死边界区域的CM增殖的增强限制不利的重构,从而如EDV和LVEF所测量的,导致从8周开始观察到心脏功能的保留。这表明CM通过Tjp1缺失而增殖部分地恢复了损伤后CM的损失,从而保护小鼠心脏免受不利的LV重构和心脏功能丧失。
总之,Tjp1抑制ErbB4信号传导,并且其去除使ErbB4对其配体Nrg1敏感并维持受体激活。这触发了下游效应物Mek-Erk、Stat3、Akt和Wnt信号传导以诱导CM增殖。MI后Tjp1的缺失保留了心脏功能,因此提供了通过CM再生来控制不利的重构和随后的HF的新靶标。Tjp1的CM特异性沉默可以避免与全身和长期Nrg1给药相关的可能的致癌风险。或者,可以认为Tjp1的心脏特异性失活,或与其他ErbB受体配体组合,增强Nrg1的功效。
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Val
Claims (24)
1.一种治疗受试者中的心脏病的方法,包括向所述受试者施用治疗有效量的Tjp1抑制剂的步骤。
2.根据权利要求1所述的方法,其中所述Tjp1抑制剂是核酸。
3.根据权利要求1或2所述的方法,其中所述核酸选自由siRNA、shRNA、反义寡核苷酸、gapmer和短发夹反义寡核苷酸(shAON)组成的组。
4.根据前述权利要求中任一项所述的方法,其中所述核酸与编码Tjp1的mRNA结合并形成核酸-mRNA复合体。
5.根据前述权利要求中任一项所述的方法,其中所述核酸-mRNA复合物体中的mRNA被切割和/或不被翻译。
6.根据前述权利要求中任一项所述的方法,其中编码所述抑制剂的核酸与选自由以下组成的组的序列具有至少90%的同一性:
SEQ ID NO:1
(CCGGGCCTGCATACAATAAAGCAAACTCGAGTTTGCTTTATTGTATGCAGGCTTTTTG),
SEQ ID NO:2
(CCGGGGAACCACTCTATCAAGTATTCTCGAGAATACTTGATAGAGTGGTTCCTTTTTG),
SEQ ID NO:3
(CCGGCGTGGATTGAACTTACTAAATCTCGAGATTTAGTAAGTTCAATCCACGTTTTTG),和
SEQ ID NO:4
(CCGGCCGCGAAGTTATGAGCAAGTTCTCGAGAACTTGCTCATAACTTCGCGGTTTTTG)。
7.根据前述权利要求中任一项所述的方法,其中所述方法还包括与所述Tjp1抑制剂一起或单独施用选自由多肽和核酸组成的组的其他因子。
8.根据权利要求7所述的方法,其中所述核酸编码细胞周期蛋白A2。
9.根据权利要求7所述的方法,其中所述多肽与ErbB4受体结合。
10.根据权利要求9所述的方法,其中所述结合ErbB4受体的多肽选自由神经调节蛋白-1、神经调节蛋白-2(NRG2)、神经调节蛋白-3(NRG3)、乙胞素(BTC)、上皮调节蛋白(EPR)、肝素结合EGF样生长因子(HB-EGF)、表皮生长因子(EGF)、β-纤维素、转化生长因子α(TGFα)和双调蛋白(AR)组成的组。
11.根据权利要求10所述的方法,其中所述结合ErbB4受体的多肽是神经调节蛋白-1(NRG1)。
12.根据权利要求7所述的方法,其中所述多肽激活Wnt信号传导。
13.根据权利要求12所述的方法,其中所述激活Wnt信号传导的多肽选自由Wnt、Norrin和R-spondin组成的组。
14.根据权利要求7所述的方法,其中所述多肽是生长因子或分泌因子。
15.根据权利要求14所述的方法,其中所述生长因子是成纤维细胞生长因子(FGF)或血管内皮生长因子(VEGF)。
16.根据权利要求14所述的方法,其中所述分泌因子是滤泡稳定蛋白样1(Fstl1)。
17.根据前述权利要求中任一项所述的方法,其中当所述抑制剂是核酸抑制剂时,所述方法包括病毒介导的递送系统。
18.根据权利要求17所述的方法,其中所述病毒选自由逆转录病毒、腺病毒、腺伴随病毒和单纯疱疹病毒组成的组。
19.根据权利要求18所述的方法,其中所述病毒是腺伴随病毒。
20.根据权利要求19所述的方法,其中所述腺伴随病毒是AAV血清型9。
21.Tjp1抑制剂在制造用于治疗心脏病的药物中的用途。
22.根据权利要求1-20中任一项所述的方法或根据权利要求21所述的用途,其中所述心脏病选自由以下组成的组:心肌梗死、急性心肌梗死(AMI)、心力衰竭、心肌症、先天性心脏病、获得性心血管疾病、心肌细胞缺乏、心脏缺血再灌注损伤、心脏创伤和在心脏细胞再生有益的患者中的其他心脏损伤。
23.一种贴剂,其中所述贴剂包含如权利要求1-20中任一项所定义的Tjp1抑制剂。
24.一种编码Tjp1抑制剂的核酸,其中所述核酸与选自由以下组成的组的序列具有至少90%的同一性:
SEQ ID NO:1
(CCGGGCCTGCATACAATAAAGCAAACTCGAGTTTGCTTTATTGTATGCAGGCTTTTTG),
SEQ ID NO:2
(CCGGGGAACCACTCTATCAAGTATTCTCGAGAATACTTGATAGAGTGGTTCCTTTTTG),
SEQ ID NO:3
(CCGGCGTGGATTGAACTTACTAAATCTCGAGATTTAGTAAGTTCAATCCACGTTTTTG),和
SEQ ID NO:4
(CCGGCCGCGAAGTTATGAGCAAGTTCTCGAGAACTTGCTCATAACTTCGCGGTTTTTG)。
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CN104736709A (zh) * | 2012-03-27 | 2015-06-24 | 科达治疗公司 | 基于钙粘蛋白调节的组合物和治疗 |
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