Roles of Hsp20-Exosomes in Myocardial Angiogenesis

描述（由申请人提供）：目前，通过直接递送生长因子的治疗性血管生成在动物模型中显示出很大的希望，但迄今为止尚未在人体中取得成功。因此，迫切需要继续寻找促进心肌血管生成的有效治疗方法。我们最近的研究有趣地表明，过表达hsp20的心肌细胞（Hsp20-myocytes）具有促血管生成能力，这与外泌体的分泌增加有关，外泌体是一组自然产生的纳米囊泡（30-100nm），从细胞中主动释放。此外，我们的最新数据表明：1)来源于Hsp20-肌细胞的外泌体中含有大量的Hsp20、VEGFR2和p-Akt，它们被有效地转移到内皮细胞中，从而激活促血管生成信号通路；2) Hsp20与Tsg101相互作用，Tsg101是一种已知参与外泌体生物发生的蛋白；3)通过siRNA敲除Tsg101可减少hsp20肌细胞的外泌体分泌。因此，检测外泌体的数量（tsg101依赖性外泌体生物生成）或质量（hsp20重编程外泌体）对hsp20肌细胞诱导的促血管生成至关重要。我们假设外泌体和Hsp20都是心肌细胞催化血管生成所必需的，并且Hsp20重编程的外泌体对糖尿病动物心脏具有治疗益处，糖尿病动物心脏表现出缺血后血管生成和微血管稀疏受损。这些假设将通过追求三个特定目标来验证：1)确定外泌体是否对Hsp20-肌细胞诱导的血管生成至关重要。目前，我们正在构建一种心脏特异性缺失Tsg101以抑制外泌体产生的小鼠模型。该模型将与hsp20转基因小鼠杂交，以检测阻断外泌体的产生是否会减弱hsp20 -肌细胞诱导的促血管生成作用。2)确定外泌体介导的心肌血管生成是否需要Hsp20。我们正在创建一个心脏特异性缺失Hsp20的诱导小鼠模型，以测试Hsp20是否在重编程促血管生成外泌体中起重要作用。3)检测hsp20干细胞来源的外泌体是否促进糖尿病动物心脏血管生成。我们建议使用骨髓来源的间充质干细胞作为hsp20重编程外泌体的治疗来源，因为它们比心肌细胞更容易获得。我们将使用链脲佐菌素（STZ）诱导的糖尿病大鼠和Goto-Kakizaki （GK）大鼠这两种成熟的糖尿病动物模型来进行这项转化研究。总之，拟议的研究有望揭示Hsp20在外泌体重编程和促进心肌细胞诱导的血管生成中的先前未被认识到的作用。此外，它有望提供新的见解，从而导致基于原始外泌体的治疗策略的发展，用于治疗冠状动脉疾病。

DESCRIPTION (provided by applicant): Currently, therapeutic angiogenesis via direct delivery of growth factors has shown great promise in animal models, but has not been successful in humans thus far. Therefore, continuing search for effective therapeutic approaches aimed at promoting myocardial angiogenesis is desperately needed. Our recent work interestingly showed that Hsp20-overexpressing cardiomyocytes (Hsp20-myocytes) possessed pro- angiogenic capacity, which was associated with increased secretion of exosomes, a group of naturally occurring nano-vesicles (30-100nm) actively released from cells. In addition, our latest data showed that: 1) exosomes derived from Hsp20-myocytes, but not from Hsp20-knockdown myocytes, contained a large amount of Hsp20, VEGFR2 and p-Akt, which were efficiently transferred into endothelial cells, resulting in activation of the pro-angiogenic signaling pathway; 2) Hsp20 interacted with Tsg101, a protein known to be involved in exosome biogenesis; and 3) knockdown of Tsg101 by siRNA attenuated exosome secretion from Hsp20-myocytes. Thus, it will be very important to test whether exosome quantity (Tsg101-dependent exosome biogenesis) or quality (Hsp20-reprogrammed exosomes) is critical for Hsp20-myocyte-elicited pro- angiogenesis. We hypothesize that exosomes and Hsp20 are both required for cardiomyocyte-catalyzed angiogenesis, and that Hsp20-reprogrammed exosomes have therapeutic benefits to diabetic animal hearts, which exhibit an impaired post-ischemic angiogenesis and microvascular rarefaction. These hypotheses will be tested by pursuing three specific aims: 1) Determine whether exosomes are essential for Hsp20- myocyte-induced angiogenesis. Currently, we are generating a mouse model with cardiac-specific deletion of Tsg101 to inhibit exosome production. This model will be crossed with Hsp20-transgenic mice for testing whether Hsp20-myocyte-elicited pro-angiogenic effects are attenuated by blockade of exosome generation. 2) Determine whether Hsp20 is required for exosome-mediated myocardial angiogenesis. We are creating an inducible mouse model with cardiac-specific deletion of Hsp20 to test whether Hsp20 plays an essential role in reprogramming pro-angiogenic exosomes. 3) Test whether Hsp20-stem cell- derived exosomes improve angiogenesis in diabetic animal hearts. We propose to use bone marrow- derived mesenchymal stem cells as a therapeutic source of Hsp20-reprogrammed exosomes, because they are more readily obtained than cardiomyocytes. We will use streptozotocin (STZ)-induced diabetic rats and the Goto-Kakizaki (GK) rats, two well-established diabetic animal models, to pursue this translational research. Together, the proposed studies are expected to unveil a previously unrecognized role of Hsp20 in exosomal reprogramming and in the promotion of cardiomyocyte-induced angiogenesis. Additionally, it is expected to provide novel insights that lead to the development of original exosome-based therapeutic strategies for the treatment of coronary artery disease.