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巨大的前景，但迄今为止在人类中尚未成功。因此，迫切需要继续寻找旨在促进心肌血管生成的有效治疗方法。我们最近的工作有趣地表明，Hsp 20过表达的心肌细胞（Hsp 20-myocytes）具有促血管生成能力，这与外泌体（exosomes）的分泌增加有关，外泌体是一组天然存在的纳米囊泡（30- 100 nm），从细胞中主动释放。此外，我们的最新研究结果表明：1）来源于Hsp 20-心肌细胞的exosomes含有大量的Hsp 20、VEGFR 2和p-Akt，并能有效地转移到内皮细胞中，激活促血管生成的信号通路;和3）通过siRNA敲低Tsg 101减弱了Hsp 20-肌细胞的外泌体分泌。因此，测试外泌体的量（Tsg 101依赖性外泌体生物发生）或质量（Hsp 20重编程的外泌体）是否对Hsp 20肌细胞引发的促血管生成至关重要将是非常重要的。我们假设外泌体和Hsp 20都是心肌细胞催化的血管生成所需的，并且Hsp 20重编程的外泌体对糖尿病动物心脏具有治疗益处，其表现出缺血后血管生成受损和微血管稀疏。这些假设将通过追求三个具体目标进行测试：1）确定外泌体是否是Hsp 20-肌细胞诱导的血管生成所必需的。目前，我们正在建立一个心脏特异性缺失Tsg 101的小鼠模型，以抑制外泌体的产生。该模型将与Hsp 20转基因小鼠杂交，以测试Hsp 20肌细胞引发的促血管生成作用是否通过阻断外泌体产生而减弱。2)确定Hsp 20是否是外泌体介导的心肌血管生成所必需的。我们正在建立一个心脏特异性缺失Hsp 20的诱导型小鼠模型，以测试Hsp 20是否在重编程促血管生成外泌体中起重要作用。3)测试Hsp 20干细胞衍生的外泌体是否改善糖尿病动物心脏中的血管生成。我们建议使用骨髓源性间充质干细胞作为Hsp 20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，两种成熟的糖尿病动物模型，进行这项转化研究。总之，这些研究有望揭示Hsp 20在外泌体重编程和促进心肌细胞诱导的血管生成中的一个先前未被认识的作用。此外，预计它将提供新的见解，导致开发用于治疗冠状动脉疾病的原始基于外泌体的治疗策略。

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.