Inhalable Stem Cell Exosomes Promote Heart Repair After Myocardial Infarction

Abstract BACKGROUND: Exosome therapy shows potential for cardiac repair after injury. However, intrinsic challenges such as short half-life and lack of clear targets hinder the clinical feasibility. Here, we report a noninvasive and repeatable method for exosome delivery through inhalation after myocardial infarction (MI), which we called stem cell–derived exosome nebulization therapy (SCENT). METHODS: Stem cell–derived exosomes were characterized for size distribution and surface markers. C57BL/6 mice with MI model received exosome inhalation treatment through a nebulizer for 7 consecutive days. Echocardiographies were performed to monitor cardiac function after SCENT, and histological analysis helped with the investigation of myocardial repair. Single-cell RNA sequencing of the whole heart was performed to explore the mechanism of action by SCENT. Last, the feasibility, efficacy, and general safety of SCENT were demonstrated in a swine model of MI, facilitated by 3-dimensional cardiac magnetic resonance imaging. RESULTS: Recruitment of exosomes to the ischemic heart after SCENT was detected by ex vivo IVIS imaging and fluorescence microscopy. In a mouse model of MI, SCENT ameliorated cardiac repair by improving left ventricular function, reducing fibrotic tissue, and promoting cardiomyocyte proliferation. Mechanistic studies using single-cell RNA sequencing of mouse heart after SCENT revealed a downregulation of Cd36 in endothelial cells (ECs). In an EC-Cd36fl/− conditional knockout mouse model, the inhibition of CD36, a fatty acid transporter in ECs, led to a compensatory increase in glucose utilization in the heart and higher ATP generation, which enhanced cardiac contractility. In pigs, cardiac magnetic resonance imaging showed an enhanced ejection fraction (Δ=11.66±5.12%) and fractional shortening (Δ=5.72±2.29%) at day 28 after MI by SCENT treatment compared with controls, along with reduced infarct size and thickened ventricular wall. CONCLUSIONS: In both rodent and swine models, our data proved the feasibility, efficacy, and general safety of SCENT treatment against acute MI injury, laying the groundwork for clinical investigation. Moreover, the EC-Cd36fl/− mouse model provides the first in vivo evidence showing that conditional EC-CD36 knockout can ameliorate cardiac injury. Our study introduces a noninvasive treatment option for heart disease and identifies new potential therapeutic targets. To characterize gene expression changes underlying the treatment efficacy of SCENT in MI mice, we performed droplet-based single-cell RNA sequencing on whole mouse hearts from the treatment group and control group

摘要
## 背景
外泌体（exosome）疗法在损伤后心脏修复领域展现出应用潜力，但存在半衰期短、缺乏明确靶点等固有挑战，制约了其临床转化可行性。本研究报道了一种无创且可重复的心肌梗死（myocardial infarction, MI）后外泌体递送方法——干细胞源性外泌体雾化疗法（stem cell-derived exosome nebulization therapy, SCENT）。
## 方法
本研究首先对干细胞源性外泌体的粒径分布及表面标志物进行表征。将构建心肌梗死模型的C57BL/6小鼠通过雾化器连续7天接受外泌体吸入治疗。通过超声心动图（echocardiography）监测干细胞源性外泌体雾化疗法（SCENT）治疗后的心脏功能，并采用组织学分析探究心肌修复情况。对全心脏进行单细胞RNA测序（single-cell RNA sequencing），以解析SCENT的作用机制。最后，借助三维心脏磁共振成像（3-dimensional cardiac magnetic resonance imaging），在猪心肌梗死模型中验证了SCENT的可行性、有效性及总体安全性。
## 结果
通过离体IVIS成像（IVIS imaging）及荧光显微镜检测，可观察到SCENT治疗后外泌体向缺血心脏的募集现象。在小鼠心肌梗死模型中，SCENT可通过改善左心室功能、减少纤维化组织生成、促进心肌细胞增殖，从而改善心脏修复。对SCENT治疗后小鼠心脏的单细胞RNA测序分析显示，内皮细胞（endothelial cells, ECs）中的Cd36表达下调。在内皮细胞特异性Cd36条件性敲除小鼠模型（EC-Cd36fl/−）中，作为脂肪酸转运蛋白的CD36受到抑制后，可引发心脏葡萄糖摄取的代偿性增加及ATP生成增多，进而增强心脏收缩功能。在猪模型中，与对照组相比，SCENT治疗后第28天的心脏磁共振成像结果显示射血分数提升（Δ=11.66±5.12%）、短轴缩短率提升（Δ=5.72±2.29%），同时梗死面积减小、心室壁厚度增加。
## 结论
在啮齿类及猪类动物模型中，本研究数据证实了SCENT治疗急性心肌梗死损伤的可行性、有效性及总体安全性，为后续临床研究奠定了基础。此外，EC-Cd36fl/−小鼠模型首次提供了体内证据，证明内皮细胞特异性CD36敲除可改善心脏损伤。本研究为心脏疾病提供了一种无创治疗选择，并鉴定出全新的潜在治疗靶点。
为了表征SCENT治疗心肌梗死小鼠的疗效背后的基因表达变化，我们对治疗组与对照组的全小鼠心脏进行了液滴式单细胞RNA测序（droplet-based single-cell RNA sequencing）。