Preservation and Vascularization of Cardiac Extracellular Matrix after Myocardial Infarction

心肌梗死（MI）后，心肌细胞外基质（ECM）主要通过上调基质金属蛋白酶2/9 （MMP-2/9）降解，肌成纤维细胞形成后心肌纤维化进展，心功能逐渐恶化。因此，抑制MMP-2/9的生物活性和抑制肌成纤维细胞的形成将改善心脏功能。然而，同时实现这两个目标的理想治疗策略仍有待建立。目前，广谱MMP抑制剂的系统递送在临床试验中没有显示出一致的结果。心肌梗死后心肌梗死中MMP-2/9的表达具有时空性。然而，目前的系统给药方法不能在时空上将MMP抑制剂递送到梗死区域。为了减轻心脏纤维化，全身递送TGFβ抑制剂或抗TGFβ抗体是一种主要方法。然而，它只降低活性tgf - β的含量。它不能抑制tgf - β信号通路来阻止肌成纤维细胞的形成。此外，小的有机MMP和TGFβ抑制剂有毒性问题。该项目的目标是创建药物输送系统，可以专门输送到梗塞的心脏，同时保存心脏ECM，防止心脏纤维化。局部给药将消除限制剂量的副作用。该系统将时空释放MMP-2/9特异性和无毒抑制剂肽CTTHWGFTLC (CTT)，以特异性调节局部MMP-2/9的生物活性。该系统还将逐渐释放具有抗纤维化和促进血管生成功能的多功能生长因子bFGF。因此，保留的ECM将被血管化。血管化对心脏ECM至关重要，否则其结构和成分会随着时间的推移而改变。在我们的初步工作中，我们已经创造了一种快速凝胶和可降解的水凝胶释放系统，能够有效地将药物保留在跳动的心脏中。系统可以释放CTT 4周。注射到梗死心脏后，释放的CTT保存了胶原蛋白，增加了组织厚度，改善了心功能。CTT比许多其他小的有机MMP抑制剂更好，不诱导心脏纤维化。此外，在心肌梗死后tgf - β上调的情况下，CTT促进内皮细胞迁移。这些结果表明，CTT可能是一种比那些小的有机抑制剂更好的心脏治疗MMP抑制剂。我们进一步创建了一个持续发布CTT和bFGF的发布系统。bFGF以其血管生成作用而闻名。我们发现bFGF能够通过TGFβ/Erk1/2途径抑制TGFβ诱导的心肌成纤维细胞向肌成纤维细胞分化。植入4周后，CTT/bFGF释放系统不仅增加了组织厚度和保存的胶原成分，而且促进了高密度毛细血管的形成，显著减少了心脏纤维化，导致心功能的增加。基于我们的初步研究，我们假设将CTT和bFGF局部和时空输送到梗死心脏，可以同时减弱心脏ECM降解，使保存的ECM血管化，防止心脏纤维化，从而显著提高心功能。AIM 1将测试假设，最佳CTT释放谱将有效减弱MMP-2的生物活性，以防止MMP-2介导的ECM降解。AIM 2将验证最佳bFGF释放水平同时促进内皮细胞形态发生和阻止心脏成纤维细胞向肌成纤维细胞分化的假设。AIM 3将验证心肌梗死后CTT和bFGF释放系统的传递是否能同时保护和血管化心脏ECM，并预防心脏纤维化。该项目具有创新性，因为它创建了转化药物输送系统，以建立：1)高效的MMP-2/MMP-9抑制剂CTT在心脏治疗中的作用和功效；2) bFGF抑制心肌纤维化促进血管生成的机制及作用；3) CTT和bFGF的持续释放如何同时实现这三个目标。该系统相对简单，功能齐全。因此，它是可翻译的。

Following myocardial infarction (MI), the degradation of cardiac extracellular matrix (ECM) mainly by upregulated matrix metalloproteinase-2/9 (MMP-2/9), and the progression of cardiac fibrosis after myofibroblast formation, progressively deteriorate cardiac function. As such, impeding MMP-2/9 bioactivity, and inhibiting myofibroblast formation will improve cardiac function. However, the ideal therapeutic strategies to simultaneously achieve both goals remain to be established. Currently, systemic delivery of broad spectrum MMP inhibitors did not show consistent outcomes in clinical trials. MMP-2/9 expression is spatiotemporal in infarcted hearts over the course of post-MI. Yet current systemic delivery approach cannot spatiotemporally deliver MMP inhibitors to the infarcted area. To attenuate cardiac fibrosis, systemic delivery of TGFβ inhibitors or anti-TGFβ antibodies represents a major approach. However, it only decreases the content of active TGFβ. It cannot inhibit TGFβ signaling pathway to prevent myofibroblast formation. Furthermore, the small organic MMP and TGFβ inhibitors have toxicity concerns. The objective of this project is to create drug delivery systems that can be specifically delivered into infarcted hearts to concurrently preserve cardiac ECM, and prevent cardiac fibrosis. Localized delivery will eliminate dose-limiting side effects. The systems will spatiotemporally release MMP-2/9 specific and non-toxic inhibitor, peptide CTTHWGFTLC (CTT), to specifically modulate local MMP-2/9 bioactivity. The systems will also gradually release a multifunctional growth factor bFGF that have anti-fibrotic and proangiogenesis functions. The preserved ECM will thus be vascularized. Vascularization is critical for cardiac ECM as otherwise its structure and composition change over time. In our preliminary work, we have created a fast gelation and degradable hydrogel-based release system capable of efficiently retaining drugs in beating hearts. The system can release CTT for 4 weeks. After being injected into infarcted hearts, the released CTT preserved collagen, increased tissue thickness, and improved cardiac function. Better than many other small organic MMP inhibitors, CTT did not induce cardiac fibrosis. Besides, CTT promoted endothelial cell migration in the presence of TGFβ that is upregulated after MI. These results demonstrate that CTT is potentially a better MMP inhibitor for cardiac therapy than those small organic inhibitors. We have further created a release system that continuously releases both CTT and bFGF. bFGF is known for its angiogenic effect. We found that bFGF is capable of inhibiting TGFβ-induced cardiac fibroblast differentiation into myofibroblast through TGFβ/Erk1/2 pathway. After 4 weeks of implantation, the CTT/bFGF release systems not only increased tissue thickness and preserved collagen composition, but also promoted the formation of a high density of capillaries and remarkably reduced cardiac fibrosis, leading to the increase of cardiac function. Based on our preliminary studies, we hypothesize that localized and spatiotemporal delivery of CTT and bFGF into infarcted hearts, will concurrently attenuate cardiac ECM degradation, vascularize the preserved ECM, and prevent cardiac fibrosis, leading to a significant increase in cardiac function. AIM 1 will test the hypothesis that optimal CTT release profiles will efficiently attenuate MMP-2 bioactivity to prevent MMP-2 mediated ECM degradation. AIM 2 will test the hypothesis that optimal bFGF release profiles will simultaneously promote endothelial cell morphogenesis and prevent cardiac fibroblasts from differentiating into myofibroblasts. AIM 3 will test the hypothesis that delivery of CTT and bFGF release systems after MI will concurrently preserve and vascularize cardiac ECM, and prevent cardiac fibrosis. This project is innovative because it creates translational drug delivery systems to establish: 1) role and efficacy of an efficient MMP-2/MMP-9 inhibitor CTT in cardiac therapy; 2) mechanism and efficacy of bFGF in inhibiting cardiac fibrosis while promoting angiogenesis; and 3) how sustained release of CTT and bFGF simultaneously achieves these three goals. The system is relatively simple and multifunctional. Therefore, it is translational.