Preservation and Vascularization of Cardiac Extracellular Matrix after Myocardial Infarction

心肌梗死(MI)后，心脏细胞外基质(ECM)的降解主要由基质金属蛋白酶-2/9(MMP2/9)上调，心肌成纤维细胞形成后的心肌纤维化进展，使心功能进行性恶化。因此，阻断基质金属蛋白酶-2/9的生物活性，抑制肌成纤维细胞的形成将改善心功能。然而，同时实现这两个目标的理想治疗策略仍有待确定。目前，全身给药的广谱基质金属蛋白酶抑制剂在临床试验中没有显示出一致的结果。基质金属蛋白酶-2/9在心肌梗死后的病程中的表达是时空的。然而，目前的全身给药方法不能在时空上将基质金属蛋白酶抑制剂输送到梗死区。为了减轻心肌纤维化，全身应用转化生长因子β抑制剂或抗转化生长因子β抗体是一种主要的方法。但仅降低活性转化生长因子β的含量。不能通过抑制转化生长因子β信号通路来阻止肌成纤维细胞的形成。此外，小分子有机基质金属蛋白酶和转化生长因子β抑制剂也有毒性问题。该项目的目标是创造可以被特异性地输送到梗死心脏的药物输送系统，以同时保存心脏ECM，并防止心脏纤维化。局部给药将消除剂量限制的副作用。该系统将在时空上释放MMP2/9特异的无毒抑制物CTTHWGFTLC(CTT)，以特异地调节局部MMP2/9的生物活性。这些系统还将逐渐释放具有抗纤维化和促血管生成功能的多功能生长因子碱性成纤维细胞生长因子。因此，保存下来的ECM将被血管化。血管形成是心脏ECM的关键，否则它的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，能够有效地保留心脏跳动中的药物。该系统可释放CTT 4周。在注射到梗塞的心脏后，释放的CTT保留了胶原蛋白，增加了组织厚度，并改善了心功能。CTT比许多其他小的有机基质金属蛋白酶抑制剂更好，不会导致心脏纤维化。此外，在心肌梗死后表达上调的转化生长因子β的作用下，CTT可促进内皮细胞迁移。这些结果表明，CTT是一种潜在的更好的心脏治疗的基质金属蛋白酶抑制剂，比那些小的有机抑制剂。我们进一步创建了一种持续释放CTT和bFGFs的释放系统。碱性成纤维细胞生长因子以其血管生成作用而闻名。我们发现碱性成纤维细胞生长因子能够通过转化生长因子β/ERK1/2通路抑制转化生长因子β诱导的心脏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，保留了胶原成分，而且促进了毛细血管的高密度形成，明显减轻了心肌纤维化，从而提高了心功能。根据我们的初步研究，我们假设CTT和bFGF的局部和时空输送将同时减轻心脏ECM的降解，使保存的ECM血管化，并防止心脏纤维化，导致心功能的显著增加。目的1验证一种假设，即最佳的CTT释放谱将有效地减弱基质金属蛋白酶-2的生物活性，以防止基质金属蛋白酶-2介导的细胞外基质降解。目的2将验证这样一种假设，即最佳的碱性成纤维细胞生长因子释放谱将同时促进内皮细胞的形态形成并阻止心脏成纤维细胞分化为肌成纤维细胞。目的3验证心肌梗死后注射CTT和bFGF释放系统可同时保护和血管化心脏ECM，防止心肌纤维化的假说。该项目具有创新性，因为它创建了转译给药系统，以建立：1)有效的基质金属蛋白酶-2/基质金属蛋白酶-9抑制剂CTT在心脏治疗中的作用和有效性；2)碱性成纤维细胞生长因子在抑制心脏纤维化同时促进血管生成的机制和有效性；以及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，功能较多。因此，它是翻译的。

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.