An infusible decellularized extracellular matrix material binds to vasculature in infarcted myocardium and induces pro-reparative gene expression following acute myocardial infarction through inherent avidity and bioactive signaling

Figures

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  Figure 1.. Characterization of iECM molecular weight distribution and separation of iECM components into two fractions. a) Fabrication of iECM by 1) mincing porcine LV tissue, 2) decellularizing with SDS, 3) lyophilizing and milling into a powder, 4) partially digesting with pepsin, 5) centrifuging to pellet out large particulate matter (red arrow), 6) sterile filtering the supernatant (blue arrow) and lyophilizing for storage. SEC traces of b) complete iECM and c) iECM separated into High MW components (blue) and Low MW components (purple). d) Quantification from SEC traces showing approximate molecular weight of iECM components. d) SDS-PAGE of complete iECM, High MW components, Low MW components, and collagen type I demonstrating efficient separation of iECM into two components.

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  Figure 2.. Compositional, morphological and biochemical analysis of iECM and its components. The fractions were analyzed via label free quantification of trypsin digested mass spectrometry for protein content with the relative spectral intensity shown for the a) High MW components and b) Low MW components. The morphologies were also visualized via TEM for morphology: c) high MW and d) low MW. Biochemical assays measuring the e) thiol content, f) free amine content and g) sGAG content of iECM, low MW and high MW components. Red arrows denote nanofibrils and cyan circles denote globular and nanofibril peptides. Only p < 0.05 displayed, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

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  Figure 3.. Evaluation of binding of iECM and its components to immobilized ECM proteins and cell receptors via quartz crystal microbalance. Display of the average 3rd harmonic frequency shift values with standard deviations to show variance for binding to a) ECM components and b) cell surface receptors. Lower frequency represents greater binding of the flowed iECM or components to the immobilized protein of interest. Comparisons were only made between the same chemical conjugated coating. One-way ANOVA with Tukey’s post-hoc between plotted groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 4.. Biodistribution, retention and effect of the iECM and its fractions on vascular permeability 1 hour and 3 days post-MI. Quantified fluorescent signal of iECM and iECM fractions a) 1 hour post reperfusion and b) 3 days post reperfusion. The LV retention signal normalized to the fluorophore conjugation efficiency for complete iECM and its fractions at c) 1 hour and d) 3 days. e) Vascular permeability of infarct as determined by residual BSA in LV at 1 hour. Immunofluorescent stains of the f) iECM, g) high MW, and h) low MW in red and isolectin for vessels in green within the infarct. White arrows denote example points of colocalization with vasculature. One-way ANOVA with Tukey’s post-hoc. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Figure 5.. Single nucleus RNA sequencing integration, differential gene expression and gene ontology enrichment analysis of endothelial cells, lymphatic endothelial cells and mural cells for iECM, high MW and low MW compared to saline treatment. The UMAPs, volcano plot and enriched GO terms for iECM, high MW and low MW vs. saline for a-c) endothelial cells, d-f) lymphatic endothelial cells and g-i) mural cells suggest that all treatments resulted in angiogenic and vasculature development related responses.

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  Figure 6.. Single nucleus RNA sequencing integration, differential gene expression and gene ontology enrichment analysis of B cells, T cells, and macrophages for iECM, high MW and low MW treatment compared to saline treatment. The UMAPs, volcano plots and enriched GO terms for iECM, high MW and low MW vs. saline for B cells (a, b, c), T cells (d, e, f) and macrophages (g, h, i) respectively. All treatments generated similar responses versus saline via activation of the immune cell type of captured. Overall, the M2-macrophage response was sustained at day 7 by all treatments vs. saline.

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  Figure 7.. Single nucleus RNA sequencing integration, differential gene expression and gene ontology enrichment analysis of cardiomyocytes for iECM, high MW and low MW compared to saline treatment. The UMAPs, volcano plots and enriched GO terms for iECM, high MW and low MW vs. saline for a-c) cardiomyocytes, d-f) fibroblasts and g-i) neuronal cells. The overall response of the cardiac function related cell types was cardioprotection.

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  Figure 8.. Summary of GO enrichment for each treatment vs. saline per cell type. The enriched pathways were similar between the treatments suggesting an angiogenic response for vascular related cell types, immune cell activation, and pro-reparative phenotypes for cell types related to cardiac function.