Evaluation of the Tissue Specific Effects of an Infusible Decellularized Extracellular Matrix Material in the Treatment of Acute Myocardial Infarction

Introduction: : Current treatments for myocardial infarction (MI) and ischemia reperfusion (I/R) injury fail to address the overreactive inflammatory response, resulting in negative left ventricular (LV) remodeling and heart failure. We recently developed an infusible ECM material (iECM) derived from porcine LV myocardium, which in contrast with other decellularized ECM materials, can be delivered through intracoronary infusion for the treatment of acute MI. This material localizes to the leaky vasculature of the infarct and exerts immunomodulatory and pro-regenerative effects, resulting in long term improvements in cardiac function. Decellularized extracellular matrix (ECM) biomaterials are a promising class of materials for treating inflammation and promoting tissue regeneration due to their potent bioactivity arising from their complex mixture of components derived from their source tissue. While previous work has demonstrated tissue specific biological effects with regard to the source tissue of decellularized ECM materials, these have only been done with injectable and implantable materials such as hydrogels and coatings. To elucidate the specific mechanism of action of iECM, we manufactured iECM from alternative tissue sources (lung, skeletal muscle, and purified collagen) to alter the composition of iECM, allowing us to determine whether iECM’s localization and therapeutic effects are due to the specific source tissue or if therapeutic effects are conserved between source tissue types.

Materials and

Methods: : iECM variants were made by first decellularizing porcine LV myocardium, lung, and skeletal muscle (SKM) using sodium dodecyl sulfate, with type 1 collagen purchased already purified from bovine tendon. Decellularized materials were milled into a fine powder and partially digested with pepsin for 48 hours, with the starting material analyzed using global mass spectrometry to determine the initial protein composition. Following digestion, solutions were centrifuged to separate large particulates and the supernatants were filtered through a 0.2 µm filter. Protein compositions of iECM variants were analyzed using size exclusion chromatography (SEC) and SDS-PAGE. Morphology of iECM variants was assessed using transmission electron microscopy (TEM). iECM variants were tested for their biochemical properties, including amounts of free amines, free thiols, and sulfated glycosaminoglycans. To determine retention in the infarcted myocardium and the effect of iECM tissue source on vascular permeability following MI, iECM variants were delivered through a simulated intracoronary infusion immediately following reperfusion in a rat model of MI. 30 minutes post reperfusion, 1 mL of fluorescently labeled bovine serum albumin (BSA) was administered through a tail vein injection to act as a tracer of vascular permeability. 30 minutes later, rats were euthanized, perfused with PBS, and hearts were collected for analysis through near infrared imaging to determine localization of iECM variants and presence of BSA in the LV.

Results, Conclusions, and Discussions::

Results: Proteomic analysis of initial decellularized material revealed differences in the initial protein content of decellularized tissues, with decellularized lung having the greatest variety of proteins, followed by cardiac, SKM, and collagen (figure 1a). All iECM variants retained the same protein distribution, with three major peaks corresponding to high molecular weight (HMW) (MW > 1000 kDa), medium molecular weight (MW = ~60 kDa), and peptide (MW = ~4 kDa) fractions, with the relative ratio of these components differing between variants (figure 1b). HMW components were seen in all variants using TEM, with all variants having collagen like fibrils. Decellularization was complete for all batches of iECM, as seen through residual double stranded DNA contents being less than 3 ng/mg of material. All batches had comparable amounts of free amines (figure 1c), though lung derived iECM contained significantly more free thiols (figure 1d) and sulfated glycosaminoglycans (figure 1e) than other batches. All iECM variants localized preferentially to the infarcted LV myocardium, with minimal signal seen in healthy portions of the heart (figure 1f,g). Similarly, all iECM variants significantly reduced vascular permeability 1 hour post reperfusion to similar degrees, with cardiac iECM showing greater decreases than other variants (figure 1h).

Conclusions: Our infusible formulation of decellularized ECM, iECM, can be made from other decellularized tissues, including cardiac, SKM, and lung tissues as well as purified type 1 collagen. The general protein distribution between iECM variations is the same, while differences in the protein composition of the initial decellularized material translated to differences in the relative proteins distribution and biochemical properties. Furthermore, once delivered immediately after reperfusion in a rat model of MI, all iECM variants localized preferentially to the infarcted myocardium, suggesting that the ability to localize is due to components shared between all variants. While all variants were able to reduce vascular permeability, cardiac iECM appeared to reduce it to the greatest degree, despite comparable retention. Ongoing work will explore differences in biological response due to differences in iECM tissue source, specifically looking at effects on inflammation and myocardial repair.

Acknowledgements and/or References (Optional)::