Hydrogel delivery of purinergic enzymes improves cardiac ischemia/reperfusion injury.

RATIONALE: The innate immune response contributes to cardiac injury in myocardial ischemia/reperfusion (MI/R). Neutrophils are an important early part of the innate immune response to MI/R. Adenosine, an endogenous purine, is a known innate immune modulator and inhibitor of neutrophil activation. However, its delivery to the heart is limited by its short half-life (<30 s) and off-target side effects. CD39 and CD73 are anti-inflammatory homeostatic enzymes that can generate adenosine from phosphorylated adenosine substrate such as ATP released from injured tissue. OBJECTIVE: We hypothesize that hydrogel-delivered CD39 and CD73 target the local early innate immune response, reduce neutrophil activation, and preserve cardiac function in MI/R injury. METHODS AND RESULTS: We engineered a poly(ethylene) glycol (PEG) hydrogel loaded with the adenosine-generating enzymes CD39 and CD73. We incubated the hydrogels with neutrophils in vitro and showed a reduction in hydrogen peroxide production using Amplex Red. We demonstrated availability of substrate for the enzymes in the myocardium in MI/R by LC/MS, and tested release kinetics from the hydrogel. On echocardiography, global longitudinal strain (GLS) was preserved in MI/R hearts treated with the loaded hydrogel. Delivery of purinergic enzymes via this synthetic hydrogel resulted in lower innate immune infiltration into the myocardium post-MI/R, decreased markers of macrophage and neutrophil activation (NETosis), and decreased leukocyte-platelet complexes in circulation. CONCLUSIONS: In a rat model of MI/R injury, CD39 and CD73 delivered via a hydrogel preserve cardiac function by modulating the innate immune response.

A Hydrogel Strategy to Augment Tissue Adenosine to Improve Hindlimb Perfusion.

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Adenosine Production by Biomaterial-Supported Mesenchymal Stromal Cells Reduces the Innate Inflammatory Response in Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: During myocardial ischemia/reperfusion (MI/R) injury, there is extensive release of immunogenic metabolites that activate cells of the innate immune system. These include ATP and AMP, which upregulate chemotaxis, migration, and effector function of early infiltrating inflammatory cells. These cells subsequently drive further tissue devitalization. Mesenchymal stromal cells (MSCs) are a potential treatment modality for MI/R because of their powerful anti-inflammatory capabilities; however, the manner in which they regulate the acute inflammatory milieu requires further elucidation. CD73, an ecto-5'-nucleotidase, may be critical in regulating inflammation by converting pro-inflammatory AMP to anti-inflammatory adenosine. We hypothesized that MSC-mediated conversion of AMP into adenosine reduces inflammation in early MI/R, favoring a micro-environment that attenuates excessive innate immune cell activation and facilitates earlier cardiac recovery. METHODS AND RESULTS: Adult rats were subjected to 30 minutes of MI/R injury. MSCs were encapsulated within a hydrogel vehicle and implanted onto the myocardium. A subset of MSCs were pretreated with the CD73 inhibitor, α,ß-methylene adenosine diphosphate, before implantation. Using liquid chromatography/mass spectrometry, we found that MSCs increase myocardial adenosine availability following injury via CD73 activity. MSCs also reduce innate immune cell infiltration as measured by flow cytometry, and hydrogen peroxide formation as measured by Amplex Red assay. These effects were dependent on MSC-mediated CD73 activity. Finally, through echocardiography we found that CD73 activity on MSCs was critical to optimal protection of cardiac function following MI/R injury. CONCLUSIONS: MSC-mediated conversion of AMP to adenosine by CD73 exerts a powerful anti-inflammatory effect critical for cardiac recovery following MI/R injury.