Role of Cardiosphere‐Derived Cell Extracellular Vesicles (CDC‐EVs) in Modulating CCR2 Mediated Pro‐inflammatory Signaling following Cardiac Injury

Abstract

BackgroundFollowing myocardial injury, CCL2 (a cytokine released by the injured cardiac tissue) recruits CCR2+ monocytes to the site of injury to begin the repair process. Heightened CCL2 levels, however, over‐activate the CCR2+ mediated pro‐inflammatory response which in turn leads to poor cardiac healing and gives rise to downstream complications like congestive heart failure (CHF) and arrhythmias. Cardiosphere‐derived cell extracellular vesicles (CDC‐EVs) have been shown to down regulate this CCL2‐mediated inflammatory response by decreasing CCR2 expression on monocytes in healthy patients. We hypothesized that patients with acute myocardial infarction (MI) and decompensated heart failure would have higher levels of peripheral monocyte CCR2 expression compared with healthy controls and treatment with CDC‐EVs would suppress CCR2 expression regardless of patient presentation.MethodsA prospective single arm observational study was performed to investigate the immune response in patients presenting with cardiac injury. We recruited inpatients at Buffalo General Medical Center who met the following inclusion criteria: 1) > 18 years of age AND 2) a type I MI or 3) acute on chronic CHF exacerbation or 4) chronic compensated CHF. Subjects were excluded from the study for the following: 1) fever in last 2 days/infection, 2) is or may be pregnant, and 3) currently undergoing cancer treatment. For part 1 of the study, blood samples from 32 patients with recent MI, acute CHF, or compensated CHF (between day 1 to 3 of admission) were drawn. Human monocytes were isolated from peripheral blood using Ficoll/centrifugation followed by magnetically activated cell sorting. Isolated monocytes were incubated with either PBS, normal human dermal fibroblast‐EVs (NHDF‐EVs) or CDC‐EVs and analyzed for CCR2 expression by flow cytometry. Multi‐variate analysis was performed to investigate the relationship of CCR2 expression and specific clinical risk factors for heart disease.ResultsCDC‐EVs significantly reduced classical monocyte (CD14hiCD16‐) surface expression of CCR2 expression by 35% as quantified by flow cytometry when compared with PBS and NHDF‐EVs (p<0.005, Kruskal‐Wallis, n=31) in patients with recent MI, acute CHF exacerbation and chronic compensated CHF. This reduction was seen regardless of patients’ pre‐existing co‐morbidities like hypertension, diabetes, hyperlipidemia, chronic kidney disease, ongoing treatment with chronic medications (Aspirin, statins or ACE inhibitors), cardiac ejection fraction, prior history of coronary stents, CCR2 genotype, sex, or age. Smoking was associated with decreased response to CDC‐EV mediated CCR2 suppression when compared to PBS (linear regression model R ‐0.93, p<0.05, n=26).ConclusionsCDC‐EVs down regulated CCR2 expression in non‐smoking patients with recent MI, acute CHF, or chronic compensated CHF. This immune‐modulatory effect of CDC‐EVs may be used in the future as an adjunct to current clinical therapy to improve cardiac healing.