Fatih Arslan

Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury.

Human ESC-derived mesenchymal stem cell (MSC)-conditioned medium (CM) was previously shown to mediate cardioprotection during myocardial ischemia/reperfusion injury through large complexes of 50-100 nm. Here we show that these MSCs secreted 50- to 100-nm particles. These particles could be visualized by electron microscopy and were shown to be phospholipid vesicles consisting of cholesterol, sphingomyelin, and phosphatidylcholine. They contained coimmunoprecipitating exosome-associated proteins, e.g., CD81, CD9, and Alix. These particles were purified as a homogeneous population of particles with a hydrodynamic radius of 55-65 nm by size-exclusion fractionation on a HPLC. Together these observations indicated that these particles are exosomes. These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.

Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury.

We have previously identified exosomes as the paracrine factor secreted by mesenchymal stem cells. Recently, we found that the key features of reperfusion injury, namely loss of ATP/NADH, increased oxidative stress and cell death were underpinned by proteomic deficiencies in ischemic/reperfused myocardium, and could be ameliorated by proteins in exosomes. To test this hypothesis in vivo, mice (C57Bl6/J) underwent 30 min ischemia, followed by reperfusion (I/R injury). Purified exosomes or saline was administered 5 min before reperfusion. Exosomes reduced infarct size by 45% compared to saline treatment. Langendorff experiments revealed that intact but not lysed exosomes enhanced viability of the ischemic/reperfused myocardium. Exosome treated animals exhibited significant preservation of left ventricular geometry and contractile performance during 28 days follow-up. Within an hour after reperfusion, exosome treatment increased levels of ATP and NADH, decreased oxidative stress, increased phosphorylated-Akt and phosphorylated-GSK-3β, and reduced phosphorylated-c-JNK in ischemic/reperfused hearts. Subsequently, both local and systemic inflammation were significantly reduced 24h after reperfusion. In conclusion, our study shows that intact exosomes restore bioenergetics, reduce oxidative stress and activate pro-survival signaling, thereby enhancing cardiac function and geometry after myocardial I/R injury. Hence, mesenchymal stem cell-derived exosomes are a potential adjuvant to reperfusion therapy for myocardial infarction.

Prognostic Value of Fractional Flow Reserve: Linking Physiologic Severity to Clinical Outcomes

BACKGROUND Fractional flow reserve (FFR) has become an established tool for guiding treatment, but its graded relationship to clinical outcomes as modulated by medical therapy versus revascularization remains unclear. OBJECTIVES The study hypothesized that FFR displays a continuous relationship between its numeric value and prognosis, such that lower FFR values confer a higher risk and therefore receive larger absolute benefits from revascularization. METHODS Meta-analysis of study- and patient-level data investigated prognosis after FFR measurement. An interaction term between FFR and revascularization status allowed for an outcomes-based threshold. RESULTS A total of 9,173 (study-level) and 6,961 (patient-level) lesions were included with a median follow-up of 16 and 14 months, respectively. Clinical events increased as FFR decreased, and revascularization showed larger net benefit for lower baseline FFR values. Outcomes-derived FFR thresholds generally occurred around the range 0.75 to 0.80, although limited due to confounding by indication. FFR measured immediately after stenting also showed an inverse relationship with prognosis (hazard ratio: 0.86, 95% confidence interval: 0.80 to 0.93; p < 0.001). An FFR-assisted strategy led to revascularization roughly half as often as an anatomy-based strategy, but with 20% fewer adverse events and 10% better angina relief. CONCLUSIONS FFR demonstrates a continuous and independent relationship with subsequent outcomes, modulated by medical therapy versus revascularization. Lesions with lower FFR values receive larger absolute benefits from revascularization. Measurement of FFR immediately after stenting also shows an inverse gradient of risk, likely from residual diffuse disease. An FFR-guided revascularization strategy significantly reduces events and increases freedom from angina with fewer procedures than an anatomy-based strategy.

Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction

Recent studies suggest that the therapeutic effects of stem cell transplantation following myocardial infarction (MI) are mediated by paracrine factors. One of the main goals in the treatment of ischemic heart disease is to stimulate vascular repair mechanisms. Here, we sought to explore the therapeutic angiogenic potential of mesenchymal stem cell (MSC) secretions. Human MSC secretions were collected as conditioned medium (MSC-CM) using a clinically compliant protocol. Based on proteomic and pathway analysis of MSC-CM, an in vitro assay of HUVEC spheroids was performed identifying the angiogenic properties of MSC-CM. Subsequently, pigs were subjected to surgical left circumflex coronary artery ligation and randomized to intravenous MSC-CM treatment or non-CM (NCM) treatment for 7 days. Three weeks after MI, myocardial capillary density was higher in pigs treated with MSC-CM (645 ± 114 vs 981 ± 55 capillaries/mm(2); P = 0.021), which was accompanied by reduced myocardial infarct size and preserved systolic and diastolic performance. Intravenous MSC-CM treatment after myocardial infarction increases capillary density and preserves cardiac function, probably by increasing myocardial perfusion.

Myocardial Ischemia/Reperfusion Injury Is Mediated by Leukocytic Toll-Like Receptor-2 and Reduced by Systemic Administration of a Novel Anti–Toll-Like Receptor-2 Antibody

Background— Reperfusion therapy for myocardial infarction is hampered by detrimental inflammatory responses partly via Toll-like receptor (TLR) activation. Targeting TLR signaling may optimize reperfusion therapy and enhance cell survival and heart function after myocardial infarction. Here, we evaluated the role of TLR2 as a therapeutic target using a novel monoclonal anti-TLR2 antibody. Method and Results— Mice underwent 30 minutes of ischemia followed by reperfusion. Compounds were administered 5 minutes before reperfusion. Cardiac function and dimensions were assessed at baseline and 28 days after infarction with 9.4-T mouse magnetic resonance imaging. Saline and IgG isotype treatment resulted in 34.5±3.3% and 31.4±2.7% infarction, respectively. Bone marrow transplantation experiments between wild-type and TLR2-null mice revealed that final infarct size is determined by circulating TLR2 expression. A single intravenous bolus injection of anti-TLR2 antibody reduced infarct size to 18.9±2.2% (P=0.001). Compared with saline-treated mice, anti-TLR2–treated mice exhibited less expansive remodeling (end-diastolic volume 68.2±2.5 versus 76.8±3.5 &mgr;L; P=0.046) and preserved systolic performance (ejection fraction 51.0±2.1% versus 39.9±2.2%, P=0.009; systolic wall thickening 3.3±6.0% versus 22.0±4.4%, P=0.038). Anti-TLR2 treatment significantly reduced neutrophil, macrophage, and T-lymphocyte infiltration. Furthermore, tumor necrosis factor-&agr;, interleukin-1&agr;, granulocyte macrophage colony-stimulating factor, and interleukin-10 were significantly reduced, as were phosphorylated c-jun N-terminal kinase, phosphorylated p38 mitogen-activated protein kinase, and caspase 3/7 activity levels. Conclusions— Circulating TLR2 expression mediates myocardial ischemia/reperfusion injury. Antagonizing TLR2 just 5 minutes before reperfusion reduces infarct size and preserves cardiac function and geometry. Anti-TLR2 therapy exerts its action by reducing leukocyte influx, cytokine production, and proapoptotic signaling. Hence, monoclonal anti-TLR2 antibody is a potential candidate as an adjunctive for reperfusion therapy in patients with myocardial infarction.

Proteolytic Potential of the MSC Exosome Proteome: Implications for an Exosome-Mediated Delivery of Therapeutic Proteasome

Mesenchymal stem cells (MSCs) are used in many of the current stem cell-based clinical trials and their therapeutic efficacy has increasingly been attributed to secretion of paracrine factors. We have previously demonstrated that a therapeutic constituent of this secretion is exosome, a secreted bilipid membrane vesicle of ~50–100 nm with a complex cargo that is readily internalized by H9C2 cardiomyocytes. It reduces infarct size in a mouse model of myocardial ischemia/reperfusion (MI/R) injury. We postulate that this therapeutic efficacy is derived from the synergy of a select permutation of individual exosome components. To identify protein candidates in this permutation, the proteome was profiled and here we identified 20S proteasome as a protein candidate. Mass spectrometry analysis detected all seven α and seven β chains of the 20S proteasome, and also the three beta subunits of “immunoproteasome” with a very high confidence level. We demonstrated that a functional proteasome copurified with MSC exosomes with a density of 1.10–1.18 g/mL, and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Circulating proteasomes in human blood also copurified with exosomes. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents to ameliorate tissue damage.

Lack of Fibronectin-EDA Promotes Survival and Prevents Adverse Remodeling and Heart Function Deterioration After Myocardial Infarction

Rationale: The extracellular matrix may induce detrimental inflammatory responses on degradation, causing adverse cardiac remodeling and heart failure. The extracellular matrix protein fibronectin-EDA (EIIIA; EDA) is upregulated after tissue injury and may act as a “danger signal” for leukocytes to cause adverse cardiac remodeling after infarction. Objective: In the present study, we evaluated the role of EDA in regulation of postinfarct inflammation and repair after myocardial infarction. Methods and Results: Wild-type and EDA−/− mice underwent permanent ligation of the left anterior coronary artery. Despite equal infarct size between groups (38.2±4.6% versus 38.2±2.9% of left ventricle; P=0.985), EDA−/− mice exhibited less left ventricular dilatation and enhanced systolic performance compared with wild-type mice as assessed by serial cardiac MRI measurements. In addition, EDA−/− mice exhibited reduced fibrosis of the remote area without affecting collagen production, cross-linking, and deposition in the infarct area. Subsequently, ventricular contractility and relaxation was preserved in EDA−/−. At tissue level, EDA−/− mice showed reduced inflammation, metalloproteinase 2 and 9 activity, and myofibroblast transdifferentiation. Bone marrow transplantation experiments revealed that myocardium-induced EDA and not EDA from circulating cells regulates postinfarct remodeling. Finally, the absence of EDA reduced monocyte recruitment as well as monocytic Toll-like receptor 2 and CD49d expression after infarction. Conclusions: Our study demonstrated that parenchymal fn-EDA plays a critical role in adverse cardiac remodeling after infarction. Absence of fn-EDA enhances survival and cardiac performance by modulating matrix turnover and inflammation via leukocytes and fibroblasts after infarction.

Derivation and characterization of human fetal MSCs: An alternative cell source for large-scale production of cardioprotective microparticles

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10(16-19) cells or 10(7-10) doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r(2)>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50-65 nm. This purified population of microparticles was cardioprotective at approximately 1/10 dosage of the crude secretion.

Intracoronary Infusion of Allogeneic Mesenchymal Precursor Cells Directly After Experimental Acute Myocardial Infarction Reduces Infarct Size, Abrogates Adverse Remodeling, and Improves Cardiac Function

Rationale: Mesenchymal precursor cells (MPCs) are a specific Stro-3+ subpopulation of mesenchymal stem cells isolated from bone marrow. MPCs exert extensive cardioprotective effects, and are considered to be immune privileged. Objective: This study assessed the safety, feasibility, and efficacy of intracoronary delivery of allogeneic MPCs directly after acute myocardial infarction in sheep. Methods and Results: Initially, intracoronary delivery conditions were optimized in 20 sheep. These conditions were applied in a randomized study of 68 sheep with an anterior acute myocardial infarction. Coronary flow was monitored during MPC infusion, and cardiac function was assessed using invasive hemodynamics and echocardiography at baseline and during 8 weeks follow-up. Coronary flow remained within thrombolysis in myocardial infarction III definitions in all sheep during MPC infusion. Global left ventricular ejection fraction as measured by pressure–volume loop analysis deteriorated in controls to 40.7±2.6% after 8 weeks. In contrast, MPC treatment improved cardiac function to 52.8±0.7%. Echocardiography revealed significant improvement of both global and regional cardiac functions. Infarct size decreased by 40% in treated sheep, whereas infarct and border zone thickness were enhanced. Left ventricular adverse remodeling was abrogated by MPC therapy, resulting in a marked reduction of left ventricular volumes. Blood vessel density increased by >50% in the infarct and border areas. Compensatory cardiomyocyte hypertrophy was reduced in border and remote segments, accompanied by reduced collagen deposition and apoptosis. No microinfarctions in remote myocardial segments or histological abnormalities in unrelated organs were found. Conclusions: Intracoronary infusion of allogeneic MPCs is safe, feasible, and markedly effective in a large animal model of acute myocardial infarction.

Treatment with OPN-305, a humanized anti-toll-like receptor-2 antibody, reduces myocardial ischemia/reperfusion injury in pigs

Background— Toll-like receptor (TLR)-2 is an important mediator of innate immunity and ischemia/reperfusion-induced cardiac injury. We have previously shown that TLR2 inhibition reduces infarct size and improves cardiac function in mice. However, the therapeutic efficacy of a clinical grade humanized anti-TLR2 antibody, OPN-305, in a large-animal model remained to be addressed. Methods and Results— Pigs (n=38) underwent 75 minutes ischemia followed by 24 hours of reperfusion. Saline or OPN-305 (12.5, 6.25, or 1.56 mg/kg) was infused intravenously 15 minutes before reperfusion. Cardiac function and geometry were assessed by echocardiography. Infarct size was calculated as the percentage of the area at risk and by serum Troponin-I levels. Flow cytometry analysis revealed specific binding of OPN-305 to porcine TLR2. In vivo, OPN-305 exhibited a secondary half-life of 8±2 days. Intravenous administration of OPN-305 before reperfusion significantly reduced infarct size (45% reduction, P=0.041) in a dose-dependent manner. In addition, pigs treated with OPN-305 exhibited a significant preservation of systolic performance in a dose-dependent fashion, whereas saline treatment completely diminished the contractile performance of the ischemic/reperfused myocardium. Conclusions— OPN-305 significantly reduces infarct size and preserves cardiac function in pigs after ischemia/reperfusion injury. Hence, OPN-305 is a promising adjunctive therapeutic for patients with acute myocardial infarction.