Matthew C L Keith

Effects of Intracoronary Infusion of Escalating Doses of Cardiac Stem Cells in Rats With Acute Myocardial Infarction

Background—Although c-kitpos cardiac stem cells (CSCs) preserve left ventricular (LV) function and structure after myocardial infarction, CSC doses have been chosen arbitrarily, and the dose–effect relationship is unknown. Methods and Results—Rats underwent a 90-minute coronary occlusion followed by 35 days of reperfusion. Vehicle or CSCs at 5 escalating doses (0.3×106, 0.75×106, 1.5×106, 3.0×106, and 6.0×106 cells/heart) were given intracoronarily 4 h after reperfusion. The lowest dose (0.3×106) had no effect on LV function and morphology, whereas 0.75, 1.5, and 3.0×106 significantly improved regional and global LV function (echocardiography and hemodynamic studies). These 3 doses had similar effects on echocardiographic parameters (infarct wall thickening fraction, LV end-systolic and end-diastolic volumes, LV ejection fraction) and hemodynamic variables (LV end-diastolic pressure, LV dP/dtmax, preload adjusted maximal power, end-systolic elastance, preload recruitable stroke work) and produced similar reductions in apoptosis, scar size, infarct wall thinning, and LV expansion index and similar increases in viable myocardium in the risk region (morphometry). Infusion of 6.0×106 CSCs markedly increased postprocedural mortality. Green fluorescent protein and 5-bromo-2′-deoxyuridine staining indicated that persistence of donor cells and formation of new myocytes were negligible with all doses. Conclusions—Surprisingly, in this rat model of acute myocardial infarction, the dose–response relationship for intracoronary CSCs is flat. A minimal dose between 0.3 and 0.75×106 is necessary for efficacy; above this threshold, a 4-fold increase in cell number does not produce greater improvement in LV function or structure. Further increases in cell dose are harmful.

Safety of Intracoronary Infusion of 20 Million C-Kit Positive Human Cardiac Stem Cells in Pigs

Background There is mounting interest in using c-kit positive human cardiac stem cells (c-kitpos hCSCs) to repair infarcted myocardium in patients with ischemic cardiomyopathy. A recent phase I clinical trial (SCIPIO) has shown that intracoronary infusion of 1 million hCSCs is safe. Higher doses of CSCs may provide superior reparative ability; however, it is unknown if doses >1 million cells are safe. To address this issue, we examined the effects of 20 million hCSCs in pigs. Methods Right atrial appendage samples were obtained from patients undergoing cardiac surgery. The tissue was processed by an established protocol with eventual immunomagnetic sorting to obtain in vitro expanded hCSCs. A cumulative dose of 20 million cells was given intracoronarily to pigs without stop flow. Safety was assessed by measurement of serial biomarkers (cardiac: troponin I and CK-MB, renal: creatinine and BUN, and hepatic: AST, ALT, and alkaline phosphatase) and echocardiography pre- and post-infusion. hCSC retention 30 days after infusion was quantified by PCR for human genomic DNA. All personnel were blinded as to group assignment. Results Compared with vehicle-treated controls (n=5), pigs that received 20 million hCSCs (n=9) showed no significant change in cardiac function or end organ damage (assessed by organ specific biomarkers) that could be attributed to hCSCs (P>0.05 in all cases). No hCSCs could be detected in left ventricular samples 30 days after infusion. Conclusions Intracoronary infusion of 20 million c-kit positive hCSCs in pigs (equivalent to ~40 million hCSCs in humans) does not cause acute cardiac injury, impairment of cardiac function, or liver and renal injury. These results have immediate translational value and lay the groundwork for using doses of CSCs >1 million in future clinical trials. Further studies are needed to ascertain whether administration of >1 million hCSCs is associated with greater efficacy in patients with ischemic cardiomyopathy.

C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways

A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

The Epigenetic Regulator HDAC1 Modulates Transcription of a Core Cardiogenic Program in Human Cardiac Mesenchymal Stromal Cells Through a p53‐Dependent Mechanism

Histone deacetylase (HDAC) regulation is an essential process in myogenic differentiation. Inhibitors targeting the activity of specific HDAC family members have been shown to enhance the cardiogenic differentiation capacity of discrete progenitor cell types; a key property of donor cell populations contributing to their afforded benefits in cardiac cell therapy applications. The influence of HDAC inhibition on cardiac‐derived mesenchymal stromal cell (CMC) transdifferentiation or the role of specific HDAC family members in dictating cardiovascular cell lineage specification has not been investigated. In the current study, the consequences of HDAC inhibition on patient‐derived CMC proliferation, cardiogenic program activation, and cardiovascular differentiation/cell lineage specification were investigated using pharmacologic and genetic targeting approaches. Here, CMCs exposed to the pan‐HDAC inhibitor sodium butyrate exhibited induction of a cardiogenic transcriptional program and heightened expression of myocyte and endothelial lineage‐specific markers when coaxed to differentiate in vitro. Further, shRNA knockdown screens revealed CMCs depleted of HDAC1 to promote the induction of a cardiogenic transcriptional program characterized by enhanced expression of cardiomyogenic‐ and vasculogenic‐specific markers, a finding which depended on and correlated with enhanced acetylation and stabilization of p53. Cardiogenic gene activation and elevated p53 expression levels observed in HDAC1‐depleted CMCs were associated with improved aptitude to assume a cardiomyogenic/vasculogenic cell‐like fate in vitro. These results suggest that HDAC1 depletion‐induced p53 expression alters CMC cell fate decisions and identify HDAC1 as a potential exploitable target to facilitate CMC‐mediated myocardial repair in ischemic cardiomyopathy. Stem Cells 2016;34:2916–2929

Femoral arterial closure using ProGlide® is more efficacious and cost-effective when ambulating early following cardiac catheterization

Objective This was a prospective, single-center study evaluating the efficacy and cost-effectiveness of early ambulation (within 30 min) following femoral artery closure with the ProGlide® suture-mediated vascular closure device (PD) in patients undergoing diagnostic cardiac catheterization compared with manual compression. Background It is unclear whether early ambulation with ProGlide is safe or is associated with patient satisfaction and cost savings as compared with manual compression (MC). Methods and results Inclusion criteria were met in 170 patients (85 PD and 85 MC patients). Patients ambulated 20 ft. within 30 min (PD) or after the requisite 4 h recumbent time (MC) if feasible. Primary endpoint was time-to-ambulation (TTA) following device closure. We also directly compared the safety of closure, times-to-hemostasis (TTH), -ambulation (TTA) and -discharge (TTD) with MC and, using a fully allocated cost model, performed cost analysis for both strategies. Multivariate analysis was used to determine predictors of patient satisfaction. The primary endpoint of safe, early ambulation was achieved following closure (mean of 27.1 ± 14.9 min; 95% confidence interval [CI] 25.2–30.2). Predictors of patient satisfaction in the PD group were absence of pain during closure, decreased TTA, and drastic reductions in TTD; the latter contributed indirectly to significant cost savings in the PD group (1250.3 ± 146.4 vs. 2248.1 ± 910.2 dollars, respectively; P < 0.001) and incremental cost savings by strategy also favored closure over MC (

Histone Deacetylase 1 Depletion Activates Human Cardiac Mesenchymal Stromal Cell Proangiogenic Paracrine Signaling Through a Mechanism Requiring Enhanced Basic Fibroblast Growth Factor Synthesis and Secretion

84,807). Conclusions ProGlide is safe and effective for femoral artery closure in patients who ambulate within 30 min after cardiac catheterization; translating into improved patient satisfaction and substantial cost savings.

Response to Letter Regarding Article, “Cell Therapy for Heart Failure: A Comprehensive Overview of Experimental and Clinical Studies, Current Challenges, and Future Directions”

Background Cardiac mesenchymal cell (CMC) administration improves cardiac function in animal models of heart failure. Although the precise mechanisms remain unclear, transdifferentiation and paracrine signaling are suggested to underlie their cardiac reparative effects. We have shown that histone deacetylase 1 (HDAC1) inhibition enhances CMC cardiomyogenic lineage commitment. Here, we investigated the impact of HDAC1 on CMC cytokine secretion and associated paracrine‐mediated activities on endothelial cell function. Methods and Results CMCs were transduced with shRNA constructs targeting HDAC1 (shHDAC1) or nontarget (shNT) control. Cytokine arrays were used to assess the expression of secreted proteins in conditioned medium (CM) from shHDAC1 or shNT‐transduced CMCs. In vitro functional assays for cell proliferation, protection from oxidative stress, cell migration, and tube formation were performed on human endothelial cells incubated with CM from the various treatment conditions. CM from shHDAC1‐transduced CMCs contained more cytokines involved in cell growth/differentiation and more efficiently promoted endothelial cell proliferation and tube formation compared with CM from shNT. After evaluating key cytokines previously implicated in cell‐therapy–mediated cardiac repair, we found that basic fibroblast growth factor was significantly upregulated in shHDAC1‐transduced CMCs. Furthermore, shRNA‐mediated knockdown of basic fibroblast growth factor in HDAC1‐depleted CMCs inhibited the effects of shHDAC1 CM in promoting endothelial proliferation and tube formation—indicating that HDAC1 depletion activates CMC proangiogenic paracrine signaling in a basic fibroblast growth factor–dependent manner. Conclusions These results reveal a hitherto unknown role for HDAC1 in the modulation of CMC cytokine secretion and implicate the targeted inhibition of HDAC1 in CMCs as a means to enhance paracrine‐mediated neovascularization in cardiac cell therapy applications.

“String Theory” of c-kitpos Cardiac Cells: A New Paradigm Regarding the Nature of These Cells That May Reconcile Apparently Discrepant Results

We welcome the opportunity to comment on the size and number of the endomyocardial biopsies that were taken in the CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trial.1 In their letter, Makkar et al indicate that they generally harvested 10 specimens per patient. Because the total tissue obtained weighed 276 mg, the average weight was ≈28 mg per specimen (or greater if 453 mg, ie, the 10 specimens weighed, on average, >45 mg each. These values are several fold higher than those which are generally achieved in clinical practice. The 2 bioptomes most commonly used in clinical practice are manufactured by Cordis (J&J) and Argon. According to the Cordis catalog (http://www.cordis.com/emea/pl/product/biopsy-forceps …