Alok R. Amraotkar

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.

Identification of a plasma metabolomic signature of thrombotic myocardial infarction that is distinct from non-thrombotic myocardial infarction and stable coronary artery disease

Aims Current non-invasive diagnostics for acute myocardial infarction (MI) identify myocardial necrosis rather than the primary cause and therapeutic target—plaque disruption and resultant thrombosis. The aim of this study was to identify changes specific to plaque disruption and pathological thrombosis that are distinct from acute myocardial necrosis. Methods and results We quantified 1,032 plasma metabolites by mass spectrometry in 11 thrombotic MI, 12 non-thrombotic MI, and 15 stable coronary artery disease (CAD) subjects at two acute phase (time of catheterization [T0], six hours [T6]) and one quiescent (>3 months follow-up) time points. A statistical classifier was constructed utilizing baseline (T0) abundances of a parsimonious set of 17 qualifying metabolites. Qualifying metabolites were those that demonstrated a significant change between the quiescent phase and the acute phase and that were distinct from any change seen in non-thrombotic MI or stable CAD subjects. Classifier performance as estimated by 10-fold cross-validation was suggestive of high sensitivity and specificity for differentiating thrombotic from non-thrombotic MI and stable CAD subjects at presentation. Nineteen metabolites demonstrated an intra-subject change from time of acute thrombotic MI presentation to the quiescent state that was distinct from any change measured in both the non-thrombotic MI and stable CAD subjects undergoing cardiac catheterization over the same time course (false discovery rate <5%). Conclusions We have identified a candidate metabolic signature that differentiates acute thrombotic MI from quiescent state after MI, from acute non-thrombotic MI, and from stable CAD. Further validation of these metabolites is warranted given their potential as diagnostic biomarkers and novel therapeutic targets for the prevention or treatment of acute MI.

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

SERUM PROLIDASE ACTIVITY IN STABLE CORONARY ARTERY DISEASE AND ACUTE MYOCARDIAL INFARCTION

Background: Collagen is a major determinant of atherosclerotic plaque stability and prolidase is a rate-limiting enzyme in the breakdown and formation of collagen. We sought to determine prolidase activity at the time of acute myocardial infarction (MI) as compared to quiescent state and to patients

Circulating Prolidase Activity in Patients with Myocardial Infarction

Background Collagen is a major determinant of atherosclerotic plaque stability. Thus, identification of differences in enzymes that regulate collagen integrity could be useful for predicting susceptibility to atherothrombosis or for diagnosing plaque rupture. In this study, we sought to determine whether prolidase, the rate-limiting enzyme of collagen turnover, differs in human subjects with acute myocardial infarction (MI) versus those with stable coronary artery disease (CAD). Methods We measured serum prolidase activity in 15 patients with stable CAD and 49 patients with acute MI, of which a subset had clearly defined thrombotic MI (n = 22) or non-thrombotic MI (n = 12). Prolidase activity was compared across study time points (at cardiac catheterization, T0; 6 h after presentation, T6; and at a quiescent follow-up, Tf/u) in acute MI and stable CAD subjects. We performed subgroup analyses to evaluate prolidase activity in subjects presenting with acute thrombotic versus non-thrombotic MI. Results Although prolidase activity was lower at T0 and T6 versus the quiescent phase in acute MI and stable CAD subjects (p < 0.0001), it was not significantly different between acute MI and stable CAD subjects at any time point (T0, T6, and Tf/u) or between thrombotic and non-thrombotic MI groups. Preliminary data from stratified analyses of a small number of diabetic subjects (n = 8) suggested lower prolidase activity in diabetic acute MI subjects compared with non-diabetic acute MI subjects (p = 0.02). Conclusion Circulating prolidase is not significantly different between patients with acute MI and stable CAD or between patients with thrombotic and non-thrombotic MI. Further studies are required to determine if diabetes significantly affects prolidase activity and how this might relate to the risk of MI.

Platelet Count and Mean Platelet Volume at the Time of and After Acute Myocardial Infarction

Platelet count has been shown to be lower and mean platelet volume (MPV) to be higher in acute myocardial infarction (MI). However, it is not known whether these changes persist post-MI or if these measures are able to distinguish between acute thrombotic and non-thrombotic MI. Platelet count and MPV were measured in 80 subjects with acute MI (thrombotic and non-thrombotic) and stable coronary artery disease (CAD) at cardiac catheterization (acute phase) and at >3-month follow-up (quiescent phase). Subjects were stratified using stringent clinical, biochemical, histological, and angiographic criteria. Outcome measures were compared between groups by analysis of variance. Forty-seven subjects met criteria for acute MI with clearly defined thrombotic (n = 22) and non-thrombotic (n = 12) subsets. Fourteen subjects met criteria for stable CAD. No significant difference was observed in platelet count between subjects with acute MI and stable CAD at the acute or quiescent phase. MPV was higher in acute MI (9.18 ± 1.21) compared to stable CAD (8.13 ± 0.66; P = 0.003) at the acute phase but not at the quiescent phase (8.48 ± 0.58 vs 8.94 ± 1.42; P = 0.19). No difference in platelet count or MPV was detected between thrombotic and non-thrombotic subsets at acute or quiescent phases. The power to detect differences in these measures between thrombotic and non-thrombotic subsets was 58%. Higher MPV at the time of acute MI is not observed by 3 months post-MI (quiescent phase). Platelet count and MPV do not differ in subjects with thrombotic versus non-thrombotic MI. Further investigation is warranted to evaluate the utility of these measures in the diagnosis of acute MI.

Presence of multiple coronary angiographic characteristics for the diagnosis of acute coronary thrombus

BACKGROUND Coronary angiography is frequently employed to aid in the diagnosis of acute coronary thrombosis, but there is limited data to support its efficacy. The aim of the study was to evaluate sensitivity and specificity of five commonly used angiographic characteristics for diagnosis of acute coronary thrombosis: Ambrose complex lesion morphology; spherical, ovoid, or irregular filling defect; abrupt vessel cutoff; intraluminal staining; and any coronary filling defect. METHODS Coronary angiography of 80 acute myocardial infarction or stable coronary artery disease subjects were assessed in blinded fashion, for the presence or absence of five angiographic characteristics. Only lesions of ≥ 10% stenosis were included in the analysis. Presence or absence of each angiographic characteristic was compared between lesions with or without the following study defined outcomes: 1) histologically confirmed thrombus, 2) highly probable thrombus, and 3) highly unlikely thrombus. RESULTS A total of 323 lesions were evaluated. All studied angiographic characteristics were associated with histologically confirmed and highly probable thrombotic lesions vs. lesions not meeting criteria for these outcomes (p < 0.03), except for complex Ambrose morphology which was not associated with any of the study outcomes (p > 0.05). Specificity for identifying histologically confirmed or highly probable thrombotic lesion was high (92-100%), especially for spherical, ovoid, or irregular filling defect (99-100%) and intraluminal staining (99%). Sensitivity for identification of histologically confirmed or highly probable thrombotic lesions was low for all tested angiographic characteristics (17-60%). CONCLUSIONS The presence of spherical, ovoid, or irregular filling defect or intraluminal staining was highly suggestive of coronary thrombus. However, none of the evaluated angiographic characteristics were useful for ruling out the presence of coronary thrombus. If confirmed in an independent cohort, these angiographic characteristic will be of significant value in confirming the diagnosis of acute coronary thrombosis.

Rapid Extracorporeal Membrane Oxygenation Overcomes Fulminant Myocarditis Induced by 5‑Fluorouracil

Fulminant myocarditis is a rare but potentially life-threatening illness caused by 5-fluorouracil cardiotoxicity. Data supporting the use of extracorporeal membrane oxygenation for the treatment of fulminant myocarditis are limited. A 49-year-old, previously healthy white man, recently diagnosed with anal squamous cell carcinoma, developed severe chest pain hours after completing his first 96-hour intravenous 5-fluorouracil treatment. Over a period of 3 days from onset of symptoms, the patient developed cardiogenic shock secondary to fulminant myocarditis induced by 5-fluorouracil cardiotoxicity. This required emergency initiation of extracorporeal membrane oxygenation. The patients systolic function recovered by day 5, and on the 17th day he was discharged in hemodynamically stable condition, without symptoms of heart failure. This case shows the importance of prompt recognition of cardiogenic shock secondary to 5-fluorouracil-induced myocarditis and how the immediate initiation of extracorporeal membrane oxygenation can restore adequate tissue perfusion, leading to myocardial recovery and ultimately the survival of the patient.

EVALUATION OF MULTIPLE CORONARY ANGIOGRAPHIC CHARACTERISTICS FOR THE DIAGNOSIS OF ACUTE CORONARY THROMBUS

Coronary angiography is often employed for the diagnosis of acute coronary syndrome; however, little data exists on its accuracy for this diagnosis. We evaluated five angiographic characteristics: Ambrose morphology; spherical, ovid or irregular filling defect; abrupt vessel cutoff; intraluminal