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.

A New Method to Stabilize c-kit Expression in Reparative Cardiac Mesenchymal Cells

Cell therapy improves cardiac function. Few cells have been investigated more extensively or consistently shown to be more effective than c-kit sorted cells; however, c-kit expression is easily lost during passage. Here, our primary goal was to develop an improved method to isolate c-kitpos cells and maintain c-kit expression after passaging. Cardiac mesenchymal cells (CMCs) from wild-type mice were selected by polystyrene adherence properties. CMCs adhering within the first hours are referred to as rapidly adherent (RA); CMCs adhering subsequently are dubbed slowly adherent (SA). Both RA and SA CMCs were c-kit sorted. SA CMCs maintained significantly higher c-kit expression than RA cells; SA CMCs also had higher expression endothelial markers. We subsequently tested the relative efficacy of SA versus RA CMCs in the setting of post-infarct adoptive transfer. Two days after coronary occlusion, vehicle, RA CMCs, or SA CMCs were delivered percutaneously with echocardiographic guidance. SA CMCs, but not RA CMCs, significantly improved cardiac function compared to vehicle treatment. Although the mechanism remains to be elucidated, the more pronounced endothelial phenotype of the SA CMCs coupled with the finding of increased vascular density suggest a potential pro-vasculogenic action. This new method of isolating CMCs better preserves c-kit expression during passage. SA CMCs, but not RA CMCs, were effective in reducing cardiac dysfunction. Although c-kit expression was maintained, it is unclear whether maintenance of c-kit expression per se was responsible for improved function, or whether the differential adherence property itself confers a reparative phenotype independently of c-kit

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

<div><p>Background</p><p>There is mounting interest in using c-kit positive human cardiac stem cells (c-kit^pos 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.</p><p>Methods</p><p>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 <i>in vitro</i> 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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

Intracoronary infusion of 20 million human c-kit^pos CSCs does not impair left ventricular (LV) function or morphology.

<p>The line graphs in the top panel show individual values of each pig’s progress over time (baseline, 6, 12, 24 h, 1 week, and 1 month). Individual plots are in yellow, and group means are illustrated by the red line plots. <b>A.</b> ejection fraction (EF). <b>B.</b> LV end-diastolic diameter (EDD). <b>C.</b> LV end-diastolic volume (EDV), <b>D.</b> LV anterior wall fractional thickening. The bottom panel shows group mean±SEM at each time point for respective LV functional and morphologic indices. Green and red bars indicate hCSC-treated and vehicle groups respectively. <b>E.</b> ejection fraction, <b>F.</b> fractional shortening, <b>G.</b> LV end-systolic area, <b>H.</b> LV end-diastolic area, <b>I.</b> LV end-systolic volume, <b>J.</b> LV end-diastolic volume, <b>K.</b> LV anterior wall thickness in systole, <b>L.</b> LV anterior wall thickness in diastole, <b>M.</b> LV end-systolic diameter, <b>N.</b> LV end-diastolic diameter, <b>O.</b> LV posterior wall thickness in systole, <b>P.</b> LV posterior wall thickness in diastole. Data are mean±SEM. There were no significant differences between groups with respect to any parameter at respective time points (P > 0.05).</p

Flow cytometric validation and immunocytochemistry of c-kit^pos hCSCs.

<p>Representative flow cytometric analyses of isotype control (left) and c-kit-labeled cell flow plots (center) are shown. Suspension immunocytochemistry of c-kit^<b>pos</b> hCSCs showing positive anti-c-kit labeling is shown in the right panels, with DAPI labeled nuclei in blue.</p

Intracoronary infusion of 20 million human c-kit^pos CSCs does not impair liver function.

<p>Liver function was assessed by serum AST, ALT, alkaline phosphatase, and total CK levels at serial time points (baseline, 6, 12, 24 h, 1 week, and 1 month). <b>A.</b> Serum aspartate aminotransferase (AST), <b>B.</b> Serum creatine phosphokinase (CPK), <b>C.</b> Serum alkaline phosphatase (Alk. Phos.), <b>D.</b> Serum alanine aminotransferase (ALT). Upper limits of normal) and lower limits of normal (LLN) in each graph are depicted by dashed lines respectively. Data are mean±SEM. There were no significant differences in serum AST, ALT, alkaline phosphatase, or total CK levels over the 1 month follow up between groups (P > 0.05 at each time point).</p

Cumulative c-kit positivity by flow cytometry and Trypan blue cell product viability.

<p>The left panel shows c-kit positivity in seven cell lines utilized for the study, which averaged 85.6%±1.9% (mean±SEM). The right panel shows viability of c-kit^<b>pos</b> hCSCs measured by cellular exclusion of Trypan blue staining prior to intracoronary infusion. Trypan negative, viable cells averaged 97.8±0.4%. Data are mean±SEM.</p

Intracoronary infusion of 20 million human c-kit^pos CSCs does not cause myocardial damage as assessed by cardiac CK-MB release.

<p><b>A.</b> Individual serum CK-MB levels (ng/ml) over serial time points (baseline, 6, 12, 24 h, 1 week, and 1 month). Green and red plots identify hCSC-treated and vehicle control pigs, respectively. <b>B.</b> Group means at each time point are shown. The green and red plots identify hCSC-treated and vehicle control groups, respectively. The inset in panel B shows cumulative CK-MB levels. Data are mean±SEM. There were no significant differences in plasma CK-MB levels over the 1 month follow up between groups (P > 0.05 at each time point).</p

Detection of human CSCs in control versus hCSC-treated pig hearts.

<p>Genomic DNA isolated from representative LV sections from control (lanes 1–5) and human CSC-treated pigs (lanes 6–14) were analyzed by PCR for the presence of human genomic DNA (HLA-DMA). Samples were also analyzed for the presence of pig genomic DNA (Gapdh) as a control for DNA quality. Genomic DNA isolated from human heart sections was used as both positive and negative control. None of the samples, including CSC-treated ones, show detectable levels of human DNA.</p