Beth A. Palka

Temporal and Spatial Variations in Structural Protein Expression During the Progression From Stunned to Hibernating Myocardium

Background—Dysfunctional and normally perfused remote regions show equal myolysis and glycogen accumulation in pig hibernating myocardium. We tested the hypothesis that these arose secondary to elevations in preload rather than ischemia. Methods and Results—Expression of structural protein (desmin, desmoplakin, titin, cardiotin, α-smooth muscle actin, lamin-A/C, and lamin-B2) in viable dysfunctional myocardium was analyzed by immunohistochemistry. We performed blinded analysis of paired dysfunctional left anterior descending coronary artery and normal remote subendocardial samples from stunned (24 hours; n=6), and hibernating (2 weeks; n=6) myocardium versus sham controls pigs (n=7). Within 24 hours, cardiac myocytes globally reexpressed α-smooth muscle actin. In stunned myocardium, cardiotin was globally reduced, whereas reductions in desmin were restricted to the dysfunctional region. Alterations progressed with the transition to hibernating myocardium, in which desmin, cardiotin, and titin were globally reduced. A qualitatively similar reorganization of cytoskeletal proteins occurred 3 hours after transient elevation of left ventricular end-diastolic pressure to 33±3 mm Hg. Conclusions—Qualitative cardiomyocyte remodeling similar to that in humans with chronic hibernation occurs rapidly after a critical coronary stenosis is applied, as well as after transient elevations in left ventricular end-diastolic pressure in the absence of ischemia. Thus, reorganization of cytoskeletal proteins in patients with viable dysfunctional myocardium appears to reflect chronic and/or cyclical elevations in preload associated with episodes of spontaneous regional ischemia.

LDL cholesterol modulates human CD34+ HSPCs through effects on proliferation and the IL-17 G-CSF axis.

Background Hypercholesterolemia plays a critical role in atherosclerosis. CD34+ CD45dim Lineage- hematopoietic stem/progenitor cells (HSPCs) give rise to the inflammatory cells linked to atherosclerosis. In mice, high cholesterol levels mobilize HSPCs into the bloodstream, and promote their differentiation to granulocytes and monocytes. The objective of our study was to determine how cholesterol levels affect HSPC quantity in humans. Methods We performed a blinded, randomized hypothesis generating study in human subjects (n=12) treated sequentially with statins of differing potencies to vary lipid levels. CD34+ HSPC levels in blood were measured by flow cytometry. Hematopoietic colony forming assays confirmed the CD34+ population studied as HSPCs with multlineage differentiation potential. Mobilizing cytokine levels were measured by ELISA. Results The quantity of HSPCs was 0.15 ± 0.1% of buffy coat leukocytes. We found a weak, positive correlation between CD34+ HSPCs and both total and LDL cholesterol levels (r2=0.096, p < 0.025). Additionally, we tested whether cholesterol modulates CD34+ HSPCs through direct effects or on the levels of mobilizing cytokines. LDL cholesterol increased cell surface expression of CXCR4, G-CSFR affecting HSPC migration, and CD47 mediating protection from phagocytosis by immune cells. LDL cholesterol also increased proliferation of CD34+ HSPCs (28 ± 5.7%, n=6, p < 0.03). Finally, the HSPC mobilizing cytokine G-CSF (r2=0.0683, p < 0.05), and its upstream regulator IL-17 (r2=0.0891, p < 0.05) both correlated positively with LDL cholesterol, while SDF-1 levels were not significantly affected. Conclusions Our findings support a model where LDL cholesterol levels positively correlate with CD34+ HSPC levels in humans through effects on the levels of G-CSF via IL-17 promoting mobilization of HSPCs, and by direct effects of LDL cholesterol on HSPC proliferation. The findings are provocative of further study to determine if HSPCs, like cholesterol levels, are linked to CVD events.

Cell surface glycoengineering improves selectin-mediated adhesion of mesenchymal stem cells (MSCs) and cardiosphere-derived cells (CDCs): Pilot validation in porcine ischemia-reperfusion model.

Promising results are emerging in clinical trials focused on stem cell therapy for cardiology applications. However, the low homing and engraftment of the injected cells to target tissue continues to be a problem. Cellular glycoengineering can address this limitation by enabling the targeting of stem cells to sites of vascular injury/inflammation. Two such glycoengineering methods are presented here: i. The non-covalent incorporation of a P-selectin glycoprotein ligand-1 (PSGL-1) mimetic 19Fc[FUT7(+)] via lipid-protein G fusion intermediates that intercalate onto the cell surface, and ii. Over-expression of the α(1,3)fucosyltransferse FUT7 in cells. Results demonstrate the efficient coupling of 19Fc[FUT7(+)] onto both cardiosphere-derived cells (CDCs) and mesenchymal stem cells (MSCs), with coupling being more efficient when using protein G fused to single-tailed palmitic acid rather than double-tailed DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). This non-covalent cellular modification was mild since cell proliferation and stem-cell marker expression was unaltered. Whereas coupling using 19Fc[FUT7(+)] enhanced cell capture on recombinant P-selectin or CHO-P cell surfaces, α(1,3)fucosylation was necessary for robust binding to E-selectin and inflamed endothelial cells under shear. Pilot studies confirm the safety and homing efficacy of the modified stem cells to sites of ischemia-reperfusion in the porcine heart. Overall, glycoengineering with physiological selectin-ligands may enhance stem cell engraftment.

Effects of statins on TH1 modulating cytokines in human subjects

Background. Activation of the innate immune system by cholesterol accelerates atherosclerosis. High levels or modified forms of cholesterol stimulate release of the inflammatory cytokines IL-12 and IL-18 that synergistically stimulate T lymphocytes to produce the atherogenic cytokine interferon-γ. While activation of the innate immune system by cholesterol is well-described in animal models and human subjects with high cholesterol levels or known atherosclerotic disease, the interaction of cholesterol and lipoproteins with the innate immune system in human subjects without known atherosclerosis is less well-described. The goal of our study was to assess the TH1 modulating cytokines IL-12 p40 and IL-18, and their counter regulatory cytokines IL-18 binding protein and IL-27, to determine if their levels are linked to cholesterol levels or other factors. Methods. We performed a blinded, randomized hypothesis-generating study in human subjects without known atherosclerotic disease. We measured serum lipids, lipoprotein levels, and collected plasma samples at baseline. Subjects were randomized to two weeks of therapy with atorvastatin, pravastatin, or rosuvastatin. Lipids and cytokine levels were measured after two weeks of statin treatment. Subjects were given a four-week statin-free period. At the end of the four-week statin-free period, venous blood was sampled again to determine if serum lipids returned to within 5% of their pre-statin levels. When lipid levels returned to baseline, subjects were again treated with the next statin in the randomization scheme. IL-12, IL-18, IL-18 binding protein, and IL-27 were measured at baseline and after each statin treatment to determine effects of statin treatment on their blood levels, and identify correlations with lipids and lipoproteins. Results. Therapy with statins revealed no significant change in the levels of IL-12, IL-18, IL-18 binding protein or IL-27 levels. We found that IL-18 levels positively correlate with total cholesterol levels (r2 = 0.15, p < 0.03), but not HDL or LDL cholesterol. In contrast, IL-12 p40 levels inversely correlated with total cholesterol (r2 = −0.17, p < 0.008), HDL cholesterol (r2 = −0.22, p < 0.002), and apolipoprotein A1 (r2 = −0.21, p < 0.002). Similarly, IL-18 binding protein levels inversely correlated with apolipoprotein A1 levels (r2 = −0.13, p < 0.02). Conclusions. Our findings suggest that total cholesterol levels positively regulate IL-18, while HDL cholesterol and apolipoprotein A1 may reduce IL-12 p40 and IL-18 binding protein levels. Additional studies in a larger patient population are needed to confirm these findings, and verify mechanistically whether HDL cholesterol can directly suppress IL-12 p40 and IL-18 binding protein levels in human subjects.