Heme oxygenase-1 restores impaired GARPCD4⁺CD25⁺ regulatory T cells from patients with acute coronary syndrome by upregulating LAP and GARP expression on activated T lymphocytes.

BACKGROUND: Accumulating evidence shows that the pathological autoreactive immune response is responsible for plaque rupture and the subsequent onset of acute coronary syndrome (ACS). Naturally occurring CD4(+)CD25(+)regulatory T cells (nTregs) are indispensable in suppressing the pathological autoreactive immune response and maintaining immune homeostasis. However, the number and the suppressive function of glycoprotein-A repetitions predominant (GARP) (+) CD4(+) CD25(+) activated nTregs were impaired in patients with ACS. Recent evidence suggests that heme oxygenase-1 (HO-1) can regulate the adaptive immune response by promoting the expression of Foxp3. We therefore hypothesized that HO-1 may enhance the function of GARP(+) CD4(+) CD25(+)Tregs in patients with ACS and thus regulate immune imbalance. METHODS: T lymphocytes were isolated from healthy volunteers (control, n=30) and patients with stable angina (SA, n=40) or ACS (n=51). Half of these cells were treated with an HO-1 inducer (hemin) for 48 h, and the other half were incubated with complete RPMI-1640 medium. The frequencies of T-helper 1 (Th1), Th2, Th17 and latency-associated peptide (LAP) (+)CD4(+) T cells and the expression of Foxp3 and GARP by CD4(+)CD25(+)T cells were then assessed by measuring flow cytometry after stimulation in vitro. The suppressive function of activated Tregs was measured by thymidine uptake. The levels of transforming growth factor-1 (TGF-ß1) in the plasma were measured using enzyme-linked immunosorbent assay (ELISA). The expression levels of the genes encoding these proteins were analyzed by real-time polymerase chain reaction. RESULTS: Patients with ACS exhibited an impaired number and suppressive function of GARP(+) CD4(+) CD25(+)Tregs and a mixed Th1/Th17-dominant T cell response when compared with the SA and control groups. The expression of LAP in T cells was also lower in patients with ACS compared to patients with SA and the control individuals. Treatment with an HO-1 inducer enhanced the biological activity of GARP(+) CD4(+) CD25(+)Tregs and resulted in increased expression of LAP and GARP by activated T cells. CONCLUSIONS: The reduced number and impaired suppressive function of GARP(+) CD4(+) CD25(+)Tregs result in excess effector T cell proliferation, leading to plaque instability and the onset of ACS. HO-1 can effectively restore impaired GARP(+) CD4(+) CD25(+)Tregs from patients with ACS by promoting LAP and GARP expression on activated T cells.

MicroRNA-208a Dysregulates Apoptosis Genes Expression and Promotes Cardiomyocyte Apoptosis during Ischemia and Its Silencing Improves Cardiac Function after Myocardial Infarction.

AIMS: miR-208a is associated with adverse outcomes in several cardiac pathologies known to have increased apoptosis, including myocardial infarction (MI). We investigated if miR-208a has proapoptotic effects on ischemic cardiomyocytes and if its silencing has therapeutic benefits in MI. METHODS AND RESULTS: The effect of miR-208a on apoptosis during ischemia was studied in cultured neonatal mice myocytes transfected with agomir or antagomir. Differential gene expression was assessed using microarrays. MI was induced in male C57BL/6 mice randomly assigned to antagomir (n = 6) or control group (n = 7), while sham group (n = 7) had sham operation done. Antagomir group received miR208a antagomir, while control and sham group mice received vehicle only. At 7 and 28 days, echocardiography was done and thereafter hearts were harvested for analysis of apoptosis by TUNEL method, fibrosis using Masson's trichrome, and hypertrophy using hematoxylin and eosin. miR-208a altered apoptosis genes expression and increased apoptosis in ischemic cardiomyocytes. Therapeutic inhibition of miR-208a decreased cardiac fibrosis, hypertrophy, and apoptosis and significantly improved cardiac function 28 days after MI. CONCLUSION: miR-208a alters apoptosis genes expression and promotes apoptosis in ischemic cardiomyocytes, and its silencing attenuates apoptosis, fibrosis, and hypertrophy after MI, with significant improvement in cardiac function.

Curcumin Downregulates Phosphate Carrier and Protects against Doxorubicin Induced Cardiomyocyte Apoptosis.

Aim. To explore the effects of curcumin on phosphate carrier (PiC) and its role in protection against doxorubicin induced myocyte toxicity. Methods. Using H9c2 cell line, the cardiotoxic effect of doxorubicin and its mitigation by curcumin were studied. H9c2 cells were cultured with doxorubicin and/or curcumin at various concentrations. Analysis for apoptosis of H9c2 was done using flow cytometry. Confocal laser scanning microscopy was used to record the fluorescence intensity ratios and to determine the mitochondrial permeability transition pore (MPTP) opening state. Oxidative stress was measured using glutathione level, superoxide dismutase activities, and malondialdehyde content. The effect of doxorubicin and curcumin on PiC gene expression was measured by real-time PCR. Results. Curcumin decreased mRNA of PiC and was partly protective against oxidative stress, loss of mitochondrial transmembrane potential, and apoptosis induced by doxorubicin. Interestingly, the effect was not clearly dose dependent and the concentration of 12 mg/L was more efficient than 15 and 10 mg/L. Conclusion. Curcumin downregulates PiC and partly protects against doxorubicin induced oxidative stress and myocyte apoptosis.

MicroRNA-208a Silencing Attenuates Doxorubicin Induced Myocyte Apoptosis and Cardiac Dysfunction.

AIMS: GATA4 depletion is a distinct mechanism by which doxorubicin leads to cardiomyocyte apoptosis, and preservation of GATA4 mitigates doxorubicin induced myocyte apoptosis and cardiac dysfunction. We investigated a novel approach of attenuating doxorubicin induced cardiac toxicity by silencing miR-208a, a heart specific microRNA known to target GATA4. METHODS AND RESULTS: Eight-week-old female Balb/C mice were randomly assigned to sham, antagomir, and control groups. Antagomir group were pretreated with miR-208a antagomir 4 days before doxorubicin administration. At day 0, control and antagomir groups received 20 mg/kg of doxorubicin, while sham mice received phosphate buffered solution. Echocardiography was done at day 7, after which animals were sacrificed and hearts harvested and assessed for apoptosis and expression of miR-208a, GATA4, and BCL-2. Doxorubicin significantly upregulated miR-208a, downregulated GATA4, and increased myocyte apoptosis, with resulting decrease in cardiac function. In contrast, therapeutic silencing of miR-208a salvaged GATA4 and BCL-2 and decreased apoptosis, with improvement in cardiac function. CONCLUSION: Doxorubicin upregulates miR-208a and promotes cardiomyocyte apoptosis, while therapeutic silencing of miR-208a attenuates doxorubicin induced myocyte apoptosis with subsequent improvement in cardiac function. These novel results highlight the therapeutic potential of targeting miR-208a to prevent doxorubicin cardiotoxicity.

Among Ectasia Patients with Coexisting Coronary Artery Disease, TIMI Frame Count Correlates with Ectasia Size and Markis Type IV Is the Commonest.

Background. Coronary artery ectasia (CAE) occurs in 0.3 to 5.3% of patients undergoing coronary angiography. TIMI frame count (TFC) is an index of coronary flow that correlates with flow velocity. In ectasia patients, there is delayed coronary flow with increased TFC. Methods. We evaluated angiograms of 789 patients for presence of CAE, coronary artery disease (CAD), and Markis type of CAE. We measured ectasia size and length and their correlation with TFC in ectatic right coronary arteries (RCA) of patients with CAE and CAD. Results. 30 patients had CAE (3.8%). Of these 16.7% had isolated CAE, while 83.87% had CAE and CAD. Among CAE and CAD patients, the RCA was most involved (70.4%), and Markis type IV CAE was the commonest (64%). In isolated CAE, the RCA, LAD, and LCx were equally involved (33.3%). Patients with CAE and CAD had significantly higher TFC compared to controls, P = 0.035. There was a positive correlation of moderate strength, between ectasia size and TFC, r(17) = 0.598, P = 0.007. Ectasia length was not significantly correlated with TFC, rho (17) = 0.334, P = 0.163. Conclusion. Among patients undergoing angiography, CAE has a prevalence of 3.8% and Markis type IV is the commonest. Larger ectasias are associated with slower coronary flow.

Disruption of the TSLP-TSLPR-LAP signaling between epithelial and dendritic cells through hyperlipidemia contributes to regulatory T-Cell defects in atherosclerotic mice.

Regulatory T-Cells (Tregs) play a protective role against the development of atherosclerosis. Moreover, thymic stromal lymphopoietin (TSLP)/thymic stromal lymphopoietin receptor (TSLPR) signaling in myeloid dendritic cells (DCs) promote Treg differentiation. Here, we examined the potential role of TSLP/TSLPR on Treg homeostasis in atherosclerosis. The frequencies of both latency-associated peptide (LAP)(+) and Foxp3(+) Tregs were reduced in the thymus and spleen of ApoE(-/-) mice compared with C57BL/6 mice, and this effect was associated with decreased thymic output. The tolerogenic function of DCs obtained from ApoE(-/-) mice was compromised compared with those from C57BL/6 mice. The expression of TSLP and TSLPR was also inhibited in ApoE(-/-) mice. In addition, we found that ox-LDL attenuated TSLP expression in cultured thymic epithelial cells (TECs) through the activation of retinoid X receptor alpha (RXRA) and IL-1ß and decreased LAP and PD-L1 expression in oxLDL-activated DCs while both were up-regulated in TSLP-activated DCs. We also observed that the TSLP-DCs mediated differentiation of Tregs was abrogated through LAP neutralization. Furthermore, TSLP injection rescued Treg defects in ApoE(-/-) mice. These findings suggest that Treg defects in ApoE(-/-) mice might partially be attributed to the disruption of TSLP-TSLPR-LAP signaling in epithelial cells (ECs) and DCs.