Xiaoxiang Yan

Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction

Acute myocardial infarction (MI) causes sterile inflammation, which is characterized by recruitment and activation of innate and adaptive immune system cells. Here we delineate the temporal dynamics of immune cell accumulation following MI by flow cytometry. Neutrophils increased immediately to a peak at 3 days post-MI. Macrophages were numerically the predominant cells infiltrating the infarcted myocardium, increasing in number over the first week post-MI. Macrophages are functionally heterogeneous, whereby the first responders exhibit high expression levels of proinflammatory mediators, while the late responders express high levels of the anti-inflammatory cytokine IL-10; these macrophages can be classified into M1 and M2 macrophages, respectively, based on surface-marker expression. M1 macrophages dominated at 1-3 days post-MI, whereas M2 macrophages represented the predominant macrophage subset after 5 days. The M2 macrophages expressed high levels of reparative genes in addition to proinflammatory genes to the same levels as in M1 macrophages. The predominant subset of dendritic cells (DCs) was myeloid DC, which peaked in number on day 7. Th1 and regulatory T cells were the predominant subsets of CD4(+) T cells, whereas Th2 and Th17 cells were minor populations. CD8(+) T cells, γδT cells, B cells, natural killer (NK) cells and NKT cells peaked on day 7 post-MI. Timely reperfusion reduced the total number of leukocytes accumulated in the post-MI period, shifting the peak of innate immune response towards earlier and blunting the wave of adaptive immune response. In conclusion, these results provide important knowledge necessary for developing successful immunomodulatory therapies.

Deleterious Effect of the IL-23/IL-17A Axis and γδT Cells on Left Ventricular Remodeling After Myocardial Infarction

Background Left ventricular (LV) remodeling leads to chronic heart failure and is a main determinant of morbidity and mortality after myocardial infarction (MI). At the present time, therapeutic options to prevent LV remodeling are limited. Methods and Results We created a large MI by permanent ligation of the coronary artery and identified a potential link between the interleukin (IL)–23/IL-17A axis and γδT cells that affects late-stage LV remodeling after MI. Despite the finsinf that infarct size 24 hours after surgery was similar to that in wild-type mice, a deficiency in IL-23, IL-17A, or γδT cells improved survival after 7 days, limiting infarct expansion and fibrosis in noninfarcted myocardium and alleviating LV dilatation and systolic dysfunction on day 28 post-MI. M1 macrophages and neutrophils were the major cellular source of IL-23, whereas >90% of IL-17A-producing T cells in infarcted heart were CD4− TCRγδ+ (γδT) cells. Toll-like receptor signaling and IL-1β worked in concert with IL-23 to drive expansion and IL-17A production in cardiac γδT cells, whereas the sphingosine-1-phosphate receptor and CCL20/CCR6 signaling pathways mediated γδT cell recruitment into infarcted heart. IL-17A was not involved in the acute inflammatory response, but it functioned specifically in the late remodeling stages by promoting sustained infiltration of neutrophils and macrophages, stimulating macrophages to produce proinflammatory cytokines, aggravating cardiomyocyte death, and enhancing fibroblast proliferation and profibrotic gene expression. Conclusions The IL-23/IL-17A immune axis and γδT cells are potentially promising therapeutic targets after MI to prevent progression to end-stage dilated cardiomyopathy.

Plasma concentrations of osteopontin, but not thrombin-cleaved osteopontin, are associated with the presence and severity of nephropathy and coronary artery disease in patients with type 2 diabetes mellitus

BackgroundThe aim of the present cross-sectional study was to assess possible associations between osteopontin (OPN), and thrombin-cleaved (N-half) OPN, and nephropathy and coronary artery disease (CAD) in patients with type 2 diabetes mellitus (T2DM).MethodsPlasma levels of OPN, N-half OPN, and high-sensitivity C-reactive protein (hsCRP) were determined in 301 diabetic patients with (n = 226) or without (n = 75) angiographically documented CAD (luminal diameter narrowing >50%), as well as in 75 non-diabetic controls with normal angiography. The estimated glomerular filtration rate (eGFR) was calculated in all patients.ResultsPlasma levels of OPN and hsCRP were significantly higher in patients with T2DM compared with controls. In addition, there was a higher occurrence of moderate renal insufficiency and lower eGFR in patients with T2DM (all P < 0.01). T2DM patients in whom OPN levels were greater than the median value had higher serum creatinine levels, a greater prevalence of mild or moderate renal insufficiency, a higher incidence of CAD, and lower eGFR (all P < 0.05) than T2DM patients in whom OPN levels were the same as or lower than the median value. However, there were no differences in these parameters when patients were stratified according to plasma N-half OPN levels. Furthermore, there was a significant correlation between OPN, but not N-half OPN, and the severity of nephropathy and CAD in diabetes. After adjustment for potential confounders and treatments, multiple linear regression analysis demonstrated an independent association between OPN, but not N-half OPN, and eGFR. Multivariate logistic regression revealed that higher OPN levels conferred a fourfold greater risk of renal insufficiency and CAD in patients with T2DM.ConclusionsThe results of the present study demonstrate that there is an independent association between plasma levels of OPN, but not N-half OPN, and the presence and severity of nephropathy and CAD in diabetes.

Adventitial CXCL1/G-CSF expression in response to acute aortic dissection triggers local neutrophil recruitment and activation leading to aortic rupture

Rationale: In-hospital outcomes are generally acceptable in patients with type B dissection; however, some patients present with undesirable complications, such as aortic expansion and rupture. Excessive inflammation is an independent predictor of adverse clinical outcomes. Objective: We have investigated the underlying mechanisms of catastrophic complications after acute aortic dissection (AAD) in mice. Methods and Results: When angiotensin II was administered in lysyl oxidase inhibitor–preconditioned mice, AAD emerged within 24 hours. The dissection was initiated at the proximal site of the descending thoracic aorta and propagated distally into an abdominal site. Dissection of the aorta caused dilatation, and ≈70% of the mice died of aortic rupture. AAD triggered CXCL1 and granulocyte-colony stimulating factor expression in the tunica adventitia of the dissected aorta, leading to elevation of circulating CXCL1/granulocyte-colony stimulating factor levels. Bone marrow CXCL12 was reduced. These chemokine changes facilitated neutrophil egress from bone marrow and infiltration into the aortic adventitia. Interference of CXCL1 function using an anti-CXCR2 antibody reduced neutrophil accumulation and limited aortic rupture post AAD. The tunica adventitia of the expanded dissected aorta demonstrated high levels of interleukin-6 (IL-6) expression. Neutrophils were the major sources of IL-6, and CXCR2 neutralization significantly reduced local and systemic levels of IL-6. Furthermore, disruption of IL-6 effectively suppressed dilatation and rupture of the dissected aorta without any influence on the incidence of AAD and neutrophil mobilization. Conclusions: Adventitial CXCL1/granulocyte-colony stimulating factor expression in response to AAD triggers local neutrophil recruitment and activation. This leads to adventitial inflammation via IL-6 and results in aortic expansion and rupture.

Endogenous Prostaglandin D2 and Its Metabolites Protect the Heart Against Ischemia–Reperfusion Injury by Activating Nrf2

We recently demonstrated that glucocorticoids markedly upregulate the expression of cyclooxygenase-2 in cardiomyocytes and protect hearts from ischemia–reperfusion (I/R) injury by activating lipocalin-type prostaglandin D (PGD) synthase (L-PGDS)–derived PGD2 biosynthesis. We examined a downstream mechanism of cardioprotection elicited by PGD2 biosynthesis. Acute PGD2 treatment did not protect hearts against I/R injury. We then speculated that PGD2 and its metabolite 15-deoxy-&Dgr;12,14-PGJ2 activate gene expression networks to mediate the glucocorticoid-mediated cardioprotection. Using an unbiased approach, we identified that glucocorticoids induce a number of well-known erythroid-derived 2–like 2 (Nrf2) target genes in the heart in an L-PGDS–dependent manner and that the cardioprotective effect of glucocorticoids against I/R injury was not seen in Nrf2-knockout hearts. We showed relatively low expression of PGD2 receptors (ie, DP1 and DP2) in the heart but abundant expression of PGF2&agr; receptor (FP), which binds PGF2&agr; and PGD2 with equal affinity. Glucocorticoids also failed to induce the expression of L-PGDS–dependent Nrf2 target genes in FP-knockout hearts. PGD2 acted through its metabolite 15-deoxy-&Dgr;12,14-PGJ2 in the heart as evidenced by the glucocorticoid-mediated activation of peroxisome proliferator-activated receptor-&ggr;. In turn, glucocorticoids failed to induce the expression of L-PGDS–dependent Nrf2 target genes in hearts pretreated with peroxisome proliferator-activated receptor-&ggr; antagonist GW9662, and glucocorticoid-mediated cardioprotection against I/R injury was compromised in FP-knockout mice and GW9662-treated mice. In conclusion, PGD2 protects heart against I/R injury by activating Nrf2 predominantly via FP receptor. In addition, we propose activation of peroxisome proliferator-activated receptor-&ggr; by the dehydrated metabolite of PGD2 (15-deoxy-&Dgr;12,14-PGJ2) as another mechanism by which glucocorticoids induce cardioprotection.

PGD2-CRTH2 Pathway Promotes Tubulointerstitial Fibrosis

Urinary excretion of lipocalin-type PGD(2) synthase (L-PGDS), which converts PG H(2) to PGD(2), increases in early diabetic nephropathy. In addition, L-PGDS expression in the tubular epithelium increases in adriamycin-induced nephropathy, suggesting that locally produced L-PGDS may promote the development of CKD. In this study, we found that L-PGDS-derived PGD(2) contributes to the progression of renal fibrosis via CRTH2-mediated activation of Th2 lymphocytes. In a mouse model, the tubular epithelium synthesized L-PGDS de novo after unilateral ureteral obstruction (UUO). L-PGDS-knockout mice and CRTH2-knockout mice both exhibited less renal fibrosis, reduced infiltration of Th2 lymphocytes into the cortex, and decreased production of the Th2 cytokines IL-4 and IL-13. Furthermore, oral administration of a CRTH2 antagonist, beginning 3 days after UUO, suppressed the progression of renal fibrosis. Ablation of IL-4 and IL-13 also ameliorated renal fibrosis in the UUO kidney. Taken together, these data suggest that blocking the activation of CRTH2 by PGD(2) might be a strategy to slow the progression of renal fibrosis in CKD.

Biphasic Time Course of the Changes in Aldosterone Biosynthesis Under High-Salt Conditions in Dahl Salt-Sensitive Rats

Objective—The comorbidity of excess salt and elevated plasma aldosterone has deleterious effects in cardiovascular disease. We evaluated the mechanisms behind the paradoxical increase in aldosterone biosynthesis in relation to dietary intake of salt. Methods and Results—Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed a high-salt diet, and plasma and tissue levels of aldosterone in the adrenal gland and heart were quantified by liquid chromatography–electrospray ionization–tandem mass spectrometry. In Dahl-S rats, we found that the delayed and paradoxical increase in aldosterone biosynthesis after the initial and appropriate response to high salt. The late rise in aldosterone biosynthesis was accompanied by upregulation of CYP11B2 expression in the zona glomerulosa and increased adrenal angiotensin II levels and renin-angiotensin system components. It preceded the appearance of left ventricular systolic dysfunction and renal insufficiency. Blockade of angiotensin AT1 receptors reversed the paradoxical increase in aldosterone biosynthesis. In contrast, Dahl-R rats maintained the initial suppression of aldosterone biosynthesis. Aldosterone levels in the heart closely paralleled those in the plasma and adrenal gland and disappeared after bilateral adrenalectomy. Conclusion—Chronic salt overload in Dahl-S rats stimulates aberrant aldosterone production via activation of the local renin-angiotensin system in the adrenal gland, thereby creating the comorbidity of excess salt and elevated plasma aldosterone.

Indispensable role of endothelial nitric oxide synthase in caloric restriction-induced cardioprotection against ischemia-reperfusion injury

Caloric restriction (CR) confers cardioprotection against ischemia-reperfusion injury (IRI). We previously found that treatment with N(G)-nitro-l-arginine methyl ester completely abrogates CR-induced cardioprotection and increases nuclear sirtuin 1 (Sirt1) expression. However, it remains unclear whether endothelial nitric oxide (NO) synthase (eNOS) plays a role in CR-induced cardioprotection and Sirt1 activation. We subjected eNOS-deficient (eNOS(-/-)) mice to either 3-mo ad libitum (AL) feeding or CR (-40%). Isolated perfused hearts were subjected to 25-min global ischemia followed by 60-min reperfusion. The degree of myocardial IRI in AL-fed eNOS(-/-) mice was more severe than that in AL-fed wild-type mice. Furthermore, CR did not exert cardioprotection in eNOS(-/-) mice. eNOS(-/-) mice exhibited elevated blood pressure and left ventricular hypertrophy compared with wild-type mice, although they underwent CR. Although nuclear Sir1 content was increased, the increases in cardiac Sirt1 activity with CR was absent in eNOS(-/-) mice. In eNOS(-/-) mice treated with hydralazine, blood pressure and left ventricular weight became comparable with CR-treated wild-type mice. However, CR-induced cardioprotection was not observed. Resveratrol enhanced cardiac Sirt1 activity but failed to mimic CR-induced cardioprotection in eNOS(-/-) mice. Finally, combination therapy with resveratrol and hydralazine attenuated myocardial IRI and reduced infarct size in eNOS(-/-) mice, and their effects were comparable with those observed in CR-treated wild-type mice. These results demonstrate the essential roles of eNOS in the development of CR-induced cardioprotection and Sirt1 activation during CR. The combination of a relatively low dose of resveratrol with an adequate vasodilator therapy might be useful for managing patients with endothelial dysfunction associated with impaired NO bioavailability.

Activation of pyruvate dehydrogenase by dichloroacetate has the potential to induce epigenetic remodeling in the heart

Dichloroacetate (DCA) promotes pyruvate entry into the Krebs cycle by inhibiting pyruvate dehydrogenase (PDH) kinase and thereby maintaining PDH in the active dephosphorylated state. DCA has recently gained attention as a potential metabolic-targeting therapy for heart failure but the molecular basis of the therapeutic effect of DCA in the heart remains a mystery. Once-daily oral administration of DCA alleviates pressure overload-induced left ventricular remodeling. We examined changes in the metabolic fate of pyruvate carbon (derived from glucose) entering the Krebs cycle by metabolic interventions of DCA. (13)C6-glucose pathway tracing analysis revealed that instead of being completely oxidized in the mitochondria for ATP production, DCA-mediated PDH dephosphorylation results in an increased acetyl-CoA pool both in control and pressure-overloaded hearts. DCA induces hyperacetylation of histone H3K9 and H4 in a dose-dependent manner in parallel to the dephosphorylation of PDH in cultured cardiomyocytes. DCA administration increases histone H3K9 acetylation in in vivo mouse heart. Interestingly, DCA-dependent histone acetylation was associated with an up-regulation of 2.3% of genes (545 out of 23,474 examined). Gene ontology analysis revealed that these genes are highly enriched in transcription-related categories. This evidence suggests that sustained activation of PDH by DCA results in an overproduction of acetyl-CoA, which exceeds oxidation in the Krebs cycle and results in histone acetylation. We propose that DCA-mediated PDH activation has the potential to induce epigenetic remodeling in the heart, which, at least in part, forms the molecular basis for the therapeutic effect of DCA in the heart.

Cardiac Sirt1 mediates the cardioprotective effect of caloric restriction by suppressing local complement system activation after ischemia-reperfusion

Caloric restriction (CR) confers cardioprotection against ischemia-reperfusion (I/R) injury. We previously found the essential roles of endothelial nitric oxide synthase in the development of CR-induced cardioprotection and Sirt1 activation during CR (Shinmura K, Tamaki K, Ito K, Yan X, Yamamoto T, Katsumata Y, Matsuhashi T, Sano M, Fukuda K, Suematsu M, Ishii I. Indispensable role of endothelial nitric oxide synthase in caloric restriction-induced cardioprotection against ischemia-reperfusion injury.Am J Physiol Heart Circ Physiol 308: H894-H903, 2015). However, the exact mechanism by which Sirt1 in cardiomyocytes mediates the cardioprotective effect of CR remains undetermined. We subjected cardiomyocyte-specific Sirt1 knockout (CM-Sirt1(-/-)) mice and the corresponding control mice to either 3-mo ad libitum feeding or CR (-40%). Isolated perfused hearts were subjected to 25-min global ischemia, followed by 60-min reperfusion. The recovery of left ventricle function after I/R was improved, and total lactate dehydrogenase release into the perfusate during reperfusion was attenuated in the control mice treated with CR, but a similar cardioprotective effect of CR was not observed in the CM-Sirt1(-/-)mice. The expression levels of cardiac complement component 3 (C3) at baseline and the accumulation of C3 and its fragments in the ischemia-reperfused myocardium were attenuated by CR in the control mice, but not in the CM-Sirt1(-/-)mice. Resveratrol treatment also attenuated the expression levels of C3 protein in cultured neonatal rat ventricular cardiomyocytes. Moreover, the degree of myocardial I/R injury in conventional C3 knockout (C3(-/-)) mice treated with CR was similar to that in the ad libitum-fed C3(-/-)mice, although the expression levels of Sirt1 were enhanced by CR. These results demonstrate that cardiac Sirt1 plays an essential role in CR-induced cardioprotection against I/R injury by suppressing cardiac C3 expression. This is the first report suggesting that cardiac Sirt1 regulates the local complement system during CR.