Zequan Yang

Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase

Corticosteroids have been shown to exert beneficial effects in the treatment of acute myocardial infarction, but the precise mechanisms underlying their protective effects are unknown. Here we show that high-dose corticosteroids exert cardiovascular protection through a novel mechanism involving the rapid, non-transcriptional activation of endothelial nitric oxide synthase (eNOS). Binding of corticosteroids to the glucocorticoid receptor (GR) stimulated phosphatidylinositol 3-kinase and protein kinase Akt, leading to eNOS activation and nitric oxide–dependent vasorelaxation. Acute administration of pharmacological concentrations of corticosteroids in mice led to decreased vascular inflammation and reduced myocardial infarct size following ischemia and reperfusion injury. These beneficial effects of corticosteroids were abolished by GR antagonists or eNOS inhibitors in wild-type mice and were completely absent in eNOS-deficient (Nos3−/−) mice. The rapid activation of eNOS by the non-nuclear actions of GR, therefore, represents an important cardiovascular protective effect of acute high-dose corticosteroid therapy.

Simultaneous Evaluation of Infarct Size and Cardiac Function in Intact Mice by Contrast-Enhanced Cardiac Magnetic Resonance Imaging Reveals Contractile Dysfunction in Noninfarcted Regions Early After Myocardial Infarction

Background—The objective of this study was to noninvasively determine the effects of reperfused myocardial infarction (MI) on regional and global left-ventricular (LV) function 24 hours after MI in intact mice with contrast-enhanced cardiac MRI and a single, gradient-echo pulse sequence. Methods and Results—Twenty-three mice received baseline MRI scans followed by either 60 minutes of coronary occlusion (MI group, n=15) or thoracotomy without occlusion (sham group, n=8). Gadolinium-DTPA–enhanced magnetic resonance (MR) images were acquired 24 hours after surgery. Hearts were then excised for conventional infarct size determination via 2,3,5-triphenyl tetrazolium chloride (TTC) staining. In addition to infarct size, analysis of the MR images yielded left ventricular (LV) mass, LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), LV ejection fraction (LVEF), cardiac output, and percent LV wall thickening (%WTh). Twenty-four hours after surgery, infarct size was 28.1±1.8% of LV mass by MRI and 27.5±1.7% by TTC (P =NS). Bland-Altman analysis revealed close agreement between the results obtained by the 2 methods. MI had little effect on LVEDV but caused a 98% increase in LVESV (from 11.3 to 22.4 &mgr;L, P <0.05), which resulted in a significant reduction in LVEF (from 70% to 37%, P <0.05). Compared with LV regional function at baseline, %WTh 24 hours after MI was significantly depressed, not only in infarcted myocardium but also in regions remote from the infarct zone. In contrast, sham-operated mice showed a small but significant increase in %WTh 24 hours after surgery (P <0.05). Conclusions—MRI can accurately assess both infarct size and cardiac function in intact mice early after large, reperfused MI, revealing the existence of contractile dysfunction in noninfarcted regions of the heart.

Myocardial Infarct–Sparing Effect of Adenosine A2A Receptor Activation Is due to Its Action on CD4+ T Lymphocytes

Background— We previously used adenosine A2A receptor (A2AR) knockout (KO) mice and bone marrow transplantation to show that the infarct-sparing effect of A2AR activation at reperfusion is primarily due to effects on bone marrow–derived cells. In this study we show that CD4+ but not CD8+ T lymphocytes contribute to myocardial ischemia/reperfusion injury. Method and Results— After a 45-minute occlusion of the left anterior descending coronary artery and reperfusion, T cells accumulate in the infarct zone within 2 minutes. Addition of 10 &mgr;g/kg of the A2AR agonist ATL146e 5 minutes before reperfusion produces a significant reduction in T-cell accumulation and a significant reduction in infarct size (percentage of risk area) measured at 24 hours. In Rag1 KO mice lacking mature lymphocytes, infarct size is significantly smaller than in C57BL/6 mice. Infarct size in Rag1 KO mice is increased to the level of B6 mice by adoptive transfer of 50 million CD4+ T lymphocytes derived from C57BL/6 or A2AR KO but not interferon-&ggr; KO mice. ATL146e completely blocked the increase in infarct size in Rag1 KO mice reconstituted with B6 but not A2AR KO CD4+ T cells. The number of neutrophils in the reperfused heart at 24 hours after infarction correlated well with the number of lymphocytes and infarct size. Conclusions— These results strongly suggest that the infarct-sparing effect of A2AR activation is primarily due to inhibition of CD4+ T-cell accumulation and activation in the reperfused heart.

Infarct-sparing effect of A2A-adenosine receptor activation is due primarily to its action on lymphocytes.

Background—A2A-adenosine receptor (A2AAR) activation on reperfusion after ischemia reduces the size of myocardial infarction, but the mechanism of action has not been fully defined. Methods and Results—We created chimeric mice by bone marrow transplantation from A2AAR-knockout or green fluorescent donor mice to irradiated congenic C57BL/6 (B6) recipients. In the GFP chimeras, we were unable to detect green fluorescent–producing cells in the vascular endothelium, indicating that bone marrow–derived cells were not recruited to endothelium at appreciable levels after bone marrow transplantation and/or acute myocardial infarction. Injection of 5 or 10 &mgr;g/kg of a potent and selective agonist of A2AAR, ATL146e, had no effect on hemodynamic parameters but reduced infarct size in B6 mice after 45 minutes of left anterior descending artery occlusion followed by 24 hours of reperfusion to 42.5±3.0% and 39.3±4.7% of risk region, respectively, compared with 61.0±2.3% in vehicle-treated B6 mice (P<0.05). Myocardial myeloperoxidase activity in the risk region measured at 4 hours after reperfusion was significantly reduced by ATL146e. The salutary effects of ATL146e were absent in A2AAR-knockout mice or in mice treated with a selective A2AAR antagonist, ZM241385. ATL146e also reduced infarct size and myeloperoxidase in B6/B6 (donor/recipient) chimeras (P<0.05) but not in A2AAR-knockout/B6 chimeras. In immunocompromised Rag-1–KO mice, infarct size was significantly reduced compared with B6 mice but was not further reduced by ATL146e. Conclusions—The results indicate that A2AAR activation on bone marrow–derived cells, specifically T or B lymphocytes, is responsible for the infarct-sparing and antiinflammatory effects of ATL146e administered at the time of reperfusion after coronary occlusion.

Angiotensin II Type 2 Receptor Overexpression Preserves Left Ventricular Function After Myocardial Infarction

Background—The role of the angiotensin II type 2 receptor (AT2-R) in left ventricular (LV) remodeling may depend on the underlying stimulus. We hypothesized that cardiac AT2-R overexpression in transgenic (TG) mice would attenuate remodeling after myocardial infarction (MI). Methods and Results—Ten wild-type (WT) C57BL/6 mice and 12 TG mice that overexpress the AT2-R in the heart were studied by cardiac MRI at baseline and days 1, 7, and 28 post-MI induced by 1 hour of occlusion of the LAD followed by reperfusion. Short-axis imaging from apex to base was used to determine LV mass index, end-diastolic and end-systolic volume indices (EDVI, ESVI), regional wall thickness and thickening, and ejection fraction (EF). Gadolinium-DTPA was infused 20 minutes before day 1 imaging to assess infarct size. At baseline, heart rate, blood pressure, LV mass index, and EDVI were similar between groups. Baseline ESVI was lower (0.20±0.07 versus 0.45±0.15 &mgr;L/g, P <0.001) and EF higher (82.3±4.9% versus 67.7±5.3%, P <0.001) in TG than WT. Infarct size was similar (36.6±7.2% in WT, 34.0±7.8% in TG, P =NS). When controlled for baseline differences, ESVI was significantly less and EF significantly higher at all time points in TG versus WT. At day 28, ESVI was 1.05±0.32 &mgr;L/g in TG and 1.63±0.41 &mgr;L/g in WT, P <0.03, and EF was 47.3±5.8% versus 34.1±9.2%, P <0.003, respectively. Regional wall thickness and thickening were greater in TG both at baseline and at day 28. At day 28, blood pressure and LV dP/dt were higher in TG. Conclusions—Cardiac AT2-R overexpression improves LV systolic function at baseline and preserves function during post-MI remodeling.

MR tagging early after myocardial infarction in mice demonstrates contractile dysfunction in adjacent and remote regions

The purpose of this study was to use MR myocardial tagging to assess regional cardiac function after myocardial infarction (MI) in mice. Eight mice were imaged before and 1 day after MI. MRI included cine imaging, myocardial tagging, and contrast‐enhanced imaging. Regional percent circumferential shortening (%CS) was measured from the tagged images, and the region of hyperenhancement on the contrast‐enhanced images was used to determine the infarcted, adjacent, and remote zones. Ejection fraction (EF) fell from 59% ± 6% at baseline to 32% ± 6% after MI (P < 0.01). At baseline, %CS was 14.5% ± 3.4%. After MI, %CS was 0.7% ± 4.4% in the infarcted zone, 7.4% ± 4.4% in the adjacent zone, and 11.8% ± 4.2% in the remote zone. %CS was statistically different for all comparisons between the infarcted, adjacent, remote, and baseline groups (P < 0.01). MR tagging can detect regional differences in myocardial function post‐MI in mice. Magn Reson Med 48:399–403, 2002.

Smooth muscle phenotypic modulation is an early event in aortic aneurysms.

OBJECTIVES Vascular smooth muscle cells can undergo profound changes in phenotype, defined by coordinated repression of smooth muscle cell marker genes and production of matrix metalloproteinases in response to injury. However, little is known of the role of smooth muscle cells in aortic aneurysms. We hypothesized that smooth muscle cells undergo phenotypic modulation early in the development of aortic aneurysms. METHODS Abdominal aortas from C57B6 mice (n = 79) were perfused with elastase or saline (control) and harvested at 1, 3, 7, or 14 days. Aortas were analyzed by means of quantitative polymerase chain reaction and immunohistochemistry for smooth muscle cell marker genes, including SM22A, smooth muscle alpha-actin, and matrix metalloproteinases 2 and 9. In complimentary experiments human aneurysms (n = 10) and control aorta (n = 10) were harvested at the time of surgical intervention and analyzed. RESULTS By 14 days, aortic diameter was larger after elastase perfusion compared with control diameter (100% +/- 9.6% vs 59.5% +/- 18.9%, P = .0002). At 7 days, elastase-perfused mice had a 78% and 85% reduction in SM22 alpha and smooth muscle alpha-actin expression, respectively, compared with that seen in control animals well before aneurysms were present, and these values remained repressed at 14 days. Immunohistochemistry confirmed less SM22 alpha and smooth muscle alpha-actin in experimental aneurysms at 14 days in concert with increased matrix metalloproteinase 2 and 9 expression at 7 and 14 days. Similarly, human aneurysms had less SM22 alpha and smooth muscle alpha-actin and increased matrix metalloproteinase 2 and 9 staining, compared with control values, as determined by means of quantitative polymerase chain reaction. CONCLUSIONS Aneurysms demonstrate smooth muscle cell phenotypic modulation characterized by downregulation of smooth muscle cell marker genes and upregulation of matrix metalloproteinases. These events in experimental models occur before aneurysm formation. Targeting smooth muscle cells to a reparative phenotype might provide a novel therapy in the treatment of aortic aneurysms.

Serial MRI evaluation of cardiac structure and function in mice after reperfused myocardial infarction

This study evaluated the utility of cardiac MRI for assessing the impact of myocardial infarction (MI) on cardiac structure and function in mice following reperfused 1‐ or 2‐hr occlusions of the left anterior descending coronary artery (LAD). When assessed 1 day after MI, the left ventricular ejection fraction (LVEF) had declined by more than half, and remained depressed for the duration of the study. Furthermore, MI initiated dramatic increases in both LV end‐systolic volume (LVESV) and end‐diastolic volume (LVEDV), with a greater than threefold increase in LVESV and a twofold increase in LVEDV by 4 weeks post‐MI. Transmural LV wall thickening (WTh) analysis revealed that noninfarcted myocardium in the remote septal region exhibited an early deficit in contractile function after MI that transiently resolved by day 7, only to be followed by a late phase of dysfunction that became fully manifest by day 28 post‐MI. In conclusion, MRI allows the serial assessment of cardiac structure and function after MI in mice, with a resolution adequate to document both regional and temporal changes. The application of these imaging techniques in transgenic and knock‐out mice will greatly expedite research aimed at defining the functional roles of individual genes in the pathophysiology of LV remodeling (LVR) after reperfused MI. Magn Reson Med 47:1158–1168, 2002.

Robust Cardiomyocyte-Specific Gene Expression Following Systemic Injection of AAV: In Vivo Gene Delivery Follows a Poisson Distribution

Newly isolated serotypes of AAV readily cross the endothelial barrier to provide efficient transgene delivery throughout the body. However, tissue-specific expression is preferred in most experimental studies and gene therapy protocols. Previous efforts to restrict gene expression to the myocardium often relied on direct injection into heart muscle or intracoronary perfusion. Here, we report an AAV vector system employing the cardiac troponin T (cTnT) promoter. Using luciferase and enhanced green fluorescence protein (eGFP), the efficiency and specificity of cardiac reporter gene expression using AAV serotype capsids: AAV-1, 2, 6, 8 or 9 were tested after systemic administration to 1-week-old mice. Luciferase assays showed that the cTnT promoter worked in combination with each of the AAV serotype capsids to provide cardiomyocyte-specific gene expression, but AAV-9 followed closely by AAV-8 was the most efficient. AAV9-mediated gene expression from the cTnT promoter was 640-fold greater in the heart compared with the next highest tissue (liver). eGFP fluorescence indicated a transduction efficiency of 96% using AAV-9 at a dose of only 3.15 × 1010 viral particles per mouse. Moreover, the intensity of cardiomyocyte eGFP fluorescence measured on a cell-by-cell basis revealed that AAV-mediated gene expression in the heart can be modeled as a Poisson distribution, requiring an average of nearly two vector genomes per cell to attain an 85% transduction efficiency.

Complementary displacement‐encoded MRI for contrast‐enhanced infarct detection and quantification of myocardial function in mice

MRI is emerging as an important modality for assessing myocardial function in transgenic and knockout mouse models of cardiovascular disease, including myocardial infarction (MI). Displacement encoding with stimulated echoes (DENSE) measures myocardial motion at high spatial resolution using phase‐reconstructed images. The current DENSE technique uses inversion recovery (IR) to suppress T1‐relaxation artifacts; however, IR is ill‐suited for contrast‐enhanced infarct imaging in the heart, where multiple T1 values are observed. We have developed a modified DENSE method employing complementary acquisitions for T1‐independent artifact suppression. With this technique, displacement and strain are measured in phase‐reconstructed images, and contrast‐enhanced regions of infarction are depicted in perfectly coregistered magnitude‐reconstructed images. The displacement measurements and T1‐weighted image contrast were validated with the use of a rotating phantom. Modified DENSE was performed in mice (N = 9) before and after MI. Circumferential (Ecc) and radial (Err) strain were measured, and contrast‐enhanced infarcted myocardium was detected by DENSE. At baseline, Ecc was −0.16 ± 0.01 and Err was 0.39 ± 0.07. After MI, Ecc was 0.04 ± 0.02 and Err was 0.03 ± 0.04 in infarcted regions, whereas Ecc was −0.12 ± 0.02 and Err was 0.38 ± 0.09 in noninfarcted regions. In vivo Ecc as determined by DENSE correlated well with Ecc obtained by conventional tag analysis (R = 0.90). Magn Reson Med 51:744–752, 2004.