Description: Background— Two endophenotypes of arterial calcification, calcification on arterial wall and calcification in atherosclerotic plaques, are associated with different types of cardiovascular events. Mgp -deficient mice showed matrix Gla protein (MGP) is strongly associated with calcification on arterial wall without atherosclerotic plaques, and MGP variants were not significantly associated with myocardial infarction. MGP may play different roles in the 2 endophenotypes. Methods and Results— We analyzed the associations of MGP variants rs4236, rs1800801, and rs1800802 with the 2 endophenotypes determined by multidetector computed tomography angiography. A total of 585 with calcification on coronary artery wall, 675 with calcification in coronary atherosclerotic plaques, 454 with calcification on aortic wall, and 725 controls were enrolled. After Bonferroni correction, rs4236 and rs1800801 were still associated with calcification on arterial wall, the odds ratios were 0.708 (95% confidence interval, 0.540–0.928) for rs4236 and 0.652 (95% confidence interval, 0.479–0.888) for rs1800801 in coronary artery wall calcification, and 0.699 (95% confidence interval, 0.525–0.931) for rs4236 and 0.650 (95% confidence interval, 0.467–0.905) for rs1800801 in aortic wall calcification, respectively. The variants were correlated with calcification severity by ln(CAC Agatston score+1) in coronary artery wall calcification but not in atherosclerotic plaque calcification. In accordance with their associations with calcification on arterial wall, rs4236C and rs1800801A were associated with higher MGP plasma levels, whereas rs1800802C was associated with lower MGP levels in normal controls. Because of the role of calcification in plaque vulnerability, their associations with acute myocardial infarction were also determined in 771 controls and 752 patients, no association was found. Conclusions— MGP genetic variants showed association with calcification on arterial wall but not with calcification in atherosclerotic plaques.

Description: Primary hypertension is a chronic disease with high morbidity, and the rate of controlled blood pressure is far from satisfactory, worldwide. Vaccination provides a promising approach for treatment of hypertension and improvement in compliance. Here, the ATRQβ-001 vaccine, a peptide (ATR-001) derived from human angiotensin II (Ang II) receptor type 1 conjugated with Qβ bacteriophage virus-like particles, was developed and evaluated in animal models of hypertension. The ATRQβ-001 vaccine significantly decreased the blood pressure of Ang II–induced hypertensive mice up to 35 mm Hg (143±4 versus 178±6 mm Hg; P =0.005) and that of spontaneously hypertensive rats up to 19 mm Hg (173±2 versus 192±3 mm Hg; P =0.003) and prevented remodeling of vulnerable hypertensive target organs. No obvious feedback activation of circulating or local renin-angiotensin system was observed. Additionally, no significant immune-mediated damage was detected in vaccinated hypertensive and nonhypertensive animals. The half-life of the anti-ATR-001 antibody was 14.4 days, surpassing that of existing chemical drugs. In vitro, the anti–ATR-001 antibody specifically bound to Ang II receptor type 1 and inhibited Ca 2+ -dependent signal transduction events, including protein kinase C-α translocation, extracellular signal-regulated kinase 1/2 phosphorylation (72% decrease; P =0.013), and elevation of intracellular Ca 2+ (68% decrease; P =0.017) induced by Ang II, but without inhibiting Ang II binding to the receptor. In conclusion, the ATRQβ-001 vaccine decreased the blood pressure of Ang II–induced hypertensive mice and spontaneously hypertensive rats effectively through diminishing the pressure response and inhibiting signal transduction initiated by Ang II. Thus, the ATRQβ-001 vaccine may provide a novel and promising method for the treatment of primary hypertension.

Description: Glucagonlike peptide (GLP-1) and its receptor (GLP-1R) exhibit cardioprotective effects after myocardial ischemia and reperfusion (MI/R) in both animal studies and clinical trials. However, the kinetics of GLP-1R expression in the infarcted/ischemic myocardium has not yet been explored. The purpose of this study was to monitor the presence and time course of regional myocardial GLP-1R expression after MI/R with noninvasive PET. Methods: Male Sprague–Dawley rats underwent a 45-min transient left coronary artery occlusion, followed by reperfusion. The myocardial infarction was confirmed by electrocardiogram and cardiac ultrasound. In vivo PET was performed to determine myocardial uptake of 18 F-FBEM-Cys 40 -exendin-4 at different time points after reperfusion. The localization of 18 F-FBEM-Cys 40 -exendin-4 accumulation was determined by coregistering 18 F-FDG PET and CT images. Ex vivo autoradiography, GLP-1R immunohistochemical staining, and Western blot analysis were performed to confirm the PET results. Results: Myocardial origin and infarcted/ischemic area localization of 18 F-FBEM-Cys 40 -exendin-4 accumulation was confirmed by coregistration of small-animal CT and 18 F-FDG images. At 8 h after MI/R, tracer uptake in the infarcted/ischemic region was 0.37 ± 0.05 percentage injected dose per gram, significantly higher than that in the control group ( P 〈 0.01). The localized tracer uptake decreased, relative to the 8-h time point, but was still significantly higher than the control group on days 1 and 3 after MI/R. At 2 wk after MI/R, the tracer uptake in the affected area showed no significant difference, compared with that in the healthy myocardium. Autoradiography showed the same trend of 18 F-FBEM-Cys 40 -exendin-4 uptake in the myocardial infarcted/ischemic area. The specificity of tracer uptake into ischemic myocardium was supported by decreased tracer uptake after the rats were pretreated with an excess amount of unlabeled exendin-4. Immunohistochemical staining and Western blotting of GLP-1R protein of excised cardiac sections confirmed that the change in uptake observed by PET corresponded to a change in GLP-1R expression. Conclusion: Noninvasive PET using 18 F-FBEM-Cys 40 -exendin-4 revealed a dynamic pattern of GLP-1R upregulation in the infarcted/ischemic area after MI/R. The imaging results will deepen our understanding of the mechanism of the cardioprotective effect of GLP-1 and its analogs and potentially provide guidance for optimization of the time frame of therapeutic intervention.

Description: Objective— The development of a murine model of spontaneous atherosclerotic plaque rupture with luminal thrombus. Methods and Results— Combined partial ligation of the left renal artery and left common carotid artery in 8-week-old apolipoprotein E–deficient mice induced endogenous renovascular hypertension and local low oscillatory shear stress in the left common carotid artery. After 8 weeks, a fresh left common carotid artery lumen thrombus associated with severe plaque burden was found in 50% (10/20) of the mice. Histological analyses indicated that all left common carotid artery lesions had vulnerable features, and 50% (5/10) of the mice showed plaque rupture with a lumen thrombus. Multiple layers with layering discontinuity and intraplaque hemorrhages were found in 80% (8/10) of the mice. Further experiments showed that both increased blood pressure, and angiotensin-II contributed to plaque progression and vulnerability. Decreased intimal collagen associated with increased collagenase activity and matrix metalloproteinase expression also resulted in plaque disruption. Conclusion— We demonstrate a murine model of spontaneous plaque rupture with a high incidence of luminal thrombus. The model not only nicely recapitulates the pathophysiological processes of human plaque rupture but it is also simple, fast, and highly efficient to generate.

Description: Rationale: Transplantation of stem cells into damaged hearts has had modest success as a treatment for ischemic heart disease. One of the limitations is the poor stem cell survival in the diseased microenvironment. Prolyl hydroxylase domain protein 2 (PHD2) is a cellular oxygen sensor that regulates 2 key transcription factors involved in cell survival and inflammation: hypoxia-inducible factor and nuclear factor-B. Objective: We studied whether and how PHD2 silencing in human adipose-derived stem cells (ADSCs) enhances their cardioprotective effects after transplantation into infarcted hearts. Methods and Results: ADSCs were transduced with lentiviral short hairpin RNA against prolyl hydroxylase domain protein 2 (shPHD2) to silence PHD2. ADSCs, with or without shPHD2, were transplanted after myocardial infarction in mice. ADSCs reduced cardiomyocyte apoptosis, fibrosis, and infarct size and improved cardiac function. shPHD2-ADSCs exerted significantly more protection. PHD2 silencing induced greater ADSC survival, which was abolished by short hairpin RNA against hypoxia-inducible factor-1α. Conditioned medium from shPHD2-ADSCs decreased cardiomyocyte apoptosis. Insulin-like growth factor-1 (IGF-1) levels were significantly higher in the conditioned medium of shPHD2-ADSCs versus ADSCs, and depletion of IGF-1 attenuated the cardioprotective effects of shPHD2-ADSC–conditioned medium. Nuclear factor-B activation was induced by shPHD2 to induce IGF-1 secretion via binding to IGF-1 gene promoter. Conclusions: PHD2 silencing promotes ADSCs survival in infarcted hearts and enhances their paracrine function to protect cardiomyocytes. The prosurvival effect of shPHD2 on ADSCs is hypoxia-inducible factor-1α dependent, and the enhanced paracrine function of shPHD2-ADSCs is associated with nuclear factor-B–mediated IGF-1 upregulation. PHD2 silencing in stem cells may be a novel strategy for enhancing the effectiveness of stem cell therapy after myocardial infarction.

Description: Background and Purpose— A novel quantitative susceptibility mapping (QSM) processing technology has been developed to map tissue susceptibility property without blooming artifacts. We hypothesize that hematoma volume measurement on QSM is independent of imaging parameters, eliminating its echo time dependence on gradient echo MRI. Methods— Gradient echo MRI of 16 patients with intracerebral hemorrhage was processed with susceptibility-weighted imaging, R 2 * (=1/T2*) mapping, and QSM at various echo times. Hematoma volumes were measured from these images. Results— Linear regression of hematoma volume versus echo time showed substantial slopes for gradient echo magnitude (0.45±0.31 L/s), susceptibility-weighted imaging (0.52±0.46), and R 2 * (0.39±0.30) but nearly zero slope for QSM (0.01±0.05). At echo time=20 ms, hematoma volume on QSM was 0.80 x that on gradient echo magnitude image ( R 2 =0.99). Conclusions— QSM can provide reliable measurement of hematoma volume, which can be performed rapidly and accurately using a semiautomated segmentation tool.

Description: Objective— This study determined the role of angiotensin-converting enzyme (ACE) on the development of angiotensin I–induced atherosclerosis and the contribution of leukocyte-specific expression of this enzyme. Approach and Results— To define the contribution of ACE-dependent activity to angiotensin II synthesis in atherosclerotic development, male low-density lipoprotein receptor –/– mice were fed a fat-enriched diet and infused with either angiotensin I or angiotensin II. The same infusion rate of these peptides had equivalent effects on atherosclerotic development. Coinfusion of an ACE inhibitor, enalapril, ablated angiotensin I–augmented atherosclerosis but had no effect on angiotensin II–induced lesion development. ACE protein was detected in several cell types in atherosclerotic lesions, with a predominance in macrophages. This cell type secreted angiotensin II, which was ablated by ACE inhibition. To study whether leukocyte ACE contributed to atherosclerosis, irradiated male low-density lipoprotein receptor –/– mice were repopulated with bone marrow–derived cells from either ACE +/+ or ACE –/– mice and fed the fat-enriched diet for 12 weeks. Chimeric mice with ACE deficiency in bone marrow–derived cells had modestly reduced atherosclerotic lesions in aortic arches but had no effects in aortic roots. Conclusions— ACE mediates angiotensin I–induced atherosclerosis, and ACE expression in leukocytes modestly contributes to atherosclerotic development in hypercholesterolemic mice.

Description: The purpose of this study was to develop a novel in vivo albumin-labeling method to allow PET of cardiac function after myocardial infarction and vascular leakage and increased permeability in inflammatory diseases and malignant tumors. Methods: To label albumin in vivo, we synthesized a NOTA (1,4,7-triazacyclononane- N,N',N'' -triacetic acid)-conjugated truncated form of Evans blue (NEB). 18 F labeling was achieved by the formation of an 18 F-aluminum fluoride ( 18 F-AlF) complex, and 64 Cu labeling was obtained by a standard chelation method. Sixty-minute dynamic PET imaging was performed on normal mice to evaluate the distribution of 18 F-AlF-NEB, which was compared with in vitro–labeled mouse serum albumin ( 18 F-fluorobenzyl-MSA). Electrocardiography-gated PET imaging was performed in a mouse model of myocardial infarction. Both dynamic and static PET scans were obtained in a mouse inflammation model induced by local injection of turpentine to evaluate vascular leakage. Tumor permeability was studied by dynamic and late-point static PET using 64 Cu-NEB in a UM-22B xenograft model. Results: NEB was successfully synthesized, and 18 F labeling including work-up took about 20–30 min, with a radiochemical purity greater than 95% without the need for high-performance liquid chromatography purification. Most of the radioactivity was retained in the circulation system at 60 min after injection (26.35 ± 1.52 percentage injected dose per gram [%ID/g]). With electrocardiography-gated PET, ventricles of the heart and major arteries were clearly visualized. The myocardial infarction mice showed much lower left ventricular ejection fraction than the control mice. Inflammatory muscles showed significantly higher tracer accumulation than the contralateral healthy ones. UM-22B tumor uptake of 64 Cu-NEB gradually increased with time (5.73 ± 1.11 %ID/g at 1 h and 8.03 ± 0.77 %ID/g at 2 h after injection). Conclusion: The distribution and local accumulation of serum albumin can be noninvasively visualized and quantified by 18 F-AlF-NEB and 64 Cu-NEB PET. The simple labeling and broad applications make these imaging probes attractive for clinical translation.

Description: Interferon regulatory factor 1 (IRF1), a critical member of the IRF family, was previously shown to be associated with the immune system and to be involved in apoptosis and tumor suppression. However, the role of IRF1 in pressure overload–induced cardiac remodeling has remained unclear. Using genetic approaches, we established a central role for the IRF1 transcription factor in the regulation of cardiac remodeling both in vivo and in vitro, and we determined the mechanism underlying this process. The expression level of IRF1 was remarkably altered in both failing human hearts and hypertrophic murine hearts. Transgenic mice with cardiac-specific IRF1 overexpression exacerbated aortic banding–induced cardiac hypertrophy, ventricular dilation, fibrosis, and dysfunction, whereas IRF1-deficient (knockout) mice exhibited a significant reduction in the hypertrophic response. Similar results were observed in a global IRF1-knockout rat model. Mechanistically, the prohypertrophic effects elicited by IRF1 in response to pathological stimuli were associated with the direct activation of inducible nitric oxide synthase (iNOS). Furthermore, we identified 1 IRF1-binding site in the promoter region of the iNOS gene, which was essential for its transcription. To examine the IRF1-iNOS axis in vivo, we generated IRF1-transgenic/iNOS-knockout mice. IRF1 exerted profoundly detrimental effects in these mice; however, these effects were nullified by iNOS ablation. These data suggest the IRF1–iNOS axis as a crucial regulator of cardiac remodeling and that IRF1 could be a potent therapeutic target for cardiac remodeling.

Description: Rationale: Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β super family of secreted factors. A recent study showed that reduced GDF11 blood levels with aging was associated with pathological cardiac hypertrophy (PCH) and restoring GDF11 to normal levels in old mice rescued PCH. Objective: To determine whether and by what mechanism GDF11 rescues aging dependent PCH. Methods and Results: Twenty-four–month-old C57BL/6 mice were given a daily injection of either recombinant (r) GDF11 at 0.1 mg/kg or vehicle for 28 days. rGDF11 bioactivity was confirmed in vitro. After treatment, rGDF11 levels were significantly increased, but there was no significant effect on either heart weight or body weight. Heart weight/body weight ratios of old mice were not different from 8- or 12-week-old animals, and the PCH marker atrial natriuretic peptide was not different in young versus old mice. Ejection fraction, internal ventricular dimension, and septal wall thickness were not significantly different between rGDF11 and vehicle-treated animals at baseline and remained unchanged at 1, 2, and 4 weeks of treatment. There was no difference in myocyte cross-sectional area rGDF11 versus vehicle-treated old animals. In vitro studies using phenylephrine-treated neonatal rat ventricular myocytes, to explore the putative antihypertrophic effects of GDF11, showed that GDF11 did not reduce neonatal rat ventricular myocytes hypertrophy, but instead induced hypertrophy. Conclusions: Our studies show that there is no age-related PCH in disease-free 24-month-old C57BL/6 mice and that restoring GDF11 in old mice has no effect on cardiac structure or function.