Description: Objective— Although the connection of oxidative stress and inflammation has been long recognized in diabetes mellitus, the underlying mechanisms are not fully elucidated. This study defined the role of 26S proteasomes in promoting vascular inflammatory response in early diabetes mellitus. Methods and Results— The 26S proteasome functionality, markers of autophagy, and unfolded protein response were assessed in (1) cultured 26S proteasome reporter cells and endothelial cells challenged with high glucose, (2) transgenic reporter (Ub G76V –green fluorescence protein) and wild-type (C57BL/6J) mice rendered diabetic, and (3) genetically diabetic (Akita and OVE26) mice. In glucose-challenged cells, and also in aortic, renal, and retinal tissues from diabetic mice, enhanced 26S proteasome functionality was observed, evidenced by augmentation of proteasome (chymotrypsin-like) activities and reduction in 26S proteasome reporter proteins, accompanied by increased nitrotyrosine-containing proteins. Also, whereas inhibitor of the nuclear factor -light-chain-enhancer of activated B cells α proteins were decreased, an increase was found in nuclear factor -light-chain-enhancer of activated B cells (NF-B) nucleus translocation, which enhanced the NF-B–mediated proinflammatory response, without affecting markers of autophagy or unfolded protein response. Importantly, the alterations were abolished by MG132 administration, small interfering RNA knockdown of PA700 (proteasome activator protein complex), or superoxide scavenging in vivo. Conclusion— Early hyperglycemia enhances 26S proteasome functionality, not autophagy or unfolded protein response, through peroxynitrite/superoxide-mediated PA700-dependent proteasomal activation, which elevates NF- B-mediated endothelial inflammatory response in early diabetes mellitus.

Description: Background and Purpose— The association between hypertension (HTN) and stroke recurrence is unclear, but may be influenced by different subtypes of stroke. This study aims to explore whether HTN contributes to the recurrence of certain subtypes of ischemic stroke (IS). Methods— Data from the China National Stroke Registry was examined and 1-year follow-up data for stroke recurrence was analyzed. Trial of Org 10172 in Acute Stroke Treatment criteria was used to classify the subtypes of all IS. HTN was defined when resting blood pressure was ≥140/90 mm Hg on repeated measurements during hospitalization or a patient had been on antihypertensive medication. Recurrent stroke was defined as a new neurological deficit compatible to IS or intracerebral hemorrhage. The association between HTN and stroke recurrence in patients with different IS subtypes was analyzed by using univariable and multivariable logistic regression models. Results— Of 11 560 patients with IS, 8409 (72.7%) had HTN and 2050 (17.7%) experienced a recurrent stroke within 1 year. Patients with HTN had an insignificantly higher stroke recurrence rate than those without (18.0% versus 17.0%; P =0.21). After stratification by Trial of Org 10172 in Acute Stroke Treatment subtypes, multivariable analysis revealed a significant association between HTN and stroke recurrence in small-artery occlusion subtype (odds ratio, 1.52; 95% confidence interval, 1.03–2.31), but not in the other subtypes (large-artery atherosclerosis: odds ratio, 0.99; 95% confidence interval, 0.81–1.21; cardioembolic: odds ratio, 1.14; 95% confidence interval, 0.75–1.73; other: odds ratio, 0.88; 95% confidence interval, 0.71–1.09). Conclusions— Our results showed that HTN is specifically related to the recurrent strokes in patients with small-vessel diseases, not other subtypes of IS.

Description: Background and Purpose— The effect of flow diverter (FD) on hemodynamic changes observed in aneurysms is inevitably affected by the actual structural configuration of deployed FD. We studied the resultant hemodynamic changes after implantation of FDs using computational fluid dynamic simulations based on micro–computed tomography reconstructions in rabbit aneurysm model. Methods— The FDs by micro–computed tomography images and vascular model based on rabbit-specific angiograms in 14 rabbits were reconstructed for computational fluid dynamic studies, and rabbit-specific inlet flow waveforms were used as boundary conditions. The occluded group (n=10) and unoccluded group (n=4) were divided according to the follow-up angiography. Hemodynamic parameters were separately evaluated for significance with respect to FD implantation and healing. Results— The normalized mean wall shear stress of the aneurysm sac and inflow volume were significantly reduced after FD deployment, and the relative residence time was significantly increased after treatment, without significant differences in mean pressure of aneurysm sac. When compared with the unoccluded group, the average relative residence time increment and percentage of inflow volume reduction in occluded group were higher. Additionally, the inlet of stream after FD deployment in the occluded group was more prevalent near the central region of the neck, whereas in the unoccluded group, it was more likely to occur near the proximal part of the neck. Conclusions— This study provided the real structural configurations of fully deployed FDs in vivo. We demonstrated the decrease of wall shear stress, inflow volume, increase of relative residence time, and change of inflow stream induced by FD implantation. The higher relative residence time increment, percentage of inflow volume reduction, and location of stream inlet near the central part of the neck may be closely related to healing.

Description: Elevated interleukin-4 (IL-4) levels are associated with cardiac fibrosis in hypertension and heart failure in both patients and experimental animals. We hypothesized that chronically elevated IL-4 induces cardiac fibrosis, resulting in a predisposition of the heart to angiotensin II–induced damage. Wild-type Balb/c (WT, high circulating IL-4) and IL-4–deficient Balb/c mice (IL-4 –/– ) were used. WT mice exhibited cardiac fibrosis (evidenced by an increase in expression of procollagen genes/interstitial collagen fraction), enlarged left ventricle chamber, and declined cardiac function associated with a greater number of mast cells and macrophages in the heart compared with IL-4 –/– . In contrast, IL-4 –/– mice had normal cardiac architecture/function while showing a 57.9% reduction in heart interstitial collagen compared with WT, despite elevated proinflammatory cytokines in heart tissue. In response to angiotensin II administration, IL-4 –/– had reduced interstitial myocardial fibrosis and were protected from developing dilated cardiomyopathy, which was seen in WT mice. This was associated with increased macrophage infiltration into the hearts of WT mice, despite a similar degree of hypertension and increased cardiac transforming growth factor-β1 in both groups. In vitro data demonstrated that IL-4 upregulates procollagen genes and stimulates collagen production in mouse cardiac fibroblasts. This process is mediated by signal transducer and activator of transcription 6 signaling pathway via IL-4 receptor alpha. This study not only establishes a causal relationship between IL-4 and cardiac fibrosis/dysfunction, but also reveals a critical role for IL-4 in angiotensin II–induced cardiac damage. IL-4 could serve as an additional target for the treatment of cardiac fibrosis.

Description: Rationale: Wnt/β-catenin signaling has an important role in the angiogenic activity of endothelial cells (ECs). Bach1 is a transcription factor and is expressed in ECs, but whether Bach1 regulates angiogenesis is unknown. Objective: This study evaluated the role of Bach1 in angiogenesis and Wnt/β-catenin signaling. Methods and Results: Hind-limb ischemia was surgically induced in Bach1 –/– mice and their wild-type littermates and in C57BL/6J mice treated with adenoviruses coding for Bach1 or GFP. Lack of Bach1 expression was associated with significant increases in perfusion and vascular density and in the expression of proangiogenic cytokines in the ischemic hindlimb of mice, with enhancement of the angiogenic activity of ECs (eg, tube formation, migration, and proliferation). Bach1 overexpression impaired angiogenesis in mice with hind-limb ischemia and inhibited Wnt3a-stimulated angiogenic response and the expression of Wnt/β-catenin target genes, such as interleukin-8 and vascular endothelial growth factor, in human umbilical vein ECs. Interleukin-8 and vascular endothelial growth factor were responsible for the antiangiogenic response of Bach1. Immunoprecipitation and GST pull-down assessments indicated that Bach1 binds directly to TCF4 and reduces the interaction of β-catenin with TCF4. Bach1 overexpression reduces the interaction between p300/CBP and β-catenin, as well as β-catenin acetylation, and chromatin immunoprecipitation experiments confirmed that Bach1 occupies the TCF4-binding site of the interleukin-8 promoter and recruits histone deacetylase 1 to the interleukin-8 promoter in human umbilical vein ECs. Conclusions: Bach1 suppresses angiogenesis after ischemic injury and impairs Wnt/β-catenin signaling by disrupting the interaction between β-catenin and TCF4 and by recruiting histone deacetylase 1 to the promoter of TCF4-targeted genes.

Description: Background The "obesity paradox" reflects an observed relationship between obesity and decreased morbidity and mortality, suggesting improved health outcomes for obese individuals. Studies examining the relationship between high body mass index (BMI) and adverse outcomes after cardiac surgery have reported conflicting results. Methods and Results The study population (N=78 762) was comprised of adult patients who had undergone first-time coronary artery bypass (CABG) or combined CABG/aortic valve replacement (AVR) surgery from April 1, 1998 to October 31, 2011 in Ontario (data from the Institute for Clinical Evaluative Sciences). Perioperative outcomes and 5-year mortality among pre-defined BMI (kg/m 2 ) categories (underweight 〈20, normal weight 20 to 24.9, overweight 25 to 29.9, obese 30 to 34.9, morbidly obese 〉34.9) were compared using Bivariate analyses and Cox multivariate regression analysis to investigate multiple confounders on the relationship between BMI and adverse outcomes. A reverse J-shaped curve was found between BMI and mortality with their respective hazard ratios. Independent of confounding variables, 30-day, 1-year, and 5-year survival rates were highest for the obese group of patients (99.1% [95% Confidence Interval {CI}, 98.9 to 99.2], 97.6% [95% CI, 97.3 to 97.8], and 90.0% [95% CI, 89.5 to 90.5], respectively), and perioperative complications lowest. Underweight and morbidly obese patients had higher mortality and incidence of adverse outcomes. Conclusions Overweight and obese patients had lower mortality and adverse perioperative outcomes after cardiac surgery compared with normal weight, underweight, and morbidly obese patients. The "obesity paradox" was confirmed for overweight and moderately obese patients. This may impact health resource planning, shifting the focus to morbidly obese and underweight patients prior to, during, and after cardiac surgery.

Description: Background Toll-like receptor 3 (TLR3) was originally identified as the receptor for viral RNA and represents a major host antiviral defense mechanism. TLR3 may also recognize extracellular RNA (exRNA) released from injured tissues under certain stress conditions. However, a role for exRNA and TLR3 in the pathogenesis of myocardial ischemic injury has not been tested. This study examined the role of exRNA and TLR3 signaling in myocardial infarction (MI), apoptosis, inflammation, and cardiac dysfunction during ischemia-reperfusion (I/R) injury. Methods and Results Wild-type (WT), TLR3 –/– , Trif –/– , and interferon (IFN) α/β receptor-1 deficient (IFNAR1 –/– ) mice were subjected to 45 minutes of coronary artery occlusion and 24 hours of reperfusion. Compared with WT, TLR3 –/– or Trif –/– mice had smaller MI and better preserved cardiac function. Surprisingly, unlike TLR(2/4)-MyD88 signaling, lack of TLR3-Trif signaling had no impact on myocardial cytokines or neutrophil recruitment after I/R, but myocardial apoptosis was significantly attenuated in Trif –/– mice. Deletion of the downstream IFNAR1 had no effect on infarct size. Importantly, hypoxia and I/R led to release of RNA including microRNA from injured cardiomyocytes and ischemic heart, respectively. Necrotic cardiomyocytes induced a robust and dose-dependent cytokine response in cultured cardiomyocytes, which was markedly reduced by RNase but not DNase, and partially blocked in TLR3-deficient cardiomyocytes. In vivo, RNase administration reduced serum RNA level, attenuated myocardial cytokine production, leukocytes infiltration and apoptosis, and conferred cardiac protection against I/R injury. Conclusion TLR3-Trif signaling represents an injurious pathway during I/R. Extracellular RNA released during I/R may contribute to myocardial inflammation and infarction.

Description: Rationale: AMP-activated protein kinase is a master regulator of cell metabolism and an attractive drug target for cancer and metabolic and cardiovascular diseases. Point mutations in the regulatory 2-subunit of AMP-activated protein kinase (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular preexcitation, and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the use of AMP-activated protein kinase as a drug target. Objective: We sought to identify the pro–growth-signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage. Methods and Results: In a mouse model of N488I mutation of the Prkag2 gene (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate–stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular preexcitation but did not affect the excessive cardiac growth in R2M mice. The progrowth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mammalian target of rapamycin and inactivation of forkhead box O transcription factor–signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mammalian target of rapamycin activity by rapamycin or restoration of forkhead box O transcription factor activity by overexpressing forkhead box O transcription factor 1 rescued the abnormal cardiac growth. Conclusions: Our study reveals a novel mechanism for Prkag2 cardiomyopathy, independent of glycogen storage. The role of 2-AMP-activated protein kinase in cell growth also has broad implications in cardiac development, growth, and regeneration.

Description: The activation of angiotensin II type 2 receptor (AT 2 R) has been considered cardioprotective. However, there are controversial findings regarding the role of overexpressing AT 2 R in the heart. Using transgenic mice with different levels of AT 2 R gene overexpression in the heart (1, 4, or 9 copies of the AT 2 R transgene: Tg 1 , Tg 4 , or Tg 9 ), we studied the effect of AT 2 R overexpression on left ventricular remodeling and dysfunction post–myocardial infarction (MI). Tg 1 , Tg 4 , Tg 9 , and their wild-type littermates were divided into (1) sham MI, (2) MI plus vehicle, and (3) MI plus AT 2 R antagonist. Treatments were started 4 weeks after MI and continued for 8 weeks. AT 2 R protein and mRNA expression in the heart was significantly increased in transgenic mice, and the increase positively correlated with copies of the transgene. AT 1 R protein and mRNA expression remained unchanged in Tg 1 and Tg 4 but slightly increased in Tg 9 mice. Systolic blood pressure and cardiac phenotypes did not differ among strains under basal conditions. MI caused myocardial hypertrophy, interstitial fibrosis, ventricular dilatation, and dysfunction associated with increased protein expression of Nox2 and transforming growth factor β 1 . These pathological responses were diminished in Tg 1 and Tg 4 mice. Moreover, the protective effects of AT 2 R were abolished by AT 2 R antagonist and also absent in Tg 9 mice. We thus conclude that whether overexpression of AT 2 R is beneficial or detrimental to the heart is largely dependent on expression levels and possibly via regulations of Nox2 and transforming growth factor β 1 signaling pathways.