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Aims/hypothesis: A hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier. Methods: sEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors. Results: Using intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell–cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state. Conclusions/interpretation: Diabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes. [ABSTRACT FROM AUTHOR]

Introduction: People with type 2 diabetes (T2D) have a higher risk of stroke and worse outcomes than those without T2D. Pooled data from randomized controlled trials indicate that the glucagon-like peptide 1 receptor agonist semaglutide is associated with stroke risk reduction in people with T2D at high cardiovascular risk. We compared real-world stroke risk in people with T2D or T2D plus atherosclerotic cardiovascular disease (ASCVD) initiating either semaglutide or a dipeptidyl peptidase 4 inhibitor (DPP4i). Methods: Adults (≥ 18 years old) in a US claims database with a claim indicating initiation of either semaglutide or a DPP4i (index date) during the index period (1 January 2018–30 September 2020), a diagnosis code for T2D on or before the index date and at least 12 months' continuous enrolment in the database pre-index were included and propensity score matched 1:1 on baseline demographic and clinical characteristics. The primary outcome was time to first stroke event during follow-up. Healthcare resource utilization was also compared between groups. Results: The analysis included 17,920 matched pairs with T2D and 4234 matched pairs with T2D and ASCVD. The groups were well matched on baseline characteristics. People initiating semaglutide had a lower risk of stroke over short-term follow-up than those initiating a DPP4i (T2D: hazard ratio 0.63 [95% confidence interval 0.41–0.95], p = 0.029; T2D plus ASCVD: 0.45 [0.24–0.86], p = 0.015). Semaglutide was also associated with a lower rate of inpatient, outpatient and emergency room visits compared with a DPP4i. Conclusion: This proof-of-concept analysis indicates that semaglutide has the potential to reduce the risk of stroke in people with T2D when prescribed in clinical practice. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis is a key aspect of heart failure, leading to reduced ventricular compliance and impaired electrical conduction in the myocardium. Various pathophysiologic conditions can lead to fibrosis in the left ventricle (LV) and/or right ventricle (RV). Despite growing evidence to support the transcriptomic heterogeneity of cardiac fibroblasts (CFs) in healthy and diseased states, there have been no direct comparisons of CFs in the LV and RV. Given the distinct natures of the ventricles, we hypothesized that LV- and RV-derived CFs would display baseline transcriptomic differences that influence their proliferation and differentiation following injury. Bulk RNA sequencing of CFs isolated from healthy murine left and right ventricles indicated that LV-derived CFs may be further along the myofibroblast transdifferentiation trajectory than cells isolated from the RV. Single-cell RNA-sequencing analysis of the two populations confirmed that Postn+ CFs were more enriched in the LV, whereas Igfbp3+ CFs were enriched in the RV at baseline. Notably, following pressure overload injury, the LV developed a larger subpopulation of pro-fibrotic Thbs4+/Cthrc1+ injury-induced CFs, while the RV showed a unique expansion of two less-well-characterized CF subpopulations (Igfbp3+ and Inmt+). These findings demonstrate that LV- and RV-derived CFs display baseline subpopulation differences that may dictate their diverging responses to pressure overload injury. Further study of these subpopulations will elucidate their role in the development of fibrosis and inform on whether LV and RV fibrosis require distinct treatments. [ABSTRACT FROM AUTHOR]

The high NLR tertile was associated with a 74% increased risk of expanded MACE and a twofold increased risk of CV death and all-cause death (secondary outcomes) vs. the low NLR tertile (Figure S2A). Antidiabetic drug, cardiovascular disease, GLP-1 analogue, randomized trial, type 2 diabetes Keywords: antidiabetic drug; cardiovascular disease; GLP-1 analogue; randomized trial; type 2 diabetes EN antidiabetic drug cardiovascular disease GLP-1 analogue randomized trial type 2 diabetes 2398 2401 4 07/04/23 20230801 NES 230801 INTRODUCTION Inflammation plays a critical role in the pathobiology of cardiometabolic diseases.[[1]] Neutrophils participate in this inflammatory response by releasing inflammatory mediators that cause vascular wall dysfunction; lymphocytes can augment or mute inflammatory responses, depending on the subtype.[2] Studies suggest that the neutrophil to lymphocyte ratio (NLR) may more accurately predict cardiovascular (CV) events than leukocyte counts,[3] and that NLR can predict CV disease (CVD) risk and all-cause mortality in diverse populations,[2], [4], [5], [6] including people with type 2 diabetes (T2D).[7], [8], [9], [10] The SUSTAIN 6[11] and PIONEER 6[12] trials investigated CV outcomes with subcutaneous and oral semaglutide, respectively, versus placebo in people with T2D at high CV risk. [Extracted from the article]

Aim: Glucagon‐like peptide‐1 receptor agonists improve glycaemic control: some are now available as oral and subcutaneous formulations, and some have indications for reducing cardiovascular risk. The expanded scope for these therapies warrants comprehensive safety evaluations. We report the safety/tolerability of subcutaneous and oral semaglutide from the SUSTAIN and PIONEER clinical trial programmes, respectively. Materials and methods: Adverse events (AEs) from 16 randomized placebo‐ or active‐controlled phase IIIa trials in patients with type 2 diabetes (n = 11 159) including once‐weekly subcutaneous semaglutide (n = 3150; SUSTAIN trials) or once‐daily oral semaglutide (n = 4116; PIONEER trials) were analysed. Data pools were analysed for each programme, with separate analyses of cardiovascular outcomes trials (CVOTs; n = 6480). Results: In the phase IIIa pools, gastrointestinal disorders were reported in 41.9%/39.1% of patients with subcutaneous/oral semaglutide, respectively (most prevalent during initiation/escalation) versus 22.0%/24.8% with comparators. Rates of kidney disorders, acute pancreatitis, malignant neoplasms, hypoglycaemia, diabetic retinopathy, heart failure and other cardiovascular events were similar for semaglutide versus comparators. Cholelithiasis incidence was higher with subcutaneous and oral semaglutide versus placebo. Diabetic retinopathy incidence was higher with subcutaneous semaglutide versus placebo in SUSTAIN 6. Small pulse rate increases occurred with both formulations; there was no increased rate of arrhythmias. Fatal AE incidence was similar between semaglutide and comparators. Versus placebo, CVOTs showed a reduced risk of major adverse cardiovascular events with subcutaneous semaglutide and non‐inferiority criteria were met with oral semaglutide. Conclusions: The most common AEs with semaglutide were gastrointestinal disorders, which decreased with continued therapy. These comprehensive safety/tolerability data may better inform patient selection and guidance in care. [ABSTRACT FROM AUTHOR]

Previous studies have established that transmural gradients of the fast transient outward K+ current (Ito,f) correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high Ito,f expression in the epimyocardium (EPI) being associated with short APs and low contractility and vice versa. Herein, we investigated the effects of altering the Ito,f gradients on transmural contractile properties using mice lacking Irx5 (Irx5-KO) or lacking Kcnd2 (KV4.2-KO) or both. Irx5- KO mice exhibited decreased global LV contractility in association with elevated Ito,f, as well as reduced cell shortening and Ca2+ transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of Irx5 ablation on ECC-related genes was to increase Ito,f gene expression (i.e., Kcnd2 and Kcnip2) in the ENDO, but not the EPI. By contrast, KV4.2-KO mice showed selective increases in cell shortening and Ca2+ transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both Irx5 and Kcnd2 displayed elevated ventricular contractility, comparable to KV4.2-KO mice, demonstrating a dominant role of Irx5- dependent modulation of Ito,f in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the Irx5-dependent Ito,f gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart. [ABSTRACT FROM AUTHOR]

Background: Cardiovascular outcome trials (CVOTs) are conducted on a background of standard of care including metformin. These analyses sought to determine whether the cardiovascular (CV) effects of semaglutide and other glucagon-like peptide-1 receptor agonists (GLP-1RAs) vary according to baseline metformin use. Methods: A post hoc analysis was conducted using pooled SUSTAIN 6 and PIONEER 6 CVOT data in subjects with and without metformin use at baseline. Additionally, a trial-level meta-analysis was conducted using data from seven CVOTs with GLP-1RAs–SUSTAIN 6, PIONEER 6, HARMONY OUTCOMES, LEADER, REWIND, EXSCEL and AMPLITUDE-O–including adults with type 2 diabetes at high CV risk, and a primary endpoint of time to first major adverse CV event (MACE). Results: In the post hoc analysis, the no-metformin subgroup was older, with a higher body mass index, lower estimated glomerular filtration rate and higher CV risk at baseline vs the metformin subgroup. Hazard ratios (95% confidence intervals) for the reduction in risk of MACE with semaglutide vs placebo in the metformin and no-metformin subgroups were 0.70 (0.55;0.89) and 0.86 (0.60;1.22), respectively. No significant interaction between the treatment effect on MACE and metformin subgroup was observed. Findings for other CV endpoints were similar. In the meta-analysis, treatment effect (GLP-1RA vs placebo) on CV outcomes was no different with vs without baseline metformin (overall ratio between the hazard ratios for metformin vs no-metformin 1.09 [0.96;1.22]). Conclusion: These findings indicate that the CV outcomes for semaglutide were similar regardless of baseline metformin use, which may also apply to all GLP-1RAs. Trial registration SUSTAIN 6 (NCT01720446), PIONEER 6 (NCT02692716). [ABSTRACT FROM AUTHOR]

Objectives: The present cost-consequence analysis compared estimated hospitalization costs in a Canadian setting with insulin degludec (degludec) versus insulin glargine 100 units/mL (glargine U100) in patients with type 2 diabetes (T2D) at high cardiovascular (CV) risk. Methods: Medical terms were mapped across the different vocabularies, in order to assign unit costs from eligible hospital abstracts in Canadian Institute for Health Information data (International Statistical Classification of Diseases and Related Health Problems, 10th Revision, Canada) to serious adverse events (SAEs; Medical Dictionary for Regulatory Activities) from the randomized DEVOTE trial comparing the two insulins degludec and glargine. Mean annual costs of SAE-related hospitalizations were estimated by treatment, the cost difference (degludec-glargine U100) was bootstrapped to compute confidence intervals (CIs) and p-values, and the cost ratio (degludec/glargine U100) was estimated using a Tweedie distribution. Results: The mean annual cost per patient for SAE-related hospitalizations was 4,074 CAD with degludec and 4,569 CAD with glargine U100 (cost difference: -495, 95% confidence interval [CI]: -966; -24, p=.039), for a cost ratio of 0.89 (95% CI: 0.81; 0.98, p=.016). Overall, cost ratios from sensitivity analyses varying individual methodological assumptions were consistent with the main analysis. Of the system organ classes from DEVOTE SAEs, cardiac disorders were the largest contributor to the costs savings with degludec versus glargine U100. Conclusions: In patients with T2D at high CV risk, our findings suggest that there are likely to be lower hospitalization costs with degludec versus glargine U100 based on the SAEs observed in DEVOTE and in a Canadian setting. [ABSTRACT FROM AUTHOR]

In the REWIND trial, dulaglutide reduced cardiovascular (CV) risk versus placebo in patients with type 2 diabetes in both the "established CV disease" (CVD) and "CV risk factor" subgroups. The SUSTAIN 6 and PIONEER 6 trials of semaglutide used different criteria for established CVD from those used in REWIND. The present post hoc analysis assessed the effect of semaglutide on major adverse CV events (MACE) in a pooled population of SUSTAIN 6 and PIONEER 6 patients, re‐categorized into CV risk subgroups using the REWIND CVD criteria. In the pooled analysis (n = 6480), a lower percentage of patients were in the established CVD subgroup, when using the REWIND CVD criteria, compared with the original trial CVD criteria (66.5% vs. 83.8%, respectively). After re‐categorization, the risk of MACE was significantly lower with semaglutide versus placebo in the established CVD subgroup (hazard ratio [HR] 0.74, 95% confidence interval [CI] 0.59, 0.92) and nonsignificantly lower in the CV risk factor subgroup (HR 0.84, 95% CI 0.55, 1.28) (P‐interaction = 0.60). These results suggest that the CV effects of semaglutide may extend to patients with type 2 diabetes across the CV risk continuum. [ABSTRACT FROM AUTHOR]

Sodium‐glucose cotransporter‐2 (SGLT2) inhibitors have been shown to reduce the risk of cardiovascular death or worsening heart failure (HF), and improve symptom burden, physical function and quality of life in patients with HF and reduced ejection fraction. The mechanisms of the HF benefits of SGLT2 inhibitors, however, remain unclear. In this substudy of the DEFINE‐HF trial, patients randomized to dapagliflozin or placebo had lung fluid volumes (LFVs) measured by remote dieletric sensing at baseline and after 12 weeks of therapy. A significantly greater proportion of dapagliflozin‐treated patients (as compared with placebo) experienced improvement in LFVs and fewer dapagliflozin‐treated patients had no change or deterioration in LFVs after 12 weeks of treatment. To our knowledge, this is the first study to suggest a direct effect of dapagliflozin (or any SGLT2 inhibitor) on more effective "decongestion", contributing in a meaningful way to the ongoing debate regarding the mechanisms of SGLT2 inhibitor HF benefits. [ABSTRACT FROM AUTHOR]

Background: Insulin exerts vasculoprotective effects on endothelial cells (ECs) and growth-promoting effects on vascular smooth muscle cells (SMCs) in vitro, and suppresses neointimal growth in vivo. Here we determined the role of ECs and SMCs in the effect of insulin on neointimal growth. Methods: Mice with transgene CreERT2 under the control of EC-specific Tie2 (Tie2-Cre) or SMC-specific smooth muscle myosin heavy chain promoter/enhancer (SMMHC-Cre) or littermate controls were crossbred with mice carrying a loxP-flanked insulin receptor (IR) gene. After CreERT2-loxP-mediated recombination was induced by tamoxifen injection, mice received insulin pellet or sham (control) implantation, and underwent femoral artery wire injury. Femoral arteries were collected for morphological analysis 28 days after wire injury. Results: Tamoxifen-treated Tie2-Cre+ mice showed lower IR expression in ECs, but not in SMCs, than Tie2-Cre− mice. Insulin treatment reduced neointimal area after arterial injury in Tie2-Cre− mice, but had no effect in Tie2-Cre+ mice. Tamoxifen-treated SMMHC-Cre+ mice showed lower IR expression in SMCs, but not in ECs, than SMMHC-Cre− mice. Insulin treatment reduced neointimal area in SMMHC-Cre− mice, whereas unexpectedly, it failed to inhibit neointima formation in SMMHC-Cre+ mice. Conclusion: Insulin action in both ECs and SMCs is required for the "anti-restenotic" effect of insulin in vivo. [ABSTRACT FROM AUTHOR]

The present cost–consequence analysis compared estimated hospitalization costs in a Canadian setting with insulin degludec (degludec) versus insulin glargine 100 units/mL (glargine U100) in patients with type 2 diabetes (T2D) at high cardiovascular (CV) risk. Medical terms were mapped across the different vocabularies, in order to assign unit costs from eligible hospital abstracts in Canadian Institute for Health Information data (International Statistical Classification of Diseases and Related Health Problems, 10th Revision, Canada) to serious adverse events (SAEs; Medical Dictionary for Regulatory Activities) from the randomized DEVOTE trial comparing the two insulins degludec and glargine. Mean annual costs of SAE-related hospitalizations were estimated by treatment, the cost difference (degludec − glargine U100) was bootstrapped to compute confidence intervals (CIs) and p-values, and the cost ratio (degludec/glargine U100) was estimated using a Tweedie distribution. The mean annual cost per patient for SAE-related hospitalizations was 4,074 CAD with degludec and 4,569 CAD with glargine U100 (cost difference: −495, 95% confidence interval [CI]: −966; −24, p =.039), for a cost ratio of 0.89 (95% CI: 0.81; 0.98, p =.016). Overall, cost ratios from sensitivity analyses varying individual methodological assumptions were consistent with the main analysis. Of the system organ classes from DEVOTE SAEs, cardiac disorders were the largest contributor to the costs savings with degludec versus glargine U100. In patients with T2D at high CV risk, our findings suggest that there are likely to be lower hospitalization costs with degludec versus glargine U100 based on the SAEs observed in DEVOTE and in a Canadian setting. [ABSTRACT FROM AUTHOR]

Background: Mortality from COVID-19 is high among hospitalized patients and effective therapeutics are lacking. Hypercoagulability, thrombosis and hyperinflammation occur in COVID-19 and may contribute to severe complications. Therapeutic anticoagulation may improve clinical outcomes through anti-thrombotic, anti-inflammatory and anti-viral mechanisms. Our primary objective is to evaluate whether therapeutic-dose anticoagulation with low-molecular-weight heparin or unfractionated heparin prevents mechanical ventilation and/or death in patients hospitalized with COVID-19 compared to usual care. Methods: An international, open-label, adaptive randomized controlled trial. Using a Bayesian framework, the trial will declare results as soon as pre-specified posterior probabilities for superiority, futility, or harm are reached. The trial uses response-adaptive randomization to maximize the probability that patients will receive the more beneficial treatment approach, as treatment effect information accumulates within the trial. By leveraging a common data safety monitoring board and pooling data with a second similar international Bayesian adaptive trial (REMAP-COVID anticoagulation domain), treatment efficacy and safety will be evaluated as efficiently as possible. The primary outcome is an ordinal endpoint with three possible outcomes based on the worst status of each patient through day 30: no requirement for invasive mechanical ventilation, invasive mechanical ventilation or death. Conclusion: Using an adaptive trial design, the Anti-Thrombotic Therapy To Ameliorate Complications of COVID-19 trial will establish whether therapeutic anticoagulation can reduce mortality and/or avoid the need for mechanical ventilation in patients hospitalized with COVID-19. Leveraging existing networks to recruit sites will increase enrollment and mitigate enrollment risk in sites with declining COVID-19 cases. [ABSTRACT FROM AUTHOR]

Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. However, culture of aCMs in particular is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance. Together, these refinements to aCM culture present a toolbox with which to advance our understanding of myocardial physiology. Callaghan et al. present a combinatory approach to culturing harvested adult mouse cardiomyocytes (aCMs). Under traditional culture protocols, aCMs rapidly lose their phenotype and undergo cell death. With their protocol, the authors show aCMs remain viable and retain their phenotype for 7 days, enough time to do genetic manipulation and small molecule screening. [ABSTRACT FROM AUTHOR]

Aim: To investigate the effects of semaglutide versus comparators on major adverse cardiovascular events (MACE: cardiovascular [CV] death, nonfatal myocardial infarction [MI] and nonfatal stroke) and hospitalization for heart failure (HF) in the SUSTAIN (subcutaneous semaglutide) and PIONEER (oral semaglutide) trials across subgroups of varying CV risk. Methods: Post hoc analyses of individual patient‐level data combined from SUSTAIN 6 and PIONEER 6 were performed to assess MACE and HF. MACE were analysed in subjects with and without: established CV disease and/or chronic kidney disease; prior MI or stroke; and prior HF. MACE in the SUSTAIN and PIONEER glycaemic efficacy trials were also assessed. Results: In SUSTAIN 6 and PIONEER 6 combined, the hazard ratio (HR) for effect of semaglutide versus placebo on overall MACE was 0.76 (95% CI 0.62, 0.92), which was mainly driven by the effect on nonfatal stroke (HR 0.65 [95% CI 0.43, 0.97]). The HR for hospitalization for HF was 1.03 (95% CI 0.75, 1.40). The HRs for MACE were <1.0 in all subgroups, except for those with prior HF (HR 1.06 [95% CI 0.72, 1.57]); P‐values for interaction of subgroup on treatment effect were >0.05, except for HF (0.046). In the combined glycaemic efficacy trials, the HR for effect of semaglutide versus comparators on MACE was 0.85 (95% CI 0.55, 1.33). Conclusions: In SUSTAIN and PIONEER combined, glucagon‐like peptide‐1 analogue semaglutide showed consistent effects on MACE versus comparators across varying CV risk. No effect of semaglutide on MACE was observed in subjects with prior HF. [ABSTRACT FROM AUTHOR]

Background: Heart failure (HF) is a common cardiovascular complication of type 2 diabetes (T2D). This secondary analysis investigated baseline factors and treatment differences associated with risk of hospitalization for HF (hHF), and the possible association between severe hypoglycemia and hHF. Methods: DEVOTE was a treat-to-target, double-blind cardiovascular outcomes trial in patients (n = 7637) with T2D and high cardiovascular risk randomized to insulin degludec (degludec) or insulin glargine 100 units/mL (glargine U100). The main endpoint of this secondary analysis was time to first hHF (standardized MedDRA Query definition). Severe hypoglycemia was adjudicated (American Diabetes Association definition). The main endpoint and the temporal association between severe hypoglycemia and hHF were analyzed with a Cox proportional hazards regression model. Predictors of time to first hHF were identified using baseline variables. Results: Overall, 372 (4.9%) patients experienced hHF (550 events). There was no significant difference in the risk of hHF between treatments (hazard ratio [HR] 0.88 [0.72;1.08]95% CI, p = 0.227). Prior HF (HR 4.89 [3.90;6.14]95% CI, p ≤ 0.0001) was the strongest predictor of future hHF events. The risk of hHF significantly increased after (HR 2.2), and within a week after (HR 11.1), experiencing a severe hypoglycemic episode compared with before an episode. Conclusions: In patients with T2D and high cardiovascular risk there were no treatment differences in terms of hHF. Prior HF was the strongest predictor of future hHF events, and there was an association between severe hypoglycemia and subsequent hHF. Further research should evaluate whether the risk of hHF can be modified by treatments aimed at reducing hypoglycemia. Trial Registration NCT01959529 [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miR) are small non-coding RNAs that regulate diverse biological functions. The bicistronic gene miR-143/145 determines cell fate and phenotype of vascular smooth muscle cells (VSMC), in part, by destabilizing Elk-1 mRNA. The transcription factor c-Myb also regulates differentiation and proliferation of VSMC, and here we test whether these effects may be mediated by miR-143/145. Methods & results: Flow cytometry of cardiovascular-directed d3.75 embryoid bodies (EBs) isolated smooth muscle progenitors with specific cell surface markers. In c-myb knockout (c-myb -/-) EB, these progenitors manifest low levels of miR-143 (19%; p<0.05) and miR-145 (6%; p<0.01) expression as compared to wild-type (wt) EB. Primary VSMC isolated from transgenic mice with diminished expression (c-myblx/lx) or reduced activity (c-mybh/h) of c-Myb also manifest low levels of miR-143 (c-myblx/lx: 50%; c-mybh/h: 41%), and miR-145 (c-myblx/lx: 49%; c-mybh/h: 56%), as compared to wt (P<0.05). Sequence alignment identified four putative c-Myb binding sites (MBS1-4) in the proximal promoter (PP) of the miR-143/145 gene. PP-reporter constructs revealed that point mutations in MBS1 and MBS4 abrogated c-Myb-dependent transcription from the miR-143/145 PP (P<0.01). Chromatin immunoprecipitation (ChIP) revealed preferential c-Myb binding at MBS4 (p<0.001). By conjugating Elk-1 3’-untranslated region (UTR) to a reporter and co-transducing wt VSMC with this plus a miR-143-antagomir, and co-transducing c-myblx/lx VSMC with this plus a miR-143-mimic, we demonstrate that c-Myb’s ability to repress Elk-1 is mediated by miR-143. Conclusion: c-Myb regulates VSMC gene expression by transcriptional activation of miR-143/145. [ABSTRACT FROM AUTHOR]

Background: Transient ischemic dilatation (TID) of the left ventricle is a potential marker of high risk obstructive coronary artery disease on stress myocardial perfusion imaging (MPI). There is, however, interstudy variation in the diagnostic performance of TID for identification of severe and extensive coronary disease anatomy, and varied prognostic implications in the published literature.Methods: We searched MEDLINE, EMBASE, and COCHRANE databases for studies where TID was compared with invasive or CT coronary angiography for evaluation of coronary artery stenosis. Two reviewers independently evaluated and abstracted data from each study. A bivariate random effects model was used to derive pooled sensitivities and specificities, in order to account for correlation between TID in MPI and anatomic disease severity.Results: A total of 525 articles were reviewed, of which 51 met inclusion criteria. Thirty-one studies contributed to the analysis, representing a total of 2037 patients in the diagnostic meta-analysis and 9003 patients in the review of prognosis. The ratio above which TID was deemed present ranged from 1.13 to 1.38. Pooled sensitivity was 44% (95% CI 30%-60%) and specificity was 88% (95% CI 83%-92%) for the detection of extensive or severe anatomic coronary artery disease. Analysis of outcome data demonstrated increased cardiac event rates in patients with TID and an abnormal MPI. In otherwise normal perfusion, TID is an indicator of poor prognosis in patients with diabetes and/or a history of coronary disease.Conclusions: Among patients undergoing MPI, the presence of TID is specific for the detection of extensive or severe coronary artery disease. [ABSTRACT FROM AUTHOR]

Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca2+-ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo and HF following experimental myocardial infarction (MI) in vivo. Methods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca2+-regulatory genes, and induced hypertrophy without significant differences in Ca2+- transients or diastolic Ca2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo. In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF. Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF. [ABSTRACT FROM AUTHOR]

Doxorubicin is a widely used chemotherapeutic with deleterious cardiotoxic side effects. HDL has been shown to protect cardiomyocytes in vitro against doxorubicin-induced apoptosis. Scavenger receptor class B type 1 (SR-B1), a high-affinity HDL receptor, mediates cytoprotective signaling by HDL through Akt. Here, we assessed whether increased HDL levels protect against doxorubicin-induced cardiotoxicity in vivo and in cardiomyocytes in culture and explored the intracellular signaling mechanisms involved, particularly the role of SRB1. Transgenic mice with increased HDL levels through overexpression of human apolipoprotein A1 (apoA1Tg/Tg) and wild-type mice (apoA1+/+) with normal HDL levels were treated repeatedly with doxorubicin. After treatment, apoA1+/+ mice displayed cardiac dysfunction, as evidenced by reduced left ventricular end-systolic pressure and +dP/dt, and histological analysis revealed cardiomyocyte atrophy and increased cardiomyocyte apoptosis after doxorubicin treatment. In contrast, apoA1Tg/Tg mice were protected against doxorubicin- induced cardiac dysfunction and cardiomyocyte atrophy and apoptosis. When SR-B1 was knocked out, however, overexpression of apoA1 did not protect against doxorubicin-induced cardiotoxicity. Using primary neonatal mouse cardiomyocytes and human immortalized ventricular cardiomyocytes in combination with genetic knockout, inhibitors, or siRNA-mediated knockdown, we demonstrated that SR-B1 is required for HDL-mediated protection of cardiomyocytes against doxorubicin-induced apoptosis in vitro via a pathway involving phosphatidylinositol 3-kinase and Akt1/2. Our findings provide proof of concept that raising apoA1 to supraphysiological levels can dramatically protect against doxorubicin-induced cardiotoxicity via a pathway that is mediated by SR-B1 and involves Akt1/2 activation in cardiomyocytes. [ABSTRACT FROM AUTHOR]

In experimental models of diabetes, augmented sodium-glucose cotransport-2 (SGLT2) activity diminishes sodium (Na+) delivery at the macula densa. As a result, less vasoconstrictive adenosine is generated, leading to afferent arteriolar vasodilatation and hyperfiltration. The measurement and significance of urinary adenosine in humans has not been examined extensively in states of renal hemodynamic impairment like that of diabetes. Our aim was to validate a method for urine adenosine quantification in humans and perform an exploratory post hoc analysis to determine whether urinary adenosine levels change dynamically in response to natriuresis in patients with type 1 diabetes (T1D) before and after treatment with the SGLT2 inhibitor (SGLT2i) empagliflozin. We hypothesized that SGLT2i, which reduces renal hyperfiltration through increased Na+ delivery to the macula densa, would increase urinary adenosine excretion. Urine adenosine corrected for creatinine was measured using our validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in 40 healthy participants and 40 patients with T1D. In the T1D cohort, measurements were performed during clamped euglycemic and hyperglycemic conditions before and following 8 wk of SGLT2i therapy. Urinary adenosine was detectable in healthy subjects (0.32±0.11μmol/mmol Cr) and patients with T1D. In response to SGLT2i, urine adenosine increased during clamped hyperglycemia (0.40± 0.11 vs. 0.45±0.12 μmol/mmol Cr, P = 0.005). Similar trends were observed during clamped euglycemia (P =0.08). In conclusion, SGLT2i increases urinary adenosine excretion under clamped hyperglycemic conditions in patients with T1D. The potentially protective role of SGLT2i against glomerular hyperfiltration and its mediation by adenosine in diabetes merits further study. [ABSTRACT FROM AUTHOR]

Objective: Few methods enable molecular and cellular studies of vascular aging or Type 2 diabetes (T2D). Here, we report a new approach to studying human vascular smooth muscle cell (VSMC) pathophysiology by examining VSMCs differentiated from progenitors found in skin. Approach and results: Skin-derived precursors (SKPs) were cultured from biopsies (N=164, ~1 cm2) taken from the edges of surgical incisions of older adults (N=158; males 72%; mean age 62.7 ± 13 years) undergoing cardiothoracic surgery, and differentiated into VSMCs at high efficiency (>80% yield). The number of SKPs isolated from subjects with T2D was ~50% lower than those without T2D (cells/g: 0.18 ± 0.03, N=58 versus 0.40 ± 0.05, N=100, P<0.05). Importantly, SKP-derived VSMCs from subjects with T2D had higher Fluo-5F-determined baseline cytosolic Ca2+ concentrations (AU: 1,968 ± 160, N=7 versus 1,386 ± 170, N=13, P<0.05), and a trend toward greater Ca2+ cycling responses to norepinephrine (NE) (AUC: 177,207 ± 24,669, N=7 versus 101,537 ± 15,881, N=20, P<0.08) despite a reduced frequency of Ca2+ cycling (events s-1 cell-1: 0.011 ± 0.004, N=8 versus 0.021 ± 0.003, N=19, P<0.05) than those without T2D. SKP-derived VSMCs from subjects with T2D also manifest enhanced sensitivity to phenylephrine (PE) in an impedance-based assay (EC50 nM: 72.3± 63.6, N=5 versus 3,684± 3,122, N=9, P<0.05), and impairedwound closure in vitro (% closure: 21.9 ± 3.6, N=4 versus 67.0 ± 10.3, N=4, P<0.05). Compared with aortic- and saphenous vein-derived primary VSMCs, SKP-derived VSMCs are functionally distinct, but mirror defects of T2D also exhibited by primary VSMCs. Conclusion: Skin biopsies from older adults yield sufficient SKPs to differentiate VSMCs, which reveal abnormal phenotypes of T2D that survive differentiation and persist even after long-term normoglycemic culture. [ABSTRACT FROM AUTHOR]

The incidence and prevalence of Type-2 diabetes mellitus (T2DM) is increasing worldwide. Cardiovascular disease (CVD) is the major cause of morbidity and mortality among subjects with T2DM. The relationship linking diabetes and CVD is complex and multifactorial, and a thorough understanding of the pathophysiological mechanisms underlying diabetes-induced cardiac damage is crucial to the development and improvement of treatment strategies for patients with T2DM at high risk of major adverse cardiovascular events (MACE). Heart failure is more frequent and has an especially adverse outcome in subjects with diabetes. Heart muscle disease not explained by either coronary heart disease or hypertension (diabetic cardiomyopathy), is an important contributing factor to the development of heart failure. This review summarizes the molecular mechanisms of certain key contributors to the progression of diabetic cardiomyopathy including insulin resistance, metabolic disturbances, oxidative stress, altered Ca2+ handling, and neurohormonal activation. [ABSTRACT FROM AUTHOR]

Background: While renal transplantation is increasingly performed for end-stage renal disease, there is a paucity of data on cardiac screening and prognostication post-transplant. We determined the prognostic value of SPECT-MPI in a cohort who underwent renal transplantation.Methods: Among 4933 renal transplant recipients identified from the Canadian Organ Replacement Register, we examined outcomes of patients who underwent SPECT-MPI in Ontario, Canada. We determined morbidity and mortality using hospitalization and vital statistics registries, according to SPECT-MPI findings.Results: We studied 282 renal transplant recipients (median age 46 years [25th, 75th percentile 37, 58]) with detailed SPECT-MPI results available, followed for a median of 5.7 (3.3, 7.7) years. Among those undergoing SPECT-MPI (66% pharmacologic stress), 41% had an abnormal summed stress score (SSS > 0) and 31% demonstrated abnormal summed difference score (SDS > 0). Rates of cardiovascular death were 0.4 per 100 person-years among those with normal stress perfusion (SSS = 0) and 0.4 per 100 person-years with SDS = 0. After adjusting for age, sex, prior myocardial infarction (MI), and cardiac risk factors, an SSS ≥ 4 conferred increased risk of cardiovascular death or cardiovascular hospitalization with adjusted hazard ratios of 2.52 (95% CI 1.41, 4.52, P = .002) for SSS 4-6 and 2.61 (95% CI 1.52, 4.49, P < .001) for SSS ≥ 7. SDS was a significant predictor of cardiovascular death or hospitalization, with adjusted hazard ratios of 2.96 (95% CI 1.72, 5.09, P < .001) for SDS 4-6 and 3.26 (95% CI 1.64, 6.50, P < .001) for SDS ≥ 7.Conclusion: Among renal transplant recipients, SPECT-MPI predicted risk of cardiovascular death and cardiovascular hospitalization events. [ABSTRACT FROM AUTHOR]

Diabetes strongly associates with microvascular complications that ultimately promote multiorgan failure. Altered myogenic responsiveness compromises tissue perfusion, aggravates hypertension, and sets the stage for later permanent structural changes to the microcirculation. We demonstrate that skeletal muscle resistance arteries isolated from patients with diabetes have augmented myogenic tone, despite reasonable blood glucose control. To understand the mechanisms, we titrated a standard diabetes mouse model (high-fat diet plus streptozotocin [HFD/STZ]) to induce a mild increase in blood glucose levels. HFD/STZ treatment induced a progressive myogenic tone augmentation in mesenteric and olfactory cerebral arteries; neither HFD nor STZ alone had an effect on blood glucose or resistance artery myogenic tone. Using gene deletion models that eliminate tumor necrosis factor (TNF) or sphingosine kinase 1, we demonstrate that vascular smooth muscle cell TNF drives the elevation of myogenic tone via enhanced sphingosine-1-phosphate (S1P) signaling. Therapeutically antagonizing TNF (etanercept) or S1P (JTE013) signaling corrects this defect. Our investigation concludes that vascular smooth muscle cell TNF augments resistance artery myogenic vasoconstriction in a diabetes model that induces a small elevation of blood glucose. Our data demonstrate that microvascular reactivity is an early disease marker and advocate establishing therapies that strategically target the microcirculation. [ABSTRACT FROM AUTHOR]

Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)-targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r(-/-)), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r(-/-) mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events. [ABSTRACT FROM AUTHOR]

Background: Distal coronary embolization (DCE) of thrombotic material occurs frequently during percutaneous interventions for acute myocardial infarction and can alter coronary flow grades. The significance of DCE on infarct size and myocardial function remains unsettled. The aims of this study were to evaluate the effects of DCE sufficient to cause no-reflow on infarct size, cardiac function and ventricular remodeling in a porcine acute myocardial infarction model. Methods and results: Female Yorkshire pigs underwent 60 min balloon occlusion of the left anterior descending coronary artery followed by reperfusion and injection of either microthrombi (prepared from autologous porcine blood) sufficient to cause no-reflow (DCE), or saline (control). Animals were sacrificed at 3 h (n = 5), 3 days (n = 20) or 6 weeks (n = 20) post-AMI. Cardiovascular magnetic resonance (CMR), serum troponin-I, and cardiac gelatinase (MMP) and survival kinase (Akt) activities were assessed. At 3d, DCE increased infarct size (CMR: 18.8 % vs. 14.5 %, p = 0.04; serum troponin-I: 13.3 vs. 6.9 ng/uL, < 0.05) and MMP-2 activity levels (0.81 vs. 0.49, = 0.002), with p reduced activation of Akt (0.06 versus 0.26, p = 0.02). At 6 weeks, there were no differences in infarct size, ventricular volume or ejection fraction between the two groups, although infarct transmurality (70 % vs. 57 %, p < 0.04) and ventricular thinning (percent change in mid anteroseptal wall thickness:-25.6 % vs. 0.7 %, p = 0.03) were significantly increased in the DCE group. Conclusions: DCE increased early infarct size, but without affecting later infarct size, cardiac function or ventricular volumes. The significance of the later remodelling changes (ventricular thinning and transmurality) following DCE, possibly due to changes in MMP-2 activity and Akt activation, merits further study. [ABSTRACT FROM AUTHOR]

Objective: To understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR). Background: Lifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we reveal the pathways that are modulated by short-term CR, which are associated with protection of the mouse heart from ischemia. Methods: Male 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food for 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. At d8, the left ventricles (LV) of AL and CR mice were collected for Western blot, mRNA and microRNA (miR) analyses to identify cardioprotective gene expression signatures. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI. Results: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function. [ABSTRACT FROM AUTHOR]

Context: Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca2+ signalling in vascular smooth muscle cells (VSMC). Objective: RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca2+ concentration [Ca2+]i. RGS2 involvement in VSMC proliferation had not been examined. Methods: Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca2+]i before and after UTP and thapsigargin. [3H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca2+ transporters was performed in primary aortic VSMC. Results and discussion: Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca2+ homeostasis, RGS2KO VSMC showed lower resting [Ca2+]i but higher thapsigargin-induced [Ca2+]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca2+ ATPase-4 (PMCA4) and Na+ Ca2+ Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca2+ ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca2+ efflux activity, 45Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La3+, but not by replacing extracellular Na+ with choline, implicating differences in the activity of PMCA and not NCX. Conclusion: RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca2+-handling mechanisms in response to PDGF stimulation. [ABSTRACT FROM AUTHOR]

Atherothrombotic cardiovascular events are a leading cause of morbidity and mortality in patients with type 2 diabetes (T2D). A number of factors beyond hyperglycemia contribute to this increased risk of cardiovascular events in T2D, including elevated blood pressure, dyslipidemia, inflammation, endothelial dysfunction, and enhanced platelet activation. Importantly, most currently available antihyperglycemic treatments for T2D do not address these additional mechanisms. Indeed, we posit that this may explain why more intensive treatment of hyperglycemia has not contributed to a reduced incidence of cardiovascular events in subjects with T2D. Incretin-targeted therapies, such as dipeptidyl peptidase 4 inhibitors, are a relatively new class of antidiabetic treatments, and preclinical as well as small mechanistic clinical studies suggest that they exert beneficial cardiovascular effects. This review focuses specifically on the potential antiatherothrombotic effects of dipeptidyl peptidase 4 inhibitors. [ABSTRACT FROM AUTHOR]

Chronic exposure to high glucose leads to diabetic nephropathy characterized by increased mesangial matrix protein (e.g., collagen) accumulation. Altered cell signaling and gene expression accompanied by oxidative stress have been documented. The contribution of the tyrosine kinase, c-Src (Src), which is sensitive to oxidative stress, was examined. Cultured rat mesangial cells were exposed to high glucose (25 mmol/L) in the presence and absence of Src inhibitors (PP2, SU6656), Src small interfering RNA (siRNA), and the tumor necrosis factor-α-converting enzyme (TACE) inhibitor, TAPI-2. Src was investigated in vivo by administration of PP2 to streptozotocin (STZ)-induced diabetic DBA2/J mice. High glucose stimulated Src, TACE, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2, p38), and collagen IV accumulation in mesangial cells. PP2 and SU6656 blocked high glucose-stimulated phosphorylation of Src Tyr-416, EGFR, and MAPKs. These inhibitors and Src knockdown by siRNA, as well as TAPI-2, also abrogated high glucose-induced phosphorylation of these targets and collagen IV accumulation. In STZ-diabetic mice, albuminuria, increased Src pTyr-416, TACE activation, ERK and EGFR phosphorylation, glomerular collagen accumulation, and podocyte loss were inhibited by PP2. These data indicate a role for Src in a high glucose-Src-TACE-heparin-binding epidermal growth factor-EGFR-MAPK-signaling pathway to collagen accumulation. Thus, Src may provide a novel therapeutic target for diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Chronic exposure to high glucose leads to diabetic nephropathy characterized by increased mesangial matrix protein (e.g., collagen) accumulation. Altered cell signaling and gene expression accompanied by oxidative stress have been documented. The contribution of the tyrosine kinase, c-Src (Src), which is sensitive to oxidative stress, was examined. Cultured rat mesangial cells were exposed to high glucose (25 mmol/L) in the presence and absence of Src inhibitors (PP2, SU6656), Src small interfering RNA (siRNA), and the tumor necrosis factor-α-converting enzyme (TACE) inhibitor, TAPI-2. Src was investigated in vivo by administration of PP2 to streptozotocin (STZ)-induced diabetic DBA2/J mice. High glucose stimulated Src, TACE, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2, p38), and collagen IV accumulation in mesangial cells. PP2 and SU6656 blocked high glucose-stimulated phosphorylation of Src Tyr-416, EGFR, and MAPKs. These inhibitors and Src knockdown by siRNA, as well as TAPI-2, also abrogated high glucose-induced phosphorylation of these targets and collagen IV accumulation. In STZ-diabetic mice, albuminuria, increased Src pTyr-416, TACE activation, ERK and EGFR phosphorylation, glomerular collagen accumulation, and podocyte loss were inhibited by PP2. These data indicate a role for Src in a high glucose-Src-TACE-heparin-binding epidermal growth factor-EGFR-MAPK-signaling pathway to collagen accumulation. Thus, Src may provide a novel therapeutic target for diabetic nephropathy. [ABSTRACT FROM AUTHOR]

During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall. The observation that circulating monocytes give rise to lesional macrophages has reinforced the concept that monocyte infiltration dictates macrophage buildup. Recent work has indicated, however, that macrophage accumulation does not depend on monocyte recruitment in some inflammatory contexts. We therefore revisited the mechanism underlying macrophage accumulation in atherosclerosis. In murine atherosclerotic lesions, we found that macrophages turn over rapidly, after 4 weeks. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation through the involvement of scavenger receptor A (SR-A). Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease. [ABSTRACT FROM AUTHOR]

Aims Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation. Methods and results Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT–PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein -expressing hESCs integrated in and contributed to new vessel formation in vivo. Conclusion The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies. [ABSTRACT FROM PUBLISHER]