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Background Angiotensin converting enzyme 2 (ACE2) is expressed in the human myocardium and blood vessels and degrades the vasoconstrictor peptide angiotensin (Ang) II. Plasma ACE2 activity is elevated in patients with cardiovascular disease (CVD) and is a predictor of major adverse cardiovascular events (MACE) in obstructive coronary artery disease. However, it is unknown whether acute coronary syndrome (ACS) causes dynamic changes in plasma ACE2 activity. Purpose We investigated dynamic changes in serial troponin-T and plasma ACE2 activity in patients presenting with ACS who underwent invasive coronary angiography (ICA). Methods Consecutive patients admitted with ACS from October-November 2019 were screened. Those meeting the Fourth Universal Definition of Myocardial Infarction who had both ICA and serial troponin-T testing were included. The study was approved by the hospitals Human Research Ethics Committee. All patients had routine plasma samples taken over 3 time-points for measurement of troponin-T; the same sample was used to measure plasma ACE2 activity. Catalytic ACE2 activity was measured using a validated, sensitive quenched fluorescent substrate-based assay. Serial median troponin and ACE2 activity levels were analysed using the Friedman test for repeated measures. Results Forty-nine patients were included. The mean age of participants was 63.9±11.0 years, and 36 (74%) patients were male. Overall, 16 (36%) patients presented with ST-elevation myocardial infarction (STEMI) and 29 (74%) with non-ST-elevation myocardial infarction (NSTEMI). Twenty-nine (59%) patients had a history of hypertension and 14 (29%) a history of ischaemic heart disease; 13 (27%) with priorMI, 11 (22%) had previous PCI and 2 (4%) had prior coronary artery bypass grafting. Over the 3 time points, there was a clear rise in median troponin-T levels representing myocardial injury (p Conclusions Patients with ACS had higher plasma ACE2 levels compared to levels previously reported in healthy controls. There were no dynamic changes in ACE2 activity in the setting of ACS, despite a significant rise in troponin-T. These results suggest that plasma ACE2 levels reflect underlying endothelial dysfunction rather than acute myocardial injury or infarction. Studies are now underway to assess if plasma ACE2 activity in ACS predicts MACE. Funding Acknowledgement Type of funding sources: None. Table 1

Background and aims Endothelial dysfunction is a precursor to atherosclerosis and is implicated in the coexistence between cardiovascular disease (CVD) and chronic kidney disease (CKD). We examined whether retinal microvascular dysfunction is present in subjects with renal impairment and predictive of long-term CKD progression in patients with CVD. Methods In a single centre prospective observational study, 253 subjects with coronary artery disease and CVD risk factors underwent dynamic retinal vessel analysis. Retinal microvascular dysfunction was quantified by measuring retinal arteriolar and venular dilatation in response to flicker light stimulation. Serial renal function assessment was performed over a median period of 9.3 years using estimated GFR (eGFR). Results Flicker light-induced retinal arteriolar dilatation (FI-RAD) was attenuated in patients with baseline eGFR Conclusions Retinal arteriolar endothelial dysfunction is present in patients with CVD who have early-stage CKD, and serves as an indicator of long-term CKD progression in those with normal renal function.

BACKGROUND: The apolipoprotein E (APOE) gene ɛ4 allele is a risk factor for Alzheimer's disease and cardiovascular disease. However, its relationship with cognition and brain volume after stroke is not clear. OBJECTIVE: We compared cognition and medial temporal lobe volumes in APOEɛ4 carriers and non-carriers in the first year after ischemic stroke. METHODS: We sampled 20 APOEɛ4 carriers and 20 non-carriers from a larger cohort of 135 ischemic stroke participants in the longitudinal CANVAS study. Participants were matched on a range of demographic and stroke characteristics. We used linear mixed-effect models to compare cognitive domain z-scores (attention, processing speed, executive function, verbal and visual memory, language, visuospatial function) and regional medial temporal lobe volumes (hippocampal, entorhinal cortex) between groups at each time-point (3, 12-months post-stroke), and within groups across time-points. APOE gene single nucleotide polymorphisms (SNPs; rs7412, rs429358) were genotyped on venous blood. RESULTS: APOEɛ4 carriers and non-carriers did not differ on any demographic, clinical, or stroke variable. Carriers performed worse than non-carriers in verbal memory at 3 months post-stroke (p = 0.046), but were better in executive function at 12 months (p = 0.035). Carriers demonstrated a significant improvement in verbal memory (p = 0.012) and executive function (p = 0.015) between time-points. Non-carriers demonstrated a significant improvement in visual memory (p = 0.0005). Carriers had smaller bilateral entorhinal cortex volumes (p

Endothelin-1 (ET-1) is a vasoconstrictor associated with cardiovascular disease, whereas adrenomedullin (ADM) is a vasorelaxant with cardioprotective properties. We sought to determine the relationship between plasma ET-1 and ADM with coronary circulatory function and long-term major adverse cardiovascular events (MACE).Thirty-two patients undergoing coronary angiography for chest pain were recruited. Baseline plasma ET-1 and ADM levels were measured. The index of microcirculatory resistance (IMR), coronary flow mediated dilatation (cFMD) and coronary flow reserve (CFR) were measured in a non-obstructed coronary artery. Patients were assessed for MACE over a median period of 8.8 years.Plasma ET-1 levels correlated with IMR (r = 0.57; p 0.01) and ADM levels correlated with CFR (r = 0.50; p = 0.04) and cFMD (r = 0.62; p = 0.01). After adjustment for age, gender and cardiovascular risk factors, the association between ADM and cFMD (β = 0.79; p 0.01) and between ET-1 and IMR (β = 5.7; p = 0.01) remained significant. IMR was higher, although not statistically significant, in patients with long-term MACE (17.9 ± 5.3 vs. 13.1 ± 6.0 units; p = 0.14). In patients free of MACE, cFMD (9.3 ± 7.6 vs. 2.8 ± 5.0%; p = 0.01) and plasma ADM levels (7.6 ± 5.3 vs. 4.0 ± 1.9 pmol/L; p = 0.07) were higher.Plasma ET-1 and ADM were associated with measures of coronary microvascular and coronary conduit vessel function, respectively. Increased cFMD and elevated plasma ADM were associated with a cardioprotective effect.

Background Recent evidence from animal studies suggests that elastin degradation accelerates atherosclerosis and increases risk of plaque rupture and subsequent myocardial infarction and stroke. Desmosine is an elastin-specific degradation product. We analysed the prognostic value of plasma desmosine (pDES) in a cohort of patients with coronary artery disease (CAD). Methods Patients with CAD (n=400) undergoing elective coronary angiography were prospectively recruited over 3 years and had bloods drawn for analysis of pDES using a validated stable isotope dilution liquid chromatography-tandem mass spectrometry method. Patients were followed up for 12 months for major adverse cardiovascular events (MACE: composite of death, myocardial infarction, target vessel repeat revascularisation, target lesion revascularisation, and heart failure hospital admission). The upper limit of normal for pDES is 0.35 ng/mL. The predictive value of pDES for MACE was analysed with Cox-proportional hazards ratio (HR) model and Kaplan-Meier survival analysis. Results During follow-up, there were 36 MACE events. Median pDES level across the entire cohort was 0.3 ng/mL (IQR 0.23–0.41 ng/mL). Patients with a pDES level >0.35 ng/mL were more likely to be male and older with a mean age of 69.7±10.3 years, have a history of prior stroke or transient ischaemic attack (TIA) and COPD. In univariable analysis, a pDES level of >0.35 ng/mL was associated with an increased risk of MACE (HR 4.76, 95% CI: 2.34–9.68, p0.35 ng/mL was associated with risk of MACE after adjustment for age, sex, COPD status and previous stroke and TIA (HR 3.97; 95% CI: 1.82–8.67, p=0.001) (Figure). Conclusion Increased elastin degradation as measured by elevated pDES levels, predicts outcomes in patients with CAD independent of traditional cardiovascular risk factors and may play a role as a future biomarker in these patients. Kaplan-Meier survival analysis Funding Acknowledgement Type of funding source: None

Aims Endothelial dysfunction is a precursor to the development of symptomatic atherosclerosis. Retinal microvascular reactivity to flicker light stimulation is a marker of endothelial function and can be quantified in vivo. We sought to determine whether retinal microvascular endothelial dysfunction predicts long-term major adverse cardiovascular events (MACE). Methods and results In a single center prospective observational study, patients with coronary artery disease (CAD) or cardiovascular risk factors underwent dynamic retinal vessel assessment in response to flicker light stimulation and were followed up for MACE. Retinal microvascular endothelial dysfunction was quantified by measuring maximum flicker light-induced retinal arteriolar (FI-RAD) and venular dilatation (FI-RVD). In total, 252 patients underwent dynamic retinal vessel assessment and 242 (96%) had long-term follow-up. Of the 242 patients, 88 (36%) developed MACE over a median period of 8.6 years (IQR 6.0-9.1). After adjustment for traditional risk factors, patients within the lowest quintile of FI-RAD had the highest risk of MACE (OR 5.21; 95% CI 1.78, 15.28). Patients with lower FI-RAD were also more likely to die (OR 2.09; 95% CI 1.00, 4.40, per standard deviation decrease in FI-RAD). In Kaplan-Meier analysis, patients with FI-RAD responses below the cohort median of 1.4% exhibited reduced MACE-free survival (55.5 vs. 71.5%; log-rank p = 0.004). FI-RVD was not predictive of MACE. Conclusions Retinal arteriolar endothelial dysfunction is an independent predictor of MACE in patients with CAD or cardiovascular risk factors. Dynamic retinal vessel analysis may provide added benefit to traditional risk factors in stratifying patients at risk for cardiovascular events. Translational perspective Subclinical endothelial dysfunction precedes cardiovascular diseases and can be assessed non-invasively using the retinal microvascular network. Retinal arteriolar endothelial dysfunction is an independent predictor of MACE and all-cause mortality in patients with established coronary artery disease or cardiovascular risk factors. Validation studies and investigation into the lifestyle and pharmacological modifiability of endothelial dysfunction could enhance risk prediction and guide intensification of therapy.

Context Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in patients with type 2 diabetes mellitus (T2DM) and associated with significant morbidity and mortality. Thyroid hormone (TH) increases β-oxidation of fatty acids and decreases intrahepatic lipid content (IHLC) in rodents with NAFLD. Objective We investigated the possibility of low intrahepatic TH concentration in NAFLD and studied the effect of TH treatment in humans. Design/setting This was a phase 2b single-arm study in six hospitals in Singapore. Intrahepatic thyroid hormone concentrations were measured in rats with induced NAFLD. Patients Euthyroid patients with T2DM and steatosis measured by ultrasonography. Intervention Levothyroxine was titrated to reach a thyroid-stimulating hormone level of 0.34 to 1.70 mIU/L before a 16-week maintenance phase. Main outcome measures The primary outcome measure was change in IHLC measured by proton magnetic resonance spectroscopy after treatment. Results Twenty male patients were included in the per-protocol analysis [mean ± SD: age, 47.8 ± 7.8 years; body mass index (BMI), 30.9 ± 4.4 kg/m2; baseline IHLC, 13% ± 4%]. After treatment, IHLC was decreased 12% (±SEM, 26%) relative to baseline (absolute change, -2%; 95% CI, -3 to 0; P = 0.046). Small decreases in BMI (P = 0.044), visceral adipose tissue volume (P = 0.047), and subcutaneous adipose tissue volume (P = 0.045) were observed. No significant changes in glucose regulation or lipid profile occurred. Conclusion This study demonstrated the efficacy and safety of low-dose TH therapy for NAFLD in men. TH or TH analogs may be beneficial for this condition.

Background ACE2 activity levels correlate with adverse left atrial remodelling in patients with atrial fibrillation (AF). Several biochemical and structural markers have been associated with embolic stroke of undetermined source (ESUS). The relationship between ACE2 activity and ESUS is unknown. Purpose Randomised controlled trials failed to demonstrate a clear benefit of oral anticoagulation in an unselected ESUS population. As selective use of oral anticoagulation guided by biomarker risk-profiling may benefit these patients, we evaluated the association between ACE2 activity and ESUS. Methods This prospective case control study compared patients with ESUS against a control group matched for vascular risk factors. ESUS was diagnosed following cerebral vascular imaging and 24 hours of cardiac monitoring to exclude AF. Blood samples were collected for measurement of ACE2 activity, D-Dimer and high sensitivity troponin T (hsTnT). Results A total of 51 patients in the ESUS group were compared with 47 patients in the Control group. ACE2 activity and D-Dimer levels were significantly higher in the ESUS group. There was a significant but weak positive correlation between ACE2 activity and hsTnT (r=0.20, p Participant characteristics Control (n=47) ESUS (n=51) P value Age (years) 65.65±6.78 67.20±6.89 0.26 Female gender 22 (45.8) 19 (38.0) 0.43 Hypertension 22 (45.8) 24 (48.0) 0.83 Diabetes mellitus 9 (18.8) 12 (24.0) 0.53 CHA2DS2VASc score 2 (1–3) 2 (1–3) 0.50 LA size & Biomarkers LA volume index (ml/m2) 36.5 (32.6–42.5) 39.1 (36.2–46.0) 0.04 ACE2 (pmol/ml/min) 7.24 (2.66–14.64) 10.16 (4.54–18.80) 0.04 D-Dimer (mg/L) 0.35 (0.3–0.5) 0.40 (0.30–0.60) 0.02 hsTroponin T (ng/L) 7.0 (5–10) 9.00 (6.0–13.5) 0.05 Values are expressed as mean ± standard deviation, median (IQR), or n (%). Median ACE2 activity Conclusion(s) ACE2 activity is associated with ESUS independent of left atrial volume and correlate with elevated Troponin. Further studies are warranted to investigate the utility of ACE2 activity in identifying ESUS patients that may benefit from oral anticoagulation. Acknowledgement/Funding This study received project funding from the Eastern Health Foundation

Objective: Australia is experiencing ever more frequent/provocative weather and environmental challenges, including more extreme heatwaves and catastrophic bushfires. Concurrently, the annual challenge of wintry conditions to a population adapted to warmer conditions persists. Remarkably, however, there are no proven interventions to reduce seasonal challenges to the cardiovascular health of vulnerable individuals. In a world-first, the REsilience to Seasonal ILlness and Increased Emergency admissioNs CarE (RESILIENCE) Trial will test the hypothesis that an individually tailored, intervention program will reduce the risk of re-hospitalisation and mortality in vulnerable individuals. Design and method: 300 adult patients admitted to the Austin Hospital in Melbourne, Australia with heart disease and multimorbidity will be recruited and randomised (1:1) to standard care (SC) or the RESILIENCE program (RP) over 12-months. Applying a COVID-19 adapted protocol, the RP group will have their bio-behavioural profile and home environment assessed post-discharge, to determine their vulnerability to seasonal events. An individualised case-management program, including a virtual clinic review with a dedicated RP cardiac nurse and physician, will be applied to promote seasonal resilience. The primary end-point is all-cause days alive out of hospital (DAOH) during 12-month follow-up. Results: With study recruitment delayed due to COVID-19 restrictions, virtual screening of medical in-patients has confirmed the need and potential for the RP. Of 630 potential participants identified over a 6 week period, 196 patients (31%) met eligibility criteria-85 women and 79 men, mean (±SD) age 79 ± 11 years. Non-eligibility was largely due to non-chronic form of heart disease (34%), no comorbidity (23 %), and inability to give informed consent (15%). Conclusions: Preliminary data suggest that once commenced, we will rapidly recruit the requisite number of trial participants and depending on the results, we will be able to determine the cost-effectiveness of the RP to reduce seasonallyinduced admissions and mortality.

Objective: COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), utilises the catalytic site of membrane-bound angiotensin converting enzyme 2 (ACE2) for cell entry. It is thought that endocytosis of ACE2 results in a decrease in membrane bound ACE2 expression, and disruption of the local tissue renin angiotensin system protection. In this study, we hypothesised that SARS-CoV-2 infection would be associated with shedding of ACE2 leading to increased plasma ACE2 activity. Design and method: Australians aged >18 years (n=66) who had recovered from SARS-CoV-2 infection (positive result by PCR testing) and uninfected controls (n=70) were recruited. Serial samples were available in 23 recovered SARS-CoV-2 patients. Plasma ACE2 activity was measured using a fluorescent substrate-based assay and levels were compared using the Mann-Whitney or Kruskal-Wallis test. Serial ACE2 activity were analysed using the Friedman test for repeated measures. Post-hoc analysis was performed with a Bonferroni correction. Two-tailed P-values 0.05) in the proportion of hypertension, obesity, diabetes, cardiovascular disease, or use of anti hypertensive, lipid lowering, and anti-platelet medications between the controls and SARSCoV-2 patients. Plasma ACE2 activity at median 35 days post-infection [interquartile range 30-38 days] was 97-fold higher in SARS-CoV-2 patients compared to controls (5.8 [2-11.3] vs. 0.06 [0.02-2.2] pmol/min/ml, p 0.05). Conclusions: Plasma ACE2 activity is elevated after SARS-CoV-2 infection and remains elevated post-infection. Our findings indicate the need for ongoing investigation to determine if ACE2 levels identify people at risk of prolonged illness following COVID-19.

COVID-19 causes persistent endothelial inflammation, lung and cardiovascular complications. SARS-CoV-2 utilises the catalytic site of full-length membrane-bound angiotensin converting enzyme 2 (ACE2) for cell entry causing downregulation of tissue ACE2. We reported downregulation of cardiac ACE2 is associated with increased plasma ACE2 activity. In this prospective observational study in recovered COVID-19 patients, we hypothesised that SARS-CoV-2 infection would be associated with shedding of ACE2 from cell membranes and increased plasma ACE2 activity.MethodsWe measured plasma ACE2 catalytic activity using a validated, sensitive quenched fluorescent substrate-based assay in a cohort of Australians aged ≥18 years (n=66) who had recovered from mild, moderate or severe SARS-CoV-2 infection (positive result by PCR testing) and age and gender matched uninfected controls (n=70). Serial samples were available in 23 recovered SARS-CoV-2 patients.ResultsPlasma ACE2 activity at a median of 35 days post-infection [interquartile range 30-38 days] was 97-fold higher in recovered SARS-CoV-2 patients compared to controls (5.8 [2-11.3] vs. 0.06 [0.02-2.2] pmol/min/ml, p0.05).DiscussionThis is the first description that plasma ACE2 activity is elevated after COVID-19 infection, and the first with longitudinal data indicating plasma ACE2 activity remains elevated out to a median of 114 days post-infection. Larger studies are now needed to determine if persistent elevated plasma ACE2 activity identifies people at risk of prolonged illness following COVID-19.

Angiotensin converting enzyme 2 (ACE2) is an important regulator of the renin-angiotensin system through actions to degrade angiotensin II. Loss of ACE2 can contribute to the development and progression of cardiovascular disease, and experimental studies have highlighted a beneficial role for novel therapeutic approaches that activate or replenish tissue ACE2. This review focuses on experimental studies that have used the off-target effects of the antitrypanosomal agent, diminazene aceturate (DIZE) to activate ACE2.In cardiovascular disease, activation of the classical renin-angiotensin system and depletion of ACE2 leads to pathophysiological changes. One approach to activate ACE2 involves the drug DIZE, which has been shown to have beneficial effects in experimental models of hypertension, pulmonary hypertension, myocardial infarction, stroke, atherosclerosis, type 1 diabetes, and eye disease. The precise mechanism of action of DIZE to activate ACE2 remains under scrutiny.Activation of ACE2 may represent an important therapeutic approach in cardiovascular disease. To date, most studies have focused on the off-target actions of DIZE, in experimental models of disease. More research is required to determine the exact mechanism of action of DIZE and evaluate its therapeutic potential in comparison with currently available clinical interventions. There are no clinical studies of DIZE, and its side-effects, and toxicity make such studies unlikely. Hence, new methods of selectively activating or replenishing ACE2 will be needed in the future if this approach is to be used in a clinical context.

Background Left ventricular hypertrophy (LVH) increases the risk of death in chronic kidney disease (CKD). The transcription factor Kruppel-like factor 15 (KLF15) is expressed in the heart and regulates cardiac remodelling through inhibition of hypertrophy and fibrosis. It is unknown if KLF15 expression is changed in CKD induced LVH, or whether expression is modulated by blood pressure reduction using angiotensin converting enzyme (ACE) inhibition. Methods CKD was induced in Sprague–Dawley rats by subtotal nephrectomy (STNx), and rats received vehicle (n = 10) or ACE inhibition (ramipril, 1 mg/kg/day, n = 10) for 4 weeks. Control, sham-operated rats (n = 9) received vehicle. Cardiac structure and function and expression of KLF15 were assessed. Results STNx caused impaired kidney function (P

Background Angiotensin converting enzyme 2 (ACE2) is an endogenous regulator of the renin angiotensin system. Increased circulating ACE2 predicts adverse outcomes in patients with heart failure (HF), but it is unknown if elevated plasma ACE2 activity predicts major adverse cardiovascular events (MACE) in patients with obstructive coronary artery disease (CAD). Methods We prospectively recruited patients with obstructive CAD (defined as ≥50% stenosis of the left main coronary artery and/or ≥70% stenosis in ≥ 1 other major epicardial vessel on invasive coronary angiography) and measured plasma ACE2 activity. Patients were followed up to determine if circulating ACE2 activity levels predicted the primary endpoint of MACE (cardiovascular mortality, HF or myocardial infarction). Results We recruited 79 patients with obstructive coronary artery disease. The median (IQR) plasma ACE2 activity was 29.3 pmol/ml/min [21.2–41.2]. Over a median follow up of 10.5 years [9.6–10.8years], MACE occurred in 46% of patients (36 events). On Kaplan-Meier analysis, above-median plasma ACE2 activity was associated with MACE (log-rank test, p = 0.035) and HF hospitalisation (p = 0.01). After Cox multivariable adjustment, log ACE2 activity remained an independent predictor of MACE (hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.24–4.72, p = 0.009) and HF hospitalisation (HR: 4.03, 95% CI: 1.42–11.5, p = 0.009). Conclusions Plasma ACE2 activity independently increased the hazard of adverse long-term cardiovascular outcomes in patients with obstructive CAD.

Left ventricular hypertrophy (LVH) is an independent risk factor for adverse cardiovascular events and is often present in patients with hypertension. Treatment to reduce blood pressure and regress LVH is key to improving health outcomes, but currently available drugs have only modest cardioprotective effects. Improved understanding of the molecular mechanisms involved in the development of LVH may lead to new therapeutic targets in the future. There is now compelling evidence that the transcription factor Kruppel-like factor 15 (KLF15) is an important negative regulator of cardiac hypertrophy in both experimental models and in man. Studies have reported that loss or suppression of KLF15 contributes to LVH, through lack of inhibition of pro-hypertrophic transcription factors and stimulation of trophic and fibrotic signaling pathways. This review provides a summary of the experimental and human studies that have investigated the role of KLF15 in the development of cardiac hypertrophy. It also discusses our recent paper that described the contribution of genetic variants in KLF15 to the development of LVH and heart failure in high-risk patients.

Endothelial dysfunction is a key feature of atherosclerosis. Retinal microvascular endothelial function can be assessed using noninvasive dynamic vessel imaging techniques. Whether it is impaired in subjects with coronary artery disease (CAD) is unknown. The aim of this study was to examine the relation of retinal microvascular endothelial function with CAD. Vascular studies were performed in 197 prospectively recruited subjects divided into 2 groups: those without CAD but ≥2 cardiovascular risk factors (non-CAD controls; n = 119) and those with stable CAD (n = 78). Retinal microvascular endothelial dysfunction was assessed by measuring retinal arteriolar and venular dilatation to flicker light, a nitric oxide-dependent phenomenon, expressed as percentage increase over baseline diameter. Fingertip pulse-volume amplitude was measured to calculate reactive hyperaemia index and brachial artery flow-mediated dilatation assessed as measures of peripheral microvascular and conduit vessel endothelial function, respectively. Mean retinal arteriolar dilatation was attenuated in patients with CAD compared with non-CAD controls (1.51 ± 1.51% vs 2.37 ± 1.95%, p = 0.001). Retinal arteriolar dilatation was independently associated with CAD after adjustment for age, gender, cardiovascular risk factors, and medication use (odds ratio 1.60, 95% confidence interval 1.14 to 2.25, p = 0.007). Reactive hyperaemia index and flow-mediated dilatation were not different. In conclusion, the capacity of retinal arterioles to dilate in response to flicker light is an independent predictor of the presence of CAD and suggests that retinal microvascular endothelial dysfunction is a marker for underlying CAD.

Background Cardiac hypertrophy increases the risk of developing heart failure and cardiovascular death. The neutrophil inflammatory protein, lipocalin‐2 (LCN2/NGAL), is elevated in certain forms of cardiac hypertrophy and acute heart failure. However, a specific role for LCN2 in predisposition and etiology of hypertrophy and the relevant genetic determinants are unclear. Here, we defined the role of LCN2 in concentric cardiac hypertrophy in terms of pathophysiology, inflammatory expression networks, and genomic determinants. Methods and Results We used 3 experimental models: a polygenic model of cardiac hypertrophy and heart failure, a model of intrauterine growth restriction and Lcn2‐knockout mouse; cultured cardiomyocytes; and 2 human cohorts: 114 type 2 diabetes mellitus patients and 2064 healthy subjects of the YFS (Young Finns Study). In hypertrophic heart rats, cardiac and circulating Lcn2 was significantly overexpressed before, during, and after development of cardiac hypertrophy and heart failure. Lcn2 expression was increased in hypertrophic hearts in a model of intrauterine growth restriction, whereas Lcn2‐knockout mice had smaller hearts. In cultured cardiomyocytes, Lcn2 activated molecular hypertrophic pathways and increased cell size, but reduced proliferation and cell numbers. Increased LCN2 was associated with cardiac hypertrophy and diastolic dysfunction in diabetes mellitus. In the YFS, LCN2 expression was associated with body mass index and cardiac mass and with levels of inflammatory markers. The single‐nucleotide polymorphism, rs13297295, located near LCN2 defined a significant cis‐eQTL for LCN2 expression. Conclusions Direct effects of LCN2 on cardiomyocyte size and number and the consistent associations in experimental and human analyses reveal a central role for LCN2 in the ontogeny of cardiac hypertrophy and heart failure., published version, peerReviewed

Background Gout is an inflammatory arthropathy characterised by elevated serum uric acid levels. In Australia, gout has a prevalence of 1.7 - 4%. 1 This increased to ∼10% in community based Australian patients with type 2 diabetes, although elevated serum uric acid did not predict cardiovascular (CV) or all-cause mortality. 2 To date, the long-term outcomes of patients with diabetes and comorbid gout being followed up in the hospital out-patient setting have not been studied. Objectives To compare cardiovascular risk factors and long-term outcomes and mortality in patients with type 2 diabetes according to the presence or absence of gout. Methods 1,405 patients with type 2 diabetes were prospectively recruited from the outpatient setting at Austin Hospital. Baseline cardiovascular risk factors and comorbidities were identified. Patients were classified as having gout if they gave a history of gout or were taking medication for the treatment of gout. For statistical analysis, patients with diabetes (Group 1) were compared to those with diabetes and gout (Group 2). Cardiovascular events and long-term CV mortality were assessed over a 10 year period. Results There were 1,329 patients with diabetes (Group 1; 95%) and 76 with diabetes and gout (Group 2; 5%). Patients with gout were older (68±11 vs. 64±12y, p=0.004), more likely to be male (80% vs. 59%, p Conclusions Although patients with comorbid gout and type 2 diabetes have a worse cardiovascular risk factor profile compared to those with diabetes alone, this was not associated with increased cardiovascular morbidity or all-cause mortality. These results suggest that elevated uric acid and gout are markers rather than determinants of CV mortality. References Robinson PC, Taylor WJ, Merriman TR. Systemic review of the prevalence of gout and hyperuricaemia in Australia. Internal Med J 2012; 42(9):997–1007. Ong G, Davis WA, Davis TME. Serum uric acid does not predict cardiovascular or all-cause mortality in type 2 diabetes: the Fremantle Diabetes Study. Diabetologia 2010; 53: 1288–129. Disclosure of Interest None declared

Background We aimed to investigate the incidence, precipitants, and outcomes of acute decompensated heart failure (ADHF) that develops during the inpatient stay. Methods We undertook a case-control study in the medical, oncology, surgical, and orthopaedic wards of a tertiary referral hospital (February–May, 2016). Patients aged ≥18 years who developed ADHF during their inpatient stay were enrolled as cases. One control patient was matched to each case by age, gender, presenting complaint/surgery performed and co-morbidities. Multivariate regression was employed to determine variables associated with ADHF. Results The incidence of ADHF was 1.0% of patients. Eighty cases were well-matched to 80 controls (p > 0.05). ADHF precipitants comprised infection (30%), inappropriate intravenous (IV) fluid and medication management (23.8% and 8.8%, respectively), tachyarrhythmia (12.5%), ischaemic heart disease (8.8%), renal failure (1.3%), and other/unclear causes (15%). Three variables were associated with ADHF: not having English as the preferred language (OR 3.5, 95%CI 1.2–9.8), a history of ischaemic heart disease (OR 3.3, 95%CI 1.2–9.1), and the administration of >2000 ml of IV fluid on the day before the ADHF (OR 8.3, 95%CI 1.5–48.0). The day before the ADHF, cases were administered significantly more IV fluids than controls (median 2,757.5 versus 975 ml, p = 0.001). Medication errors mostly related to failure to restart regular diuretics. Cases had significantly greater length of stay (median 15 versus 6 days, p Conclusions New onset ADHF is common and a substantial proportion of cases are iatrogenic. Cases experience significantly increased length of hospital stay, morbidity, and mortality.

Cognitive impairment is common in patients with hypertension. Left ventricular hypertrophy (LVH) is recognised as a marker of hypertension-related organ damage and is a strong predictor of coronary artery disease, heart failure and stroke. There is evidence that LVH is independently associated with cognitive impairment, even after adjustment for the presence of hypertension. We conducted a systematic review that examined cognitive impairment in adults with LVH. Independent searches were performed in Ovid MEDLINE, Ovid psycInfo and PubMed with the terms left ventricular hypertrophy and cognition. Seventy-three studies were identified when both searches were combined. After limiting the search to studies that were: (1) reported in English; (2) conducted in humans; (3) in adults aged 50 years and older; and (4) investigated the relationship between LVH and cognitive performance, nine papers were included in this systematic review. The majority of studies found an association between LVH and cognitive performance. Inspection of results indicated that individuals with LVH exhibited a lower performance in cognitive tests, when compared to individuals without LVH. Memory and executive functions were the cognitive domains that showed a specific vulnerability to the presence of LVH. A possible mechanism for the relationship between LVH and cognition is the presence of cerebral white matter damage. White matter lesions occur frequently in patients with LVH and may contribute to cognitive dysfunction. Together, the results of this review suggest that memory impairment and executive dysfunction are the cognitive domains that showed a particular association with the presence of LVH.

Background Cognitive impairment is common in type 2 diabetes mellitus, and there is a strong association between type 2 diabetes and Alzheimer’s disease. However, we do not know which type 2 diabetes patients will dement or which biomarkers predict cognitive decline. Left ventricular hypertrophy (LVH) is potentially such a marker. LVH is highly prevalent in type 2 diabetes and is a strong, independent predictor of cardiovascular events. To date, no studies have investigated the association between LVH and cognitive decline in type 2 diabetes. The Diabetes and Dementia (D2) study is designed to establish whether patients with type 2 diabetes and LVH have increased rates of brain atrophy and cognitive decline. Methods The D2 study is a single centre, observational, longitudinal case control study that will follow 168 adult patients aged >50 years with type 2 diabetes: 50% with LVH (case) and 50% without LVH (control). It will assess change in cardiovascular risk, brain imaging and neuropsychological testing between two time-points, baseline (0 months) and 24 months. The primary outcome is brain volume change at 24 months. The co-primary outcome is the presence of cognitive decline at 24 months. The secondary outcome is change in left ventricular mass associated with brain atrophy and cognitive decline at 24 months. Discussion The D2 study will test the hypothesis that patients with type 2 diabetes and LVH will exhibit greater brain atrophy than those without LVH. An understanding of whether LVH contributes to cognitive decline, and in which patients, will allow us to identify patients at particular risk. Trial registration Australian New Zealand Clinical Trials Registry (ACTRN12616000546459), date registered, 28/04/2016 Electronic supplementary material The online version of this article (doi:10.1186/s12902-017-0173-7) contains supplementary material, which is available to authorized users.

Left ventricular (LV) hypertrophy (LVH) is a heritable trait that is common in type 2 diabetes and is associated with the development of heart failure. The transcriptional factor Kruppel like factor 15 (KLF15) is expressed in the heart and acts as a repressor of cardiac hypertrophy in experimental models. This study investigated if KLF15 gene variants were associated with LVH in type 2 diabetes. In stage 1 of a 2-stage approach, patients with type 2 diabetes and no known cardiac disease were prospectively recruited for a transthoracic echocardiographic assessment (Melbourne Diabetes Heart Cohort) (n = 318) and genotyping of two KLF15 single nucleotide polymorphisms (SNPs) (rs9838915, rs6796325). In stage 2, the association of KLF15 SNPs with LVH was investigated in the Genetics of Diabetes Audit and Research in Tayside Scotland (Go-DARTS) type 2 diabetes cohort (n = 5631). The KLF15 SNP rs9838915 A allele was associated in a dominant manner with LV mass before (P = 0.003) and after (P = 0.001) adjustment for age, gender, body mass index (BMI) and hypertension, and with adjusted septal (P, Highlights • KLF15 SNP rs9838915 A allele is associated with increased LV mass in patients with 2 diabetes. • KLF15 SNP rs9838915 predicts incident heart failure hospitalization. • Genotyping KLF15 SNP rs9838915 allowed more precise stratification of the risk of heart failure hospitalization. Left ventricular hypertrophy (LVH) is a heritable trait that is common in patients with diabetes. The Kruppel like factor 15 (KLF15) is expressed in the heart and acts as a repressor of cardiac hypertrophy and fibrosis. Our study provides evidence that genetic variation in KLF15 is associated with LVH in patients with type 2 diabetes and these findings were then replicated in an independent cohort of patients with type 2 diabetes. The KLF15 genetic variant was also associated with first heart failure hospitalization. These findings add to our understanding of the molecular mechanisms that contribute to increased LV mass.

We previously reported that exogenous angiotensin (Ang) 1-7 has adverse cardiac effects in experimental kidney failure due to its action to increase cardiac angiotensin converting enzyme (ACE) activity. This study investigated if the addition of an ACE inhibitor (ACEi) to Ang 1-7 infusion would unmask any beneficial effects of Ang 1-7 on the heart in experimental kidney failure. Male Sprague-Dawley rats underwent subtotal nephrectomy (STNx) and were treated with vehicle, the ACEi ramipril (oral 1mg/kg/day), Ang 1-7 (subcutaneous 24 μg/kg/h) or dual therapy (all groups, n = 12). A control group (n = 10) of sham-operated rats were also studied. STNx led to hypertension, renal impairment, cardiac hypertrophy and fibrosis, and increased both left ventricular ACE2 activity and ACE binding. STNx was not associated with changes in plasma levels of ACE, ACE2 or angiotensin peptides. Ramipril reduced blood pressure, improved cardiac hypertrophy and fibrosis and inhibited cardiac ACE. Ang 1-7 infusion increased blood pressure, cardiac interstitial fibrosis and cardiac ACE binding compared to untreated STNx rats. Although in STNx rats, the addition of ACEi to Ang 1-7 prevented any deleterious cardiac effects of Ang 1-7, a limitation of the study is that the large increase in plasma Ang 1-7 with ramipril may have masked any effect of infused Ang 1-7.

Objective Retinal microvascular signs are markers of cardiovascular disease risk. There are limited data, on relationships between retinal microvascular signs and retinal microvascular endothelial function. We sought to determine the relationship of retinal vascular signs with retinal microvascular endothelial function in patients with or at high risk of coronary artery disease. Methods Participants with atherosclerosis risk factors and coronary disease ( n = 258; mean age 57 ± 11 years) were recruited to have static and dynamic retinal vascular assessment. Retinal arteriolar dilatation in response to flicker light (FI–RAD) was measured using the Digital Vessel Analyser and expressed as percentage increase over baseline diameter. Static retinal photographs were acquired utilising a digital fundus camera for measurement of central retinal artery and vein equivalent (CRAE and CRVE), arteriovenous nicking (AVN) and focal arteriolar narrowing (FAN). Results Intra-class correlation coefficient was 0.82 for flicker-light induced retinal arteriolar dilatation. There were modest associations in retinal vascular measurements between eyes. For each 10 μm decrease in retinal arteriolar diameter, the absolute increase in FI–RAD was 0.28% (95% CI 0.11, 0.45; p = 0.002) independent of age, gender and atherosclerosis risk factors. AVN and FAN were associated with attenuated FI–RAD ( β = −0.67%; 95% CI −1.20, −0.15; p = 0.012) and ( β = −0.83%; 95% CI −1.44, −0.23; p = 0.007) respectively after adjustment for age and gender. Conclusion Assessment of retinal microvascular endothelial function is reproducible and correlated with retinal microvascular structure and signs, independent of atherosclerosis risk factors. Assessment of retinal vascular structure and function may provide insights into atherosclerotic disease.

The aims of this observational study were to determine the prevalence and predictors of an abnormal echocardiogram in adults with type 1 diabetes, and to assess the evolution of changes in a subset of subjects.Cardiac function and structure were prospectively investigated by comprehensive transthoracic echocardiographic techniques in asymptomatic adults with type 1 diabetes seen in the ambulatory care setting.We recruited 136 subjects (mean age 39 years, SD 14 years) with a median diabetes duration of 21 years [25(th), 75(th) interquartile range; 11, 29]. An abnormal echocardiogram was present in 29% of subjects; diastolic dysfunction in 69%, left ventricular hypertrophy in 38% and systolic dysfunction in 10%. The independent predictors of an abnormal echocardiogram were age, with a 9-fold increase in those ≥40 years (OR 9.40 [95% CI 2.68-33.04], P0.0001), and increased body mass index (BMI), with a 17% increase in risk (P=0.04). A second echocardiogram was available in 65 subjects (3.8±1.7 years later). The results showed that one in five with a normal first study had developed an abnormal second study, mainly diastolic dysfunction, with age being the only independent predictor of progression (P=0.006).Subclinical echocardiographic abnormalities are common in asymptomatic type 1 diabetes adults, and changes are progressive. The addition of an echocardiogram to complication surveillance programs in those with type 1 diabetes aged ≥40 years may represent a cost-effective way to screen for, and aggressively treat, occult cardiac disease.

The renin-angiotensin system plays a major role in the pathophysiology of cardiovascular disease (CVD). The enzyme angiotensin-converting enzyme (ACE) converts angiotensin (Ang) I into the vasoconstrictor AngII and was thought, until recently, to be the main effector of the system. The enzyme ACE2, discovered in 2000, can counterbalance the effects of ACE through degradation of AngII and generation of Ang-(1-7). Angiotensin-converting enzyme 2 is abundantly expressed in the heart and localized to the endothelial cells of coronary vessels and smooth muscle cells. Its catalytically active ectodomain undergoes shedding, resulting in ACE2 in the circulation. There are 10 studies to date that have measured circulating ACE2 activity in humans, including in healthy subjects and those with heart failure, Type 1 diabetes, implantable cardioverter/defibrillator, elderly subjects undergoing emergency orthopaedic surgery and kidney transplant patients. The results suggest that circulating ACE2 activity may be a marker of CVD, with low levels in healthy individuals and increased levels in those with cardiovascular risk factors or disease. Whether increased plasma ACE2 activity reflects increased synthesis from tissue ACE2 mRNA or increased shedding of tissue ACE2 remains to be determined. Angiotensin-converting enzyme 2 is located on the X-chromosome and circulating ACE2 levels are higher in men than in women. Large clinical studies in CVD are needed to more precisely clarify the role of ACE2 as a biomarker of CVD, determine the prognostic significance of circulating ACE2 activity and assess whether the measurement of ACE2 will improve CVD risk prediction.

Objective Upregulation of the receptor for advanced glycation end products (RAGE) has been proposed as a pathophysiological mechanism underlying the development of atrial fibrillation (AF). We sought to investigate if soluble RAGE levels are associated with AF in Caucasian patients. Methods Patients (n = 587) were prospectively recruited and serum levels of soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE) measured. The patients included 527 with sinus rhythm, 32 with persistent AF (duration >7 days, n = 32) and 28 with paroxysmal AF (duration

Angiotensin converting enzyme (ACE) 2 is a negative regulator of the renin angiotensin system (RAS) through its role to degrade angiotensin II. In rats with subtotal nephrectomy (STNx), adverse cardiac remodelling occurs despite elevated cardiac ACE2 activity. We hypothesised that diminazene aceturate (DIZE), which has been described as having an off-target effect to activate ACE2, would have beneficial cardiac effects in STNx rats. STNx led to hypertension, diastolic dysfunction, left ventricular hypertrophy, cardiac fibrosis, and increased cardiac ACE, ACE2, Ang II and Ang 1-7 levels. Cardiac gene expression of ADAM17 was also increased. In STNx, two-weeks of subcutaneous DIZE (15mg/kg/d) had no effect on blood pressure but improved diastolic dysfunction and cardiac fibrosis, reduced ADAM17 mRNA and shifted the cardiac RAS balance to a cardioprotective profile with reduced ACE and Ang II. There was no change in cardiac ACE2 activity or in cardiac Ang 1-7 levels with DIZE. In conclusion, our results suggest that DIZE exerts a protective effect on the heart under the pathological condition of kidney injury. This effect was not due to improved kidney function, a fall in blood pressure or a reduction in LVH but was associated with a reduction in cardiac ACE and cardiac Ang II levels. As in vitro studies showed no direct effect of DIZE on ACE2 or ACE activity, the precise mechanism of action of DIZE remains to be determined.

The RAS (renin–angiotensin system) plays an important role in the pathophysiology of CVD (cardiovascular disease), and RAS blockade is an important therapeutic strategy in the management of CVD. A new counterbalancing arm of the RAS is now known to exist in which ACE (angiotensin-converting enzyme) 2 degrades Ang (angiotensin) II, the main effector of the classic RAS, and generates Ang-(1–7). Altered ACE2 expression is associated with cardiac and vascular disease in experimental models of CVD, and ACE2 is increased in failing human hearts and atherosclerotic vessels. In man, circulating ACE2 activity increases with coronary heart disease, as well as heart failure, and a large proportion of the variation in plasma ACE2 levels has been attributed to hereditary factors. The ACE2 gene maps to chromosome Xp22 and this paper reviews the evidence associating ACE2 gene variation with CVD and considers clues to potential functional ACE2 variants that may alter gene expression or transcriptional activity. Studies to date have investigated ACE2 gene associations in hypertension, left ventricular hypertrophy and coronary artery disease, but the results have been inconsistent. The discrepancies may reflect the sample size of the studies, the gender or ethnicity of subjects, the cardiovascular phenotype or the ACE2 SNP investigated. The frequent observation of apparent sex-dependence might be of special importance, if confirmed. As yet, there are no studies to concurrently assess ACE2 gene polymorphisms and circulating ACE2 activity. Large-scale carefully conducted clinical studies are urgently needed to clarify more precisely the potential role of ACE2 in the CVD continuum.

The RAS (renin–angiotensin system) is activated after MI (myocardial infarction), and RAS blockade with ACEis [ACE (angiotensin-converting enzyme) inhibitors] or ARBs (angiotensin receptor blockers) slows but does not completely prevent progression to heart failure. Cardiac ACE is increased after MI and leads to the formation of the vasoconstrictor AngII (angiotensin II). The enzyme ACE2 is also activated after MI and degrades AngII to generate the vasodilator Ang-(1–7) [angiotensin-(1–7)]. Overexpression of ACE2 offers cardioprotective effects in experimental MI, but there is conflicting evidence as to whether the benefits of ACEis and ARBs are mediated through increasing ACE2 after MI. In the present study, we assessed the effect of an ACEi and ARB, alone and in combination, on cardiac ACE2 in a rat MI model. MI rats received vehicle, ACEi (ramipril; 1 mg/kg of body weight), ARB (valsartan; 10 mg/kg of body weight) or combination (ramipril at 1 mg/kg of body weight and valsartan at 10 mg/kg of body weight) orally for 28 days. Sham-operated rats were also studied and received vehicle alone. MI increased LV (left ventricular) mass (P

Aims To assess the capacity of global longitudinal strain (GLS) in patients with aortic stenosis (AS) to (i) detect the subclinical left ventricular (LV) dysfunction [LV ejection fraction (LVEF) ≥50% patients]; (ii) predict all-cause mortality and major adverse cardiac events (MACE) (all patients), and (iii) provide incremental prognostic information over current risk markers. Methods and results Patients with AS ( n = 146) and age-matched controls ( n = 12) underwent baseline echocardiography to assess AS severity, conventional LV parameters and GLS via speckle tracking echocardiography. Baseline demographics, symptom severity class and comorbidities were recorded. Outcomes were identified via hospital record review and subject/physician interview. The mean age was 75 ± 11, 62% were male. The baseline aortic valve (AV) area was 1.0 ± 0.4 cm2 and LVEF was 59 ± 11%. In patients with a normal LVEF ( n = 122), the baseline GLS was controls −21 ± 2%, mild AS −18 ± 3%, moderate AS −17 ± 3% and severe AS −15 ± 3% ( P < 0.001). GLS correlated with the LV mass index, LVEF, AS severity, and symptom class ( P < 0.05). During a median follow-up of 2.1 (inter-quartile range: 1.8–2.4) years, there were 20 deaths and 101 MACE. Unadjusted hazard ratios (HRs) for GLS (per %) were all-cause mortality (HR: 1.42, P < 0.001) and MACE (HR: 1.09, P < 0.001). After adjustment for clinical and echocardiographic variables, GLS remained a strong independent predictor of all-cause mortality (HR: 1.38, P < 0.001). Conclusions GLS detects subclinical dysfunction and has incremental prognostic value over traditional risk markers including haemodynamic severity, symptom class, and LVEF in patients with AS. Incorporation of GLS into risk models may improve the identification of the optimal timing for AV replacement.

Renin-angiotensin system blockade slows but does not prevent the cardiovascular complications of chronic kidney disease (CKD). Angiotensin-converting enzyme (ACE) 2 is differentially regulated in acute kidney injury, with increased cardiac ACE2 but decreased kidney ACE2 levels. This study investigated the effect of long-term ACE inhibition on cardiac and renal ACE2 in rats with CKD induced by subtotal nephrectomy (STNx). Sprague-Dawley rats had sham (control) or STNx surgery. Control rats received vehicle (n = 9) and STNx rats ramipril (1 mg kg(-1) day(-1); n = 10) or vehicle (n = 10) for 28 days. Subtotal nephrectomy resulted in impaired creatinine clearance (P < 0.05), proteinuria (P < 0.05), renal fibrosis (P < 0.05) and reduced renal cortical ACE2 mRNA (P < 0.05) and activity (P < 0.05). In rats with CKD, ramipril improved creatinine clearance (P < 0.05) and was associated with an increase in cortical but not medullary ACE2 activity (P < 0.05). Compared with control rats, STNx rats were hypertensive (P < 0.01), with increased left ventricular end-diastolic pressure (LVEDP; P < 0.01), left ventricular hypertrophy (LVH; P < 0.05) and interstitial (P < 0.05) and perivascular fibrosis (P < 0.01). In rats with CKD, ramipril decreased blood pressure (P < 0.001) and reduced LVEDP (P < 0.01), LVH (P < 0.01) and perivascular fibrosis (P < 0.05) but did not significantly reduce interstitial fibrosis. There was no change in cardiac ACE2 in rats with CKD compared with control rats. In rats with CKD, ACE inhibition had major benefits to reduce blood pressure and cardiac hypertrophy and to improve creatinine clearance, but did not significantly impact on cardiac ACE2, cardiac interstitial fibrosis, renal fibrosis or proteinuria. Thus, in rats with CKD, renal ACE2 deficiency and lack of activation of cardiac ACE2 may contribute to the progression of cardiac and renal tissue injury. As long-term ACE inhibition only partly ameliorated the adverse cardio-renal effects of CKD, adjunctive therapies that lead to further increases in ACE2 activity may be needed to combat the cardio-renal complications of CKD.

Peguero et al. [(1)][1] have described new electrocardiographic (ECG) criteria (the SD + SV4 –Peguero-Lo Presti) for the diagnosis of left ventricular hypertrophy (LVH), which improved the sensitivity and accuracy of the test. We addressed 2 outstanding issues with regard to the new criteria in

The accumulation of advanced glycation end products is thought to be a key factor in the initiation and progression of diabetic nephropathy. Here we determined whether the size of the ligands for the receptor for advanced glycation end products (RAGEs) that were present in the serum of patients with type 2 diabetes modulates their pathogenic potential. Serum was collected from control subjects and patients with type 2 diabetes with varying degrees of renal disease (normo-, micro-, or macroalbuminuria). The titers of the RAGE ligands N-carboxymethyllysine (CML), S100A, S100B, and high-mobility group box 1 (HMGB1) were measured by enzyme-linked immunosorbent assay in serum as well as in pooled size-fractionated serum. We also measured cellular binding of serum fractions to mesangial cells transfected with RAGE and examined the downstream signaling pathways. Circulating CML was increased in patients with type 2 diabetes, whereas HMGB1 was decreased. S100A8, S100BA9, and soluble RAGE were unchanged. The high-molecular-weight (over 50 kDa) serum fraction contained the greatest proportion of RAGE ligands, with all immunoreactivity and cellular binding observed only with serum fractions over 30 kDa. High-molecular-weight serum from macroalbuminuric patients showed greater RAGE binding capacity, modulation of cell-surface RAGE expression, increased phospho-protein kinase C-alpha, and p65 nuclear factor kappaB DNA-binding activity, which were competitively inhibited by soluble RAGE or CML neutralizing antibodies. These data show that ligands that activate RAGE present in the circulation of patients with type 2 diabetes and nephropathy are predominantly of high molecular weight.

Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. There was a significant negative correlation between mtDNA copy number and islet donor age (r = –0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged ≥50 years (n = 8) compared with those aged

OBJECTIVE— Type 2 diabetes is characterized by impaired pancreatic β-cell function and decreased insulin sensitivity. Genome-wide association studies have identified common, novel type 2 diabetes susceptibility loci within the FTO, CDKAL1, CDKN2A/CDKN2B, IGF2BP2, HHEX/IDE, and SLC30A8 gene regions. Our objective was to explore the relationships between the diabetes-associated alleles and measures of β-cell function and whole-body insulin sensitivity.RESEARCH DESIGN AND METHODS— A total of 1,276 healthy subjects of European ancestry were studied at 19 centers. Indexes of β-cell function (including 30-min insulin response and glucose sensitivity) were derived from a 75-g oral glucose tolerance test, and whole-body insulin sensitivity (M/I) was assessed by hyperinsulinemic-euglycemic clamp. Genotype/phenotype relationships were studied by linear trend analysis correcting for age, sex, and recruitment center.RESULTS— CDKAL1 and HHEX/IDE diabetes-associated alleles were both associated with decreased 30-min insulin response (both P = 0.0002) and decreased pancreatic β-cell glucose sensitivity (P = 9.86 × 10−5 and 0.009, respectively), and these relationships remained after correction for M/I. The FTO susceptibility allele showed a weak but consistent association with increased adiposity, which in turn was linked to a decrease in M/I. However, none of the other novel diabetes susceptibility alleles were associated with insulin sensitivity.CONCLUSIONS— CDKAL1 and HHEX/IDE diabetes-associated alleles are associated with decreased pancreatic β-cell function, including decreased β-cell glucose sensitivity that relates insulin secretion to plasma glucose concentration. We confirmed the association between the FTO allele and increased adiposity, but none of the other novel susceptibility alleles were associated with whole-body insulin sensitivity.

Cardiovascular disease, including cardiac hypertrophy, is common in patients with kidney disease and can be partially attenuated using blockers of the renin-angiotensin system (RAS). It is unknown whether cardiac microRNAs contribute to the pathogenesis of cardiac hypertrophy or to the protective effect of RAS blockade in kidney disease. Using a subtotal nephrectomy rat model of kidney injury, we investigated changes in cardiac microRNAs that are known to have direct target genes involved in the regulation of apoptosis, fibrosis, and hypertrophy. The effect of treatment with the angiotensin-converting enzyme (ACE) inhibitor ramipril on cardiac microRNAs was also investigated. Kidney injury led to a significant increase in cardiac microRNA-212 and microRNA-132 expression. Ramipril reduced cardiac hypertrophy, attenuated the increase in microRNA-212 and microRNA-132, and significantly increased microRNA-133 and microRNA-1 expression. There was altered expression of caspase-9, B cell lymphoma-2, transforming growth factor-β, fibronectin 1, collagen type 1A1, and forkhead box protein O3, which are all known to be involved in the regulation of apoptosis, fibrosis, and hypertrophy in cardiac cells while being targets for the above microRNAs. ACE inhibitor treatment increased expression of microRNA-133 and microRNA-1. The inhibitory action of ACE inhibitor treatment on increased cardiac NADPH oxidase isoform 1 expression after subtotal nephrectomy surgery suggests that inhibition of oxidative stress is also one of mechanism of ACE inhibitor-mediated cardioprotection. These finding suggests the involvement of microRNAs in the cardioprotective action of ACE inhibition in acute renal injury, which is mediated through an inhibitory action on profibrotic and proapoptotic target genes and stimulatory action on antihypertrophic and antiapoptotic target genes.