A Liquid Biopsy-Based Approach to Isolate and Characterize Adipose Tissue-Derived Extracellular Vesicles from Blood.

Obesity is a major risk factor for multiple chronic diseases. Anthropometric and imaging approaches are primarily used to assess adiposity, and there is a dearth of techniques to determine the changes in adipose tissue (AT) at the molecular level. Extracellular vesicles (EVs) have emerged as a novel and less invasive source of biomarkers for various pathologies. Furthermore, the possibility of enriching cell or tissue-specific EVs from the biofluids based on their unique surface markers has led to classifying these vesicles as "liquid biopsies", offering valuable molecular information on hard-to-access tissues. Here, we isolated small EVs from AT (sEVAT) of lean and diet-induced obese (DIO) mice, identified unique surface proteins on sEVAT by surface shaving followed by mass spectrometry, and developed a signature of five unique proteins. Using this signature, we pulled out sEVAT from the blood of mice and validated the specificity of isolated sEVAT by measuring the expression of adiponectin, 38 adipokines on an array, and several adipose tissue-related miRNAs. Furthermore, we provided evidence of sEV applicability in disease prediction by characterizing sEVAT from the blood of lean and DIO mice. Interestingly, sEVAT-DIO cargo showed a stronger pro-inflammatory effect on THP1 monocytes compared to sEVAT-Lean and a significant increase in obesity-associated miRNA expression. Equally important, sEVAT cargo revealed an obesity-associated aberrant amino acid metabolism that was subsequently validated in the corresponding AT. Lastly, we show a significant increase in inflammation-related molecules in sEVAT isolated from the blood of nondiabetic obese (>30 kg/m2) individuals. Overall, the present study offers a less-invasive approach to characterize AT.

The ß-Secretase 1 Enzyme as a Novel Therapeutic Target for Prostate Cancer.

Recent studies have demonstrated the association of APP and Aß with cancer, suggesting that BACE1 may play an important role in carcinogenesis. In the present study, we assessed BACE1's usefulness as a therapeutic target in prostate cancer (PCa). BACE1 expression was observed in human PCa tissue samples, patient-derived xenografts (PDX), human PCa xenograft tissue in nude mice, and transgenic adenocarcinoma of the mouse prostate (TRAMP) tissues by immunohistochemistry (IHC) analysis. Additionally, the downstream product of BACE1 activity, i.e., Aß1-42 expression, was also observed in these PCa tissues by IHC as well as by PET imaging in TRAMP mice. Furthermore, BACE1 gene expression and activity was confirmed in several established PCa cell lines (LNCaP, C4-2B-enzalutamide sensitive [S], C4-2B-enzalutamide resistant [R], 22Rv1-S, 22Rv1-R, PC3, DU145, and TRAMP-C1) by real-time PCR and fluorometric assay, respectively. Treatment with a pharmacological inhibitor of BACE1 (MK-8931) strongly reduced the proliferation of PCa cells in in vitro and in vivo models, analyzed by multiple assays (MTT, clonogenic, and trypan blue exclusion assays and IHC). Cell cycle analyses revealed an increase in the sub-G1 population and a significant modulation in other cell cycle stages (G1/S/G2/M) following MK-8931 treatment. Most importantly, in vivo administration of MK-8931 intraperitoneal (30 mg/kg) strongly inhibited TRAMP-C1 allograft growth in immunocompetent C57BL/6 mice (approximately 81% decrease, p = 0.019). Furthermore, analysis of tumor tissue using the prostate cancer-specific pathway array revealed the alteration of several genes involved in PCa growth and progression including Forkhead O1 (FOXO1). All together, these findings suggest BACE1 as a novel therapeutic target in advanced PCa.

Mass Spectrometry-Based Proteome Profiling of Extracellular Vesicles Derived from the Cerebrospinal Fluid of Adult Rhesus Monkeys Exposed to Cocaine throughout Gestation.

Cocaine use disorder has been reported to cause transgenerational effects. However, due to the lack of standardized biomarkers, the effects of cocaine use during pregnancy on postnatal development and long-term neurobiological and behavioral outcomes have not been investigated thoroughly. Therefore, in this study, we examined extracellular vesicles (EVs) in adult (~12 years old) female and male rhesus monkeys prenatally exposed to cocaine (n = 11) and controls (n = 9). EVs were isolated from the cerebrospinal fluid (CSF) and characterized for the surface expression of specific tetraspanins, concentration (particles/mL), size distribution, and cargo proteins by mass spectrometry (MS). Transmission electron microscopy following immunogold labeling for tetraspanins (CD63, CD9, and CD81) confirmed the successful isolation of EVs. Nanoparticle tracking analyses showed that the majority of the particles were <200 nm in size, suggesting an enrichment for small EVs (sEV). Interestingly, the prenatally cocaine-exposed group showed ~54% less EV concentration in CSF compared to the control group. For each group, MS analyses identified a number of proteins loaded in CSF-EVs, many of which are commonly listed in the ExoCarta database. Ingenuity pathway analysis (IPA) demonstrated the association of cargo EV proteins with canonical pathways, diseases and disorders, upstream regulators, and top enriched network. Lastly, significantly altered proteins between groups were similarly characterized by IPA, suggesting that prenatal cocaine exposure could be potentially associated with long-term neuroinflammation and risk for neurodegenerative diseases. Overall, these results indicate that CSF-EVs could potentially serve as biomarkers to assess the transgenerational adverse effects due to prenatal cocaine exposure.

Prevalence of type 2 diabetes mellitus and association of HbA1c with severity of coronary artery disease in patients presenting as non-diabetic acute coronary syndrome.

BACKGROUND: Acute coronary syndrome (ACS) indicates the serious clinical manifestation of coronary artery disease (CAD) and is closely associated with cardiovascular prognosis in patients with ACS. This study was aimed to study the prevalence of type 2 diabetes mellitus (T2DM) and the relation of HbA1c with the severity of CAD in patients presenting as non-diabetic ACS. Diabetic status of the patients was assessed with fasting blood sugar (FBS) and HbA1c levels, and coronary artery disease burden was assessed by coronary angiography. RESULTS: Out of 208 patients, 85.1% were males, and 14.9% were females; 73.56% cases were hypertensive. 80.77% of cases had STEMI, 17.79% had NSTEMI, and 1.44% had unstable angina. Out of 168 STEMI patients, 64.3% were thrombolysed, 21.42% presented late, 2.38% had contraindications to thrombolysis, and 11.9% underwent primary PCI. FBS in diabetic range was found in 44.23% of cases, impaired FBS in 36.54%, and 19.23% of patients had FBS in non-diabetic range. According to HbA1c, 41.8% were diabetic, 39.4% were pre-diabetic, and 18.8% were non-diabetic. A significant positive correlation was found between HbA1c and Gensini score and between HbA1c and the number of vessels involved. CONCLUSION: This study emphasises the importance of evaluating the presence of diabetes in patients presenting as non-diabetic acute coronary syndrome in developing countries. Acute coronary syndrome may be considered as one of the presentations of diabetes mellitus.

Primary percutaneous coronary intervention with diagnostic catheter in an anomalous origin right coronary artery-a case report.

BACKGROUND: Although rare, the possibility of encountering an anomalous coronary artery is a reality. The outcome of such a procedure is greatly influenced by the awareness of the operator about the anatomical variations and the technique required. CASE PRESENTATION: A 50-year-old female patient presented with chest pain. On evaluation, she was found to have an inferior wall myocardial infarction. Left coronary angiography showed non-obstructive disease in the left anterior descending (LAD) and left circumflex artery (LCX). The right coronary artery could not be hooked despite multiple attempts and catheter changes. A non-specific aortic angiogram revealed anomalous origin of the right coronary artery (RCA) above the sinotubular junction on the left side. RCA was hooked with the AL-2 diagnostic catheter, and the percutaneous coronary intervention (PCI) procedure was completed via the same diagnostic catheter. CONCLUSION: In a life-threatening difficult situation like acute coronary syndrome with anomalous origin of coronary arteries, PCI can be done using a diagnostic catheter.

The effect of percutaneous coronary revascularization on plasma N-terminal pro-B-type natriuretic peptide levels in stable coronary artery disease.

BACKGROUND: This study was conducted to assess the effect of percutaneous coronary revascularization (PCR) on plasma NT-proBNP concentration in patients with chronic stable angina (CSA). METHODS: This prospective open label interventional study included 22 patients with moderate to severe CSA, normal left ventricular (LV) systolic functions and critical (>90%) proximal stenosis in one of the three major epicardial coronary arteries. After stabilization of medications for 8 weeks, resting supine plasma NT-proBNP levels were measured and patients underwent PCR of the involved vessels. Eight weeks later, with medications unaltered; plasma NT-proBNP levels were repeated and compared with the baseline levels. LV systolic and diastolic functions were assessed before and after PCR. RESULTS: The mean age of the patients was 61.27±8.87years. Out of 22 patients, 20 were male and 2 were female. PCR was performed on left anterior descending coronary artery (LAD) in 12 patients and in a non-LAD vessel in 10 patients. After 8 weeks of successful PCR, there was a significant overall reduction in mean plasma NT-proBNP levels (from 244.36±218.99 to 168.68±161.61pg/mL, p=0.016). The patients who underwent PCR of LAD demonstrated significantly reduced NT-pro-BNP levels after PCR (p=0.009). In the non-LAD group, NT-proBNP levels also decreased, albeit insignificantly (p=0.432). Reduction in NT-proBNP was independent of change in LV systolic functions. CONCLUSION: Successful PCR, by relieving myocardial ischemia, significantly reduced plasma NT-proBNP levels in majority of the patients with chronic stable angina secondary to critical epicardial coronary artery stenosis.

Contrast venography vs. microwire assisted axillary venipuncture for cardiovascular implantable electronic device implantation.

Aims: This study was conducted to compare the safety and efficacy of microwire assisted technique with contrast venography guided axillary venipuncture in patients undergoing cardiovascular implantable electronic device (CIED) implantation. Methods and results: This prospective randomized study included 212 consecutive adult patients undergoing CIED implantation at our institute between 2013 and 2015. Patients were randomized to either venography guided technique (Group I; n = 105) or microwire assisted technique (Group II; n = 107) for axillary venipuncture. In Group I axillary venogram was used as a roadmap for guiding the puncture. In Group II, a 0.014 inch hydrophilic coronary guidewire ('microwire') was introduced through the ipsilateral antecubital vein and puncture needle was aimed to hit the microwire over the first or second rib. Outcome measures including technical success rates; number of attempts to successful puncture; puncture duration; fluoroscopy times and adverse events were compared in the two groups. Overall success rates were similar in both groups (97.4% in Group I and 100% in Group II, P = 0.061). We demonstrated significantly higher first attempt success rates; shorter puncture durations and fluoroscopy times; and lower number of attempts to successful puncture with microwire assisted technique (89.3% vs. 65.6%; 36.7 ± 23.1 s vs. 67.8 ± 44.9 s; 62.4 ± 35.3 s vs. 118.9 ± 63.2 s; and 1.21 ± 0.82 vs. 2.16 ± 1.54 respectively, P < 0.001). Adverse event rates were significantly lower with microwire assisted technique (0.9% vs. 8.6%, P = 0.009). Conclusion: Microwire assisted technique is a simple, quicker, safer and more efficacious alternative to contrast venography guided axillary venipuncture.

Clinico-angiographic profile and procedural outcomes in patients undergoing percutaneous coronary interventions: The Srinagar registry.

BACKGROUND: This study was aimed at exploring the clinical profile, angiographic characteristics and procedural outcomes in patients undergoing PCI at our institute. METHODS: This prospective observational study included all consecutive patients who underwent PCI at our hospital between January 2014 and December 2015. Data including clinico-demographic profile, angiographic details and lesion characteristics were recorded in all patients. Procedural details including devices and drugs used, procedure related complications, and in-hospital outcomes of these patients were analysed. RESULTS: A total of 624 patients (mean age- 59.30±11.17years) with 84.8% males and 15.2% females were included in the study. Smoking and hypertension were the most common risk factors, present in 79.8% and 74.8% patients respectively. Diabetes mellitus, dyslipidemia, and obesity were observed in 24.5%, 26.1%, and 25.0% patients respectively. Anterior wall MI was the most common mode of presentation (32.1%). Single Vessel Disease (SVD) was most common angiographic pattern, observed in 50.3% patients; left anterior descending artery (LAD) was the most frequently involved vessel (65.9%); and type B lesions were most prevalent (52.3%). Most of the procedures were elective (61.4%) and femoral route was used in the majority (82.6%). Drug eluting stents were deployed in 99.1% of the cases. The overall procedural success rate was 93.6%. Procedural mortality was 1.0% and periprocedural complications occurred in 9.9% patients. CONCLUSION: This first prospective PCI registry from the state of Jammu & Kashmir provides an insight into the patterns of CAD among Kashmiri population, and highlights the spectrum of PCIs performed with their outcomes.

An Innovation in Pacemaker Lead Implantation via Persistent Left Superior Vena Cava: The "3D Alpha Curve" Stylet.

Persistent left superior vena cava (PLSVC) draining into coronary sinus is typically detected incidentally during transcatheter interventions using left subclavian venous approach. In our experience, we have encountered this anomaly on a few occasions and in all these cases we successfully implanted leads in the right ventricle (RV) by shaping the stylet into a "U-shaped" or "pigtail-shaped" curve. Herein, we report a case of an adult male who underwent successful dual-chamber pacemaker implantation via PLSVC through left axillary venous approach. In this case, we were unable to deliver the lead into RV using aforementioned stylets. As an innovation, we used a "three-dimensional alpha curve"-shaped stylet that facilitated an easy entry of pacing lead into the RV.

Components and determinants of therapeutic delay in patients with acute ST-elevation myocardial infarction: A tertiary care hospital-based study.

BACKGROUND: Delayed reperfusion is associated with worse outcomes in ST-segment elevation myocardial infarction (STEMI). This study was conducted to assess the components and determinants of therapeutic delay in STEMI patients of our state. METHODS: This study included consecutive patients of STEMI admitted to the coronary care units of two tertiary care hospitals in Srinagar, between 2012 and 2015. Various components of treatment delay including the patient's decision to delay, referral delay, transportation delay, prehospital delay, and door-to-needle time were calculated. Factors associated with delayed treatment and clinico-demographic correlates of late presentation were identified. RESULTS: During a period of 3 years, 523 patients (mean age, 57.6 ± 10.5 years) were enrolled in this study. Thrombolysis was administered to 60.2% patients, while 39.8% of patients could not be thrombolysed because of late presentation. The median treatment delay was 250 minutes. Prehospital delay constituted about 83.8% of total treatment delay. Patient's decision to delay, referral delay, and transport delay constituted 59%, 16%, and 25% of prehospital delay, respectively. Median door-to-needle time was 40 minutes. Residence in rural areas [odds ratio (OR), 2.35; 95% confidence interval (CI), 1.60-3.46], absence of prior coronary artery disease (OR, 1.54; 95% CI, 1.00-2.39), and negative family history of coronary artery disease (OR; 2.76; 95% CI, 1.86-4.10), were identified as independent predictors of delayed presentation (p < 0.001). Interestingly, 44.7% of the patients presented late due to misdiagnosis by local healthcare providers. CONCLUSION: The standard of STEMI management in our state is far from ideal, and calls for a lot of improvement. Major efforts to reduce prehospital and in-hospital treatment delays are urgently needed.

Does plasma copeptin level at admission predict final infarct size in ST-elevation myocardial infarction.

BACKGROUND: Copeptin is a novel biomarker of potential diagnostic and prognostic value in patients with ST-elevation myocardial infarction (STEMI). This study was conducted to investigate the relationship between plasma copeptin levels at admission and final infarct size in STEMI patients. MATERIALS AND METHODS: This observational study was conducted in Sher-i-Kashmir Institute of Medical sciences, Srinagar, for a period of 1year. 60 patients with STEMI admitted within 24h of symptom onset were included in the study. Plasma copeptin concentrations were determined by ELISA from blood samples drawn at the time of admission. Infarct size was estimated on cardiac MRI after 5-14days of admission, in successfully reperfused patients. Correlations between plasma copeptin levels, infarct size and various clinico-hemodynamic variables were studied. RESULTS: Plasma copeptin concentrations showed a significant positive correlation with MRI determined infarct size (r=0.957; p≤0.0001). Copeptin levels were significantly higher in patients with anterior wall infarction (p≤0.0001), longer symptom duration (p=0.018), advanced Killip class (p≤0.0001), higher body mass index (p=0.019) and extensive coronary artery disease (p≤0.0001). On multivariate analysis, copeptin levels at admission independently predicted final infarct size, irrespective of the clinico-hemodynamic profile of patients or mode of reperfusion (p≤0.0001). The only independent predictor of copeptin level was symptom duration (p=0.018). CONCLUSION: Copeptin level at admission predicts final infarct size in STEMI patients. Further evidence is however needed before implementation of this biomarker into routine clinical practice.

Prevalence and characterization of coronary artery disease in patients with symptomatic bradyarrhythmias requiring pacemaker implantation.

BACKGROUND: This study was conducted to assess the prevalence and characterization of CAD in high risk patients requiring pacemaker implantation for symptomatic bradyarrhythmias. METHODS: This study included 100 patients with symptomatic sinus node dysfunction or atrioventricular block, who were at high risk of CAD or had previously documented atherosclerotic vascular disease (ASCVD). Coronary angiography was performed before pacemaker implantation. CAD was defined as the presence of any degree of narrowing in at least one major coronary artery or its first order branch. Obstructive CAD was defined as ≥50% diameter stenosis. CAD was categorized as single vessel disease (SVD), double vessel disease (DVD), or triple vessel disease (TVD); and obstructive CAD in the arteries supplying the conduction system was sub-classified according to Mosseri's classification. RESULTS: Out of 100 patients (mean age 64.6±10.7 years), 45 (45%) had CAD. 29% patients had obstructive CAD while 16% had non-obstructive CAD. 53.3% patients had SVD, 15.6% had DVD and 31.1% had TVD. Among patients with obstructive CAD; Type I, II, III and IV coronary anatomies were present in 6.9%, 34.5%, 10.3% and 48.3% patients respectively. Presence of CAD significantly correlated with dyslipidemia (p=0.047), history of smoking (p=0.025), and family history of CAD (p=0.002). CONCLUSION: Angiographic CAD is observed in a substantial proportion of patients with symptomatic bradyarrhythmias and risk factors for CAD. It could be argued that such patients should undergo a coronary work-up before pacemaker implantation. Treatment of concomitant CAD is likely to improve the long term prognosis of these patients.

Immediate effect of percutaneous transvenous mitral commissurotomy on atrial electromechanical delay and P-wave dispersion in patients with severe mitral stenosis.

BACKGROUND: Mitral stenosis (MS) is associated with prolonged inter- and intra-atrial electromechanical delays and increased P-wave dispersion, which are markers of atrial fibrillation (AF) risk. This study was conducted to assess the immediate effect of successful percutaneous transvenous mitral commissurotomy (PTMC) on these parameters. METHODS: This single center observational study included 25 patients with severe MS (aged 34.1 ± 7.1 years, with mean mitral valve area (MVA) of 0.74 ± 0.13 cm(2)), in sinus rhythm, who underwent successful PTMC at our hospital. P-wave dispersion (PWD) was calculated by subtracting minimum P-wave duration (P min) from maximum P-wave duration (Pmax), measured on a 12-lead surface ECG obtained from each patient in supine position at a paper speed of 50mm/s and 20mm/mV. Inter-atrial (AEMD), left intra-atrial (L-IAEMD), and right intra-atrial (R-IAEMD) electromechanical delays were measured on tissue Doppler imaging. PTMC was performed using the standard Inoue Balloon technique. All these parameters were evaluated and compared before and 24-48 h after PTMC. RESULTS: Successful PTMC led to significant reduction in AEMD (p < 0.001), L-IAEMD (p < 0.001), and R-IAEMD (p < 0.001). There were no changes in Pmax, Pmin, and PWD immediately after PTMC. CONCLUSIONS: Successful PTMC has a favorable early impact on inter- and intra-atrial electromechanical delays, which are considered as novel parameters of atrial electromechanical remodeling in MS patients. Prospective large-scale studies are required to confirm whether improvement in these markers translates into reduced long-term AF risk.

Left ventricular mass and functions in normotensive offspring of hypertensive parents: an echocardiographic study.

OBJECTIVE: Published observations about cardiovascular alterations in normotensive individuals genetically predisposed to develop essential hypertension are conflicting. We tested the hypothesis that abnormalities in left ventricular mass and/or functions may be present in normotensive children of hypertensive parents. METHODS: One hundred normotensive offsprings (6 to 18 year age) of hypertensive parents (OHP) and an equal number of age- and sex-matched normotensive offsprings of normotensive parents (ONP) were studied with 2-dimensionally guided M-mode and Doppler echocardiography for left ventricular (LV) dimensions, mass, and systolic and diastolic functions. RESULTS: Both the groups had similar body mass index and blood pressure levels. LV dimensions and LV mass in OHP were higher than the corresponding values in ONP but the differences were not statistically significant. LV mass in male OHP was higher than in female OHP; LV mass was also higher when the mother rather than father was hypertensive. None of these differences were statistically significant, however. LV systolic functions were normal and identical in the two subject groups. Indices of LV diastolic function (peak early filling velocity and its deceleration time and late filling velocity) were also normal and similar in the two groups. CONCLUSION: We conclude that children with a family history of essential hypertension have modest alterations in LV mass and these alterations might have a genetic basis separate from but possibly co-inherited with the trait of essential hypertension.