Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation.

Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.

Proinflammatory Effect of Endothelial Microparticles Is Mitochondria Mediated and Modulated Through MAPKAPK2 (MAPK-Activated Protein Kinase 2) Leading to Attenuation of Cardiac Hypertrophy.

Objective- This study investigates the functional significance of mitochondria present in endothelial microparticles (EMP) and how MK2 (MAPKAPK2 [MAPK-activated protein kinase 2]) governs EMP production and its physiological effect on cardiac hypertrophy. Approach and Results- Flow cytometric analysis, confocal imaging, oxygen consumption rate measurement through Seahorse were used to confirm the presence of functionally active mitochondria in nontreated EMP (EMP derived from untreated control cells), lipopolysaccharide, and oligomycin treatment increased mitochondrial reactive oxygen species activity in EMP (EMP derived from cells treated with lipopolysaccharide and EMP derived from cells treated with oligomycin, respectively). The dysfunctional mitochondria contained in EMP derived from cells treated with lipopolysaccharide and EMP derived from cells treated with oligomycin induced the expression of proinflammatory mediators in the target endothelial cells leading to the augmented adhesion of human monocytic cell line on EA.hy926 cells. Multiphoton real-time imaging detected the increased adherence of EMP derived from cells treated with oligomycin at the site of carotid artery injury as compared to EMP derived from untreated control cells. MK2 regulates EMP generation during inflammation by reducing E-selectin expression and regulating the cytoskeleton rearrangement through ROCK-2 (Rho-associated coiled-coil containing protein kinase 2) pathway. MK2-deficient EMP reduced the E-selectin and ICAM-1 (intracellular adhesion molecule-1) expression on target endothelial cells leading to reduced monocyte attachment and reduced cardiac hypertrophy in mice. Conclusions- MK2 promotes the proinflammatory effect of EMP mediated through dysfunctional mitochondria. MK2 modulates the inflammatory effect induced during cardiac hypertrophy through EMP.

Curcuma oil reduces endothelial cell-mediated inflammation in postmyocardial ischemia/reperfusion in rats.

Endothelial cells initiated inflammation persisting in postmyocardial infarction needs to be controlled and moderated for avoiding fatal complications. Curcuma oil (C.oil, Herbal Medicament), a standardized hexane soluble fraction of Curcuma longa has possessed neuroprotective effect. However, its effect on myocardial ischemia/reperfusion (MI/RP) and endothelial cells remains incompletely defined. Here, using in vivo rat MI/RP injury model and in vitro cellular approaches using EA.hy926 endothelial cells, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and myograph, we provide evidence that with effective regimen and preconditioning of rats with C.oil (250 mg/kg, PO), before and after MI/RP surgery protects rats from MI/RP-induced injury. C.oil treatment reduces left ventricular ischemic area and endothelial cell-induced inflammation, specifically in the ischemic region (*P < 0.0001) and improved endothelial function by reducing the expression of proinflammatory genes and adhesion factors on endothelial cells both in vitro and in vivo. Furthermore, mechanistic studies have revealed that C.oil reduced the expression of adhesion factors like E-selectin (#P = 0.0016) and ICAM-1 ($P = 0.0069) in initiating endothelial cells-induced inflammation. In line to the real-time polymerase chain reaction expression data, C.oil reduced the adhesion of inflammatory cells to endothelial cells as assessed by the interaction of THP-1 monocytes with the endothelial cells using flow-based adhesion and under inflammatory conditions. These studies provide evidence that salutary effect of C.oil on MI/RP could be achieved with pretreatment and posttreatment of rats, C.oil reduced MI/RP-induced injury by reducing the endothelial cell-mediated inflammation, specifically in the ischemic zone of MI/RP rat heart.

Generation of two iPSC lines from vascular Ehlers-Danlos Syndrome (vEDS) patients carrying a missense mutation in COL3A1 gene.

Vascular Ehlers-Danlos Syndrome (vEDS) is an inherited connective tissue disorder caused by COL3A1 gene, mutations that encodes type III collagen, a crucial component of blood vessels. vEDS can be life-threatening as these patients can have severe internal bleeding due to arterial rupture. Here, we generated induced pluripotent stem cell (iPSC) lines from two vEDS patients carrying a missense mutation in the COL3A1 (c.226A > G, p.Asn76Asp) gene. These lines exhibited typical iPSC characteristics including morphology, expression of pluripotency markers, and could differentiate to all three germ layer. These iPSC lines can serve as valuable tools for elucidating the pathophysiology underlying vEDS.

Generation of induced pluripotent stem cell line from a patient suffering from arterial calcification due to deficiency of CD73 (ACDC).

Arterial calcification due to deficiency of CD73 (ACDC) is an adult onset, rare genetic vascular disorder signified by calcium deposition in lower extremity arteries and joints of hands and feet. Mutations in NT5E gene has been shown to be responsible for the inactivation of enzyme CD73 causing calcium buildup. Here, we report a iPSC line generated from a patient showing signs of ACDC and carrying a missense mutation in NT5E (c.1126A→G,p.T376A) gene. This iPSC line shows normal morphology, pluripotency, karyotype, and capability to differentiate into three germ layers, making it useful for disease modeling and investigating pathological mechanisms of ACDC.

Involvement of HIF1α/Reg protein in the regulation of HMGB3 in myocardial infarction.

AIMS: Myocardial ischemia and infarction are the number one cause of cardiovascular disease associated mortality. Cardiomyocyte death during ischemia leads to the loss of cardiac tissue and initiates a signaling cascade between the infarct zone and the area at risk of the myocardium. Here, we sought to determine the involvement of one of the damage-associated molecular patterns HMGB3 in myocardial ischemia and infarction. METHODS AND RESULTS: We used the left anterior descending coronary artery ligation model to study the involvement of HMGB3 in myocardial ischemia and infarction. Our results indicated the presence of HMGB3 at a low level under normal conditions, while myocardial injury caused a robust increase in HMGB3 levels in the heart. Further, intra-cardiac injection of mabHMGB3 had improved cardiac function at day 3 by downregulating HMGB3 levels. In contrast, injection of recombinant rat HMGB3 for 7 days during the adaptation phase of myocardial ischemia improved cardiac functional parameters by increasing regenerative protein family expression. Further, to mimic the disease condition, neonatal rat ventricle cardiomyocytes and fibroblasts were exposed to hypoxia; we observed a significant upregulation in the HMGB3, HIF1α, and Reg1α levels. Endothelial cells exposed to recombinant HMGB3 increased the tubule length. Further, the mitochondrial oxygen consumption rate was reduced with the acute induction of recombinant HMGB3 on cardiomyocytes and fibroblasts. CONCLUSION: HMGB3 plays a dual role during the progression of myocardial ischemia and infarction. Clinically, post-myocardial infarction HMGB3-induced sterile inflammation needs to be tightly controlled, as it plays both a pro-inflammatory role and improves cardiac function during the cardiac remodeling phase.

Elective Tracheostomy During Covid-19 Pandemic- A Tertiary Care Centre Experience.

Aims and Objectives: : The aim of our study was to present an experience of elective tracheostomy in COVID-19 patients at our institute. Materials and methods: The present prospective study was conducted, after approval by Institutional Ethics Committee, in the Department of ENT, SMGS Hospital, GMC Jammu from May 2020 to March 2021 over 60 patients having need for prolonged mechanical ventilation and having tested positive for COVID-19 with nasopharyngeal swab on rtPCR assay testing. Detailed information regarding following aspects was gathered :Age, Gender, Comorbidities (Diabetes, Cardiovascular disease, Pulmonary disease, Malignancy), time of endotracheal intubation to tracheostomy, time to wean sedation after tracheostomy, time to wean mechanical ventilation after tracheostomy, surgical complications, mortality, any health care worker in operating team getting infected by SARS-CoV-2. All 60 patients underwent Elective Open Tracheostomy Bed-side in the ICU section of our institute. Results: The mean age of presentation was 55.9 ± 2.34 years, with male preponderance. The most common indication for tracheostomy was ARDS (Acute Respiratory Distress Syndrome) (56.6%). Out of 60 patients, co-morbidities were present in 44 patients (73.3%). The mean time between endotracheal intubation and tracheostomy was 12.2 ± 4.9 days. The mean time to wean mechanical ventilation after tracheostomy was 10.4 ± 2.31 days. The mean time to wean sedation was 2.2 ± 0.83 days. There were no deaths during the procedure. Out of 60 patients, 5 patients (8.3%) died due to complications of COVID-19. Conclusion: Our study provides important clinical data (such as timing of tracheostomy, pre-operative evaluation of patients, recommendations during procedure, outcomes of tracheostomy and postoperative care) on this threatening issue of tracheostomy in COVID-19 patients and might be of immense help to various Otorhinolaryngologists who are dealing with the same situation.

SGLT2 inhibitor ameliorates endothelial dysfunction associated with the common ALDH2 alcohol flushing variant.

The common aldehyde dehydrogenase 2 (ALDH2) alcohol flushing variant known as ALDH2*2 affects ∼8% of the world's population. Even in heterozygous carriers, this missense variant leads to a severe loss of ALDH2 enzymatic activity and has been linked to an increased risk of coronary artery disease (CAD). Endothelial cell (EC) dysfunction plays a determining role in all stages of CAD pathogenesis, including early-onset CAD. However, the contribution of ALDH2*2 to EC dysfunction and its relation to CAD are not fully understood. In a large genome-wide association study (GWAS) from Biobank Japan, ALDH2*2 was found to be one of the strongest single-nucleotide polymorphisms associated with CAD. Clinical assessment of endothelial function showed that human participants carrying ALDH2*2 exhibited impaired vasodilation after light alcohol drinking. Using human induced pluripotent stem cell-derived ECs (iPSC-ECs) and CRISPR-Cas9-corrected ALDH2*2 iPSC-ECs, we modeled ALDH2*2-induced EC dysfunction in vitro, demonstrating an increase in oxidative stress and inflammatory markers and a decrease in nitric oxide (NO) production and tube formation capacity, which was further exacerbated by ethanol exposure. We subsequently found that sodium-glucose cotransporter 2 inhibitors (SGLT2i) such as empagliflozin mitigated ALDH2*2-associated EC dysfunction. Studies in ALDH2*2 knock-in mice further demonstrated that empagliflozin attenuated ALDH2*2-mediated vascular dysfunction in vivo. Mechanistically, empagliflozin inhibited Na+/H+-exchanger 1 (NHE-1) and activated AKT kinase and endothelial NO synthase (eNOS) pathways to ameliorate ALDH2*2-induced EC dysfunction. Together, our results suggest that ALDH2*2 induces EC dysfunction and that SGLT2i may potentially be used as a preventative measure against CAD for ALDH2*2 carriers.

Generation of two induced pluripotent stem cell lines carrying the phospholamban R14del mutation for modeling ARVD/C.

The phospholamban (PLN) R14del mutation is associated with arrhythmogenic right ventricular dysplasia (ARVD/C). ARVD/C is a cardiac disease characterized by arrhythmias and structural abnormalities in the right ventricle. Because PLN is a regulator of calcium release, this mutation can have deleterious effects on tissue integrity and contraction. This mutation is a trinucleotide (AGA) deletion that leads to an arginine deletion at position 14 of the PLN structure. Here we show two lines carrying this mutation with typical iPSC morphology, pluripotency, karyotype, ability to differentiate into the three germ layers in vitro, and readily availability for studying pathological mechanisms or ARVD/C.

Engineered Nanoparticle-Protein Interactions Influence Protein Structural Integrity and Biological Significance.

Engineered nanoparticles (ENPs) are artificially synthesized particles with unique physicochemical properties. ENPs are being extensively used in several consumer items, elevating the probability of ENP exposure to biological systems. ENPs interact with various biomolecules like lipids, proteins, nucleic acids, where proteins are most susceptible. The ENP-protein interactions are mostly studied for corona formation and its effect on the bio-reactivity of ENPs, however, an in-depth understanding of subsequent interactive effects on proteins, such as alterations in their structure, conformation, free energy, and folding is still required. The present review focuses on ENP-protein interactions and the subsequent effects on protein structure and function followed by the therapeutic potential of ENPs for protein misfolding diseases.

Fabrication, characterization and in vivo assessment of cardiogel loaded chitosan patch for myocardial regeneration.

Cell therapy is one of the promising approaches for cardiac repair, subsequently after infarction or injury. However, contemporary mesenchymal stromal/stem cell (MSCs) delivery strategies result in low retention and poor engraftment of donor cells, thus limiting the therapeutic efficacy. Here, we developed an engineered biomimetic cardiogel patch (EBCP) comprising of the native decellularized cardiac extracellular matrix (ECM) "cardiogel" and chitosan, leading to the efficient regeneration of injured myocardium. We also developed novel bio-adhesive that is capable of suture-free epicardial placement of EBCP to injured myocardium. We have illustrated the potential of the mussels-inspired bioadhesive system, which comprises gelatin catechol and partially oxidized chitosan, which relies on self-crosslinking capability, to promote wet adhesion. In vitro studies with isolated cardiogel promoted cell proliferation, adhesion, and migration while aiding cardiomyogenic differentiation. The EBCP's ability to protect cells from abrasion due to surrounding tissues in the myocardial infarction (MI) rat model makes it more desirable. Furthermore, the epicardial implantation of the EBCP loaded with MSCs improves the initial retention of cells and subsequent functional cardiac recovery with enhanced myocardial tissue restoration. Histological examination showed the presence of EBCP and infiltration of cells to the infarcted heart tissue. The fast and facile synthesis of bioadhesive and major therapeutic benefits of EBCP make it a potential candidate for recuperating the ailing heart.

Incidence of Olfactory and Gustatory Dysfunctions in the Early Stages of COVID-19: An Objective Evaluation.

Introduction Coronavirus disease 2019 (COVID-19) is a dangerous infectious disease caused by a newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has various clinical presentations. Numerable cases with non-specific olfactory and gustatory dysfunctions in COVID-19 have been reported from all over the globe. This is important as awareness will let people to self-isolate and help in limiting disease spread. Objective To objectively evaluate the frequency of olfactory and gustatory dysfunction, which may occur independently or with other symptoms, in laboratory confirmed COVID-19 patients at an early stage of the disease. Methods Objective evaluation of olfactory and gustatory function of 322 COVID-19 patients treated at our hospital, (SMGS, Government Medical College, Jammu), from August 2020 until November 2020. Results Our study population included 127 (39.4%) males and 195 (60.6%) females. Two hundred and twenty-six (70.2%) COVID-19 patients experienced olfactory and gustatory disorders. One hundred and sixty-five (51.2%) cases experienced both olfactory and gustatory disorders. Isolated olfactory dysfunction was reported in 34 (10.6%) patients, while 27 (8.4%) patients experienced only gustatory dysfunction. Conclusion The olfactory and gustatory dysfunctions, without any nasal obstruction or rhinorrhea, are significant symptoms in the clinical presentation of early COVID-19 patients. This presentation can be recognized at the earliest one, and it can reduce the high communicability of the COVID-19 disease.

Generation of two iPSC lines from hypertrophic cardiomyopathy patients carrying MYBPC3 and PRKAG2 variants.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disorder characterized by a thick left ventricular wall and an increased risk of arrhythmias, heart failure, and sudden cardiac death. The MYBPC3 and PRAKG2 are known causal genes for HCM. Here we generated two human-induced pluripotent stem cell lines from two HCM patients carrying two heterozygous mutations in MYBPC3 (c.459delC) and PRKAG2 (c.1703C > T). Both iPSC lines expressed pluripotent markers, had a normal karyotype, and were able to differentiate into three germ layers, making them potentially valuable tools for modeling HCM in vitro and investigating the pathological mechanisms related to these two variants.

Otological Assessment in Head Injury Patients: A Prospective Study and Review of Literature.

Head injuries constitute a tragic problem invariably in under-developed, developed and developing countries. The concomitant otological injuries often go unnoticed. The purpose of this study was to assess the various otological manifestations following head injuries. Prospective study with review of literature using PubMed database was done. All the patients were evaluated for their presenting symptoms and signs. Audiological investigations including PTA (Pure tone audiometry), OAE (Otoacoustic emission), Impedance-Audiometry and BERA were done. HRCT temporal bone was advised in cases of suspicion. Relevant literature was reviewed to calculate the pooled prevalence rates. Random-effects model to synthesize overall effects was used. Heterogeneity was evaluated with the I2 statistic. Of 53 patients enrolled in the study, RTA was the most common mode of injury. The audiometric findings showed SNHL, CHL and mixed HL in 34, 20 and 18% of patients respectively. HRCT showed Longitudinal fracture (n = 17; 53.12%); isolated mastoid bone fracture (n = 9; 28.12%), transverse (n = 3; 9.37%) and isolated EAC fracture in (n = 3; 9.37%) patients. The pooled prevalence (n = 1106 patients) of SNHL, CHL, Mixed HL and Normal hearing were-35% (95%CI, 18-55%; I2 = 95.20%; P < 0.00), 24% (95%CI, 16-33%; I2 = 80.01%; P < 0.00), 15%(95%CI, 9-23%; I2 = 79.64%; P < 0.00) and 30% (95%CI, 3-66%; I2 = 98.71%; P < 0.00) respectively. The pooled prevalence (n = 4191 patients) of longitudinal, Transverse, mixed and other fractures were-44% (95%CI, 3-66%;I2 = 99.48%; P < 0.00), 9% (95% CI, 4-16%; I2 = 95.95%; P < 0.00), 4% (95%CI, 1-8%; I2 = 94.13%; P < 0.00) and 1% (95%CI, 0-4%; I2 = 90.37%; P < 0.00) respectively. In patients with head injury coordination between the trauma-surgeon, neurosurgeon and otologist is must to improve the long-term outcomes.

Determinants of plasma vitamin D levels in patients with acute coronary syndromes.

BACKGROUND: Vitamin D is implicated in various biological functions ranging from cellular proliferation to immunity. Vitamin D deficiency is associated with an increased risk of several diseases including coronary atherosclerosis. MATERIALS AND METHODS: We measured plasma 25(OH)D3 level in 224 patients with acute coronary syndromes (ACS) and 209 control individuals by ELISA. We genotyped the study populations for 11 single nucleotide polymorphisms (SNPs) in seven genes involved in vitamin D biosynthesis and metabolism by 5' nuclease assays. RESULTS: The mean and median plasma 25(OH)D3 levels were not significantly different between patients with ACS and controls (median: 22·06 vs. 22·24 ng mL(-1) , respectively, P = 0·618). Plasma 25(OH)D3 level was < 20 ng mL(-1) in 175/433 (40%) and < 30 ng mL(-1) in 333/433 (77%) participants. Only four individuals had plasma 25(OH)D3 levels of above 60 ng mL(-1) . African-American and Hispanic populations, women and those with diabetes mellitus had significantly lower plasma 25(OH)D3 levels. In multivariable regression analysis, age, sex, diabetes mellitus, body weight, rs2762933 (CYP24A1) and rs6055987 (PLCB1) SNPs were independent predictors of plasma 25(OH)D3 level in the Caucasian population. CONCLUSIONS: We found no difference in mean plasma vitamin D levels between patients with ACS and controls. Differences in population characteristics between the two study groups including medications use and the lack of data on vitamin D, calcium and multivitamin supplements intake as well as the relatively small sample size of the populations could confound the results. Ethnic background, sex, age, body weight and SNPs in CYP24A1 and PLCB1 were independent determinants of plasma vitamin D levels.

Multidisciplinary collaborative approach for management of adrenal tumors: Outcomes of minimally invasive adrenalectomy at a single center.

INTRODUCTION: Laparoscopy is currently the gold standard for the management of adrenal tumors as it is associated with less morbidity. Owing to technological advances, even large adrenal tumors are currently amenable to laparoscopic removal. In this work, we describe our multidisciplinary collaborative approach for management of adrenal tumors at a single center. MATERIAL AND METHODS: Between January 2017 and January 2020, 18 patients with adrenal lesions were operated at our center. Five of these patients had incidentalomas. All patients were evaluated in coordination with endocrinologists and anesthetists. All patients underwent transperitoneal laparoscopic adrenalectomy. The surgical complications were classified as per Clavein-Dindo system. All patients with pheochromocytoma and Cushing syndrome were followed up by the endocrinologist for further evaluation and titration of glucocorticoids and antihypertensive medication. RESULTS: Two out of the 18 patients had American Society of Anesthesiologists (ASA) physical status III. Out of the 18 patients, only one required conversion to open surgery. Five patients developed intraoperative hypertensive crisis while three patients developed hypotensive crisis. All patients were ambulated on the first postoperative day and were discharged on the third post operative day. None of the patients developed any major (Clavein-Dindo III-V) intra operative or post operative complications. CONCLUSION: Laparoscopic adrenalectomy is the procedure of choice for adrenal tumors. A multi-disciplinary approach involving the endocrinologist, anesthesiologist, and laparoscopic surgeon can help achieve favorable outcomes.

Injectable hydrogel for co-delivery of 5-azacytidine in zein protein nanoparticles with stem cells for cardiac function restoration.

Heart failure is major cause of mortality associated with mostly Myocardial infarction (MI). Transplanting mesenchymal stem cells (MSC) have exhibited potential role in myocardial regeneration. Secretion of immune-modulatory cytokines and various growth factors after transplantation plays significant role in remodelling process of MI region. However, low retention, higher shear stress during administration and rejection at host infarct environment hinders therapeutic efficacy. Myocardial regeneration demands for accurate spatio-temporal delivery of MSCs with supportive vascular network that leads to improvement of cardiac function. In this study, injectable alginate based microporous hydrogel has been used to deliver 5-Azacytidine (5-Aza) in zein protein nanoparticle with MSCs for attenuating adverse cardiac remodelling after MI. Zein nanoparticles loaded with 5-Aza were prepared by liquid-liquid dispersion, and it was found that 35% of drug was released in 7 days supported with mathematical modelling. The presence of 5-Aza and zein in developed hydrogel supported in vitro MSC proliferation, migration and angiogenesis. Significant increased expression of cardiac specific markers, GATA4, MEF2C, MLC, SERCA and NKX2.5 was observed in vitro. 5-Aza loaded protein nanoparticle with MSCs encapsulated hydrogels in rat MI model also exhibited substantial improvement of functional cardiac parameters such as cardiac output and ejection fraction. Histopathological analysis showed reduced fibrosis, attenuated infarct expansion and cardiac tissue restoration and angiogenesis. In brief, we developed nanocarrier-hydrogel system a promising strategy for co-delivering 5-Aza as cardiac differentiation cue with MSCs to achieve higher cell retention and enhanced improvement in myocardial regeneration after MI.