DNA methylation signatures on vascular differentiation genes are aberrant in vessels of human cerebral arteriovenous malformation nidus.

Arteriovenous malformation (AVM) is a tangle of arteries and veins, rupture of which can result in catastrophic hemorrhage in vulnerable sites such as the brain. Cerebral AVM is associated with a high mortality rate in humans. The causative factor or the stimulus at the artery-venous junction and the molecular basis of the development and progression of cerebral AVM remain unknown. While it is known that aberrant hemodynamic forces in the artery-vein junction contribute to the development of AVMs, the mechanistic pathways are unclear. Given that various environmental stimuli modulate epigenetic modifications on the chromatin of cells, we speculated that misregulated DNA methylome could lead to cerebral AVM development. To identify the aberrant epigenetic signatures, we used AVM nidus tissues and analyzed the global DNA methylome using the Infinium DNA methylome array. We observed significant alterations of DNA methylation in the genes associated with the vascular developmental pathway. Further, we validated the DNA hypermethylation by DNA bisulfite sequencing analysis of selected genes from human cerebral AVM nidus. Taken together, we provide the first experimental evidence for aberrant epigenetic signatures on the genes of vascular development pathway, in human cerebral AVM nidus.

Shear Stress Alterations Activate BMP4/pSMAD5 Signaling and Induce Endothelial Mesenchymal Transition in Varicose Veins.

Chronic venous diseases, including varicose veins, are characterized by hemodynamic disturbances due to valve defects, venous insufficiency, and orthostatism. Veins are physiologically low shear stress systems, and how altered hemodynamics drives focal endothelial dysfunction and causes venous remodeling is unknown. Here we demonstrate the occurrence of endothelial to mesenchymal transition (EndMT) in human varicose veins. Moreover, the BMP4-pSMAD5 pathway was robustly upregulated in varicose veins. In vitro flow-based assays using human vein, endothelial cells cultured in microfluidic chambers show that even minimal disturbances in shear stress as may occur in early stages of venous insufficiency induce BMP4-pSMAD5-based phenotype switching. Furthermore, low shear stress at uniform laminar pattern does not induce EndMT in venous endothelial cells. Targeting the BMP4-pSMAD5 pathway with small molecule inhibitor LDN193189 reduced SNAI1/2 expression in venous endothelial cells exposed to disturbed flow. TGFß inhibitor SB505124 was less efficient in inhibiting EndMT in venous endothelial cells exposed to disturbed flow. We conclude that disturbed shear stress, even in the absence of any oscillatory flow, induces EndMT in varicose veins via activation of BMP4/pSMAD5-SNAI1/2 signaling. The present findings serve as a rationale for the possible use of small molecular mechanotherapeutics in the management of varicose veins.

Cyclophilin A Impairs Efferocytosis and Accelerates Atherosclerosis by Overexpressing CD 47 and Down-Regulating Calreticulin.

Impairment of efferocytosis in apoptotic macrophages is a known determinant of the severity of atherosclerosis and the vulnerability of plaques to rupture. The precise mechanisms involved in impaired efferocytosis are unclear. Given the well-recognized role of the inflammatory cytokine cyclophilin A (Cyp A) in modulating several atherogenic mechanisms in high-glucose primed monocytes, we investigated the role of Cyp A in macrophage efferocytosis. The efficiency of efferocytosis in RAW 264.7 macrophages grown in vitro and primed with cyclophilin A was assessed using flow cytometry and confocal assays. Cholesterol content in cells was measured using cell-based cholesterol efflux assay. Proteomic analysis and bioinformatics tools were employed to decipher the link between cyclophilin A and the known ligand receptors involved in efferocytosis. Cyclophilin A was found to impair efferocytosis in apoptotic macrophages by reducing ABCA1-mediated cholesterol efflux in foam cells derived from macrophages. Cyclophilin A-primed macrophages showed an increase in expression of the don't-eat-me signal CD 47 and a decrease in the expression of the eat-me signal, calreticulin. Phagocytosis was restored upon silencing of cyclophilin A. New Zealand white rabbits were fed a high-fat diet, and lesions in their aortae were analyzed histologically for evidence of atherosclerosis and the expression of Cyp A, CD 47 and calreticulin, the ligand receptor involved in efferocytosis. Gene and protein expressions in aortae and macrophages were analyzed by real-time PCR and Western blotting. Cyclophilin A, via its effects on the expression of CD 47 and calreticulin, impairs efferocytosis in apoptotic macrophages. Together with its impact on cholesterol efflux from macrophages, these effects can amplify other mechanisms of Cyp A in accelerating the progression of atherosclerosis.

Proteomics Analysis Reveals Diverse Molecular Characteristics between Endocardial and Aortic-Valvular Endothelium.

The variations in the protein profile of aortic-valvular (AVE) and endocardial endothelial (EE) cells are currently unknown. The current study's objective is to identify differentially expressed proteins and associated pathways in both the endothelial cells. We used endothelial cells isolated from the porcine (Sus scrofa) aortic valve and endocardium for the profiling of proteins. Label-free proteomics was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our proteomics analysis revealed that 29 proteins were highly expressed, and 25 proteins were less expressed in the valve than the endocardial endothelium. The cell surface markers, such as CD63, ICAM1, PECAM1, PROCR, and TFRC, were highly expressed in EE. In contrast, CD44 was highly expressed in AVE. The pathway analysis showed that metabolic process-related proteins and extracellular matrix-related proteins were enriched in valves. Differential enrichment of signaling pathways was observed in the endocardium. The hemostasis function-related proteins were increased in both endothelial cells. The proteins and pathways enriched in aortic-valvular and endocardial endothelial cells revealed the distinct phenotype of these two closely related cells.

Aberrant regulation of retinoic acid signaling genes in cerebral arterio venous malformation nidus and neighboring astrocytes.

BACKGROUND: Cerebral arterio venous malformations (AVM) are a major causal factor for intracranial hemorrhage, which result in permanent disability or death. The molecular mechanisms of AVM are complex, and their pathogenesis remains an enigma. Current research on cerebral AVM is focused on characterizing the molecular features of AVM nidus to elucidate the aberrant signaling pathways. The initial stimuli that lead to the development of AVM nidus structures between a dilated artery and a vein are however not known. METHODS: In order to understand the molecular basis of development of cerebral AVM, we used in-depth RNA sequencing with the total RNA isolated from cerebral AVM nidus. Immunoblot and qRT-PCR assays were used to study the differential gene expression in AVM nidus, and immunofluorescence staining was used to study the expression pattern of aberrant proteins in AVM nidus and control tissues. Immunohistochemistry was used to study the expression pattern of aberrant proteins in AVM nidus and control tissues. RESULTS: The transcriptome study has identified 38 differentially expressed genes in cerebral AVM nidus, of which 35 genes were upregulated and 3 genes were downregulated. A final modular analysis identified an upregulation of ALDH1A2, a key rate-limiting enzyme of retinoic acid signaling pathway. Further analysis revealed that CYR61, a regulator of angiogenesis, and the target gene for retinoic acid signaling is upregulated in AVM nidus. We observed that astrocytes associated with AVM nidus are abnormal with increased expression of GFAP and Vimentin. Triple immunofluorescence staining of the AVM nidus revealed that CYR61 was also overexpressed in the abnormal astrocytes associated with AVM tissue. CONCLUSION: Using high-throughput RNA sequencing analysis and immunostaining, we report deregulated expression of retinoic acid signaling genes in AVM nidus and its associated astrocytes and speculate that this might trigger the abnormal angiogenesis and the development of cerebral AVM in humans.

Spectrum of cardiac involvement in patients with dengue fever.

BACKGROUND: Dengue fever (DF) is an infectious disease of viral origin common in the tropics. Studies on a large number of patients with dengue infection to assess associated cardiac involvement are rare. METHODS: We analyzed the incidence and spectrum of cardiac abnormalities in 320 patients with dengue fever admitted to our hospital located in an endemic area for dengue infection. All patients were evaluated following the WHO guidelines. Those confirmed to have dengue infection by serology had detailed clinical evaluation, 12­lead electrocardiography (ECG), assay for cardiac markers (troponin T, CK-MB, NT Pro BNP) and 2-D echocardiography. RESULTS: Among the 320 patients selected for the study 112 (35%) had changes of cardiac involvement as detected by investigations. Changes in ECG were seen in all of them. Sinus bradycardia in spite of fever was the most common abnormality (n = 63;19.7%). Forty-two (13.1%) patients had left ventricular ejection fraction less than 40%. Forty-eight patients (15%) had increased serum levels of troponin-T. Serum levels of CK-MB were elevated in 34 (10.6%) and serum levels of NT-pro BNP was increased in 19 (5.9%). Fourteen patients died and all of them had abnormalities in electrocardiogram, echocardiogram and serum markers. CONCLUSION: Our study reveals that cardiac involvement in patients with dengue infection is not uncommon. We found that ECHO or ECG abnormalities or elevated serum levels of markers of cardiac injury are predictors of risk for adverse outcome. Absence of these abnormalities has a 100% negative predictive value.

Plasma leptin level mirrors metabolome alterations in young adults.

INTRODUCTION: Leptin is known to regulate pathways of energy metabolism, reproduction, and control appetite. Whether plasma leptin levels reflect changes in metabolites of these pathways is unknown. OBJECTIVES: We aimed to find whether there is an association between leptin levels and levels of metabolites of energy and hormone metabolism. METHODS: We performed an untargeted metabolomics analysis of plasma from 110 healthy adults (men: women = 1:1; aged 18-40 years), using liquid chromatography-tandem mass spectrometry. Blood samples were collected from all the study subjects in the fasting state. Clinical features and markers of obesity and Type 2 diabetes mellitus (T2DM) were assessed in all. The association between levels of metabolites and clinical and biochemical parameters was identified using the multivariable-adjusted linear regression model and PLS-DA analysis. RESULTS: The leptin level was found to have a significant association with a substantial number of metabolites in women and men. Leptin level was positively associated with glycocholic acid and arachidic acid, metabolites related to energy metabolisms, pregnanediol-3-glucuronide, a metabolite of progesterone metabolism, and quercetin 3'-sulfate, a diet-derived metabolite. Leptin level was negatively associated with ponasteroside A and barringtogenol C levels. Leptin level was positively correlated with adiponectin and negatively with total calorie intake and levels of triglyceride and very-low-density lipoprotein. Leptin levels were associated with lipid and sex hormone metabolism in women, while metabolites involved in amino acid metabolism were correlated to leptin in men. CONCLUSION: Our study indicates that leptin level reflects metabolome alterations and hence could be a useful marker to detect early changes in energy and hormone metabolisms.

Postprandial Metabolism is Impaired in Overweight Normoglycemic Young Adults without Family History of Diabetes.

While the risk factors for Type 2 diabetes (T2DM) are known, early predictive markers of transition from normal to a prediabetes state are unidentified. We studied the basal metabolism and metabolic response to a mixed-meal challenge in 110 healthy subjects in the age group of 18 to 40 years (Male:Female = 1:1); grouped into first degree relatives of patients with T2DM (n = 30), those with a body mass index >23 kg/m2 but <30 kg/m2 (n = 30), those with prediabetes (n = 20) and normal controls (n = 30). We performed an untargeted metabolomics analysis of plasma and related that with clinical and biochemical parameters, markers of inflammation, and insulin sensitivity. Similar to prediabetes subjects, overweight subjects had insulin resistance and significantly elevated levels of C-peptide, adiponectin and glucagon and lower level of ghrelin. Metabolites such as MG(22:2(13Z, 16Z)/0:0/0:0) and LysoPC (15:0) were reduced in overweight and prediabetes subjects. Insulin sensitivity was significantly lower in men. Fasting levels of uric acid, xanthine, and glycochenodeoxycholic-3-glucuronide were elevated in men. However, both lysophospholipids and antioxidant defense metabolites were higher in women. Impaired postprandial metabolism and insulin sensitivity in overweight normoglycemic young adults indicates a risk of developing hyperglycemia. Our results also indicate a higher risk of diabetes in young men.

Untargeted metabolomics reveals alterations in metabolites of lipid metabolism and immune pathways in the serum of rats after long-term oral administration of Amalaki rasayana.

Amalaki rasayana, a traditional preparation, is widely used by Ayurvedic physicians for the treatment of inflammatory conditions, cardiovascular diseases, and cancer. Metabolic alterations induced by Amalaki rasayana intervention are unknown. We investigated the modulations in serum metabolomic profiles in Wistar rats following long-term oral administration of Amalaki rasayana. Global metabolic profiling was performed of the serum of rats administered with either Amalaki rasayana (AR) or ghee + honey (GH) for 18 months and control animals which were left untreated. Amalaki rasayana components were confirmed from AR extract using HR-LCMS analysis. Significant reductions in prostaglandin J2, 11-dehydrothromboxane B2, and higher levels of reduced glutathione and glycitein metabolites were observed in the serum of AR administered rats compared to the control groups. Eleven different metabolites classified as phospholipids, glycerophospholipids, glucoside derivatives, organic acids, and glycosphingolipid were exclusively observed in the AR administered rats. Pathway analysis suggests that altered metabolites in AR administered rats are those associated with different biochemical pathways of arachidonic acid metabolism, fatty acid metabolism, leukotriene metabolism, G-protein mediated events, phospholipid metabolism, and the immune system. Targeted metabolomics confirmed the presence of gallic acid, ellagic acid, and arachidonic acid components in the AR extract. The known activities of these components can be correlated with the altered metabolic profile following long-term AR administration. AR also activates IGF1R-Akt-Foxo3 signaling axis in heart tissues of rats administered with AR. Our study identifies AR components that induce alterations in lipid metabolism and immune pathways in animals which consume AR for an extended period.

Binimetinib, encorafenib and cetuximab (BEACON Trial) combination therapy for patients with BRAF V600E-mutant metastatic colorectal cancer.

BRAF V600E mutations are associated with 8-10% of metastatic colorectal cancers (mCRC) and carry a poor prognosis with limited therapeutic options. In contrast to metastatic melanoma, BRAF inhibition alone or in combination with mitogen-activated protein kinase kinase (MEK) inhibitors has shown little utility in the treatment of BRAF V600E-mutant mCRC. This is secondary to upstream activation of the epidermal growth factor receptor (EGFR) pathway and other escape mechanisms. Combining RAF and MEK inhibitors with inhibition of the EGFR pathway through an anti-EGFR receptor antibody (cetuximab) led to the BEACON clinical trial (binimetinib, encorafenib and cetuximab). Trial patients had undergone at least one prior line of chemotherapy. The trial met all its endpoints and is now included in NCCN (National Comprehensive Cancer Network) guidelines. Herein we provide updates in treatment options for patients with BRAF V600E-mutant mCRC, focusing on the practice-changing BEACON-triplet regimen, the first chemotherapy-free combination regimen for mCRC. This combination is being explored frontline in the ANCHOR clinical trial.

Intellectual property management at the National Animal Science Research Institute in India: A case study.

AIM: The National Institute of Veterinary Epidemiology and Disease Informatics is an animal science research institute under the aegis of the Indian Council of Agricultural Research. The intellectual property management system (IPMS) of the institute oversees technology creation, protection, and transfer/commercialization. This study reviews the effectiveness of the IPMS using traditional strengths, weaknesses, opportunities, and threats (SWOT) evaluation. MATERIALS AND METHODS: A comprehensive repository was developed to compile the SWOT pertaining to the IPMS based on relevant document reviews and the inputs of experts and stakeholders. The repository was shared among scientists of the institute for rating. The rating process revealed the top ten key SWOT associated with the structure and operation of the IPMS. The weighted SWOT matrix technique was used to identify the best strategies to improve and develop the IPMS further. This included strategies derived from the best combinations of key strengths and opportunities (S-O strategies), key weaknesses and opportunities (W-O strategies), key strengths and threats (S-T strategies), and key weaknesses and threats (W-T strategies). RESULTS: The top-ranked strengths included "possession of patented technology" and "state-of-the-art biosafety laboratory facilities," while "lack of in-house faculty with legal expertise in intellectual property rights (IPR)" and "lack of technology incubation facilities" were the key weaknesses. The key opportunities included "external funding for research projects" and "market demand for onsite diagnostic tools." The major threats were "lack of market for veterinary diagnostics" and "broad-based patents on research tools and technologies." CONCLUSION: The strengths of the system, such as a state-of-the-art biosafety laboratory and technology-marketing collaboration with Agrinnovate India Ltd., could be employed effectively to gain from the opportunities tendered by the market demand for on-site disease diagnostic tools (S-O strategies). The limitation arising from a dearth of technical staff could be overcome by technological backstopping through international linkages in the area of disease monitoring and surveillance. Funding from externally supported projects could also be utilized for recruitment of personnel (W-O strategies). Limitations arising from the combination of inadequate in-house IPR expertise and the threat arising from broad-based patents on research tools warrant vigilance (W-T strategies).

Correction to: Genetic and epigenetic mechanisms in the development of arteriovenous malformations in the brain.

Upon publication of the original article [1] the authors noticed that the affiliation Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India was missing.

A Splicing-Independent Function of RBM10 Controls Specific 3' UTR Processing to Regulate Cardiac Hypertrophy.

RNA binding motif protein 10 (RBM10) is a regulator of alternative splicing in apoptosis and inflammation. We discovered a splicing-independent function of RBM10 critical for the regulation of heart failure (HF). RBM10 is enriched in the heart and associates with Star-PAP (TUT1) to control the expression and 3' end processing of cardiac mRNAs. The RBM10 RRM2 domain binds the Star-PAP catalytic domain, which directs Star-PAP activity toward polyadenylation. RBM10 binds the pre-mRNA UTR, assembles the Star-PAP complex, and guides this complex specifically to mRNAs encoding anti-hypertrophy regulators. Accordingly, we tested cellular hypertrophy in rat cardiomyoblasts and cardiac hypertrophy (CH) and the subsequent progression to HF in Wistar rat hearts. We demonstrated downregulation of RBM10 during CH and HF. Ectopic re-expression of RBM10 rescued cardiomyocyte hypertrophy. RBM10 depletion evoked a hypertrophic response in H9c2 cells. Our results establish an anti-hypertrophy mechanism mediated by RBM10 in the heart that is directly linked to HF.

Establishment of an in vitro organoid model of dermal papilla of human hair follicle.

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three-dimensional (3D) DP spheroids (DPS) with or without a silk-gelatin (SG) microenvironment, which showed enhanced DP-specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil-treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell-cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial-mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.

Gene expression analysis of nidus of cerebral arteriovenous malformations reveals vascular structures with deficient differentiation and maturation.

OBJECTIVE: Arteriovenous malformations (AVMs) are characterised by tangles of dysplastic blood vessels which shunt blood from arteries to veins with no intervening capillary bed. It is not known at what stage of development and differentiation, AVM vessels became aberrant. To address this, we have analysed the expression of vascular differentiation, vascular maturation and brain capillary specific genes in AVM nidus. METHODOLOGY: We performed immunohistochemistry and western blot analysis of vascular differentiation (HEY2, DLL4, EFNB2, and COUP-TFII), vascular maturation (ENG and KLF2) and brain capillary specific genes (GGTP and GLUT1) on ten surgically excised human brain AVMs and ten normal human brain tissues. RESULTS: Immunohistochemical analysis revealed that AVM vessels co-express both artery and vein differentiation genes. H-score analysis revealed that there is statistically significant (P < 0.0001) increase in expression of these proteins in AVM vessels compared to control vessels. These findings were further confirmed by western blot analysis and found to be statistically significant (P < 0.0001 and P < 0.001) for all proteins except Hey2. Both immunostaining and western blot analysis revealed that AVM vessels express GGTP and GLUT1, markers specific to brain capillaries. Immunofluorescent staining demonstrated that expression of KLF2, a vascular maturation marker is significantly (P <0.001) decreased in AVM vessels and was further confirmed by western blot analysis (P < 0.001). Immunohistochemical and western blot analysis demonstrated that another vascular maturation protein Endoglin had high expression in AVM vessels compared to control vessels. The results were found to be statistically significant (P < 0.0001). SUMMARY: Our findings suggest that vascular structures of AVMs co-express markers specific for arteries, veins and capillaries. We conclude that AVM nidus constitutes of aberrant vessels which are not terminally differentiated and inadequately matured.

Genetic and epigenetic mechanisms in the development of arteriovenous malformations in the brain.

Vascular malformations are developmental congenital abnormalities of the vascular system which may involve any segment of the vascular tree such as capillaries, veins, arteries, or lymphatics. Arteriovenous malformations (AVMs) are congenital vascular lesions, initially described as "erectile tumors," characterized by atypical aggregation of dilated arteries and veins. They may occur in any part of the body, including the brain, heart, liver, and skin. Severe clinical manifestations occur only in the brain. There is absence of normal vascular structure at the subarteriolar level and dearth of capillary bed resulting in aberrant arteriovenous shunting. The causative factor and pathogenic mechanisms of AVMs are unknown. Importantly, no marker proteins have been identified for AVM. AVM is a high flow vascular malformation and is considered to develop because of variability in the hemodynamic forces of blood flow. Altered local hemodynamics in the blood vessels can affect cellular metabolism and may trigger epigenetic factors of the endothelial cell. The genes that are recognized to be associated with AVM might be modulated by various epigenetic factors. We propose that AVMs result from a series of changes in the DNA methylation and histone modifications in the genes connected to vascular development. Aberrant epigenetic modifications in the genome of endothelial cells may drive the artery or vein to an aberrant phenotype. This review focuses on the molecular pathways of arterial and venous development and discusses the role of hemodynamic forces in the development of AVM and possible link between hemodynamic forces and epigenetic mechanisms in the pathogenesis of AVM.

Cationic, amphiphilic dextran nanomicellar clusters as an excipient for dry powder inhaler formulation.

Effective delivery of drugs to alveoli in a controlled manner using hydrophobic polymers as carriers has already been reported. Preclinical studies revealed that toxicity and hydrophobicity are related to each other in pulmonary delivery. Here, we are reporting a chemically modified dextran having amphiphilicity and cationicity achieved by controlled grafting of stearyl amine. Two proportions of lipopolymers were synthesized and physico-chemical characterization was carried out. In vivo evaluation of sub-acute toxicity of the synthesized lipopolymer in Sprague-Dawley rats was carried out for three months. This was followed by a histological evaluation of the sacrificed animal's lung. Further, the synthesized lipopolymer was formulated with drug (Rifampicin) loaded inhalable microparticles through spray drying. The final drug formulation was tested for toxicity and proinflammatory responses in human cell lines. Dose deposition efficiency of the formulation was determined using Anderson Cascade Impactor.

Forkhead box C2 promoter variant c.-512C>T is associated with increased susceptibility to chronic venous diseases.

Chronic venous disease (CVD) is one of the most prevalent yet underrated disorders worldwide. High heritability estimates of CVD indicate prominent genetic components in its etiology and pathology. Mutations in human forkhead box C2 (FoxC2) gene are strongly associated with valve failure in saphenous and deep veins of lower extremities. We explored the association of genetic variants of FoxC2 as well as FoxC2 mRNA and protein expression levels with CVD of lower limbs. We systematically sequenced the single coding exon, 5' and 3' flanking regions of FoxC2 gene in 754 study subjects which includes 382 patients with CVD and 372 healthy subjects. Four novel and three reported polymorphisms were identified in our cohort. Three variants in 5' flanking region and one in 3' flanking region of FoxC2 gene were significantly associated with CVD risk. FoxC2 mRNA in vein tissues from 22 patients was 4±1.42 fold increased compared to saphenous veins from 20 normal subjects (p<0.01). FoxC2 protein was also significantly upregulated in varicose veins compared to control samples. The c.-512C>T (rs34221221: C>T) variant which is located in the FoxC2 putative promoter region was further analyzed. Functional analysis of c.-512C>T revealed increased mRNA and protein expression in patients with homozygous TT genotype compared to heterozygous CT and wild CC genotypes. Luciferase assay indicated higher transcriptional activity of mutant compared to wild genotype of this variant. These findings suggested that c.-512C>T variant of FoxC2 was strongly associated with susceptibility to CVD and also that this variant resulted in FoxC2 overexpression. To obtain a mechanistic insight into the role of upregulated FoxC2 in varicosities, we overexpressed FoxC2 in venous endothelial cells and observed elevated expression of arterial markers Dll4 and Hey2 and downregulation of venous marker COUP-TFII. Our study indicates altered FoxC2-Notch signaling in saphenous vein wall remodeling in patients with varicose veins.

Lack of association between plasma levels of non-nucleoside reverse transcriptase inhibitors & virological outcomes during rifampicin co-administration in HIV-infected TB patients.

BACKGROUND & OBJECTIVES: Among patients with HIV-associated tuberculosis (TB), reduced plasma non-nucleoside reverse transcriptase inhibitors (NNRTI) concentrations during rifampicin (RMP) co-administration could lead to HIV treatment failure. This study was undertaken to examine the association between plasma nevirapine (NVP) and efavirenz (EFV) concentrations and virological outcomes in patients infected with HIV-1 and TB. METHODS: This was a nested study undertaken in a clinical trial of patients with HIV-1 and TB, randomized to two different once-daily antiretroviral treatment (ART) regimens along with anti-TB treatment (ATT). Trough concentrations of plasma NVP and EFV were estimated at months 1 (during ATT and ART) and 6 months (ART only) by HPLC. Plasma HIV-1 RNA level >400 copies/ml or death within 6 months of ART were considered as unfavourable outcomes. Genotyping of CYP2B6 516G>T polymorphism was performed. RESULTS: Twenty nine per cent of patients in NVP arm had an unfavourable outcome at 6 months compared to 9 per cent in EFV arm (P<0.08). The mean NVP and EFV levels estimated at 1 and 6 months did not significantly differ between favourable and unfavourable responders. Logistic regression analysis showed CYP2B6 516G>T polymorphism significantly associated with virologic outcome in patients receiving EFV-based regimen. INTERPRETATION & CONCLUSIONS: Trough plasma concentrations of NVP and EFV did not show any association with response to ART in patients on ATT and once-daily ART. CYP2B6 516G>T polymorphism was associated with virologic outcome among patients on EFV.