Peer-Reviewed Research

Journal Article, Research Support, Non-U.S. Gov't

Neutralizing antibody responses to SARS-CoV-2 Omicron variants: Post six months following two-dose & three-dose vaccination of ChAdOx1 nCoV-19 or BBV152.

Yadav PD, Sardana V, Deshpande GR, Shinde PV, Thangaraj JWV, George LS, Sapkal GN, Patil DY, Sahay RR, Shete AM, Joshi M, Murhekar M, Godbole S, Gupta N, Prakash S, Rathore M, Ujjainiya R, Singh AP, Mishra A, Dash D, Chaudhary K, Sengupta S, ICMR Booster Dose Study Group

The Indian journal of medical research · 2024

BACKGROUND OBJECTIVES: The Omicron sub-lineages are known to have higher infectivity, immune escape and lower virulence. During December 2022 - January 2023 and March - April 2023, India witnessed increased SARS-CoV-2 infections, mostly due to newer Omicron sub-lineages. With this unprecedented rise in cases, we assessed the neutralization potential of individuals vaccinated with ChAdOx1 nCoV (Covishield) and BBV152 (Covaxin) against emerging Omicron sub-lineages. METHODS: Neutralizing antibody responses were measured in the sera collected from individuals six months post-two doses (n=88) of Covishield (n=44) or Covaxin (n=44) and post-three doses (n=102) of Covishield (n=46) or Covaxin (n=56) booster dose against prototype B.1 strain, lineages of Omicron; XBB.1, BQ.1, BA.5.2 and BF.7. RESULTS: The sera of individuals collected six months after the two-dose and the three-dose demonstrated neutralizing activity against all variants. The neutralizing antibody (NAbs) level was highest against the prototype B.1 strain, followed by BA5.2 (5-6 fold lower), BF.7 (11-12 fold lower), BQ.1 (12 fold lower) and XBB.1 (18-22 fold lower). INTERPRETATION CONCLUSIONS: Persistence of NAb responses was comparable in individuals with two- and three-dose groups post six months of vaccination. Among the Omicron sub-variants, XBB.1 showed marked neutralization escape, thus pointing towards an eventual immune escape, which may cause more infections. Further, the correlation of study data with complete clinical profile of the participants along with observations for cell-mediated immunity may provide a clear picture for the sustained protection due to three-dose vaccination as well as hybrid immunity against the newer variants.

Journal Article

Hypobaric hypoxia modulated structural characteristics of circulating cell-free DNA in high-altitude pulmonary edema.

Ali M, Choudhary R, Singh K, Kumari S, Kumar R, Graham BB, Pasha MAQ, Rabyang S, Thinlas T, Mishra A

American journal of physiology. Lung cellular and molecular physiology · 2024

The utility of cell-free (cf) DNA has extended as a surrogate or clinical biomarker for various diseases. However, a more profound and expanded understanding of the diverse cfDNA population and its correlation with physiological phenotypes and environmental factors is imperative for using its full potential. The high-altitude (HA; altitude > 2,500 m above sea level) environment characterized by hypobaric hypoxia offers an observational case-control design to study the differential cfDNA profile in patients with high-altitude pulmonary edema (HAPE) (number of subjects,= 112) and healthy HA sojourners (= 111). The present study investigated cfDNA characteristics such as concentration, fragment length size, degree of integrity, and subfractions reflecting mitochondrial-cfDNA copies in the two groups. The total cfDNA level was significantly higher in patients with HAPE, and the level increased with increasing HAPE severity (= 0.0036). A lower degree of cfDNA integrity of 0.346 in patients with HAPE (= 0.001) indicated the prevalence of shorter cfDNA fragments in circulation in patients compared with the healthy HA sojourners. A significant correlation of cfDNA characteristics with the peripheral oxygen saturation levels in the patient group demonstrated the translational relevance of cfDNA molecules. The correlation was further supported by multivariate logistic regression and receiver operating characteristic curve. To our knowledge, our study is the first to highlight the association of higher cfDNA concentration, a lower degree of cfDNA integrity, and increased mitochondrial-derived cfDNA population with HAPE disease severity. Further deep profiling of cfDNA fragments, which preserves cell-type specific genetic and epigenetic features, can provide dynamic physiological responses to hypoxia.This study observed altered cell-free (cf) DNA fragment patterns in patients with high-altitude pulmonary edema and the significant correlation of these patterns with peripheral oxygen saturation levels. This suggests deep profiling of cfDNA fragments in the future may identify genetic and epigenetic mechanisms underlying physiological and pathophysiological responses to hypoxia.

Journal Article

Unraveling Epigenetic Interplay between Inflammation, Thrombosis, and Immune-Related Disorders through a Network Meta-analysis.

Chanchal S, Sharma S, Mohd S, Sultan A, Mishra A, Ashraf MZ

TH open : companion journal to thrombosis and haemostasis · 2024

Inflammation and thrombosis are two distinct yet interdependent physiological processes. The inflammation results in the activation of the coagulation system that directs the immune system and its activation, resulting in the initiation of the pathophysiology of thrombosis, a process termed immune-thrombosis. Still, the shared underlying molecular mechanism related to the immune system and coagulation has not yet been explored extensively. Inspired to answer this, we carried out a comprehensive gene expression meta-analysis using publicly available datasets of four diseases, including venous thrombosis, systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. A total of 609 differentially expressed genes (DEGs) shared by all four datasets were identified based on the combined effect size approach. The pathway enrichment analysis of the DEGs showed enrichment of various epigenetic pathways such as histone-modifying enzymes, posttranslational protein modification, chromatin organization, chromatin-modifying enzymes, HATs acetylate proteins. Network-based protein-protein interaction analysis showed epigenetic enzyme coding genes dominating among the top hub genes. The miRNA-interacting partner of the top 10 hub genes was determined. The predomination of epitranscriptomics regulation opens a layout for the meta-analysis of miRNA datasets of the same four diseases. We identified 30 DEmiRs shared by these diseases. There were 9 common DEmiRs selected from the list of miRNA-interacting partners of top 10 hub genes and shared significant DEmiRs from microRNAs dataset acquisition. These common DEmiRs were found to regulate genes involved in epigenetic modulation and indicate a promising epigenetic aspect that needs to be explored for future molecular studies in the context of immunothrombosis and inflammatory disease.

Review, Journal Article, Research Support, Non-U.S. Gov't

Respiratory tract infection: an unfamiliar risk factor in high-altitude pulmonary edema.

Choudhary R, Kumari S, Ali M, Thinlas T, Rabyang S, Mishra A

Briefings in functional genomics · 2024

The dramatic changes in physiology at high altitude (HA) as a result of the characteristic hypobaric hypoxia condition can modify innate and adaptive defense mechanisms of the body. As a consequence, few sojourners visiting HA with mild or asymptomatic infection may have an enhanced susceptibility to high-altitude pulmonary edema (HAPE), an acute but severe altitude sickness. It develops upon rapid ascent to altitudes above 2500 m, in otherwise healthy individuals. Though HAPE has been studied extensively, an elaborate exploration of the HA disease burden and the potential risk factors associated with its manifestation are poorly described. The present review discusses respiratory tract infection (RTI) as an unfamiliar but important risk factor in enhancing HAPE susceptibility in sojourners for two primary reasons. First, the symptoms of RTI s resemble those of HAPE. Secondly, the imbalanced pathways contributing to vascular dysfunction in HAPE also participate in the pathogenesis of the infectious processes. These pathways have a crucial role in shaping host response against viral and bacterial infections and may further worsen the clinical outcomes at HA. Respiratory tract pathogenic agents, if screened in HAPE patients, can help in ascertaining their role in disease risk and also point toward their association with the disease severity. The microbial screenings and identifications of pathogens with diseases are the foundation for describing potential molecular mechanisms underlying host response to the microbial challenge. The prior knowledge of such infections may predict the manifestation of disease etiology and provide better therapeutic options.

Clinical Trial, Journal Article

Dexamethasone prophylaxis protects from acute high-altitude illness by modifying the peripheral blood mononuclear cell inflammatory transcriptome.

Kumar R, Chanana N, Sharma K, Palmo T, Lee MH, Mishra A, Nolan K, Fonseca Balladares DC, Mickael C, Gupta MD, Thinlas T, Pasha Q, Graham BB

Bioscience reports · 2023

Acute high-altitude (HA) exposure can induce several pathologies. Dexamethasone (DEX) can be taken prophylactically to prevent HA disease, but the mechanism by which it acts in this setting is unclear. We studied the transcriptome of peripheral blood mononuclear cells (PBMCs) from 16 subjects at low altitude (LA, 225 m) and then 3 days after acute travel to HA (3500 m) during the India-Leh-Dexamethasone-Expedition-2020 (INDEX2020). Half of the participants received oral DEX prophylaxis 4 mg twice daily in an unblinded manner, starting 1 day prior to travel to HA, and 12 h prior to the first PBMC collection. PBMC transcriptome data were obtained from 16 subjects, half of whom received DEX. The principal component analysis demonstrated a clear separation of the groups by altitude and treatment. HA exposure resulted in a large number of gene expression changes, particularly in pathways of inflammation or the regulation of cell division, translation, or transcription. DEX prophylaxis resulted in changes in fewer genes, particularly in immune pathways. The gene sets modulated by HA and DEX were distinct. Deconvolution analysis to assess PBMC subpopulations suggested changes in B-cell, T-cell, dendritic cell, and myeloid cell numbers with HA and DEX exposures. Acute HA travel and DEX prophylaxis induce significant changes in the PBMC transcriptome. The observed benefit of DEX prophylaxis against HA disease may be mediated by suppression of inflammatory pathways and changing leukocyte population distributions.

Journal Article

Association of transient mitochondrial functional impairment with acute heat exposure in children from Muzaffarpur region of Bihar, India.

Singh K, Kumari S, Ali M, Das MK, Mishra A, Singh AK

International journal of biometeorology · 2023

Over the past several years, the Muzaffarpur district of Bihar (India) has witnessed recurrent outbreaks of acute encephalitis illness of unknown etiology, called acute encephalitis syndrome (AES) among young children, especially during the peak-summer season. Pesticide exposure, viral encephalitis, and litchi toxin intake have all been postulated as potential sources of the ailment. However, no conclusive etiology for AES has been identified in the affected children. During recent rounds of the outbreak, metabolic abnormalities have been documented in these children, and a direct correlation was observed between higher environmental temperature during the peak-summer month and AES caseload. The clinical and metabolic profiles of these children suggested the possible involvement of mitochondrial dysfunction during heat stress as one of the several contributory factors leading to multisystem metabolic derangement. The present study observed that mitochondrial function parameters such as cell death, mitochondrial membrane potential, oxidative stress, and mitochondrial pathway-related gene expression in peripheral blood mononuclear cells (PBMCs) isolated from children were affected in peak-summer when compared to post-summer months. Similar observations of mitochondrial function parameters along with impaired bioenergetic parameters were demonstrated in the heat-exposed model of PBMCs isolated from healthy adult individuals. In conclusion, the results suggested that there is an association of transient mitochondrial dysfunction when exposed to sustained heat during the summer months. One may consider mitochondrial dysfunction as one of the important factors leading to an outbreak of AES among the children from affected regions though this needs to be substantiated with further studies.

Journal Article

Differential methylation in EGLN1 associates with blood oxygen saturation and plasma protein levels in high-altitude pulmonary edema.

Sharma K, Mishra A, Singh H, Thinlas T, Pasha MAQ

Clinical epigenetics · 2022

BACKGROUND: High-altitude (HA, 2500 m) hypoxic exposure evokes a multitude of physiological processes. The hypoxia-sensing genes though influence transcriptional output in disease susceptibility; the exact regulatory mechanisms remain undetermined in high-altitude pulmonary edema (HAPE). Here, we investigated the differential DNA methylation distribution in the two genes encoding the oxygen-sensing HIF-prolyl hydroxylases, prolyl hydroxylase domain protein 2 (PHD2) and factor inhibiting HIF-1α and the consequent contributions to the HAPE pathophysiology. METHODS: Deep sequencing of the sodium bisulfite converted DNA segments of the two genes, Egl nine homolog 1 (EGLN1) and Hypoxia Inducible Factor 1 Subunit Alpha Inhibitor (HIF1AN), was conducted to analyze the differential methylation distribution in three study groups, namely HAPE-patients (HAPE-p), HAPE-free sojourners (HAPE-f) and healthy HA natives (HLs). HAPE-p and HAPE-f were permanent residents of low altitude (< 200 m) of North India who traveled to Leh (3500 m), India, and were recruited through Sonam Norboo Memorial (SNM) hospital, Leh. HLs were permanent residents of altitudes at and above 3500 m. In addition to the high resolution, bisulfite converted DNA sequencing, gene expression of EGLN1 and HIF1AN and their plasma protein levels were estimated. RESULTS: A significantly lower methylation distribution of CpG sites was observed in EGLN1 and higher in HIF1AN (P < 0.01) in HAPE-p compared to the two control groups, HAPE-f and HLs. Of note, differential methylation distribution of a few CpG sites, 231,556,748, 231,556,804, 231,556,881, 231,557,317 and 231,557,329, in EGLN1 were significantly associated with the risk of HAPE (OR = 4.79-10.29; P = 0.048-004). Overall, the methylation percentage in EGLN1 correlated with upregulated plasma PHD2 levels (R = - 0.36, P = 0.002) and decreased peripheral blood oxygen saturation (SpO) levels (R = 0.34, P = 0.004). We also identified a few regulatory SNPs in the DNA methylation region of EGLN1 covering chr1:231,556,683-231,558,443 suggestive of the functional role of differential methylation distribution of these CpG sites in the regulation of the genes and consequently in the HIF-1α signaling. CONCLUSIONS: Significantly lower methylation distribution in EGLN1 and the consequent physiological influences annotated its functional epigenetic relevance in the HAPE pathophysiology.

Journal Article

COVID-19 Induced Coagulopathy (CIC): Thrombotic Manifestations of Viral Infection.

Sharma S, Mishra A, Ashraf Z

TH open : companion journal to thrombosis and haemostasis · 2022

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may result in an overactive coagulative system, thereby resulting in serious cardiovascular consequences in critically affected patients. The respiratory tract is a primary target for COVID-19 infection, which is manifested as acute lung injury in the most severe form of the viral infection, leading to respiratory failure. A proportion of infected patients may progress to serious systemic disease including dysfunction of multiple organs, acute respiratory distress syndrome (ARDS), and coagulation abnormalities, all of which are associated with increased mortality, additionally depending on age and compromised immunity. Coagulation abnormalities associated with COVID-19 mimic other systemic coagulopathies otherwise involved in other severe infections, such as disseminated intravascular coagulation (DIC) and may be termed COVID-19 induced coagulopathy (CIC). There is substantial evidence that patients with severe COVID-19 exhibiting CIC can develop venous and arterial thromboembolic complications. In the initial stages of CIC, significant elevation of D-dimer and fibrin/fibrinogen degradation products is observed. Alteration in prothrombin time, activated partial thromboplastin time, and platelet counts are less common in the early phase of the disease. In patients admitted to intensive care units (ICUs), coagulation test screening involving the measurement of D-dimer and fibrinogen levels, has been recommended. Prior established protocols for thromboembolic prophylaxis are also followed for CIC, including the use of heparin and other standard supportive care measures. In the present review, we summarize the characteristics of CIC and its implications for thrombosis, clinical findings of coagulation parameters in SARS-CoV-2 infected patients with incidences of thromboembolic events and plausible therapeutic measures.

Journal Article, Review

Therapeutic Role of Sirtuins Targeting Unfolded Protein Response, Coagulation, and Inflammation in Hypoxia-Induced Thrombosis.

Sadia K, Ashraf MZ, Mishra A

Frontiers in physiology · 2021

Thrombosis remains one of the leading causes of morbidity and mortality across the world. Many pathological milieus in the body resulting from multiple risk factors escort thrombosis. Hypoxic condition is one such risk factor that disturbs the integrity of endothelial cells to cause an imbalance between anticoagulant and procoagulant proteins. Hypoxia generates reactive oxygen species (ROS) and triggers inflammatory pathways to augment the coagulation cascade. Hypoxia in cells also activates unfolded protein response (UPR) signaling pathways in the endoplasmic reticulum (ER), which tries to restore ER homeostasis and function. But the sustained UPR linked with inflammation, generation of ROS and apoptosis stimulates the severity of thrombosis in the body. Sirtuins, a group of seven proteins, play a vast role in bringing down inflammation, oxidative and ER stress and apoptosis. As a result, sirtuins might provide a therapeutic approach towards the treatment or prevention of hypoxia-induced thrombosis. Sirtuins modulate hypoxia-inducible factors (HIFs) and counteract ER stress-induced apoptosis by attenuating protein kinase RNA-like endoplasmic reticulum kinase (PERK)/Eukaryotic translation initiation factor 2α (eIF2α) pathway activation. It prevents ER-stress mediated inflammation by targeting X-Box Binding Protein 1 (XBP1) and inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ) signaling through deacetylation. Sirtuins also obstruct nucleotide-binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome activation to reduce the expression of several pro-inflammatory molecules. It protects cells against oxidative stress by targeting nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione (GSH), forkhead box O3 (FOXO3), superoxide dismutase (SOD), catalase (CAT), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), glucose-6-phosphate dehydrogenase (G6PD), phosphoglucomutase-2 (PGAM2), and NF-κB, to name few. This review, thus, discusses the potential role of sirtuins as a new treatment for hypoxia-induced thrombosis that involves an intersection of UPR and inflammatory pathways in its pathological manifestation.

Journal Article

Hypertensive Patients Exhibit Enhanced Thrombospondin-1 Levels at High-Altitude.

Sharma K, Chanana N, Mohammad G, Thinlas T, Gupta M, Syed MA, Das RS, Pasha Q, Mishra A

Life (Basel, Switzerland) · 2021

Thrombospondin-1 (THBS1) levels elevate under hypoxia and have relevance in several cardiovascular disorders. The association of THBS1 with endothelial dysfunction implies its important role in hypertension. To establish the hypothesis, we screened patients with hypertension and their respective controls from the two different environmental regions. Cohort 1 was composed of Ladakhis, residing at 3500 m above sea level (ASL), whereas Cohort 2 was composed of north-Indians residing at ~200 m ASL. Clinical parameters and circulating THBS1 levels were correlated in the case-control groups of the two populations. THBS1 levels were significantly elevated in hypertension patients of both cohorts; however, the levels were distinctly enhanced in the hypertensive patients of HA as compared to normoxia (< 0.002). The observation was supported by the receiver operating curve analysis with an area under curve of 0.7007 (0.627-0.774) demonstrating the discriminatory effect of hypobaric hypoxia on the levels as compared to normoxia (< 0.011). Significant correlation of THBS1 and mean arterial pressure was observed with upraised positive correlations in the hypertensive highlanders as compared to the hypertensive patients from sea-level. The prevalence of differential distribution ofandgenes variants, their interactions, and association with the THBS1 levels were also determined. Genotype-interactions betweenrs2228263 andrs9879947 were relevant and the regression analysis highlighted the association of risk genotype-interactions with increased THBS1 levels in hypertension. Genetic studies of additional thrombospondin pathway-related genes suggest the complex role of THBS1 in the presence of its family members and the related receptor molecules at HA.

Journal Article, Research Support, Non-U.S. Gov't

High-altitude pulmonary edema is aggravated by risk loci and associated transcription factors in HIF-prolyl hydroxylases.

Sharma K, Mishra A, Singh HN, Parashar D, Alam P, Thinlas T, Mohammad G, Kukreti R, Syed MA, Pasha MAQ

Human molecular genetics · 2021

High-altitude (HA, >2500 m) hypoxic exposure evokes several physiological processes that may be abetted by differential genetic distribution in sojourners, who are susceptible to various HA disorders, such as high-altitude pulmonary edema (HAPE). The genetic variants in hypoxia-sensing genes influence the transcriptional output; however the functional role has not been investigated in HAPE. This study explored the two hypoxia-sensing genes, prolyl hydroxylase domain protein 2 (EGLN1) and factor inhibiting HIF-1α (HIF1AN) in HA adaptation and maladaptation in three well-characterized groups: highland natives, HAPE-free controls and HAPE-patients. The two genes were sequenced and subsequently validated through genotyping of significant single nucleotide polymorphisms (SNPs), haplotyping and multifactor dimensionality reduction. Three EGLN1 SNPs rs1538664, rs479200 and rs480902 and their haplotypes emerged significant in HAPE. Blood gene expression and protein levels also differed significantly (P < 0.05) and correlated with clinical parameters and respective alleles. The RegulomeDB annotation exercises of the loci corroborated regulatory role. Allele-specific differential expression was evidenced by luciferase assay followed by electrophoretic mobility shift assay, liquid chromatography with tandem mass spectrometry and supershift assays, which confirmed allele-specific transcription factor (TF) binding of FUS RNA-binding protein (FUS) with rs1538664A, Rho GDP dissociation inhibitor 1 (ARHDGIA) with rs479200T and hypoxia upregulated protein 1 (HYOU1) with rs480902C. Docking simulation studies were in sync for the DNA-TF structural variations. There was strong networking among the TFs that revealed physiological consequences through relevant pathways. The two hydroxylases appear crucial in the regulation of hypoxia-inducible responses.

Letter, Comment

Response: Comment and Update on "Using Artificial Intelligence to Manage Thrombosis Research, Diagnosis, and Clinical Management".

Mishra A, Ashraf MZ

Seminars in thrombosis and hemostasis · 2021

Journal Article

Host-Viral Interactions Revealed among Shared Transcriptomics Signatures of ARDS and Thrombosis: A Clue into COVID-19 Pathogenesis.

Mishra A, Chanchal S, Ashraf MZ

TH open : companion journal to thrombosis and haemostasis · 2020

Severe novel corona virus disease 2019 (COVID-19) infection is associated with a considerable activation of coagulation pathways, endothelial damage, and subsequent thrombotic microvascular injuries. These consistent observations may have serious implications for the treatment and management of this highly pathogenic disease. As a consequence, the anticoagulant therapeutic strategies, such as low molecular weight heparin, have shown some encouraging results. Cytokine burst leading to sepsis which is one of the primary reasons for acute respiratory distress syndrome (ARDS) drive that could be worsened with the accumulation of coagulation factors in the lungs of COVID-19 patients. However, the obscurity of this syndrome remains a hurdle in making decisive treatment choices. Therefore, an attempt to characterize shared biological mechanisms between ARDS and thrombosis using comprehensive transcriptomics meta-analysis is made. We conducted an integrated gene expression meta-analysis of two independently publicly available datasets of ARDS and venous thromboembolism (VTE). Datasets GSE76293 and GSE19151 derived from National Centre for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) database were used for ARDS and VTE, respectively. Integrative meta-analysis of expression data (INMEX) tool preprocessed the datasets and effect size combination with random effect modeling was used for obtaining differentially expressed genes (DEGs). Network construction was done for hub genes and pathway enrichment analysis. Our meta-analysis identified a total of 1,878 significant DEGs among the datasets, which when subjected to enrichment analysis suggested inflammation-coagulation-hypoxemia convolutions in COVID-19 pathogenesis. The top hub genes of our study such as tumor protein 53 (TP53), lysine acetyltransferase 2B (KAT2B), DExH-box helicase 9 (DHX9), REL-associated protein (RELA), RING-box protein 1 (RBX1), and proteasome 20S subunit beta 2 (PSMB2) gave insights into the genes known to be participating in the host-virus interactions that could pave the way to understand the various strategies deployed by the virus to improve its replication and spreading.

Journal Article, Research Support, Non-U.S. Gov't

Susceptibility to high-altitude pulmonary edema is associated with circulating miRNA levels under hypobaric hypoxia conditions.

Alam P, Agarwal G, Kumar R, Mishra A, Saini N, Mohammad G, Pasha MAQ

American journal of physiology. Lung cellular and molecular physiology · 2020

Hypobaric hypoxia poses stress to sojourners traveling to high-altitude. A cascade of physiological changes occurs to cope with or adapt to hypobaric hypoxia. However, an insufficient physiological response to the hypoxic condition resulting from imbalanced vascular homeostasis pathways results in high-altitude pulmonary edema (HAPE). The present study aims to identify the implication of miRNAs associating with HAPE and adaptation. We analyzed the expression of 1,113 miRNAs in HAPE-patients (HAPE-p), HAPE-free controls (HAPE-f), and highland natives (HLs). Based on miRNA profiling and in silico analyses, miR-124-3p emerged relevantly. We observed a significant overexpression of miR-124-3p in HAPE-p. In silico analyses revealed a direct interaction of miR-124-3p with vascular homeostasis and hypoxia-associated genes(endothelial nitric oxide synthase),, and(V-Ets avian erythroblastosis virus E2 oncogene homolog 1). Moreover, the transcript and biolevel expression of these genes were significantly decreased in HAPE-p when compared with HAPE-f or HLs. Our in vitro analysis in human umbilical vein endothelial cells demonstrated a significant knockdown of these genes both at transcript and protein levels following miR-124-3p overexpression. Conclusively, our results showed that miR-124-3p might play a plausible role in HAPE pathophysiology by inhibiting the expression of,, and.

Journal Article, Review

Involvement of Epigenetic Control and Non-coding RNAs in Cardiovascular System.

Sharma S, Mishra A, Ashraf MZ

Advances in experimental medicine and biology · 2020

Cardiovascular Diseases (CVDs) as a leading cause of death worldwide inflict major stress on morbidity and societal costs. Though the studies pertaining to pathophysiology and genetics of CVDs have helped in prevention, diagnosis and treatment of diseases, there are still lacunas in our knowledge. So, novel tools that can define genomic regulation under different conditions are needed to bridge this gap. 'Epigenetic' mechanism helps the cells to quickly respond to ever changing environment by molecular mechanisms like methylation, histone modifications, nc-RNAs. These mechanisms act as a new layer of regulation in CVDs. The role of epigenetics as a key regulatory player in prevention, diagnosis and treatment of CVDs is emerging. Thus, the focus of present chapter is to decipher the role of epigenetics in CVDs and its potential to be used in risk assessment or as biomarkers in devising and deploying better diagnosis and treatment for different CVDs.

Journal Article, Review

Understanding Inflammatory Responses in the Manifestation of Prothrombotic Phenotypes.

Chanchal S, Mishra A, Singh MK, Ashraf MZ

Frontiers in cell and developmental biology · 2020

Inflammasome complex is a multimeric protein comprising of upstream sensor protein of nucleotide-binding oligomerization domain (NOD)-like receptor family. It has an adaptor protein apoptosis-associated speck-like protein and downstream effector cysteine protease procaspase-1. Activation of inflammasome complex is body's innate response to pathogen attack but its abnormal activation results in many inflammatory and cardiovascular disorders including thrombosis. It has displayed a prominent role in the clot formation advocating an interplay between inflammation and coagulation cascades. Therefore, elucidation of inflammasome and its molecular mechanisms in the manifestation of prothrombotic phenotypes becomes pertinent. Thrombosis is the formation and propagation of blood clot in the arterial or venous system due to several interactions of vascular and immune factors. It is a prevalent pathology underlying disorders like venous thromboembolism, stroke and acute coronary syndrome; thus, making thrombosis, a major contributor to the global disease burden. Recently studies have established a strong connection of inflammatory processes with this blood coagulation disorder. The hemostatic balance in thrombosis gets altered by the inflammatory mechanisms resulting in endothelial and platelet activation that subsequently increases secretion of several prothrombotic and antifibrinolytic factors. The upregulation of these factors is the critical event in the pathogenesis of thrombosis. Among various inflammasome, nucleotide-binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) is one of the best-studied sterile inflammasome strengthening a link between inflammation and coagulation in thrombosis. NLRP3 activation results in the catalytic conversion of procaspase-1 to active caspase-1, which facilitate the maturation of interleukin-1β (IL-1β) and interleukin-18. These cytokines are responsible for immune cells activation critical for immune responses. These responses further results in endothelial and platelet activation and aggregation. However, the exact molecular mechanism related to the pathogenesis of thrombosis is still elusive. There have been several reports that demonstrate Tissue factor (TF)-mediated signaling in the production of pro-inflammatory cytokines enhancing inflammation by activating protease-activated receptors on various cells, which lead to additional cytokine expression. Therefore, it would be illuminating to interpret the inflammasomes regulation in coagulation and inflammation. This review, thus, tries to comprehensively compile emerging regulatory roles of the inflammasomes in thrombosis and discusses their molecular pathways in the manifestation of thrombotic phenotypes.

Journal Article

Using Artificial Intelligence to Manage Thrombosis Research, Diagnosis, and Clinical Management.

Mishra A, Ashraf MZ

Seminars in thrombosis and hemostasis · 2020

Thrombosis development in either arterial or venous system remains a major cause of death and disability worldwide. This poorly controlled in vivo clotting could result in many severe complications including myocardial infarction, venous thromboembolism, stroke, and cerebral venous thrombosis, to name a few. These conditions are collectively known as thromboembolic disorders (TEDs). Appropriate understanding of TEDs is challenging, as they are multifactorial and involve several and often different risk factors. Hence, it requires a collective effort and data from numerous research studies to fully comprehend molecular mechanisms for prediction, prevention, treatment, and overall management of these conditions. To accomplish this arduous feat, a comprehensive approach is required that can compile thousands of available experimental data and transform these into more applicable and purposeful findings. Thus, large datasets could be utilized to generate models that could be predictive of how an individual would respond when subjected to any kind of additional risk factors or surgery, hospitalization, etc., or in the presence of some susceptible genetic variations. Artificial intelligence-based methods harness the capabilities of computer software to imitate human behaviors such as language translation, visual perception, and, most importantly, decision making. These emerging tools, if appropriately explored, might assist in processing of large data and tackle the complexities of identifying novel or interesting pathways that could otherwise be hidden due to their enormity. This narrative review attempts to compile the applications of various subfields of artificial intelligence and machine learning in the context of thrombosis research to date. It further reflects on the potential of artificial intelligence in transforming enormous research data into translational application in the form of predictive computational models.

Journal Article, Research Support, Non-U.S. Gov't, Review

Emerging Role of Vitamin D and its Associated Molecules in Pathways Related to Pathogenesis of Thrombosis.

Mohammad S, Mishra A, Ashraf MZ

Biomolecules · 2019

Vitamin D, besides having an essential role in calcium and bone metabolism, also acts as a mediator of many non-calcemic effects through modulations of several biological responses. Vitamin D exists in its two major forms, vitamin D, or commonly known as ergocalciferol, and vitamin D, or commonly known as cholecalciferol. Both of these forms bind to vitamin D-binding protein to get transported to all vital target organs, where it serves as a natural ligand to vitamin D receptors for enabling their biological actions. Clinical reports corroborating vitamin D deficiency with an increase in thrombotic episodes implicate the role of vitamin D and its associated molecule in the regulation of thrombosis-related pathways. Thrombosis is the formation and propagation of a blood clot, known as thrombus. It can occur either in the arterial or the venous system resulting in many severe complications, including myocardial infarction, stroke, ischemia, and venous thromboembolism. Vitamin D, directly or indirectly, controls the expression of several genes responsible for the regulation of cellular proliferation, differentiation, apoptosis, and angiogenesis. All of these are the processes of potential relevance to thrombotic disorders. This review, thus, discussed the effects of vitamin D on pathways involved in thrombosis, such as hemostatic process, inflammatory pathway, and endothelial cell activation, with a focus on the molecular mechanisms associated with them.

Journal Article, Research Support, Non-U.S. Gov't

Genetic differences and aberrant methylation in the apelin system predict the risk of high-altitude pulmonary edema.

Mishra A, Kohli S, Dua S, Thinlas T, Mohammad G, Pasha MA

Proceedings of the National Academy of Sciences of the United States of America · 2015

Hypoxia-inducible factor stimulates the expression of apelin, a potent vasodilator, in response to reduced blood arterial oxygen saturation. However, aberrations in the apelin system impair pulmonary vascular function, potentially resulting in the development of high-altitude (HA)-related disorders. This study aimed to elucidate the genetic and epigenetic regulation of apelin, apelin receptor (APLNR), and endothelial nitric oxide synthase (NOS3) in HA adaptation and HA pulmonary edema (HAPE). A genome-wide association study and sequencing identified variants of apelin, APLNR, and NOS3 that were validated in a larger sample size of HAPE-patients (HAPE-p), HAPE-free controls (HAPE-f), and healthy highland natives (HLs). Apelin-13 and nitrite levels and apelin and NOS3 expression were down-regulated in HAPE-p (P < 0.001). Among the several studied polymorphisms, apelin rs3761581, rs2235312, and rs3115757; APLNR rs11544374 and rs2282623; and NOS3 4b/4a, rs1799983, and rs7830 were associated with HAPE (P < 0.03). The risk allele rs3761581G was associated with a 58.6% reduction in gene expression (P = 0.017), and the risk alleles rs3761581G and rs2235312T were associated with low levels of apelin-13 and nitrite (P < 0.05). The latter two levels decreased further when both of these risk alleles were present in the patients (P < 0.05). Methylation of the apelin CpG island was significantly higher in HAPE-p at 11.92% than in HAPE-f and HLs at ≤ 7.1% (P < 0.05). Moreover, the methylation effect was 9% stronger in the 5' UTR and was associated with decreased apelin expression and apelin-13 levels. The rs3761581 and rs2235312 polymorphisms and methylation of the CpG island influence the expression of apelin in HAPE.

Journal Article, Research Support, Non-U.S. Gov't, Review

Lungs at high-altitude: genomic insights into hypoxic responses.

Mishra A, Mohammad G, Norboo T, Newman JH, Pasha MA

Journal of applied physiology (Bethesda, Md. : 1985) · 2015

Hypobaric hypoxia at high altitude (HA) results in reduced blood arterial oxygen saturation, perfusion of organs with hypoxemic blood, and direct hypoxia of lung tissues. The pulmonary complications in the cells of the pulmonary arterioles due to hypobaric hypoxia are the basis of the pathophysiological mechanisms of high-altitude pulmonary edema (HAPE). Some populations that have dwelled at HA for thousands of years have evolutionarily adapted to this environmental stress; unadapted populations may react with excessive physiological responses that impair health. Individual variations in response to hypoxia and the mechanisms of HA adaptation provide insight into physiological responses. Adaptive and maladaptive responses include alterations in pathways such as oxygen sensing, hypoxia signaling, K(+)- and Ca(2+)-gated channels, redox balance, and the renin-angiotensin-aldosterone system. Physiological imbalances are linked with genetic susceptibilities, and nonhomeostatic responses in gene regulation that occur by small RNAs, histone modification, and DNA methylation predispose susceptible humans to these HA illnesses. Elucidation of the interaction of these factors will lead to a more comprehensive understanding of HA adaptations and maladaptations and will lead to new therapeutics for HA disorders related to hypoxic lungs.

Journal Article, Research Support, Non-U.S. Gov't

Interactions between the genes of vasodilatation pathways influence blood pressure and nitric oxide level in hypertension.

Kumar R, Kohli S, Mishra A, Garg R, Alam P, Stobdan T, Nejatizadeh A, Gupta M, Tyagi S, Pasha MA

American journal of hypertension · 2015

BACKGROUND: This study investigates the contribution of genetic interactions between the β-2 adrenergic receptor (ADRB2) and nitric oxide synthase (NOS3) genes to the complex etiology of hypertension. METHODS: Using single nucleotide polymorphism (SNP) markers, we studied potential interactions between ADRB2 and NOS3 variants and their correlation with clinical, biochemical, and expression levels in 546 individuals with hypertension and 884 age-, sex-, and ethnicity-matched unrelated control subjects. Generalized multifactor dimensionality reduction (GMDR) analysis identified the models for genotype interaction. RESULTS: The best models to represent association of genotypes with augmented hypertension susceptibility were the 4- and 5-locus interacting GMDR models of ADRB2 and NOS3 compared with within-gene 6-locus ADRB2 and 2-locus NOS3 (odds ratio (OR) = 4.8, P = 0.04; OR = 5.6, P = 0.02, respectively). Stratification of 4- and 5-locus GMDR models on the basis of risk alleles (in increasing order) increased the ORs from 1.26 to 14.17 and from 0.81 to 14.18, respectively, and correlated linearly with increased systolic blood pressure, diastolic blood pressure, and mean arterial pressure and decreased nitric oxide level (P ≤ 0.0004). We performed various analyses, such as single-locus, genetic interactions, sliding-window, and comparative analysis. Each analysis consistently revealed the 46A allele of ADRB2 46G/A SNP and 4a allele of NOS3 4b/4a SNP to be associated with risk of hypertension. These risk-conferring markers were associated with decreased ADRB2 and NOS3 expression and decreased nitric oxide level in the patients (P ≤ 0.04). CONCLUSIONS: Evidence of interaction between the genetic loci of ADRB2 and NOS3 points to varied clinical, biochemical, and expression levels and a role in hypertension susceptibility.

Journal Article

EGLN1 variants influence expression and SaO2 levels to associate with high-altitude pulmonary oedema and adaptation.

Mishra A, Mohammad G, Thinlas T, Pasha MA

Clinical science (London, England : 1979) · 2013

EGLN1 [encoding HIF (hypoxia-inducible factor)-prolyl hydroxylase 2] plays a pivotal role in the HIF pathway and has emerged as one of the most intriguing genes with respect to physiology at HA (high altitude). EGLN1, being an actual oxygen sensor, appears to have a potential role in the functional adaptation to the hypobaric hypoxic environment. In the present study, we screened 30 polymorphisms of EGLN1, evaluated its gene expression and performed association analyses. In addition, the role of allelic variants in altering TF (transcription factor)-binding sites and consequently the replacement of TFs at these loci was also investigated. The study was performed in 250 HAPE-p [HAPE (HA pulmonary oedema)-patients], 210 HAPE-f (HAPE-free controls) and 430 HLs (healthy Ladakhi highland natives). The genotypes of seven polymorphisms, rs1538664, rs479200, rs2486729, rs2790879, rs480902, rs2486736 and rs973252, differed significantly between HAPE-p and HAPE-f (P<0.008). The genotypes AA, TT, AA, GG, CC, AA and GG of rs1538664, rs479200, rs2486729, rs2790879, rs480902, rs2486736 and rs973252, prevalent in HAPE-p, were identified as risk genotypes and their counterpart homozygotes, prevalent in HLs, were identified as protective. EGLN1 expression was up-regulated 4.56-fold in HAPE-p (P=0.0084). The risk genotypes, their haplotypes and interacting genotypes were associated with up-regulated EGLN1 expression (P<0.05). Similarly, regression analysis showed that the risk alleles and susceptible haplotypes were associated with decreased SaO2 (arterial oxygen saturation) levels in the three groups. The significant inverse correlation of SaO2 levels with PASP (pulmonary artery systolic pressure) and EGLN1 expression and the association of these polymorphisms with SaO2 levels and EGLN1 expression contributed to uncovering the molecular mechanism underlying hypobaric hypoxic adaptation and maladaptation.

Journal Article, Research Support, Non-U.S. Gov't

Interactions among vascular-tone modulators contribute to high altitude pulmonary edema and augmented vasoreactivity in highlanders.

Ali Z, Mishra A, Kumar R, Alam P, Pandey P, Ram R, Thinlas T, Mohammad G, Pasha MA

PloS one · 2012

BACKGROUND: The interactions among various biomarkers remained unexplored under the stressful environment of high-altitude. Present study evaluated interactions among biomarkers to study susceptibility for high altitude pulmonary edema (HAPE) in HAPE-patients (HAPE-p) and adaptation in highland natives (HLs); both in comparison to HAPE-free sojourners (HAPE-f). METHODOLOGY/PRINCIPAL FINDINGS: All the subjects were recruited at 3500 m. We measured clinical parameters, biochemical levels in plasma and gene expression using RNA from blood; analyzed various correlations between and among the clinical parameters, especially arterial oxygen saturation (SaO(2)) and mean arterial pressure (MAP) and biochemical parameters like, asymmetric dimethylarginine (ADMA), serotonin (5-HT), 8-iso-prostaglandin F2α (8-isoPGF2α), endothelin-1 (ET-1), plasma renin activity (PRA), plasma aldosterone concentration (PAC), superoxide dismutase (SOD) and nitric oxide (NO) in HAPE-p, HAPE-f and HLs. ADMA, 5-HT, 8-isoPGF2α, ET-1 levels, and PAC were significantly higher (p<0.0001, each), whereas SOD activity and NO level were significantly lower in HAPE-p than HAPE-f (p ≤ 0.001). Furthermore, ADMA, 5-HT, 8-isoPGF2α, NO levels and PAC were significantly higher (p<0.0001), whereas ET-1 level significantly (p<0.0001) and SOD activity non-significantly (p>0.05) lower in HLs than HAPE-f. The expression of respective genes differed in the three groups. In the correlations, SaO(2) inversely correlated with ADMA, 5-HT and 8-isoPGF2α and positively with SOD in HAPE-p (p≤0.009). MAP correlated positively with 5-HT and 8-isoPGF2α in HAPE-p and HLs (p ≤ 0.004). A strong positive correlation was observed between ADMA and 5-HT, 5-HT and 8-isoPGF2α (p≤0.001), whereas inverse correlation of SOD with ET-1 in HAPE-p and HLs (p ≤ 0.004), with 5-HT and 8-isoPGF2α in HAPE-p (p = 0.01) and with 5-HT in HLs (p = 0.05). CONCLUSIONS/SIGNIFICANCE: The interactions among these markers confer enhanced vascular activity in HLs and HAPE in sojourners.

Journal Article, Research Support, Non-U.S. Gov't

CYBA and GSTP1 variants associate with oxidative stress under hypobaric hypoxia as observed in high-altitude pulmonary oedema.

Mishra A, Ali Z, Vibhuti A, Kumar R, Alam P, Ram R, Thinlas T, Mohammad G, Pasha MA

Clinical science (London, England : 1979) · 2012

HAPE (high-altitude pulmonary oedema) is characterized by pulmonary hypertension, vasoconstriction and an imbalance in oxygen-sensing redox switches. Excess ROS (reactive oxygen species) contribute to endothelial damage under hypobaric hypoxia, hence the oxidative-stress-related genes CYBA (cytochrome b-245 α polypeptide) and GSTP1 (glutathione transferase Pi 1) are potential candidate genes for HAPE. In the present study, we investigated the polymorphisms -930A/G and H72Y (C/T) of CYBA and I105V (A/G) and A114V (C/T) of GSTP1, individually and in combination, in 150 HAPE-p (HAPE patients), 180 HAPE-r (HAPE-resistant lowland natives) and 180 HLs (healthy highland natives). 8-Iso-PGF2α (8-iso-prostaglandin F2α) levels were determined in plasma and were correlated with individual alleles, genotype, haplotype and gene-gene interactions. The relative expression of CYBA and GSTP1 were determined in peripheral blood leucocytes. The genotype distribution of -930A/G, H72Y (C/T) and I105V (A/G) differed significantly in HAPE-p compared with HAPE-r and HLs (P≤0.01). The haplotypes G-C of -930A/G and H72Y (C/T) in CYBA and G-C and G-T of I105V (A/G) and A114V (C/T) in GSTP1 were over-represented in HAPE-p; in contrast, haplotypes A-T of -930A/G and H72Y (C/T) in CYBA and A-C of I105V (A/G) and A114V (C/T) in GSTP1 were over-represented in HAPE-r and HLs. 8-Iso-PGF2α levels were significantly higher in HAPE-p and in HLs than in HAPE-r (P=2.2×10(-16) and 1.2×10(-14) respectively) and the expression of CYBA and GSTP1 varied differentially (P<0.05). Regression analysis showed that the risk alleles G, C, G and T of -930A/G, H72Y (C/T), I105V (A/G) and A114V (C/T) were associated with increased 8-iso-PGF2α levels (P<0.05). Interaction between the two genes revealed over-representation of most of the risk-allele-associated genotype combinations in HAPE-p and protective-allele-associated genotype combinations in HLs. In conclusion, the risk alleles of CYBA and GSTP1, their haplotypes and gene-gene interactions are associated with imbalanced oxidative stress and, thereby, with high-altitude adaptation and mal-adaptation.

Journal Article, Research Support, Non-U.S. Gov't

CYP1A1, CYP1A2 and CYBA gene polymorphisms associated with oxidative stress in COPD.

Vibhuti A, Arif E, Mishra A, Deepak D, Singh B, Rahman I, Mohammad G, Pasha MA

Clinica chimica acta; international journal of clinical chemistry · 2010

BACKGROUND: The genetic susceptibility to chronic obstructive pulmonary disease (COPD) depends on detoxification and antioxidant enzymes, which detoxify cigarette smoke reactive components that, otherwise, generate oxidative stress. METHODS: In a case-control study of 346 subjects with and without COPD, we examined the polymorphisms 462Ile/Val, 3801T/C of CYP1A1, -3860G/A of CYP1A2 and -930A/G, 242C/T of CYBA individually or in combination and their contribution to oxidative stress markers by measuring malondialdehyde (MDA), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx). RESULTS: COPD patients had significantly increased MDA concentration (p<0.001) and decreased CAT activity, GSH concentration, GPx activity (p< or =0.01). The patients were over-represented by the alleles 462Val, 3801C of CYP1A1 and -930G, 242C of CYBA (p<0.001, p=0.003, p=0.030 and p=0.031, respectively) and consequently the haplotypes of same alleles i.e. 462Val:3801C, 462Val:3801T and -930G:242C (p=0.048, p=0.016 and p=0.039, respectively). Similarly, CYP1A1 and CYP1A2 haplotypes, 462Val:3860G and 462Val:3801T:3860G were significantly over-represented (p=0.001 and p=0.003), respectively in patients. The same alleles-associated genotype-combinations between genes were more prevalent in patients. Of note, the genotypes, 462Ile/Val+Val/Val, 3801TC+CC of CYP1A1 and -930AG+GG of CYBA associated with increased MDA concentration (p=0.018, p=0.045 and p=0.017, respectively), decreased CAT activity (p<0.0001, p=0.080 and p<0.0001, respectively) and GSH concentration (p<0.0001, p=0.0002 and p=0.011, respectively) in patients. CONCLUSION: The identified alleles, its haplotypes and the genotype-combination along with increased oxidative stress, signify the importance in susceptibility to COPD.