Biology subjects

Dhingra, R.

Publications and source records attributed to Dhingra, R..

10 recordsLinked to original sources

bioRxiv2023

Upregulated GRP78 and sFlt-1 in preeclampsia induces IRE1 and ATF6 arms of UPR leading to ER stress in placental cells

IntroductionAmongst vivid and diversified stresses which placenta in preeclampsia undergoes, ER stress has been a hal of the fame which is an insinuation of ill-fated UPR. In our previous study, we have already reported activation of PERK arm due to sFlt-1 present in preeclamptic mothers. The present study is an attempt to interpret rest of the two arms (IRE1 and ATF6) of UPR and ER stress subsequently by in vitro study using BeWo cells after upregulation of master regulator of UPR (GRP78) in placental tissue. Materials and MethodsPart I: Serum analysis of circulating levels of GRP78 and sFlt-1 [in 50 pairs of preeclamptic and normotensive, non-proteinuric (control) pregnant women)] using ELISA. Part II: Correlation analysis of levels of GRP78 and sFlt-1 in preeclamptic and control groups. Part III: Status of expression of GRP78 in placentae (n=10 each, preeclamptic and control groups) was reported using immunofluorescence. Part IV: In vitro experiments using BeWo cells were carried out to analyse the effect of GRP78 and sFlt-1 on IRE1 and ATF6 arms of UPR at protein (immunofluorescence and western blot) and transcript (qRT-PCR) levels. ResultsProtein expressions of GRP78 and sFlt-1 were found significantly elevated in preeclamptic patients as compared to controls. Enhanced expression of master regulator of UPR (GRP78) in placental tissue of preeclamptic women was reported. Up-regulated expression of XBP1 (IRE1 arm) and ATF6 markers of UPR was observed in trophoblast cells. ConclusionThe upregulated expression of GRP78 in preeclamptic placentae and enhanced expression of XBP1 and ATF6 markers in BeWo cells at both protein and transcript levels connote role played by raised circulating levels of GRP78 and sFlt-1 in preeclampsia.

developmental biology↗

bioRxiv2023

Abnormal trophoblast invasion in early onset preeclampsia: involvement of cystathionine β-synthase, specificity protein 1 and miR-22

IntroductionImpaired trophoblast invasion has been observed in early onset Preeclampsia patients (EOPE). Trophoblast cell invasion during human placentation is majorly regulated by the balance between MMPs 2, 9 and their inhibitors [tissue inhibitors of matrix metalloproteinases (TIMPs 1, 2)]. Exogenous NaHS (hydrogen sulphide donor) treatment was shown to significantly increase the expression levels of matrix metalloproteinases (MMPs 2, 9) in human bladder cancer EJ cells. Epigentically, the gene expression of hydrogen sulphide synthesising enzyme CBS (cystathionine {beta}-synthase) could be further regulated by various mi-RNAs via the transcription factors like Sp1. Specificity protein 1 (Sp1) has been identified as a target gene for miR-22 to regulate the invasion and metastasis of gastric cancer cells. However, the mechanism of MMPs regulation by either CBS, Sp1 and miRNA-22 in the pregnancies having EOPE is not known. Aims and ObjectivesTo determine and compare the expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp1 and miRNA-22 in EOPE patients and normotensive, non-proteinuric controls. Materials and methods100 pregnant women were enrolled from Department of Obstetrics and Gynaecology, AIIMS, New Delhi, India. EOPE women (n=50) after clinical diagnosis as per ACOG guidelines were enrolled as cases and normotensive, non-proteinuric pregnant women (n=50) were enrolled as controls. Protocol of the study was approved by Institute Ethics Committee, AIIMS, New Delhi. 5 ml of venous blood was collected from all the recruited women (2.5 ml in each EDTA and sera vial) followed by plasma and sera separation. Plasma samples were used subsequently to determine gene expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp1 and miRNA-22 by qRT-PCR and sera samples were used to estimate their protein levels by ELISA. Data were analyzed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly down regulated expression of MMPs 2, 9, CBS and Sp1 whereas up regulated expression for that of TIMPs 1, 2 was observed in EOPE patients as compared to healthy pregnant women at both transcription and translation levels. Expression of miR-22 (pre miR-22 and miR-22-3p) was found to be significantly elevated in EOPE patients as compared to normotensive, non-proteinuric controls. ConclusionThis is the very first study of its kind which implicates that down regulated MMPs 2, 9, CBS, Sp1 levels and simultaneously upregulation of miR-22 expression in EOPE patients could have some association. In vitro experiments are needed to prove their association which if proven may provide a new, potential therapeutic target to treat early onset Preeclampsia.

developmental biology↗

bioRxiv2023

MicroRNA-22, Specificity protein-1 and Cystathionine β-synthase in early onset Preeclampsia: significance in trophoblast invasion

IntroductionTrophoblast cell invasion during human placentation is majorly regulated by the balance between MMPs 2, 9 and their inhibitors [tissue inhibitors of matrix metalloproteinases (TIMPs 1, 2)]. Exogenous NaHS (hydrogen sulphide donor) treatment was shown to significantly increase the expression levels of matrix metalloproteinases (MMPs 2, 9) in human bladder cancer EJ cells. Epigentically, the gene expression of hydrogen sulphide synthesising enzyme CBS (cystathionine {beta}-synthase) could be further regulated by various mi-RNAs via the transcription factors like Sp1. Specificity protein 1 (Sp1) has been identified as a target gene for miR-22 to regulate the invasion and metastasis of gastric cancer cells. However, the mechanism of MMPs regulation by either CBS, Sp1 and miRNA-22 in the pregnancies having early onset preeclampsia is not known. Aim of the studyTo determine and compare the expression of MMPs 2, 9, TIMPs 1, 2, CBS, Sp-1 and miR-22 in early onset preeclamptic patients and normotensive, non-proteinuric controls at both transcription and translation levels. Materials and methods30 pregnant women were enrolled from Department of Obstetrics and Gynaecology, AIIMS, New Delhi, India. EOPE women (n=15) after clinical diagnosis as per ACOG guidelines were enrolled as cases and normotensive, non-proteinuric pregnant women (n=15) were enrolled as controls. Protocol of the study was approved by Institute Ethics Committee, AIIMS, New Delhi. 30 caesarean delivered placentae (15 each of patients and controls) were collected to analyse the mRNA and protein expression/levels of MMPs 2, 9, TIMPs 1, 2, CBS, and Sp1. Protein activity of MMP-2 and 9 was determined. Gene expression of miR-22 was observed in placentae of the recruited patients and controls. Data were analysed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly reduced mRNA and protein expression/levels of MMPs 2, 9, CBS and Sp1 whereas elevated for those of TIMP-1 and TIMP-2 was observed in EOPE patients as compared to controls. Gelatinolytic activity of MMP-2 and 9 was also found significantly reduced in placentae from EOPE patients whereas gene expression of miR-22 was found significantly up regulated in the early onset preeclamptic patients in comparison to controls. ConclusionThis is the first study of its kind which implicates that insufficient trophoblastic invasion may be because of down regulation of MMPs 2, 9, CBS, Sp1 and concomitant up regulation of TIMPs 1, 2 and miR-22 in the early onset preeclamptic patients as compared to controls.

developmental biology↗

bioRxiv2023

Role of MicroRNA-22 (miR-22)/Specificity protein 1 (Sp1)/Cystathionine beta-synthase (CBS) axis in regulating trophoblast invasion: an in vitro study

IntroductionPlacenta expresses many ubiquitous as well as specific miRNAs which regulate trophoblast cell differentiation, proliferation, apoptosis, invasion, migration, and angiogenesis. Aberrant miRNAs expression (miR-22) has been linked to pregnancy complications, such as preeclampsia. Specificity protein 1 (Sp1) was identified a direct target of miR-22 and an inverse linear correlation between expression of miR-22 and Sp1 mRNA in gastric tumour has been reported. Also, Sp1 has a critical and indispensable role in the regulation of cystathionine {beta}-synthase (H2S synthesizing enzyme). Exogenous H2S could promote cell proliferation and invasion by upregulating the expression of MMP-2 and MMP-9 in human bladder cancer EJ cells. Worldwide studies reported significant role of MMP-2 and MMP-9 in the regulation of trophoblast invasion. Aims and ObjectivesTo determine the role of MicroRNA-22 (miR-22)/Specificity protein 1 (Sp1)/Cystathionine {beta}-synthase (CBS) axis in regulating trophoblast invasion in the first-trimester trophoblast cells. Materials and methodsThe human first trimester trophoblast cell line (HTR-8/SVneo Cell line) was procured from American Type Culture Collection. Gain/Loss of function studies were conducted on HTR-8/SVneo cells and the expression of miR-22 (pre miR-22 and miR-22-3p), Sp1, CBS, MMP-2 and MMP-9 was observed. Invasion capacity of the cells was assessed by transwell invasion assay. Data were analyzed by STATA 14 and Graph Pad Prism 8. ResultsSignificantly down regulated regulated mRNA and protein expression of Sp1, CBS and and MMPs 2, 9 was observed when the cells were transfected with miR-22-3p mimic in comparison to miR-22-3p inhibitor transfected cells. Expression of CBS and MMPs 2, 9 was found to be significantly up regulated when the cells were transfected with Sp1 plasmid as compared to Sp1 inhibitor (2,4,5 trifluoroaniline) treated cells at both transcription and translation levels. MMP-2 and MMP-9 levels were found to be significantly elevated when the cells were subjected to NaHS [sodium hydrogen sulphide (H2S donor)] treatment as compared to AOAA (aminooxyacetic acid) treated cells. ConclusionThis is the first study which implies that miR-22 via targeting Sp1 must have influenced the mRNA and protein expression of CBS, MMP-2 and MMP-9 in order to regulate the trophoblast invasion.

developmental biology↗

bioRxiv2023

Altered expression of MMP9 and TIMP1 leads to impaired placentation in early onset preeclamptic patients: a case control study

IntroductionMatrix metalloproteinases (MMPs) specifically MMP-9 is a key regulator of vascular and uterine spiral artery remodelling and its activity is controlled at multiple levels, including gene transcription, activation of its latent forms and endogenous inhibition by tissue inhibitors of metalloproteinases [TIMPs (specifically TIMP-1)]. Alteration in MMP-9 and TIMP-1 expression may contribute to uterine and vascular dysfunction leading to adverse pregnancy outcomes such as Preeclampsia (PE). Depending on time, PE is classified as early-onset preeclampsia, which is diagnosed before 34 weeks of gestation, or late-onset preeclampsia, which is diagnosed after 34 weeks. Early-onset preeclampsia (EOPE) is a severe obstetrics disease which threatens mother and foetus. Therefore, in the present study, we aimed to investigate the levels of MMP-9 and its inhibitor TIMP-1 in the placentae of EOPE patients and their maternal age matched normotensive, non-proteinuric controls at both mRNA and protein levels. MethodsA total of 30 caesarean delivered placentae (15 EOPE patients and 15 controls) were collected from Department of Obstetrics and Gynaecology, AIIMS, New Delhi after taking permission from Institute Ethics Committee. MMP-9 and TIMP-1 protein expression was observed by immunohistochemistry and immunofluorescence stainings. Gelatin Gel Zymography was done to evaluate MMP-9 activity and Western Blot was done for the quantification of MMP-9 and TIMP-1 proteins. mRNA levels of MMP-9 and TIMP-1 were determined by qRT-PCR ResultsImmunohistochemistry and Immunofluorescence staining discerned stronger expression of MMP-9 in normotensive placentae as compared to EOPE placentae whereas stronger expression of TIMP-1 was seen in EOPE placentae in comparison to those of normotensive placentae. Gelatin Gel Zymography reflected that MMP-9 activity was found elevated in maternal placental side in normotensive placentae as compared to EOPE placentae. Western Blot analysis revealed that protein expression of MMP-9 was elevated whereas for that of TIMP-1 was reduced in normotensive placentae in comparison to EOPE placentae. mRNA expression of MMP-9 was found up-regulated whereas for that of TIMP-1 was down-regulated in normotensive placentae as compared to EOPE placentae. ConclusionThe present study implies that aberrant functioning of MMP-9 and TIMP-1 in EOPE patients contribute to impaired placentation which might be relevant for possible future screening programs in order to predict and to design therapies for early onset preeclamptic patients.

developmental biology↗

bioRxiv2020

Neighborhood environment, social cohesion, and epigenetic aging

Living in adverse neighborhood environments have been linked to increased risk of aging-related diseases and mortality; however, the biological mechanisms explaining this observation remain poorly understood. DNA methylation (DNAm), a proposed biomarker of biological aging responsive to environmental stressors, offers promising insight into molecular pathways. We examined associations of three measures of neighborhood conditions (poverty, quality, and social cohesion) with three different epigenetic clocks (Horvath, Hannum, and Levine) using data from the Detroit Neighborhood Health Study (n=158). Using linear regression models, we evaluated associations in the total sample and stratified by gender and social cohesion. Differential effects by gender were found between men and women. Neighborhood poverty was associated with PhenoAge acceleration among women, but not among men (women: {beta} = 1.4; 95% CI: -0.4, 3.3 vs. men: {beta} = -0.3; 95% CI: -2.2, 1.5) in fully adjusted models. In models stratified on social cohesion, association of neighborhood poverty and quality with accelerated DNAm aging remained elevated for residents living in neighborhoods with lower social cohesion, but were null for those living in neighborhoods with higher social cohesion. Our study suggests that living in adverse neighborhood conditions can speed up epigenetic aging, while positive neighborhood characteristics may buffer effects.

genomics↗

bioRxiv2018

Evaluating DNA methylation age on the Illuminas methylationEPIC BeadChip

DNA methylation age (DNAm age) has become a widely utilized epigenetic biomarker for the aging process. The Horvath method for determining DNAm age is perhaps the most widely utilized and validated DNA methylation age assessment measure. Horvath DNAm age is calculated based on methylation measurements at 353 loci which were present on Illuminas 450k and 27k DNA methylation microarrays. With the increasing use of the more recently developed Illumina MethylationEPIC (850k) microarray, it is worth revisiting this widely used aging measure to evaluate differences in DNA methylation age estimation based on array design. Of the requisite 353 loci, 17 are missing from the current 850k microarray. Using 17 datasets with 27k, 450k, and/or 850k methylation data, we calculated and compared each samples epigenetic age estimated from all 353 loci required from the Horvath DNAm age calculator (full), and using only the 336 loci present on the 27k, 450k, and 850k arrays (reduced). In 450k/27k data, missing loci caused underestimation of epigenetic age when compared with the full clock. Underestimation of full epigenetic age grew from ages 0 to ~20, remaining stable thereafter (mean= -3.46 y, SD=1.13) years for individuals [≥]20 years. Underestimation of DNAm age by the reduced 450k/27k data was similar to the underestimation observed in the 850k data indicating that array differences in DNAm age estimation are primarily driven by missing probes. Correlations between age and DNAm age were not dependent on missing probes or on array designs and consequently associations between DNAm age and outcomes such as sex remained the same independent of missing probes and probe design. In conclusion, DNAm age estimations are array dependent driven by missing probes between arrays. Though correlations and associations with DNAm age may remain the same, researchers should exercise caution when interpreting results based on absolute differences in DNAm age or when mixing samples assayed on different arrays.\n\nDisclaimerThis paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. EPA, and is approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The authors declare they have no competing financial interests.

bioinformatics↗

bioRxiv2018

Direct Small Molecule Activation of Mitofusins

Mitochondrial fusion is a physiological process that is regulated by mitofusins on the outer mitochondrial membrane. Conformational plasticity between anti- and pro-tethering conformations of mitofusins permits mitochondrial tethering and subsequent fusion. Here we developed a pharmacophore-based model to rationally manipulate the conformational plasticity of mitofusin 2 and perfomed an in silico small-molecule screen. This enabled the discovery of a direct activator of mitofusins, MASM7, capable of potently promoting mitochondrial fusion. The specificity of the MASM7-mitofusin 2 interaction is highlighted by structure-activity relationships of MASM7 analogues, FRET, NMR and mitochondrial fusion studies using mitofusin mutants. Our study identified the first-in-class direct activator of mitofusins, demonstrating a new paradigm for chemical modulation of mitochondrial fusion and downstream processes.

biochemistry↗

bioRxiv2018

sFlt-1 commutes unfolded protein response into endoplasmic reticulum stress in trophoblast cells in preeclamptic pregnancies

Preeclampsia (PE) and its subtypes (early and late onset) are serious concerns all across the globe affecting about 8% of total pregnancies and accounts for approximately 60,000 deaths annually with a predominance in developing under-developed and countries. The two-stage model in the progression of this disease, deficient spiral artery remodelling and an imbalance between angiogenic (VEGF) and anti-antigenic factor(s) (sFlt-1) are well established facts pertaining to this disease. The presence of increased sFlt-1, high oxidative stress and Endoplasmic reticulum stress (ER stress) have been proposed in preeclamptic pregnancies. Recently, the role of endoplasmic reticulum stress in the onset of the variant forms of PE highlighted a new window to explore further. In our previous studies, we demonstrated that sFlt-1 can induce apoptosis and oxidative stress in trophoblast cells. However the role of sFlt-1, in inducing ER stress is not known so far. In the present study, we for the first time demonstrated significant ER stress in the placental cells (BeWo Cells) (in vitro) when exposed to sera from preeclamptic pregnancies having increased concentration of sFlt-1. The expression of ER stress markers (GRP78, eIF2, XBP1, ATF6 and CHOP) at both transcript and protein levels were compared (between preeclamptic and normotensive non-proteinuric women) at three different time points (8h, 14h and 24hrs), analyzed and found to be significant (p<0.05).\n\nConclusionOur results suggested that sFlt-1, released from placental cells in preeclampsia may be one of the various factors having potential to induce endoplasmic reticulum stress in BeWo cells.

developmental biology↗

bioRxiv2017

sFlt-1 (sVEGFR1) induces placental endoplasmic reticulum stress in trophoblast cell: implications for the complications in preeclampsia - an in vitro study

BackgroundThe concentration of sFlt-1, a major anti-angiogenic protein in maternal circulation has been seen to be raised in preeclamptic pregnancies. Endoplasmic reticulum (ER) stress represents one of the three (immunological, oxidative and ER stress) major stresses which placenta undergoes during pregnancies. The present study is designed to investigate the role of sFlt-1 in induction of ER stress in trophoblast cells.\n\nMaterials and MethodsMaternal serum levels of anti-angiogenic protein sFlt-1 and central regulator of unfolded protein response GRP78 was measured using sandwich ELISA. The expression of various ER stress markers (GRP78, eIF2, XBP1, ATF6 and apoptotic protein CHOP) were analyzed depending on various treatments given to the trophoblast cells using Immunofluorescence, western blot and q-RT PCR.\n\nResultsIncreased expression of ER stress markers (GRP78, eIF2, XBP1 ATF6 and apoptotic protein CHOP) was detected in the placental trophoblast cells treated with raised concentration of sFlt-1.\n\nConclusionSignificant upregulated expression of ER stress markers in trophoblast cells exposed with increased concentration of sFlt-1 suggested that it may be one of the anti-angiogenic factors present in maternal sera which not only contributes to oxidative stress but also may cause endoplasmic reticulum stress.

developmental biology↗