MicroRNA-126 contributes to renal microvascular heterogeneity of VCAM-1 protein expression in acute inflammation

2012 ◽  
Vol 302 (12) ◽  
pp. F1630-F1639 ◽  
Author(s):  
S. A. Ásgeirsdóttir ◽  
C. van Solingen ◽  
N. F. Kurniati ◽  
P. J. Zwiers ◽  
P. Heeringa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Molecular Control ◽  
Protein Levels ◽  
Tnf Α ◽  
Inflammatory Stimuli ◽  
Endothelial Cell Adhesion Molecules ◽  
Differential Responsiveness

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291–299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.

Mechanisms of MAdCAM-1 gene expression in human intestinal microvascular endothelial cells

2005 ◽  
Vol 288 (2) ◽  
pp. C272-C281 ◽  
Author(s):  
Hitoshi Ogawa ◽  
David G. Binion ◽  
Jan Heidemann ◽  
Monica Theriot ◽  
Pamela J. Fisher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Primary Cultures ◽  
Microvascular Endothelial Cells ◽  
Cellular Density ◽  
Tnf Α ◽  
Gene And Protein Expression ◽  
Microvascular Endothelial

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a homing receptor preferentially expressed on gut-associated endothelial cells that plays a central role in leukocyte traffic into the mucosal immune compartment. Although the molecular mechanisms underlying endothelial ICAM-1 or E-selectin expression have been intensively investigated, the mechanisms that regulate human MAdCAM-1 expression have not been defined. We report MAdCAM-1 gene and protein expression in primary cultures of human intestinal microvascular endothelial cells (HIMEC) that was not demonstrated in human umbilical vein endothelial cells. Similar to ICAM-1 and E-selectin expression, MAdCAM-1 gene expression in HIMEC was inducible with TNF-α, IL-1β, or LPS activation. However, in striking contrast to ICAM-1 and E-selectin expression, MAdCAM-1 mRNA and protein expression in HIMEC was heavily dependent on culture duration and/or cellular density, suggesting a prominent role for cell-cell interaction among these endothelial cells in the expression of the mucosal addressin. MAdCAM-1 expression was inhibited by both SN-50 (NF-κB inhibitor) and LY-294002 [phosphatidylinositol 3-kinase (PI3-K) inhibitor], whereas ICAM-1 and E-selectin expression was inhibited by SN-50 but not by LY-294002. The Akt phosphorylation by TNF-α or LPS was greater at higher cell density, demonstrating a pattern similar to that of MAdCAM-1 expression. NF-κB activation was not affected by cellular density in HIMEC. MAdCAM-1 expression in human gut endothelial cells is regulated by distinct signaling mechanisms involving both NF-κB and PI3-K/Akt. These data also suggest that PI3-K/Akt is involved in the gut-specific differentiation of HIMEC, which results in expression of the mucosal addressin MAdCAM-1.

scholarly journals Target prediction of anti-chronic liver injury of Kangxian pill based on network pharmacology

2021 ◽  
Author(s):  
Min Cao ◽  
Yiyang Wang ◽  
Haizhao Liu ◽  
Xueqian Dong ◽  
Mengxue Dong ◽  
...  
Keyword(s):  
Liver Injury ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Target Prediction ◽  
Vital Role ◽  
Chronic Liver ◽  
Network Pharmacology ◽  
Chronic Liver Injury ◽  
Tnf Α

Abstract BackgroundThe present study aimed to validate the protect effect of Kangxian pill (KXP) on chronic hepatic injury (CHI) and investigate its potential mechanism by network pharmacology-based prediction and experimental verification in vivo . MethodsThe effect of KXP in the treatment of carbon tetrachloride (CCL 4 )-induced CHI is investigated by calculating liver index, measuring AST and ALT levels and performing HE staining. Targets of active ingredients of KXP were predicted in TCSMP and targets of chronic liver injury were searched in DisGeNET, OMIM and GeneCards databases. We obtain some pivotal targets of KXP for the treatment of CHI by intersecting the targets of KXP and CHI. Subsequently, we performed gene ontology (GO) functional and pathways enrichment analyses, as well as conducted networks based on potential targets to determine the core targets and representative pathways.We further validated expressions of IL-6, IL-1β, TNF-α, Bax, Bcl2, PI3K, Akt, and pAkt according to the potential molecular mechanisms analyzed based on network pharmacology analysis.ResultsThe results showed that the levels of AST and ALT in serum decreased after treatment with KXP. HE staining also revealed that KXP could improve hepatocyte abnormality in vivo . A total of 81 potential targets of KXP in the treatment of CHI were identified through network pharmacology analysis. After integrating potential targets, function enrichment, representative pathways and networks, we identified PI3K, AKT1,BCL2, TNF-α, IL-1β, and IL-6 as potential targets, which may play a vital role in the KXP treatment. The experimental results also showed that KXP could down-regulate the mRNA and protein expression of IL-1β, IL-6, TNF-α and Bax, and up-regulate the PI3K and p-Akt protein expression i n vivo .ConclusionsOur results suggest that KXP could alleviate CHI through regulating inflammation and apoptosis and provide deep insight into the hepato-protective mechanisms.

scholarly journals CD31 signals confer immune privilege to the vascular endothelium

2015 ◽  
Vol 112 (43) ◽  
pp. E5815-E5824 ◽  
Author(s):  
Kenneth Cheung ◽  
Liang Ma ◽  
Guosu Wang ◽  
David Coe ◽  
Riccardo Ferro ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Vascular Endothelial Cells ◽  
Forkhead Transcription Factor ◽  
Extrinsic Pathway ◽  
Immune Mediated ◽  
Tnf Α ◽  
Inflammatory Stimuli ◽  
Caspase 7

Constitutive resistance to cell death induced by inflammatory stimuli activating the extrinsic pathway of apoptosis is a key feature of vascular endothelial cells (ECs). Although this property is central to the maintenance of the endothelial barrier during inflammation, the molecular mechanisms of EC protection from cell-extrinsic, proapoptotic stimuli have not been investigated. We show that the Ig-family member CD31, which is expressed by endothelial but not epithelial cells, is necessary to prevent EC death induced by TNF-α and cytotoxic T lymphocytes in vitro. Combined quantitative RT-PCR array and biochemical analysis show that, upon the engagement of the TNF receptor with TNF-α on ECs, CD31 becomes activated and, in turn, counteracts the proapoptotic transcriptional program induced by TNF-α via activation of the Erk/Akt pathway. Specifically, Akt activation by CD31 signals prevents the localization of the forkhead transcription factor FoxO3 to the nucleus, thus inhibiting transcription of the proapoptotic genes CD95/Fas and caspase 7 and de-repressing the expression of the antiapoptotic gene cFlar. Both CD31 intracellular immunoreceptor tyrosine-based inhibition motifs are required for its prosurvival function. In vivo, CD31 gene transfer is sufficient to recapitulate the cytoprotective mechanisms in CD31− pancreatic β cells, which become resistant to immune-mediated rejection when grafted in fully allogeneic recipients.

scholarly journals Inflammation induces lymphangiogenesis through up-regulation of VEGFR-3 mediated by NF-κB and Prox1

Blood ◽  
2010 ◽  
Vol 115 (2) ◽  
pp. 418-429 ◽  
Author(s):  
Michael J. Flister ◽  
Andrew Wilber ◽  
Kelly L. Hall ◽  
Caname Iwata ◽  
Kohei Miyazono ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Lymphatic Vessels ◽  
Growth Factor Receptor ◽  
Receptor Expression ◽  
Lymphatic Endothelial Cells ◽  
Inflammatory Stimuli ◽  
And Migration

Abstract The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-κB) family; however, the role of NF-κB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-κB pathway. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-κB followed by sequential up-regulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-κB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells, resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-κB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-κB pathway by inflammatory stimuli activates Prox1, and both NF-κB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of preexisting lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

scholarly journals Novel Treatment of Hypertension by Specifically Targeting E2F for Restoration of Endothelial Dihydrofolate Reductase and eNOS Function Under Oxidative Stress

Hypertension ◽  
2019 ◽  
Vol 73 (1) ◽  
pp. 179-189 ◽  
Author(s):  
Hong Li ◽  
Qiang Li ◽  
Yixuan Zhang ◽  
Wenting Liu ◽  
Bo Gu ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Endothelial Cells ◽  
Protein Expression ◽  
Dihydrofolate Reductase ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Therapeutic Option ◽  
Protein Levels ◽  
Treatment Of Hypertension ◽  
Mrna And Protein Expression

We have shown that hydrogen peroxide (H 2 O 2 ) downregulates tetrahydrobiopterin salvage enzyme DHFR (dihydrofolate reductase) to result in eNOS (endothelial NO synthase) uncoupling and elevated blood pressure. Here, we aimed to delineate molecular mechanisms underlying H 2 O 2 downregulation of endothelial DHFR by examining transcriptional pathways hypothesized to modulate DHFR expression and effects on blood pressure regulation of targeting these novel mechanisms. H 2 O 2 dose and time dependently attenuated DHFR mRNA and protein expression and enzymatic activity in endothelial cells. Deletion of E2F-binding sites, but not those of Sp1 (specificity protein 1), abolished H 2 O 2 attenuation of DHFR promoter activity. Overexpression of E2F1/2/3a activated DHFR promoter at baseline and alleviated the inhibitory effect of H 2 O 2 on DHFR promoter activity. H 2 O 2 treatment diminished mRNA and protein expression of E2F1/2/3a, whereas overexpression of E2F isoforms increased DHFR protein levels. Chromatin immunoprecipitation assay indicated direct binding of E2F1/2/3a to the DHFR promoter, which was weakened by H 2 O 2 . E2F1 RNA interference attenuated DHFR protein levels, whereas its overexpression elevated tetrahydrobiopterin levels and tetrahydrobiopterin/dihydrobiopterin ratios in vitro and in vivo. In Ang II (angiotensin II)–infused mice, adenovirus-mediated overexpression of E2F1 markedly abrogated blood pressure to control levels, by restoring endothelial DHFR function to improve NO bioavailability and vasorelaxation. Bioinformatic analyses confirmed a positive correlation between E2F1 and DHFR in human endothelial cells and arteries, and downregulation of both by oxidized phospholipids. In summary, endothelial DHFR is downregulated by H 2 O 2 transcriptionally via an E2F-dependent mechanism, and that specifically targeting E2F1/2/3a to restore DHFR and eNOS function may serve as a novel therapeutic option for the treatment of hypertension.

scholarly journals Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qilu Wei ◽  
Ning Kong ◽  
Xiaohui Liu ◽  
Run Tian ◽  
Ming Jiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fast Green ◽  
Expression Levels ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Safranin O ◽  
Tgf Β1

Abstract Background Osteoarthritis (OA) is a disease of the entire joint involving synovial fibrosis and inflammation. Pathological changes to the synovium can accelerate the progression of OA. Pirfenidone (PFD) is a potent anti-fibrotic drug with additional anti-inflammatory properties. However, the influence of PFD on OA is unknown. Methods Proliferation of human fibroblast-like synoviocytes (FLSs) after treatment with TGF-β1 or PFD was evaluated using a Cell Counting Kit-8 assay and their migration using a Transwell assay. The expression of fibrosis-related genes (COL1A1, TIMP-1, and ACTA-2) and those related to inflammation (IL-6 and TNF-α) was quantified by real-time quantitative PCR. The protein expression levels of COL1A1, α-SMA (coded by ACTA-2), IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. A rabbit model of OA was established and then PFD was administered by gavage. The expression of genes related to fibrosis (COL1A1, TIMP-1, and ADAM-12) and inflammation (IL-6 and TNF-α) was measured using RNA extracted from the synovium. Synovial tissue was examined histologically after staining with H&E, Masson’s trichrome, and immunofluorescence. Synovitis scores, the volume fraction of collagen, and mean fluorescence intensity were calculated. Degeneration of articular cartilage was analyzed using a Safranin O-fast green stain and OARSI grading. Results The proliferation of FLSs was greatest when induced with 2.5 ng/ml TGF-β1 although it did not promote their migration. Therefore, 2.5 ng/ml TGF-β1 was used to stimulate the FLSs and evaluate the effects of PFD, which inhibited the migration of FLSs at concentrations as low as 1.0 mg/ml. PFD decreased the expression of COL1A1 while TGF-β1 increased both mRNA and protein expression levels of IL-6 but had no effect on α-SMA or TNF-α expression. PFD decreased mRNA expression levels of COL1A1, IL-6, and TNF-α in vivo. H&E staining and synovitis scores indicated that PFD reduced synovial inflammation, while Masson’s trichrome and immunofluorescence staining suggested that PFD decreased synovial fibrosis. Safranin O-Fast Green staining and the OARSI scores demonstrated that PFD delayed the progression of OA. Conclusions PFD attenuated synovial fibrosis and inflammation, and postponed the progression of osteoarthritis in a modified Hulth model of OA in rabbits, which was related to its anti-fibrotic and anti-inflammatory properties.

Expression of P-selectin on Endothelial Cells is Upregulated by LPS and TNF-α in Vivo

1994 ◽  
Vol 2 (1) ◽  
pp. 7-14 ◽  
Author(s):  
Ursula Gotsch ◽  
Ute Jäger ◽  
Mara Dominis ◽  
Dietmar Vestweber
Keyword(s):  
Endothelial Cells ◽  
Tnf Α

Abstract 116: Arterial Endothelial Cell Has Stronger Angiogenic Potential Compared To Venous Endothelial Cell Through Up-regulation Of Endogenous FGF2 And FGF5 Expression In 3D Microfluidic System

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Ha-Rim Seo ◽  
Hyo Eun Jeong ◽  
Hyung Joon Joo ◽  
Seung-Cheol Choi ◽  
Jong-Ho Kim ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Molecular Mechanisms ◽  
Umbilical Vein ◽  
Assay System ◽  
Vascular Density ◽  
Angiogenic Potential ◽  
Angiogenesis Assay

Background: Human body contains many kinds of different type of endothelial cells (EC). However, cellular difference of their angiogenic potential has been hardly understood. We compared in vitro angiogenic potential between arterial EC and venous EC and investigated its underlying molecular mechanisms. Method: Used human aortic endothelial cells (HAEC) which was indicated from arterial EC and human umbilical vein endothelial cells (HUVEC) indicated from venous EC. To explore angiogenic potential in detail, we adopted a novel 3D microfluidic angiogenesis assay system, which closely mimic in vivo angiogenesis. Results: In 3D microfluidic angiogenesis assay system, HAEC demonstrated stronger angiogenic potential compared to HUVEC. HAEC maintained its profound angiogenic property under different biophysical conditions. In mRNA microarray sorted on up- regulated or down-regulated genes, HAEC demonstrated significantly higher expression of gastrulation brain homeobox 2 (GBX2), fibroblast grow factor 2 (FGF2), FGF5 and collagen 8a1. Angiogenesis-related protein assay revealed that HAEC has higher secretion of endogenous FGF2 than HUVEC. HAEC has only up-regulated FGF2 and FGF5 in this part of FGF family. Furthermore, FGF5 expression under vascular endothelial growth factor-A (VEGF-A) stimulation was higher in HAEC compared to HUVEC although VEGF-A augmented FGF5 expression in both HAEC and HUVEC. Those data suggested that FGF5 expression in both HAEC and HUVEC is partially dependent to VEGF-A stimulate. HUVEC and HAEC reduced vascular density after FGF2 and FGF5 siRNA treat. Conclusion: HAEC has stronger angiogenic potential than HUVEC through up-regulation of endogenous FGF2 and FGF5 expression

Abstract 531: Tie-2/Cre--Mediated Conditional Deletion of Lipid Phosphate Phosphatase-3 Reveals Its Role in Endothelial Cell Apoptosis

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Ishita Chatterjee ◽  
Kishore K Wary
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Genome Wide Association Study ◽  
Vascular Endothelial Cell ◽  
Protein Levels ◽  
Conditional Deletion ◽  
Transmission Electron ◽  
Increased Risk

Rationale: A recent genome-wide association study (GWAS) has linked a frequently occurring variation in the LPP3 (also known as PPAP2b) loci to increased risk of coronary heart disease (CAD). However, the in vivo function of LPP3 in vascular endothelial cell is incompletely understood. Goal: To address the endothelial cell (EC) specific function of Lpp3 in mice. Results: Tie-2/Cre mediated Lpp3 deletion did not affect normal vasculogenesis in early embryonic development, in contrast, in late embryonic stages it led to impaired angiogenesis associated with hemorrhage, edema and late embryonic lethal phenotype. Immunohistochemical staining followed by microscopic analyses of mutant embryos revealed reduced fibronectin and VE-cadherin expression throughout different vascular bed, and increased apoptosis in CD31+ vascular structures. Transmission electron microscopy (TEM) showed the presence of apoptotic endothelial cells and disruption of adherens junctions in mutant embryos. LPP3-knockdown in vitro showed an increase in p53 and p21 protein levels, with concomitant decrease in cell proliferation. LPP3-knockdown also decreased transendothelial electrical resistance (TER), interestingly re-expression of ß-catenin cDNA into LPP3-depleted endothelial cells partially restored the effect of loss of LPP3. Conclusion: These results suggest the ability of LPP3 to regulate survival and apoptotic activities of endothelial cells during patho/physiological angiogenesis.

scholarly journals Knockdown of Long Non-Coding RNA XIST Inhibited Doxorubicin Resistance in Colorectal Cancer by Upregulation of miR-124 and Downregulation of SGK1

2018 ◽  
Vol 51 (1) ◽  
pp. 113-128 ◽  
Author(s):  
Jia Zhu ◽  
Rui Zhang ◽  
Dongxiang Yang ◽  
Jibin Li ◽  
Xiaofei Yan ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Western Blot ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Glutathione S Transferase ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Ic50 Value

Background/Aims: Doxorubicin (DOX) is a widely used chemotherapeutic agent for colorectal cancer (CRC). However, the acquirement of DOX resistance limits its clinical application for cancer therapy. Mounting evidence has suggested that aberrantly expressed lncRNAs contribute to drug resistance of various tumors. Our study aimed to explore the role and molecular mechanisms of lncRNA X-inactive specific transcript (XIST) in chemoresistance of CRC to DOX. Methods: The expressions of XIST, miR-124, serum and glucocorticoid-inducible kinase 1 (SGK1) mRNA in DOX-resistant CRC tissues and cells were detected by qRT-PCR or western blot analysis. DOX sensitivity was assessed by detecting IC50 value of DOX, the protein levels of P-glycoprotein (P-gp) and glutathione S-transferase-π (GST-π) and apoptosis. The interactions between XIST, miR-124 and SGK1 were confirmed by luciferase reporter assay, qRT-PCR and western blot. Xenograft tumor assay was used to verify the role of XIST in DOX resistance in CRC in vivo. Results: XIST expression was upregulated and miR-124 expression was downregulated in DOX-resistant CRC tissues and cells. Knockdown of XIST inhibited DOX resistance of CRC cells, as evidenced by the reduced IC50 value of DOX, decreased P-gp and GST-π levels and enhanced apoptosis in XIST-silenced DOX-resistant CRC cells. Additionally, XIST positively regulated SGK1 expression by interacting with miR-124 in DOX-resistant CRC cells. miR-124 suppression strikingly reversed XIST-knockdown-mediated repression on DOX resistance in DOX-resistant CRC cells. Moreover, SGK1-depletion-elicited decrease of DOX resistance was greatly restored by XIST overexpression or miR-124 inhibition in DOX-resistant CRC cells. Furthermore, XIST knockdown enhanced the anti-tumor effect of DOX in CRC in vivo. Conclusion: XIST exerted regulatory function in resistance of DOX possibly through miR-124/SGK1 axis, shedding new light on developing promising therapeutic strategy to overcome chemoresistance in CRC patients.

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