Fli1 Deficiency Induces CXCL6 Expression in Dermal Fibroblasts and Endothelial Cells, Contributing to the Development of Fibrosis and Vasculopathy in Systemic Sclerosis

2017 ◽  
Vol 44 (8) ◽  
pp. 1198-1205 ◽  
Author(s):  
Takashi Taniguchi ◽  
Yoshihide Asano ◽  
Kouki Nakamura ◽  
Takashi Yamashita ◽  
Ryosuke Saigusa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Systemic Sclerosis ◽  
Mrna Expression ◽  
Target Genes ◽  
Critical Role ◽  
Dermal Fibroblasts ◽  
Vascular Involvement ◽  
Digital Ulcers ◽  
Mrna Levels ◽  
Predisposing Factor

Objective.CXCL6, a chemokine with proangiogenic property, is reported to be involved in vasculopathy associated with systemic sclerosis (SSc). We investigated the contribution of CXCL6 to SSc development by focusing on the association of friend leukemia virus integration 1 (Fli1) deficiency, a potential predisposing factor of SSc, with CXCL6 expression and clinical correlation of serum CXCL6 levels.Methods.mRNA levels of target genes and the binding of Fli1 to the CXCL6 promoter were evaluated by quantitative reverse transcription-PCR and chromatin immunoprecipitation, respectively. Serum CXCL6 levels were determined by ELISA.Results.FLI1 siRNA significantly enhanced CXCL6 mRNA expression in human dermal fibroblasts and human dermal microvascular endothelial cells, while Fli1 haploinsufficiency significantly suppressed CXCL6 mRNA expression in murine peritoneal macrophages stimulated with lipopolysaccharide. Supporting a critical role of Fli1 deficiency to induce SSc-like phenotypes, CXCL6 mRNA expression was higher in SSc dermal fibroblasts than in normal dermal fibroblasts. Importantly, Fli1 bound to the CXCL6 promoter in dermal fibroblasts, endothelial cells, and THP-1 cells. In patients with SSc, serum CXCL6 levels correlated positively with the severity of dermal and pulmonary fibrosis and were elevated in association with cardiac and pulmonary vascular involvement and cutaneous vascular symptoms, including Raynaud phenomenon, digital ulcers (DU)/pitting scars, and telangiectasia. Especially, serum CXCL6 levels were associated with DU/pitting scars and heart involvement by multiple regression analysis.Conclusion.CXCL6 expression is upregulated by Fli1 deficiency in fibroblasts and endothelial cells, potentially contributing to the development of fibrosis and vasculopathy in the skin, lung, and heart of SSc.

scholarly journals Endothelial CCR6 expression due to FLI1 deficiency contributes to vasculopathy associated with systemic sclerosis

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Tetsuya Ikawa ◽  
Takuya Miyagawa ◽  
Yuki Fukui ◽  
Satoshi Toyama ◽  
Jun Omatsu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Systemic Sclerosis ◽  
Vascular Permeability ◽  
Dermal Fibroblasts ◽  
Microvascular Endothelial Cells ◽  
Clinical Samples ◽  
Mrna Levels ◽  
Angiogenic Activity ◽  
Microvascular Endothelial

Abstract Background We have recently demonstrated that serum CCL20 levels positively correlate with mean pulmonary arterial pressure in patients with systemic sclerosis (SSc). Considering a proangiogenic effect of CCL20 on endothelial cells via CCR6, the CCL20/CCR6 axis may contribute to the development of SSc vasculopathy. Therefore, we explored this hypothesis using clinical samples, cultured cells, and murine SSc models. Methods The expression levels of CCL20 and CCR6 in the skin, mRNA levels of target genes, and the binding of transcription factor FLI1 to the target gene promoter were evaluated by immunostaining, quantitative reverse transcription PCR, and chromatin immunoprecipitation, respectively. Vascular permeability was evaluated by Evans blue dye injection in bleomycin-treated mice. Angiogenic activity of endothelial cells was assessed by in vitro angiogenesis assay. Results CCL20 expression was significantly elevated in dermal fibroblasts of patients with early diffuse cutaneous SSc, while CCR6 was significantly up-regulated in dermal small vessels of SSc patients irrespective of disease subtypes and disease duration. In human dermal microvascular endothelial cells, FLI1 siRNA induced the expression of CCR6, but not CCL20, and FLI1 bound to the CCR6 promoter. Importantly, vascular permeability, a representative SSc-like vascular feature of bleomycin-treated mice, was attenuated by Ccr6 siRNA treatment, and CCR6 siRNA suppressed the angiogenic activity of human dermal microvascular endothelial cells assayed by in vitro tube formation. Conclusions The increased expression of endothelial CCR6 due to FLI1 deficiency may contribute to the development of SSc vasculopathy.

scholarly journals Endothelial CCR6 Expression Due to FLI1 Deficiency Contributes to Vasculopathy Associated with Systemic Sclerosis

2021 ◽  
Author(s):  
Tetsuya Ikawa ◽  
Takuya Miyagawa ◽  
Yuki Fukui ◽  
Satoshi Toyama ◽  
Jun Omatsu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Systemic Sclerosis ◽  
Vascular Permeability ◽  
Dermal Fibroblasts ◽  
Microvascular Endothelial Cells ◽  
Clinical Samples ◽  
Mrna Levels ◽  
Angiogenic Activity ◽  
Microvascular Endothelial

Abstract Background: We have recently demonstrated that serum CCL20 levels positively correlate with mean pulmonary arterial pressure in patients with systemic sclerosis (SSc). Considering a proangiogenic effect of CCL20 on endothelial cells via CCR6, the CCL20/CCR6 axis may contribute to the development of SSc vasculopathy. Therefore, we explored this hypothesis using clinical samples, cultured cells and murine SSc models.Methods: The expression levels of CCL20 and CCR6 in the skin, mRNA levels of target genes and the binding of transcription factor FLI1 to the target gene promoter were evaluated by immunostaining, quantitative reverse transcription PCR and chromatin immunoprecipitation, respectively. Vascular permeability was evaluated by Evans blue dye injection in bleomycin-treated mice. Angiogenic activity of endothelial cells was assessed by in vitro angiogenesis assay.Results: CCL20 expression was significantly elevated in dermal fibroblasts of patients with early diffuse cutaneous SSc, while CCR6 was significantly up-regulated in dermal small vessels of SSc patients irrespective of disease subtypes and disease duration. In human dermal microvascular endothelial cells, FLI1 siRNA induced the expression of CCR6, but not CCL20, and FLI1 bound to the CCR6 promoter. Importantly, vascular permeability, a representative SSc-like vascular feature of bleomycin-treated mice, was attenuated by Ccr6 siRNA treatment, and CCR6 siRNA suppressed the angiogenic activity of human dermal microvascular endothelial cells assayed by in vitro tube formation.Conclusions: The increased expression of endothelial CCR6 due to FLI1 deficiency may contribute to the development of SSc vasculopathy.

scholarly journals Reassessing the Role of the Active TGF-β1 as a Biomarker in Systemic Sclerosis: Association of Serum Levels with Clinical Manifestations

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Andréa Tavares Dantas ◽  
Sayonara Maria Calado Gonçalves ◽  
Anderson Rodrigues de Almeida ◽  
Rafaela Silva Guimarães Gonçalves ◽  
Maria Clara Pinheiro Duarte Sampaio ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Clinical Manifestations ◽  
Serum Levels ◽  
Vascular Involvement ◽  
Digital Ulcers ◽  
Serum Samples ◽  
Peripheral Blood Mononuclear ◽  
Pbmc Culture ◽  
Significant Difference ◽  
Skin Biopsies

Objective. To determine active TGF-β1 (aTGF-β1) levels in serum, skin, and peripheral blood mononuclear cell (PBMC) culture supernatants and to understand their associations with clinical parameters in systemic sclerosis (SSc) patients.Methods. We evaluated serum samples from 56 SSc patients and 24 healthy controls (HC). In 20 SSc patients, we quantified spontaneous or anti-CD3/CD28 stimulated production of aTGF-β1 by PBMC. The aTGF-β1 levels were measured by ELISA. Skin biopsies were obtained from 13 SSc patients and six HC, and TGFB1 expression was analyzed by RT-PCR.Results. TGF-β1 serum levels were significantly higher in SSc patients than in HC (p< 0.0001). Patients with increased TGF-β1 serum levels were more likely to have diffuse subset (p= 0.02), digital ulcers (p= 0.02), lung fibrosis (p< 0.0001), positive antitopoisomerase I (p= 0.03), and higher modified Rodnan score (p= 0.046). Most of our culture supernatant samples had undetectable levels of TGF-β1. No significant difference in TGFB1 expression was observed in the SSc skin compared with HC skin.Conclusion. Raised active TGF-β1 serum levels and their association with clinical manifestations in scleroderma patients suggest that this cytokine could be a marker of fibrotic and vascular involvement in SSc.

scholarly journals Birdsong Decreases Protein Levels of FoxP2, a Molecule Required for Human Speech

2008 ◽  
Vol 100 (4) ◽  
pp. 2015-2025 ◽  
Author(s):  
Julie E. Miller ◽  
Elizabeth Spiteri ◽  
Michael C. Condro ◽  
Ryan T. Dosumu-Johnson ◽  
Daniel H. Geschwind ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Target Genes ◽  
Expression Patterns ◽  
Vocal Learning ◽  
Brain Regions ◽  
Motor Deficits ◽  
Mrna Levels ◽  
Adult Males ◽  
Protein Levels ◽  
Area X

Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability.

Abstract 261: Endothelial Cells Contribute to RAS Activation in Microvascular Smooth Muscle Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kayoko Miyata ◽  
Ryousuke Satou ◽  
L Gabriel Navar
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Mrna Expression ◽  
Culture Medium ◽  
Primary Cultures ◽  
Mrna Levels ◽  
Ang Ii ◽  
Relative Ratio ◽  
Renin Mrna ◽  
Afferent Arterioles

Introduction: We have demonstrated that Ang II augments angiotensinogen (AGT) expression in rat preglomerular vascular smooth muscle cells (VSMCs). However, it is unclear if endothelial cells (ECs) are involved in augmentation of AGT in renal afferent arterioles. Hypothesis: We assessed the hypothesis that the ECs respond to paracrine signals that Ang II contribute to AGT augmentation in VSMCs. Objective: We established primary cultures of preglomerular ECs and examined the effects of Ang II and/or culture medium from ECs on AGT expression in preglomerular VSMCs. Methods and Results: We established primary cultures of preglomerular ECs, isolated from afferent arterioles of Sprague-Dawley rats. The cells were identified as ECs by being positive for a marker, CD34 and endothelial NOS and negative for alpha-SMA (a marker for VSMCs) and P4H-b (a marker for Fibroblasts) by immnostaining. The expression levels of AGT mRNA and renin mRNA in preglomerular ECs were examined by real-time RT-PCR. Ang II (100 pmol/L) increased AGT mRNA levels (1.34 +/- 0.16, by 100 pmol/L, N=4) and Renin mRNA levels (6.16 +/- 0.96, by 100 nmol/L, N=4) in ECs. On the other hand, the same dose of Ang II suppressed Renin mRNA expression in isolated Juxtaglomerular cells (JGs). These results indicate that preglomerular ECs are respond to Ang II and exclude the possible contamination of JGs into ECs. 100 pmol/L of Ang II increased AGT mRNA expression levels (1.37 +/- 0.03, relative ratio, N=4) in preglomerular VSMCs and the culture medium of ECs without Ang II treatment also more increased AGT mRNA expression (1.62 +/- 0.13, relative ratio, N=4) in preglomerular VSMCs. The AGT mRNA expression augmentation was enhanced when preglomerular VSMCs were treated with culture medium of Ang II-treated preglomerular ECs (2.39 +/- 0.41, relative ratio, N=4). The synergistic effects of Ang II and preglomerular ECs were also observed in PAI-1 expression in preglomerular VSMCs. Conclusion: These data demonstrate that preglomerular ECs contribute to Ang II-upregulation of AGT in renal afferent arterioles leading to further Ang II augmentation, which leads to increases in inflammatory and sclerotic factors in preglomerular VSMCs.

scholarly journals Three rare variants of SOX7 impairing its interaction with GATA4 may be a predisposing factor to complete AVSD

2020 ◽  
Author(s):  
Baolei Li ◽  
Zhuoyan Li ◽  
Jianping Yang ◽  
Nanchao Hong ◽  
Lihui Jin ◽  
...  
Keyword(s):  
Target Genes ◽  
Rare Variants ◽  
Umbilical Vein ◽  
Heart Defects ◽  
Critical Role ◽  
Cardiovascular Development ◽  
Chinese Han ◽  
Predisposing Factor ◽  
Target Sequencing ◽  
Atrioventricular Cushions

Abstract Background Atrioventricular septal defects (AVSD) are a complicated subtype of congenital heart defects for which the genetic basis is poorly understood. Many evidences have demonstrated that transcription factor SOX7 which can interact with GATA4 plays a pivotal role in the cardiovascular development. The critical role of GATA4 in the morphogenesis of atrioventricular septum implies SOX7 a potential involvement of AVSD. However, whether SOX7 variants are involved in the pathogenesis of AVSD needs to be explored. Methods 100 sporadic non-syndromic AVSD Chinese Han patients were recruited and the variants of SOX7 in 100 patients were screened by target sequencing. Functional assays were performed to testify the potential pathogenicity of nonsynonymous variants of SOX7 found in these AVSD patients. Results Through target sequencing, we identified three rare variants c.40C>G, c.542G>A, and c.743C>T of SOX7 in 100 sporadic non-syndromic AVSD Chinese Han patients. All mutant sites were highly conserved in mammals. Compared to the wildtype, these variants of SOX7 increased mRNA expression and decreased protein. In the developing murine hearts, SOX7 along with GATA4 expressed highly in the region of atrioventricular cushions. Moreover, SOX7 overexpression promoted the expression of GATA4 in human umbilical vein endothelial cells. Chromatin immunoprecipitation assay uncovered that SOX7 could directly bind to the region of GATA4 promoter. Luciferase assays demonstrated that SOX7 activated GATA4 promoter and the variants impaired the transcriptional activity of SOX7. Furthermore, the variants of SOX7 altered the regulation to the activity of GATA4 on its target genes. Conclusions Our studies provide evidence that deleterious variants in SOX7 are potential contributors to human AVSD and provide novel insights into the etiology of AVSD.

Chronic intrauterine pulmonary hypertension decreases calcium-sensitive potassium channel mRNA expression

2000 ◽  
Vol 279 (5) ◽  
pp. L857-L862 ◽  
Author(s):  
David N. Cornfield ◽  
Ernesto R. Resnik ◽  
Jean M. Herron ◽  
Steven H. Abman
Keyword(s):  
Gene Expression ◽  
Pulmonary Hypertension ◽  
Mrna Expression ◽  
Internal Control ◽  
Vascular Reactivity ◽  
Ductus Arteriosus ◽  
Critical Role ◽  
Mrna Levels ◽  
Channel Gene ◽  
Mrna Content

Calcium-sensitive potassium (KCa) channels play a critical role in mediating perinatal pulmonary vasodilation. Because infants with persistent pulmonary hypertension of the newborn (PPHN) have blunted vasodilator responses to birth-related stimuli, we hypothesized that lung KCachannel gene expression is decreased in PPHN. To test this hypothesis, we measured KCa channel gene expression in distal lung homogenates from both fetal lambs with severe pulmonary hypertension caused by prolonged compression of the ductus arteriosus and age-matched, sham-operated animals (controls). After at least 9 days of compression of the ductus arteriosus, fetal lambs were killed. To determine lung KCa channel mRNA levels, primers were designed against the known sequence of the KCa channel and used in semiquantitative RT-PCR, with lung 18S rRNA content as an internal control. Compared to that in control lambs, lung KCa channel mRNA content in the PPHN group was reduced by 26 ± 6% ( P < 0.02), whereas lung voltage-gated K+ 2.1 mRNA content was unchanged. We conclude that lung KCa channel mRNA expression is decreased in an ovine model of PPHN. Decreased KCa channel gene expression may contribute to the abnormal pulmonary vascular reactivity associated with PPHN.

scholarly journals IL-1β dysregulates cGMP signaling in the newborn lung

2020 ◽  
Vol 319 (1) ◽  
pp. L21-L34
Author(s):  
Ying Zhong ◽  
Kristina Bry ◽  
Jesse D. Roberts
Keyword(s):  
Mrna Expression ◽  
Transforming Growth Factor ◽  
Critical Role ◽  
Lung Epithelial Cells ◽  
Lung Fibroblasts ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Iκb Kinase ◽  
Primary Mouse ◽  
Cgmp Signaling

Cyclic guanosine monophosphate (cGMP) signaling is an important regulator of newborn lung function and development. Although cGMP signaling is decreased in many models of newborn lung injury, the mechanisms are poorly understood. We determined how IL-1β regulates the expression of the α1-subunit of soluble guanylate cyclase (sGCα1), a prime effector of pulmonary cGMP signaling. Physiologic levels of IL-1β were discovered to rapidly decrease sGCα1 mRNA expression in a human fetal lung fibroblast cell line (IMR-90 cells) and protein levels in primary mouse pup lung fibroblasts. This sGCα1 expression inhibition appeared to be at a transcriptional level; IL-1β treatment did not alter sGCα1 mRNA stability, although it reduced sGCα1 promoter activity. Transforming growth factor-β (TGFβ)-activated kinase-1 (TAK1) was determined to be required for IL-1β’s regulation of sGCα1 expression; TAK1 knockdown protected sGCα1 mRNA expression in IL-1β-treated IMR-90 cells. Moreover, heterologously expressed TAK1 was sufficient to decrease sGCα1 mRNA levels in those cells. Nuclear factor-κB (NF-κB) signaling played a critical role in the IL-1β-TAK1-sGCα1 regulatory pathway; chromatin immunoprecipitation studies demonstrated enhanced activated NF-κB subunit (RelA) binding to the sGCα1 promoter after IL-1β treatment unless treated with an IκB kinase-2 inhibitor. Also, this NF-κB signaling inhibition protected sGCα1 expression in IL-1β-treated fibroblasts. Lastly, using transgenic mice in which active IL-1β was conditionally expressed in lung epithelial cells, we established that IL-1β expression is sufficient to stimulate TAK1 and decrease sGCα1 protein expression in the newborn lung. Together these results detail the role and mechanisms by which IL-1β inhibits cGMP signaling in the newborn lung.

Lysophosphatidylcholine Decreases the Synthesis of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

1998 ◽  
Vol 79 (01) ◽  
pp. 217-221 ◽  
Author(s):  
Koichi Kokame ◽  
Toshiyuki Miyata ◽  
Naoaki Sato ◽  
Hisao Kato
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Mrna Levels ◽  
Down Regulation ◽  
Human Umbilical Vein ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

SummaryThrombotic complications are frequently associated with atherosclerosis. Lysophosphatidylcholine (LPC), a component accumulated in oxidatively modified LDL (ox-LDL), is known to play a crucial role in the initiation and progression of atherosclerotic vascular lesions. Since a vascular anticoagulant, tissue factor pathway inhibitor (TFPI), has the function of regulating the initial reaction of tissue factor (TF)-induced coagulation, we investigated the effect of LPC on TFPI synthesis in cultured human umbilical vein endothelial cells (HUVEC). The treatment of HUVEC with LPC for 24 h decreased TFPI antigen levels in both the culture medium and the cell lysate in a dose-dependent manner. Northern blot analysis revealed that LPC caused a time-dependent decrease in the TFPI mRNA levels. The levels of TFPI antigen and mRNA were decreased to 72% and 38%, respectively, by the incubation with 50 μM LPC for 24 h. The down-regulation by LPC of TFPI mRNA expression was not observed in the presence of cycloheximide, suggesting that protein synthesis was involved in the suppression of TFPI mRNA expression. The TFPI mRNA levels in actinomycin D-treated cells were relatively stable, indicating that the down-regulation of TFPI mRNA by LPC would be partly explained by the enhanced mRNA destabilization. In contrast to the significant down-regulatory effects of LPC on TFPI expression, LPC did not induce TF mRNA expression in HUVEC. These results indicate that LPC accumulated in the atherosclerotic vascular wall would suppress endothelial TFPI synthesis, reducing the antithrombotic property of endothelial cells.

scholarly journals Low Shear Stress Upregulates CX3CR1 Expression by Inducing VCAM-1 via the NF-κB Pathway in Vascular Endothelial Cells

2020 ◽  
Vol 78 (3) ◽  
pp. 383-389 ◽  
Author(s):  
Yiwei Zhao ◽  
Peile Ren ◽  
Qiufang Li ◽  
Shafiu Adam Umar ◽  
Tan Yang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Protein Expression ◽  
Vascular Endothelial Cells ◽  
Critical Role ◽  
Mrna Levels ◽  
Vascular Endothelial ◽  
Low Shear Stress ◽  
Low Shear ◽  
Cx3cr1 Expression

Abstract Atherosclerosis is a significant cause of mortality and morbidity. Studies suggest that the chemokine receptor CX3CR1 plays a critical role in atherogenesis. Shear stress is an important mechanical force that affects blood vessel function. In this study, we investigated the effect of shear stress on CX3CR1 expression in vascular endothelial cells (VECs). First, cells were exposed to different shear stress and then CX3CR1 mRNA and protein were measured by quantitative RT-PCR and western blot analysis, respectively. CX3CR1 gene silencing was used to analyze the molecular mechanisms underlying shear stress-mediated effects on CX3CR1 expression. CX3CR1 mRNA and protein expression were significantly increased with 4.14 dyne/cm2 of shear stress compared with other tested levels of shear stress. We observed a significant increase in CX3CR1 mRNA levels at 2 h and CX3CR1 protein expression at 4 h. CX3CR1-induced VCAM-1 expression in response to low shear stress by activating NF-κB signaling pathway in VECs. Our findings demonstrate that low shear stress increases CX3CR1 expression, which increases VCAM-1 expression due to elevated NF-κB activation. The current study provides evidence of the correlation between shear stress and atherosclerosis mediated by CX3CR1.

Sign in / Sign up

Export Citation Format

Share Document