Loss of Function in Dopamine D3 Receptor Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation in vitro

Evidence suggests the existence of an intracardiac dopaminergic system that plays a pivotal role in regulating cardiac function and fibrosis through G-protein coupled receptors, particularly mediated by dopamine receptor 3 (D3R). However, the expression of dopamine receptors in cardiac tissue and their role in cardiac fibroblast function is unclear. In this brief report, first we determined expression of D1R and D3R both in left ventricle (LV) tissue and fibroblasts. Then, we explored the role of D3R in the proliferation and migration of fibroblast cell cultures using both genetic and pharmaceutical approaches; specifically, we compared cardiac fibroblasts isolated from LV of wild type (WT) and D3R knockout (D3KO) mice in response to D3R-specific pharmacological agents. Finally, we determined if loss of D3R function could significantly alter LV fibroblast expression of collagen types I (Col1a1) and III (Col3a1). Cardiac fibroblast proliferation was attenuated in D3KO cells, mimicking the behavior of WT cardiac fibroblasts treated with D3R antagonist. In response to scratch injury, WT cardiac fibroblasts treated with the D3R agonist, pramipexole, displayed enhanced migration compared to control WT and D3KO cells. Loss of function in D3R resulted in attenuation of both proliferation and migration in response to scratch injury, and significantly increased the expression of Col3a1 in LV fibroblasts. These findings suggest that D3R may mediate cardiac fibroblast function during the wound healing response. To our knowledge this is the first report of D3R's expression and functional significance directly in mouse cardiac fibroblasts.

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Loss of Function in Dopamine Receptor‐3 (D3R) Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation In Vitro

The FASEB Journal ◽

10.1096/fasebj.2019.33.1_supplement.706.2 ◽

2019 ◽

Vol 33 (S1) ◽

Author(s):

Andrew Kisling ◽

Madaniah O Zakari ◽

Musaad B Alsahly ◽

Deepthy C Melit‐Thomas ◽

Stefan Clemens ◽

...

Keyword(s):

Dopamine Receptor ◽

Left Ventricular ◽

Cardiac Fibroblast ◽

Loss Of Function ◽

Fibroblast Migration ◽

Proliferation In Vitro

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SPARC regulates collagen interaction with cardiac fibroblast cell surfaces

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01247.2010 ◽

2011 ◽

Vol 301 (3) ◽

pp. H841-H847 ◽

Cited By ~ 36

Author(s):

Brett S. Harris ◽

Yuhua Zhang ◽

Lauren Card ◽

Lee B. Rivera ◽

Rolf A. Brekken ◽

...

Keyword(s):

Cardiac Tissue ◽

Cardiac Fibroblasts ◽

Pressure Overload ◽

Fibroblast Cell ◽

Immunoblot Analysis ◽

Collagen I ◽

Cell Surfaces ◽

Insoluble Collagen ◽

Total Collagen

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [3H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α1(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

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Platelet-derived growth factor receptor-α is essential for cardiac fibroblast survival

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00054.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. H330-H344 ◽

Cited By ~ 5

Author(s):

Malina J. Ivey ◽

Jill T. Kuwabara ◽

Kara L. Riggsbee ◽

Michelle D. Tallquist

Keyword(s):

Growth Factor ◽

Cardiac Fibroblasts ◽

Growth Factor Receptor ◽

Fibroblast Cell ◽

Cardiac Fibroblast ◽

Platelet Derived Growth Factor ◽

Activating Transcription Factor 3 ◽

Rapid Reduction ◽

Functional Changes

Platelet-derived growth factor receptor α (PDGFRα), a receptor tyrosine kinase required for cardiac fibroblast development, is uniquely expressed by fibroblasts in the adult heart. Despite the consensus that PDGFRα is expressed in adult cardiac fibroblasts, we know little about its function when these cells are at rest. Here, we demonstrate that loss of PDGFRα in cardiac fibroblasts resulted in a rapid reduction of resident fibroblasts. Furthermore, we observe that phosphatidylinositol 3-kinase signaling was required for PDGFRα-dependent fibroblast maintenance. Interestingly, this reduced number of fibroblasts was maintained long-term, suggesting that there is no homeostatic mechanism to monitor fibroblast numbers and restore hearts to wild-type levels. Although we did not observe any systolic functional changes in hearts with depleted fibroblasts, the basement membrane and microvasculature of these hearts were perturbed. Through in vitro analyses, we showed that PDGFRα signaling inhibition resulted in an increase in fibroblast cell death, and PDGFRα stimulation led to increased levels of the cell survival factor activating transcription factor 3. Our data reveal a unique role for PDGFRα signaling in fibroblast maintenance and illustrate that a 50% loss in cardiac fibroblasts does not result in lethality. NEW & NOTEWORTHY Platelet-derived growth factor receptor α (PDGFRα) is required in developing cardiac fibroblasts, but a functional role in adult, quiescent fibroblasts has not been identified. Here, we demonstrate that PDGFRα signaling is essential for cardiac fibroblast maintenance and that there are no homeostatic mechanisms to regulate fibroblast numbers in the heart. PDGFR signaling is generally considered mitogenic in fibroblasts, but these data suggest that this receptor may direct different cellular processes depending on the cell’s maturation and activation status.

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LncRNA LINC00520 Aggravates Cell Proliferation and Migration in Lung Adenocarcinoma via a Positive Feedback Loop

10.21203/rs.3.rs-225838/v1 ◽

2021 ◽

Author(s):

Wen Huang ◽

Xinxing Wang ◽

Fubing Wu ◽

Fanggui Xu

Keyword(s):

Cell Proliferation ◽

Lung Adenocarcinoma ◽

Primary Lung Cancer ◽

Luciferase Reporter ◽

Loss Of Function ◽

Protein Coding ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration

Abstract Background: Lung adenocarcinoma (LUAD) is the most common histological subtype of primary lung cancer. Thus, to figure out the biomarker of diagnosis for LUAD is of great significance. Long non-coding RNAs (lncRNAs) are previously revealed to exert vital effects in numerous cancers. LncRNA long intergenic non-protein coding RNA 520 (LINC00520) served as an oncogene in certain cancers. Therefore, our report was specially designed to probe role of LINC00520 in LUAD. Results: LINC00520 expression was detected by RT-qPCR. Next, function of LINC00520 in LUAD was verified by in vitro loss-of-function experiments. As for LINC00520 regulatory mechanism in LUAD, we conducted pull down, ChIP, RIP, and luciferase reporter assays. We found that LINC00520 was upregulated in LUAD. Additionally, LINC00520 upregulation suggested the poorer prognosis for patients with LUAD. Furthermore, LINC00520 downregulation suppressed LUAD cell proliferation and migration and induced cell apoptosis. Simultaneously, forkhead box P3 (FOXP3) is identified as the transcription factor (TF) to transcriptionally activate LINC00520. Moreover, LINC00520 positively upregulated FOXP3 via sponging miR-3611 in LUAD. Subsequently, rescue experiments delineated that miR-3611 downregulation or FOXP3 overexpression could reverse the effect of silenced LINC00520 on proliferative and migratory capabilities in LUAD. Conclusion: This study ﬁrst put forward and proved that lncRNA LINC00520 facilitated cell proliferative and migratory abilities in LUAD through interacting with miR-3611 and targeting FOXP3, which may provide a potential novel insight for treatment of LUAD.

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Effects of Abnormal Savda Munzip on the Proliferation Activity and Migration Ability of Fibroblasts Derived from Hypertrophic Scar In Vitro

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/870514 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Hujun Wang ◽

Weicheng Gao ◽

Menglong Kong ◽

Nan Li ◽

Ma Shaolin

Keyword(s):

Cell Structure ◽

Fibroblast Cell ◽

Control Group ◽

Blank Control Group ◽

Migration Ability ◽

Migration Assay ◽

Proliferation Activity ◽

And Migration ◽

Proliferation And Migration

Background. To explore the effect of ASMq on proliferation and migration ability of the fibroblast derived from HS of donor (HSFbs) in vitro.Methods. The HSFbs were cultured from tissue specimens and passaged to the 3~4 generation, which were treated with the different concentrations of ASMq and 5-Fu from 1 to 11 days. The difference of HSFbs proliferation activity was analyzed by the CCK-8 method. The HSFbs migration ability in ASMq (0.4 mg/mL) was analyzed by the Cell Scratch method.Results. Transmission electron microscope result shows ASMq concentration significantly increases and fibroblast cell structure markedly change in the experimental group. The proliferation activity of the HSFbs was obviously weakened in ASMq groups than those of the group A (P<0.05) at seven days. The group C (0.4 mg/mL) is better suitable than other three ASMq treatment groups. Cell Migration Assay shows that the migration ability HSFbs was significantly reduced in ASMq (0.4 mg/mL) treatment group compared with those of blank control group at both 24 h and 48 h (P<0.05).Conclusions. These results suggest that ASMq effectively restrains the proliferation and migration ability of the HTSFbs in vitro, which can be one of the mechanisms for the prevention and treatment of HS.

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Human Cardiac Fibroblast Number and Activation State Modulate Electromechanical Function of hiPSC-Cardiomyocytes in Engineered Myocardium

Stem Cells International ◽

10.1155/2020/9363809 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16

Author(s):

Cassady E. Rupert ◽

Tae Yun Kim ◽

Bum-Rak Choi ◽

Kareen L. K. Coulombe

Keyword(s):

Cardiac Tissue ◽

Disease Modeling ◽

Contractile Function ◽

Cardiac Fibroblasts ◽

Cell Number ◽

Cardiac Fibroblast ◽

Cellular Interactions ◽

Excitation Rate ◽

Cardiac Tissues

Cardiac tissue engineering using hiPSC-derived cardiomyocytes is a promising avenue for cardiovascular regeneration, pharmaceutical drug development, cardiotoxicity evaluation, and disease modeling. Limitations to these applications still exist due in part to the need for more robust structural support, organization, and electromechanical function of engineered cardiac tissues. It is well accepted that heterotypic cellular interactions impact the phenotype of cardiomyocytes. The current study evaluates the functional effects of coculturing adult human cardiac fibroblasts (hCFs) in 3D engineered tissues on excitation and contraction with the goal of recapitulating healthy, nonarrhythmogenic myocardium in vitro. A small population (5% of total cell number) of hCFs in tissues improves tissue formation, material properties, and contractile function. However, two perturbations to the hCF population create disease-like phenotypes in engineered cardiac tissues. First, increasing the percentage of hCFs to 15% resulted in tissues with increased ectopic activity and spontaneous excitation rate. Second, hCFs undergo myofibroblast activation in traditional two-dimensional culture, and this altered phenotype ablated the functional benefits of hCFs when incorporated into engineered cardiac tissues. Taken together, the results of this study demonstrate that human cardiac fibroblast number and activation state modulate electromechanical function of hiPSC-cardiomyocytes and that a low percentage of quiescent hCFs are a valuable cell source to advance a healthy electromechanical response of engineered cardiac tissue for regenerative medicine applications.

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LncRNA LINC00520 aggravates cell proliferation and migration in lung adenocarcinoma via a positive feedback loop

BMC Pulmonary Medicine ◽

10.1186/s12890-021-01657-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Wen Huang ◽

Xinxing Wang ◽

Fubing Wu ◽

Fanggui Xu

Keyword(s):

Cell Proliferation ◽

Lung Adenocarcinoma ◽

Primary Lung Cancer ◽

Foxp3 Expression ◽

Luciferase Reporter ◽

Loss Of Function ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration

Abstract Background Lung adenocarcinoma (LUAD) is the most common histological subtype of primary lung cancer. To identify the biomarker of diagnosis for LUAD is of great significance. Long non-coding RNAs (lncRNAs) were previously revealed to exert vital effects in numerous cancers. LncRNA long intergenic non-protein coding RNA 520 (LINC00520) served as an oncogene in various cancers. Therefore, our study was specially designed to probe the role of LINC00520 in LUAD. Results LINC00520 expression was detected by RT-qPCR. Next, function of LINC00520 in LUAD was verified by in vitro loss-of-function experiments. DNA pull down, ChIP, RIP, and luciferase reporter assays were conducted to reveal the regulatory mechanism of LINC00520. We found that LINC00520 was upregulated in LUAD. Additionally, LINC00520 upregulation is associated with the poor prognosis for patients with LUAD. Furthermore, LINC00520 downregulation suppressed LUAD cell proliferation and migration and induced cell apoptosis. Forkhead box P3 (FOXP3) is identified as the transcription factor to transcriptionally activate LINC00520. Moreover, LINC00520 positively upregulated FOXP3 expression via sponging miR-3611 in LUAD cells. Subsequently, rescue experiments delineated that miR-3611 downregulation or FOXP3 overexpression reversed the effects of silenced LINC00520 on proliferative and migratory capabilities in LUAD cells. Conclusion This study innovatively indicated that lncRNA LINC00520 facilitated cell proliferative and migratory abilities in LUAD through interacting with miR-3611 and targeting FOXP3, which may provide a potential novel insight for treatment of LUAD.

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Long noncoding RNA NEAT1 promotes cardiac fibrosis in heart failure through increased recruitment of EZH2 to the Smad7 promoter region

Journal of Translational Medicine ◽

10.1186/s12967-021-03211-8 ◽

2022 ◽

Vol 20 (1) ◽

Author(s):

Zhuowang Ge ◽

Chengye Yin ◽

Yingze Li ◽

Ding Tian ◽

Yin Xiang ◽

...

Keyword(s):

Heart Failure ◽

Promoter Region ◽

Noncoding Rna ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Left Ventricular ◽

Loss Of Function ◽

Smad7 Expression ◽

Lncrna Neat1

AbstractCardiac fibrosis, a well-known major pathological process that ultimately leads to heart failure, has attracted increasing attention and focus in recent years. A large amount of research indicates that long noncoding RNAs (lncRNAs) play an important role in cardiac fibrosis, but little is known about the specific function and mechanism of the lncRNA NEAT1 in the progression of cardiac fibrosis to heart failure. In the present study, we have demonstrated that the lncRNA NEAT1 is upregulated in patients with heart failure. Similarly, the expression of Neat1 was also increased in the left ventricular tissue of transverse aortic constriction (TAC) surgery mice and cardiac fibroblasts treated with TGF-β1. Further, gain-of-function and loss-of-function experiments showed that silencing of Neat1 attenuated cardiac fibrosis, while overexpression of Neat1 with adenovirus significantly aggravated the in vitro progression of fibrosis. With regard to the underlying mechanism, our experiments showed that Neat1 recruited EZH2 to the promoter region of Smad7 through physical binding of EZH2 to the promoter region, as a result of which Smad7 expression was inhibited and the progression of cardiac fibrosis was ultimately exacerbated. We found that the introduction of shNeat1 carried by adeno-associated virus-9 significantly ameliorated cardiac fibrosis and dysfunction caused by TAC surgery in mice. Overall, our study findings demonstrate that the lncRNA Neat1 accelerates the progression of cardiac fibrosis and dysfunction by recruiting EZH2 to suppress Smad7 expression. Thus, NEAT1 may serve as a target for the treatment of cardiac fibrosis.

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Targeting 5-HT 2B Receptor Signaling Prevents Border Zone Expansion and Improves Microstructural Remodeling after Myocardial Infarction

Circulation ◽

10.1161/circulationaha.120.051517 ◽

2021 ◽

Cited By ~ 1

Author(s):

J. Caleb Snider ◽

Lance A. Riley ◽

Noah T. Mallory ◽

Matthew R. Bersi ◽

Prachi Umbarkar ◽

...

Keyword(s):

Myocardial Infarction ◽

Rna Sequencing ◽

Cardiac Fibroblasts ◽

Left Ventricular ◽

Cardiac Fibroblast ◽

Scar Formation ◽

Border Zone ◽

Ventricular Contractility ◽

Fibrotic Process

Background: Myocardial infarction (MI) induces an intense injury response which ultimately generates a collagen-dominated scar. While required to prevent ventricular rupture, the fibrotic process is often sustained in a manner detrimental to optimal recovery. Cardiac myofibroblasts are the cells tasked with depositing and remodeling collagen and are a prime target to limit the fibrotic process post-MI. Serotonin 2B receptor (5-HT 2B ) signaling has been shown to be harmful in a variety of cardiopulmonary pathologies and could play an important role in mediating scar formation after MI. Methods: We employed two pharmacologic antagonists to explore the effect of 5-HT 2B inhibition on outcomes post-MI and characterized the histological and microstructural changes involved in tissue remodeling. Inducible, 5-HT 2B ablation driven by Tcf21 MCM and Postn MCM were used to evaluate resident cardiac fibroblast- and myofibroblast-specific contributions of 5-HT 2B , respectively. RNA sequencing was used to motivate subsequent in vitro analyses to explore cardiac fibroblast phenotype. Results: 5-HT 2B antagonism preserved cardiac structure and function by facilitating a less fibrotic scar, indicated by decreased scar thickness and decreased border zone area. 5-HT 2B antagonism resulted in collagen fiber redistribution to thinner collagen fibers which were more anisotropic, enhancing left ventricular contractility, while fibrotic tissue stiffness was decreased, limiting the hypertrophic response of uninjured cardiomyocytes. Using a tamoxifen-inducible Cre, we ablated 5-HT 2B from Tcf21 -lineage resident cardiac fibroblasts and saw similar improvements to the pharmacologic approach. Tamoxifen-inducible Cre-mediated ablation of 5-HT 2B after onset of injury in Postn -lineage myofibroblasts also improved cardiac outcomes. RNA sequencing and subsequent in vitro analyses corroborate a decrease in fibroblast proliferation, migration, and remodeling capabilities through alterations in Dnajb4 expression and Src phosphorylation. Conclusions: Together, our findings illustrate that 5-HT 2B expression in either cardiac fibroblasts or activated myofibroblasts directly contributes to excessive scar formation, resulting in adverse remodeling and impaired cardiac function after MI.

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KDM3A is a Positive Regulator of Cardiac Fibroblasts Conversion that Increases Smad3 Phosphorylation Following TGFβ1 Stimulation

10.21203/rs.3.rs-1060401/v1 ◽

2021 ◽

Author(s):

Xiaopei Liu ◽

Zhou Jining ◽

Bofang Zhang ◽

Gen Liu ◽

Qi Hu ◽

...

Keyword(s):

Cell Migration ◽

Cardiovascular Diseases ◽

Cardiac Fibroblasts ◽

Specific Inhibitor ◽

Underlying Mechanism ◽

Biological Functions ◽

Loss Of Function ◽

Smad3 Phosphorylation ◽

And Migration ◽

Proliferation And Migration

Abstract The epigenetic molecule KDM3A has been shown to be involved in improving cardiovascular diseases, but its effect on cardiac fibroblasts (CFs) remains unclear. Thus, we designed the gain- and loss-of-function experiments to investigate the biological functions of KDM3A in cardiac fibroblasts (CFs). Moreover, we added a SIS3-HCL (a specific inhibitor of p-Smad3) to explore the underlying mechanism. The cells viability and migration were verified by CCK-8 and cell migration experiments, respectively, and the degree of fibrosis was measured by Western blot analysis. Our data reveal that KDM3A enhance the proliferation and migration of cardiac fibroblasts, meanwhile, increasing the fibroblast-to-myofibroblast transition, while enabling Smad3 phosphorylation response to TGFβ1 stimuli. However, these results could be abolished by SIS3-HCL, an inhibitor of the p-Smad3. Furthermore, KDM3A inhibition obviously protect cardiac fibroblasts conversion against TGFβ1 stimuli. These results identify that KDM3A may be a novel regulator of the cardiac fibroblasts conversion, through its ability to modulate phosphorylation of Smad3 following TGFβ1 stimuli.

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