Primary Human Fibroblasts in Culture Switch to a Myofibroblast-Like Phenotype Independently of TGF Beta

Fibroblasts are the prevalent cell type and main source for extracellular matrix (ECM) in connective tissue. Depending on their origin, fibroblasts play a central role in non-pathological tissue remodeling and disease like fibrosis. This study examined the effect of established culture conditions of primary human fibroblasts, from different origins on the myofibroblast-like phenotype formation. We isolated primary human fibroblasts from aortic adventitia, lung, juvenile- and adult skin and investigated the expression levels of CD90, alpha smooth muscle actin (αSMA) and procollagen I under different concentrations of fetal calf serum (FCS) and ascorbic acid (AA) in culture media by immunoblot and immunofluorescence assays. Furthermore, we determined the viability using XTT and migration/wound healing in scratch assays. Collagen 1 secretion was quantified by specific ELISA. Primary human fibroblasts show in part a myofibroblast-like phenotype even without addition of FCS. Supplemented AA reduces migration of cultured fibroblasts with no or low concentrations of FCS. Furthermore, AA and higher concentrations of FCS in culture media lead to higher levels of collagen 1 secretion instead of procollagen I accumulation. This study provides evidence for a partial switch of primary human fibroblasts of different origin to a myofibroblast-like phenotype under common culture conditions.

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An Improved Method of Maintaining Primary Murine Cardiac Fibroblasts in Two-Dimensional Cell Culture

Scientific Reports ◽

10.1038/s41598-019-49285-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Natalie M. Landry ◽

Sunil G. Rattan ◽

Ian M. C. Dixon

Keyword(s):

Cell Culture ◽

Culture Media ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Growth Factor Receptor ◽

Culture Conditions ◽

Quiescent State ◽

Alpha Smooth Muscle Actin ◽

Cell Culture Conditions

Abstract Primary cardiac fibroblasts are notoriously difficult to maintain for extended periods of time in cell culture, due to the plasticity of their phenotype and sensitivity to mechanical input. In order to study cardiac fibroblast activation in vitro, we have developed cell culture conditions which promote the quiescent fibroblast phenotype in primary cells. Using elastic silicone substrata, both rat and mouse primary cardiac fibroblasts could be maintained in a quiescent state for more than 3 days after isolation and these cells showed low expression of myofibroblast markers, including fibronectin extracellular domain A, non-muscle myosin IIB, platelet-derived growth factor receptor-alpha and alpha-smooth muscle actin. Gene expression was also more fibroblast-like vs. that of myofibroblasts, as Tcf21 was significantly upregulated, while Fn1-EDA, Col1A1 and Col1A2 were markedly downregulated. Cell culture conditions (eg. serum, nutrient concentration) are critical for the control of temporal fibroblast proliferation. We propose that eliminating mechanical stimulus and limiting the nutrient content of cell culture media can extend the quiescent nature of primary cardiac fibroblasts for physiological analyses in vitro.

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MicroRNA-145 Involves in the Pathogenesis of Renal Vascular Lesions and May Become a Potential Therapeutic Target in Patients with Juvenile Lupus Nephritis

Kidney & Blood Pressure Research ◽

10.1159/000500923 ◽

2019 ◽

Vol 44 (4) ◽

pp. 643-655 ◽

Cited By ~ 2

Author(s):

Zhaomin Cai ◽

Wei Xiang ◽

Xiaojie Peng ◽

Yan Ding ◽

Wang Liao ◽

...

Keyword(s):

Smooth Muscle ◽

Lupus Nephritis ◽

Smooth Muscle Actin ◽

Vascular Lesions ◽

Alpha Smooth Muscle Actin ◽

Migration Ability ◽

Phenotypic Transformation ◽

And Migration ◽

Renal Vascular ◽

Proliferation And Migration

Aims: The current study was conducted with the central objective of investigating the expression of microRNA-145 (miR-145) in renal vascular lesions (RVLs) in juvenile lupus nephritis (JLN) and its possible mechanism. Methods: The clinical data of 49 JLN patients confirmed by renal biopsy were collected and followed by grouping according to the RVLs score after hematoxylin-eosin staining: mild, moderate, and severe groups. In situ hybridization was used to detect the expression of miR-145 in renal vessels which was then being compared among different RVLs groups. Up-LV-miR-145 and LV-miR-NC lentiviral vectors were constructed and transfected into human vascular smooth muscle cells (HVSMCs), respectively. After HVSMCs were treated with 10.0 µg/L platelet-derived growth factor (PDGF)-BB for 24 h, the proliferation, migration, and apoptosis of endothelial cells were detected by MTT, Transwell assay, and flow cytometry, respectively. Western blot was used to detect expression of alpha-smooth muscle actin (α-SM-actin) and osteopontin (OPN). Results: The expression of miR-145 in renal vascular cells was statistically significant. The higher the inner membrane ratio, the lesser the miR-145 expression. After treatment with PDGF-BB, expression of miR-145 in HVSMCs decreased, proliferation and migration ability enhanced, apoptosis decreased, α-SM-actin decreased, and OPN increased. The proliferation and migration ability of HVSMCs in the LV-miR-145 group suppressed, apoptosis enhanced, α-SM-actin increased, and OPN decreased. Conclusions: Our study revealed that miR-145 expression decreased with the increase of vascular damage. miR-145 can inhibit proliferation, migration, and differentiation phenotypic transformation of HVSMCs induced by PDGF-BB. miR-145 may be involved in the pathogenesis of RVLs and may be a new target for treatment of RVLs in lupus nephritis.

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Effect of culture conditions on artificial activation of porcine oocytes matured in vitro

Reproduction Fertility and Development ◽

10.1071/rd9961153 ◽

1996 ◽

Vol 8 (8) ◽

pp. 1153 ◽

Cited By ~ 9

Author(s):

N Yamauchi ◽

H Sasada ◽

S Sugawara ◽

T Nagai

Keyword(s):

In Vitro Maturation ◽

Fetal Calf Serum ◽

Culture Media ◽

Calf Serum ◽

Treated Group ◽

Culture Conditions ◽

Culture Period ◽

Subsequent Response ◽

Artificial Activation

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.

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Isoliquiritigenin inhibits TGF-β1-induced fibrogenesis through activating autophagy via PI3K/AKT/mTOR pathway in MRC-5 cells

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa067 ◽

2020 ◽

Vol 52 (8) ◽

pp. 810-820 ◽

Cited By ~ 2

Author(s):

Jinjuan He ◽

Hao Peng ◽

Meifang Wang ◽

Ying Liu ◽

Xingrong Guo ◽

...

Keyword(s):

Antioxidant Activities ◽

Smooth Muscle Actin ◽

Mtor Pathway ◽

Type I ◽

Akt Inhibitor ◽

Alpha Smooth Muscle Actin ◽

Human Lung Fibroblast ◽

And Migration ◽

Tgf Β1 ◽

Proliferation And Migration

Abstract Isoliquiritigenin (ISL), a natural flavonoid derived from the root of liquorice, has been reported to possess anti-inflammatory and antioxidant activities. Previous studies have found that ISL plays a crucial role in anti-fibrosis of adipose tissue and renal tissue; however, its effect on pulmonary fibrogenesis has not been demonstrated. In this study, we aimed to explore the roles and the underlying mechanisms of ISL in TGF-β1-induced fibrogenesis using human lung fibroblast-derived MRC-5 cells. Cell proliferation and migration were determined by MTT and wound healing assay, respectively. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (COLIA1) and fibronectin (FN), microtubule-associated protein light chain 3 (LC3) and related signaling molecules were detected by quantitative real-time PCR, western blot and immunofluorescence assay, correspondingly. EGFP-LC3 transfection was used for autophagy analysis. The results showed that ISL inhibited the TGF-β1-induced proliferation and migration, and down-regulated the expressions of α-SMA, COLIA1 and FN. ISL treatment led to up-regulation of LC3 in TGF-β1-treated MRC-5 cells, accompanied by significant decrease in the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In addition, the inhibitory effects of ISL on TGF-β1-induced fibrogenic features in MRC-5 cells were enhanced by pretreatment with autophagy activator Rapmycin and PI3K/AKT inhibitor LY294002 and reversed by autophagy inhibitor 3-methyladenine and PI3K/AKT activator IGF-1. Taken together, our results demonstrated that ISL could attenuate the fibrogenesis of TGF-β1-treated MRC-5 cells by activating autophagy via suppressing the PI3K/AKT/mTOR pathway. Therefore, ISL holds a great potential to be developed as a novel therapeutic agent for the treatment of pulmonary fibrosis.

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Nintedanib suppresses expression of extracellular matrix proteins in TGF-β1-stimulated primary human fibroblasts of the Tenon

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2021-2093 ◽

2021 ◽

Vol 7 (2) ◽

pp. 367-370

Author(s):

Andreas Brietzke ◽

Rudolf F. Guthoff ◽

Niels Grabow ◽

Thomas Stahnke

Keyword(s):

Extracellular Matrix ◽

Smooth Muscle Actin ◽

Human Fibroblasts ◽

Inhibitory Effect ◽

Marginal Effect ◽

Alpha Smooth Muscle Actin ◽

Primary Fibroblasts ◽

Implant Coating ◽

Tgf Β1

Abstract The development of strategies for fibrosis prevention is a perpetual cutting the edge challenge, especially in ophthalmologic fistulating surgery. Stenosis of liquid draining implants but also fibrotic activation of implant-associated tissues are major clinical examples for fibrosis induced implant failure. Implant coating or incorporation of antifibrotic agents offer a promising approach to minimise failure rates. Nintendanib is a drug that has been successfully used for the treatment of ideopathic pulmonary fibrosis since 2015. In this study, we evaluated the suitability of Nintedanib for ophthalmic therapeutic treatment. Therefore, the antifibrotic potential of the active substance was tested with a fibrotic cell culture model on primary fibroblasts of the Tenon (hTF) in vitro. The concentration of 10μM Nintedanib demonstrated a marginal effect on cell viability but coincidently diminished cell proliferation remarkably. Both, immunocytochemical and Western blot analyses revealed a significant inhibitory effect of Nintedanib on the TGF-β1 induced expression of the extracellular matrix (ECM) components fibronectin and collagen. Moreover it supressed the expression and formation of stress fibres of the fibrotic marker protein alpha smooth muscle actin (α-SMA).

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Ultrastructural characterization of an extracellular matrix produced by Leishmania mexicana pifanoi

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124902 ◽

1992 ◽

Vol 50 (1) ◽

pp. 900-901

Author(s):

Carlos Argüello

Keyword(s):

Extracellular Matrix ◽

Culture Media ◽

Calf Serum ◽

Axenic Culture ◽

Culture Conditions ◽

Ruthenium Red ◽

Leishmania Braziliensis ◽

Leishmania Mexicana ◽

Metabolic Products

In a previous study it was shown that Leishmania braziliensis promastigotes secreted metabolic products to the culture media. These metabolites called “excreted factors” were chemically characterized as polysaccharides and glycoproteins that reacted with antileishmania sera. These observations together with the recent characterization of lipophosphoglycans and phosphoglycan lead us to investigate by ultrastructural procedues if leishmania is able to produce an extracellular matrix, in axenic culture conditions. In this study, Leishmania mexicana were cultured in minimum essential medium, supplemented with 10% fetal calf serum, at 22°C. Under these conditions, the parasites tend to associate forming small aggregates, called “rosettes” in which the flagellum is oriented inwards and the cell body outwards (Fig.1). In order to retain their association I supposed the existance of an adhesive substance that kept the parasites together. To test this possibility, rosettes were fixed with glutaraldehyde in cacodylate buffer 0.1 M, or with glutaraldehyde containing 1 mg of ruthenium red, that binds to glycoproteins or polyanionic polysaccharides of extracellular matrices.

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Heparin-binding EGF-like growth factor mRNA is upregulated in the peri-infarct region of the remnant kidney model: in vitro evidence suggests a regulatory role in myofibroblast transformation.

Journal of the American Society of Nephrology ◽

10.1681/asn.v981464 ◽

1998 ◽

Vol 9 (8) ◽

pp. 1464-1473

Author(s):

G Kirkland ◽

K Paizis ◽

L L Wu ◽

M Katerelos ◽

D A Power

Keyword(s):

Endothelial Cells ◽

In Situ Hybridization ◽

Growth Factor ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Human Fibroblasts ◽

Renal Infarction ◽

Heparin Binding ◽

Alpha Smooth Muscle Actin

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent fibroblast and epithelial cell mitogen that may be important in wound healing. The aim of this study was to determine its distribution and possible function in segmental renal infarction. At day 1 postinfarction, in situ hybridization showed that HB-EGF mRNA was markedly increased by tubular epithelial cells bordering the infarcted zone. At day 3, typical myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA) were present in large numbers at the peri-ischemic border and, over succeeding days, were also seen within the infarcted area. Some of these cells expressed HB-EGF mRNA by in situ hybridization suggesting possible autocrine stimulation. Endothelial cells appeared to be more resistant to ischemia than tubules because some capillaries at the periphery of the infarct, surrounded by infarcted tubules, also expressed HB-EGF mRNA. The staining intensity of HB-EGF mRNA in individual tubules and endothelial cells was maximal at day 5 after infarction, although Northern blots of tissue from the peri-infarct area only showed significantly increased expression of HB-EGF mRNA at days 1 and 3, perhaps reflecting a smaller area of greater intensity of expression at day 5. Because tubular cells expressing high levels of HB-EGF mRNA were directly apposed to myofibroblasts, an attempt was made to determine whether HB-EGF contributed to upregulation of alpha-SMA by human fibroblasts. Although stimulation of the fibroblast cell line MRC-5 with transforming growth factor-beta1 (TGF-beta1) increased alpha-SMA, HB-EGF reduced expression. HB-EGF also strongly inhibited the increased expression of alpha-SMA due to TGF-beta1. Because HB-EGF is a potent fibroblast mitogen and TGF-beta is usually antiproliferative, this study suggests that HB-EGF may contribute to a local balance between fibroblast proliferation and differentiation into myofibroblasts during scarring.

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Metformin attenuates carotid neointimal hyperplasia by modulating the vascular smooth muscle cell phenotype transformation through upregulation of TET2, Nur77 and calponin

Archives of Biological Sciences ◽

10.2298/abs201103009l ◽

2021 ◽

Vol 73 (1) ◽

pp. 135-144

Author(s):

Hao Lin ◽

Shuo Cheng ◽

Zhichao Yuan ◽

Zhiqiang Yan ◽

Jifa Zhang

Keyword(s):

Smooth Muscle ◽

Carotid Artery ◽

Vascular Smooth Muscle ◽

Smooth Muscle Actin ◽

Neointimal Hyperplasia ◽

Nuclear Antigen ◽

Cell Phenotype ◽

Alpha Smooth Muscle Actin ◽

And Migration ◽

Proliferation And Migration

Metformin is a drug used to treat type 2 diabetes based on its effectiveness as well as cardiovascular safety. Metformin has been shown to modulate proliferation and migration of vascular smooth muscle cells (VSMCs), but the underlying mechanisms of the effect of metformin on VSMC function remains unclear. We found that metformin inhibits VSMC proliferation and migration and upregulates the expression of nuclear receptor subfamily 4 group A member 1 (Nur77), ten-eleven translocation 2 (TET2), and calponin in vitro. In the carotid artery balloon injury model of rats, metformin effectively prevented neointimal hyperplasia in the carotid artery, including neointimal thickness, increased neointimal area, and the neointimal area/medial area ratio. It also reduced the number of proliferating cell nuclear antigen (PCNA)+ cells and increased the expression of Nur77, calponin and alpha-smooth muscle actin (?-SMA). These results show that metformin attenuates neointimal hyperplasia in balloon-injured carotid arteries via increased expression of TET2, Nur77 and calponin, and reduced expression of matrix metallopeptidase 9 (MMP-9).

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