Myofibroblast Viability Research Articles

Novel Synthetic Polymer-Based 3D Contraction Assay: A Versatile Preclinical Research Platform for Fibrosis.

The driving factors causing fibrosis and scar formation include fibroblast differentiation into myofibroblasts and hampered myofibroblast apoptosis, which ultimately results in collagen accumulation and tissue contraction. Currently, only very few drugs are available for fibrosis treatment, and there is an urgent demand for new pharmaceutical products. High-throughput in vitro fibrosis models are necessary to develop such drugs. In this study, we developed such a novel model based on synthetic polyisocyanide (PIC-RGD) hydrogels. The model not only measures contraction but also allows for subsequent molecular and cellular analysis. Fibroblasts were seeded in small (10 μL) PIC-RGD gels in the absence or presence of TGFβ1, the latter to induce myofibroblast differentiation. The contraction model clearly differentiates fibroblasts and myofibroblasts. Besides a stronger contraction, we also observed α-smooth muscle actin (αSMA) production and higher collagen deposition for the latter. The results were supported by mRNA expression experiments of αSMA, Col1α1, P53, and Ki67. As proof of principle, the effects of FDA-approved antifibrotic drugs nintedanib and pirfenidone were tested in our newly developed fibrosis model. Both drugs clearly reduce myofibroblast-induced contraction. Moreover, both drugs significantly decrease myofibroblast viability. Our low-volume synthetic PIC-RGD hydrogel platform is an attractive tool for high-throughput in vitro antifibrotic drug screening.

Lycium barbarum Polysaccharide Suppresses Expression of Fibrotic Proteins in Primary Human Corneal Fibroblasts.

(1) Objective: To study the anti-fibrotic effects of Lycium barbarum polysaccharides (LBP) on corneal stromal fibroblasts and assess LBP’s effect on cell viability. (2) Methods: Primary human corneal keratocytes of passage 3 to 6 were used for all experiments. Cells are pretreated with LBP solution for 24 h and then transforming growth factor beta 1 (TGFβ1) for 48 h and collected for experiments. Fibrotic protein analysis was performed using immunofluorescence and Western blot. The effect of LBP on cell viability was assessed using the MTS assay. (3) Results: LBP significantly reduced the expression of fibrotic proteins, including α-SMA and extracellular matrix proteins (collagen type I and III). LBP significantly decreased the viability of myofibroblasts but not the fibroblasts. Conclusions: In this study, LBP was effective in the prevention of fibrosis gene expression. Further studies to assess the underlying mechanism and pharmacological properties will facilitate the formation of a topical LBP solution for in vivo studies.

TGFβ1 and TGFβ2 proteins in corneas with and without stromal fibrosis: Delayed regeneration of apical epithelial growth factor barrier and the epithelial basement membrane in corneas with stromal fibrosis

The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) β1 and TGFβ2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, −4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFβ1 or TGFβ2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to −4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFβ1 and tear TGFβ2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFβ1 and/or TGFβ2 in the stroma in some corneas. TGFβ1 and TGFβ2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four −4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in −4.5D PRK corneas without fibrosis, compared to −4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFβ1 and TGFβ2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.

Postinfarction Hearts Are Protected by Premature Senescent Cardiomyocytes Via GATA 4-Dependent CCN 1 Secretion.

BackgroundStress‐induced cell premature senescence participates in a variety of tissue and organ remodeling by secreting such proteins as proinflammatory cytokines, chemokines, and growth factors. However, the role of cardiomyocyte senescence in heart remodeling after acute myocardial infarction has not been thoroughly elucidated to date. Therefore, we sought to clarify the impact of premature myocardial senescence on postinfarction heart function.Methods and ResultsSenescence markers, including p16INK 4a, p21CIP 1/ WAF 1, and SA‐β‐gal staining, were analyzed in several heart disease models by immunostaining. Both postinfarction mouse hearts and ischemic human myocardium demonstrated increased senescence markers. Additionally, senescence‐related secretory phenotype was activated after acute myocardial infarction, which upregulated senescence‐related secretory phenotype factors, including CCN family member 1 (CCN1), interleukin‐1α, tumor necrosis factor α, and monocyte chemoattractant protein‐1. In vivo, a tail vein injection of AAV9‐Gata4‐shRNA significantly attenuated senescence‐related secretory phenotype secretion and aggravated postinfarction heart dysfunction. Furthermore, among activated senescence‐related secretory phenotype factors, CCN1 administration reduced myofibroblast viability in vitro and rescued the deleterious effect of AAV9‐Gata4‐shRNA in vivo.ConclusionsMyocardial premature senescence was observed in the ischemic hearts and improved postinfarction heart function, partly through the GATA‐binding factor 4‐CCN1 pathway.

Transmission Electron Microscopy Analysis of Epithelial Basement Membrane Repair in Rabbit Corneas With Haze

To assess the ultrastructure of the epithelial basement membrane using transmission electron microscopy (TEM) in rabbit corneas with and without subepithelial stroma opacity (haze). Two groups of eight rabbits each were included in this study. Photorefractive keratectomy (PRK) was performed using an excimer laser. The first group had -4.5-diopter (-4.5D) PRK and the second group had -9.0D PRK. Contralateral eyes were unwounded controls. Rabbits were sacrificed at 4 weeks after surgery. Immunohistochemical analysis was performed to detect the myofibroblast marker α-smooth muscle actin (SMA). TEM was performed to analyze the ultrastructure of the epithelial basement membrane and stroma. At 4 weeks after PRK, α-SMA+ myofibroblasts were present at high density in the subepithelial stroma of rabbit eyes that had -9.0D PRK, along with prominent disorganized extracellular matrix, whereas few myofibroblasts and little disorganized extracellular matrix were noted in eyes that had -4.5D PRK. The epithelial basement membrane was irregular and discontinuous and lacking typical morphology in all corneas at 1 month after -9D PRK compared to corneas at 1 month in the -4.5D PRK group. The epithelial basement membrane acts as a critical modulator of corneal wound healing. Structural and functional defects in the epithelial basement membrane correlate to both stromal myofibroblast development from precursor cells and continued myofibroblast viability, likely through the modulation of epithelial-stromal interactions mediated by cytokines. Prolonged stromal haze in the cornea is associated with abnormal regeneration of the epithelial basement membrane.

Interleukin-1 receptor role in the viability of corneal myofibroblasts

The purpose of this study was to investigate the role of interleukin-1 (IL-1) in modulating myofibroblast viability in mouse corneas with stromal opacity. Twenty-four female B6; 129S1-Il1r1tm1Roml/J homozygous IL-1RI knockout mice and 24 control B6129SF2/J mice were included in this study. Each mouse had opacity-generating irregular phototherapeutic keratectomy (PTK) performed with an excimer laser in one eye. Groups of 8 mice from each group were euthanized at one month, three months and six months after surgery and the eyes cryo-preserved. The contralateral eye served as unwounded control. Immunohistochemistry was performed for α-smooth muscle actin (SMA) in central sections of all corneas. The TUNEL assay for apoptosis was performed on 8 sections of four eyes from each group. No SMA+ cells were detected in the stroma of unwounded control or knockout corneas. SMA+ myofibroblast density was significantly higher (p < 0.001) in the IL-1RI knockout group than in the control group at one month, three and six months after irregular PTK. Mean TUNEL+ stromal cells in the anterior 50 μm of stroma was significantly lower in the IL-1RI knockout group compared to the control group at six months after irregular PTK (p = 0.04). These results corroborate the findings of recent in vitro work that demonstrated an antagonistic effect of TGFβ and IL-1 on myofibroblast viability, and found that IL-1-triggered myofibroblast apoptosis was suppressed by TGFβ. Thus, IL-1 is an important modulator of myofibroblast viability during corneal wound healing.

The influence of extracts from Potentilla species on normal human colon cells

The biological activity of extracts obtained from aerial parts of Potentilla species: P. erecta (L.) Rauschel, P. anserina L., P. argentea L., P. rupestris L., P. grandiflora L. was analyzed. Extracts were tested using MTT, NR and DPPH tests on two human normal cell lines: CCD 841 CoTr and CCD-18Co. Fluorescence staining of the cellular cytoskeleton after rhodamine-phalloidyne addition and IL-6, IL-10 (ELISA) in culture supernatants after 24h of incubation with Potentilla extracts and nitric oxide (NO) analysis with a Griess method were performed. Extracts were tested at the range of 25–225µg/mL concentrations while to the ELISA two non-toxic doses (15 and 30µg/mL) were chosen. We found that all extracts stimulated metabolism of epithelial cells while myofibroblasts' mitochondrial dehydrogenase activity was stimulated at concentrations higher than 125µg/mL. The exception was P. grandiflora which activated succinyl dehydrogenase just at low extract dose (25µg/mL). Extracts from P. erecta and P. argentea had no toxic effect on colon epithelial cells while other extracts significantly decreased viability of cells even when added at 25µg/mL concentration. Only P. grandiflora and P. rupestris significantly decreased viability of myofibroblasts. All extracts showed free radical scavenging effect in a concentration dependent manner. Potentilla extracts inhibited IL-6 and IL-10 production by myofibroblasts while in epithelial cells slightly induced or had no effect on the cytokine level. Potentilla extracts influenced F-actin filament composition and changed the cellular cytoskeleton and morphology of cells. Modulation of NO production after plant extracts addition has also been observed. Keywords: Potentilla, cytotoxicity, normal human colon cells

Simvastatin induces apoptosis by a Rho-dependent mechanism in cultured cardiac fibroblasts and myofibroblasts

Several clinical trials have shown the beneficial effects of statins in the prevention of coronary heart disease. Additionally, statins promote apoptosis in vascular smooth muscle cells, in renal tubular epithelial cells and also in a variety of cell lines; yet, the effects of statins on cardiac fibroblast and myofibroblast, primarily responsible for cardiac tissue healing are almost unknown. Here, we investigated the effects of simvastatin on cardiac fibroblast and myofibroblast viability and studied the molecular cell death mechanism triggered by simvastatin in both cell types. Methods Rat neonatal cardiac fibroblasts and myofibroblasts were treated with simvastatin (0.1–10 μM) up to 72 h. Cell viability and apoptosis were evaluated by trypan blue exclusion method and by flow cytometry, respectively. Caspase-3 activation and Rho protein levels and activity were also determined by Western blot and pull-down assay, respectively. Results Simvastatin induces caspase-dependent apoptosis of cardiac fibroblasts and myofibroblasts in a concentration- and time-dependent manner, with greater effects on fibroblasts than myofibroblasts. These effects were prevented by mevalonate, farnesylpyrophosphate and geranylgeranylpyrophosphate, but not squalene. These last results suggest that apoptosis was dependent on small GTPases of the Rho family rather than Ras. Conclusion Simvastatin triggered apoptosis of cardiac fibroblasts and myofibroblasts by a mechanism independent of cholesterol synthesis, but dependent of isoprenilation of Rho protein. Additionally, cardiac fibroblasts were more susceptible to simvastatin-induced apoptosis than cardiac myofibroblasts. Thus simvastatin could avoid adverse cardiac remodeling leading to a less fibrotic repair of the damaged tissues.

Stromal interleukin-1 expression in the cornea after haze-associated injury

The purpose of this study was to determine whether myofibroblasts or other cells in the stroma in the cornea produce interleukin (IL)-1α or IL-1β that could modulate myofibroblast viability in corneas with haze after photorefractive keratectomy (PRK). Twenty-four female rabbits had haze-generating PRK for 9 diopters of myopia and were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were removed, frozen in OCT at −80 °C, and analyzed by immunocytochemistry using primary antibodies to IL-1α, IL-1β and alpha smooth muscle actin (SMA). Double immunostaining was performed for the co-localization of SMA with IL-1α or IL-1β. Central dense haze and peripheral slight haze regions of each cornea were analyzed. SMA+ cells that expressed IL-1α protein were detected in both regions of the corneas at most time points following PRK. However, in the haze region at the 1, 3 and 4 week time points, significantly more ( p < 0.01) SMA+ cells did not express IL-1α. Also, in the haze region at all three time points, significantly more ( p < 0.01) SMA- cells than SMA+ cells expressed interleukin-1α protein. IL-1β expression patterns in SMA+ and SMA- stromal cells was similar to that of IL-1α after PRK. Previous studies have demonstrated that IL-1α or IL-1β triggers myofibroblast apoptosis in vitro, depending on the available concentration of apoptosis-suppressive TGFβ. This study demonstrates that SMA- cells such as corneal fibroblasts, keratocytes, or inflammatory cells may produce IL-1α and/or IL-1β that could act in paracrine fashion to regulate myofibroblast apoptosis—especially in the region where there is haze in the cornea after PRK was performed and SMA+ myofibroblasts are present at higher density. However, some SMA+ myofibroblasts themselves produce IL-1α and/or IL-1β, suggesting that myofibroblast viability could also be regulated via autocrine mechanisms.

Expression of PDGF receptor-α in corneal myofibroblasts in situ

The purpose of this study was to investigate the expression of platelet-derived growth factor receptor-alpha (PDGFR-α) in the myofibroblasts of corneas with stromal haze. Central corneal sections from rabbit eyes that had −9 diopter PRK were analyzed by immunocytochemistry (IHC) for the expression of PDGFR-α at 4 week after surgery. PDGFR-α was expressed immediately beneath the epithelial basement membrane in the anterior stroma. Double IHC studies revealed the expression of PDGFR-α in the anterior stroma co-localized with alpha-smooth muscle actin (SMA) marker for myofibroblasts. In vitro studies have suggested that PDGF is important in the development and viability of myofibroblasts after corneal injury. Expression of PDGFR-α in myofibroblasts supports these findings.

Corneal myofibroblast viability: Opposing effects of IL-1 and TGF β1

The purpose of this study was to test the effect of corneal epithelial scrape on myofibroblasts associated with haze and elucidate the effect of interleukin-1 and transforming growth factor β1 on corneal stromal myofibroblasts viability and death in vitro. Corneal epithelial scrape was performed in rabbit eyes with severe haze at one month after −9 diopter photorefractive keratectomy. Corneas were processed for immunocytochemistry for myofibroblast marker α-smooth muscle actin (α-SMA) and the TUNEL assay to detect apoptosis. Rabbit corneal fibroblasts were cultured with 2 ng/ml of transforming growth factor β1 (TGF β1) to induce myofibroblast differentiation confirmed by monitoring α-SMA expression. Fluorescence-based TUNEL assay was performed to analyze the apoptotic response of myofibroblasts to IL-1α or IL-1β, in the presence or absence of TGF β1. Dose response experiments were performed after withdrawal of TGF β1 and exposure to 1, 5, or 10 ng/ml of IL-1α or IL-1β for 1 h. Subsequent experiments were performed with myofibroblasts exposed to 5 ng/ml of IL-1α or IL-1β in conjunction with 0, 1, 5, or 10 ng/ml of TGF β1. Corneal epithelial scrape with a scalpel blade produced myofibroblast apoptosis. Exposure to TGF β1 in vitro resulted in greater than 99% transformation of corneal fibroblasts to α-SMA+ myofibroblasts. There was a statistically significant dose-dependent increase in the percentage of TUNEL+ cells with either IL-1α or IL-1β initiated at concentrations as low as 1 ng/ml. For example, after withdrawal of TGF β1, the % TUNEL+ cells at 1 h after exposure to IL-1α increased significantly with increasing concentration (0 ng/ml, 2.4 ± 0.8% [S.E.M.]; 1 ng/ml, 15.4 ± 1.8%; 5 ng/ml, 47.4 ± 3.9%; or 10 ng/ml, 70.3 ± 3.2%). Similar results were obtained with IL-1β. The differences between the means of apoptotic myofibroblasts for the different concentrations of cytokine for either IL-1α or IL-1β were significantly different (ANOVA, p < 0.001). When myofibroblasts were exposed to 5 ng/ml of IL-1α or IL-1β, the % TUNEL+ cells at 1 h were reduced in a significant dose-dependent manner when TGF β1 at a concentration of 5 ng/ml or 10 ng/ml was present in the medium (ANOVA p < 0.01). IL-1α or IL-1β triggers the death of myofibroblasts in vitro and TGF β1 reduces the IL-1 effect on cell death. TGF β1 and IL-1 have opposing effects on myofibroblast viability and likely interact to modulate haze generation after corneal injury.

Involvement of selenoprotein P in the regulation of redox balance and myofibroblast viability in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease of unknown etiology, is characterized by the expansion of myofibroblasts and abnormal deposition of extracellular matrix in the lung parenchyma. To elucidate the molecular mechanisms that lead to IPF, we analyzed myofibroblasts established from patients with IPF by oligonucleotide microarrays. Gene expression profiles clearly suggested that lipid peroxidation is enhanced in myofibroblasts, which was confirmed by measuring cellular lipid hydroperoxides. One of the most highly up-regulated proteins in myofibroblasts was selenoprotein P, an antioxidant protein not previously associated with IPF. Subsequent analysis demonstrated that selenoprotein P reduces lipid hydroperoxides and maintains the viability of myofibroblasts. Thus, our results reveal a novel pathophysiologic function of myofibroblasts as a generator of lipid hydroperoxides, and a self-defense mechanism against self-generated oxidative stress.