scholarly journals Engineering a 3D hydrogel system to study optic nerve head astrocyte morphology and behavior in response to glaucomatous insult

2022 ◽  
Author(s):  
Ana N Strat ◽  
Alexander Kirschner ◽  
Hannah Yoo ◽  
Ayushi Singh ◽  
Tyler Bague ◽  
...  
Keyword(s):  
Optic Nerve ◽  
Optic Nerve Head ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Uv Light ◽  
Collagen Type I ◽  
Data Sets ◽  
Type I ◽  
Extracellular Matrix Components

In glaucoma, astrocytes within the optic nerve head (ONH) rearrange their actin cytoskeleton, while becoming reactive and upregulating intermediate filament glial fibrillary acidic protein (GFAP). Increased transforming growth factor beta 2 (TGFβ2) levels have been implicated in glaucomatous ONH dysfunction. A key limitation of using conventional 2D culture to study ONH astrocyte behavior is the inability to faithfully replicate the in vivo ONH microenvironment. Here, we engineer a 3D ONH astrocyte hydrogel to better mimic in vivo mouse ONH astrocyte (MONHA) morphology, and test induction of MONHA reactivity using TGFβ2. Primary MONHAs were isolated from C57BL/6J mice and cell purity confirmed. To engineer 3D cell-laden hydrogels, MONHAs were mixed with photoactive extracellular matrix components (collagen type I, hyaluronic acid) and crosslinked for 5 minutes using a photoinitiator (0.025% riboflavin) and UV light (405-500 nm, 10.3 mW/cm2). MONHA-encapsulated hydrogels were cultured for 3 weeks, and then treated with TGFβ2 (2.5, 5.0 or 10 ng/ml) for 7 days to assess for reactivity. Following encapsulation, MONHA retained high cell viability in hydrogels and continued to proliferate over 4 weeks as determined by live/dead staining and MTS assays. Sholl analysis demonstrated that MONHAs within hydrogels developed increasing process complexity with longer process length over time. Cell processes connected with neighboring cells, coinciding with Connexin43 expression within astrocytic processes. Treatment with TGFβ2 induced reactivity in MONHA-encapsulated hydrogels as determined by altered F-actin cytoskeletal morphology, increased GFAP expression, and elevated fibronectin and collagen IV deposition. Our data sets the stage for future use of this 3D biomimetic ONHA-encapsulated hydrogel to investigate ONHA behavior in response to glaucomatous insult.

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

2020 ◽  
Vol 9 (11) ◽  
pp. 3572
Author(s):  
Sum Sum Kwok ◽  
Francisca Siu-Yin Wong ◽  
Kendrick Co Shih ◽  
Yau-Kei Chan ◽  
Yashan Bu ◽  
...  
Keyword(s):  
Cell Viability ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Underlying Mechanism ◽  
Collagen Type I ◽  
In Vivo Studies ◽  
Type I ◽  
Lycium Barbarum ◽  
Stromal 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.

In vivo analysis using variants of zebrafish BMPR-IA: range of action and involvement of BMP in ectoderm patterning

Development ◽  
1999 ◽  
Vol 126 (1) ◽  
pp. 181-190 ◽  
Author(s):  
M. Nikaido ◽  
M. Tada ◽  
H. Takeda ◽  
A. Kuroiwa ◽  
N. Ueno
Keyword(s):  
Cell Fate ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Active Form ◽  
Ectopic Expression ◽  
Signal Propagation ◽  
Early Embryo ◽  
Type I ◽  
Cell Fate Conversion

It has been an intriguing problem whether the polypeptide growth factors belonging to the transforming growth factor-beta (TGF-beta) superfamily function as direct and long-range signaling molecules in pattern formation of the early embryo. In this study, we examined the mechanism of signal propagation of bone morphogenetic protein (BMP) in the ectodermal patterning of zebrafish embryos, in which BMP functions as an epidermal inducer and a neural inhibitor. To estimate the effective range of zbmp-2, we first performed whole-mount in situ hybridization analysis. The zbmp-2-expressing domain and the neuroectoderm, marked by otx-2 expression, were complementary, suggesting that BMP has a short-range effect in vivo. Moreover, mosaic experiments using a constitutively active form of a zebrafish BMP type I receptor (CA-BRIA) demonstrated that the cell-fate conversion, revealed by ectopic expression of gata-3 and repression of otx-2, occurred in a cell-autonomous manner, denying the involvement of the relay mechanism. We also found that zbmp-2 was induced cell autonomously within the transplanted cells in the host ectoderm, suggesting that BMP cannot influence even the neighboring cells. This result is consistent with the observation that there is no gap between the expression domains of zbmp-2 and otx-2. Taken together, we propose that, in ectodermal patterning, BMP exerts a direct and cell-autonomous effect to fate uncommitted ectodermal cells to become epidermis.

Paeoniflorin inhibits TGF-β1-mediated collagen production bySchistosoma japonicumsoluble egg antigenin vitro

Parasitology ◽  
2007 ◽  
Vol 134 (11) ◽  
pp. 1611-1621 ◽  
Author(s):  
D. CHU ◽  
Q. LUO ◽  
C. LI ◽  
Y. GAO ◽  
L. YU ◽  
...  
Keyword(s):  
Hepatic Fibrosis ◽  
Transforming Growth Factor Beta ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Collagen Type I ◽  
Peritoneal Macrophages ◽  
Type I ◽  
Smad3 Expression ◽  
Egg Antigen ◽  
Tgf Β1

SUMMARYThe main pathological characteristics of hepatic fibrosis in schistosomiasis are the proliferation of hepatic stellate cells (HSCs) and the deposition of collagen type I (Col I) and collagen type III (Col III). Transforming growth factor beta-1 (TGF-β1) plays an important role in hepatic fibrosis. Paeoniflorin (PAE) has been reported to have immunoregulatory effects; however, the mechanism of its anti-hepatic fibrosis inS. japonicumhas not been elucidated. In the present study, we found that mouse peritoneal macrophages (PMφs) stimulated by soluble egg antigen (SEA) ofS. japonicumcould secrete TGF-β1, and the TGF-β1 in the peritoneal macrophage-conditioned medium (PMCM) could induce proliferation of HSCs and secretion of Col I and III. We selected PMCM at 1:2 dilution as the optimum PMCM (OPMCM). Then we treated HSCs pre-incubated with OPMCM with PAE, and found that the inhibition of HSC proliferation or Col I and III production were closely correlated with the concentration of PAE. Further investigation found that PAE significantly decreased the Smad3 transcription and phosphorylation in HSCs stimulated by OPMCM. In conclusion, SEA plays a key role in hepatic fibrosis by inducing TGF-β1 from PMφs. PAE can exert anti-fibrogenic effects by inhibiting HSCs proliferation and down-regulating Smad3 expression and phosphorylation through TGF-β1 signalling.

scholarly journals Calvaria Bone Transcriptome in Mouse Models of Osteogenesis Imperfecta

2021 ◽  
Vol 22 (10) ◽  
pp. 5290
Author(s):  
Pierre Moffatt ◽  
Iris Boraschi-Diaz ◽  
Juliana Marulanda ◽  
Ghalib Bardai ◽  
Frank Rauch
Keyword(s):  
Osteogenesis Imperfecta ◽  
Mouse Models ◽  
Transforming Growth Factor Beta ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Collagen Type I ◽  
Bone Fragility ◽  
Type I ◽  
Growth Factor Beta ◽  
Upregulated Genes

Osteogenesis imperfecta (OI) is a bone fragility disorder that is usually caused by mutations affecting collagen type I. We compared the calvaria bone tissue transcriptome of male 10-week-old heterozygous Jrt (Col1a1 mutation) and homozygous oim mice (Col1a2 mutation) to their respective littermate results. We found that Jrt and oim mice shared 185 differentially expressed genes (upregulated: 106 genes; downregulated: 79 genes). A total of seven genes were upregulated by a factor of two or more in both mouse models (Cyp2e1, Slc13a5, Cgref1, Smpd3, Ifitm5, Cthrc1 and Rerg). One gene (Gypa, coding for a blood group antigen) was downregulated by a factor of two or more in both OI mouse models. Overrepresentation analyses revealed that genes involved in ‘ossification’ were significantly overrepresented among upregulated genes in both Jrt and oim mice, whereas hematopoietic genes were downregulated. Several genes involved in Wnt signaling and transforming growth factor beta signaling were upregulated in oim mice, but less so in Jrt mice. Thus, this study identified a set of genes that are dysregulated across various OI mouse models and are likely to play an important role in the pathophysiology of this disorder.

scholarly journals Mechanosensitive channel inhibition attenuates TGFβ2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes.

2021 ◽  
Author(s):  
Alexander Kirschner ◽  
Ana N Strat ◽  
John Yablonski ◽  
Tyler Bague ◽  
Haiyan Li ◽  
...  
Keyword(s):  
Optic Nerve ◽  
Optic Nerve Head ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Stress Fibers ◽  
Mechanosensitive Channel ◽  
Dependent Manner ◽  
Cytoskeletal Remodeling ◽  
Channel Inhibition ◽  
Actin Stress Fibers

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFβ2) levels within astrocytes have been described in glaucoma, and TGFβ signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFβ2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFβ2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFβ2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining for GFAP. Levels of fibronectin deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (Iba1) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFβ2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFβ2 treatment and the presence of f-actin stress fibers. TGFβ2 treatment significantly increased GFAP and fibronectin fluorescence intensity, which were decreased with GsMTx4 treatment. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

scholarly journals Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression

1994 ◽  
Vol 304 (3) ◽  
pp. 817-824 ◽  
Author(s):  
J Armendariz-Borunda ◽  
C P Simkevich ◽  
N Roy ◽  
R Raghow ◽  
A H Kang ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Binding Proteins ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Collagen Type I ◽  
Type I ◽  
Tgf Beta ◽  
Ito Cells ◽  
Nuclear Extracts ◽  
I Gene

Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Pro alpha 1(I) gene in FIC. LTIC expressed abundant transcripts of Pro alpha 1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Pro alpha 1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor beta (TGF beta)-treated LTIC had a preferential increase in the rate of Pro alpha 1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) [Thompson, Simkevich, Holness, Kang and Raghow (1991) J. Biol. Chem. 266, 2549-2556] was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGF beta treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGF beta. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGF beta-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGF beta-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.

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