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PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0262046
Author(s):  
Allison A. Fuchs ◽  
Praveen K. Balne ◽  
Elizabeth A. Giuliano ◽  
Nishant R. Sinha ◽  
Rajiv R. Mohan

Corneal injury and aberrant wound healing commonly result in corneal fibrosis and subsequent vision loss. Intermediate-conductance calmodulin/calcium-activated K+ channels (KCa3.1) have been shown to promote fibrosis in non-ocular and ocular tissues via upregulation of transforming growth factor beta (TGFβ). TRAM-34 is a selective inhibitor of KCa3.1 and reduces fibrosis by downregulation of TGFβ-induced transdifferentiation of stromal fibroblasts to myofibroblasts. Ascorbic acid has been demonstrated to be effective in promoting corneal re-epithelialization and reduction of neovascularization via anti-VEGF and anti-MMP mechanisms. This study evaluates tolerability and efficacy of a novel combination of TRAM-34 (25μM) and ascorbic acid (10%) topical treatment for corneal fibrosis using an established in vivo rabbit model and conducting clinical eye examinations. Markers of corneal fibrosis were evaluated in all corneas at study endpoint via histopathology, immunofluorescence, and quantitative real-time PCR. The eyedrop treated eyes showed significantly improved clinical outcomes based on modified McDonald Shadduck scores, reduction of clinical haze on Fantes scores, and reduction of central corneal thickness (CCT). At cellular and molecular levels, eyedrop treatment also significantly reduced expression of alpha smooth muscle actin (α-SMA) mRNA and protein, collagen III mRNA, and fibronectin mRNA compared to non-treated eyes. Our study suggests that a tested new bimodal eyedrop is well tolerated and effectively reduces corneal fibrosis/haze in rabbits in vivo.


2022 ◽  
Vol 2022 ◽  
pp. 1-19
Author(s):  
Baining Cui ◽  
Yan Wang ◽  
Jiahui Jin ◽  
Zhen Yang ◽  
Ruoxi Guo ◽  
...  

UVB exposure is one of the primary factors responsible for the development of photoaging, and the aim of this study was to investigate the mechanism involved in the photoprotective properties of resveratrol (RES) in UVB-induced photoaging. Photoaging models of Hacat cells and ICR mice were established by UVB irradiation. The effect of RES on cell viability was then assessed using the MTT assay. The effect of RES on reactive oxygen species (ROS) production was detected through a fluorescent probe assay. The effect of RES on oxidized glutathione (GSSH) content, and superoxide dismutase (SOD) activity in photoaging Hacat cells, were measured separately, using kits. An enzyme-linked immunosorbent assay (ELISA) was used to measure the effect of RES on IL-6 secretion. The effect of VEGF-B on RES photoprotection was examined through the RT-qPCR method, after silencing VEGF-B through siRNA transfection. For animal experiments, the relative water content of the skin of ICR mice was determined using the Corneometer CM825 skin moisture tester. Starting from the third week of the study, the back skin of photoaging ICR mice was photographed weekly using the TIVI700 camera, and the depth of skin wrinkles in photoaging ICR mice was also analyzed. The thickness of the epidermis in photoaging ICR mice was assessed by the hematoxylin-eosin (HE) staining method. The content of collagen fibers in the skin dermis of photoaging ICR mice was measured by the Masson trichrome staining method. The content of collagen III in the dermis of the skin in photoaging ICR mice was measured through immunohistochemistry (IHC) techniques. The effect of RES on the mRNA expression levels of MMP-1, MMP-9, HO-1, GPX-4, IL-6, TNF-α, VEGF-B, caspase9, and caspase3 in photoaging Hacat cells, and that of MMP-3, Nrf2, HO-1, NQO1, SOD1, GPX-4, caspase9, caspase3, and IL-6 in the skin of photoaging ICR mice, was measured by RT-qPCR. The effects of RES on caspase3, Nrf2 (intranuclear), COX-2, P-ERK1/2, ERK1/2, P-P38MAPK, and P38MAPK in photoaging Hacat cells, and on MMP-9, caspase3, COX-2, P-JNK, P-ERK1/2, and P-P38MAPK protein expression in the skin of photoaging ICR mice, were assayed by the WB method. The results of this study, therefore, show that RES has a protective effect against UVB-induced photoaging in both Hacat cells and ICR mice. Its mechanism of action may include reducing the expression of MMPs and the secretion of collagen and inflammatory factors by inhibiting the ROS-mediated MAPK and COX-2 signaling pathways, balancing oxidative stress in the skin of Hacat cells and ICR mice by promoting the Nrf2 signaling pathway, inducing antiapoptotic effects by inhibiting caspase activation, and exerting antioxidant and antiapoptotic effects by targeting the VEGF-B, demonstrating its photoprotective effects against UVB irradiation-induced photoaging.


2022 ◽  
Vol 12 (1) ◽  
pp. 10-18
Author(s):  
Yi Du ◽  
Yifan Wu

To explore the repairing effect of platelet-rich plasma (PRP) on tendon (AT) healing, and provide evidence for PRP therapy to treat tendon-related diseases, 32 New Zealand white rabbits were selected to construct tendinopathy animal model. Leukocyte-rich Platelet-Rich Fibrin (Lr-PRP) and Leukocyte-poor Platelet-Rich Fibrin (Lp-PRP) were prepared, whose biological effects on tendon stem cells (TSCs) were explored. Rabbits were divided into control group, low, medium, and high dose groups regrading concentration of Lp-PRP. The number of fibroblasts, collagen fiber content, tubuloglomerular feedback-β1 (TGF-β1) expression, and biomechanical properties were compared at 15 and 30 days after operation. The results showed that Collagen-III (CoI-III) protein expression levels, interleukin-β (IL-β), and interleukin-6 (IL-6) levels in the Lp-PRP group were significantly higher than Lr-PRP group (P < 0.05). Fibroblasts and collagen fibers in group II and III were significantly higher versus group C 15 and 30 days after operation (P < 0.05). Fibroblasts and of collagen fibers in group III were significantly higher versus group II (P < 0.05). Expression of TGF-β1 in groups II and III was significantly higher than that in group C 15 days after operation (P < 0.05). Tensile load of AT repair site in group III was significantly higher than group C 30 days after operation (P < 0.05). Platelet plasma concentration had a certain repair effect on tendon injury and can effectively improve the quality of healing. In addition, Lp-PRP was better than Lr-PRP in tissue healing. When the concentration of Lp-PRP was 100%, the repair effect was the best.


2021 ◽  
Vol 23 (1) ◽  
pp. 174
Author(s):  
Laia Yáñez-Bisbe ◽  
Anna Garcia-Elias ◽  
Marta Tajes ◽  
Isaac Almendros ◽  
Antonio Rodríguez-Sinovas ◽  
...  

Information about heart failure with reduced ejection fraction (HFrEF) in women and the potential effects of aging in the female heart is scarce. We investigated the vulnerability to develop HFrEF in female elderly mice compared to young animals, as well as potential differences in reverse remodeling. First, HF was induced by isoproterenol infusion (30 mg/kg/day, 28 days) in young (10-week-old) and elderly (22-month-old) female mice. In a second set of animals, mice underwent isoproterenol infusion followed by no treatment during 28 additional days. Cardiac remodeling was assessed by echocardiography, histology and gene expression of collagen-I and collagen-III. Following isoproterenol infusion, elderly mice developed similar HFrEF features compared to young animals, except for greater cell hypertrophy and tissue fibrosis. After beta-adrenergic withdrawal, young female mice experienced complete reversal of the HFrEF phenotype. Conversely, reversed remodeling was impaired in elderly animals, with no significant recovery of LV ejection fraction, cardiomyocyte hypertrophy and collagen deposition. In conclusion, chronic isoproterenol infusion is a valid HF model for elderly and young female mice and induces a similar HF phenotype in both. Elderly animals, unlike young, show impaired reverse remodeling, with persistent tissue fibrosis and cardiac dysfunction even after beta-adrenergic withdrawal.


2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Bin Yan ◽  
Canjun Zeng ◽  
Yuhui Chen ◽  
Minjun Huang ◽  
Na Yao ◽  
...  

Mechanical stress promotes human ligamentum flavum cells (LFCs) to synthesize multitype collagens, leading to ligamentum flavum hypertrophy (LFH). However, the mechanism of mechanical stress in the formation of collagen remains unclear. Therefore, we investigated the relationship between mechanical stress and collagen synthesis in the present study. First, LFCs were isolated from 9 patients and cultured with or without mechanical stress exposure for different times. IGF-1, collagen I (col-I), and collagen III (col-III) protein and mRNA levels were then detected via ELISA and qPCR, respectively. Moreover, the activation of pIGF-1R, pAKT, and pS6 was examined by Western blot analysis. To further explore the underlying mechanism, an IGF-1 neutralizing antibody, NVP-AEW541, and rapamycin were used. IGF-1, col-I, and col-III were significantly increased in stressed LFCs compared to nonstressed LFCs. In addition, the activation of pIGF-1R, pAKT, and pS6 was obviously enhanced in stressed LFCs. Interestingly, col-I protein, col-I mRNA, col-III protein, col-III mRNA, and IGF-1 protein, but not IGF-1 mRNA, were inhibited by IGF-1 neutralizing antibody. In addition, col-I and col-III protein and mRNA, but not IGF-1, were inhibited by both NVP-AEW541 and rapamycin. Moreover, the activation of pIGF-1R, pAKT, and pS6 was reduced by the IGF-1 neutralizing antibody and NVP-AEW541, and the activation of pS6 was reduced by rapamycin. In summary, these results suggested that mechanical stress promotes LFCs to produce IGF-1, which facilitates col-I and col-III synthesis via the IGF-1R/AKT/mTORC1 signaling pathway.


2021 ◽  
Author(s):  
Brijesh Kumar Verma ◽  
Aritra Chatterjee ◽  
Paturu Kondaiah ◽  
Namrata Gundiah

Biomaterials, like polydimethylsiloxane (PDMS), are soft, biocompatible, and tuneable, which makes them useful to delineate specific substrate factors that regulate the complex landscape of cell-substrate interactions. We used a commercial formulation of PDMS to fabricate substrates with moduli 40 kPa, 300 kPa, and 1.5 MPa, and cultured HMF3S fibroblasts on them. Gene expression analysis was performed by RT-PCR and Western blotting. Cellular and nuclear morphologies were analyzed using confocal imaging, and MMP-2 and MMP-9 activities were determined with gelatin zymography. Results, comparing mechanotransduction on PDMS substrates with control petridishes, show that substrate stiffness modulates cell morphologies and proliferations. Cell nuclei were rounded on compliant substrates and correlated with increased tubulin expression. Proliferations were higher on stiffer substrates with cell cycle arrest on softer substrates. Integrin alpha5 expression decreased on lower stiffness substrates, and correlated with inefficient TGF-beta; activation. Changes to the activated state of the fibroblast on higher stiffness substrates were linked to altered TGF-beta; secretion. Collagen I, collagen III, and MMP-2 expression levels were lower on compliant PDMS substrates as compared to stiffer ones; there was little MMP-9 activity on substrates. These results demonstrate critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts which is highly relevant in diseases like tissue fibrosis.


2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Min Zhao ◽  
Zhijun Weng ◽  
Yan Huang ◽  
Handan Zheng ◽  
Dong Han ◽  
...  

Background and Aims. Intestinal fibrosis is one of the severe and common complications of Crohn’s disease (CD), but the etiology and pathogenesis remain uncertain. The study intended to examine whether the effect of herb-partitioned moxibustion on rats with CD-associated intestinal fibrosis is associated with the RhoA/ROCK1/MLC pathway. Methods. All experimental rats were randomly allocated into the normal control group (NC), model control group (MC), and herb-partitioned moxibustion group (HPM). Intestinal fibrosis was established in rats with CD by repeated rectal administrations of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Herb-partitioned moxibustion was applied at the Qihai (CV6) and Tianshu (ST25) acupoints once daily for 10 days in the HPM group. In this study, histological changes were examined by hematoxylin and eosin (HE) staining; then, Masson’s trichrome staining was used to assess the degree of fibrosis in each group. Experimental methods of immunohistochemistry, western blotting, and real-time PCR were applied to detect the levels of α-SMA, collagen III, RhoA, ROCK1, and p-MLC. Moreover, the double immunofluorescent staining for the colocalization of both α-SMA and ROCK1 was performed. Results. Contrasted with the normal controls, the collagen deposition and fibrosis scores were increased in colonic tissue of model rats, and HPM decreased the collagen deposition and fibrosis scores. The protein of α-SMA and collagen III in the MC group exceeds that of the NC group; HPM decreased the expression of α-SMA and collagen III in rats with intestinal fibrosis. Similarly, the expression of RhoA, ROCK1, and p-MLC in model rats was obviously increased compared with normal controls; the expression of RhoA, ROCK1, and p-MLC was decreased after HPM. The coexpression of α-SMA and ROCK1 in rats with intestinal fibrosis was higher than normal rats. Conclusion. HPM improves CD-associated intestinal fibrosis by suppressing the RhoA/ROCK1/MLC pathway.


2021 ◽  
Vol 22 (21) ◽  
pp. 11893
Author(s):  
Yue Zhang ◽  
Weiwei Gong ◽  
Mengting Xu ◽  
Shuping Zhang ◽  
Jieru Shen ◽  
...  

Myocardial ischemia or hypoxia can induce myocardial fibroblast proliferation and myocardial fibrosis. Hydrogen sulfide (H2S) is a gasotransmitter with multiple physiological functions. In our present study, primary cardiac fibroblasts were incubated with H2S donor sodium hydrosulfide (NaHS, 50 μM) for 4 h followed by hypoxia stimulation (containing 5% CO2 and 1% O2) for 4 h. Then, the preventive effects on cardiac fibroblast proliferation and the possible mechanisms were investigated. Our results showed that NaHS reduced the cardiac fibroblast number, decreased the hydroxyproline content; inhibited the EdU positive ratio; and down-regulated the expressions of α-smooth muscle actin (α-SMA), the antigen identified by monoclonal antibody Ki67 (Ki67), proliferating cell nuclear antigen (PCNA), collagen I, and collagen III, suggesting that hypoxia-induced cardiac fibroblasts proliferation was suppressed by NaHS. NaHS improved the mitochondrial membrane potential and attenuated oxidative stress, and inhibited dynamin-related protein 1 (DRP1), but enhanced optic atrophy protein 1 (OPA1) expression. NaHS down-regulated receptor interacting protein kinase 1 (RIPK1) and RIPK3 expression, suggesting that necroptosis was alleviated. NaHS increased the sirtuin 3 (SIRT3) expressions in hypoxia-induced cardiac fibroblasts. Moreover, after SIRT3 siRNA transfection, the inhibitory effects on cardiac fibroblast proliferation, oxidative stress, and necroptosis were weakened. In summary, necroptosis inhibition by exogenous H2S alleviated hypoxia-induced cardiac fibroblast proliferation via SIRT3.


2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A967-A967
Author(s):  
Marcel Deken ◽  
Karolina Niewola ◽  
Elisa Matas-Rico ◽  
Ragini Medhi ◽  
Alan Carruthers ◽  
...  

BackgroundAutotaxin (ATX) is a secreted glycoprotein that hydrolyzes lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). The expression of both ATX and LPA is elevated in most solid tumors and plasma. LPA signaling directly modulates tumor cell function and contributes to the development of the fibrotic tumor microenvironment, a mechanism by which tumors evade host immunity and impairs response to therapy. IOA-289 is a potent, orally available autotaxin inhibitor which is being developed as a novel treatment of solid tumours burdened with a high degree of fibrosis.MethodsInhibition of ATX activity in human plasma was determined by measuring reduction in LPA species as quantified by LC-MS/MS. In vitro activity on biomarkers of fibrosis was assessed using the BioMAP screen and fibroblast cell cultures. T cell migration was measured using 48-well chemotaxis chambers. PK/PD studies were performed following a single oral dose of IOA-289 in mice, and plasma LPA was used as a PD biomarker. In vivo efficacy was studied in two models of breast cancer, 4T1 and E0771. Bioinformatics used TCGA and GTEX publicly available datasets.ResultsIOA-289 inhibits plasma LPA18:2 with an IC50 of 36nM, with similar results for other LPA species. IOA-289 inhibited fibrosis relevant factors in the BioMAP phenotypic screen, including sIL-6, MCP-1, αSMA, collagen-III, and sVEGF. In further studies, IOA-289 inhibited the secretion of PAI-1 and IL-6 by stimulated fibroblasts. LPA and cancer cell conditioned media inhibited T cell chemotaxis in vitro and the effect was overcome in the presence of IOA-289. The efficacious human dose of IOA-289 was determined following PK/PD studies using plasma LPA as a biomarker of response to ATX inhibition. In vivo studies showed that IOA-289 inhibited metastasis of 4T1 cells, enhanced the infiltration of T cells into 4T1 s.c. implanted tumors and prevented the growth of primary, orthotopically implanted E0771 tumors. Bioinformatics analysis demonstrated elevated ATX expression in pancreatic cancer (PDAC), and PDAC patient plasma showed a correlation of ATX levels with CA-19-9.ConclusionsThe ATX/LPA pathway represents a novel target for anti-cancer therapy with actions on the tumor, immune cell and stromal environment. IOA-289 is a highly potent and selective inhibitor of ATX with demonstrated monotherapy activity in cancer models. Based on the mechanism of action we are investigating combinations of IOA-289 with chemotherapy, immunotherapy and novel agents in ongoing preclinical studies. An acceptable safety and PK profile support the clinical development of IOA-289 which is currently in a phase I clinical trial.Ethics ApprovalThe 4T1 study was approved by The University Claude Bernard Lyon 1 Ethics Board; approval number DR2014-38 (vM). The E0771 study was reviewed and approved by the Institutional Animal Care and Use Committee of the contract research organization (Covance, Ann Arbor, MI, USA), an AAALAC International accredited program.


Nephron ◽  
2021 ◽  
pp. 1-11
Author(s):  
Liangliang Bi ◽  
Yanjie Huang ◽  
Jing Li ◽  
Xiaoqing Yang ◽  
Gailing Hou ◽  
...  

<b><i>Background:</i></b> Our previous studies had shown pirfenidone (PFD) not only improved tubulointerstitial fibrosis (TIF) but also inhibited the expression of microRNA-21 (miR-21) in the renal tissue of unilateral urethral obstruction (UUO) rats. This study aims to investigate whether PFD can attenuate TIF through inhibiting miR-21 in UUO rats. <b><i>Methods:</i></b> Sprague Dawley rats were divided randomly into sham-operated group, UUO group, and PFD and olmesartan (Olm) treatment groups. Samples were collected on day 14. Expression of miR-21, TGF-β1, Smad3, and Smad7 mRNA in the renal tissue was detected using real-time quantitative PCR. Immunohistochemistry was performed to assess the protein expressions of collagen III, E-cadherin, and α-SMA. Automated capillary Western blotting was used to detect the quantitative expression of TGF-β1, Smad3, p-Smad3, Smad7, collagen III, E-cadherin, and α-SMA in renal tissues. The expression of miR-21 and Smad7 mRNA and the protein levels of collagen III and α-SMA were examined in the miR-21-overexpressing cell line, NRK-52E. <b><i>Results:</i></b> Compared with the UUO group, both PFD and Olm inhibited renal tubular dilation, diffused epithelial cell degeneration and necrosis, and reduced renal interstitial edema, inflammatory cell infiltration, and collagen fiber deposition, while no significant difference between PFD group and Olm group. Informatics-based approaches identified Smad7 as a likely candidate for regulation by miR-21. Compared with the sham group, miR-21 expression was upregulated in the UUO group resulting in the downregulation of Smad7 expression due to degradation. The overexpression of miR-21 in the in vitro model downregulated Smad7 and promoted EMT and ECM accumulation. Protein levels of TGF-β1, Smad3, p-Smad3, collagen III, and α-SMA were upregulated, while E-cadherin protein was downregulated in the UUO group than in the sham group. PFD rather than Olm decreased the expression of miR-21 and increased the expression level of Smad7 mRNA and then inhibited the TGF-β1/Smad3 signaling pathway. Olm only downregulated the TGF-β1/Smad3 signaling pathway. <b><i>Conclusions:</i></b> PFD improves TIF by downregulating the expression of miR-21, then elevating Smad7, and finally inhibiting the activation of the TGF-β1/Smad3 signaling pathway in UUO rats.


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