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Article ; Online: FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro.

Yeung, Vincent / Sriram, Sriniwas / Tran, Jennifer A / Guo, Xiaoqing / Hutcheon, Audrey E K / Zieske, James D / Karamichos, Dimitrios / Ciolino, Joseph B

Biomolecules

2021 Volume 11, Issue 11

Abstract: Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as ... ...

Abstract	Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis-
MeSH term(s)	Cell Differentiation ; Fibroblasts ; Humans ; Myofibroblasts ; Transforming Growth Factor beta1
Chemical Substances	TGFB1 protein, human ; Transforming Growth Factor beta1
Language	English
Publishing date	2021-11-12
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2701262-1
ISSN	2218-273X ; 2218-273X
ISSN (online)	2218-273X
ISSN	2218-273X
DOI	10.3390/biom11111682
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Article: Cornea As a Model for Testing CTGF-Based Antiscarring Drugs.

Sriram, Sriniwas / Tran, Jennifer A / Zieske, James D

Bone and tissue regeneration insights

2016 Volume 7

Abstract: Scarring remains a serious complication of the wound healing process that can lead to the formation of excessive fibrous connective tissue in an organ or tissue leading to pain and loss of function. This process is mainly regulated by Transforming growth ...

Abstract	Scarring remains a serious complication of the wound healing process that can lead to the formation of excessive fibrous connective tissue in an organ or tissue leading to pain and loss of function. This process is mainly regulated by Transforming growth factor β1 (TGF-β1), which binds to receptors and induces its downstream mediator, Connective tissue growth factor (CTGF). The number of drugs targeting CTGF for treating scars has been on the rise in the past few years. The purpose of this article is to suggest the possibility of using cornea as a model for testing anti-CTGF therapies for scarring.
Language	English
Publishing date	2016-06-20
Publishing country	United States
Document type	Journal Article
ZDB-ID	2659433-X
ISSN	1179-061X
ISSN	1179-061X
DOI	10.4137/BTRI.S19954
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Article ; Online: Initiation of fibrosis in the integrin Αvβ6 knockout mice.

Wu, Wenjing / Hutcheon, Audrey E K / Sriram, Sriniwas / Tran, Jennifer A / Zieske, James D

Experimental eye research

2018 Volume 180, Page(s) 23–28

Abstract: We previously demonstrated that β6 knockout mice showed impaired wound repair in corneal debridement and keratectomy wounds. In the current investigation, we continued our examination of integrin αvβ6 in order to determine if it was required for the ... ...

Abstract	We previously demonstrated that β6 knockout mice showed impaired wound repair in corneal debridement and keratectomy wounds. In the current investigation, we continued our examination of integrin αvβ6 in order to determine if it was required for the initiation of wound healing in a corneal wound model that normally heals in a fibrotic manner. A full-thickness corneal incision was made in C57BL/6 J wild type (WT) and C57BL/6-Itgb6 KO (β6
MeSH term(s)	Actins/metabolism ; Animals ; Antigens, Neoplasm/physiology ; Cornea/pathology ; Corneal Injuries/metabolism ; Corneal Injuries/physiopathology ; Debridement ; Disease Models, Animal ; Eye Injuries, Penetrating/physiopathology ; Female ; Fibrosis/pathology ; Fluorescent Antibody Technique, Indirect ; Integrins/physiology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Thrombospondin 1/metabolism ; Wound Healing/physiology
Chemical Substances	Acta2 protein, mouse ; Actins ; Antigens, Neoplasm ; Integrins ; Thrombospondin 1 ; integrin alphavbeta6 ; Thbs1 protein, mouse
Language	English
Publishing date	2018-11-28
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	80122-7
ISSN	1096-0007 ; 0014-4835
ISSN (online)	1096-0007
ISSN	0014-4835
DOI	10.1016/j.exer.2018.11.027
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Article ; Online: Inhibition of Human Corneal Myofibroblast Formation.

Guo, Xiaoqing / Sriram, Sriniwas / Tran, Jennifer A / Hutcheon, Audrey E K / Zieske, James D

Investigative ophthalmology & visual science

2018 Volume 59, Issue 8, Page(s) 3511–3520

Abstract: Purpose: Transforming growth factor-beta (TGF-β) isoform 1 (T1) is involved in corneal fibrotic wound healing by stimulating myofibroblast transformation and altering fibrotic gene expression. In this study, two specific inhibitors were used to dissect ... ...

Abstract	Purpose: Transforming growth factor-beta (TGF-β) isoform 1 (T1) is involved in corneal fibrotic wound healing by stimulating myofibroblast transformation and altering fibrotic gene expression. In this study, two specific inhibitors were used to dissect the relationship between myofibroblast generation and the TGF-β/Smad- or TGF-β/p38-signaling pathway in human corneal fibroblasts (HCF). Methods: In HCF, Trx-SARA (Smad-pathway inhibitor) was used to block the TGF-β/Smad-signaling pathway, and the p38 inhibitor (p38inh, SB202190) was used to inhibit p38MAPK, thus blocking the TGF-β/p38-signaling pathway. HCF ± Trx-SARA or Trx-GA (SARA control) were serum starved overnight in Eagle's minimum essential medium (EMEM) ± p38inh, grown in EMEM ± T1 ± p38inh for 24 hours, and then processed for indirect-immunofluorescence, Western blot, or quantitative real-time polymerase chain reaction to examine α-smooth muscle actin (αSMA) and other fibrotic genes, such as fibronectin, thrombospondin1, and type III collagen. In addition, the morphology and the effect of p38inh on myofibroblast phenotype after myofibroblast formation were examined. Results: We observed that Trx-SARA had little effect on αSMA expression, indicating that blocking the Smad pathway did not significantly inhibit myofibroblast formation. However, p38inh did significantly inhibit αSMA and other fibrotic genes, thus efficiently preventing the transition of HCFs to myofibroblasts. In addition, morphology changed and αSMA decreased in myofibroblasts exposed to p38inh medium, as compared with controls. Conclusions: HCF transition to myofibroblasts was mainly through the p38 pathway. Therefore, blocking the p38 pathway may be a potential therapeutic tool for human corneal fibrosis prevention/treatment, because it controls myofibroblast formation in human corneal cells, while leaving other functions of T1 unaffected.
MeSH term(s)	Actins/genetics ; Blotting, Western ; Cell Line ; Cell Transdifferentiation/physiology ; Cells, Cultured ; Corneal Keratocytes/cytology ; Corneal Keratocytes/metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme Inhibitors/pharmacology ; Fluorescent Antibody Technique, Indirect ; Humans ; Imidazoles/pharmacology ; MAP Kinase Signaling System/physiology ; Myofibroblasts/cytology ; Myofibroblasts/metabolism ; Pyridines/pharmacology ; Real-Time Polymerase Chain Reaction ; Smad Proteins/metabolism ; Transforming Growth Factor beta/metabolism
Chemical Substances	ACTA2 protein, human ; Actins ; Enzyme Inhibitors ; Imidazoles ; Pyridines ; Smad Proteins ; Transforming Growth Factor beta ; 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole (PVX798P8GI)
Language	English
Publishing date	2018-07-19
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	391794-0
ISSN	1552-5783 ; 0146-0404
ISSN (online)	1552-5783
ISSN	0146-0404
DOI	10.1167/iovs.18-24239
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Article ; Online: Connective tissue growth factor is not necessary for haze formation in excimer laser wounded mouse corneas.

Xiaodi Feng / Liya Pi / Sriniwas Sriram / Gregory S Schultz / Daniel J Gibson

PLoS ONE, Vol 12, Iss 2, p e

2017 Volume 0172304

Abstract: We sought to determine if connective tissue growth factor (CTGF) is necessary for the formation of corneal haze after corneal injury. Mice with post-natal, tamoxifen-induced, knockout of CTGF were subjected to excimer laser phototherapeutic keratectomy ( ... ...

Abstract	We sought to determine if connective tissue growth factor (CTGF) is necessary for the formation of corneal haze after corneal injury. Mice with post-natal, tamoxifen-induced, knockout of CTGF were subjected to excimer laser phototherapeutic keratectomy (PTK) and the corneas were allowed to heal. The extent of scaring was observed in non-induced mice, heterozygotes, and full homozygous knockout mice and quantified by macrophotography. The eyes from these mice were collected after euthanization for re-genotyping to control for possible Cre-mosaicism. Primary corneal fibroblasts from CTGF knockout corneas were established in a gel plug assay. The plug was removed, simulating an injury, and the rate of hole closure and the capacity for these cells to form light reflecting cells in response to CTGF and platelet-derived growth factor B (PDGF-B) were tested and compared to wild-type cells. We found that independent of genotype, each group of mice was still capable of forming light reflecting haze in the cornea after laser ablation (p = 0.40). Results from the gel plug closure rate in primary cell cultures of knockout cells were not statistically different from serum starved wild-type cells, independent of treatment. Compared to the serum starved wild-type cells, stimulation with PDGF-BB significantly increased the KO cell culture's light reflection (p = 0.03). Most interestingly, both reflective cultures were positive for α-SMA, but the cellular morphology and levels of α-SMA were distinct and not in proportion to the light reflection seen. This new work demonstrates that corneas without CTGF can still form sub-epithelial haze, and that the light reflecting phenotype can be reproduced in culture. These data support the possibilities of growth factor redundancy and that multiple pro-haze pathways exist.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
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Article ; Online: Connective tissue growth factor is not necessary for haze formation in excimer laser wounded mouse corneas.

Feng, Xiaodi / Pi, Liya / Sriram, Sriniwas / Schultz, Gregory S / Gibson, Daniel J

PloS one

2017 Volume 12, Issue 2, Page(s) e0172304

Abstract	We sought to determine if connective tissue growth factor (CTGF) is necessary for the formation of corneal haze after corneal injury. Mice with post-natal, tamoxifen-induced, knockout of CTGF were subjected to excimer laser phototherapeutic keratectomy (PTK) and the corneas were allowed to heal. The extent of scaring was observed in non-induced mice, heterozygotes, and full homozygous knockout mice and quantified by macrophotography. The eyes from these mice were collected after euthanization for re-genotyping to control for possible Cre-mosaicism. Primary corneal fibroblasts from CTGF knockout corneas were established in a gel plug assay. The plug was removed, simulating an injury, and the rate of hole closure and the capacity for these cells to form light reflecting cells in response to CTGF and platelet-derived growth factor B (PDGF-B) were tested and compared to wild-type cells. We found that independent of genotype, each group of mice was still capable of forming light reflecting haze in the cornea after laser ablation (p = 0.40). Results from the gel plug closure rate in primary cell cultures of knockout cells were not statistically different from serum starved wild-type cells, independent of treatment. Compared to the serum starved wild-type cells, stimulation with PDGF-BB significantly increased the KO cell culture's light reflection (p = 0.03). Most interestingly, both reflective cultures were positive for α-SMA, but the cellular morphology and levels of α-SMA were distinct and not in proportion to the light reflection seen. This new work demonstrates that corneas without CTGF can still form sub-epithelial haze, and that the light reflecting phenotype can be reproduced in culture. These data support the possibilities of growth factor redundancy and that multiple pro-haze pathways exist.
MeSH term(s)	Animals ; Connective Tissue Growth Factor/physiology ; Cornea/metabolism ; Cornea/pathology ; Corneal Injuries/etiology ; Corneal Injuries/metabolism ; Corneal Injuries/pathology ; Corneal Opacity/etiology ; Corneal Opacity/metabolism ; Corneal Opacity/pathology ; Lasers, Excimer/adverse effects ; Mice ; Mice, Knockout ; Wound Healing
Chemical Substances	CCN2 protein, mouse ; Connective Tissue Growth Factor (139568-91-5)
Language	English
Publishing date	2017-02-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0172304
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Article ; Online: Development of wound healing models to study TGFβ3's effect on SMA.

Sriram, Sriniwas / Tran, Jennifer A / Guo, Xiaoqing / Hutcheon, Audrey E K / Kazlauskas, Andrius / Zieske, James D

Experimental eye research

2017 Volume 161, Page(s) 52–60

Abstract: The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFβ3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in ... ...

Abstract	The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFβ3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFβ1 or β3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle's minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3's action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy.
MeSH term(s)	Actins/genetics ; Animals ; Cell Culture Techniques ; Cornea/drug effects ; Cornea/metabolism ; Corneal Keratocytes/drug effects ; Corneal Keratocytes/metabolism ; Corneal Stroma/cytology ; Disease Models, Animal ; Fluorescent Antibody Technique, Indirect ; Organ Culture Techniques ; Platelet-Derived Growth Factor/metabolism ; RNA, Messenger/genetics ; Rabbits ; Real-Time Polymerase Chain Reaction ; Transforming Growth Factor beta1/pharmacology ; Transforming Growth Factor beta3/pharmacology ; Wound Healing/physiology
Chemical Substances	Actins ; Platelet-Derived Growth Factor ; RNA, Messenger ; Transforming Growth Factor beta1 ; Transforming Growth Factor beta3
Language	English
Publishing date	2017-06-06
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	80122-7
ISSN	1096-0007 ; 0014-4835
ISSN (online)	1096-0007
ISSN	0014-4835
DOI	10.1016/j.exer.2017.06.005
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Article ; Online: PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts.

Sriram, Sriniwas / Tran, Jennifer A / Guo, Xiaoqing / Hutcheon, Audrey E K / Lei, Hetian / Kazlauskas, Andrius / Zieske, James D

Investigative ophthalmology & visual science

2017 Volume 58, Issue 2, Page(s) 1179–1186

Abstract: Purpose: The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) ... ...

Abstract	Purpose: The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-β3's response to wounding. Methods: A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-β1 or -β3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results: In HCF, TGF-β3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-β1, while in HCF-P, both TGF-β1 and -β3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-β3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. Conclusions: These results show that TGF-β3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-β3 treatment. Understanding the role of PDGFRα in TGF-β3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing.
MeSH term(s)	Actins/metabolism ; Analysis of Variance ; Cell Movement/drug effects ; Cells, Cultured ; Cornea/cytology ; Fibroblasts/drug effects ; Fibroblasts/metabolism ; Humans ; Polymerase Chain Reaction ; Receptor, Platelet-Derived Growth Factor alpha/physiology ; Transforming Growth Factor beta1/pharmacology ; Transforming Growth Factor beta1/physiology ; Transforming Growth Factor beta3/pharmacology ; Transforming Growth Factor beta3/physiology ; Wound Healing/physiology
Chemical Substances	ACTA2 protein, human ; Actins ; Transforming Growth Factor beta1 ; Transforming Growth Factor beta3 ; Receptor, Platelet-Derived Growth Factor alpha (EC 2.7.10.1)
Language	English
Publishing date	2017-02-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	391794-0
ISSN	1552-5783 ; 0146-0404
ISSN (online)	1552-5783
ISSN	0146-0404
DOI	10.1167/iovs.16-20016
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Article ; Online: Reduction of corneal scarring in rabbits by targeting the TGFB1 pathway with a triple siRNA combination

Sriniwas Sriram / Daniel Gibson / Paulette Robinson / Sonal Tuli / Alfred S. Lewin / Gregory Schultz

Advances in Bioscience and Biotechnology, Vol 04, Iss 10, Pp 47-

2013 Volume 55

Abstract: Purpose: The transforming growth factor beta1 (TGFB1) pathway has been linked to fibrosis in several tissues including skin, liver, kidney and the cor nea. In this study, a RNA interference-based approach using siRNAs targeting three critical scarring ... ...

Abstract	Purpose: The transforming growth factor beta1 (TGFB1) pathway has been linked to fibrosis in several tissues including skin, liver, kidney and the cor nea. In this study, a RNA interference-based approach using siRNAs targeting three critical scarring genes, TGFB1, TGFB receptor 2 (TGFBR2) and connective tissue growth factor (CTGF), was tested for effects on reducing alpha smooth muscle actin (SMA) and corneal scarring (haze) in excimer laser ablated rabbit corneas. Methods: Levels of TGFB1 and CTGF mRNAs were measured using qRT-PCR in the epithelial and endothelial cell layers of normal and excimer ablated rabbit corneas at 30 minutes, 1 day and 2 days after ablation. Two different scarring models were utilized to assess the effects of the triple siRNA combination on corneal scarring. In the first model, rabbit corneas were unevenly ablated creating a mesh pattern then treated immediately with the triple siRNA combination. After 1 day the ablated areas of corneas were collected and levels of mRNAs for TGFB1, TGFBR2 and CTGF were measured. After 14 days, levels of mRNA for SMA were measured and SMA protein im munolocalized in frozen sections. In the second model, rabbit corneas were uniformly ablated to a depth of 155 microns followed by three daily doses of the triple combination of siRNA. After 14 days, corneas were photographed and images were analyzed using Image J software to assess corneal scarring. Corneas were also analyzed for levels of SMA mRNA. Results: In both unwounded and wounded corneas, levels of TGFB1 and CTGF mRNA were always significantly higher in endothelial cells than in epithelial cells (10 to 30 fold). Thirty minutes after injury, levels of both TGFB1 and CTGF mRNAs increased approximately 20-fold in both epithelial and endothelial cells, and further increased approximately 60-fold in 2 days. In the first therapeutic experiment with a single siRNA dose, two of three rabbits showed substantial reductions of all three target genes after 1 day with a maximum knock down of 80% of TGFb 1, 50% reduction of TGFBR2 and 40% reduction of CTGF mRNA levels and reduc ed SMA mRNA at day 14. In the second therapeutic experiment with multiple doses of siRNA treatment, both rabbits showed a ~22% reduction in scar formation at day 14 as calculated by image analysis. There was also a corresponding 70% and 60% reduction of SMA RNA expression. Conclusion: These results demonstrate that both TGFB1 and CTGF dramatically increase in rabbit corneal epithelial and endothelial cells after injury. Treatment of excimer ablated rabbit corneas with a triple combination of siRNAs effec tively reduced levels of the target genes and SMA, lead ing to reduced corneal scarring at 14 days, suggesting that this triple siRNA combination may be an effective new approach to reducing scarring in cornea and other tissues.
Keywords	RNA Interference ; siRNA Combination ; Corneal Scarring ; TGFB1 ; CTGF ; Biology (General) ; QH301-705.5 ; Science ; Q ; DOAJ:Biology ; DOAJ:Biology and Life Sciences
Subject code	333
Language	English
Publishing date	2013-09-01T00:00:00Z
Publisher	Scientific Research Publishing
Document type	Article ; Online
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Article ; Online: Triple combination of siRNAs targeting TGFβ1, TGFβR2, and CTGF enhances reduction of collagen I and smooth muscle actin in corneal fibroblasts.

Sriram, Sriniwas / Robinson, Paulette / Pi, Liya / Lewin, Alfred S / Schultz, Gregory

Investigative ophthalmology & visual science

2013 Volume 54, Issue 13, Page(s) 8214–8223

Abstract: Purpose: Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic ... ...

Abstract	Purpose: Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA). Methods: Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict "effective" and "ineffective" triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry. Results: Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%-80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability. Conclusions: Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.
MeSH term(s)	Actins/metabolism ; Animals ; Cells, Cultured ; Collagen/metabolism ; Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/metabolism ; Cornea/metabolism ; Cornea/pathology ; Corneal Diseases/genetics ; Corneal Diseases/pathology ; Corneal Diseases/therapy ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts/metabolism ; Fibroblasts/pathology ; Genetic Therapy/methods ; Immunohistochemistry ; RNA, Small Interfering/genetics ; RNA, Small Interfering/metabolism ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta1/genetics ; Transforming Growth Factor beta1/metabolism ; Transforming Growth Factor beta2/genetics ; Transforming Growth Factor beta2/metabolism
Chemical Substances	Actins ; RNA, Small Interfering ; Transforming Growth Factor beta1 ; Transforming Growth Factor beta2 ; Connective Tissue Growth Factor (139568-91-5) ; Collagen (9007-34-5)
Language	English
Publishing date	2013-12-17
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	391794-0
ISSN	1552-5783 ; 0146-0404
ISSN (online)	1552-5783
ISSN	0146-0404
DOI	10.1167/iovs.13-12758
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In stock of ZB MED Cologne/Königswinter

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