PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts

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

doi:10.1167/iovs.16-20016

Cited by 11 publications

(12 citation statements)

References 28 publications

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“…Sidney and Hopkinson showed that TGF-β3 led to lower levels of ALDH3A1 and vimentin and higher levels α-SMA gene expression when compared to cells cultured under serum and growth factor free conditions. 12 Another study recently showed positive α-SMA staining resulting from the addition of TGF-β3 although the amount of staining was significantly less than at the same concentration of TGF-β1 55 From our results it would appear that both TGF-β1 and TGF-β3 promote a myofibroblastic phenotype.…”

Section: Discussionsupporting

confidence: 49%

Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype

Fernández‐Pérez

Ahearne

2018

Current Eye Research

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Purpose: To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration. Methods: Proliferation was assessed by PrestoBlue ™ , morphology was monitored by phase contrast microscopy, phenotype was analyzed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay. Results: Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-β1) and 3 (TGF-β3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression. Conclusions: Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.

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Section: Discussionsupporting

confidence: 49%

Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype

Fernández‐Pérez

Ahearne

2018

Current Eye Research

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“…Additional file 1: Supplementary Table S1. Primer pair sequences used for the gene expression profiling [35][36][37][38][39][40][41][42][43].…”

Section: Supplementary Informationmentioning

confidence: 99%

Comprehensive analysis of skeletal muscle- and bone-derived mesenchymal stem/stromal cells in patients with osteoarthritis and femoral neck fracture

et al. 2020

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Background: Mesenchymal stem/stromal cells (MSCs) can replenish the aged cells of the musculoskeletal system in adult life. Stem cell exhaustion and decrease in their regenerative potential have been suggested to be hallmarks of aging. Here, we investigated whether muscle-and bone-derived MSCs of patients with osteoarthritis and osteoporosis are affected by this exhaustion, compared to healthy donors. Methods: Patients with primary osteoarthritis, femoral neck fractures due to osteoporosis, and healthy donors (controls) were included. MSCs were isolated from the skeletal muscle and subchondral bone from each patient and compared using ex vivo and in vitro analyses, including immunophenotyping, colony-forming unit fibroblast assays, growth kinetics, cell senescence, multilineage potential, and MSC marker gene expression profiling. Results: Freshly isolated cells from muscle from patients with osteoarthritis showed a lower proportion of CD45/ CD19/CD14/CD34-negative cells compared to patients with osteoporosis and healthy donors. Freshly isolated muscle cells from patients with osteoarthritis and osteoporosis also showed higher clonogenicity compared to healthy donors. MSCs from both tissues of osteoarthritis patients showed significantly reduced osteogenesis and MSCs from the bone also reduced adipogenesis. Chondrogenic pellet diameter was reduced in bone-derived MSCs from both patient groups compared to healthy donors. A significant positive correlation was observed between adipogenesis and CD271 expression in muscle-derived MSCs. CD73 was significantly lower in bone-derived MSCs from osteoarthritis patients, compared to osteoporosis patients. Gene expression profiling showed significantly lower expression of MSC marker gene leptin receptor, LEPR, previously identified as the major source of the bone and adipocytes in the adult bone marrow, in bone-derived MSCs from patients with osteoarthritis in comparison with osteoporotic patients and healthy donors.

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“…In addition to the TGFβ family, there is extensive literature available in different models associating platelet-derived growth factors (PDGFs) and their receptors (PDGFR) to fibrosis(Kaur et al, 2009; Sriram et al, 2017). The PDGF/PDGFR family includes four genes encoding PDGF isoforms (A, B, C, D) and two genes for PDGFR subunits (α and β).…”

Section: Introductionmentioning

confidence: 99%

Development of wound healing models to study TGFβ3's effect on SMA

Sriram

Tran

Guo

et al. 2017

Experimental Eye Research

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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.

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PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts

Cited by 11 publications

References 28 publications

Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype

Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype

Comprehensive analysis of skeletal muscle- and bone-derived mesenchymal stem/stromal cells in patients with osteoarthritis and femoral neck fracture

Development of wound healing models to study TGFβ3's effect on SMA

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