Jennifer A Tran

Pulmonary Inflammation Induced by Subacute Ozone Is Augmented in Adiponectin-Deficient Mice: Role of IL-17A

Pulmonary responses to ozone, a common air pollutant, are augmented in obese individuals. Adiponectin, an adipose-derived hormone that declines in obesity, has regulatory effects on the immune system. To determine the role of adiponectin in the pulmonary inflammation induced by extended (48–72 h) low-dose (0.3 parts per million) exposure to ozone, adiponectin-deficient (Adipo−/−) and wild-type mice were exposed to ozone or to room air. In wild-type mice, ozone exposure increased total bronchoalveolar lavage (BAL) adiponectin. Ozone-induced lung inflammation, including increases in BAL neutrophils, protein (an index of lung injury), IL-6, keratinocyte-derived chemokine, LPS-induced CXC chemokine, and G-CSF were augmented in Adipo−/− versus wild-type mice. Ozone also increased IL-17A mRNA expression to a greater extent in Adipo−/− versus wild-type mice. Moreover, compared with control Ab, anti–IL-17A Ab attenuated ozone-induced increases in BAL neutrophils and G-CSF in Adipo−/− but not in wild-type mice, suggesting that IL-17A, by promoting G-CSF release, contributed to augmented neutrophilia in Adipo−/− mice. Flow cytometric analysis of lung cells revealed that the number of CD45+/F4/80+/IL-17A+ macrophages and γδ T cells expressing IL-17A increased after ozone exposure in wild-type mice and further increased in Adipo−/− mice. The IL-17+ macrophages were CD11c− (interstitial macrophages), whereas CD11c+ macrophages (alveolar macrophages) did not express IL-17A. Taken together, the data are consistent with the hypothesis that adiponectin protects against neutrophil recruitment induced by extended low-dose ozone exposure by inhibiting the induction and/or recruitment of IL-17A in interstitial macrophages and/or γδ T cells.

Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization

Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.

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

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-β3s 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-β3s ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing.

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

ABSTRACT The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGF&bgr;3) in reducing &agr;‐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 TGF&bgr;1 or &bgr;3 (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 Eagles 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 T3s action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti‐scarring therapy. HIGHLIGHTS3D cell cultures and ex vivo organ culture models show a difference in function between – TGFB1 and TGFB3.While TGFB1 increases scarring, TGFB3 improves corneal transparency.The difference in fibrotic functions of the TGFB isoforms may be due to PDGF signaling.

Cornea As a Model for Testing CTGF-Based Antiscarring Drugs

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.

Inhibition of Human Corneal Myofibroblast Formation

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 Eagles 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.

TGF-β-target genes are differentially regulated in corneal epithelial cells and fibroblasts

PURPOSE Transforming growth factor-beta (TGF-β) activates the canonical Smad pathway, which includes the Smad family of proteins and SARA (Smad Anchor for Receptor Activation) and other less understood pathways, including one involving p38MAPK. The goal of the current research was to determine if corneal epithelial cells and fibroblasts used the classical or alternative TGF-β-signaling pathways. To examine this question, we made use of Trx-SARA, which inhibits native SARA, thus blocking the Smad pathway. METHODS A human corneal epithelial cell line (HCE-TJ), and stromal fibroblasts (HCF) were infected with retroviruses (RTV) containing either Trx-SARA or Trx-GA (a control plasmid). The effect of Trx-SARA on thrombospondin-1 (TSP-1) expression in both cell types, p15ink4b expression in HCE-TJ, and cellular fibronectin (cFN) expression in HCF was determined. In addition, the effect of p38MAPK inhibitor on TSP-1 and p15ink4b were examined. RESULTS In HCE-TJ with TGF-β1, TSP-1-protein levels increased and peaked at 24 hours. Trx-SARA reduced TSP-1 expression in HCE-TJ, but had no effect on p15ink4b. With HCF, Trx-SARA failed to reduce TSP-1 expression; however, cFN expression decreased and proliferation was inhibited. By blocking the p38MAPK pathway, TSP-1 expression was reduced in HCF and p15ink4b expression was decreased in HCE-TJ. CONCLUSIONS Surprisingly, TSP-1 was regulated through the Smad pathway in HCE-TJ and the p38MAPK pathway in HCF. The p38MAPK pathway also induced p15ink4b in HCE-TJ. Our results indicate that not all TGF-β-target proteins require the Smad pathway, and it may be possible to block certain TGF-β-target proteins without blocking the expression of all the TGF-β-target proteins.