Browsing by Subject "TGFβ2"

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    Age and sex affect TGFβ2-induced ocular hypertension in C57BL/6J mice
    (Elsevier, 2022) Sugali, Chenna Kesavulu; Rayana, Naga Pradeep; Dai, Jiannong; Peng, Michael; Mao, Weiming; Ophthalmology, School of Medicine
    Glaucoma is a leading cause of blindness worldwide. The loss of vision in glaucoma patients is due to optic nerve damage. The most important risk factor of glaucoma is elevated intraocular pressure (IOP) which is due to glaucomatous changes in the trabecular meshwork. Animal models, especially mouse models for ocular hypertension (OHT), are important for studying glaucoma. Published studies showed that 2.5X107 PFU adenoviral vectors expressing the biologically active form of human TGFβ2 elevate IOP in female C57BL/6J mice when they are intravitreally delivered. In this study, we found that 2.5X107 PFU adenoviral TGFβ2 vector did not elevate IOP in 3- or 5-month old male C57BL/6J mice. In contrast, 5X107 PFU of the same viral vectors elevated IOP in both 3- and 5-month old male C57BL/6J mice. Also, 5-month old mice showed earlier OHT and higher IOP compared to 3-month old mice. In summary, our data showed that age and sex play roles in adenoviral vector-mediated TGFβ2-induced OHT in C57BL/6J mice.
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    The application of lentiviral vectors for the establishment of TGFβ2-induced ocular hypertension in C57BL/6J mice
    (Elsevier, 2022) Peng, Michael; Margetts, Tyler J.; Rayana, Naga Pradeep; Sugali, Chenna Kesavulu; Dai, Jiannong; Mao, Weiming; Biochemistry and Molecular Biology, School of Medicine
    Elevated levels of TGFβ2 in the aqueous humor is associated with the pathological changes in the trabecular meshwork (TM). These changes lead to ocular hypertension (OHT), the most important risk factor for the development and progression of primary open angle glaucoma (POAG), a leading cause of blindness worldwide. Therefore, TGFβ2 is frequently used to develop OHT models including in perfusion cultured eyes and in mouse eyes. Adenovirus-mediated overexpression of human mutant TGFβ2 has demonstrated great success in increasing intraocular pressure (IOP) in mouse eyes. However, adenoviruses have limited capacity for a foreign gene, induce transient expression, and may cause ocular inflammation. Here, we explored the potential of using lentiviral vectors carrying the mutant human TGFβ2C226S/C228S (ΔhTGFβ2C226S/C228S) gene expression cassette for the induction of OHT in C57BL/6J mice. Lentiviral vectors using CMV or EF1α promoter to drive the expression of ΔhTGFβ2C226S/C228S were injected into one of the mouse eyes and the fellow eye was injected with the same vector but expressing GFP/mCherry as controls. Both intravitreal and intracameral injection routes were tested in male and female mice. We did not observe significant IOP changes using either promoter or injection route at the dose of 8×105 PFU/eye. Immunostaining showed normal anterior chamber angle structures and a slight increase in TGFβ2 expression in the TM of the eyes receiving intracameral viral injection but not in those receiving intravitreal viral injection. At the dose of 2×106 PFU/eye, intracameral injection of the lentiviral vector with the CMV-ΔhTGFβ2C226S/C228S cassette induced significant IOP elevation and increased the expression of TGFβ2 and fibronectin isoform EDA in the TM. Our data suggest that lentiviral doses are important for establishing the TGFβ2-induced OHT model in the C57BL/6J strain.
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    Using CRISPR Interference as a Therapeutic Approach to Treat TGFβ2-Induced Ocular Hypertension and Glaucoma
    (Association for Research in Vision and Ophthalmology, 2021-09-02) Rayana, Naga Pradeep; Sugali, Chenna Kesavulu; Dai, Jiannong; Peng, Michael; Liu, Shaohui; Zhang, Yucheng; Wan, Jun; Mao, Weiming; Ophthalmology, School of Medicine
    Purpose: Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide with elevated intraocular pressure (IOP) as the most important risk factor. POAG IOP elevation is due to pathological changes in the trabecular meshwork (TM). Elevated TGFβ2 contributes to these changes and increases IOP. We have shown that histone hyperacetylation is associated with TGFβ2 elevation in the TM. In this study, we determined if clustered regularly interspaced short palindromic repeats (CRISPR) interference could specifically deacetylate histones and decrease TGFβ2 in the TM. Methods: We tested the efficiency of different promoters in driving KRAB-dCAS9 expression in human TM cells. We also screened and determined the optimal sgRNA sequence in the inhibition of TGFβ2. Chromatin immunoprecipitation-qPCR was used to determine the binding of KRAB-dCAS9. An adenovirus-mediated TGFβ2-induced ocular hypertension (OHT) mouse model was used to determine the effect of the CRISPR interference system in vivo. Results: We found that the CRISPR interference system inhibited TGFβ2 expression in human TM cells, and properly designed sgRNA targeted the promoter of the TGFβ2 gene. Using sgRNA targeting the CMV promoter of the Ad5-CMV-TGFβ2 viral vector, we found that lentivirus-mediated KRAB-dCAS9 and sgRNA expression was able to inhibit Ad5-CMV-TGFβ2-induced OHT in C57BL/6J female and male mice eyes. This inhibition of OHT was associated with decreased levels of TGFβ2 and extracellular matrix proteins in the mouse eye. Conclusions: Our results indicate that CRISPR interference is a useful tool for gene inhibition and may be a therapeutic approach to treat TGFβ2-induced OHT.