A Novel Mouse Model of TGFβ2-Induced Ocular Hypertension Using Lentiviral Gene Delivery

Shruti V. Patil, Ramesh B. Kasetti, J. Cameron Millar, Gulab S. Zode

Research output: Contribution to journalArticlepeer-review

Abstract

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) β2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFβ2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFβ2 in the TM. We developed an LV vector-encoding active hTGFβ2C226, 228Sunder the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2C226, 228S. We observed a significant increase in IOP 3 weeks postinjection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFβ2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFβ2C226, 228Sexhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFβ2C226, 228Sthat induces TM dysfunction and outflow resistance.

Original languageEnglish
Article number6883
JournalInternational journal of molecular sciences
Volume23
Issue number13
DOIs
StatePublished - 1 Jul 2022

Keywords

  • TGFβ2
  • intraocular pressure
  • lentiviruses
  • rodent mouse models of ocular hypertension
  • trabecular meshwork

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