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

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 hTGFβ2^C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2^C226,228S. We observed a significant increase in IOP 3 weeks post-injection 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_hTGFβ2^C226,228S exhibited 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 hTGFβ2^C226,228S that induces TM dysfunction and outflow resistance.     

Effect of process parameters on phase behavior and particle size of aspirin during freeze concentration

Abstract

The present work investigated effect of process parameters on phase behavior of aspirin (ASP) solution and ASP particle size during freeze concentration. ASP solution (3% w/w in 40% w/w TBA in water) was studied using differential scanning calorimetry (DSC) and cold stage polarized light microscopy (CSPLM) at cooling rates of 0.1, 0.5, 1, 3, 5, 10, 20, and 30?°C/min. Significant reduction in D₉₀ of ASP crystals (from 45.6?µm to 3.0?µm) was observed for ASP crystals in frozen solution with increase in cooling rate. Non-isothermal crystallization kinetics (NITC) of ASP in frozen solution using CSPLM revealed linear increase and an exponential increase in Avrami exponent and crystallization rate constant, respectively. NITC results indicated generation of higher nucleation sites and localized multidimensional crystallization with increase in cooling rate. Moreover, the high degree of supercooling and higher supersaturation achieved with increasing the cooling rate further supported the generation of more nuclei and smaller ASP crystals. Finally, the implication of phase behavior was established using lab scale freeze dryer, where particle size (D₉₀) of 41.8, 14.8, and 7.1?µm was obtained with cooling rate of 1?°C/min, 15?°C/min and quench cooling, respectively. The proposed strategy can be applied for development of lyophilized product that can generate parenteral suspension upon reconstitution.     

Sodium 4-Phenylbutyrate Reduces Ocular Hypertension by Degrading Extracellular Matrix Deposition via Activation of MMP9

Ocular hypertension (OHT) is a serious adverse effect of the widely prescribed glucocorticoid (GC) therapy and, if left undiagnosed, it can lead to glaucoma and complete blindness. Previously, we have shown that the small chemical chaperone, sodium-4-phenylbutyrate (PBA), rescues GC-induced OHT by reducing ocular endoplasmic reticulum (ER) stress. However, the exact mechanism of how PBA rescues GC-induced OHT is not completely understood. The trabecular meshwork (TM) is a filter-like specialized contractile tissue consisting of TM cells embedded within extracellular matrix (ECM) that controls intraocular pressure (IOP) by constantly regulating aqueous humor (AH) outflow. Induction of abnormal ECM deposition in TM is a hallmark of GC-induced OHT. Here, we investigated whether PBA reduces GC-induced OHT by degrading abnormal ECM deposition in TM using mouse model of GC-induced OHT, ex vivo cultured human TM tissues and primary human TM cells. We show that topical ocular eye drops of PBA (1%) significantly lowers elevated IOP in mouse model of GC-induced OHT. Importantly, PBA prevents synthesis and deposition of GC-induced ECM in TM. We report for the first time that PBA can degrade existing abnormal ECM in normal human TM cells/tissues by inducing matrix metalloproteinase (MMP)9 expression and activity. Furthermore, inhibition of MMPs activity by chemical-inhibitor (minocycline) abrogated PBA’s effect on ECM reduction and its associated ER stress. Our study indicates a non-chaperone activity of PBA via activation of MMP9 that degrades abnormal ECM accumulation in TM.