Several population-based studies have identified elevated intraocular pressure (IOP) as a major causative risk factor associated with primary open angle glaucoma (POAG), the most common form of glaucoma that affects millions of people worldwide. Moreover, multi-ethnic clinical trials in several different countries over the last few decades have provided overwhelming evidence showing correlation between lowering of IOP and reduced progression of vision loss. As a result, IOP reducing therapeutic interventions are the gold standard in glaucoma therapy. Although the role of IOP is evident in pathology of POAG, very few studies have delved into the complex physiological mechanisms that regulate IOP homeostasis. From continuous telemetry recordings in nonhuman primates, we now know that IOP is a dynamic variable that fluctuates throughout the day. However, despite the fluctuations, the mean IOP is still maintained within a narrow physiological range. The level of IOP elevation at any given time depends on the resistance to aqueous humor outflow encountered in the conventional outflow pathway consisting of the trabecular meshwork (TM), Schlemm’s canal (SC), and the distal episcleral vessels. Recent studies have suggested that the cells of the outflow pathway have intrinsic ability to detect biomechanical stimuli in their environment (like shear stress) and convert these stimuli into biochemical signals to elicit specific cellular responses. Although mechanotransduction at the TM is deemed critical for IOP homeostasis, we are yet to conclusively identify the exact signaling pathway involved.
In this study, we identify the role of transient receptor potential vanilloid IV channels (TRPV4) in sensing mechanical stress on the TM. We show that shear stress activates TRPV4 channels in human primary TM cells, which leads to endothelial nitric oxide synthase (eNOS)-dependent nitric oxide (NO) production. NO, itself has been identified as a key regulator of IOP. Exogenous NO delivery to the eye has been shown to reduce IOP in humans. However, the underlying mechanism that regulates endogenous levels of NO still remains unknown. To this end, we demonstrate that TRPV4 channels regulate eNOS-dependent NO production in primary human TM cells and ex vivo cultured human TM tissues. We show that TRPV4 activation by mechanical shear leads to activation of eNOS signaling and NO production. Furthermore, pharmacological activation of TRPV4 channels via a selective agonist GSK1016790A (GSK101) leads to eNOS phosphorylation and NO production. In animal models, we demonstrate a role of TRPV4 channels in regulating physiological IOP. Treatment of C57BL/6J mouse eyes with TRPV4 agonist GSK101 leads to reduction in baseline night-time IOP and nominal improvement in outflow facility. We also show that conditional knockout of TRPV4 channels in Ad5-Cre injected TRPV4f/f mice leads to increase in IOP. We use the NOS3-/- (eNOS) to further show that TRPV4 mediated lowering of IOP is eNOS dependent. Dysregulation of the TM cells leads to increase in resistance and IOP elevation. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in lowering the resting IOP. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.
|Advisor:||Zode, Gulab S.|
|Commitee:||Clark, Abe, Pang, Iok-Hou, Krishnamoorthy, Raghu, Rickards, Caroline|
|School:||University of North Texas Health Science Center at Fort Worth|
|Department:||Pharmacology and Neuroscience|
|School Location:||United States -- Texas|
|Source:||DAI-B 82/5(E), Dissertation Abstracts International|
|Subjects:||Ophthalmology, Cellular biology, Physiology, Pathology|
|Keywords:||Glaucoma, Intraocular pressure, Nitric oxide, Shear stress, TRPV4, Therapeutic interventions, Distal episcleral vessels, Trabecular meshwork|
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