Glaucoma Dialogue IGR 22-1
See also comment(s) by Heather McGowan & Louis Pasquale
• Darryl Overby & Daniel Stamer
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The authors thank the commentators for their constructive feedback. The authors would like to clarify few comments made by Overby and Stamer related to our recent PNAS manuscript.
- eNOS expression in TM: Overby and Stamer questioned the validity of our conclusion that eNOS is expressed in TM cells. We have utilized multiple approaches to demonstrate that eNOS is expressed in human TM cells/tissues. Using Western blot for phosphorylated and total eNOS and immunostaining for total eNOS, we have shown the presence of phosphorylated eNOS and total eNOS in both human primary TM cells and tissues. The specificity of antibodies used for phosphorylated eNOS and total eNOS was characterized using eNOS knock out mice (Supplementary information). We utilized 6 different donor eyes to examine eNOS protein levels in outflow pathway and all donors showed eNOS expression in TM and SC cells. We have also utilized multiple strains of primary human TM cells and ex-vivo corneoscleral segment tissues to further support eNOS expression in TM. Similar eNOS expression has been shown by other labs as well. Studies by Nathanson and M McKee, 1995 demonstrated the presence of eNOS in both TM and SC cells in human donor eyes.1 eNOS expression in TM cells was also observed by Fernández-Durango et al 2008.2 In contrast to our studies, which examined protein levels, studies cited by Overby and Stamer utilized single-cell RNA expression. It is conceivable that mRNA transcripts are not accurately detected by single cell RNA analysis. Nonetheless, detection of eNOS protein is more functionally relevant in this case.
- TRVP4-mediated IOP mechanosensation is more likely occurring within SC, and not the TM: We do not agree with this opinion. Our findings that TRPV4-eNOS signaling in TM plays important role in IOP regulation is based on strong mouse and human data presented in the manuscript. Importantly, adenoviral expression of Cre resulted in loss of TRPV4 in mouse TM, elevating IOP in TRPV4f/f mice. Adenoviral injections have selective tropism for TM cells in mice as previously described. In contrast, loss of TRPV4 in SC did not elevate IOP significantly in TRPV4f/f mice (data not published). TRPV4f/f mice were crossed with Cdh5(endothelial promoter)-driven Cre-ERT2 mice and tamoxifen eye drops were given to induce Cre. These data further establish the role of TRPV4-eNOS signaling in TM cells. As discussed in the manuscript, it is likely that SC plays a critical role in shear stress-sensing of IOP via mechanisms independent of TRPV4, or SC cells may not need TRPV4 channels to activate eNOS. Given the focus of our study on the TM, we only assessed pharmacological activation of TRPV4 channels in SC cells and not flow-induced activation. In the future, we would like to perform a more thorough comparison of TRPV4 channels in TM and SC cells in shear-stress inducing flow setting.
- We would also like to address the question whether mechanisms involving flow/shear mediated mechanosensation are physiologically relevant in the TM. The expression of mechanosensory ion channels like TRPV4 in the TM has already been shown.5-7Our study and a previously published independent report has shown TM cells are capable of sensing flow/shear. Therefore, we now know the capacity of TM cells to sense flow/shear in vitro. We also know that activation of these channels results in Ca2+ entry in TM cells. Recently published data from other groups suggest that TM cells have Ca2+-regulated smooth muscle-like contractile machinery and TRPV4 channels play a role in cytoskeletal remodeling.7 We acknowledge that there are multiple players involved in IOP homeostasis. For example, TRPV4 activation leads to immediate entry of extracellular Ca2+ that is known to contract TM cells. As discussed in our manuscript, perhaps TRPV4 activation leads to an initial contraction. However, after a lag phase, Ca2+ entry through TRPV4 leads to the activation of eNOS and production of NO, a negative regulator of cell contraction (relaxing TM). We postulate that this oscillatory system maintains the tone of the TM, and facilitates the clearance of aqueous humor out of the eye as suggested by studies from Murray Johnstone.8 Our future work will investigate this oscillatory behavior in response to TRPV4 channel activation in details.
- Nathanson JA, McKee M. Identification of an extensive system of nitric oxide-producing cells in the ciliary muscle and outflow pathway of the human eye. Invest Ophthalmol Vis Sci. 1995;36(9):1765-73. Epub 1995/08/01. PubMed PMID: 7543462.
- Fernandez-Durango R, Fernandez-Martinez A, Garcia-Feijoo J, Castillo A, de la Casa JM, Garcia-Bueno B, Perez-Nievas BG, Fernandez-Cruz A, Leza JC. Expression of nitrotyrosine and oxidative consequences in the trabecular meshwork of patients with primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 2008;49(6):2506-11. Epub 2008/02/26. doi: 10.1167/iovs.07-1363. PubMed PMID: 18296660.
- Kasetti RB, Patel PD, Maddineni P, Patil S, Kiehlbauch C, Millar JC, Searby CC, Raghunathan V, Sheffield VC, Zode GS. ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load. Nat Commun. 2020;11(1):5594. Epub 2020/11/07. doi: 10.1038/s41467-020-19352-1. PubMed PMID: 33154371; PMCID: PMC7644693.
- Millar JC, Pang IH, Wang WH, Wang Y, Clark AF. Effect of immunomodulation with anti-CD40L antibody on adenoviral-mediated transgene expression in mouse anterior segment. Mol Vis. 2008;14:10-9. Epub 2008/02/05. PubMed PMID: 18246028; PMCID: PMC2267727.
- Yarishkin O, Phuong TTT, Baumann JM, De Ieso ML, Vazquez-Chona F, Rudzitis CN, Sundberg C, Lakk M, Stamer WD, Krizaj D. Piezo1 channels mediate trabecular meshwork mechanotransduction and promote aqueous fluid outflow. J Physiol. 2021;599(2):571-92. Epub 2020/11/24. doi: 10.1113/JP281011. PubMed PMID: 33226641; PMCID: PMC7849624.
- Patel PD, Chen YL, Kasetti RB, Maddineni P, Mayhew W, Millar JC, Ellis DZ, Sonkusare SK, Zode GS. Impaired TRPV4-eNOS signaling in trabecular meshwork elevates intraocular pressure in glaucoma. Proc Natl Acad Sci U S A. 2021;118(16). Epub 2021/04/16. doi: 10.1073/pnas.2022461118. PubMed PMID: 33853948; PMCID: PMC8072326.
- Ryskamp DA, Frye AM, Phuong TT, Yarishkin O, Jo AO, Xu Y, Lakk M, Iuso A, Redmon SN, Ambati B, Hageman G, Prestwich GD, Torrejon KY, Krizaj D. TRPV4 regulates calcium homeostasis, cytoskeletal remodeling, conventional outflow and intraocular pressure in the mammalian eye. Sci Rep. 2016;6:30583. Epub 2016/08/12. doi: 10.1038/srep30583. PubMed PMID: 27510430; PMCID: PMC4980693.
- Johnstone M, Xin C, Tan J, Martin E, Wen J, Wang RK. Aqueous outflow regulation - 21st century concepts. Prog Retin Eye Res. 2021;83:100917. Epub 2020/11/21. doi: 10.1016/j.preteyeres.2020.100917. PubMed PMID: 33217556; PMCID: PMC8126645.