Activation of the ROCK/MYLK Pathway Affects Complex Molecular and Morphological Changes of the Trabecular Meshwork Associated With Ocular Hypertension

Abstract

Purpose: The Rho-associated protein kinase and myosin light chain kinase (ROCK/MYLK) pathway undeniably plays a pivotal role in the pathophysiology of primary open-angle glaucoma (POAG). In our study, we utilized both ocular hypertension (OHT) rabbit models and clinical investigations to gain invaluable insights that propel the development of novel treatments targeting proteins and genes associated with the trabecular meshwork (TM), thereby offering promising avenues for the management of POAG.

Methods: Following microbead injections into the anterior chamber of the ocular cavity of rabbits, we observed elevated histiocyte numbers and immune scores for MYLK-4/ pMLC-2, alongside a reduction in the void space within the TM. Notably, treatment was performed with 0.1% ITRI-E-(S)-4046, a compound with dual kinase inhibitor (highly specific inhibitor of ROCK1/2 and MYLK4), significantly reduced intraocular pressure (IOP; P < 0.05) and expanded the void space within the TM (P < 0.0001) compared with OHT rabbits. In clinical investigations, we utilized whole transcriptome sequencing to analyze gene expression specifically related to the TM, obtained from patients (5 early-onset and 5 late-onset) undergoing trabeculectomy.

Results: Our findings revealed 103 differential expression genes (DEGs) out of 265 molecules associated with the Rho family GTPase pathway, exhibiting a P value of 1.25E-10 and a z-score of -2.524. These results underscore significant differences between the early-onset and late-onset POAG and highlight the involvement of the ROCK/MYLK pathway.

Conclusions: These findings underscore the critical involvement of the ROCK/MYLK pathway in both OHT-related and different onsets of POAG, providing valuable insights into the TM-related molecular mechanisms underlying the disease.

Figure 1.

Introducing a simplified plot structure for our study on microbead-induced OHT in rabbits, focusing on the activation of ROCK/MYLK pathway-associated proteins, leads to the phosphorylation of myosin light chain (p-MLC), increasing contraction in the TM and subsequently raising IOP. Conversely, myosin light chain phosphatase (MLCP) reverses this effect by dephosphorylating MLC, thereby reducing TM contraction and helping to lower IOP. The dynamic expression of ROCK/MYLK pathway-associated proteins are critical markers in the molecular mechanisms of OHT and the development of anti-glaucoma drugs.

Figure 2.

IOP is measured at multiple time points to monitor changes over time. Mean IOP in microbead-injected eyes and non-injected eyes. (A) IOP elevated acutely on day 1 following the injection of magnetic microbeads into the eye. The IOP reaches plateau around day 2 post-injection and gradually decreased after day 7 post-injection. The notation **** (P < 0.0001) indicates that the differences in IOP between the microbead-injected eyes (n = 7) and the control eyes (n = 7) are statistically significant as determined by a 2-way ANOVA, suggesting a direct impact of the microbeads on IOP. (B) External eye photography observed on days 1, 3, 7, 10, and 14 post-injection of magnetic microbeads in the right eye (OD) and control eye (OS).

Figure 3.

IOP lowering effect of vehicle, and three topical administration distinct treatment groups (included 0.05% ITRI-E-(S)4046, 0.1% ITRI-E-(S)4046, and 0.02% AR-13324, n = 5 in each group) were given to each microbead-injected OHT eye once daily for 2 consecutive days (at 0 and 24 hours). The ∆IOP was measured at 0, 2, 4, 6, 8, 24, 26, 28, 30, 32, and 48 hours. The ∆ IOP (mm Hg) was the change of IOPs from time zero IOP, vertical bars represent means ± standard error of the mean (SEM) of data obtained from 5 rabbits by 2-way ANOVA. The 0.1% of ITRI-E-(S)4046 was significantly more effective in reducing IOP compared to 0.05% ITRI-E-(S)4046 (*, P < 0.05); 0.1% ITRI-E-(S)4046 also showed a significantly greater IOP reduction compared to 0.02% AR-13324 (‡, P < 0.05); 0.02% AR-13324 significantly lowered IOP compared to the vehicle control (#, P < 0.05).

Figure 4.

IHC analysis. (A) For pMLC-2, in eyes injected with microbeads (n = 26), the median of histiocyte count in a, b, and c was found to be approximately 20-fold higher compared to non-induced eyes (n = 10), indicating significant differences in histiocytes ratio between the two groups and a severe inflammatory response (****, P < 0.0001). Additionally, the median of p-MLC2 immune score in d, e, and f was observed to be approximately 4-fold higher (****, P < 0.0001), suggests enhanced enzymatic activity that contributes to increased muscle contraction, as depicted in (A). (B) For MYLK-4, in eyes injected with microbeads (n = 22), the median of histiocyte count in a, b, and c was found to be 12-fold higher (****, P < 0.0001) in microbead-injected OHT eyes (n = 9), with the median of MYLK-4 immune score in d, e, and f being 1.7-fold higher (****, P < 0.0001), as depicted in (B). Both parts of this analysis used the Mann-Whitney test, a nonparametric method suitable for comparing two independent samples that may not follow a normal distribution.

Figure 5.

Illustrates the morphological changes, specifically the percentage of the void space, in the TM of the OHT rabbit model. It presents two different experimental groups: the control group (on the left side in color yellow, n = 8) and the microbead-induced OHT group (on the right side in color blue, n = 15). On the yellow box, representing the control group, no significant differences were observed in the mean rank percentage of void space within the TM compared to administration of 0.1% ITRI-E-(S)4046 (P5L) (n = 9). However, prior to administration of 0.1% ITRI-E-(S) 4046 (upper in the blue box), microbead-induced OHT (n = 15) resulted in a significant increase in mean rank of the percentage of void space within the TM compared to the control group (P < 0.0001, ****) (bottom in the blue box). Right after application of 0.1% ITRI-E-(S) 4046 (n = 19), the mean rank of the percentage of void space within the TM increased by 3-fold in the microbead-induced OHT group (bottom one) (P = 0.0011, **) using the Kruskal-Wallis test.

Figure 6.

(A) In the upper panel, the Principal Component Analysis (PCA) plot, based on expression data, reveals differing gene expression dispersion between these two subtypes. The patients with late-onset (red) POAG demonstrates higher dispersion in gene expression, whereas the patients with early-onset (green) POAG displays lower dispersion. The Volcano Plot illustrated in the lower panel of (A), along the y-axis, it plots the negative log of the P value against the log fold change between the two groups, helping identify genes that are significantly upregulated or downregulated, whereas genes higher on the y-axis have lower P values, indicating higher statistical significance. Genes located further to the left or right on the x-axis represent greater fold changes in expression, the genes involved in the ROCK/MYLK pathways, such as RhoA, ROCK1/2, MYLK, MYL6, and CALM2 are marked in red and are all upregulated in patients with early-onset POAG. (B) Potential differential expression genes (DEGs) in TM, such as MYL6, MYLK, CALM2, and ROCK1/2 exhibit higher expression in patients with early-onset POAG compared with patients with late-onset POAG. Along the y-axis, transcripts per million (TPM) is utilized to represent the expression levels of various genes.

Figure 7.

The significantly increased expression of ROCK1 (P < 1.16 × 10-4, n = 5), ROCK2 (P < 2.3 × 10⁻⁴, n = 5), MYLK (P < 0.05, n = 5), as well as CALM2 (P < 0.0006, n = 5) in patients with early-onset POAG compared with patients with late-onset POAG is a compelling finding. These results strengthen the hypothesis that alterations in the ROCK/MYLK pathway play a crucial role in the pathology of early-onset POAG through the enhanced activation of phosphorylated myosin light chain (p-MLC). Therefore, the ROCK/MYLK path is activated. In summary, the pathogenic mechanism in patients with early-onset POAG involves a higher activation level of the ROCK/MYLK signaling pathway compared to patients with late-onset POAG, similar to the pathway activation observed in microbead-induced OHT rabbits.