Switch to Autophagy the Key Mechanism for Trabecular Meshwork Death in Severe Glaucoma

Figures & data

Table 1 Demographic and Clinical Profile of Patients with Adult Primary Glaucoma

Variables	POAG (n = 30)	PACG (n = 34)	Controls (n = 25)	P value*
Age (Years)	56±5.7	58±4.6	55±3.8	0.9
Male:Female (%)	44:56	32:68	42:58	0.06
Baseline IOP (mm Hg)	24±6.8	26±8.1	8±4.6	<0.001
Mean deviation (dB)	−19±6.9	−18±7.9	−0.2±4.1	<0.001
Pattern standard deviation (dB)	12±8.1	12±10.2	5±1.3	<0.001
Number of Medications	2medicines=16>3medicines=12	2medicines=12>3medicines=22	NA	<0.001

Notes: IOP, intraocular pressure, *one-way Anova with post-hoc Bonferroni.

Table 2 List of Proteins Related to Apoptosis, Autophagy and TGF Beta Signaling Pathways in the Trabecular Meshwork of Patients with Severe Adult Primary Glaucoma

Cell Death Proteins	POAG*	PACG*
1. Autophagy proteins
-Autophagy related	LAMC1, CTSD	LAMC1, CTSD
-Endoplasmic reticulum stress markers	EIF4A2, GRP78	EIF4A2, GRP78
-Senescence and autophagy	LAMP1	LAMP1
Apoptosis proteins	Caspase14	Caspase14
	ANXA5	ANXA5
	ANXA6	ANXA6
	NID2	NID2
Proteins specific to trabecular meshwork tissue	AQP1	AQP1
	MYOC	MYOC
	ANGPTL7	ANGPTL7
	SERPINA3	SERPINA3
	HBB	HBB
	APOD	APOD
	PTGDS	PTGDS
	KRT5 and KRT13, MYH11	KRT5 and KRT13, MYH11

Notes: See appendix for description of proteins; *data of log2 fold change values of all proteins have been deposited to the ProteomeXchange Consortium.

Figure 1 (A) shows the box plot of the protein expressions in triplicate samples of each of primary open angle glaucoma, primary angle closure glaucoma and controls showing very little variability among the triplicate experiments. (B) shows the venn diagram of differential expression of proteins among glaucoma and controls while (C) shows the heat map of the significantly differentially expressed proteins primarily belonging to two clusters as shown.

Figure 2 (A) MTT assay of Human trabecular meshwork cells after oxidative stress with H₂O₂. (B) shows the representation of FACS analysis of cells at 72 hours compared with control (Q1: necrotic cells, Q2: early apoptotic cells Q3: healthy cell, Q4: late apoptotic cells). (C) showing percentage of early and late apoptotic cells maximum at 72 hours in 600 uM of H₂O₂ concentration. Means ± SEM shown, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 one way ANOVA post hoc t test with Tukey correction.

Figure 3 Relative gene expression of apoptosis (A), autophagy (B), and necrosis (C) related genes in in-vitro primary human trabecular meshwork cells after 48 hours and 72 hours of oxidative stress with H2O2 relatively compared with control and normalized with the average CT of ACTB, GAPDH, HPRT1, RPLP20 and HGDC. (D and E) protein expression of apoptosis and autophagy markers and densitometry analysis in HTM cells after 600uM H202 treatment in HTM cells at 24, 48 and 72 hours by western. (F and G) epigenetic modifications of histones ie, H2 and H3 and densitometry analysis after 600 uM of H₂O₂ treatment in HTM cells at 24, 48 and 72 hours. Means ± SEM shown, *p<0.05, **p<0.01, ***p<0.001 one way ANOVA post hoc t test with Tukey correction.