Caveolin-1 modulates intraocular pressure: implications for caveolae mechanoprotection in glaucoma

Figures

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  Figure 1. Caveolae proteins localize to human outflow tissue and respond to mechanical stress in human TM cells. (a,b) Cav-1 (green) and Cav-2 (red) colocalize in the TM and SC of the human conventional outflow pathway. The white box in (a) delineates the region shown at higher magnification in (b). Abbreviations: TM, trabecular meshwork; SC, Schlemm’s canal; CM, ciliary muscle; AC, anterior chamber. (c) PTRF/cavin-1 (green), a protein essential for caveolae formation, partially localizes with Cav-2 (red) but is also present in nuclei of the TM and SC. Scale bars = 100 μm in (a), 50 μm in (b), and 30 μm in (c). (d,e) Stretch-induced dissociation of Cav-1 and PTRF/cavin-1 was assessed by coimmunoprecipitation using Cav-1 antibody for immunoprecipitation. Cyclic stretch (15%, 1 Hz, for 24 h) resulted in reduced recovery of PTRF/cavin-1 with Cav-1 (upper panel) with no change in the association of Cav-2 (lower panel). Lane labeled “Mock” represents the caveolin-1 antibody incubated without cell lysate to indicate position of IgG and any bands resulting from the antibody alone. (e) Densitometric analysis of the stretch-induced dissociation of PTRF/cavin-1 and Cav-1 (**p ≤ 0.01, one sample…

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  Figure 2. Loss of Cav-1 results in increased IOP and reduced aqueous humor drainage. (a) Localization of Cav-1 (green) and Cav-2 (red) in murine outflow pathway is similar to that observed in human tissue. CD31 (blue) labels the SC endothelium. (b) Intraocular pressure was significantly increased in Cav1−/− mice compared to controls as measured by rebound tonometry at indicated ages (*p ≤ 0.05, ** p ≤ 0.01, one-way ANOVA and Newman-Keuls post hoc analysis, n = 13–22 mice for each group). (c) Conventional, pressure-dependent outflow is significantly reduced in Cav1−/− eyes. Outflow facility was measured in perfused, enucleated eyes subjected to sequential pressure steps. Pressure-flow relationships (left panel) for Cav1−/− and control eyes. The slope of the regression line (conventional outflow facility) is fit through the mean ± SEM values for n = 8 control and n = 9 Cav1−/− eyes (from n = 5 control and n = 6 Cav1−/− mice. (d) Comparison of conventional outflow facility between Cav1−/− and control eyes (*p ≤ 0.05, unpaired two-tailed t-test).

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  Figure 3. Morphological and ultrastructural analyses of Cav-1−/− conventional outflow pathway. (a) Semithin sections (Richardson’s stain) through the iridocorneal angle of representative control (upper left panel) and Cav-1−/− (upper right panel) eyes. Middle and lower panels in (a) show higher magnifications by transmission electron microscopy (TEM). The chamber angle is open in control and Cav-1−/− eyes while obvious abnormalities of ciliary body (CB), trabecular meshwork (TM), and Schlemm’s canal (SC) are absent. At higher magnification (middle and lower panels), giant vacuoles (GV) are detectable in both genotypes. (b–e) Quantitative ultrastructural analyses of control and Cav-1−/− eyes. No significant differences in the number of GVs (b) or their sizes (c) were observed. (d) Endothelial nuclei diameter, a measurement of endothelial thickness, was not significantly different between genotypes. (e) The JCT depth was also not significantly different between Cav-1−/− and control eyes. Eyes from 4 Cav-1−/− mice and 7 littermate controls were used for these quantitative analyses. Numbers of GVs were quantified in 86 non-overlapping images from Cav-1−/− eyes and 151 images from controls.…

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  Figure 4. Caveolae protect SC endothelial membranes from IOP-induced damage. (A) Representative maximum projections of z-stacks from anterior segment wholemounts that encompass the entire SC diameter from Cav-1−/− and littermate controls subjected to IOP elevation (upper panels) or maintained at standing IOP (lower panels). The SC endothelium was co-stained with CD31 (green) and PI-stained nuclei are labeled in red. Scale bar = 100 μm. (B) Quantification of PI-stained nuclei in each image stack (from n = 5 mice/genotype). For statistical analysis, multiple images from each eye were averaged to yield a single value of PI cells/mm3 per mouse. ***p ≤ 0.001, one-way ANOVA and Newman-Keuls post hoc analysis.

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  Figure 5. Cav1−/− eyes are more sensitive to NOS inhibition than control eyes. Outflow facility was measured in paired eyes perfused with 10 μM L-NAME or PBS vehicle in control (a) and Cav1−/− (b) eyes. (c) Cav1−/− eyes treated with L-NAME had significant reduction in conventional outflow facility compared to control eyes (p ≤ 0.05, one-way ANOVA with Newman-Keuls post hoc test, n = 7–9). Outflow facilities (mean ± SEM) were 0.019 ± 0.003 for control + vehicle, 0.014 ± 0.002 for control + L-NAME, 0.015 ± 0.002 for Cav1−/− + vehicle, and 0.008 ± 0.001 for Cav1−/− + L-NAME.