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, 30 (4), 1670-82

Cathepsin D: An Mϕ-derived Factor Mediating Increased Endothelial Cell Permeability With Implications for Alteration of the Blood-Retinal Barrier in Diabetic Retinopathy

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Cathepsin D: An Mϕ-derived Factor Mediating Increased Endothelial Cell Permeability With Implications for Alteration of the Blood-Retinal Barrier in Diabetic Retinopathy

Finny Monickaraj et al. FASEB J.

Abstract

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). We have previously reported increased monocyte (Mono) trafficking into the retinas of diabetic animals. In this study, we have examined the effect of activated Monos on retinal endothelial cells (ECs). The U937 Mϕ-conditioned medium (CM) significantly decreased the transendothelial resistance of EC monolayers as measured by electric cell-substrate impedance sensing (P= 0.007). The CM was fractioned, and the effective fraction (30-100 kDa) was analyzed by liquid chromatography-mass spectrometry, and cathepsin D (CD) was identified as a major secreted product. Immunoprecipitated CD resulted in decreased resistance in ECs (P= 0.006). The specificity of CD in mediating alterations of the EC barrier was confirmed using small interfering RNA. The decreased resistance correlated with a significantly increased gap between ECs. CD altered the Ras homolog gene family, member A/Rho-associated kinase pathway with increased stress actin filament formation in the EC layer. Increased CD levels were found in the retinas of diabetic mice (3-fold) and serum samples of patients with diabetic macular edema (1.6-fold) measured by Western blot and ELISA. These findings suggest an important role for Mϕ-derived CD in altering the blood-retinal barrier and reveal a potential therapeutic target in the treatment of DR.-Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.

Keywords: DME; Rho/ROCK pathway; inflammation.

Figures

Figure 1.
Figure 1.
Mϕs secrete a factor causing a change in EC monolayer permeability. A) Normalized resistance of HREC monolayers treated with a 1:5 dilution of CM. A significant decrease is seen in the resistance of cells treated with U937 Mϕ CM compared to HRECs treated with Ctrl, or the CM from U937 Monos. *P = 0.007, significantly less than cells in Ctrl. B) Schematic representation of the method used for the preparation of U937 Mono and Mϕ CM. Mass Spec., mass spectrometry; Mol. Wt., molecular mass. C) Normalized resistance of HREC monolayers treated with a 1:5 dilution of fractionated U937 Mϕ CM: 3–10, 10–30, 30–100, and 100–300 kDa. Data are the means ± sd. *P = 0.004, significantly less than cells in Ctrl.
Figure 2.
Figure 2.
The 30–100 kDa fraction contains CD, which alters the EC barrier. A) CD levels associated with agarose beads as measured by ELISA following IP. *P = 0.02, significantly greater than beads plus antibody (ab) without exposure to the 30–100 kDa fraction. B) Silver-stained gel. Mol. Wt. (molecular mass) marker is in lane 1, IP no sample is in lane 2, and IP CM is in lane 3. C) Representative ECIS tracings of HRECs treated with CD beads demonstrate significantly decreased resistance in comparison to cells treated, beads plus CD antibody (ab), or Ctrl. *P = 0.006, significantly less than cells grown in Ctrl. D) Representative image of VE-cadherin immunofluorescence of HRECs grown in Ctrl and in the presence of CD beads. White arrows indicate gaps between adjacent cells treated with CD beads. Quantitation demonstrates significantly increased gap width (micrometers) between adjacent cells. Data are means ± sd. *P = 0.0001, significantly greater than cells grown in Ctrl.
Figure 3.
Figure 3.
CD siRNA inhibits increased permeability induced by activated U937 cells. A) Representative Western blot image of CD and β-tubulin protein in PMA-stimulated U937 cells (U937 Mϕ; Mac), U937 Mϕ treated with CD siRNA, and U937 Mϕ treated with scrambled siRNA. B) The CD beads from U937 Mϕ cells treated with siRNA (siRNA CD beads) caused a significant reduction in the degree of induced HREC barrier alteration as compared to the CD beads from untreated U937 Mϕ cells (CD beads). *P = 0.005, significantly greater than untreated U937 Mϕ cells (CD beads). C) Representative image of VE-cadherin (cad) immunofluorescence in HRECs grown in the presence of CD beads or siRNA CD beads. siRNA CD beads induced fewer gaps between adjacent cells. White arrows indicate gaps between adjacent cells treated with CD beads. *P = 0.0001, significantly greater than cells grown in Ctrl; **P = 0.0005, significantly less than HRECs grown in the presence of CD beads.
Figure 4.
Figure 4.
Permeability effect of CD is mediated by an M6P-dependent mechanism. A) ECIS tracings of HRECs treated with CD beads, CD beads with 20 μM PepA, CD beads with 50 μM PepA, or Ctrl. *P = 0.001, significantly less than cells grown in Ctrl. B) ECIS tracings of HRECs treated with CD beads, CD beads plus 100 μM M6P, or Ctrl. *P = 0.02, significantly less than cells treated with CD beads alone.
Figure 5.
Figure 5.
Involvement of Rho/ROCK in permeability alteration and gap formation. A) Representative immunofluorescence images of HRECs stained for stress actin filaments in cells treated with CD beads or siRNA CD beads. Right panel demonstrates the quantitation of the stress actin filaments/cells. No difference was seen in cells treated with scrambled siRNA beads in comparison with CD beads. *P < 0.0001, significantly greater than cells grown in normal medium (Ctrl); **P = 0.0001, significantly less than cells grown in the presence of U937 Mϕ CD beads. B) RhoA activity was significantly increased in HRECs treated with CD beads. This response was dampened by siRNA CD beads or CD beads along with excess of M6P. OD, optical density. *P = 0.004, significantly greater than cells grown in Ctrl; **P = 0.003, significantly less than cells grown in CD beads; #P = 0.05, significantly less than cells grown in CD beads. C) Relative gene expression levels of ROCK2 normalized to glyceraldehyde 3-phosphate dehydrogenase as measured by real-time PCR. Data are means ± sd. *P = 0.007, significantly greater than cells grown in Ctrl; **P = 0.01, significantly less than cells grown in the presence of CD beads.
Figure 6.
Figure 6.
ROCK inhibitor Y-27632 prevents CD-induced permeability and intercellular gap formation. A) HRECs treated with CD beads along with the ROCK inhibitor, Y-27632 (25 μM), demonstrate resistance similar to cells grown in Ctrl. *P = 0.01, significantly greater than cells grown in the presence of CD beads. B) There is less stress actin fiber formation in cells treated with the ROCK inhibitor, Y-27632 (25 μM). *P = 0.0001, significantly greater than cells grown in Ctrl; **P = 0.001, significantly less than cells treated with CD beads. C) Intercellular gap formation is decreased in HRECs treated with the ROCK inhibitor, Y-27632 (25 μM). VE-cad, VE-cadherin. Data are represented as means ± sd. *P = 0.0001, significantly greater than cells grown in Ctrl; **P = 0.001, significantly less than cells treated with CD beads.
Figure 7.
Figure 7.
CD levels are increased in the retinas of diabetic mice and serum of patients with DME. A) Albumin levels in retinal tissue as measured by Western blot in diabetic animals compared with nondiabetic animals. *P = 0.04, significantly greater than nondiabetic animals (n = 4). B) Representative Western blot image of CD and β-tubulin in the retinas of nondiabetic and 4-mo-old diabetic mice. Band density quantitation demonstrates significantly elevated levels of CD protein in the retinas of diabetic mice compared to nondiabetic animals. *P = 0.003, significantly greater than nondiabetic animals (n = 4). C) CCL2 levels measured by ELISA from diabetic mouse retinas were significantly higher than CCL2 levels in the retinas of nondiabetic mice (n = 4). *P = 0.001, significantly greater than nondiabetic mouse retinas. D) ROCK2 gene expression levels, measured by real-time PCR, show significantly increased levels in the retinas of diabetic mice compared to nondiabetic animals (n = 4). *P = 0.03, significantly greater than nondiabetic animals. E) Age-adjusted mean values of CD levels measured by ELISA from human serum samples showed significantly elevated levels in patients with DR (DME) compared to nondiabetic subjects or diabetic subjects without DME (n = 11). Data are represented as means ± sd. *P < 0.0001, significantly greater than nondiabetic subjects; #P < 0.0001, significantly greater than diabetic subjects without DME.
Figure 8.
Figure 8.
A schematic diagram highlights the cause and effect of CD in altered vascular permeability in retinal capillaries in diabetes. In diabetes, there is increased Mono trafficking in retinal tissues, and Monos are further differentiated into Mϕs that secrete CD. The CD then binds to the CIMPR on the EC surface, activating the Rho/ROCK pathway, which results in a significant increase in actin stress fiber density and thickness. This leads to EC separation and increased endothelial permeability, and alteration of the BRB.

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