The unconventional role of acid sphingomyelinase in regulation of retinal microangiopathy in diabetic human and animal models - PubMed (original) (raw)

doi: 10.2337/db10-0550. Epub 2011 Jul 19.

Maria Tikhonenko, Svetlana Bozack, Todd A Lydic, Gavin E Reid, Kelly M McSorley, Andrew Sochacki, Gloria I Perez, Walter J Esselman, Timothy Kern, Richard Kolesnick, Maria B Grant, Julia V Busik

Affiliations

The unconventional role of acid sphingomyelinase in regulation of retinal microangiopathy in diabetic human and animal models

Madalina Opreanu et al. Diabetes. 2011 Sep.

Abstract

Objective: Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study.

Research design and methods: Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies.

Results: We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α-induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina; however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM(-/-) mice or inhibition of ASM activity by DHA prevents acellular capillary formation.

Conclusions: This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

ASM expression in human retinal cells. ASM activity (A) and protein expression (B; immunoblot and quantitative analysis) level is the highest in HREC compared with HRPE and HMC. The results are means ± SD of three independent experiments performed in triplicate. *P < 0.05 compared with HREC. C: ASM, caveolin-1, and LAMP1 colocalization in HREC was assessed by immunohistochemistry. There is 56.38 ± 5.85% colocalization of ASM and caveolin-1 (top) and a strong punctate perinuclear staining, negative for caveolin-1 that associates with LAMP1 (bottom; yellow color in the MERGED panel). D: Immunofluorescent staining and quantification of ASM cellular distribution (E) in nonpermeabilized (left) and permeabilized (right) HREC, HRPE, and HMC. Only HREC express plasma membrane ASM as demonstrated by staining in nonpermeabilized cells (D, top left, and quantitation in E). ASM staining in permeabilized cells corresponding to ASM in the intracellular compartment is equal in HREC, HRPE, and HMC (D, right column, and quantitation in E). The results are means ± SD of at least three independent experiments performed in triplicate with eight images in each. RFU, relative fluorescence units. nd, not detected. Red bar = 27.5 μm; blue bar = 55 μm; white bar = 110 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

FIG. 2.

The effect of DHA and cholesterol depletion on ASM expression in HREC. A: DHA and MCD pretreatment of HREC significantly decreased cholesterol content of the caveolar fractions compared with control. B: The inhibitory effect of DHA on ASM activity was not mimicked by MCD, and cholesterol replenishment did not restore ASM activity in DHA-treated HREC in whole-cell lysates. However, cholesterol depletion inhibited ASMase activity, and cholesterol replenishment reversed the inhibitory effect of DHA on ASM activity in the plasma membrane fraction. C: ASM protein expression in caveolar fractions was isolated from control and DHA-treated HREC. The results are means ± SD of three independent experiments performed in triplicate. *P < 0.01 compared with control. D: Decrease in ceramide-to-sphingomyelin ratio in caveolae from DHA-treated HREC compared with control. There was no change in ceramide-to-sphingomyelin ratio in total plasma membrane. DHA prevents TNF-α–induced increase in ceramide-to-sphingomyelin ratio in the caveolae. The results are means ± SD of three independent experiments performed in triplicate. *P < 0.05 compared with control. E and F: DHA or ASMase small interfering RNA (siRNA) treatment prevents IL-1β–induced increase in ICAM-1 expression in HREC. Random siRNA was used as control for ASM siRNA experiments. The results are means ± SD of at least three independent experiments performed in triplicate. *P < 0.05 compared with control; #P < 0.05 compared with IL-1β–stimulated HREC.

FIG. 3.

FIG. 3.

Increase in vascular ASM expression and apoptosis of neuroretina after retinal I/R injury in rats. A: Colocalization of ASM and caveolin-1 in normal rat retina flat mount. B: ASM expression in normal rat retina showing ASM-positive staining of retinal vessels (black arrow) and perinuclear lysosomal compartment of retinal ganglion cells (black arrowhead). C: Caveolin-1 expression in normal rat retina showing caveolin-1-positive staining of retinal vessels (black arrow). D: Specific increase in ASM expression in the rat retinal vessels (black arrow) from I/R-injured eye compared with control. DHA supplementation prevents the increase in vascular ASM expression in I/R-injured eye. E: Quantification of ASM staining (normalized to background) in retinal vessels (black arrow) and neuroretina (retinal GCL; black arrowhead). TUNEL staining of retinal sections from control and DHA-supplemented animals after transient retinal ischemia (F) is shown. Number of TUNEL-labeled cells in GCL and INL after retinal I/R (G) is shown. The data are presented as means ± SE from two independent sets of animals, with five animals, 11–17 retinal vessels per group. *P < 0.05 compared with all other groups. RDU, relative densitometry units. ONL, outer nuclear layer. Red bar = 55 μm; black bar = 220 μm; white bar = 40 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

FIG. 4.

Retinal gene expression and capillary degeneration in mice undergoing retinal I/R injury. Quantitative PCR analysis of ASM, ICAM-1, VCAM-1, IL-1β, TNF-α, and VEGF (A) in retinas isolated from ASM+/+ and ASM−/− mice 2 days after retinal I/R injury is shown. The data are presented as means ± SE from three independent sets of mice performed in triplicate with a total of five mice per group. B: Acellular capillary occurrence (black arrows) in the retina isolated from ASM+/+ and ASM−/− mice 7 days after retinal I/R injury. Quantification of the number of acellular capillaries from five independent sets of mice, with a total of seven mice per ASM+/+ group and six mice per ASM−/− group, is shown. At least eight fields of retina were counted in duplicates by two independent investigators. *P < 0.05; black bar = 110 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

FIG. 5.

Effect of DHA-supplemented diet on short-term diabetes-induced changes in rat retina. Retinas isolated 1 month after induction of diabetes were analyzed by quantitative PCR and Western blotting for inflammatory/angiogenic molecule expression levels. Quantitative PCR analysis of ASM, ICAM-1, VEGF, and IL-1β (A) from retinas isolated from rats on standard diet (control, white bar_;_ diabetic, black bar) and a DHA-enriched diet (diabetic, striped bar) is shown. The results are means ± SE from three independent sets of animals, with five to eight animals in each group. *P < 0.05 compared with control. Immunoblot (B) and quantitative analysis (C) of ASM and VEGF protein levels in retinas isolated from control rats on standard diet and from diabetic rats on standard diet or DHA-enriched diet. The results are means ± SD of four animals in each group, performed in triplicate. *P < 0.05 compared with control; #P < 0.05 compared with diabetes.

FIG. 6.

FIG. 6.

Effect of DHA-supplemented diet on long-term diabetes-induced degenerative changes in rat retina. Retinas isolated 9 months after induction of diabetes were analyzed by quantitative PCR and immunobloting for inflammatory/angiogenic molecule expression. Quantitative PCR analysis of ASM, ICAM-1, VEGF, and IL-1β (A) of retinas isolated from rats subjected to a standard diet (control, white bar_;_ diabetic, black bar_)_ or a DHA-enriched diet (diabetic, striped bar) is shown. The results are means ± SE from one set of animals, with five to eight animals in each group. *P < 0.05 compared with control. Immunoblot (B) and quantitative analysis (C) of ASM protein levels in retinas isolated from control rats on standard diet and diabetic rats on standard and DHA-enriched diet. The results are means ± SD of four animals in each group, performed in triplicate. *P < 0.05 compared with control; #P < 0.05 compared with diabetes. D: Retinal vasculature from control, diabetic, or diabetic supplemented with DHA animals was prepared using trypsin digestion and stained with hematoxylin and periodic acid–Schiff. Dramatically increased number of acellular capillaries (black arrows) was observed in retinal vasculature isolated from diabetic animals; however, diabetic animals supplemented with DHA were protected from acellular capillaries formation. D: Quantification of the total number of acellular capillaries. The results are means ± SD from one set of animals, with eight animals per group. At least eight fields of retina were counted in duplicates by two independent investigators. *P < 0.05 compared with control; black bar = 110 μm. (A high-quality digital representation of this figure is available in the online issue.)

References

    1. Imai H, Singh RS, Fort PE, Gardner TW. Neuroprotection for diabetic retinopathy. Dev Ophthalmol 2009;44:56–68 - PubMed
    1. Adamis AP, Berman AJ. Immunological mechanisms in the pathogenesis of diabetic retinopathy. Semin Immunopathol 2008;30:65–84 - PubMed
    1. Kern TS. Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy. Exp Diabetes Res 2007;2007:95103. - PMC - PubMed
    1. Joussen AM, Poulaki V, Mitsiades N, et al. Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression. FASEB J 2002;16:438–440 - PubMed
    1. Yang LP, Sun HL, Wu LM, et al. Baicalein reduces inflammatory process in a rodent model of diabetic retinopathy. Invest Ophthalmol Vis Sci 2009;50:2319–2327 - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources