Preventing vision loss in murine diabetes by vascular stabilization

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Judd M. Cahoon1,2,3, Hironori Uehara1, Ling Luo1, Jacquelyn M. Simonis1,3, Britt Dubil1, Tadashi Miya1,3, Paul R. Olson1, Kortnie Walker1, Bonnie Archer1,3, Peter Barabas1, David Krizaj1, Gou Young Koh4, Guangping Gao5, Balamurali K. Ambati1,3

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Figure 1 AAV2.COMP-Ang1 mitigates diabetic retinal capillary dropout(a) Retinal flatmounts of 6 month-old mice and stained for isolectin (endothelial cell marker, green) and α-SMA (pericyte marker, red). Ins2Akita mice experienced endothelial and pericyte dropout compared to C57BL/6J mice. Endothelial cell loss was prevented by AAV2.COMP-Ang1.

Figure 3 AAV2.COMP-Ang1 prevents diabetes-induced retinal ganglion cell layer degeneration(a) Cross sections of 6 month old mouse retina stained for VE-cadherin (red) or nuclei (DAPI, blue). Right, magnified view of the ganglion cell layer from AAV2.COMP-Ang1 and AAV2.AcGFP treated mice demonstrat-ing increased VE-cadherin staining. (b) Optical coherence tomography (OCT) measuring retinal thickness. AAV2.COMP-Ang1 prevented diabetes-induced retinal thinning, n=5 mice/group, *P=0.03 vs. C57BL/6J, #P=0.03 vs. AAV2.AcGFP

Figure 4 AAV2.COMP-Ang1 prevents visual function loss in diabetic retinopathyVisual acuity was determined by testing optomotor responsiveness. (a) Ins2Akita mice exhibited decreased tracking response and AAV2.COMP-Ang1 prevented the decrease in visual acuity; n=6 mice/group. (b) Rep-resentative ERG. (c) Decreased b-wave amplitudes in Ins2Akita mice treated with PBS or AAV2.AcGFP compared to C57BL/6JL6 mice; AAV2.COMP-Ang1 prevented the decrease in amplitude. n=5 mice/group, data are mean ± s.e.m. *P=0.0001 vs. C57BL/6J. #P=0.01 vs. AAV2.AcGFP.

Figure 2 COMP-Ang1 increases vascular barrier function(a) COMP-Ang1 (100 ng/mL) increased resistance of HrMVECs (n=3) but did not overcome VEGF (50 ng/mL)-induced decreases in resistance. (b) AAV2.COMP-Ang1 returned vascular hyperpermeabilty to control levels. (c) AAV2.COMP-Ang1 increased VE-cadherin expression in Ins2Akita mouse retinas. (d) AAV2.COMP-Ang1 decreased VEGF-A in Ins2Akita mouse retinas. n=3 mice/group, data are mean ± s.e.m. *P<0.001 vs.C57BL/6-J, #P=0.02 vs. AAV2.AcGFP.

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IntroductionDiabetic retinopathy is the leading cause of blindness among the working age population and a 50% increase in its preva-lence is expected by 20301. Pericyte loss, vascular hyperper-meability, and increased vascular endothelial growth factor-A (VEGF-A) production are critical to its pathogenesis2,3. Angio-poietin 1 (Ang1), secreted by pericytes, maintains a stable and mature vasculature by preventing VEGF-A-induced vascular hyperpermeability and promoting vascular endothelial (VE)-cadherin stabilization4.

normoglycemia

Tie2VE-cadherin

Pericyte Endothelial cell

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HypothesisStabilizing the vasculature with COMP-Ang1 will prevent reti-nal structural and functional deficits in diabetic retinopathy

Ang1

hyperglycemia

hyperpermeability

Tie2

Ang1 VE-cadherin internalization

Diabetes

Methods

Mice. Mice heterozygous for the Ins2 mutation (C57BL/6-Ins2Akita/J (Ins2Akita)) experience hypoinsulinemia and hyperglycemia by 4 weeks of age. We used only male Ins2Akita with blood sugar levels greater than 280 mg/dL or age-matched controls (background strain C57BL/6J).

COMP-Ang1. Cartilage oligo matrix protein (COMP)-Ang1 is an Ang1 variant with enhanced solubility and stability.

AAV2. Consitutive expression of COMP-Ang1 (or control) was ac-complished with adeno-associated viral vector serotype 2 (AAV2.COMP-Ang1 and AAV2.AcGFP).

Retinal vasopermeability. Vascular hyperpermeability was estab-lished with Evans Blue method.

Visual Acuity. Optomotor head tracking response was used to de-termine visual acuity.

Retinal electrical function. Electroretinography (ERG) was per-formed on anesthetized mice under scotopic conditions.

References1. Wild, S., et al., Global prevalence of diabetes: estimates for the year 2000 and projec tions for 2030. Diabetes Care, 2004. 27(5): p. 1047-53.2. Hammes, H.-P., et al., Pericytes and the pathogenesis of diabetic retinopathy. Diabe tes, 2002. 51(10): p. 3107-12.3. Frank, R.N., Diabetic Retinopathy. New England Journal of Medicine, 2004. 350: p. 48-58.4. Gavard, J., V. Patel, and J.S. Gutkind, Angiopoietin-1 Prevents VEGF-Induced Endo thelial Permeability by Sequestering Src through mDia Developmental Cell, 2008. 14: p. 25-36.SupportThis work partly supported by:NEI 5R01EY017950, NEI 5R01EY017182, and the University of Utah T-32 Neurosci-ence Training Grant 5T32DC008553-05This poster generously supported by the ARVO/National Eye Institute Travel Grant

COMP-Ang1 prevents endothelial loss in diabetes COMP-Ang1 prevents ganglion cell layer loss and retinal thinning in diabetes

COMP-Ang1 increases endothelial resistance and decreases hyperpermeabilty COMP-Ang1 preserves visual acuity and retinal function in diabetes

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COMP-Ang1 prevents retinal hyperpermeability in diabetic mice

COMP-Ang1 increases VE-cadherin in the mouse retina COMP-Ang1 decreases VEGF in the mouse retina d

COMP-Ang1 prevents visual acuity decrease in diabetic mice

DiscussionThe Ins2Akita mouse exhibits several hallmarks of non-proliferative diabetic retinopathy including increased VEGF-A protein expression, vascular hyperpermeability and hypoper-fusion.

Current therapy for diabetic retinopathy focuses on suppress-ing VEGF either through ablating large portions of the retina with laser photocoagulation or administering anti-VEGF anti-bodies, neither of which addresses the underlying cause.

We hypothesized that stabilizing the vasculature would pro-mote proper perfusion and prevent the retinal ischemia re-sponsible for increased VEGF production.

AAV2.COMP-Ang1 prevents diabetes-induced: endothelial dropout vascular hyperpermeability (despite persistent pericyte dropout) retinal thinning and ganglion cell loss visual acuity and retinal function deficits

We propose that AAV2.COMP-Ang1 normalizes the vascula-ture by increasing VE-cadherin stability and preventing the endothelial cell loss seen in Ins2Akita mice. The normalized vasculature results in enhanced perfusion, reduced hypoxia-driven VEGF production (further contributing to vascular sta-bility) and reduced ganglion cell layer loss. This is turn pre-vents decreases in visual acuity and ERG responsiveness, which decrease in diabetic patients due to ischemia.

Scotopic ERG response COMP-Ang1 prevents diabetes-induced decreases in b-wave amplitude

Intravitreal injection2 months

in vivo retinal assessment (visual acuity, ERG, optical coherence tomography)

End point6 months

Diabetes

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Diabetes + COMP-Ang1

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Ischemia-induced VEGF Adequate perfusion

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Ang1 VE-cadherin internalization

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1 John A. Moran Eye Center, University of Utah; 2 Intperdepartmental Program in Neuroscience MD/PhD Program University of Utah; 3 Department of Veterans Affairs Salt Lake Healthcare System; Korean Advanced Institute of Science and Technology; 5 University of Massachusetts

judd.cahoon@hsc.utah.edu