Inflammatory basis of diabetic retinopathy. Tim Kern, PhD Case Western Reserve University and Stokes VA Hospital, Cleveland, OH

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1 Inflammatory basis of diabetic retinopathy Tim Kern, PhD Case Western Reserve University and Stokes VA Hospital, Cleveland, OH

2 Diabetic retinopathy currently is diagnosed based on vascular abnormalities

4 Pericyte ghost Acellular capillary Microaneurysm

8 CURRENT THERAPIES AGAINST DIABETIC RETINOPATHY Laser photocoagulation Anti-VEGF injections (intra-vitreal) Corticosteroids Improved glycemic control Fibrates Blood pressure medications

9 DIABETES ELEVATED BLOOD HEXOSE METABOLIC ABNORMALITIES NONPROLIFERATIVE RETINOPATHY (especially capillary nonperfusion & degeneration) RETINAL ISCHEMIA PROLIFERATIVE RETINOPATHY

10 DIABETES ELEVATED BLOOD HEXOSE METABOLIC INFLAMMATION ABNORMALITIES NONPROLIFERATIVE RETINOPATHY (especially capillary nonperfusion & degeneration)

11 What is the molecular cause of capillary degeneration in diabetic retinopathy? What cell types are involved in the development of diabetic retinopathy, and do those cells offer new ways to inhibit the retinopathy? Leukocytes Photoreceptors

12 Study design Glycemic control Histology 8-10 months diabetes inos, NO and nitrotyrosine 2-3 months diabetes ICAM Leukostasis Superoxide generation by isolated retina NF-κB and C/EBP activation Cell culture studies Induce diabetes Assign to groups 12 wks; biochemical & physiological assays 36 wks; histopathology

13 Leukostasis

14 Examples of glycemia in experimental animals: Effect of PJ-34 therapy Group (n) Duration (month) Body weight (g) Non-fast blood glucose (mg/l) Glycated Hemoglobin(%) Normal (10) ± ± ± 1.0 Diabetes (10) ± ± ± 2.8 D+PJ-34 (7) ± ± ± 1.1 Normal (28) ± ± ± 0.7 Diabetes (19) ± ± ± 1.4 D+PJ-34 (19) ± ± ± 1.0

15 PJ-34 inhibited diabetes-induced capillary cell apoptosis, and formation of acellular capillaries and pericyte ghosts TUNEL+ capillary cells/ retina TUNEL-positive capillary cells Non-diabetes Diabetes Diabetes+PJ-34 9 mos diabetes Acellular capillaries / mm 2 ret Acellular capillaries * * ** ** Ghosts per 1000 capillary cells Pericyte ghosts * * ** ** Zheng, Szabó, and Kern. Diabetes 53:2960, 2004

16 Expression (% of nondiabetic) PJ34 inhibited the diabetes-induced increase in retinal ICAM expression and leukostasis 350% 300% 250% 200% ICAM expression 150% 100% 50% 0% Nondiab normal Diab diabetes D+PJ-34 PARPi 70.0 * Non-diabetes ** Diabetes Adherent leukocytes/retina Diabetes+PJ-34 Leukostasis * * ** ** Arteriolar Venular Microvascular *

17 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

18 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β PARP inhibitors IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

19 ? Aldose reductase 5 lipoxygenase Superoxide generation Salicylates NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

20 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor Aminoguanidine inos -/- mice prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

21 5 lipoxygenase Diabetes RAGE Pro IL-1β? Aldose reductase Superoxide generation NO & ONOO - COX inhibitors p38 PARP activation NF-κB activation inos COX IL-1β IL-1β receptor TNFα CD40 prostaglandins ICAM Vasoocclusion? Capillary cell death Capillary degeneration

22 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM ICAM -/- mice Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

23 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes Lipoxygenase RAGE inhibitors p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

24 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 IL-1β inhibitors Vasoocclusion? Capillary cell death Capillary degeneration

25 ? 5 lipoxygenase Superoxide generation Diabetes RAGE p38 PARP activation NF-κB activation Aldose Antioxidants Mn SOD overexpression reductas inos Cu,Zn SOD overexpression e NO & ONOO - COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

26 ? Aldose reductase 5 lipoxygenase RAGE p38 MAPK inhibitor p38 Superoxide generation PARP activation NO & ONOO - Diabetes NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

27 ? Aldose reductase 5 lipoxygenase RAGE inhibitor Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

28 ? Aldose reductase 5 lipoxygenase Superoxide generation AR deletion NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

29 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 CD40 deletion Vasoocclusion? Capillary cell death Capillary degeneration

30 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα TNF inhibition CD40 Vasoocclusion? Capillary cell death Capillary degeneration

31 ? Aldose reductase 5 lipoxygenase Superoxide generation NO & ONOO - Diabetes RAGE p38 PARP activation NF-κB activation inos COX ICAM Pro IL-1β IL-1β IL-1β receptor prostaglandins TNFα CD40 Vasoocclusion? Capillary cell death Capillary degeneration

32 What cell types have the biochemical abnormalities that cause capillary degeneration/dysfunction in diabetes? Vascular Neuroglial Blood cells

33 Wildtype C57Bl/6J mouse (Recipient) Genetically modified mouse (inos -/-, PARP -/-, etc) Irradiate (whole body) to kill bone marrow Isolate bone marrow Inject into irradiated recipient Chimeric mouse inos -/- WT only marrow-derived cells lack inos WT inos -/- only marrow-derived cells have inos

34 5 lipoxygenase Diabetes RAGE Pro Il-1β? p38 Superoxide generation PARP activation NF-κB activation inos NO & ONOO - COX-2 IL-1β IL-1β receptor TNFα prostaglandins ICAM Vasoocclusion? Capillary cell death Capillary degeneration

35 Wildtype C57Bl/6J mouse (Recipient) Genetically modified mouse (inos -/-, PARP -/-, etc) Irradiate (whole body) to kill bone marrow Isolate bone marrow Inject into irradiated recipient Chimeric mouse inos -/- WT only marrow-derived cells lack inos WT inos -/- only marrow-derived cells have inos

36 Wildtype C57Bl/6J mouse (Recipient) Genetically modified mouse (inos -/-, PARP -/-, etc) Irradiate (whole body) to kill bone marrow Isolate bone marrow Inject into irradiated recipient Chimeric mouse inos -/- WT only marrow-derived cells lack inos WT inos -/- only marrow-derived cells have inos

37 Degenerate capillaries (per mm 2 retina) Degenerate capillaries per mm2 retina Irradiation does not inhibit the process leading to diabetic retinopathy Nondiab Diabetic Leukostasis (% of nondiab) abetes-induced inflammation (mrna; fold above nondiab) Nondiabetic Diabetic * N D wt wt inos -/- PARP -/- Control wt wt * inos -/- WT PARP -/- WT * inos TNFα COX-2 inos TNFα COX2 Diabetes 2012;61:

38 Degenerate capillaries (per mm 2 retina) Degenerate capillaries per mm2 retina Deletion of inos from marrow-derived cells only inhibits DR but deletion of inos from retinal cells only does not Leukostasis (% of nondiab) abetes-induced inflammation (mrna; fold above nondiab) Nondiabetic Diabetic * N D wt wt inos -/- PARP -/- Control wt wt * inos -/- WT PARP -/- WT * inos TNFα COX-2 inos TNFα COX2 Diabetes 2012;61:

39 Degenerate capillaries (per mm 2 retina) Degenerate capillaries per mm2 retina Deletion of inos from marrow-derived cells only inhibits DR but deletion of inos from retinal cells only does not Leukostasis (% of nondiab) abetes-induced inflammation (mrna; fold above nondiab) Nondiabetic Diabetic * N D wt wt inos -/- PARP -/- Control wt wt * inos -/- WT PARP -/- WT * inos TNFα COX-2 inos TNFα COX2 Diabetes 2012;61:

Degenerate capillaries (per mm 2 retina) Degenerate capillaries per mm2 retina Deletion of inos from marrow-derived cells only inhibits DR but deletion of

(mrna; fold above nondiab) 5 Oxidative stress in the retina in the retina 3 2 1 0 Nondiabetic Diabetic * N D wt wt inos -/- PARP -/- Control wt wt * inos -/-

40 Degenerate capillaries (per mm 2 retina) Degenerate capillaries per mm2 retina Deletion of inos from marrow-derived cells only inhibits DR but deletion of inos from retinal cells only does not Leukostasis (% of nondiab) Deletion of inos or PARP only from leukocytes also inhibited: abetes-induced inflammation (mrna; fold above nondiab) 5 Oxidative stress in the retina in the retina Nondiabetic Diabetic * N D wt wt inos -/- PARP -/- Control wt wt * inos -/- WT PARP -/- WT * Production of inflammatory proteins Leukostasis in the retina inos TNFα COX-2 inos TNFα COX2 Diabetes 2012;61: Diabetes 2012;61:

41 Co-culture of marrow-derived cells or leukocytes with retinal endothelial cells to measure leukocyte-mediated killing of endothelial cells Leukocytes from diabetic or nondiabetic mice Transformed retinal endothelial cells 5 mm glucose 30 mm glucose 1:5 ratio of leukocytes Ratio of WBC to endothelial cells 24 hr co-culture Count endothelial cells that are dead (stained with trypan blue) relative to number of healthy (unstained) endothelial cells Diabetes 2012;61:

42 Leukocytes from diabetic mice kill more retinal endothelial cells in high glucose in vitro, and this is inhibited in leukocytes from inos-deficient mice Retinal endothelial cells killed by overnight incubation with WBC Leukocyte mediated endothelial death (% of total endothelial count) all p< Endothelial cells incubated in: 5mM glucose 30 mm glucose Donor phenotype N N D D SD SD SD SD SD Donor genotype WT WT WT WT WT WT inos -/- inos -/- inos -/- In vitro treatment Lipoic anti- - Lipoic antiacid FasL acid FasL Diabetes 2012;61:

43 Leukocytes from diabetic mice kill more retinal endothelial cells in high glucose in vitro, and this is inhibited in leukocytes from inos-deficient mice Retinal endothelial cells killed by overnight incubation with WBC Leukocyte mediated endothelial death (% of total endothelial count) all p< Endothelial cells incubated in: 5mM glucose 30 mm glucose Endothelial cells Donor phenotype N N D D SD SD SD SD SD Leukocytes Donor genotype WT WT WT WT WT WT inos -/- inos -/- inos -/- In vitro treatment Lipoic anti- - Lipoic antiacid FasL acid FasL Diabetes 2012;61:

44 Leukocytes from diabetic mice kill retinal endothelial cells in high glucose in vitro Retinal endothelial cells killed by overnight incubation with WBC Leukocytes from nondiabetic mice Endothelial cells incubated in: 5mM glucose 30 mm glucose Donor phenotype ND ND SD SD SD SD SD SD SD Donor genotype WT WT WT WT WT WT inos -/- inos -/- inos -/- In vitro treatment Lipoic anti- - Lipoic antiacid FasL acid FasL Diabetes 2012;61:

Leukocytes from diabetic mice kill more retinal endothelial cells in high glucose in vitro, and this is inhibited in leukocytes from inos-deficient mice Retinal endothelial cells killed by overnight

45 Leukocytes from diabetic mice kill more retinal endothelial cells in high glucose in vitro, and this is inhibited in leukocytes from inos-deficient mice Retinal endothelial cells killed by overnight incubation with WBC Leukocytes from nondiabetic mice Leukocytes from diabetic mice Endothelial cells incubated in: 5mM glucose 30 mm glucose Donor phenotype N N D D SD SD SD SD SD Donor genotype WT WT WT WT WT WT inos -/- inos -/- inos -/- In vitro treatment Lipoic anti- - Lipoic antiacid FasL acid FasL Diabetes 2012;61:

46 The animal studies indicate that capillary degeneration that occurs in diabetic retinopathy and possibly neuropathy are caused by leukocytes. The same leukocyte-mediated killing of endothelial cells occurs in diabetic patients.

47 Mol Vis : Leukocytes Leukocytes from from diabetic diabetic humans humans likewise kill retinal endothelial cells via direct contact likewise kill retinal endothelial cells human human co-culture Nondiabetic Diabetic L; human retinal endothelial cells incubated in 5mM glucose H; human retinal endothelial cells incubated in 25mM glucose N; leukocytes from nondiabetic patients D; leukocytes from diabetic patients

48 Mol Vis : Leukocytes from from diabetic diabetic humans humans likewise kill retinal endothelial cells via direct contact likewise kill retinal endothelial cells human human co-culture L; human retinal endothelial cells incubated in 5mM glucose H; human retinal endothelial cells incubated in 25mM glucose N; leukocytes from nondiabetic patients D; leukocytes from diabetic patients

49 In vivo inhibition of leukocyte-mediated killing of retinal endothelial cells in diabetic patients 1. Collect blood from diabetic patients, and measure leukocytemediated killing of human retinal endothelial cells (Before). 2. Ask patients to consume the antioxidant and anti-inflammatory agent, berberine, for one month. 3. Recollect blood from the same patients, and re-measure leukocyte-mediated killing of endothelial cells (After). Leukocyte-mediated killing of endo cells (%) Molecular Vision 2013; 19:

50 In vivo inhibition of leukocyte-mediated killing of retinal endothelial cells in diabetic patients 1. Collect blood from diabetic patients, and measure leukocytemediated killing of human retinal endothelial cells (Before). 2. Ask patients to consume the antioxidant and anti-inflammatory agent, berberine, for one month. 3. Recollect blood from the same patients, and re-measure leukocyte-mediated killing of endothelial cells (After). Leukocyte-mediated killing of endo cells (%) Molecular Vision 2013; 19:

51 In vivo inhibition of leukocyte-mediated killing of retinal endothelial cells in diabetic patients 1. Collect blood from diabetic patients, and measure leukocytemediated killing of human retinal endothelial cells (Before). 2. Ask patients to consume the antioxidant and anti-inflammatory agent, berberine, for one month. 3. Recollect blood from the same patients, and re-measure leukocyte-mediated killing of endothelial cells (After). Leukocyte-mediated killing of endo cells (%) Molecular Vision 2013; 19:

52 What is the molecular cause of capillary degeneration in diabetic retinopathy? What cell types are involved in the development of diabetic retinopathy, and do those cells offer new ways to inhibit the retinopathy? Leukocytes Photoreceptors

Photoreceptors and diabetic retinopathy Laser photocoagulation has beneficial effects on advanced diabetic retinopathy in patients Patients having retinitis pigmentosa have been reported to have less

53 Photoreceptors and diabetic retinopathy Laser photocoagulation has beneficial effects on advanced diabetic retinopathy in patients Patients having retinitis pigmentosa have been reported to have less diabetic retinopathy. Br J Ophthalmol. 2001;85(3): Continuous low intensity light all night inhibits retinal edema. Eye (Lond). 2010;24: and 2011;25: Density of retinal vasculature was preserved in diabetic rhodopsin -/- animals. Invest. Ophthalmol. Vis. Sci. 47: , 2006

54 PNAS U S A :

55 Photoreceptor cells cause the oxidative stress and secondary inflammatory response in diabetes Photoreceptor degeneration occurs in animals lacking opsin

56 Low-intensity far-red light inhibits early lesions that contribute to diabetic retinopathy: in vivo and in vitro

57 Low intensity far-red light (photobiomodulation): effects on the retina in diabetic rats Light treatment is only 4 minutes per day 670 nm 6 joules cm 2 per session 7days/wk 10wks therapy Diabetes was induced approx 8 days before LED therapy was initiated

58 Light therapy inhibited the diabetesinduced increase in leukostasis in retinal microvessels and induction of ICAM

59 Light therapy inhibits the diabetesinduced increase in retinal production of superoxide and inhibits loss of SOD

60 Light therapy inhibited diabetes-induced apoptosis of retinal ganglion cells in vivo TUNEL Ganglion Cell Layer 8 6 TUNEL Cells ND ND +LED SD SD +LED

61 amplitude ( V) Light therapy partially inhibited diabetes-induced loss in retinal function Normal Normal+LED Diabetic+LED Diabetic B Photopic (photopic response) Nondiabetic Nondiabetic +LED Diabetic +LED Diabetic intensities (log cd s m-2)

62 Far-red light also inhibits leukocytemediated degeneration of First day retinal endothelial cells Mice WBCs Co-culture with mrecs Cell Death Ratio 9 p<0.01 p< ND SD SD+LED Light treatment is only 4 minutes per day

63 Optical coherence tomography assessment of macular edema in patients 003

64 Reversal of non-center involved macular edema using 3 min/day of far-red light 7 mos 7 mos 9 mos 2 mos

65 Reversal of non-center involved macular edema using 3 min/day of far-red light 7 mos 7 mos 9 mos 2 mos

66 Conclusions Immune/inflammatory processes play a critical role in the development of at least the vascular lesions of early diabetic retinopathy. The inflammation seems to be derived, or at least initiated by, marrow-derived cells (notably neutrophils). Retinal cells contribute also, but their contribution needs to be studied more. Future investigations on the pathogenesis of diabetic complications need to expand beyond the traditional vascularspecific view of complications, to include also immune cells. We are investigating the possibility that leukocyte studies might be used in the future to predict the likelihood that a particular patient will progress to advanced complications, and to predict their response to particular therapies.

THE ROLE OF POLY(ADP-RIBOSE) POLYMERASE-1 AND NF-κB IN THE DEVELOPMENT OF DIABETIC RETINOPATHY

THE ROLE OF POLY(ADP-RIBOSE) POLYMERASE-1 AND NF-κB IN THE DEVELOPMENT OF DIABETIC RETINOPATHY by LING ZHENG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy