DYSLIPIDEMIA AND DIABETIC RETINOPATHY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1015198
• Project Start Date: Mar 1, 2018
• Project End Date: Feb 28, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Physiology

Non Technical Summary
Diabetic retinopathy, a leading cause of blindness, occurs due to accumulated damage to the blood vessels in the retina. Based on our preliminary data, we believe that diabetes-induced retinal damage is contributed by an increase in retinal cholesterol levels. We have shown that diabetes causes a decrease in pathways that promote cholesterol out of the retina. The goal of our current work is to investigate the beneficial effects that activation of cholesterol export pathways have on the diabetic retina. We plan to accomplish these objectives by studying cholesterol metabolism in human retinal cells, and in an animal model of diabetes. Also, we will use the world-class cholesterol analysis facility at MSU to uniquely measure/identify cholesterol metabolites in healthy and diabetic milieus.Although there are strong associations between lipid control and diabetic vascular complications, the nature of these associations are not understood. This gap in knowledge stems from limitations in methodological and analytical tools available to study lipid metabolism. Further understanding of the role of lipid dysregulation in diabetic vascular complications will help to i) identify novel, clinically relevant mechanism of cholesterol elimination; ii) establish whether this mechanism represents a viable approach for prevention/reversal of sight threatening diabetic retinopathy; and iii) facilitate novel approaches in drug development. Importantly, the strategies identified here using retinal vascular damage, will have broader impact on overall vascular health in diabetic patients.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
701	3840	1020	100%

Knowledge Area
701 - Nutrient Composition of Food;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1020 - Physiology;

Keywords

dyslipidemia

diabetic retinopathy

fatty acids

cholesterol

Goals / Objectives
In this proposal, we will test the hypothesis that diabetes-induced downregulation of LXR expression and activity in the retina results in abnormal lipid metabolism, inflammation and vascular damage. Strategies to stimulate LXR will result in activation of cellular and tissue cholesterol removal with normalization of cholesterol homeostasis and repression of the inflammatory genes, such as iNOS, IL-1β, ICAM-1 and prevention of retinal vascular damage To test our hypothesis, we put forth the following Objectives:Objective 1: To determine if LXR systemic activation by pharmacological agonist (DMHCA) or intravitreal injection of AAV2 LXRα/β reverses diabetes-induced pro-inflammatory gene expression in the retina (iNOS, IL-1β, ICAM-1) through normalization of cholesterol homeostasis and prevents diabetes-induced retinal vascular damage.Objective 2: To determine if LXR activation by pharmacological agonist (DMHCA) or AAV2 LXRα/β normalizes cholesterol metabolism, pro-inflammatory gene expression and function of retinal RPE, HRECs, and HMC isolated from diabetic donors.Impact: This study will establish whether LXR activation represents a viable approach for prevention/reversal of DR and facilitate novel drug development.

Project Methods
We propose to determine if, in the retinas from db/m and db/db mice, the LXR controls i) cholesterol elimination through RTC genes ABCA1, ABCG1 and ApoE; ii) NFkB and inflammatory gene expression; and iii) systemic and retinal-specific LXR activation to reverse diabetes-induced cholesterol metabolism and inflammatory changes in the retina.For these proposed studies four key models will be utilized: (1) db/db diabetic mice treated with 8mg/kg body weight/day DMHCA, or (2) intravitreally injected with LXRα and b AAV2. The results will be compared to untreated db/m (3) and db/db (4) mice.As several aspects of mouse cholesterol homeostasis differ from that in humans,it is critical to validate key findings in the human retina. We will perform studies using human donor retinas, as well as carry out mechanistic in vitro investigation in retinal cell cultures to determine if LXR activation can normalize diabetes-induced changes in cholesterol metabolism and inflammatory gene expression in these cells.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The studies performed under this project are of interest for American Diabetes Association and Association for Research in Vision and Ophthalmology members. The results were presented at the annual meetings of these societies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The studies performed under this project were presented at the virtualAssociation for Research in Vision and Ophthalmology Annual Meeting What do you plan to do during the next reporting period to accomplish the goals?We will continue to exanime the effects of cholesterol uptake, accumulation and reverse cholesterol transport on the development of diabetic retinal pathology. The role of SIRT1, LXR and blood retinal barrier integrity for cholesterol accumulation in the diabetic retina and retinal cells will be evaluated under the Objectives 1 and 2.

Impacts
What was accomplished under these goals? In the last funding cycle, we produced and tested AAV2 LXRα/β. In this cycle, we used AAV2 LXRα/β to determine the effect on diabetes-induced pro-inflammatory gene expression in the retina (iNOS, IL-1β, ICAM-1) through normalization of cholesterol homeostasis and prevention of diabetes-induced retinal vascular damage (Objective 1) and on cholesterol metabolism, pro-inflammatory gene expression and function of retinal endothelial cells isolated from diabetic donors (Objective 2). the studies reporting these data are now included in the manuscript under revision for Diabetologia. In addition to AAV2 studies, we performed fasting and fasting mimicking conditions studies and SITR1 activation studies using SRT1720. These studies are also included in Diabetologia manuscript.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Levitsky Y, Pegouske DJ, Hammer SS, Frantz NL, Fisher KP, Muchnik AB, Saripalli AR, Kirschner P, Bazil JN, Busik JV, Proshlyakov DA. Micro-respirometry of whole cells and isolated mitochondria. RSC Adv. 2019;9(57):33257-67. Epub 2019/01/01. doi: 10.1039/c9ra05289e. PubMed PMID: 32123561; PMCID: PMC7051014.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Saadana A, Mast N, Trichonas G, Chakraborty D, Hammer S, Busik JV, Grant MB, Pikuleva IA. Retinal Vascular Abnormalities and Microglia Activation in Mice with Deficiency in Cytochrome P450 46A1-Mediated Cholesterol Removal. Am J Pathol. 2019;189(2):405-25. Epub 2018/11/19. doi: 10.1016/j.ajpath.2018.10.013. PubMed PMID: 30448403; PMCID: PMC6360352.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Huang C, Fisher KP, Hammer SS, Busik JV. Extracellular Vesicle-Induced Classical Complement Activation Leads to Retinal Endothelial Cell Damage via MAC Deposition. Int J Mol Sci. 2020;21(5). Epub 2020/03/04. doi: 10.3390/ijms21051693. PubMed PMID: 32121610; PMCID: PMC7084203.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Levitsky Y, Hammer SS, Fisher KP, Huang C, Gentles TL, Pegouske DJ, Xi C, Lydic TA, Busik JV, Proshlyakov DA. Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes. Int J Mol Sci. 2020;21(11). Epub 2020/06/03. doi: 10.3390/ijms21113830. PubMed PMID: 32481596; PMCID: PMC7312467.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Qi X, Mitter SK, Yan Y, Busik JV, Grant MB, Boulton ME. Diurnal Rhythmicity of Autophagy Is Impaired in the Diabetic Retina. Cells. 2020;9(4). Epub 2020/04/11. doi: 10.3390/cells9040905. PubMed PMID: 32272782; PMCID: PMC7226792.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Vieira CP, Fortmann SD, Hossain M, Longhini AL, Hammer SS, Asare-Bediako B, Crossman DK, Sielski MS, Adu-Agyeiwaah Y, Dupont M, Floyd JL, Li Calzi S, Lydic T, Welner RS, Blanchard GJ, Busik JV, Grant MB. Selective LXR agonist DMHCA corrects retinal and bone marrow dysfunction in type 2 diabetes. JCI Insight. 2020;5(13). Epub 2020/07/10. doi: 10.1172/jci.insight.137230. PubMed PMID: 32641586; PMCID: PMC7406260.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The studies performed under this project are of interest forAmerican Diabetes Association and Association for Research in Vision and Ophthalmology members. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The studies performed under this project were presented at theAmerican Diabetes Association Scientific Sessions, Association for Research in Vision and Ophthalmology Annual Meeting and EYE2019 meetings. What do you plan to do during the next reporting period to accomplish the goals?In this funding cycle, we produced and tested AAV2 LXRα/β. We are now using AAV2 LXRα/β to determine the effect ondiabetes-induced pro-inflammatory gene expression in the retina (iNOS, IL-1β, ICAM-1) through normalization of cholesterol homeostasis and prevents diabetes-induced retinal vascular damage (Objective 1) and oncholesterol metabolism, pro-inflammatory gene expression and function of retinalRPE, HRECs, and HMC isolated from diabetic donors (Objective 2).

Impacts
What was accomplished under these goals? Objective 1. In the previous cycle, we demonstrated that activation of LXR using the pharmacological agents, GW3965 or DMHCA restored reverse cholesterol transport, prevented inflammation, and reduced pro-inflammatory macrophages activity. GW3965 prevented the formation of diabetes-induced acellular capillaries in the Leprdb/db mouse. During this cycle,DMHCA was further evaluated for its retinal effects on normal C57BL/6J and Cyp27a1-/Cyp46a1-/- mice; the latter similar to the BBZDR rat have a higher total and esterified cholesterol in the retina. In both genotypes, the higher DMHCA doses (37-80 mg/kg of body weight/day) did not adversely increase serum triglycerides nor did it increase serum cholesterol, but it did alter the levels of retinal sterols. Total retinal cholesterol was decreased in the DMHCA-treated mice mainly due to a decrease in retinal unesterified cholesterol. This data are now published in AJP. We produced and tested AAV2 LXRa/b, the studies using the virus are undergoing. Objective 2. As direct LXRα activation of LXR increases expression of SREBP1c and ChREBPleading to liver steatosis, novel approaches for modulation of LXR activity are needed. In the current study, we utilized SIRT1 nutrient-sensing abilities to deacetylase and activate LXRa and its downstream signaling targets. SIRT1 is activated in low nutrient, calorie-reduced physiological states. Fasting-mimicking conditions (FMCs) activated SIRT1 expression and histone deacetylase (HDAC) activity in REC. Consequently, increased HDAC activity resulted in elevated total LXRa protein levelsand activity, as measured by the expression of ABCA1 and ABCG1. Moreover, total and active LXRa protein levels were significantly increased in REC treated with FMC. Administration of diabetes relevant stimuli, TNFa, increased cholesterol levels in REC while FMC lowered this diabetes-induced cholesterol accumulation. As expected, activation of LXRa via DMHCA, a steroidal LXR ligand, significantly reduced REC cholesterol levels. Treatment with FMC, in combination with DMHCA administration, significantly augmented cholesterol export and lowered REC cholesterol levels even further when compared to DMHCA treatment alone. In addition to augmenting cholesterol export, FMC treatment prevented the upregulation of NFkB dependent pro-inflammatory gene, VCAM1. These studies are in the final stages of manuscript preparation for a publication in a high profile journal.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Saadane A, Mast N, Trichonas G, Chakraborty D, Hammer S, Busik JV, Grant MB, Pikuleva IA. Retinal Vascular Abnormalities and Microglia Activation in Mice with Deficiency in Cytochrome P450 46A1-Mediated Cholesterol Removal.Am J Pathol. 2019 Feb;189(2):405-425. doi: 10.1016/j.ajpath.2018.10.013. Epub 2018 Nov 16.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Agbaga MP, Merriman DK, Brush RS, Lydic TA, Conley SM, Naash MI, Jackson S, Woods AS, Reid GE, Busik JV, Anderson RE. Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors. J Lipid Res. 2018 Sep;59(9):1586-1596. doi: 10.1194/jlr.M082495. Epub 2018 Jul 9.

Progress 03/01/18 to 09/30/18

Outputs
Target Audience:WebesEdge was commissioned by American Diabetes Association TV to record a movie about our work. The movie was featured at ADA Scientific Session in Orlando, 2018. It is also available on youtube at https://www.youtube.com/watchv=hurweaOP6pg&list=PL9CZabk3nD4EHOhMJxT1dungR11OPBV6g&index=38&t=0s Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The data were presented at American Diabetes Association meeting, Association for Research in Vision and Ophthalmology, International Society for Eye Research, and KeyStone mitochondrial metabolism meetings. In addition, ADA chose our work to be featured at the meeting. WebEdge crew made a movie about our work and broadcast it at the ADA meeting in Orlando, June 2018, it is also available on ADA web page and youtube. What do you plan to do during the next reporting period to accomplish the goals?In the next funding period we will complete the study on the role of SIRT1 deacetylase activity in the control of LXR in diabetic retina.

Impacts
What was accomplished under these goals? During this funding period, we published a key paper that demonstrates the accomplishment of our primary goals. This manuscript provides insight into the pathogenesis of diabetic retinopathy (DR) (Hammer et al) by showing that Sirtuin 1 and LXR modulate retinal cholesterol metabolism and inflammation in the diabetic retina. We found that expression of both regulators, Sirtuin 1 and LXR, were significantly decreased in diabetic human retinal samples and in a type 2 diabetic animal model (Leprdb/db mouse). Additionally, activation of LXR using the pharmacological agents, GW3965 restored reverse cholesterol transport, prevented inflammation, and reduced pro-inflammatory macrophages activity. GW3965 prevented the formation of diabetes-induced acellular capillaries in the Leprdb/db mouse. To determine the effect of diabetes on retinal cholesterol metabolism, we utilized the T2D BBZDR rat model and performed lipid analysis. In the T2D retinas, oxysterol levels were decreased by 64% (p< 0.05) and total cholesteryl ester abundance was increased compared to controls. Since diabetes induced a significant decrease in LXR activators, oxysterols, these data also support that LXR signaling and activation of downstream targets are impaired in diabetic tissue. We next tested N,N-dimethyl-3b-hydroxycholenamide (DMHCA) which is a LXR agonist, which does not induce the undesired hepatic lipogenesis as does GW 3965. DMHCA was evaluated for its retinal effects on normal C57BL/6J and Cyp27a1-/Cyp46a1-/- mice; the latter similar to the BBZDR rat have higher total and esterified cholesterol in the retina. In both genotypes, the higher DMHCA doses (37-80 mg/kg of body weight/day) did not adversely increase serum triglycerides nor did it increase serum cholesterol, but it did alter the levels of retinal sterols. Total retinal cholesterol was decreased in the DMHCA-treated mice mainly due to a decrease in retinal unesterified cholesterol. To test the hypothesis that SIRT1 deacetylation leads to LXR activation we used serum starvation as known SIRT1 activator. Serum starvation leads to increased deacetylase activityand SIRT1 mRNA. Moreover, SIRT1 activation leads to increased LXR activity as demonstrated by the increase in LXR targets ABCA1 and ABCG1.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kady NM, Liu X, Lydic TA, Syed MH, Navitskaya S, Wang Q, Hammer SS, O'Reilly S, Huang C, Seregin SS, Amalfitano A, Chiodo VA, Boye SL, Hauswirth WW, Antonetti DA, Busik JV. ELOVL4-Mediated Production of Very Long-Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability. Diabetes. 2018 April;67(4):769-781. PubMed PMID: 29362226; PubMed Central PMCID: PMC5860862.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang C, Fisher KP, Hammer SS, Navitskaya S, Blanchard GJ, Busik JV. Plasma Exosomes Contribute to Microvascular Damage in Diabetic Retinopathy by Activating the Classical Complement Pathway. Diabetes. 2018 August;67(8):1639-1649. PubMed PMID: 29866771; PubMed Central PMCID: PMC6054433.
• Type: Websites Status: Published Year Published: 2018 Citation: https://www.youtube.com/watch?v=hurweaOP6pg&t=0s&list=PL9CZabk3nD4EHOhMJx T1dungR11OPBV6g&index=38