Intrauterine and genetic risk factors for proliferative diabetic retinopathy

Diabetes is a complex progressive metabolic disorder characterized by hyperglycemia and caused by different etiopathogenic factors. Individuals with diabetes have heterogeneous clinical representation and increased risk of micro- and macrovascular complications. Diabetic retinopathy (DR) is the most frequent microvascular complication of diabetes and one of the leading causes of blindness. Currently, existing treatment modalities target a severe sight-threatening form of the disease, proliferative DR (PDR), and are characterized by significant side effects. The prevailing strategy for prevention or slowing down DR progression is glucose-lowering therapy, which is not efficient enough and might be harming to older groups of patients. Risk factors for PDR include duration of diabetes, hypertension, dyslipidemia, genetics and environment, and their interplay. The adverse intrauterine environment, particularly exposure to prenatal famine, was shown to play an important role in predisposition to diverse metabolic disorders in adults such as type 2 diabetes (T2D), hypertension, and cardiovascular diseases (CVD). In this Ph.D. thesis, we aimed to study novel diabetes subgroups based on pathophysiological characteristics of patients, highlighting subgroup(s) with an elevated risk of diabetic complications, particularly PDR. Further, we aimed to investigate the association of intrauterine exposure to famine with the risk of PDR in adult individuals with T2D. Finally, we wanted to study the molecular mechanisms linked to famine-related PDR. In paper 1, we performed a k-means cluster analysis to identify novel subgroups of individuals with new-onset and long-term diabetes, and estimated the risks of diabetic complications using logistic regression. In paper 2, we evaluated effect of intrauterine famine exposure on the risk of PDR in individuals with T2D using logistic regression adjusted for established risk factors such as age, sex, duration of diabetes and HbA1c. In paper 3, we performed candidate gene analysis using generalized estimation equation (GEE) to study the effect of interaction between SNPs and perinatal famine exposure on the risk of PDR. In paper 4, we performed genome-wide association (GWAS) and interaction (GWIS) studies using a linear mixed model (LMM) to investigate molecular underpinnings of famine-related PDR. In paper 1, we identified three subgroups with severe diabetes and two subgroups with mild diabetes. The highest risk of PDR was observed in the severe autoimmune diabetes (SAID) and severe insulin-deficient diabetes (SIDD) subgroups and the lowest in the insulin-resistant obesity-related diabetes 2 (IROD2) subgroup. In paper 2, we demonstrated that individuals with T2D, who were perinatally exposed to famine had an elevated risk of PDR in adult life. In paper 3, we demonstrated a significant association between famine-associated PDR and SNPs which were located in genes with neuronal function. In paper 4, we identified diverse pathways potentially linked to famine-related PDR, among them the most significant were lipid metabolism and inflammation pathways. In conclusion, we suggested that the altered development of neurovascular unit in the retina due to exposure to intrauterine famine may increase susceptibility to PDR later in life. Changes in metabolic adaptations during developmental programming induced by adverse early life events may affect insulin secretion and lipid metabolism, which consequently may increase predisposition to PDR under diabetes environment in adulthood. We suggested that drugs targeting these mechanisms in addition to glucose-lowering treatments may be beneficial for the prevention or slowing down the progression to PDR in the early stages of the disease.Diabetes er en kompleks progressiv metabolsk sykdom som kjennetegnes ved hyperglykemi og er forårsaket av forskjellige etiopatogenetiske faktorer. Personer med diabetes har heterogen klinisk representasjon og økt risiko for mikro- og makrovaskulære komplikasjoner. Diabetisk retinopati (DR) er den hyppigste mikrovaskulære komplikasjonen ved diabetes og en av de viktigste årsakene til blindhet. For tiden er eksisterende behandlingsmetoder rettet mot en alvorlig synstruende form av sykdommen, proliferativ DR (PDR), og medfører betydelige bivirkninger. Den rådende strategien for forebygging eller forsinking av DR-progresjon er glukosesenkende terapi, som ikke er effektiv nok og kan være skadelig for eldre pasienter. Risikofaktorer for PDR inkluderer varighet av diabetes, hypertensjon, dyslipidemi, genetikk og miljø, og deres samspill. Det ugunstige intrauterine miljøet, spesielt eksponering for prenatal hungersnød, ble vist å spille en viktig rolle i predisposisjon for ulike metabolske forstyrrelser hos voksne som type 2 diabetes (T2D), hypertensjon og kardiovaskulære sykdommer (CVD). I denne doktorgradsavhandlingen, har vi hatt som mål å studere nye diabetes-undergrupper basert på patofysiologiske egenskaper hos pasienter, og fremheve undergruppe(r) med økt risiko for diabetiske komplikasjoner, spesielt PDR. Videre hadde vi som mål å undersøke sammenheng mellom intrauterin eksponering for hungersnød og risikoen for utvikling av PDR hos voksne med T2D. Til slutt ønsket vi å studere de molekylære mekanismene knyttet til hungersnød-relatert PDR. I artikkel 1 utførte vi en k-means-klyngeanalyse for å identifisere nye undergrupper av individer med nyoppstått og langvarig diabetes, og estimerte risikoen for diabetiske komplikasjoner ved hjelp av logistisk regresjon. I artikkel 2 evaluerte vi effekten av eksponering for intrauterin hungersnød på risikoen for PDR hos individer med T2D ved bruk av logistisk regresjon justert for etablerte risikofaktorer som alder, kjønn, varighet av diabetes og HbA1c. I artikkel 3 utførte vi kandidatgenanalyse ved å bruke generalisert estimeringsligning (GEE) for å studere effekten av interaksjon mellom SNP-er og perinatal hungersnødeksponering på risikoen for PDR. I artikkel 4 utførte vi genomomfattende assosiasjonsstudier (GWAS) og interaksjonsstudier (GWIS) ved å bruke en lineær blandet modell (LMM) for å undersøke molekylært grunnlag for hungersnødrelatert PDR. I artikkel 1 identifiserte vi tre undergrupper med alvorlig diabetes og to undergrupper med mild diabetes. Den høyeste risikoen for PDR ble observert i undergruppene med alvorlig diabetes type 1 (SAID) og alvorlig diabetes type 2 (SIDD), og den laveste i undergruppen insulinresistent fedme-relatert diabetes 2 (IROD2). I artikkel 2 viste vi at personer med T2D, som ble perinatalt utsatt for hungersnød, hadde en forhøyet risiko for utvikling av PDR i voksenlivet. I artikkel 3 viste vi en signifikant sammenheng mellom hungersnødassosiert PDR og SNP-er som var lokalisert i gener med nevronal funksjon. I artikkel 4 identifiserte vi ulike veier som er potensielt knyttet til hungersnød-relatert PDR, blant dem var de mest betydningsfulle lipidmetabolisme og betennelsesveier. Avslutningsvis foreslo vi at den endrede utviklingen av nevrovaskulær kretsløp i netthinnen på grunn av eksponering for intrauterin hungersnød kan øke følsomheten for PDR senere i livet. Endringer i metabolske tilpasninger under utviklingsprogrammering indusert av uønskede hendelser i tidlig liv kan påvirke insulinsekresjon og lipidmetabolisme, som følgelig kan øke predisposisjon for PDR under diabetesmiljø i voksen alder. Vi foreslo at medikamenter som retter seg mot disse mekanismene i tillegg til glukosesenkende behandlinger kan være gunstig for forebygging eller forsinke progresjon til PDR i de tidlige stadiene av sykdommen.Doktorgradsavhandlin

Neuronal Dysfunction Is Linked to the Famine-Associated Risk of Proliferative Retinopathy in Patients With Type 2 Diabetes

Diabetic retinopathy; Famine; Neuronal functionRetinopatía diabética; Hambruna; Función neuronalRetinopatia diabètica; Fam; Funció neuronalPersons with type 2 diabetes born in the regions of famine exposures have disproportionally elevated risk of vision-threatening proliferative diabetic retinopathy (PDR) in adulthood. However, the underlying mechanisms are not known. In the present study, we aimed to investigate the plausible molecular factors underlying progression to PDR. To study the association of genetic variants with PDR under the intrauterine famine exposure, we analyzed single nucleotide polymorphisms (SNPs) that were previously reported to be associated with type 2 diabetes, glucose, and pharmacogenetics. Analyses were performed in the population from northern Ukraine with a history of exposure to the Great Ukrainian Holodomor famine [the Diagnostic Optimization and Treatment of Diabetes and its Complications in the Chernihiv Region (DOLCE study), n = 3,583]. A validation of the top genetic findings was performed in the Hong Kong diabetes registry (HKDR, n = 730) with a history of famine as a consequence of the Japanese invasion during WWII. In DOLCE, the genetic risk for PDR was elevated for the variants in ADRA2A, PCSK9, and CYP2C19*2 loci, but reduced at PROX1 locus. The association of ADRA2A loci with the risk of advanced diabetic retinopathy in famine-exposed group was further replicated in HKDR. The exposure of embryonic retinal cells to starvation for glucose, mimicking the perinatal exposure to famine, resulted in sustained increased expression of Adra2a and Pcsk9, but decreased Prox1. The exposure to starvation exhibited a lasting inhibitory effects on neurite outgrowth, as determined by neurite length. In conclusion, a consistent genetic findings on the famine-linked risk of ADRA2A with PDR indicate that the nerves may likely to be responsible for communicating the effects of perinatal exposure to famine on the elevated risk of advanced stages of diabetic retinopathy in adults. These results suggest the possibility of utilizing neuroprotective drugs for the prevention and treatment of PDR.This work was supported by the Swedish Research Council (Dnr2015-03574 and Dnr349-2006-237), Strategic Research Area Exodiab (Dnr2009-1039), the Novonordisk Foundation (NNF12OC1016467), Swedish Foundation for Strategic Research (DnrIRC15-0067), the Steno Diabetes Center Copenhagen, Bergen Research Foundation and Trond Mohn Foundation (BFS811294), and the University of Bergen

Increased Plasma Soluble Interleukin-2 Receptor Alpha Levels in Patients With Long-Term Type 1 Diabetes With Vascular Complications Associated With IL2RA and PTPN2 Gene Polymorphisms

Type 1 diabetes (T1D) is largely considered an autoimmune disease leading to the destruction of insulin-producing pancreatic beta cells. Further, patients with T1D have 3-4-fold increased risk of developing micro- and macrovascular complications. However, the contribution of immune-related factors contributing to these diabetes complications are poorly understood. Individuals with long-term T1D who do not progress to vascular complications offer a great potential to evaluate end-organ protection. The aim of the present study was to investigate the association of inflammatory protein levels with vascular complications (retinopathy, nephropathy, cardiovascular disease) in individuals with long-term T1D compared to individuals who rapidly progressed to complications. We studied a panel of inflammatory markers in plasma of patients with long-term T1D with (n = 81 and 26) and without (n = 313 and 25) vascular complications from two cross-sectional Scandinavian cohorts (PROLONG and DIALONG) using Luminex technology. A subset of PROLONG individuals (n = 61) was screened for circulating immune cells using multicolor flow cytometry. We found that elevated plasma levels of soluble interleukin-2 receptor alpha (sIL-2R) were positively associated with the complication phenotype. Risk carriers of polymorphisms in the IL2RA and PTPN2 gene region had elevated plasma levels of sIL-2R. In addition, cell surface marker analysis revealed a shift from naive to effector T cells in T1D individuals with vascular complications as compared to those without. In contrast, no difference between the groups was observed either in IL-2R cell surface expression or in regulatory T cell population size. In conclusion, our data indicates that IL2RA and PTPN2 gene variants might increase the risk of developing vascular complications in people with T1D, by affecting sIL-2R plasma levels and potentially lowering T cell responsiveness. Thus, elevated sIL-2R plasma levels may serve as a biomarker in monitoring the risk for developing diabetic complications and thereby improve patient care.Peer reviewe

Novel Reclassification of Adult Diabetes Is Useful to Distinguish Stages of β-Cell Function Linked to the Risk of Vascular Complications: The DOLCE Study From Northern Ukraine

Background: Presently, persons with diabetes are classified as having type 1 (T1D) or type 2 diabetes (T2D) based on clinical diagnosis. However, adult patients exhibit diverse clinical representations and this makes treatment approaches challenging to personalize. A recent Scandinavian study proposed a novel classification of adult diabetes into five clusters based on disease pathophysiology and risk of vascular complications. The current study aimed to characterize new subgroups of adult diabetes using this strategy in a defined population from northern Ukraine. Methods: We analyzed 2,140 patients with established diabetes from the DOLCE study (n = 887 with new-onset diabetes and n = 1,253 with long duration). We used the k-means approach to perform clustering analyses using BMI, age at onset of diabetes, HbA1c, insulin secretion (HOMA2-B), and insulin resistance (HOMA2-IR) indices and glutamic acid decarboxylase antibodies (GADA) levels. Risks of macro- (myocardial infarction or stroke) and microvascular [retinopathy, chronic kidney disease (CKD) and neuropathy] complications and associations of genetic variants with specific clusters were studied using logistic regression adjusted for age, sex, and diabetes duration. Results: Severe autoimmune diabetes (SAID, 11 and 6%) and severe insulin-deficient diabetes (SIDD, 25 and 14%) clusters were twice as prevalent in patients with long-term as compared to those with new-onset diabetes. Patients with long duration in both SAID and SIDD clusters had highest risks of proliferative retinopathy, and elevated risks of CKD. Long-term insulin-resistant obese diabetes 1 (IROD1) subgroup had elevated risks of CKD, while insulin-resistant obese diabetes 2 (IROD2) cluster exhibited the highest HOMA2-B, lowest HbA1c, and lower prevalence of all microvascular complications as compared to all other clusters. Genetic analyses of IROD2 subgroup identified reduced frequency of the risk alleles in the TCF7L2 gene as compared to all other clusters, cumulatively and individually (p = 0.0001). Conclusion: The novel reclassification algorithm of patients with adult diabetes was reproducible in this population from northern Ukraine. It may be beneficial for the patients in the SIDD subgroup to initiate earlier insulin treatment or other anti-diabetic modalities to preserve β-cell function. Long-term diabetes cases with preserved β-cell function and lower risk for microvascular complications represent an interesting subgroup of patients for further investigations of protective mechanisms

Increased Plasma Soluble Interleukin-2 Receptor Alpha Levels in Patients With Long-Term Type 1 Diabetes With Vascular Complications Associated With IL2RA and PTPN2 Gene Polymorphisms

Type 1 diabetes (T1D) is largely considered an autoimmune disease leading to the destruction of insulin-producing pancreatic β cells. Further, patients with T1D have 3–4-fold increased risk of developing micro- and macrovascular complications. However, the contribution of immune-related factors contributing to these diabetes complications are poorly understood. Individuals with long-term T1D who do not progress to vascular complications offer a great potential to evaluate end-organ protection. The aim of the present study was to investigate the association of inflammatory protein levels with vascular complications (retinopathy, nephropathy, cardiovascular disease) in individuals with long-term T1D compared to individuals who rapidly progressed to complications. We studied a panel of inflammatory markers in plasma of patients with long-term T1D with (n = 81 and 26) and without (n = 313 and 25) vascular complications from two cross-sectional Scandinavian cohorts (PROLONG and DIALONG) using Luminex technology. A subset of PROLONG individuals (n = 61) was screened for circulating immune cells using multicolor flow cytometry. We found that elevated plasma levels of soluble interleukin-2 receptor alpha (sIL-2R) were positively associated with the complication phenotype. Risk carriers of polymorphisms in the IL2RA and PTPN2 gene region had elevated plasma levels of sIL-2R. In addition, cell surface marker analysis revealed a shift from naïve to effector T cells in T1D individuals with vascular complications as compared to those without. In contrast, no difference between the groups was observed either in IL-2R cell surface expression or in regulatory T cell population size. In conclusion, our data indicates that IL2RA and PTPN2 gene variants might increase the risk of developing vascular complications in people with T1D, by affecting sIL-2R plasma levels and potentially lowering T cell responsiveness. Thus, elevated sIL-2R plasma levels may serve as a biomarker in monitoring the risk for developing diabetic complications and thereby improve patient care

Neuronal Dysfunction Is Linked to the Famine-Associated Risk of Proliferative Retinopathy in Patients With Type 2 Diabetes

Persons with type 2 diabetes born in the regions of famine exposures have disproportionally elevated risk of vision-threatening proliferative diabetic retinopathy (PDR) in adulthood. However, the underlying mechanisms are not known. In the present study, we aimed to investigate the plausible molecular factors underlying progression to PDR. To study the association of genetic variants with PDR under the intrauterine famine exposure, we analyzed single nucleotide polymorphisms (SNPs) that were previously reported to be associated with type 2 diabetes, glucose, and pharmacogenetics. Analyses were performed in the population from northern Ukraine with a history of exposure to the Great Ukrainian Holodomor famine [the Diagnostic Optimization and Treatment of Diabetes and its Complications in the Chernihiv Region (DOLCE study), n = 3,583]. A validation of the top genetic findings was performed in the Hong Kong diabetes registry (HKDR, n = 730) with a history of famine as a consequence of the Japanese invasion during WWII. In DOLCE, the genetic risk for PDR was elevated for the variants in ADRA2A, PCSK9, and CYP2C19*2 loci, but reduced at PROX1 locus. The association of ADRA2A loci with the risk of advanced diabetic retinopathy in famine-exposed group was further replicated in HKDR. The exposure of embryonic retinal cells to starvation for glucose, mimicking the perinatal exposure to famine, resulted in sustained increased expression of Adra2a and Pcsk9, but decreased Prox1. The exposure to starvation exhibited a lasting inhibitory effects on neurite outgrowth, as determined by neurite length. In conclusion, a consistent genetic findings on the famine-linked risk of ADRA2A with PDR indicate that the nerves may likely to be responsible for communicating the effects of perinatal exposure to famine on the elevated risk of advanced stages of diabetic retinopathy in adults. These results suggest the possibility of utilizing neuroprotective drugs for the prevention and treatment of PDR

Perinatal famine is associated with excess risk of proliferative retinopathy in patients with type 2 diabetes

PURPOSE: Intrauterine undernutrition is associated with increased risk of type 2 diabetes. Children born premature or small for gestational age were reported to have abnormal retinal vascularization. However, whether intrauterine famine act as a trigger for diabetes complications, including retinopathy, is unknown. The aim of the current study was to evaluate long-term effects of perinatal famine on the risk of proliferative diabetic retinopathy (PDR).METHODS: We studied the risk for PDR among type 2 diabetes patients exposed to perinatal famine in two independent cohorts: the Ukrainian National Diabetes Registry (UNDR) and the Hong Kong Diabetes Registry (HKDR). We analysed individuals born during the Great Famine (the Holodomor, 1932-1933) and the WWII (1941-1945) famine in 101 095 (3601 had PDR) UNDR participants. Among 3021 (251 had PDR) HKDR participants, we studied type 2 diabetes patients exposed to perinatal famine during the WWII Japanese invasion in 1942-1945.RESULTS: During the Holodomor and WWII, perinatal famine was associated with a 1.76-fold (p = 0.019) and 3.02-fold (p = 0.001) increased risk of severe PDR in the UNDR. The risk for PDR was 1.66-fold elevated among individuals born in 1942 in the HKDR (p < 0.05). The associations between perinatal famine and PDR remained statistically significant after corrections for HbA1c in available 18 507 UNDR (padditive interaction < 0.001) and in 3021 HKDR type 2 diabetes patients (p < 0.05).CONCLUSION: In conclusion, type 2 diabetes patients, exposed to perinatal famine, have increased risk of PDR compared to those without perinatal famine exposure. Further studies are needed to understand the underlying mechanisms and to extend this finding to other diabetes complications