Association of Metformin Treatment with Reduced Severity of Diabetic Retinopathy in Type 2 Diabetic Patients

Purpose To evaluate effects of long-term metformin on the severity of diabetic retinopathy (DR) in high-risk type 2 diabetic (T2D) patients. Methods A retrospective chart review study was conducted involving 335 DR patients with T2D ≥ 15 years from 1990 to 2013. The severity of DR was determined by Early Treatment Diabetic Retinopathy Study scale. The associations between metformin and DR severity were evaluated. Comparison with stratification for the use of sulfonylurea and insulin was performed to identify possible confounding effects. Results Severe nonproliferative diabetic retinopathy or proliferative diabetic retinopathy (SNPDR/PDR) was more often diagnosed in nonmetformin users (67/142, 47%) versus metformin users (48/193, 25%) (p < 0.001), regardless of gender and race of the patients. The odds ratio of metformin associated with SNPDR/PDR was 0.37 in all cases (p < 0.001), 0.35 in sulfonylurea use cohort (p < 0.05), 0.45 in nonsulfonylurea use cohorts (p < 0.01), and 0.42 in insulin use cohort (p < 0.01). Insulin users had a higher rate of SNPDR/PDR. Metformin had no influence on the occurrence of clinical significant diabetic macular edema. Conclusions Long-term use of metformin is independently associated with a significant lower rate of SNPDR/PDR in patients with type 2 diabetes ≥ 15 years.


Introduction
Diabetes affects 29.3 million people in the United States [1], of which 28.5% have diabetic retinopathy (DR) [2]. About one fourth of DR will progress to the stage of severe nonproliferative diabetic retinopathy or proliferative diabetic retinopathy (SNPDR/PDR). SNPDR/PDR is usually associated with significant vision loss due to the presence of macular edema, retinal angiogenesis, vitreous hemorrhage, and retinal detachment. DR is a leading cause of legal blindness in US working-age population [3], despite the efforts in systemic metabolic control and commonly applied laser photocoagulation.
Metformin has been used to treat hyperglycemia since 1950s and becomes the first-line and the most widely used oral medication for type 2 diabetes (T2D) recently [1,4]. Such increased usage of metformin as the preferred treatment in T2D is due to its newly identified protective effect against microvascular and macrovascular complications in diabetes patients beyond glycemic control. The landmark trial United Kingdom Prospective Diabetes Study (UKPDS) demonstrated a substantial beneficial effect of metformin on cardiovascular disease outcomes when compared with conventional diet treatment [5]. This finding was confirmed by several other independent clinical studies [6][7][8] and meta-analysis [9,10] that metformin reduced risks of cardiovascular complications in diabetes patients compared with any other oral hypoglycemics or placebo, independent of its hypoglycemic effects. Further, metformin was reported to effectively improve endothelial-dependent vasodilation [11] and suppress serum markers of endothelial activation such as soluble vascular adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) [12,13] in T2D patients. These evidences pointed to a potential effect of metformin on benefiting microvascular complications of diabetes such as DR. However, firsthand clinical data regarding the role of metformin in DR is scarce.
In this retrospective cross-sectional study, we assessed the relationship between long-term oral metformin and severity of DR in patients with T2D for 15 years or longer. Metformin-treated patients were found less likely to have SNPDR/PDR versus nonmetformin-treated patients, regardless of gender and race, and independent of glycated hemoglobin (HbA1c) levels. The association between metformin and a significant lower rate of SNPDR/PDR was not confounded by the use of insulin or sulfonylurea.

Participants.
This study was a retrospective, crosssectional study that included all patients with a diagnosis of DR and a history of T2D ≥ 15 years within Henry Ford Health System from January 1990 to September 2013. Cases were identified with an International Classification of Diseases-(ICD-) 9 diagnosis of DR. The index date of cases was the date of the most recent visit. Two groups were included in this study. One was the metformin-treated group that comprised all those who received oral metformin for the last 5 or more consecutive years. The other was the nonmetformin control group that consisted of cases that had no record of metformin use for T2D therapy. Concurrent use of insulin or other hypoglycemic agents was not excluded in both groups. Cases with intermittent metformin use, history of retinal detachment, and coexistence of other retinal vascular disorders, such as retinal vein occlusion and wet-type age-related macular degeneration, were excluded. A history of T2D ≥ 15 years and metformin treatment ≥ 5 years were chosen because prevalence of visual complication was notably increased when duration of diabetes exceeds 15 years [14], and it takes years for the effect of medication to become apparent. The study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of Henry Ford Health System.

Measurements.
From each case, the following information were collected: (1) demographic features, including age, gender, and race; (2) general information of T2D progression and management, such as date of T2D diagnosis, 5-year HbA1c levels (including the lowest, median, and highest values), and medications for T2D (including metformin, insulin, and other hypoglycemic agents); (3) grading of DR and prior DR treatments such as laser photocoagulation and intravitreal antivascular endothelial growth factor (VEGF) agents; and (4) ophthalmic history such as retinal vein occlusion and age-related macular degeneration.
Diagnosis of DR was made by a staff retinal specialist. The grading of DR severity and the presence of clinically significant macular edema (CSME) were determined according to the Early Treatment Diabetic Retinopathy Study (ETDRS) grading standards [15,16]. All cases were assigned into either mild or moderate NPDR or SNPDR/PDR accordingly. If both eyes had DR, the severity would be determined due to the grade in the worse eye.
The rate of SNPDR/PDR was compared between the metformin group and the nonmetformin control group, in all cases as well as in subgroups categorized according to sex and race. The odds ratio (OR) of SNPDR/PDR was calculated to assess the association between hypoglycemic treatment and severity of DR in all cases as well as in treatment-stratified cohorts.

Statistics.
Statistical analysis was performed using SAS software, version 9.2 (SAS Institute Inc., USA). Group difference of categorical variables was determined using standard chi-square test in the presence of nonsparse data, and Fisher exact test in the presence of sparse data. Sparsity was defined as the presence of expected cell counts less than 5. Group comparisons of numeric variables were made using twosample t-test in the presence of distributional normality, and Wilcoxon rank sum test in the presence of distributional nonnormality. The ORs and 95% confidence intervals were obtained from logistic regression analysis, and the statistical significance of OR was assessed by chi-square test or Fisher's exact test. Significance was considered when p < 0 05.

Demographic Features and Clinical Characteristics in the
Metformin and Nonmetformin Groups. This retrospective cross-sectional study included 335 DR cases with T2D for 15 years or longer. Of all the cases, 193 (58%) had used metformin for at least 5 consecutive years and 142 (42%) had no record of metformin use. Demographic features were comparable between the two groups regarding age, gender, and race ( Table 1). The duration of T2D was 15.1 ± 6.7 in the metformin group and 15.7 ± 7 in the nonmetformin group (p = 0 39; Table 1). The highest, lowest, and median levels of HbA1c in the most recent 5 years were well matched between the metformin group and the nonmetformin control group (p = 0 40, 0.81, and 0.58, resp.; Table 1).
Local therapies for DR including focal/grid laser photocoagulation, pan-retinal photocoagulation (PRP), and intravitreal anti-VEGF injection were applied in both groups. Significantly fewer patients in the metformin group received PRP versus the nonmetformin group (48 of 193, 25% versus 61 of 142, 43%) (p = 0 001; Table 1). There was no difference in the use of focal/grid laser photocoagulation or anti-VEGF agents between the two groups (Table 1).

Long-Term Metformin Treatment Was Associated with
Significantly Reduced Severity of DR. SNPDR/PDR was diagnosed among 48 (25%) of the patients who used metformin for 5 years or longer, compared with 67 (47%) of those who never used metformin (p < 0 001; Table 2). The rest of the patients in both groups (53% of the nonmetformin group and 75% of the metformin group) were diagnosed with   Table 2), implying a 63% reduction of SNPDR/PDR associated with metformin treatment. Gender and racial subgroup analysis further revealed that longer-term metformin was correlated with significantly lower rate of SNPDR/PDR in both female and male, as well as in black and white subgroups (Figure 1).

Metformin-Associated Less Severe DR Was Not
Confounded by the Use of Sulfonylurea or Insulin. Since there was notable difference in the use of insulin and sulfonylurea between the two groups (both p < 0 001; Table 1), association of these hypoglycemic therapies with the occurrence of SNPDR/PDR was evaluated. As summarized in  Table 2). Therefore, insulin was correlated with a higher rate of SNPDR/PDR, which was opposite to that of metformin. Further, comparison with stratification for the hypoglycemic treatments was performed to identify possible confounding effects. The association between metformin and lowered frequency of SNPDR/PDR was persistent in sulfonylurea cohort [OR 0. 35 (Figure 2). These data indicated the association between metformin, and a lower rate of SNPDR/PDR was independent of sulfonylurea, and likely independent of insulin as well.

Metformin Had No Effect on the Development of CSME.
Focal/grid laser photocoagulation was the major form of therapy for CSME before anti-VEGF therapy was available. CSME requiring focal/grid laser was identified in 83 of 193 (43%) cases among metformin users, and 69 of 142 (49%) cases among nonmetformin users. No significant difference was found between the two groups (p = 0 33).

Discussion
It is known from the United Kingdom Prospective Diabetes Study (UKPDS) that metformin significantly reduced the risk of many diabetes-related macrovascular and microvascular events when compared with other hypoglycemic therapies in diabetic patients [5]. As one of several endpoints of UKPDS, metformin lowered the risk of DR progression in overweight diabetic patients when compared with diabetic diet treatment. Inspired by UKPDS but different from it, this study focused on whether metformin influences DR severity in high-risk diabetic patients, that is, those with 15 years or longer history of T2D. We found a significant association between oral metformin for ≥5 years and less severe DR in these patients, which was not companied by a different HbA1c level. Further, this association persisted across the gender and racial subgroups and was independent of concurrent insulin or sulfonylurea treatment. The use of insulin was associated with more severe DR in our study.

Link between Metformin Treatment and Reduced DR
Severity. As a retrospective study, we were unable to control the use of insulin and sulfonylurea among the patients. A higher percentage of nonmetformin users received insulin treatment versus that of metformin users (93% versus 73%). On the contrary, more patients of the metformin group received sulfonylurea compared with those of the nonmetformin group (74% versus 38%). Insulin therapy is generally advocated when oral medications are insufficient in controlling blood glucose. It is likely that nonmetformin users had poorly control glycemia at some point, which led to prescription of insulin and might contribute to a higher risk of DR in this group. The homogeneity in the duration of diabetes and 5-year HbA1c levels among the two groups helped provide a relatively equal comparison regarding the risk factors of DR in this study, since these two clinical parameters are believed to be the most important factors that impact DR progression [14,17]. However, it is also important to note that tight glycemic control and a lower HbA1c have minimal effect on DR in T2D after prolonged follow-up, as evidenced by large-scale and long-term clinical studies including a 4.1-year ADVANCE study [18], a 5.6-year VADT trial [19], and a 10-year follow-up of UKPDS [20]. We assume that the different rates of SNPDR/PDR between the two groups were not simply a mirror of differently controlled blood glucose but rather reflected direct regulation on the pathogenic risk factors of DR by metformin.
The initiation of DR was associated with hyperglycemia, nonenzymatic glycosylation, and local oxidative and inflammatory stresses [21], while the progression of DR to the proliferative phase was more prominently a consequence of retinal inflammation, endothelial activation, and neovascularization [22][23][24]. The inflammatory cytokine ICAM-1 was 24 Figure 2: Odds ratios that indicate the association between the rate of SNPDR/PDR and hypoglycemic therapy in all cases and within each stratum of metformin, sulfonylurea, and insulin users. Metformin-or sulfonylurea-treated patients were significantly less likely to have SNPDR/PDR when all cases were assessed, while insulin users were more likely to have SNPDR/PDR versus those did not use insulin. When stratified for the use of each hypoglycemic agent, metformin showed independent association with lowered frequency of SNPDR/PDR within each stratums of hypoglycemic treatment, with statistical significance in sulfonylurea users, nonsulfonylurea users, and insulin users. However, sulfonylurea-associated significantly lower rate of SNPDR/PDR was only observed in insulin users. Insulin was associated with higher rate of SNPDR/PDR, which persisted in all the four cohorts stratified by metformin or sulfonylurea. Three of the four ORs had borderline p values. OR: odds ratio; SNPDR/PDR: severe nonproliferative diabetic retinopathy/proliferative diabetic retinopathy. one of the most studied molecules in DR pathogenesis. ICAM-1 was upregulated in the retina and vitreous of DR patients [25,26] and diabetes rodent models [27]. ICAM-1 knockout or pharmaceutical blockade attenuated DR features such as retinal leukostasis and vascular leakage in diabetic animals [24,27]. The assumption that metformin could directly modulate molecular events in DR progression, rather than through controlling blood glucose, was supported by clinical evidences that metformin significantly reduced plasma ICAM-1 in diabetes patients [12,13]. In addition, we have recently reported that long-term metformin treatment effectively reduced multiple inflammatory cytokines and their correlations in the vitreous of DR patients, including ICAM-1 and interleukins [26]. Experiments in endothelial cells and STZ-induced diabetes mice by us and others also identified significant attenuation of ICAM-1 and MCP-1 levels, as well as inflammatory responses by metformin [28,29]. The antiangiogenic activity of metformin was demonstrated in various endothelial cell lines as well as retinal neovascularization mouse models [29,30].
One major molecular target of metformin is AMPactivated protein kinase (AMPK), a crucial cellular energy sensor that regulates lipid and glucose metabolism. Activation of AMPK was intimately associated with metformin's action in regulating the metabolic processes [31] and the inflammatory responses [28]. The association between metformin and reduced severity of DR in T2D patients could be explained by metformin-induced restoration of energy balance in the retina through activation of AMPK. However, AMPK may not account for all actions of metformin, as demonstrated by the preserved metabolic effect of metformin in liver-specific AMPK-deficient mice [32]. The regulation on endothelial inflammatory and angiogenic responses by metformin also has been shown through both AMPK-dependent and AMPK-independent mechanisms [29,33].
Metformin did not change the rate of CSME in this study. Macular edema is mediated in substantial part by VEGF. Currently, the most successful treatment of macular edema is intravitreal injection of anti-VEGF agents [34]. Although metformin was weakly correlated with downregulation of hypoxia-inducible factor-1α/VEGF induced by insulin and insulin-like growth factor [35], this effect was insufficient to control the symptomatic diabetic macular edema.

Effects of Sulfonylurea and
Insulin on DR Severity. Comparison with stratification for use of insulin or sulfonylurea treatment confirmed that the association between metformin and reduced DR severity was independent of either hypoglycemic therapy. On the other hand, although sulfonylurea was also correlated with a lower rate of SNPDR/PDR in all cases, this correlation disappeared in cohorts stratified by the use of metformin. Since 74% of sulfonylurea users also used metformin in this study, it is possible that the effect of sulfonylurea on DR severity was modified by metformin. Our data previously supported findings by the ADVANCE trial that sulfonylurea does not prevent or reduce DR progression despite reaching the glucose control goals in T2D patients with vascular complications or risk factors of vascular diseases [36].
In contrast to metformin and sulfonylurea, insulin was associated with a higher rate of SNPDR/PDR in all patients with an OR of 4.35. Stratified analysis reduced the level of significance but did not reverse this trend, with the OR values varied from 1.96 to 6.91. Similar to what we found, some other clinical studies also associated insulin with increased risk of DR in T2D patients [37,38]. There were divergent findings about the effect of insulin on DR. UKPDS suggested that intensive blood glucose control with insulin could reduce risk of DR in newly diagnosed diabetes [39]. A Veterans Affairs Cooperative Study found that insulin had no effect on cardiovascular event or retinopathy in patients with established T2D [40]. Another study in diabetic mice associated insulin with increased retinal vascular permeability [41]. The exact impact of insulin on DR remains to be studied.

Strengths and Limitations of This Study.
Our finding that long-term metformin treatment was associated with significantly reduced rate of SNPDR/PDR was supported by previous clinical and basic research reports on the protective effects of metformin against microvascular complications of diabetes. In particular, this study adds firsthand clinical data on the glycemic-independent effect of metformin on the severity of DR in patients with established T2D. Although there was an imbalance in the use of insulin and sulfonylurea between the two groups, we used stratified comparison to adjust for potentially confounding effects of both agents. The consistence of the crude and adjusted OR for metformin treatment helps to rule out the confounders. The difference between the crude OR and adjusted OR for sulfonylurea suggested that its effect was modified by metformin and insulin. Insulin exhibited an opposite effect on DR severity compared with metformin and sulfonylurea. As a single-centered, retrospective, cross-sectional study, we could only control a few most prominent risk factors of DR. Unmeasured confounding may remain, such as blood pressure, blood lipid, and degree of microproteinuria. Future large-scale prospective studies are warranted for a better understanding of how metformin influences the progression of DR in T2D patients.

Conclusion
Our results should be interpreted as a significant association between long-term metformin treatment and reduced severity of DR in patients with established T2D. This association exists in the lack of a different HbA1c level, persists across the gender and racial cohorts, and is not confounded by sulfonylurea or insulin treatment. Insulin users were more likely to have severe DR versus noninsulin users. Metformin might be used for the purpose of reducing DR progression in patients with long history of T2D. Our investigation is a conceptual study which calls for large-scale future studies for a full evaluation of the exact role of metformin in the progression of DR.

Disclosure
Part of the results in the manuscript was presented as a meeting abstract [42].

Conflicts of Interest
The authors declare that there is no conflict of interests.