Association of a Pro-Inflammatory Axis Comprised By TSP1-TGF-CTGF/CCN2 with a Decrease in Mir-19a-3p in the Pathophysiology of Diabetic Retinopathy
Authors: Alvarez Dela Cadena Philippe Alexander , Calva Moreno Jose Francisco , Del Carpio-Cano Fabiola Elizabeth , Ajaykumar Rao , Foster William J , Dela Cadena Raul A 
Published: October 2019

Abstract
Diabetic retinopathy (DR) is the leading cause of legal blindness in working age adults in the United States with about 18 thousand new cases of blindness each year. Approximately 75% of patients with type-2 diabetes will develop some sign of retinopathy after 15 years. The healthcare costs associated with diabetes related vision loss has been estimated at $500 million annually and the cost rises significantly with the severity of DR. Therefore, preventing the progression of DR from mild non-progressive diabetic retinopathy (NPDR) to progressive diabetic retinopathy (PDR) could ameliorate the economic burden related to diabetes and vision loss. Our laboratory has focused on a pro-inflammatory axis comprised by thrombospondin-1 (TSP1), transforming growth factor β (TGFb) and connective tissue growth factor (CTGF/CCN2) in the pathophysiology of DR. Furthermore, the regulation of this pro-inflammatory axis during disease states in humans associated with acute and chronic inflammation such as type-2 diabetes has not been elucidated. The current prospective study documents a potential regulatory mechanism, namely a microRNA associated with the up-regulation of the pro-inflammatory axis. Patients were recruited between February 2013 to May 2014. In May of 2015 it was determined by this research team that the data was mature for final analysis and, thus the results are the topic of this abstract. The participants were patients with type-2 diabetes (n=36) with either evidence of non-proliferative diabetic retinopathy (NPDR Group, n=16), or with evidence of proliferative diabetic retinopathy (PDR Group, n=20) as well as non-diabetic human subjects (Control Group, n=21). Each patient's history and duration of diabetes was obtained from the medical electronic records.
TSP1 was found significantly increased in the NPDR (502±145.03 ng/ml, p<0.05) and PDR (1033.9±136 ng/ml, p<0.001) groups when compared to control group (167±41.9 ng/ml). TGF-β was found significantly increased in the NPDR (32.0±4.49 pg/ml, p<0.001) and PDR (7.75±1.7 pg/ml, p<0.05) groups when compared to control (0.75±0.05 pg/ml). CTGF/CCN2 was found significantly increased in the NPDR and PDR groups, namely 3.69±1.52 ng/ml (p<0.001) and 1.69±0.4 ng/ml (p<0.05) respectively when compared to the control group (0.25±0.049 ng/ml). Tumor necrosis factor-α was found significantly increased (17.3±2.1 pg/ml, p<0.05) in the NPDR group but not in the PDR group (trend for higher levels) when compared to control group (9.61±0.74 pg/ml). Interleukin-4 was found decreased in both the NPDR (1.54±0.23 pg/ml, p<0.05) and the PDR (2.02±0.26, p<0.05) groups when compared with the control group (5.42±1.63 pg/ml). IL-4 recently has been documented as an important regulator of lipid metabolism.
When comparing the three groups using an ANOVA, one microRNA, namely miR-19a-3p was found to be the most strongly differentially expressed using a cutoff p-value ≤ 0.05 after a Benjamini-Hochberg correction for multiple testing, recommended when doing panel or array profiling. Figure 1 panel A shows the expression level centered to assay average for miR-19a-3p detected in most samples. Panel B clearly illustrates a significant decrease in miR-19a-3p in both groups, namely NPDR (p<0.0012) and PDR (p<0.049).
Interestingly down-regulation of miR-19a was found independent of progression of disease suggesting that the differential expression seen in our study in the NPDR and PDR groups is inherent to the underlying disease. These last observations including the mechanistic steps involved are currently under investigation in our laboratory, since understanding, how the down-regulation of miR-19a leads to up-regulation of TSP1-TGFb-CTGF/CCN2, might have a two-fold potential value in type-2 diabetes. First, it can be used as a biomarker for early diagnosis, prognosis and therapeutic response. Second, a therapeutic approach would be able to restore the mature miRNA levels in the proper tissue/cells. In summary our results support the role of a pro-inflammatory axis in the pathophysiology of type-2 diabetes as a result of differential expression of miR-19a.
Figure 1. Panel A and Panel B Figure 1. Panel A and Panel B

Disclosures
No relevant conflicts of interest to declare.