CTRP3 is a novel biomarker for diabetic retinopathy and inhibits HGHL-induced VCAM-1 expression in an AMPK-dependent manner

Abstract

Objectives: Diabetic retinopathy (DR) is a severe complication of chronic diabetes. The C1q/TNF-related protein family (CTRPs) has been demonstrated to exert protective effects against obesity and atherosclerosis in animal studies. Heretofore, the association between circulating CTRPs and DR patients has been unexplored. In the current study, we attempt to define this association, as well as the effect of CTRPs upon DR pathophysiology.

Design: The present investigation is a case control study that enrolled control subjects and type 2 diabetes mellitus (T2DM) patients diagnosed with DR. Serum CTRPs and sVACM-1 were determined by ELISA.

Results: Serum CTRP3 and CTRP5 levels were markedly decreased in patients with T2DM compared to controls (p<0.05) and inversely associated with T2DM. Furthermore, mutivariate regression and ROC analysis revealed CTRP3 deficiency, not CTRP5, was associated with proliferative diabetic retinopathy (PDR). Spearman's rank correlation assay demonstrated an inverse association between CTRP3 and sVCAM-1. Finally, exogenous CTRP3 administration attenuated high glucose high lipid (HGHL)-induced VCAM-1 production in an AMPK-dependent manner in cultured human retinal microvascular endothelial cells (HRMECs).

Conclusion: CTRP3 may serve as a novel biomarker for DR severity. CTRP3 may represent a future novel therapeutic against DR, a common ocular complication of diabetes.

Fig 1. ROC curves, for DR/PDR diagnosis, by circulating CTRP3 level.

(A) ROC curve analysis revealed that CTRP3 distinguishes patients with and without DR, AUC = 0.900 (95% CI 0.838–0.962, P<0.001). (B) CTRP3 distinguishes PDR from NPDR, AUC = 0.919 (95% CI 0.850–0.989, P<0.001).

Fig 2. CTRP3 inhibits high glucose/high lipids (HGHL)-induced expression of VCAM-1 in a time- and dose-dependent manner.

(A) HRMECs were subjected to high glucose/high lipid endothelial growth medium for 48 hours, incubated with CTRP3 (0.3, 1, 3μg/mL) for 60 minutes. VCAM-1 was inhibited in a concentration-dependent manner. (B) HRMECs were subjected to various periods of CTRP3 treatment (30, 60, and 120 minutes). VCAM-1 significantly decreased after 15 minutes, with decreasing trend for 120 total minutes. (C) HRMECs were placed in transwell migration chambers containing filters coated with matrigel. To stimulate migration, different concentrations of CTRP3 (0, 0.3, 1, 3μg/ml) were added to the lower chamber. Transwell chambers were stained with crystal violet and imaged after 8 hours. (D) The average number of migrated cells was quantified compared to vehicle control. Results are expressed as means±SD from 5–6 independent experiments. Each experiment was repeated 3 times. ^*, P<0.05; ^##, P<0.01 vs respective control.

Fig 3. CTRP3 inhibited HGHL induced VCAM-1 production in an AMPK dependent manner.

(A) HRMECs were incubated with HGHL for 48 hours, and subjected to CTRP3 administration (doses 0.3, 1, and 3μg/ml). (B) Akt phosphorylation was determined after 15 minutes. (C) AMPK phosphorylation was determined after 48 hours of HGHL incubation, followed by 15 minutes of CTRP3 treatment (doses 0.3, 1, and 3μg/ml). (D) Western blot analysis confirmed successful knockdown of AMPK by siRNA (>80% knockdown) E) AMPK knockdown blocked CTRP3 (3μg/ml, 1 hour treatment)-mediated inhibition of VCAM-1 expression induced by HGHL (48 hours treatment). Results are expressed as means±SD from 5–6 independent experiments. Each experiment was repeated 3 times. *p<0.05; ##, p<0.01 vs respective control.