RAGE Regulating Vascular Remodeling in Diabetes by Regulating Mitochondrial Dynamics with JAK2/STAT3 Pathway

Abstract

In this research, we will explore the role and modulation of mitochondrial dynamics in diabetes vascular remodeling. Only a few cell types express the pattern recognition receptor, also known as the AGE receptor (RAGE). However, it is triggered in almost all of the cells that have been investigated thus far by events that are known to cause inflammation. Here, Type 2 diabetes was studied in both cellular and animal models. Elevated Receptor for advanced glycation end products (RAGE), phosphorylated JAK2 (p-JAK2), phosphorylated STAT3 (p-STAT3), transient receptor potential ion channels (TRPM), and phosphorylated dynamin-related protein 1 (p-DRP1) were observed in the context of diabetes. In addition, we found that inhibition of RAGE was followed by a remarkable decrease in the expression of the above proteins. It has also been demonstrated by western blotting and immunofluorescence results in vivo and in vitro. Suppressing STAT3 and DRP1 phosphorylation produced effects similar to those of RAGE inhibition on the proliferation, cell cycle, migration, invasion, and expression of TRPM in VSMCs and vascular tissues obtained from diabetic animals. These findings indicate that RAGE regulates vascular remodeling via mitochondrial dynamics through modulating the JAK2/STAT3 axis in diabetes. The findings could be crucial in gaining a better understanding of diabetes-related vascular remodeling. It also contributes to a better cytopathological understanding of diabetic vascular disease and provides a theoretical foundation for novel targets that aid in the prevention and treatment of diabetes-related cardiovascular problems.

Figure 1

RAGE regulates mitochondrial dynamics and JAK2/STAT3 pathway in diabetes. (a) Western blotting analysis of the protein expression of RAGE, Drp1, p-Drp1, Mfn2, JAK2, p-JAK2, STAT3, and p-STAT3 in different treatment groups. (b) Immunofluorescence staining of TRPM in different treatment groups. The experiments were carried out in triplicate and representative images were presented. Note. ns = non-significant, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001 compared to control, ^#p < 0.05, ^##p < 0.01 compared to CML-BSA group.

Figure 2

RAGE regulates JAK2/STAT3 pathway in diabetic mice. Western blotting analysis of the protein expression of Drp1, p-Drp1, Mfn2, JAK2, p-JAK2, STAT3, and p-STAT3 in different treatment groups. The experiments were carried in triplicate and representative images were presented. Note. ns = non-significant, ^∗∗p < 0.01, ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001 compared to control, ^#p < 0.05, ^##p < 0.01 compared to TD2M group.

Figure 3

RAGE-mediated activation of JAK2/STAT3 axis regulates the proliferation of VSMCs. (a) Western blotting analysis of the protein expression of Drp1, p-Drp1, Mfn2, JAK2, p-JAK2, STAT3, and p-STAT3 in different treatment groups. (b) Cell viability of VSMCs in different treatment groups. The experiment was carried out in triplicate and representative images were presented. Note: ns = non-significant, ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001 compared to control, ^#p < 0.05 compared to CML-BSA group, ^$p < 0.05 compared to CML-BSA + siRAGE group.

Figure 4

RAGE-mediated activation of JAK2/STAT3 axis regulates the cell cycle, migration and invasion of VSMCs. (a) Cell cycle analysis of VSMCs in different treatment groups by flow cytometry. (b) Cell migration of VSMCs in different treatment groups determined by wound-healing assay. (c) Cell invasion of VSMCs in different treatment groups determined by matrigel Transwell assay. The experiments were carried out in triplicate and representative images were presented. Note: ns = non-significant, ^∗∗p < 0.01, ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001 compared to control, ^#p < 0.05 compared to CML-BSA group, ^$p < 0.05 compared to CML-BSA + siRAGE group.

Figure 5

RAGE-mediated activation of JAK2/STAT3 axis regulates ROS and vascular remodeling of VSMCs. (a) ROS analysis of VSMCs in different treatment groups by flow cytometry. (b) Western blot analysis of vascular remodeling-related proteins (α-SMA, CD31, VEGF-A) in different treatment groups. The experiments were carried out in triplicate and representative images were presented. Note. ns = non-significant, ^∗∗p < 0.01, ^∗∗∗p < 0.001 and ^∗∗∗∗p < 0.0001 compared to control, ^#p < 0.05 compared to CML-BSA group, ^$p < 0.05 compared to CML-BSA + siRAGE group.

Figure 6

RAGE-mediated activation of JAK2/STAT3 axis regulates TRPM in VSMCs. The expression of TRPM in different groups was determined by immunofluorescence. The experiments were carried out in triplicate and representative images were presented.

Figure 7

RAGE-mediated activation of JAK2/STAT3 axis regulates the depolarization of mitochondrial membrane potential in VSMCs. (a) The depolarization of MMP was detected by JC-1 staining method (the magnification for the figure is 200×). (b) Ratio of red/green fluorescence intensity expressed as mean ± SD of triplicates. The experiments were carried out in triplicate and representative images were presented.

Figure 8

RAGE-mediated activation of JAK2/STAT3 axis regulates vascular remodeling of VSMCs in vivo. (a) Oil red O staining and HE histological analysis of aortas of mice from different treatment groups (the magnification for the figure is 200×). (b) Immunofluorescence analysis of α-SMA in aorta sections of mice from different treatment groups. The experiments were carried out in triplicate and representative images were presented. The magnification for the figure is 200×.

Figure 9

RAGE-mediated activation of JAK2/STAT3 axis regulates the expression of vascular remodeling markers in VSMCs in vivo. (a) Immunofluorescence analysis of VEGF-A in aorta sections of mice from different treatment groups. (b) Immunofluorescence analysis of CD31 in aorta sections of mice from different treatment groups.