Glycemic control prevents microvascular remodeling and increased tone in type 2 diabetes: link to endothelin-1

Abstract

Medial thickening and vascular hypertrophy of resistance arteries can lead to cardiovascular complications associated with diabetes. While previous studies have established a role of type 1 diabetes in vascular remodeling, we recently extended these observations to type 2 diabetes and reported increased collagen deposition due to alterations in matrix metalloproteinase expression and activity in mesenteric resistance arteries. These studies also showed that remodeling response was mediated by endothelin-1 (ET-1) via activation of ET(A) receptors, whereas blockade of ET(B) receptors exacerbated the remodeling. However, the effectiveness of glycemic control strategies in preventing these vascular changes, including activation of the ET system still remained unclear. Also, very little is known about whether and to what extent reorganization of the extracellular matrix (ECM) affects vascular compliance and vasomotor tone. Accordingly, this study assessed structural remodeling of mesenteric microvessels, vascular compliance, and myogenic tone, as well as the role of matrix metalloproteinases (MMP) in mediating these processes. Spontaneously diabetic, non-obese Goto-Kakizaki (GK) rats, a model for type 2 diabetes, and normoglycemic Wistar rats were used for the studies. A subset of GK rats were administered metformin to achieve euglycemia. Glycemic control normalized the increased media-to-lumen ratios (M/L) and myogenic tone seen in diabetes, as well as normalizing plasma ET-1 levels and mesenteric ET(A) receptor expression. There was increased collagen synthesis in diabetes paralleled by decreased collagenase MMP-13 activity, while glycemic control attenuated the process. These findings and our previous study taken together suggest that hyperglycemia-mediated activation of ET-1 and ET(A) receptors alter vascular structure and mechanics in type 2 diabetes.

Fig. 1.

Formalin-fixed mesenteric artery cross sections were analyzed for morphological changes and collagen deposition by Masson staining. Diabetes induced nearly a two-fold increase in M/L ratio, and glycemic control with metformin prevented this increase. Representative images (A) and combined analysis (B) are shown. *P < 0.05 vs. control or euglycemic; n = 6/group.

Fig. 2.

Collagen deposition patterns were observed from picrosirius red-stained cross sections, viewed under polarized light. There was increased new collagen in diabetes compared with control (increased green staining), and metformin treatment prevented this effect. Representative images (A) and combined analyses are shown of mature (B) and new collagen (C) for all animals. *P < 0.05 vs. control or euglycemic; n = 5/group.

Fig. 3.

Expression of type 1 collagen (A), collagenase (B), and MMP-13 (C) were determined by slot-blotting and immunoblotting, respectively. Densitometric analysis showed increased expression of type 1 collagen and MMP-13 in diabetic animals. B: increased fluorescence that is directly proportional to the proteolytic cleavage of FITC-collagen by MMP-13 was measured at 24 h, and it was decreased in diabetic animals. *P < 0.05 vs. control, n = 6–8/group.

Fig. 4.

A: mesenteric artery homogenates from diabetic animals show increased gelatinolytic activity when incubated with FITC-gelatin, which was decreased to control levels in metformin-treated rats. B: TIMP-2 levels measured by ELISA were similar in all groups. *P < 0.05 vs. control or euglycemic; n = 6–8/group.

Fig. 5.

A: microvascular tone at 60 and 80 mmHg intraluminal pressure was increased in diabetes and glycemic control with metformin prevented this phenomenon. B: β-coefficient, a measure of vessel stiffness, was decreased in diabetes and metformin treatment did not have any effect on vessel stiffness. *P < 0.05 vs. control or euglycemic; n = 6–8/group.

Fig. 6.

Plasma ET-1 and ET receptor expression in mesenteric arteries. A: ET-1 levels are increased three-fold in diabetes, which was prevented by glycemic control. B: representative immunoblot showing ET_A protein levels and densitometric analysis indicated an increase in ET_A in diabetes and metformin treatment normalized receptor expression to control values. Densitometry values reported have been normalized to β-actin levels in all samples to account for differences in loading. *P < 0.05 vs. control or euglycemic; n = 6/group.