Increase in GLUT1 in smooth muscle alters vascular contractility and increases inflammation in response to vascular injury

Abstract

Objective: The goal of this study was to test the contributing role of increasing glucose uptake in vascular smooth muscle cells (VSMCs) in vascular complications and disease.

Methods and results: A murine genetic model was established in which glucose trasporter 1 (GLUT1), the non-insulin-dependent glucose transporter protein, was overexpressed in smooth muscle using the sm22α promoter. Overexpression of GLUT1 in smooth muscle led to significant increases in glucose uptake (n=3, P<0.0001) as measured using radiolabeled 2-deoxyglucose. Fasting blood glucose, insulin, and nonesterified fatty acids were unchanged. Contractility in aortic ring segments was decreased in sm22α-GLUT1 mice (n=10, P<0.04). In response to vascular injury, sm22α-GLUT1 mice exhibited a proinflammatory phenotype, including a significant increase in the percentage of neutrophils in the lesion (n=4, P<0.04) and an increase in monocyte chemoattractant protein-1 (MCP-1) immunofluorescence. Circulating haptoglobin and glutathione/total glutathione were significantly higher in the sm22α-GLUT1 mice postinjury compared with controls (n=4, P<0.05), suggesting increased flux through the pentose phosphate pathway. sm22α-GLUT1 mice exhibited significant medial hypertrophy following injury that was associated with a significant increase in the percentage of VSMCs in the media staining positive for nuclear phosphoSMAD2/3 (n=4, P<0.003).

Conclusions: In summary, these findings suggest that increased glucose uptake in VSMCs impairs vascular contractility and accelerates a proinflammatory, neutrophil-rich lesion in response to injury, as well as medial hypertrophy, which is associated with enhanced transforming growth factor-β activity.

Figure 1

Representative microphotographs of GLUT1 immunostaining in smooth muscle layer of femoral artery in control (a) and sm22α-GLUT1 (b). Representative Western blotting of GLUT1 in aorta from control and sm22α-Glut1 mice (c). Glucose uptake in VSMCs is significantly higher in isolated aortic VSMCs from sm22α-GLUT1 mice vs. control (c.) (n=3, p < 0.0001).

Figure 2

Contractility of aortic ring segments from sm22α-GLUT1 mice was significantly reduced in the presence of phenylephrine (PE) (p<0.03) or serotonin (5-HT) (p<0.04). Aortic ring segments (control n=10 and sm22α-GLUT1 n=10) were allowed to equilibrate at a resting tension of 400 mg for at least 60 min prior to exposure to 60 mM potassium chloride (KCl) at 30 min intervals, 3x. The contractile responses to 1 µM PE and 10 µM 5-HT were then assessed in random order with washout and re-equilibration periods of 30 minutes.

Figure 3

Western analysis of proteins in aorta from control and sm22α-GLUT1 mice (a). Fibronectin expression in sm22α-GLUT1 (n=4) is significantly decreased as compared to control aorta (n=4), p<0.003 (b).

Figure 4

Inflammation induced by increased glucose uptake. Representative H & E stained photomicrographs showing lesion at 7 days post injury in control (a) and sm22α-GLUT1 (b) mice and total number of inflammatory cells in lesion (n=4, p < 0.04) (c). Representative photomicrographs showing neutrophils, as assessed by staining with anti-Ly6G antibody, in lesion at 7 days post injury in control (d) and sm22α-GLUT1 (e). Increased neutrophils in sm22α-GLUT1 lesion (n=4, p < 0.04) (f). Representative photomicrographs showing macrophages (highlighted with red arrowhead), as assessed by staining with anti-Mac3 antibody, in lesion at 7 days post injury in control (g) and sm22α-GLUT1 (h). The total number of macrophages was not significantly different between the groups (n=4, p = ns) (i).

Figure 5

MCP-1 immunofluorescence in lesion at 7 days post injury in control (a) and sm22α-GLUT1 mice (b). Isotype IgG stained section from control with DAPI positive nuclei (c). Circulating MCP-1 is higher in sm22α-GLUT1 as compared to control in response to vascular injury (d). *p<0.002 control (n=4) vs sm22α-GLUT1 (n=5) values at 7 days post injury. #p<0.003 sm22α-GLUTI value at 0 day (n=9) vs sm22α-GLUT1 (n=5) values at 7 days post injury. There was no significant difference in MCP-1 between controls at 0 day (n=16) and controls at 7 days post injury (n=4).

Figure 6

Increased phosphoSMAD2/3 in the nuclei of medial VSMC post injury in sm22αα-GLUT1 mice (a) and control mice (b). Percent of phosphoSMAD2/3 positive nuclei (white arrowheads) is increased in sm22α-GLUT1 as compared to controls at 28 days post injury (n=4, p<0.003).