Vascular protection in diabetic stroke: role of matrix metalloprotease-dependent vascular remodeling

Abstract

Temporary focal ischemia causes greater hemorrhagic transformation (HT) in diabetic Goto-Kakizaki (GK) rats, a model with increased cerebrovascular matrix metalloprotease (MMP) activity and tortuosity. The objective of the current study was to test the hypotheses that (1) diabetes-induced cerebrovascular remodeling is MMP dependent and (2) prevention of vascular remodeling by glucose control or MMP inhibition reduces HT in diabetic stroke. Control and GK rats were treated with vehicle, metformin, or minocycline for 4 weeks, and indices of remodeling including vascular tortuosity index, lumen diameter, number of collaterals, and middle cerebral artery (MCA) MMP activity were measured. Additional animals were subjected to 3 hours MCA occlusion/21 hours reperfusion, and infarct size and HT were evaluated as indices of neurovascular injury. All remodeling markers including MMP-9 activity were increased in diabetes. Infarct size was smaller in minocycline-treated animals. Both metformin and minocycline reduced vascular remodeling and severity of HT in diabetes. These results provide evidence that diabetes-mediated stimulation of MMP-9 activity promotes cerebrovascular remodeling, which contributes to greater HT in diabetes. Metformin and minocycline offer vascular protection, which has important clinical implications for diabetes patients who are at a fourfold to sixfold higher risk for stroke.

Figure 1

Diabetes promotes remodeling of cerebral vessels. (A) Representative images of the pial vessels after PU4ii injection to visualize middle, anterior, and posterior cerebral arteries trees in control (C), diabetes (D), diabetes+metformin (D+Me), control+minocycline (C+M), or diabetes+minocycline (D+M) groups. Tortuosity index (B), number of collaterals (C), number of anastomoses (D), and collateral diameter (E) were significantly higher in diabetes all of which were prevented by metformin or minocycline treatments. Mean±s.e.m., n=7 to 14, ^*P<0.0001 versus C, ^**P<0.001 versus D, ^***P<0.0002 versus C.

Figure 2

Diabetes does not increase wall thickness or M/L ratio in diabetes. Middle cerebral artery (MCA) wall remodeling parameters including (A) inner diameter, (B) cross-sectional area (CSA), (C) outer diameter, and (D) M/L ratio were measured by pressure arteriography at 80 mm Hg. Mean±s.e.m., n=6 to 9, ^*P=0.0023 and ^**P=0.01, disease by treatment interaction. M/L, media/lumen.

Figure 3

Diabetes increases myogenic tone of isolated middle cerebral arteries (MCAs). (A) The MCA myogenic tone was increased in diabetic animals and both metformin and minocycline reduced the tone. (B) There was a disease and treatment interaction such that minocycline decreased stiffness in controls but increased it in diabetic rats. Mean±s.e.m., n=7 to 14, ^*P<0.05 versus C, ^**P=0.011 versus D, ^***P=0.0046 versus C; ^****P<0.0001. C, control; D, diabetes.

Figure 4

Diabetes augments ischemia-induced stimulation of MMP-9 activity in isolated cerebral vessels. (A) The MMP-9 activity in macrovessels isolated from ischemic (I) and nonischemic (NI) hemispheres of animals subjected to middle cerebral artery occlusion (MCAO) was measured using gelatin zymography. The MMP-9 activity was greater in both the NI and I hemispheres in diabetes indicating increased baseline and ischemia-induced augmentation of MMP-9 activity. Both metformin and minocycline caused a dramatic reduction in enzyme activity. (B) The MMP-2 activity of the same groups did not show any difference. (C) When lytic activity was assessed in the same cerebral macrovessels isolated from animals not subjected to MCAO, both MMP-2 and MMP-9 baseline activities were greater in diabetes. (D) Protein levels of MMP-2 and MMP-9 were increased in diabetes. Metformin and minocycline treatments significantly reduced MMP-2 level but not MMP-9. Mean±s.e.m., n=5 to 8, ^*P<0.01 versus NI, ^**P=0.05 versus C, ^***P=0.012 versus D, ^****P<0.0001 versus vehicle C or D, ^ςP=0.031 versus D, ^ΨP=0.003 versus D. C, control; D, diabetes; MMP, matrix metalloprotease.

Figure 5

Infarct size is smaller in diabetes. (A) Representative images of 2,3,5-triphenyltetrazolium chloride-stained coronal sections of the brain after middle cerebral artery occlusion (MCAO). (B) Quantitative analysis of infarct size indicated smaller infarcts in diabetes. Although metformin had no effect on infarct size minocycline reduced infarct in both groups. Mean±s.e.m., n=6 to 9, ^*P<0.0001 versus C, ^**P=0.05 versus vehicle C or D. C, control; D, diabetes.

Figure 6

Vascular injury is greater in diabetes. (A) Occurrence of macroscopic intracerebral bleeding was significantly higher in diabetes compared with control and was reduced by chronic minocycline treatment. (B) ELISA measurements of hemoglobin show significant increase in hemorrhagic transformation (HT) in diabetes and a vasoprotective effect of minocycline and metformin. There was a disease and treatment interaction showing minocycline preventing HT in diabetes but no effect on control animals. (C) Edema was significantly higher in diabetes compared with control and both metformin and minocycline reduced it. Minocycline had no effect on edema in control animals, indicating a disease/treatment interaction. Mean±s.e.m., n=6 to 11, ^*P<0.05 versus C, ^**P=0.001 versus D. ^***P=0.0009 and ^****P=0.0024 are disease/treatment interaction for minocycline. C, control; D, diabetes; ELISA, enzyme linked immunosorbent assay.

Figure 7

Short-term (24 hours) neurologic outcomes after middle cerebral artery occlusion (MCAO) in all treatment groups. (A) A composite score for multiple neurobehavioral tests shows worse functional recovery in diabetes compared with control. Both metformin and minocycline treatment improved the score significantly. Minocycline showed a differential effect indicated by no change in control animals but improvement of score in diabetic animals. Although there was no difference in individual scores of elevated body swing test (B) and forepaw grasp (C), forepaw grasp was improved in minocycline-treated animals. Mean±s.e.m., n=6 to 13, ^*P<0.05 versus C, ^**P=0.05 versus D, ^***P=0.035 disease/treatment interaction for minocycline, ^****P=0.0031 versus vehicle C or D. C, control; D, diabetes.