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High-mobility group box-1 protein promotes angiogenesis after peripheral ischemia in diabetic mice through a VEGF-dependent mechanism.

Biscetti F, Straface G, De Cristofaro R, Lancellotti S, Rizzo P, Arena V, Stigliano E, Pecorini G, Egashira K, De Angelis G, Ghirlanda G, Flex A - Diabetes (2010)

Bottom Line: High-mobility group box-1 (HMGB1) protein is a nuclear DNA-binding protein released from necrotic cells, inducing inflammatory responses and promoting tissue repair and angiogenesis.We found that the perfusion recovery was significantly attenuated in diabetic mice compared with normoglycemic control mice.Furthermore, we observed that HMGB1 administration restored the blood flow recovery and capillary density in the ischemic muscle of diabetic mice, that this process was associated with the increased expression of vascular endothelial growth factor (VEGF), and that HMGB1-induced angiogenesis was significantly reduced by inhibiting VEGF activity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Vascular Biology and Genetics, Department of Medicine, A. Gemelli University Hospital, Catholic University School of Medicine, Rome, Italy. f.biscetti@gmail.com

ABSTRACT

Objective: High-mobility group box-1 (HMGB1) protein is a nuclear DNA-binding protein released from necrotic cells, inducing inflammatory responses and promoting tissue repair and angiogenesis. Diabetic human and mouse tissues contain lower levels of HMGB1 than their normoglycemic counterparts. Deficient angiogenesis after ischemia contributes to worse outcomes of peripheral arterial disease in patients with diabetes. To test the hypothesis that HMGB1 enhances ischemia-induced angiogenesis in diabetes, we administered HMGB1 protein in a mouse hind limb ischemia model using diabetic mice.

Research design and methods: After the induction of diabetes by streptozotocin, we studied ischemia-induced neovascularization in the ischemic hind limb of normoglycemic, diabetic, and HMGB1-treated diabetic mice.

Results: We found that the perfusion recovery was significantly attenuated in diabetic mice compared with normoglycemic control mice. Interestingly, HMGB1 protein expression was lower in the ischemic tissue of diabetic mice than in normoglycemic mice. Furthermore, we observed that HMGB1 administration restored the blood flow recovery and capillary density in the ischemic muscle of diabetic mice, that this process was associated with the increased expression of vascular endothelial growth factor (VEGF), and that HMGB1-induced angiogenesis was significantly reduced by inhibiting VEGF activity.

Conclusions: The results of this study show that endogenous HMGB1 is crucial for ischemia-induced angiogenesis in diabetic mice and that HMGB1 protein administration enhances collateral blood flow in the ischemic hind limbs of diabetic mice through a VEGF-dependent mechanism.

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A: Foot blood flow monitored in vivo by LDPI in control normoglycemic and diabetic mice. Representative evaluation of the ischemic (right) and nonischemic (left) hind limbs, immediately after, and on days 7, 14, 21, and 28 after surgery. In color-coded images, red indicates normal perfusion and blue indicates a marked reduction in blood flow in the ischemic hind limb. Blood flow recovery is impaired in diabetic mice compared with normoglycemic mice. The blood flow of the ischemic hind limb is expressed as the ratio between the perfusion of the ischemic limb and the uninjured limb. P < 0.05 and P < 0.01 vs. diabetic mice. B: Representative photomicrographs of ischemic muscle sections from control normoglycemic and diabetic mice stained with antibody directed against VEGF, 7 days after surgery, and against CD31, 28 days after surgery. Positive staining appears in brown. Magnification ×20. C: Number of vessels per cross section is significantly reduced in diabetic mice with respect to normoglycemic mice. P < 0.05 vs. diabetic mice. D: Representative Western blot of VEGF protein content in the ischemic legs of control and diabetic mice on postoperative day 7. VEGF expression is reduced in the ischemic tissue of diabetic mice compared with control mice. ns, not significant. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 1: A: Foot blood flow monitored in vivo by LDPI in control normoglycemic and diabetic mice. Representative evaluation of the ischemic (right) and nonischemic (left) hind limbs, immediately after, and on days 7, 14, 21, and 28 after surgery. In color-coded images, red indicates normal perfusion and blue indicates a marked reduction in blood flow in the ischemic hind limb. Blood flow recovery is impaired in diabetic mice compared with normoglycemic mice. The blood flow of the ischemic hind limb is expressed as the ratio between the perfusion of the ischemic limb and the uninjured limb. P < 0.05 and P < 0.01 vs. diabetic mice. B: Representative photomicrographs of ischemic muscle sections from control normoglycemic and diabetic mice stained with antibody directed against VEGF, 7 days after surgery, and against CD31, 28 days after surgery. Positive staining appears in brown. Magnification ×20. C: Number of vessels per cross section is significantly reduced in diabetic mice with respect to normoglycemic mice. P < 0.05 vs. diabetic mice. D: Representative Western blot of VEGF protein content in the ischemic legs of control and diabetic mice on postoperative day 7. VEGF expression is reduced in the ischemic tissue of diabetic mice compared with control mice. ns, not significant. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Immediately after the femoral artery ligation, blood flow in the ischemic hind limb was equally reduced in both nondiabetic and diabetic mice (Fig. 1). Laser Doppler perfusion imaging (LDPI) was performed before, immediately after, and on days 7, 14, 21, and 28 after surgery. Perfusion recovery was significantly attenuated in diabetic mice compared with normoglycemic mice on postoperative days 7, 14, 21, and 28 (Fig. 1A). In addition, histological analysis revealed that the capillary density in the ischemic limb was significantly increased in nondiabetic mice, whereas no such increase was noted in diabetic mice at 4 weeks after the hind limb ischemia (Fig. 1B and C). Furthermore, immunostaining and immunoblot analyses showed increased VEGF expression in the ischemic tissue of normoglycemic mice compared with diabetic mice on postoperative day 7 (Fig. 1B and D). In agreement with previous data (3), these findings confirm the relative inability of diabetes to mount a robust angiogenic response to ischemia after arterial occlusion (25).


High-mobility group box-1 protein promotes angiogenesis after peripheral ischemia in diabetic mice through a VEGF-dependent mechanism.

Biscetti F, Straface G, De Cristofaro R, Lancellotti S, Rizzo P, Arena V, Stigliano E, Pecorini G, Egashira K, De Angelis G, Ghirlanda G, Flex A - Diabetes (2010)

A: Foot blood flow monitored in vivo by LDPI in control normoglycemic and diabetic mice. Representative evaluation of the ischemic (right) and nonischemic (left) hind limbs, immediately after, and on days 7, 14, 21, and 28 after surgery. In color-coded images, red indicates normal perfusion and blue indicates a marked reduction in blood flow in the ischemic hind limb. Blood flow recovery is impaired in diabetic mice compared with normoglycemic mice. The blood flow of the ischemic hind limb is expressed as the ratio between the perfusion of the ischemic limb and the uninjured limb. P < 0.05 and P < 0.01 vs. diabetic mice. B: Representative photomicrographs of ischemic muscle sections from control normoglycemic and diabetic mice stained with antibody directed against VEGF, 7 days after surgery, and against CD31, 28 days after surgery. Positive staining appears in brown. Magnification ×20. C: Number of vessels per cross section is significantly reduced in diabetic mice with respect to normoglycemic mice. P < 0.05 vs. diabetic mice. D: Representative Western blot of VEGF protein content in the ischemic legs of control and diabetic mice on postoperative day 7. VEGF expression is reduced in the ischemic tissue of diabetic mice compared with control mice. ns, not significant. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: A: Foot blood flow monitored in vivo by LDPI in control normoglycemic and diabetic mice. Representative evaluation of the ischemic (right) and nonischemic (left) hind limbs, immediately after, and on days 7, 14, 21, and 28 after surgery. In color-coded images, red indicates normal perfusion and blue indicates a marked reduction in blood flow in the ischemic hind limb. Blood flow recovery is impaired in diabetic mice compared with normoglycemic mice. The blood flow of the ischemic hind limb is expressed as the ratio between the perfusion of the ischemic limb and the uninjured limb. P < 0.05 and P < 0.01 vs. diabetic mice. B: Representative photomicrographs of ischemic muscle sections from control normoglycemic and diabetic mice stained with antibody directed against VEGF, 7 days after surgery, and against CD31, 28 days after surgery. Positive staining appears in brown. Magnification ×20. C: Number of vessels per cross section is significantly reduced in diabetic mice with respect to normoglycemic mice. P < 0.05 vs. diabetic mice. D: Representative Western blot of VEGF protein content in the ischemic legs of control and diabetic mice on postoperative day 7. VEGF expression is reduced in the ischemic tissue of diabetic mice compared with control mice. ns, not significant. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Immediately after the femoral artery ligation, blood flow in the ischemic hind limb was equally reduced in both nondiabetic and diabetic mice (Fig. 1). Laser Doppler perfusion imaging (LDPI) was performed before, immediately after, and on days 7, 14, 21, and 28 after surgery. Perfusion recovery was significantly attenuated in diabetic mice compared with normoglycemic mice on postoperative days 7, 14, 21, and 28 (Fig. 1A). In addition, histological analysis revealed that the capillary density in the ischemic limb was significantly increased in nondiabetic mice, whereas no such increase was noted in diabetic mice at 4 weeks after the hind limb ischemia (Fig. 1B and C). Furthermore, immunostaining and immunoblot analyses showed increased VEGF expression in the ischemic tissue of normoglycemic mice compared with diabetic mice on postoperative day 7 (Fig. 1B and D). In agreement with previous data (3), these findings confirm the relative inability of diabetes to mount a robust angiogenic response to ischemia after arterial occlusion (25).

Bottom Line: High-mobility group box-1 (HMGB1) protein is a nuclear DNA-binding protein released from necrotic cells, inducing inflammatory responses and promoting tissue repair and angiogenesis.We found that the perfusion recovery was significantly attenuated in diabetic mice compared with normoglycemic control mice.Furthermore, we observed that HMGB1 administration restored the blood flow recovery and capillary density in the ischemic muscle of diabetic mice, that this process was associated with the increased expression of vascular endothelial growth factor (VEGF), and that HMGB1-induced angiogenesis was significantly reduced by inhibiting VEGF activity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Vascular Biology and Genetics, Department of Medicine, A. Gemelli University Hospital, Catholic University School of Medicine, Rome, Italy. f.biscetti@gmail.com

ABSTRACT

Objective: High-mobility group box-1 (HMGB1) protein is a nuclear DNA-binding protein released from necrotic cells, inducing inflammatory responses and promoting tissue repair and angiogenesis. Diabetic human and mouse tissues contain lower levels of HMGB1 than their normoglycemic counterparts. Deficient angiogenesis after ischemia contributes to worse outcomes of peripheral arterial disease in patients with diabetes. To test the hypothesis that HMGB1 enhances ischemia-induced angiogenesis in diabetes, we administered HMGB1 protein in a mouse hind limb ischemia model using diabetic mice.

Research design and methods: After the induction of diabetes by streptozotocin, we studied ischemia-induced neovascularization in the ischemic hind limb of normoglycemic, diabetic, and HMGB1-treated diabetic mice.

Results: We found that the perfusion recovery was significantly attenuated in diabetic mice compared with normoglycemic control mice. Interestingly, HMGB1 protein expression was lower in the ischemic tissue of diabetic mice than in normoglycemic mice. Furthermore, we observed that HMGB1 administration restored the blood flow recovery and capillary density in the ischemic muscle of diabetic mice, that this process was associated with the increased expression of vascular endothelial growth factor (VEGF), and that HMGB1-induced angiogenesis was significantly reduced by inhibiting VEGF activity.

Conclusions: The results of this study show that endogenous HMGB1 is crucial for ischemia-induced angiogenesis in diabetic mice and that HMGB1 protein administration enhances collateral blood flow in the ischemic hind limbs of diabetic mice through a VEGF-dependent mechanism.

Show MeSH
Related in: MedlinePlus