Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:1053–1060 DOI 10.1007/s00210-015-1138-x
ORIGINAL ARTICLE
Tumor necrosis factor inhibition increases the revascularization of ischemic hind-limbs in diabetic mice Adel M. A. Assiri 1,2 & Hatim A. El-Baz 3 & Ali H. Amin 1,4
Received: 31 March 2015 / Accepted: 21 May 2015 / Published online: 31 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Tumor necrosis factor (TNF) is first identified as a mediator of lethal endotoxin poisoning. The anti-TNF therapy in the treatment of rheumatoid arthritis is based on the recognition of the role of TNF as the master regulator. Type II diabetes is characterized with altered stem cells and reduced vasculogenesis. Therefore, we aimed to determine if TNF inhibitor would improve vasculogenesis in ischemic hind-limbs of diabetic mice. Fifty male type 2 diabetic and their control (8–10 weeks old mice) were used, and ischemia was induced in the hind-limbs of all mice for 28 days. Vessel density was assessed by high-definition microangiography at the end of the treatment period. After 4 weeks, vessel density displayed no difference between the ischemic and the non-ischemic legs in control mice. However, in diabetic mice, the ischemic hindlimb vessel density was significantly decreased. Interestingly, diabetic mice displayed a significant improved vasculogenesis when treated with TNF inhibitor. Moreover, this data was confirmed by capillary density determined by immunostaining. TNF inhibitors are able to improve the formation of microvessels in response to ischemia in type 2 diabetes.
* Ali H. Amin [email protected] 1
The Institute of Scientific Research and Revival of Islamic Heritage, Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia
2
Biochemistry Department, Faculty of Medicine, Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia
3
Biochemistry Department, Genetic Engineering and Biotechnology Division, National Research Centre, Cairo, Egypt
4
Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt
Keywords Ischemia . Tumor necrosis factor . Diabetes . Revascularization . Vasculogenesis . TNF inhibition
Introduction Diabetes is increasing in the world, causes severe cardiovascular complication, and considered the number one worldwide killer disease. Since they have hind-limbs ischemia with altered neovascularization, which leads to foot amputation, then therapeutic strategies are urgently needed. In a previous study, we found that bone marrow cells from db−/db− mice (purchased from Jackson Laboratory, Maine, USA) are altered inducing limb ischemia which leads to foot amputation. The development of new blood vessels supplying blood to ischemic tissues became one of the most important therapeutic strategies in that mice (Amin et al. 2010). Diabetes mellitus is a chronic disease marked by high levels of glucose in the blood. Type II diabetes is the most common form of diabetes. The prevalence of diabetes mellitus in the Saudi community was 34.1 % in males and 27.6 % in females, and it increases with age (Alqurashi et al. 2011). Secondary complications of diabetes are serious risk factors that might lead to vascular complication and the development of atherosclerosis, nephropathy ended by renal failure, retinopathy which leads to blindness, neuropathy, and heart disease (Flyvbjerg 2010; Khan et al. 2000). The likely keys include the careful choice of a biological agent that may be different for different indications, effective delivery modality with pharmacokinetic properties matching biological needs, and the meticulous choice of the patient population and end points for study (Simons 2005). Peripheral vascular disease (PVD) associated with impaired neovascularization is a major problem in diabetic patients. It affects 7.5 % of the diabetic patients aged between 60–64 years in the USA. Furthermore,
1054
PVD alone is responsible for 3900 lower limb amputation per million in a year (Most and Sinnock 1983). During the last decade, a growing number of studies reported abnormalities of neovascularization in diabetic patients with coronary (Mohamed et al. 2007), peripheral artery diseases (Lebherz et al. 2005), and abnormal enhanced angiogenesis (Martin et al. 2003; Nakagawa et al. 2009). Ocular neovascularization in diabetes is the worldwide leading causes of blindness (Lebherz et al. 2005). This may be due to ischemia enhances HIF-1 expression in normal condition, while it is not the case under diabetic which has a defect in transactivation of HIF-1 (Thangarajah et al. 2010). At the same time, defective arteriogenesis has also been reported in patients with diabetes mellitus (Waltenberger 2001). The diabetes management should not focused on control of hyperglycemia only, but it should focus also on the presence of abnormalities of neovascularization in diabetes (Martin et al. 2003). Insulin resistance is one of the major causes of obesity complications because of increased excretion of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and C-reactive protein (CRP) (Bal et al. 2010). Hyperglycemia induces intracellular reactive oxygen species in kidney epithelial cells (Piwkowska et al. 2011) which induces cytokines, IL-6, and TNF-α production in the diabetic kidney. Ischemia is an absolute or relative shortage of the blood supply to an organ. Ischemia results in tissue damage because of a lack of oxygen and nutrients. Ultimately, this can cause severe damage because of the potential for a build-up of metabolic wastes. Pathological hypoxia could be caused by internal factors such as anemia and ischemia. Ischemia leads to hypoxia, which is one of the most important factors that induce neovascularization. Consequently, hypoxia induces hypoxia-inducible factor-1 (HIF-1) as a key transcription factor that increases during cancer and vascular and pulmonary diseases (Harris 2002). Angiogenesis is induced by hypoxia. Hypoxic organ induces a variety of positive- and negativeacting growth factors. These factors stimulate the expansion and remodeling of the existing vasculature to enhance blood flow in oxygen-deprived tissue. The vascular network mediates the delivery of oxygen and nutrients to all cells of the organism. It also removes metabolites and carbon dioxide while maintaining an adequate hydrostatic pressure (Carmeliet 2003). Cells die when they are placed in severe hypoxic conditions for a prolonged time. To avoid this result, cells try to save themselves by a variety of biological responses: First, the initial response is that hypoxic cells undergo a shift to anaerobic metabolism instead of aerobic one (Dang and Semenza 1999; Chen et al. 2009). Afterwards, hypoxia induces erythropoietin to increase the hemoglobin production. Finally, several signaling pathways are activated to standardize proliferation, angiogenesis, and apoptosis (Harris 2002).
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:1053–1060
Under healthy conditions, normal cells response to hypoxia by producing growth factors that induce new blood vessel formation. Under pathological condition, hypoxia is associated with poor neovascularization as in diabetes, the defect in transactivation of HIF-1 (Thangarajah et al. 2010). As a result, those cells lose their advantage to respond to hypoxia by induction of angiogenesis while obtaining the disadvantage of others as apoptosis (Kung et al. 2000). Chronic critical limb ischemia is marked by painful symptoms even at rest (burning pain), non-healing wounds, and finally gangrenes. As was mentioned before, diabetic and hypertensive patients are more likely to have PAD with ischemia. The progressive gangrene with continuous ischemia can lead to amputation of the organ. Revascularization will decrease the risks of limb amputation (Santilli and Santilli 1999). Tumor necrosis factor is a key cytokine that stimulates the inflammatory cell response (Arend 2002). Most organs of the body are affected by TNF-α, and the cytokine serves a variety of functions; the cytokine possesses both growth-stimulating properties and growth-inhibitory processes, and it appears to have self-regulatory properties as well. For instance, TNF-α induces neutrophil proliferation during inflammation, but it also induces neutrophil apoptosis upon binding to the TNFR55 receptor (Murray et al. 1997). TNF-α-blocking antibody Enbrel abrogates BV6-induced cell elongation, migration, and invasion (Tchoghandjian et al. 2013). The pathological activities of TNF-α have attracted much attention. For instance, although TNF-α causes necrosis of some types of tumors, it promotes the growth of other types of tumor cells. High levels of TNF-α correlate with increased risk of mortality (Rink and Kirchner 1996). The soluble TNF receptor agents (Enbrel or Etanercept), used clinically to block the functional activity of TNF-α protein which is a dimeric fusion protein composed of an extracellular ligand-binding portion of the human (p75) TNFR linked to the Fc portion of human IgG1 (Mikuls and Moreland 2001). Enbrel inhibits the binding of both TNF-α and TNF-β (lymphotoxin alpha) to cell surface TNFRs, rendering TNF biologically inactive. TNF-α blockers are classified as biologics as they are able to stimulate or restore the ability of the immune system to fight disease (Grounds et al. 2005). Both hyperoxia-induced proapoptotic sensitization of alveolar type II cells (TII cells) and high-stretch mechanical ventilation-induced pulmonary inflammation are TNF-α mediated (Guthmann et al. 2009). Despite the lack of statistically significant correlation between the gene expression level of TNF and its receptors TNFR1 and TNFR2, Significantly decreased numbers of TNFR1 and TNFR2 gene copies may play a significant role in initiation and progression of arteriosclerosis (Dabek et al. 2012). Diabetes mellitus is associated with enhanced leukocyte accumulation in response to ischemia— reperfusion is mediated by CD11/CD18–ICAM-1 interactions and P-selectin. Diabetes causes insulin resistance which is also
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:1053–1060
responsible for endothelial dysfunction associated with increase of TNF-α level (Kolluru et al. 2012). Therefore, binding of free TNF-α should improve the formation of microvessels in response to ischemia in diabetes. The aim of present study is searching the efficacy of Enbrel as a therapy for enhanced recovery of angiogenesis and blood flow of the ischemic hind-limb of type 2 diabetic mice.
Materials and methods Experimental design Experimental design was performed as shown in Fig. 1. Antibodies Total Akt, phosphorylated Akt (Ser476), phosphorylated p40Phox (Thr154), Von Willebrand factor, α-actin, intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and the used secondary antibodies were purchased from Cell Signal and Promega (P.O. Box 257 Dammam 31411 Saudi Arabia). Drugs Etanercept (Enbre) was purchased from a pharmaceutical supplier in 1 cm3 syringes at 50 mg/mL and was used in native carrier forms.
1055
present study. These studies conformed to the principles of the handling and treatment of the animals used for research in accordance with the principles of Islam and good humanitarian practices. BKing Abdulaziz City for Science and Technology (KACST) Guide for the Care and Use of Laboratory Animals^ was approved by the Umm Al-Qura University (UQU) Institutional Animal Care and Use Committee. The hind-limbs ischemia was initiated by ligation of the left femoral artery. Before each surgery, the mouse was deeply anesthetized and shaved under sterile conditions. A longitudinal skin incision (about 1 cm) was made in the groin; the femoral triangle was explored, and the major vessels were identified and dissected. Black braided silk sutures (deknatel, USA) were used for the ligation prior to excision of the arterial vessel bed between the distal ends of the external iliac artery (Yang et al. 2009). Finally, the overlying skin was closed with stainless steel wound clips (world precision instruments, USA) and disinfected by scrubbing with Betadine solution. Body temperature maintained using isothermal pads. Mice were monitored over an experimental period. Treatment of db−/db− mice with Enbrel Enbrel was injected as two doses of 100 μg dose for each injection (for an average body weight of 25 g) intraperitoneally at 3 days and 1 day before surgery. As a control, a group of db−/db− mice were subjected to intraperitoneal injection of PBS only. Laser Doppler measurement of hind-limb blood flow
Animal model and surgery −
−
Fifty male type 2 diabetic (db /db ; with C57/Bl6 genetic background) and their control (db−/db+) between 8–10 were used. Fifty-nine old mice were used for the completion of the Fig. 1 Diagram showing experimental design. Group 1 included 10 mice C75BL6/J, Group 2 included 20 diabetic mice, and Group 3 included 20 treated diabetic mice
Hind-limb blood flow measurements over the region of interest were performed at baseline before surgery, immediately postoperatively, and serially over the 4-week period with laser Doppler perfusion imaging (LDPI) (Moor Instruments).
1056
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:1053–1060
X-ray quantification of hind-limb angiogenesis
Immunohistochemistry
Ve s s e l d e n s i t y w a s a s s e s s e d b y h i g h - d e f i n i t i o n microangiography at the end of the treatment period (Jacobi et al. 2004).
Immunohistochemistry was performed (Belmadani et al. 2008a, 2009; Su et al. 2008). Statistical analysis
Western blot analysis Western blot analysis of tissue was performed (Belmadani et al. 2008a, b; Su et al. 2008). The quantification of Western blot was determined using Fujifilm-Multi Gauge software. Fig. 2 Blood flow of hind-limbs in all groups
Results are expressed as mean±SEM. One-way or 2-way ANOVA was used to compare each parameter when there were two independent groups. Comparisons between groups were performed with post hoc Bonferroni t tests when the ANOVA test was statistically significant. Values of P
ORIGINAL ARTICLE
Tumor necrosis factor inhibition increases the revascularization of ischemic hind-limbs in diabetic mice Adel M. A. Assiri 1,2 & Hatim A. El-Baz 3 & Ali H. Amin 1,4
Received: 31 March 2015 / Accepted: 21 May 2015 / Published online: 31 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Tumor necrosis factor (TNF) is first identified as a mediator of lethal endotoxin poisoning. The anti-TNF therapy in the treatment of rheumatoid arthritis is based on the recognition of the role of TNF as the master regulator. Type II diabetes is characterized with altered stem cells and reduced vasculogenesis. Therefore, we aimed to determine if TNF inhibitor would improve vasculogenesis in ischemic hind-limbs of diabetic mice. Fifty male type 2 diabetic and their control (8–10 weeks old mice) were used, and ischemia was induced in the hind-limbs of all mice for 28 days. Vessel density was assessed by high-definition microangiography at the end of the treatment period. After 4 weeks, vessel density displayed no difference between the ischemic and the non-ischemic legs in control mice. However, in diabetic mice, the ischemic hindlimb vessel density was significantly decreased. Interestingly, diabetic mice displayed a significant improved vasculogenesis when treated with TNF inhibitor. Moreover, this data was confirmed by capillary density determined by immunostaining. TNF inhibitors are able to improve the formation of microvessels in response to ischemia in type 2 diabetes.
* Ali H. Amin [email protected] 1
The Institute of Scientific Research and Revival of Islamic Heritage, Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia
2
Biochemistry Department, Faculty of Medicine, Umm Al-Qura University, Makkah, Kingdom of Saudi Arabia
3
Biochemistry Department, Genetic Engineering and Biotechnology Division, National Research Centre, Cairo, Egypt
4
Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt
Keywords Ischemia . Tumor necrosis factor . Diabetes . Revascularization . Vasculogenesis . TNF inhibition
Introduction Diabetes is increasing in the world, causes severe cardiovascular complication, and considered the number one worldwide killer disease. Since they have hind-limbs ischemia with altered neovascularization, which leads to foot amputation, then therapeutic strategies are urgently needed. In a previous study, we found that bone marrow cells from db−/db− mice (purchased from Jackson Laboratory, Maine, USA) are altered inducing limb ischemia which leads to foot amputation. The development of new blood vessels supplying blood to ischemic tissues became one of the most important therapeutic strategies in that mice (Amin et al. 2010). Diabetes mellitus is a chronic disease marked by high levels of glucose in the blood. Type II diabetes is the most common form of diabetes. The prevalence of diabetes mellitus in the Saudi community was 34.1 % in males and 27.6 % in females, and it increases with age (Alqurashi et al. 2011). Secondary complications of diabetes are serious risk factors that might lead to vascular complication and the development of atherosclerosis, nephropathy ended by renal failure, retinopathy which leads to blindness, neuropathy, and heart disease (Flyvbjerg 2010; Khan et al. 2000). The likely keys include the careful choice of a biological agent that may be different for different indications, effective delivery modality with pharmacokinetic properties matching biological needs, and the meticulous choice of the patient population and end points for study (Simons 2005). Peripheral vascular disease (PVD) associated with impaired neovascularization is a major problem in diabetic patients. It affects 7.5 % of the diabetic patients aged between 60–64 years in the USA. Furthermore,
1054
PVD alone is responsible for 3900 lower limb amputation per million in a year (Most and Sinnock 1983). During the last decade, a growing number of studies reported abnormalities of neovascularization in diabetic patients with coronary (Mohamed et al. 2007), peripheral artery diseases (Lebherz et al. 2005), and abnormal enhanced angiogenesis (Martin et al. 2003; Nakagawa et al. 2009). Ocular neovascularization in diabetes is the worldwide leading causes of blindness (Lebherz et al. 2005). This may be due to ischemia enhances HIF-1 expression in normal condition, while it is not the case under diabetic which has a defect in transactivation of HIF-1 (Thangarajah et al. 2010). At the same time, defective arteriogenesis has also been reported in patients with diabetes mellitus (Waltenberger 2001). The diabetes management should not focused on control of hyperglycemia only, but it should focus also on the presence of abnormalities of neovascularization in diabetes (Martin et al. 2003). Insulin resistance is one of the major causes of obesity complications because of increased excretion of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and C-reactive protein (CRP) (Bal et al. 2010). Hyperglycemia induces intracellular reactive oxygen species in kidney epithelial cells (Piwkowska et al. 2011) which induces cytokines, IL-6, and TNF-α production in the diabetic kidney. Ischemia is an absolute or relative shortage of the blood supply to an organ. Ischemia results in tissue damage because of a lack of oxygen and nutrients. Ultimately, this can cause severe damage because of the potential for a build-up of metabolic wastes. Pathological hypoxia could be caused by internal factors such as anemia and ischemia. Ischemia leads to hypoxia, which is one of the most important factors that induce neovascularization. Consequently, hypoxia induces hypoxia-inducible factor-1 (HIF-1) as a key transcription factor that increases during cancer and vascular and pulmonary diseases (Harris 2002). Angiogenesis is induced by hypoxia. Hypoxic organ induces a variety of positive- and negativeacting growth factors. These factors stimulate the expansion and remodeling of the existing vasculature to enhance blood flow in oxygen-deprived tissue. The vascular network mediates the delivery of oxygen and nutrients to all cells of the organism. It also removes metabolites and carbon dioxide while maintaining an adequate hydrostatic pressure (Carmeliet 2003). Cells die when they are placed in severe hypoxic conditions for a prolonged time. To avoid this result, cells try to save themselves by a variety of biological responses: First, the initial response is that hypoxic cells undergo a shift to anaerobic metabolism instead of aerobic one (Dang and Semenza 1999; Chen et al. 2009). Afterwards, hypoxia induces erythropoietin to increase the hemoglobin production. Finally, several signaling pathways are activated to standardize proliferation, angiogenesis, and apoptosis (Harris 2002).
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:1053–1060
Under healthy conditions, normal cells response to hypoxia by producing growth factors that induce new blood vessel formation. Under pathological condition, hypoxia is associated with poor neovascularization as in diabetes, the defect in transactivation of HIF-1 (Thangarajah et al. 2010). As a result, those cells lose their advantage to respond to hypoxia by induction of angiogenesis while obtaining the disadvantage of others as apoptosis (Kung et al. 2000). Chronic critical limb ischemia is marked by painful symptoms even at rest (burning pain), non-healing wounds, and finally gangrenes. As was mentioned before, diabetic and hypertensive patients are more likely to have PAD with ischemia. The progressive gangrene with continuous ischemia can lead to amputation of the organ. Revascularization will decrease the risks of limb amputation (Santilli and Santilli 1999). Tumor necrosis factor is a key cytokine that stimulates the inflammatory cell response (Arend 2002). Most organs of the body are affected by TNF-α, and the cytokine serves a variety of functions; the cytokine possesses both growth-stimulating properties and growth-inhibitory processes, and it appears to have self-regulatory properties as well. For instance, TNF-α induces neutrophil proliferation during inflammation, but it also induces neutrophil apoptosis upon binding to the TNFR55 receptor (Murray et al. 1997). TNF-α-blocking antibody Enbrel abrogates BV6-induced cell elongation, migration, and invasion (Tchoghandjian et al. 2013). The pathological activities of TNF-α have attracted much attention. For instance, although TNF-α causes necrosis of some types of tumors, it promotes the growth of other types of tumor cells. High levels of TNF-α correlate with increased risk of mortality (Rink and Kirchner 1996). The soluble TNF receptor agents (Enbrel or Etanercept), used clinically to block the functional activity of TNF-α protein which is a dimeric fusion protein composed of an extracellular ligand-binding portion of the human (p75) TNFR linked to the Fc portion of human IgG1 (Mikuls and Moreland 2001). Enbrel inhibits the binding of both TNF-α and TNF-β (lymphotoxin alpha) to cell surface TNFRs, rendering TNF biologically inactive. TNF-α blockers are classified as biologics as they are able to stimulate or restore the ability of the immune system to fight disease (Grounds et al. 2005). Both hyperoxia-induced proapoptotic sensitization of alveolar type II cells (TII cells) and high-stretch mechanical ventilation-induced pulmonary inflammation are TNF-α mediated (Guthmann et al. 2009). Despite the lack of statistically significant correlation between the gene expression level of TNF and its receptors TNFR1 and TNFR2, Significantly decreased numbers of TNFR1 and TNFR2 gene copies may play a significant role in initiation and progression of arteriosclerosis (Dabek et al. 2012). Diabetes mellitus is associated with enhanced leukocyte accumulation in response to ischemia— reperfusion is mediated by CD11/CD18–ICAM-1 interactions and P-selectin. Diabetes causes insulin resistance which is also
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:1053–1060
responsible for endothelial dysfunction associated with increase of TNF-α level (Kolluru et al. 2012). Therefore, binding of free TNF-α should improve the formation of microvessels in response to ischemia in diabetes. The aim of present study is searching the efficacy of Enbrel as a therapy for enhanced recovery of angiogenesis and blood flow of the ischemic hind-limb of type 2 diabetic mice.
Materials and methods Experimental design Experimental design was performed as shown in Fig. 1. Antibodies Total Akt, phosphorylated Akt (Ser476), phosphorylated p40Phox (Thr154), Von Willebrand factor, α-actin, intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and the used secondary antibodies were purchased from Cell Signal and Promega (P.O. Box 257 Dammam 31411 Saudi Arabia). Drugs Etanercept (Enbre) was purchased from a pharmaceutical supplier in 1 cm3 syringes at 50 mg/mL and was used in native carrier forms.
1055
present study. These studies conformed to the principles of the handling and treatment of the animals used for research in accordance with the principles of Islam and good humanitarian practices. BKing Abdulaziz City for Science and Technology (KACST) Guide for the Care and Use of Laboratory Animals^ was approved by the Umm Al-Qura University (UQU) Institutional Animal Care and Use Committee. The hind-limbs ischemia was initiated by ligation of the left femoral artery. Before each surgery, the mouse was deeply anesthetized and shaved under sterile conditions. A longitudinal skin incision (about 1 cm) was made in the groin; the femoral triangle was explored, and the major vessels were identified and dissected. Black braided silk sutures (deknatel, USA) were used for the ligation prior to excision of the arterial vessel bed between the distal ends of the external iliac artery (Yang et al. 2009). Finally, the overlying skin was closed with stainless steel wound clips (world precision instruments, USA) and disinfected by scrubbing with Betadine solution. Body temperature maintained using isothermal pads. Mice were monitored over an experimental period. Treatment of db−/db− mice with Enbrel Enbrel was injected as two doses of 100 μg dose for each injection (for an average body weight of 25 g) intraperitoneally at 3 days and 1 day before surgery. As a control, a group of db−/db− mice were subjected to intraperitoneal injection of PBS only. Laser Doppler measurement of hind-limb blood flow
Animal model and surgery −
−
Fifty male type 2 diabetic (db /db ; with C57/Bl6 genetic background) and their control (db−/db+) between 8–10 were used. Fifty-nine old mice were used for the completion of the Fig. 1 Diagram showing experimental design. Group 1 included 10 mice C75BL6/J, Group 2 included 20 diabetic mice, and Group 3 included 20 treated diabetic mice
Hind-limb blood flow measurements over the region of interest were performed at baseline before surgery, immediately postoperatively, and serially over the 4-week period with laser Doppler perfusion imaging (LDPI) (Moor Instruments).
1056
Naunyn-Schmiedeberg's Arch Pharmacol (2015) 388:1053–1060
X-ray quantification of hind-limb angiogenesis
Immunohistochemistry
Ve s s e l d e n s i t y w a s a s s e s s e d b y h i g h - d e f i n i t i o n microangiography at the end of the treatment period (Jacobi et al. 2004).
Immunohistochemistry was performed (Belmadani et al. 2008a, 2009; Su et al. 2008). Statistical analysis
Western blot analysis Western blot analysis of tissue was performed (Belmadani et al. 2008a, b; Su et al. 2008). The quantification of Western blot was determined using Fujifilm-Multi Gauge software. Fig. 2 Blood flow of hind-limbs in all groups
Results are expressed as mean±SEM. One-way or 2-way ANOVA was used to compare each parameter when there were two independent groups. Comparisons between groups were performed with post hoc Bonferroni t tests when the ANOVA test was statistically significant. Values of P
