Mol Biol Rep (2014) 41:5813–5818 DOI 10.1007/s11033-014-3455-4
CCN1 enhances angiogenic potency of bone marrow transplantation in a rat model of hindlimb ischemia Cunping Yin • Yuan Liang • Shuguang Guo Xingli Zhou • Xinghua Pan
•
Received: 20 November 2013 / Accepted: 12 June 2014 / Published online: 29 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Implantation of autologous bone marrow mononuclear cells (BM-MNCs) has been performed in ischemic tissues, for stimulation of angiogenesis, but the limited number of BM-MNCs in patients with hindlimb ischemia disease may offset their overall therapeutic efficacy. CCN1 is a novel and essential regulator during angiogenesis. We evaluated whether CCN1 and BM-MNC are capable of promoting angiogenesis in hindlimb ischemia. In this study, we created the rat model of hindlimb ischemia, and then the rats were randomly divided into four groups: CCN1 infusion plus BM-MNC transplantation (CCN1 ? BM-MNCs group), CCN1 infusion plus PBS injection (CCN1 group), vehicle infusion plus BM-MNC transplantation (BM-MNCs group) and vehicle infusion plus PBS injection (control group). The combination of Cunping Yin and Yuan Liang authors contributed equally to this work. C. Yin (&) S. Guo X. Zhou Department of Vascular Surgery, Kunming General Hospital, Chengdu Military Command, Kunming 650032, People’s Republic of China e-mail: [email protected] C. Yin X. Pan Stem cell Engineering Laboratory of Yunnan Province, Kunming General Hospital, Chengdu Military Command, Kunming 650032, People’s Republic of China Y. Liang Department of Geriatrics, Kunming General Hospital, Chengdu Military Command, Kunming 650032, People’s Republic of China X. Pan Department of Clinic Laboratory, Kunming General Hospital, Chengdu Military Command, Kunming 650032, People’s Republic of China
CCN1 and BM-MNC therapy could increase blood perfusion, capillary/muscle fiber ratio and tissue oxygenation in ischemic hindlimb. Moreover, CCN1 could not only inhibit the apoptosis of BM-MNCs, but also enhance the adhesiveness of BM-MNCs to HUVEC. Taken together, CCN1 enhanced angiogenesis of BM-MNC transplantation, and combining CCN1 with BM-MNC transplantation is a useful alternative for ischemic limbs. Keywords CCN1 Bone marrow mononuclear cells Angiogenesis
Introduction Hindlimb ischemia due to arteriosclerosis obliterans is a disease that impacts patients’ quality of life. It is characterized by leg pain, ischemic ulcers and gangrene [1]. Current therapeutic strategies to treat arteriosclerosis obliterans include medical and surgical procedures [2]. Although these therapies have shown considerable promise in reducing patients’ complications, it remains a considerable medical challenge: surgery are not suitable for patients with severe hindlimb ischemia [3]. Therefore, there is an urgent need to develop new treatments for hindlimb ischemia. Recently, angiogenic therapeutic methods have been used to treat no-option patients with hindlimb ischemia disease. Although angiogenic cytokines, such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), promoted collateral circulation in patients with hindlimb ischemia [4], there were some negative results in clinical trials. It has been reported that implantation of bone marrow mononuclear cells (BM-MNCs) could increase neovascularization of ischemic tissue through supplying endothelial progenitor cells (EPCs) to the vasculature [5, 6].
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EPCs replaces injured mature endothelial cell by incorporating into blood vessels, and secrets many proangiogenic factors to promote the survival and proliferation of endothelial cells [7], which indicates that autologous BM-MNCs implantation induces angiogenesis. However, the number of BM-MNCs is limited in patients with hindlimb ischemia, which offsets the therapeutic efficacy. CCN1 (cyr61) is a member of the CCN (cyr61, ctgf, nov) protein family [8], and was identified as a protein encoded by an immediate-early gene induced by growth factor in fibroblasts [9]. Accumulating evidence indicates that CCN1 regulates a variety of cellular processes, such as cell migration, proliferation and apoptosis [10–12]. Mo et al. [13] have demonstrated that placental vascular insufficiency and compromised vessel integrity resulted in embryonic death in CCN1-deficient mice. In addition, as recently reported by Schutze et al. [14], CCN1 plays a critical role in the migration and differentiation of bone marrow-derived mesenchymal stem cells, a population of pluripotent progenitors with the ability to accelerate the repair of endothelial cell damage in adult blood vessels. Furthermore, CCN1 binds to and acts through integrin receptors, promotes the attachment of CD34? progenitor cells to the endothelial cells and stimulates endothelial cell migration, which results in endothelial regeneration [15]. Therefore, in this study, we evaluated whether CCN1 and BM-MNC are capable of promoting angiogenesis in hindlimb ischemia.
Materials and methods BM-MNC isolation and culture BM-MNCs were isolated from the right iliac crest of rats and centrifuged by Histopaque density gradient (Sigma, St. Louis, MO) [16]. BM-MNCs were cultured on fibronectin-coated plates in Medium 199 with 20 % fetal bovine serum (Abcam, Cambridge, UK), endothelial cell growth supplement, 10 U/mL heparin (Sigma, St. Louis, MO), and antibiotics at 37 8C in a humidified incubator containing 5 % CO2. Animal model of hindlimb ischemia All surgical procedures were performed under anesthesia with intraperitoneal sodium salt injection (80 mg/kg). The left femoral vessels were isolated without damaging the femoral nerve. The proximal portion of the femoral artery and the distal portion of the saphenous artery were ligated with 7–0 silk ligatures. The remaining branches between these two sites as well as veins were all dissected free and then excised. The right hindlimb was kept intact and used as
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the nonischemic limb. Rats were randomly divided into four groups: (1) CCN1 infusion plus BM-MNC transplantation (CCN1 ? BM-MNCs group, n = 10), (2) CCN1 infusion plus PBS injection (CCN1 group, n = 10), (3) vehicle infusion plus BM-MNC transplantation (BM-MNCs group, n = 10), (4) vehicle infusion plus PBS injection (control group, n = 10). All experimental procedures conformed to the guidelines issued by the Kunming General Hospital, Chengdu Military Command for Laboratory Animals. The present study was performed with approval from the Animal Ethics Committee of the Kunming General Hospital, Chengdu Military Command (Certification NO: 0119). BM-MNC transplantation and CCN1 infusion BM-MNCs (5 9 106 cells per rat) were transplanted at 5 different sites in the ischemic thigh muscle with a 26-gauge needle. Human recombinant CCN1 (0.01 lg kg-1 min-1) (Invitrogen, Carlsbad, CA) or vehicle was injected in the left inguinal region for 7 days with a mini-osmotic pump. Determination of blood perfusion and capillary/muscle fiber ratio A laser Doppler perfusion image (LDPI) analyzer (OMEGAZONE OZ-1, OMEGAWAVE, Inc., Tokyo, Japan) was used to measure the blood flow. The mean blood flow of each limb was determined. The ratio between the ischemic and the nonischemic limb was calculated and represents LDPI index. On day 21 after hindlimb ischemic, capillary density of ischemic hindlimbs was evaluated under light microscopy (Olympus Corporation, Tokyo, Japan). We obtained tissue specimens from the ischemic hindlimbs. The specimens were frozen in liquid nitrogen and cut into 5 m thickness, then stained for alkaline phosphatase to detect capillary endothelial cells. Transcutaneous oxygen pressure measurements On day 21 after hindlimb ischemic, the transcutaneous oxygen pressure (TcO2) was measured on the dorsum, crus and thigh of hindlimbs using a TcO2 analyzer (PF5000, Perimed, Stockholm, Sweden). Measurements were also taken on the three skin points. Then we calculated the ratio of ischemic/normal hindlimb. In vitro apoptosis assay The cell apoptosis was determined using Annexin V-FITC/ PI kit (Invitrogen, Carlsbad, CA). Briefly, cells were plated in 24-well plates at a density of 2 9 105 cells/well and then various concentrations of CCN1 were added. 10 ll of Annexin V-FITC and 10 ll of PI were added to each well.
Mol Biol Rep (2014) 41:5813–5818
Fig. 1 Results of hindlimb blood perfusion with LDPI index, ratio of ischemic to nonischemic hindlimb blood perfusion. The LDPI index was significantly higher in the CCN1 ? BM-MNCs group. *P \ 0.05 vs control group; &P \ 0.05 versus control group; #P \ 0.05 versus BM-MNCs group; $P \ 0.05 versus CCN1 group. (n = 3)
After 30 min, apoptotic cells were analyzed with flow cytometry (Beckman Coulter, Brea, CA). In vitro adhesion assay To determine whether CCN1 enhances BM-MNCs’ adhesiveness, we performed a standard adhesion assay with minor modifications [17]. In brief, human umbilical vein endothelial cells (HUVEC) (Cell Applications Inc., San Diego, CA) were treated with tumor necrosis factor- (1 ng/mL) (Sigma, St. Louis, MO) and then cultured in 6-well plates. After pretreatment with CCN1, BM-MNCs (1 9 106) labeled with PKH26 (Sigma, St. Louis, MO) were incubated on a HUVEC monolayer for 24 h. Nonadherent BM-MNCs were removed by PBS, and the number of PKH26-positive cells was counted under a microscope.
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Fig. 2 Results of capillary density in ischemic hindlimb muscles. Combination of CCN1 and BM-MNCs therapy could significantly increase the capillary/muscle fiber ratio of ischemic muscle. *P \ 0.05 compared with control group; #P \ 0.05 compared with CCN1 and BM-MNCs group. (n = 3)
LDPI analyzer. As shown in Fig. 1, in the CCN1 and BMMNC rats, blood flow recovered gradually and returned to 70 % of nonischemic hindlimb levels on day 21. Furthermore, as compared with other groups, CCN1 ? BM-MNCs obviously increased the LDPI index. Also, during 3 weeks after surgery, blood flow recovered gradually and returned to almost nonischemic hindlimb levels in the CCN1 ? BM-MNCs group. These results demonstrated that the combination of CCN1 and BM-MNC therapy could increase blood perfusion of ischemic hindlimb. Combination of CCN1 and BM-MNC therapy increases the capillary/muscle fiber ratio
Results
The capillary/muscle ratio of ischemic muscle reflects the degree of vascularization. Therefore, we determined the capillary/muscle ratio among four groups. As shown in Fig. 2, the capillary/muscle ratio of capillary/muscle ratio of ischemic muscle was markedly greater in the three treatments, compared with the control group. However, no significant difference was observed between the CCN1 and the BM-MNCs group. Furthermore, promotion was greatest for the CCN1 ? BM-MNCs group.These results demonstrated that the combination of CCN1 and BM-MNC therapy could increase the capillary/muscle fiber ratio of ischemic muscle.
Combination of CCN1 and BM-MNC therapy increases blood perfusion of ischemic hindlimb
Combination of CCN1 and BM-MNC therapy enhances tissue oxygenation in ischemic hindlimb
Blood flow recovery through ischemic skeletal muscle in rats of the four groups was evaluated for 3 weeks by an
Because TcO2 is an index of the functionality of neovascularization, we therefore measured TcO2. As shown in
Statistical analysis All results are reported as mean ± SD. Student’s unpaired t test was used to compare differences between two groups. Comparisons of multiple groups were made by 1-way ANOVA, followed by Scheffe multiple comparison test. Comparisons of the time course of the LDPI index were made by 2-way ANOVA for repeated measures, followed by Scheffe multiple comparison test. Statistical significance was set as P \ 0.05.
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Fig. 3 Results of TcO2 in ischemic hindlimb muscles. Ratio of the ischemic/normal hindlimb TcO2 was measured on day 21 after hindlimb ischemic. Combination of CCN1 and BM-MNCs therapy enhances tissue oxygenation in ischemic hindlimb. *P \ 0.05 compared with control group; #P \ 0.05 compared with CCN1 and BMMNCs group. (n = 3)
Fig. 3, CCN1 sole therapy or solo BM-MNC therapy elevated the ratio of TcO2 in the ischemic/nonischemic hindlimbs compared with the control group. Furthermore, the TcO2 ratio of the ischemic/nonischemic hindlimbs was significantly higher in the CCN1 ? BM-MNCs group than in the other groups, suggesting that the combination of CCN1 and BM-MNC therapy could improve microcirculation in ischemic hindlimb.
Mol Biol Rep (2014) 41:5813–5818
Fig. 5 The effect of CCN1 on BM-MNCs adhesiveness. CCN1 enhanced-adhesiveness of BM-MNCs to endothelial cells-like cells was induced by TNF-a. Data are mean ± SD of six dishes from three separate experiments. *P \ 0.05 compared with control group
MNCs is one of the primary factors that influence angiogenesis. As shown in Fig. 4, the results showed that CCN1 caused a dose-dependent decrease in cell apoptosis; the apoptosis rate decreased to 15.7 % when the concentration of CCN1 was 10-7 mmol/L. Effect of CCN1 on BM-MNC adhesiveness
The proliferation, migration and remodeling of endothelial cells results in neo-vascular formation, a process of angiogenesis [18]. And BM-MNCs affect the number of vascular endothelial progenitor cells, which plays an important role in angiogenesis [19]. Therefore, the number of BM-
The adhesiveness of BM-MNCs to endothelial-like cells may contribute to angiogenesis [20]. To investigate whether CCN1 enhances BM-MNC adhesiveness, we performed a cell adhesion assay. As shown in Fig. 5, as compared with the control group, CCN1 significantly increased the number of adherent BM-MNCs on a HUVEC monolayer. Furthermore, CCN1 stimulation in the presence of 1 ng/ml tumor necrosis factor-a (TNF-a) reinforced the adhesiveness of BM-MNCs to HUVEC. These results suggested that CCN1 enhanced-adhesiveness of BMMNCs to endothelial-like cells was induced by TNF-a.
Fig. 4 The anti-apoptotic effect of CCN1 on BM-MNCs. Flow cytometry analysis of CCN1 treated BM-MNCs stained with annexin V and PI. CCN1 treatment inhibits apoptosis of BM-MNCs in a dose–
dependent manner (a control group; b 10-9 mol/L CCN1 group; c 10-8 mol/L CCN1 group; d 10-7 mol/L CCN1 group). All experiments were repeated at least three times
Anti-apoptotic effect of CCN1 on BM-MNCs
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Discussion There are two independent strategies for angiogenic therapy for patients with arteriosclerosis obliterans: angiogenic gene transfer and autologous cell implantation. Angiogenic gene therapy exhibits good angiogenic effects, however, there are some issues, such as efficacy and safety remain to be resolved [21]. Autologous BM-MNC transplantation therapy for hindlimb ischemia patients has reportedly shown excellent results [22, 23]. However, the limited number of BM-MNCs in patients may offset their overall therapeutic efficacy. The present study demonstrated that infusion of CCN1 enhanced the angiogenic potency of BMMNC transplantation. In this report, a combination of CCN1 and BM-MNC therapy was able to increase blood perfusion, capillary/muscle fiber ratio and tissue oxygenation in ischemic hindlimb. Moreover, CCN1 not only inhibited BM-MNC apoptosis, but also enhanced the adhesiveness of BM-MNCs to HUVEC. During the process of angiogenesis, a harmonious interaction of cytokines, growth factors, vasoactive substances and vascular cells is very important, which ensures the promotion of functional vessels. [24]. Therefore, a single growth factor may not be sufficient to form a functioning vascular network. Previous studies have reported that overexpression CCN1 accelerated reendothelialization via a direct action on endothelial progenitor cell differentiation [10]. CCN1 also promoted endothelial cell adhesion, migration, proliferation and the formation of endothelial tubules [25]. BM-MNC transplantation causes angiogenesis by supplying endothelial progenitor cells and angiogenic cytokines such as VEGF [16, 26]. In this study, we demonstrated that a combination of CCN1 infusion and BM-MNC transplantation significantly increased blood perfusion and capillary density in the ischemic hindlimb. Aspects of our study are consistent with prior reports: a combination therapy with the angiopoietin-1 gene and BMMNCs obviously augmented angiographic scores and capillary densities [27]; adrenomedullin infusion and BMMNC transplantation also promoted angiogenesis in hindlimb ischemia. [28]. These results suggest that CCN1 and BM-MNCs may synergize in promoting neovascularization in hindlimb ischemia. Recently, some studies have shown that angiogenic genes could improve angiogenic effects through inhibiting apoptosis of BM-MNCs, which suggested that reduced apoptosis of BM-MNCs is important during the neovascularization of ischemic tissue. Iwase et al. [28] reported that adrenomedullin inhibited the apoptosis of BM-MNCs in vitro and in vivo. In this study, CCN1 inhibited apoptosis of BM-MNCs in a dose–dependent manner. We hypothesize that CCN1 prolongs BM-MNC survival and thereby enhances neovascularization in ischemic hindlimb.
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Previous studies have shown that BM-MNC adhesiveness to endothelial cells is indispensable for BM-MNC differentiation into the endothelial lineage. Fujiyama et al. [29] reported that BM-MNCs are more potent than CD34? cells and adheres on injured endothelial cells in a monocyte chemoattractant protein-1-dependent manner, resulting in vascular regeneration. In this study, CCN1 promoted the adhesion of BM-MNCs to a HUVEC monolayer. Furthermore, TNF-a is one of the pro-inflammatory cytokines that mediate various immune and inflammatory responses and induces CCN1 expression in some cells [30]. In the present study, we showed that CCN1 enhanced-adhesiveness of BM-MNCs to endothelial-like cells was induced by TNFa. Therefore, it is possible that CCN1 infusion enhances the angiogenic potency of BM-MNCs in part through promoting the adhesion of BM-MNCs to vascular endothelial cells. In summary, CCN1 enhanced the potency of angiogenesis of BM-MNC transplantation, and the combination of CCN1 with BM-MNC transplantation may represent a novel strategy for the treatment of arteriosclerosis obliterans.
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CCN1 enhances angiogenic potency of bone marrow transplantation in a rat model of hindlimb ischemia Cunping Yin • Yuan Liang • Shuguang Guo Xingli Zhou • Xinghua Pan
•
Received: 20 November 2013 / Accepted: 12 June 2014 / Published online: 29 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Implantation of autologous bone marrow mononuclear cells (BM-MNCs) has been performed in ischemic tissues, for stimulation of angiogenesis, but the limited number of BM-MNCs in patients with hindlimb ischemia disease may offset their overall therapeutic efficacy. CCN1 is a novel and essential regulator during angiogenesis. We evaluated whether CCN1 and BM-MNC are capable of promoting angiogenesis in hindlimb ischemia. In this study, we created the rat model of hindlimb ischemia, and then the rats were randomly divided into four groups: CCN1 infusion plus BM-MNC transplantation (CCN1 ? BM-MNCs group), CCN1 infusion plus PBS injection (CCN1 group), vehicle infusion plus BM-MNC transplantation (BM-MNCs group) and vehicle infusion plus PBS injection (control group). The combination of Cunping Yin and Yuan Liang authors contributed equally to this work. C. Yin (&) S. Guo X. Zhou Department of Vascular Surgery, Kunming General Hospital, Chengdu Military Command, Kunming 650032, People’s Republic of China e-mail: [email protected] C. Yin X. Pan Stem cell Engineering Laboratory of Yunnan Province, Kunming General Hospital, Chengdu Military Command, Kunming 650032, People’s Republic of China Y. Liang Department of Geriatrics, Kunming General Hospital, Chengdu Military Command, Kunming 650032, People’s Republic of China X. Pan Department of Clinic Laboratory, Kunming General Hospital, Chengdu Military Command, Kunming 650032, People’s Republic of China
CCN1 and BM-MNC therapy could increase blood perfusion, capillary/muscle fiber ratio and tissue oxygenation in ischemic hindlimb. Moreover, CCN1 could not only inhibit the apoptosis of BM-MNCs, but also enhance the adhesiveness of BM-MNCs to HUVEC. Taken together, CCN1 enhanced angiogenesis of BM-MNC transplantation, and combining CCN1 with BM-MNC transplantation is a useful alternative for ischemic limbs. Keywords CCN1 Bone marrow mononuclear cells Angiogenesis
Introduction Hindlimb ischemia due to arteriosclerosis obliterans is a disease that impacts patients’ quality of life. It is characterized by leg pain, ischemic ulcers and gangrene [1]. Current therapeutic strategies to treat arteriosclerosis obliterans include medical and surgical procedures [2]. Although these therapies have shown considerable promise in reducing patients’ complications, it remains a considerable medical challenge: surgery are not suitable for patients with severe hindlimb ischemia [3]. Therefore, there is an urgent need to develop new treatments for hindlimb ischemia. Recently, angiogenic therapeutic methods have been used to treat no-option patients with hindlimb ischemia disease. Although angiogenic cytokines, such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), promoted collateral circulation in patients with hindlimb ischemia [4], there were some negative results in clinical trials. It has been reported that implantation of bone marrow mononuclear cells (BM-MNCs) could increase neovascularization of ischemic tissue through supplying endothelial progenitor cells (EPCs) to the vasculature [5, 6].
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EPCs replaces injured mature endothelial cell by incorporating into blood vessels, and secrets many proangiogenic factors to promote the survival and proliferation of endothelial cells [7], which indicates that autologous BM-MNCs implantation induces angiogenesis. However, the number of BM-MNCs is limited in patients with hindlimb ischemia, which offsets the therapeutic efficacy. CCN1 (cyr61) is a member of the CCN (cyr61, ctgf, nov) protein family [8], and was identified as a protein encoded by an immediate-early gene induced by growth factor in fibroblasts [9]. Accumulating evidence indicates that CCN1 regulates a variety of cellular processes, such as cell migration, proliferation and apoptosis [10–12]. Mo et al. [13] have demonstrated that placental vascular insufficiency and compromised vessel integrity resulted in embryonic death in CCN1-deficient mice. In addition, as recently reported by Schutze et al. [14], CCN1 plays a critical role in the migration and differentiation of bone marrow-derived mesenchymal stem cells, a population of pluripotent progenitors with the ability to accelerate the repair of endothelial cell damage in adult blood vessels. Furthermore, CCN1 binds to and acts through integrin receptors, promotes the attachment of CD34? progenitor cells to the endothelial cells and stimulates endothelial cell migration, which results in endothelial regeneration [15]. Therefore, in this study, we evaluated whether CCN1 and BM-MNC are capable of promoting angiogenesis in hindlimb ischemia.
Materials and methods BM-MNC isolation and culture BM-MNCs were isolated from the right iliac crest of rats and centrifuged by Histopaque density gradient (Sigma, St. Louis, MO) [16]. BM-MNCs were cultured on fibronectin-coated plates in Medium 199 with 20 % fetal bovine serum (Abcam, Cambridge, UK), endothelial cell growth supplement, 10 U/mL heparin (Sigma, St. Louis, MO), and antibiotics at 37 8C in a humidified incubator containing 5 % CO2. Animal model of hindlimb ischemia All surgical procedures were performed under anesthesia with intraperitoneal sodium salt injection (80 mg/kg). The left femoral vessels were isolated without damaging the femoral nerve. The proximal portion of the femoral artery and the distal portion of the saphenous artery were ligated with 7–0 silk ligatures. The remaining branches between these two sites as well as veins were all dissected free and then excised. The right hindlimb was kept intact and used as
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the nonischemic limb. Rats were randomly divided into four groups: (1) CCN1 infusion plus BM-MNC transplantation (CCN1 ? BM-MNCs group, n = 10), (2) CCN1 infusion plus PBS injection (CCN1 group, n = 10), (3) vehicle infusion plus BM-MNC transplantation (BM-MNCs group, n = 10), (4) vehicle infusion plus PBS injection (control group, n = 10). All experimental procedures conformed to the guidelines issued by the Kunming General Hospital, Chengdu Military Command for Laboratory Animals. The present study was performed with approval from the Animal Ethics Committee of the Kunming General Hospital, Chengdu Military Command (Certification NO: 0119). BM-MNC transplantation and CCN1 infusion BM-MNCs (5 9 106 cells per rat) were transplanted at 5 different sites in the ischemic thigh muscle with a 26-gauge needle. Human recombinant CCN1 (0.01 lg kg-1 min-1) (Invitrogen, Carlsbad, CA) or vehicle was injected in the left inguinal region for 7 days with a mini-osmotic pump. Determination of blood perfusion and capillary/muscle fiber ratio A laser Doppler perfusion image (LDPI) analyzer (OMEGAZONE OZ-1, OMEGAWAVE, Inc., Tokyo, Japan) was used to measure the blood flow. The mean blood flow of each limb was determined. The ratio between the ischemic and the nonischemic limb was calculated and represents LDPI index. On day 21 after hindlimb ischemic, capillary density of ischemic hindlimbs was evaluated under light microscopy (Olympus Corporation, Tokyo, Japan). We obtained tissue specimens from the ischemic hindlimbs. The specimens were frozen in liquid nitrogen and cut into 5 m thickness, then stained for alkaline phosphatase to detect capillary endothelial cells. Transcutaneous oxygen pressure measurements On day 21 after hindlimb ischemic, the transcutaneous oxygen pressure (TcO2) was measured on the dorsum, crus and thigh of hindlimbs using a TcO2 analyzer (PF5000, Perimed, Stockholm, Sweden). Measurements were also taken on the three skin points. Then we calculated the ratio of ischemic/normal hindlimb. In vitro apoptosis assay The cell apoptosis was determined using Annexin V-FITC/ PI kit (Invitrogen, Carlsbad, CA). Briefly, cells were plated in 24-well plates at a density of 2 9 105 cells/well and then various concentrations of CCN1 were added. 10 ll of Annexin V-FITC and 10 ll of PI were added to each well.
Mol Biol Rep (2014) 41:5813–5818
Fig. 1 Results of hindlimb blood perfusion with LDPI index, ratio of ischemic to nonischemic hindlimb blood perfusion. The LDPI index was significantly higher in the CCN1 ? BM-MNCs group. *P \ 0.05 vs control group; &P \ 0.05 versus control group; #P \ 0.05 versus BM-MNCs group; $P \ 0.05 versus CCN1 group. (n = 3)
After 30 min, apoptotic cells were analyzed with flow cytometry (Beckman Coulter, Brea, CA). In vitro adhesion assay To determine whether CCN1 enhances BM-MNCs’ adhesiveness, we performed a standard adhesion assay with minor modifications [17]. In brief, human umbilical vein endothelial cells (HUVEC) (Cell Applications Inc., San Diego, CA) were treated with tumor necrosis factor- (1 ng/mL) (Sigma, St. Louis, MO) and then cultured in 6-well plates. After pretreatment with CCN1, BM-MNCs (1 9 106) labeled with PKH26 (Sigma, St. Louis, MO) were incubated on a HUVEC monolayer for 24 h. Nonadherent BM-MNCs were removed by PBS, and the number of PKH26-positive cells was counted under a microscope.
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Fig. 2 Results of capillary density in ischemic hindlimb muscles. Combination of CCN1 and BM-MNCs therapy could significantly increase the capillary/muscle fiber ratio of ischemic muscle. *P \ 0.05 compared with control group; #P \ 0.05 compared with CCN1 and BM-MNCs group. (n = 3)
LDPI analyzer. As shown in Fig. 1, in the CCN1 and BMMNC rats, blood flow recovered gradually and returned to 70 % of nonischemic hindlimb levels on day 21. Furthermore, as compared with other groups, CCN1 ? BM-MNCs obviously increased the LDPI index. Also, during 3 weeks after surgery, blood flow recovered gradually and returned to almost nonischemic hindlimb levels in the CCN1 ? BM-MNCs group. These results demonstrated that the combination of CCN1 and BM-MNC therapy could increase blood perfusion of ischemic hindlimb. Combination of CCN1 and BM-MNC therapy increases the capillary/muscle fiber ratio
Results
The capillary/muscle ratio of ischemic muscle reflects the degree of vascularization. Therefore, we determined the capillary/muscle ratio among four groups. As shown in Fig. 2, the capillary/muscle ratio of capillary/muscle ratio of ischemic muscle was markedly greater in the three treatments, compared with the control group. However, no significant difference was observed between the CCN1 and the BM-MNCs group. Furthermore, promotion was greatest for the CCN1 ? BM-MNCs group.These results demonstrated that the combination of CCN1 and BM-MNC therapy could increase the capillary/muscle fiber ratio of ischemic muscle.
Combination of CCN1 and BM-MNC therapy increases blood perfusion of ischemic hindlimb
Combination of CCN1 and BM-MNC therapy enhances tissue oxygenation in ischemic hindlimb
Blood flow recovery through ischemic skeletal muscle in rats of the four groups was evaluated for 3 weeks by an
Because TcO2 is an index of the functionality of neovascularization, we therefore measured TcO2. As shown in
Statistical analysis All results are reported as mean ± SD. Student’s unpaired t test was used to compare differences between two groups. Comparisons of multiple groups were made by 1-way ANOVA, followed by Scheffe multiple comparison test. Comparisons of the time course of the LDPI index were made by 2-way ANOVA for repeated measures, followed by Scheffe multiple comparison test. Statistical significance was set as P \ 0.05.
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Fig. 3 Results of TcO2 in ischemic hindlimb muscles. Ratio of the ischemic/normal hindlimb TcO2 was measured on day 21 after hindlimb ischemic. Combination of CCN1 and BM-MNCs therapy enhances tissue oxygenation in ischemic hindlimb. *P \ 0.05 compared with control group; #P \ 0.05 compared with CCN1 and BMMNCs group. (n = 3)
Fig. 3, CCN1 sole therapy or solo BM-MNC therapy elevated the ratio of TcO2 in the ischemic/nonischemic hindlimbs compared with the control group. Furthermore, the TcO2 ratio of the ischemic/nonischemic hindlimbs was significantly higher in the CCN1 ? BM-MNCs group than in the other groups, suggesting that the combination of CCN1 and BM-MNC therapy could improve microcirculation in ischemic hindlimb.
Mol Biol Rep (2014) 41:5813–5818
Fig. 5 The effect of CCN1 on BM-MNCs adhesiveness. CCN1 enhanced-adhesiveness of BM-MNCs to endothelial cells-like cells was induced by TNF-a. Data are mean ± SD of six dishes from three separate experiments. *P \ 0.05 compared with control group
MNCs is one of the primary factors that influence angiogenesis. As shown in Fig. 4, the results showed that CCN1 caused a dose-dependent decrease in cell apoptosis; the apoptosis rate decreased to 15.7 % when the concentration of CCN1 was 10-7 mmol/L. Effect of CCN1 on BM-MNC adhesiveness
The proliferation, migration and remodeling of endothelial cells results in neo-vascular formation, a process of angiogenesis [18]. And BM-MNCs affect the number of vascular endothelial progenitor cells, which plays an important role in angiogenesis [19]. Therefore, the number of BM-
The adhesiveness of BM-MNCs to endothelial-like cells may contribute to angiogenesis [20]. To investigate whether CCN1 enhances BM-MNC adhesiveness, we performed a cell adhesion assay. As shown in Fig. 5, as compared with the control group, CCN1 significantly increased the number of adherent BM-MNCs on a HUVEC monolayer. Furthermore, CCN1 stimulation in the presence of 1 ng/ml tumor necrosis factor-a (TNF-a) reinforced the adhesiveness of BM-MNCs to HUVEC. These results suggested that CCN1 enhanced-adhesiveness of BMMNCs to endothelial-like cells was induced by TNF-a.
Fig. 4 The anti-apoptotic effect of CCN1 on BM-MNCs. Flow cytometry analysis of CCN1 treated BM-MNCs stained with annexin V and PI. CCN1 treatment inhibits apoptosis of BM-MNCs in a dose–
dependent manner (a control group; b 10-9 mol/L CCN1 group; c 10-8 mol/L CCN1 group; d 10-7 mol/L CCN1 group). All experiments were repeated at least three times
Anti-apoptotic effect of CCN1 on BM-MNCs
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Discussion There are two independent strategies for angiogenic therapy for patients with arteriosclerosis obliterans: angiogenic gene transfer and autologous cell implantation. Angiogenic gene therapy exhibits good angiogenic effects, however, there are some issues, such as efficacy and safety remain to be resolved [21]. Autologous BM-MNC transplantation therapy for hindlimb ischemia patients has reportedly shown excellent results [22, 23]. However, the limited number of BM-MNCs in patients may offset their overall therapeutic efficacy. The present study demonstrated that infusion of CCN1 enhanced the angiogenic potency of BMMNC transplantation. In this report, a combination of CCN1 and BM-MNC therapy was able to increase blood perfusion, capillary/muscle fiber ratio and tissue oxygenation in ischemic hindlimb. Moreover, CCN1 not only inhibited BM-MNC apoptosis, but also enhanced the adhesiveness of BM-MNCs to HUVEC. During the process of angiogenesis, a harmonious interaction of cytokines, growth factors, vasoactive substances and vascular cells is very important, which ensures the promotion of functional vessels. [24]. Therefore, a single growth factor may not be sufficient to form a functioning vascular network. Previous studies have reported that overexpression CCN1 accelerated reendothelialization via a direct action on endothelial progenitor cell differentiation [10]. CCN1 also promoted endothelial cell adhesion, migration, proliferation and the formation of endothelial tubules [25]. BM-MNC transplantation causes angiogenesis by supplying endothelial progenitor cells and angiogenic cytokines such as VEGF [16, 26]. In this study, we demonstrated that a combination of CCN1 infusion and BM-MNC transplantation significantly increased blood perfusion and capillary density in the ischemic hindlimb. Aspects of our study are consistent with prior reports: a combination therapy with the angiopoietin-1 gene and BMMNCs obviously augmented angiographic scores and capillary densities [27]; adrenomedullin infusion and BMMNC transplantation also promoted angiogenesis in hindlimb ischemia. [28]. These results suggest that CCN1 and BM-MNCs may synergize in promoting neovascularization in hindlimb ischemia. Recently, some studies have shown that angiogenic genes could improve angiogenic effects through inhibiting apoptosis of BM-MNCs, which suggested that reduced apoptosis of BM-MNCs is important during the neovascularization of ischemic tissue. Iwase et al. [28] reported that adrenomedullin inhibited the apoptosis of BM-MNCs in vitro and in vivo. In this study, CCN1 inhibited apoptosis of BM-MNCs in a dose–dependent manner. We hypothesize that CCN1 prolongs BM-MNC survival and thereby enhances neovascularization in ischemic hindlimb.
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Previous studies have shown that BM-MNC adhesiveness to endothelial cells is indispensable for BM-MNC differentiation into the endothelial lineage. Fujiyama et al. [29] reported that BM-MNCs are more potent than CD34? cells and adheres on injured endothelial cells in a monocyte chemoattractant protein-1-dependent manner, resulting in vascular regeneration. In this study, CCN1 promoted the adhesion of BM-MNCs to a HUVEC monolayer. Furthermore, TNF-a is one of the pro-inflammatory cytokines that mediate various immune and inflammatory responses and induces CCN1 expression in some cells [30]. In the present study, we showed that CCN1 enhanced-adhesiveness of BM-MNCs to endothelial-like cells was induced by TNFa. Therefore, it is possible that CCN1 infusion enhances the angiogenic potency of BM-MNCs in part through promoting the adhesion of BM-MNCs to vascular endothelial cells. In summary, CCN1 enhanced the potency of angiogenesis of BM-MNC transplantation, and the combination of CCN1 with BM-MNC transplantation may represent a novel strategy for the treatment of arteriosclerosis obliterans.
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