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Pro-angiogenic actions of Salvianolic acids on in vitro cultured endothelial progenitor cells and chick embryo chorioallantoic membrane model
Journal of Ethnopharmacology 131 (2010) 562–566
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Pro-angiogenic actions of Salvianolic acids on in vitro cultured endothelial progenitor cells and chick embryo chorioallantoic membrane model Yu-Juan Li, Chang-Ling Duan 1 , Jian-Xun Liu ∗ , Yong-Gang Xu China Academy of Chinese Medical Sciences, Xiyuan Hospital, Beijing, PR China
a r t i c l e
i n f o
Article history: Received 27 April 2010 Received in revised form 17 June 2010 Accepted 18 July 2010 Available online 24 July 2010 Keywords: Endothelial progenitor cells Salvia miltiorrhiza Salvianolic acids Coronary artery disease
a b s t r a c t Aim of the study: Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely-used Chinese herbal medicines in clinical practice. In this study we aimed to demonstrate the pro-angiogenic effects of Salvianolic acids (SAs) to treat illnesses such as ischemic cardiovascular diseases, the main active components of aqueous extract of SM. Materials and methods: To do this, new-born rat SD spleen mononuclear cells were isolated and endothelial progenitor cells (EPCs) were expanded (not more than 24 h) SD. Then the pro-angiogenic activities of SAs were evaluated on in vitro cultured EPCs and chick embryo chorioallantoic membrane (CAM) model. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein (DiI-acLDL) and fluorescein Ulex Europaeus agglutinin-1 (FITC-UEA-1), and then viewed by laser scanning confocal microscope (LSCM) to confirm EPCs lineage. EPCs identification was also tested by ultrastructural analyses. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay, transwell chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Results: EPCs phenotype was confirmed by the presence of double positive cells for DiI-acLDL uptake and lectin binding and identification of Weibel–Palade body in cytoplasm by ultrastructural analyses. Incubation of EPCs with SAs increased the number of EPCs and promoted EPCs migratory, adhesive and in vitro vasculogenesis capacity. SAs also promoted angiogenesis as evidenced by CAM model. Conclusions: The results of the present study suggest that SAs may have utility for therapeutic postnatal vasculogenesis of ischemic tissue, contributing to the clinical benefit of SM therapy in patients with coronary artery disease. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The endothelium plays a pivotal role in cardiovascular homeostasis, including the regulation of vascular growth and neovascularization in response to ischemia (Celermajer, 1997). Mature endothelial cells (ECs), however, have limited regenerative capacity. Therefore, there is increasing interest in the effect of pharmacological approaches, to maintain structural and functional integrity of the endothelium, in part by promoting endothelial repair and preventing endothelial cell apoptosis. The description 10 years ago of putative endothelial progenitor cells (EPCs) that could mobilize from bone-marrow to participate in neovascularization at sites of ischemia was enthusiastically greeted because of the potential they hold for cardiovascular regeneration (Asahara et al., 1997). In this study, selected circulating CD34+ cells were
∗ Corresponding author. Tel.: +86 1062835601. 1 Equal contribution to the first author. 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.07.040
shown to differentiate into mature ECs ex vivo and to contribute to neovascularization in hindlimb ischemia. EPCs in peripheral blood function in the search for and repair of endothelial damage, indicating that dysfunction of EPCs results in impaired vascular repair. Importantly, several cardiovascular risk factors reduce circulating numbers and impair functional activity of circulating EPCs, suggesting a loss of endogenous endothelial repair capacity in patients at high risk for cardiovascular events. The number of peripheral blood EPCs is decreased in patients with cardiovascular risk factors such as hypertension (Hill et al., 2003; Vasa et al., 2001), and EPCs become dysfunctional in certain patients (Choi et al., 2004; Eizawa et al., 2004). Therefore it is of great importance to find the agents which are capable of promoting mobilization and functional activity of EPCs. Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely-used Chinese herbal medicines in clinical, containing phenolic compounds and potent antioxidant properties (Ji et al., 2003). SM had demonstrated its effectiveness in reduction of infarct size and mortality rate in rats with acute myocardial
Y.-J. Li et al. / Journal of Ethnopharmacology 131 (2010) 562–566
infarction (MI). The antioxidant effect and possible feature of angiogenesis of SM had been suggested as the main factor in protecting ischemic myocardium. Salvianolic acids (SAs), as the main active components of aqueous extract of SM, exhibit potential effects to prevent myocardial injury induced by infarction in clinical and animal experiments (Chang et al., 2006; Shi et al., 2007; Zhang and Wang, 2006). We hypothesized whether SAs could increase EPCs number and enhance functions, thus accelerating endothelial repair process, which contributes to improve the clinical symptoms and prognosis of patients with coronary artery diseases (CAD). To test this hypothesis, we studied the effects of SAs on EPCs and their corresponding effective doses. 2. Methods 2.1. Culture of EPCs For preparing spleen tissue homogenates, the whole organ was placed in cold PBS. The tissue was minced and immediately homogenized. Mononuclear cells (MNCs) were isolated by density gradient centrifugation using Histopaque-1.083 solution and washed with M199 (Gibco). Buffy coat MNCs were resuspended in endothelial growth medium (EGM-2) (lonza) consisting of endothelial basal medium, 5% fetal bovine serum, hEGF, VEGF, hFGF-B, IGF-1, ascorbic acid. Cells were seeded onto six-well tissue culture plates (5 × 106 cells per well) precoated with fibronetion (FN) and incubated at 37 ◦ C with 95% air and 5% CO2 . Under daily observation, first media change was performed ≈4 days after plating. Thereafter, media were changed every 3 days. 2.2. Cellular staining Fluorescent chemical detection of EPCs was performed on attached MNCs after 7 days in culture. Direct fluorescent staining was used to detect dual binding of FITCUlex europaeus agglutinin (UEA)-1 (Sigma) and 1-dioctadecyl3,3,3,3-tetramethylindocarbocyanine (DiI)-acetylated low-density lipoprotein (acLDL; Molecular Probe). Cells were first incubated with 2.5 g/ml acLDL at 37 ◦ C for 1 h and later fixed with 4% paraformaldehyde for 15 min. After washing, the cells were reacted with UEA-1 (10 g/ml) for 1 h. After the staining, samples were viewed with a laser cofoncal scan microscope (Zeiss). Cells demonstrating double positive fluorescence were identified as differentiating EPCs (Vasa et al., 2001). 2.3. Electron microscopy characterization of EPCs Cells were fixed with 0.1 mol/l PBS (pH 7.4) containing 2.5% glutaraldehyde at room temperature for 120 min. After washing with 0.1 mol/l cacodylate buffer (pH 7.4) and post-fixing for 10 min at room temperature with 0.1 mol/l PBS containing 1% (w/v) OsO4 , cells were then washed with 0.1 mol/l PBS and dehytrated by differing concentrations acetone and processed for embedding. Sections were analysed with a Jem1400 electron microscope. 2.4. Cell proliferation assay The effect of SAs on EPCs proliferation was determined by MTT assay. After confluence, EPCs were digested with 0.25% trypsin and then cultured in 96-well plate with 0.5% FBS medium, to which was added SAs with final concentrations ranging from 0.3 to 30 mg/l. 0.5% FBS medium served as a control. After being cultured for 24 h, 10 l MTT (5 g/l) was supplemented to the medium and incubated for 4 h. Then the supernatant was discarded and 150 l dimethyl sulfoxide was added. After shaking for 10 min, the OD value was measured at 490 nm.
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2.5. Cell migration assay EPCs migration was analysed using 24-well transwell chambers (Costar). Briefly, transwell chambers equipped with a 6.5 mmdiameter polyester membrane (pore size 8 .0 m) were used. 2 × 104 EPCs suspended in 100 l of medium containing 0.5% FBS without VEGF were seeded in the top chamber, and culture medium (500 l) supplemented with 0.5% FBS and differring concentrations SAs together with 50 g/l VEGF (peprotech Asia) was placed in the lower chamber. The transwell chamber was then incubated for 6 h at 37 ◦ C in a humidified incubator with 5% CO2 to render EPCs able to migrate through the membrane. After the incubation period, nonmigrated EPCs on the upper side of the membrane were scrape-off with a rubber. The membranes were then fixed with methanol for 10 min and stained with 0.1% crystal violet. Migrated EPCs attached to the lower side of the membrane were counted in three randomly selected microscopic fields (100×) in each chamber, and the average number of migrated cells were calculated. All experiments were performed in 5 wells, and data were expressed as the number of migrated cells/fields. 2.6. Cell adhesion assay After 1 day of incubation with SAs at concentrations ranging from 0.3 to 30 mg l−1 , EPCs were washed with PBS and gently detached with 0.25% trypsin in PBS. After centrifugation and resuspension in 0.5% FCS basal complete medium, identical cell numbers were placed onto FN-coated culture plate and incubated for 30 min at 37 ◦ C. Adherent cells were counted by independent blinded investigator (Walter et al., 2002). 2.7. In Vitro tube formation on matrigel plate Matrigel (Becton Dickinson Labware) basement membrane matrix was added to 96-well plate. After 0.5 h of incubation at 37 ◦ C to allow the matrix solution to solidify, 2 × 104 cells/ml were replated on top of the solidified matrix solution, EPCs were grown with SAs or vehicle control, and incubated at 37 ◦ C for 6 h. Tubule formation was inspected under an inverted light microscope at 400× magnification. Four representative fields were taken and the average number of tubules/400× field was determined. 2.8. CAM assay Fifty fresh fertile chicken eggs aged 8 days were obtained from a commercial source (Beijing Merial Vital Laboratory Animal Technology Co., Ltd). Eggs weighing from 55 to 65 g were incubated under standard conditions (MCO175 incubator, Sanyo, Japan; 37.5 ◦ C; 60% RH). On day 9 of the incubation, the fertile eggs were randomly allocated into five groups. Fertilized eggs are first disinfected with 70% alcohol, a window opening is punctured at the blunt end of the egg facing upwards using sterilized forceps. The eggshell above the air cell was removed and the shell membrane attached to it, was cut-off for further examination. Normal development was verified and embryos with malformations or dead embryos were excluded. CAMs (10 eggs per group) were treated as described as follows: (1) overlaying them with 20 mm2 sterilized filter discs that had been loaded with 10 l of PBS (negative control), 10 l of PBS containing 11 ∼ 300 mg/l of SAs. The window was sealed with tape and the eggs were returned to the incubator. After incubation for an additional 72 h, all samples were immediately fixed in methanol and acetone (1:1) for at least 15 min, then they were photographed using a stereomicroscope equipped with a camera and image analysis system. The angiogenic response was evaluated by counting the vessel density converging toward the filter discs.
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Fig. 1. (A) Representative EPCs binding of UEA-1. (B) Representative EPCs of uptake of acLDL. (C) Representative.
2.9. Statistical analysis All data are presented as mean ± SD. Differences between group means were assessed by an unpaired Student’s t test for single comparisons and by ANOVA for multiple comparisons. Values of P < 0.05 were considered significant. 3. Results
dose-dependently, with a maximal effect achieved at 30 mg/l (control vs. 30 mg/l, P < 0.001) (Fig. 5). 3.5. Effects of SAs on EPCs in vitro vasculogenesis Recent studies have demonstrated that circulating EPCs home into ischemic sites and differentiate into endothelial cells in situ (Asahara et al., 1999) in a manner consistent with a process termed vasculogenesis. Here in vitro tubulogenesis assay was used to inves-
3.1. Characterization of EPCs EPCs were characterized as adherent cells double positive for DiI-acLDL-uptake and lectin binding by using LSCM (Fig. 1). They were further documented by demonstrating Weibel–Palade body in cytoplasm (Fig. 2). 3.2. Effect of SAs on EPCs prolifertion The effect of SAs on EPCs proliferation activity was assayed using a MTT assay (Fig. 3). EPCs numbers showed a marked increase when they were cultured in medium supplimented with SAs at concentrations of 3 and 30 mg/l (P < 0.05, <0.05). 3.3. Effects of SAs on EPCs migration in response to VEGF The effects of SAs on EPCs migration were analysed in transwell chamber assay. Migrated cells were profoundly enhanced exposed to SAs at 3 and 30 mg/l than control (P < 0.001, <0.001) (Fig. 4). 3.4. Effects of SAs on EPCs adhesiveness To study the possibility that SAs promote adhesiveness of EPCs, EPCs were incubated with SAs for 24 h. After replating on 96-well plates, EPCs exposed to SAs exhibited a significant increase in the number of adhesiveness cells at 30 min. The increase occurred
Fig. 2. Representative transmission electron microscopy images of EPCs (25,000×), arrow shows the W–sP body.
Y.-J. Li et al. / Journal of Ethnopharmacology 131 (2010) 562–566
Fig. 3. MTT assay of rat EPCs in response to SAs. A moderate dose-dependent mitogenic response to SAs was observed when incubated with cultured EPCs for 24 h. *P < 0.05 vs. control. Data are expressed as mean ± SD, n = 5.
Fig. 4. Migration assay of rat EPCs in response to SAs. Migratory effect was augmented by SAs treatment in a dose-dependent manner. ***P < 0.001 vs. control. Data are shown as mean ± SD, n = 5.
tigate the capacity of EPCs to participate in neovascularization, which is the most important activity of EPCs. The responsiveness of tubulogenesis assay to SAs is depicted in Fig. 6. Addition of 30 mg/l SAs increased tubule formation significantly (81 ± 8 vs. control 38 ± 8, P < 0.001).
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Fig. 6. Angiogenic responsiveness of angiogenesis assay. Tubule number increased when exposed to 30 mg/l SAs. ***P < 0.001 vs. control. Data are expressed as mean ± SD, n = 5.
Fig. 7. Effects of SAs on CAM model. SAs showed pro-angiogenic effects exposure at the concentrations of 100 and 300 mg/l. *P < 0.05, **P < 0.01 vs. control. Data are expressed as mean ± SD, n = 8 ∼ 9.
3.6. Effects of SAs on CAM model Having shown the capacity for increasing the number and function promotion of EPCs, we then assessed whether SAs would augment vessel formation in CAM model. To do this, fertile 8-dayold chicken eggs were used to prepare CAM model. After exposure to SAs for 72 h at concentrations of 100 and 300 mg/l, vessel density was increased significantly compared to control (2.98 ± 0.39% and 3.07 ± 0.36%, respectively, vs. 2.47 ± 0.35%) (Fig. 7). 4. Discussion and conclusion
Fig. 5. Incubation of rat EPCs with SAs for 24 h increased the number of adhesiveness cells at 30 min dose-dependently. *P < 0.05, ***P < 0.001 vs. control. Data are expressed as mean ± SD, n = 5.
In the past, the vascularization of adult tissue was considered to occur via angiogenesis, the sprouting of new vessels from existing ones. It relies on the proliferation, migration, and remodeling of fully differentiated endothelial cells (Ausprunk and Folkman, 1977). However, the mature ECs have limited regenerative capability, recent advances in vascular biology have led scientists to revisit this conventional picture of angiogenesis, with the discovery of circulating EPCs having the capacity to home in on sites of endothelial injury (Asahara et al., 1997; Hill et al., 2003), it is now believed that a combination of angiogenesis and a modified type of vasculogenesis contributes to neovascularization. With evidence suggesting EPCs incorporation into the vasculature, EPCsdependent neovascularization in adults may represent a third means of blood vessel formation, paralleling developmental vas-
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culogenesis in the embryo. EPCs may serve as the substrate for new vessel formation. Accumulating evidence suggests that bonemarrow-derived EPCs promote endothelial repair and contribute to ischemia-induced neovascularization. Gurtner’s group demonstrated that postnatal vasculogenesis is initiated primarily as a response to local tissue ischemia. Ischemia release growth factors/cytokines, such as VEGF, which subsequently mobilize EPCs from the bone-marrow. Local tissue hypoxia alters the vascular endothelium in ischemic tissue to arrest EPCs in regions where neovascularization is needed. Adherent EPCs egress into tissue and are themselves exposed to local tissue hypoxia. Over the next few days, these hypoxic conditions stimulate EPCs proliferation and the organization of cell clusters. After 14 days of injury, the increasing pool of EPCs form cordlike vascular structures. Soon after, vascular cords newly formed from bone-marrow-derived cells canalize and connect to existing vasculature (Tepper et al., 2005). CAD and its risk factors, such as diabetes, hypercholesterolemia, hypertension and smoking, are associated with a reduced number and impaired functional activity of circulating EPCs. Moreover, initial data suggest that reduced EPCs levels are associated with endothelial dysfunction and an increased risk of cardiovascular events, compatible with the concept that impaired EPCs-mediated vascular repair promotes progression of vascular disease (Vasa et al., 2001). Therefore, the augmentation of EPCs numbers and functions by pharmacological modulation may be a novel strategy to improve neovascularization after ischemia. In the present experiments, we demonstrated for the first time that SAs could augment EPCs numbers and promote EPCs migration, adhesion and in vitro vasculogenesis capacity. Besides, SAs could also enhance angiogenesis in CAM model. The use of CAM was one of the proposed alternatives of testing models to mimic human tissue with several advantages including simplicity, rapidity, sensitivity, ease of performance, and cost-effectiveness (Saw et al., 2008). Given the well-established role of EPCs participating in neovascularization and reendothelialization, the results from this study thus indicate that SAs may have utility for therapeutic postnatal vasculogenesis of ischemic tissue, limiting neointimal formation and ultimately the occlusion of diseased vessels in part by enhancing the mobilization and homing of EPCs after vascular injury, contribute to the clinical benefit of SM therapy in patient with CAD. However, the mechanisms whereby SAs alters number and function of EPCs will have to be determined in more detail. In particular, the impact of SAs on direct incorporation of EPCs into the endothelial monolayer and the release of paracrine mediators by EPCs remain to be further explored.
Acknowledgement This research was supported by the National Natural Science Foundation of China (NO. 30830118). References Asahara, T., Murohara, T., Sullivan, A., Silver, M., van der Zee, R., Li, T., Witzenbichler, B., Schatteman, G., Isner, J.M., 1997. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964–967. Asahara, T., Masuda, H., Takahashi, T., Kalka, C., Pastore, C., Silver, M., Kearne, M., Magner, M., Isner, J.M., 1999. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circulation Research 85, 221–228. Ausprunk, D.H., Folkman, J., 1977. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvascular Research 14, 53–65. Celermajer, D.S., 1997. Endothelial dysfunction: does it matter? Is it reversible? Journal of the American College of Cardiology 30, 325–333. Chang, P.N., Mao, J.C., Huang, S.H., Ning, L., Wang, Z.J., On, T., Duan, W., Zhu, Y.Z., 2006. Analysis of cardioprotective effects using purified Salvia miltiorrhiza extract on isolated rat hearts. Journal of Pharmacological Sciences 101, 245–249. Choi, J.H., Kim, K.L., Huh, W., Kim, B., Byun, J., Suh, W., Sung, J., Jeon, E.S., Oh, H.Y., Kim, D.K., 2004. Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure. Arteriosclerosis, Thrombosis, and Vascular Biology 24, 1246–1252. Eizawa, T., Ikeda, U., Murakami, Y., Matsui, K., Yoshioka, T., Takahashi, M., Muroi, K., Shimada, K., 2004. Decrease in circulating endothelial progenitor cells in patients with stable coronary artery disease. Heart 90, 685–686. Hill, J.M., Zalos, G., Halcox, J.P., Schenke, W.H., Waclawiw, M.A., Quyyumi, A.A., Finkel, T., 2003. Circulating endothelial progenitor cells, vascular function, and caridovascular risk. The New England Journal of Medicine 348, 593–600. Ji, X., Tan, B.K., Zhu, Y.C., Linz, W., Zhu, Y.Z., 2003. Comparison of cardioprotective effects using ramipril and DanShen for the treatment of acute myocardial infarction in rats. Life Sciences 73, 1413–1426. Saw, C.L., Heng, P.W., Liew, C.V., 2008. Chick chorioallantoic membrane as an in situ biological membrane for pharmaceutical formulation development: a review. Drug Development and Industrial Pharmacy 34, 1168–1177. Shi, C.S., Huang, H.C., Wu, H.L., Kuo, C.H., Chang, B.I., Shiao, M.S., Shi, G.Y., 2007. Salvianolic acid B modulates hemostasis properties of human umbilical vein endothelial cells. Thrombosis Research 119, 769–775. Tepper, O.M., Capla, J.M., Galiano, R.D., Ceradini, D.J., Callaghan, M.J., Kleinman, M.E., Gurtner, G.C., 2005. Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow-derived cells. Blood 105, 1068–1077. Vasa, M., Fichtlscherer, S., Aicher, A., Adler, K., Urbich, C., Martin, H., Zeiher, A.M., Dimmeler, S., 2001. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circulation Research 89, El–E7. Walter, D.H., Rittig, K., Bahlmann, F.H., Kirchmair, R., Silver, M., Murayama, T., Nishimura, H., Losordo, D.W., Asahara, T., Isner, J.M., 2002. Statin therapy accelerate reendothelialization. Circulation 105, 3017–3024. Zhang, H.S., Wang, S.Q., 2006. Salvianolic acid B from Salvia miltiorrhiza inhibits tumor necrosis factor-␣(TNF-␣)-induced MMP-2 upregulation in human aortic smooth muscle cells via suppression of NAD(P)H oxidasederived reactive oxygen species. Journal of Molecular and Cellular Cardiology 41, 138–148.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Pro-angiogenic actions of Salvianolic acids on in vitro cultured endothelial progenitor cells and chick embryo chorioallantoic membrane model Yu-Juan Li, Chang-Ling Duan 1 , Jian-Xun Liu ∗ , Yong-Gang Xu China Academy of Chinese Medical Sciences, Xiyuan Hospital, Beijing, PR China
a r t i c l e
i n f o
Article history: Received 27 April 2010 Received in revised form 17 June 2010 Accepted 18 July 2010 Available online 24 July 2010 Keywords: Endothelial progenitor cells Salvia miltiorrhiza Salvianolic acids Coronary artery disease
a b s t r a c t Aim of the study: Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely-used Chinese herbal medicines in clinical practice. In this study we aimed to demonstrate the pro-angiogenic effects of Salvianolic acids (SAs) to treat illnesses such as ischemic cardiovascular diseases, the main active components of aqueous extract of SM. Materials and methods: To do this, new-born rat SD spleen mononuclear cells were isolated and endothelial progenitor cells (EPCs) were expanded (not more than 24 h) SD. Then the pro-angiogenic activities of SAs were evaluated on in vitro cultured EPCs and chick embryo chorioallantoic membrane (CAM) model. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein (DiI-acLDL) and fluorescein Ulex Europaeus agglutinin-1 (FITC-UEA-1), and then viewed by laser scanning confocal microscope (LSCM) to confirm EPCs lineage. EPCs identification was also tested by ultrastructural analyses. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay, transwell chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Results: EPCs phenotype was confirmed by the presence of double positive cells for DiI-acLDL uptake and lectin binding and identification of Weibel–Palade body in cytoplasm by ultrastructural analyses. Incubation of EPCs with SAs increased the number of EPCs and promoted EPCs migratory, adhesive and in vitro vasculogenesis capacity. SAs also promoted angiogenesis as evidenced by CAM model. Conclusions: The results of the present study suggest that SAs may have utility for therapeutic postnatal vasculogenesis of ischemic tissue, contributing to the clinical benefit of SM therapy in patients with coronary artery disease. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The endothelium plays a pivotal role in cardiovascular homeostasis, including the regulation of vascular growth and neovascularization in response to ischemia (Celermajer, 1997). Mature endothelial cells (ECs), however, have limited regenerative capacity. Therefore, there is increasing interest in the effect of pharmacological approaches, to maintain structural and functional integrity of the endothelium, in part by promoting endothelial repair and preventing endothelial cell apoptosis. The description 10 years ago of putative endothelial progenitor cells (EPCs) that could mobilize from bone-marrow to participate in neovascularization at sites of ischemia was enthusiastically greeted because of the potential they hold for cardiovascular regeneration (Asahara et al., 1997). In this study, selected circulating CD34+ cells were
∗ Corresponding author. Tel.: +86 1062835601. 1 Equal contribution to the first author. 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.07.040
shown to differentiate into mature ECs ex vivo and to contribute to neovascularization in hindlimb ischemia. EPCs in peripheral blood function in the search for and repair of endothelial damage, indicating that dysfunction of EPCs results in impaired vascular repair. Importantly, several cardiovascular risk factors reduce circulating numbers and impair functional activity of circulating EPCs, suggesting a loss of endogenous endothelial repair capacity in patients at high risk for cardiovascular events. The number of peripheral blood EPCs is decreased in patients with cardiovascular risk factors such as hypertension (Hill et al., 2003; Vasa et al., 2001), and EPCs become dysfunctional in certain patients (Choi et al., 2004; Eizawa et al., 2004). Therefore it is of great importance to find the agents which are capable of promoting mobilization and functional activity of EPCs. Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely-used Chinese herbal medicines in clinical, containing phenolic compounds and potent antioxidant properties (Ji et al., 2003). SM had demonstrated its effectiveness in reduction of infarct size and mortality rate in rats with acute myocardial
Y.-J. Li et al. / Journal of Ethnopharmacology 131 (2010) 562–566
infarction (MI). The antioxidant effect and possible feature of angiogenesis of SM had been suggested as the main factor in protecting ischemic myocardium. Salvianolic acids (SAs), as the main active components of aqueous extract of SM, exhibit potential effects to prevent myocardial injury induced by infarction in clinical and animal experiments (Chang et al., 2006; Shi et al., 2007; Zhang and Wang, 2006). We hypothesized whether SAs could increase EPCs number and enhance functions, thus accelerating endothelial repair process, which contributes to improve the clinical symptoms and prognosis of patients with coronary artery diseases (CAD). To test this hypothesis, we studied the effects of SAs on EPCs and their corresponding effective doses. 2. Methods 2.1. Culture of EPCs For preparing spleen tissue homogenates, the whole organ was placed in cold PBS. The tissue was minced and immediately homogenized. Mononuclear cells (MNCs) were isolated by density gradient centrifugation using Histopaque-1.083 solution and washed with M199 (Gibco). Buffy coat MNCs were resuspended in endothelial growth medium (EGM-2) (lonza) consisting of endothelial basal medium, 5% fetal bovine serum, hEGF, VEGF, hFGF-B, IGF-1, ascorbic acid. Cells were seeded onto six-well tissue culture plates (5 × 106 cells per well) precoated with fibronetion (FN) and incubated at 37 ◦ C with 95% air and 5% CO2 . Under daily observation, first media change was performed ≈4 days after plating. Thereafter, media were changed every 3 days. 2.2. Cellular staining Fluorescent chemical detection of EPCs was performed on attached MNCs after 7 days in culture. Direct fluorescent staining was used to detect dual binding of FITCUlex europaeus agglutinin (UEA)-1 (Sigma) and 1-dioctadecyl3,3,3,3-tetramethylindocarbocyanine (DiI)-acetylated low-density lipoprotein (acLDL; Molecular Probe). Cells were first incubated with 2.5 g/ml acLDL at 37 ◦ C for 1 h and later fixed with 4% paraformaldehyde for 15 min. After washing, the cells were reacted with UEA-1 (10 g/ml) for 1 h. After the staining, samples were viewed with a laser cofoncal scan microscope (Zeiss). Cells demonstrating double positive fluorescence were identified as differentiating EPCs (Vasa et al., 2001). 2.3. Electron microscopy characterization of EPCs Cells were fixed with 0.1 mol/l PBS (pH 7.4) containing 2.5% glutaraldehyde at room temperature for 120 min. After washing with 0.1 mol/l cacodylate buffer (pH 7.4) and post-fixing for 10 min at room temperature with 0.1 mol/l PBS containing 1% (w/v) OsO4 , cells were then washed with 0.1 mol/l PBS and dehytrated by differing concentrations acetone and processed for embedding. Sections were analysed with a Jem1400 electron microscope. 2.4. Cell proliferation assay The effect of SAs on EPCs proliferation was determined by MTT assay. After confluence, EPCs were digested with 0.25% trypsin and then cultured in 96-well plate with 0.5% FBS medium, to which was added SAs with final concentrations ranging from 0.3 to 30 mg/l. 0.5% FBS medium served as a control. After being cultured for 24 h, 10 l MTT (5 g/l) was supplemented to the medium and incubated for 4 h. Then the supernatant was discarded and 150 l dimethyl sulfoxide was added. After shaking for 10 min, the OD value was measured at 490 nm.
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2.5. Cell migration assay EPCs migration was analysed using 24-well transwell chambers (Costar). Briefly, transwell chambers equipped with a 6.5 mmdiameter polyester membrane (pore size 8 .0 m) were used. 2 × 104 EPCs suspended in 100 l of medium containing 0.5% FBS without VEGF were seeded in the top chamber, and culture medium (500 l) supplemented with 0.5% FBS and differring concentrations SAs together with 50 g/l VEGF (peprotech Asia) was placed in the lower chamber. The transwell chamber was then incubated for 6 h at 37 ◦ C in a humidified incubator with 5% CO2 to render EPCs able to migrate through the membrane. After the incubation period, nonmigrated EPCs on the upper side of the membrane were scrape-off with a rubber. The membranes were then fixed with methanol for 10 min and stained with 0.1% crystal violet. Migrated EPCs attached to the lower side of the membrane were counted in three randomly selected microscopic fields (100×) in each chamber, and the average number of migrated cells were calculated. All experiments were performed in 5 wells, and data were expressed as the number of migrated cells/fields. 2.6. Cell adhesion assay After 1 day of incubation with SAs at concentrations ranging from 0.3 to 30 mg l−1 , EPCs were washed with PBS and gently detached with 0.25% trypsin in PBS. After centrifugation and resuspension in 0.5% FCS basal complete medium, identical cell numbers were placed onto FN-coated culture plate and incubated for 30 min at 37 ◦ C. Adherent cells were counted by independent blinded investigator (Walter et al., 2002). 2.7. In Vitro tube formation on matrigel plate Matrigel (Becton Dickinson Labware) basement membrane matrix was added to 96-well plate. After 0.5 h of incubation at 37 ◦ C to allow the matrix solution to solidify, 2 × 104 cells/ml were replated on top of the solidified matrix solution, EPCs were grown with SAs or vehicle control, and incubated at 37 ◦ C for 6 h. Tubule formation was inspected under an inverted light microscope at 400× magnification. Four representative fields were taken and the average number of tubules/400× field was determined. 2.8. CAM assay Fifty fresh fertile chicken eggs aged 8 days were obtained from a commercial source (Beijing Merial Vital Laboratory Animal Technology Co., Ltd). Eggs weighing from 55 to 65 g were incubated under standard conditions (MCO175 incubator, Sanyo, Japan; 37.5 ◦ C; 60% RH). On day 9 of the incubation, the fertile eggs were randomly allocated into five groups. Fertilized eggs are first disinfected with 70% alcohol, a window opening is punctured at the blunt end of the egg facing upwards using sterilized forceps. The eggshell above the air cell was removed and the shell membrane attached to it, was cut-off for further examination. Normal development was verified and embryos with malformations or dead embryos were excluded. CAMs (10 eggs per group) were treated as described as follows: (1) overlaying them with 20 mm2 sterilized filter discs that had been loaded with 10 l of PBS (negative control), 10 l of PBS containing 11 ∼ 300 mg/l of SAs. The window was sealed with tape and the eggs were returned to the incubator. After incubation for an additional 72 h, all samples were immediately fixed in methanol and acetone (1:1) for at least 15 min, then they were photographed using a stereomicroscope equipped with a camera and image analysis system. The angiogenic response was evaluated by counting the vessel density converging toward the filter discs.
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Fig. 1. (A) Representative EPCs binding of UEA-1. (B) Representative EPCs of uptake of acLDL. (C) Representative.
2.9. Statistical analysis All data are presented as mean ± SD. Differences between group means were assessed by an unpaired Student’s t test for single comparisons and by ANOVA for multiple comparisons. Values of P < 0.05 were considered significant. 3. Results
dose-dependently, with a maximal effect achieved at 30 mg/l (control vs. 30 mg/l, P < 0.001) (Fig. 5). 3.5. Effects of SAs on EPCs in vitro vasculogenesis Recent studies have demonstrated that circulating EPCs home into ischemic sites and differentiate into endothelial cells in situ (Asahara et al., 1999) in a manner consistent with a process termed vasculogenesis. Here in vitro tubulogenesis assay was used to inves-
3.1. Characterization of EPCs EPCs were characterized as adherent cells double positive for DiI-acLDL-uptake and lectin binding by using LSCM (Fig. 1). They were further documented by demonstrating Weibel–Palade body in cytoplasm (Fig. 2). 3.2. Effect of SAs on EPCs prolifertion The effect of SAs on EPCs proliferation activity was assayed using a MTT assay (Fig. 3). EPCs numbers showed a marked increase when they were cultured in medium supplimented with SAs at concentrations of 3 and 30 mg/l (P < 0.05, <0.05). 3.3. Effects of SAs on EPCs migration in response to VEGF The effects of SAs on EPCs migration were analysed in transwell chamber assay. Migrated cells were profoundly enhanced exposed to SAs at 3 and 30 mg/l than control (P < 0.001, <0.001) (Fig. 4). 3.4. Effects of SAs on EPCs adhesiveness To study the possibility that SAs promote adhesiveness of EPCs, EPCs were incubated with SAs for 24 h. After replating on 96-well plates, EPCs exposed to SAs exhibited a significant increase in the number of adhesiveness cells at 30 min. The increase occurred
Fig. 2. Representative transmission electron microscopy images of EPCs (25,000×), arrow shows the W–sP body.
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Fig. 3. MTT assay of rat EPCs in response to SAs. A moderate dose-dependent mitogenic response to SAs was observed when incubated with cultured EPCs for 24 h. *P < 0.05 vs. control. Data are expressed as mean ± SD, n = 5.
Fig. 4. Migration assay of rat EPCs in response to SAs. Migratory effect was augmented by SAs treatment in a dose-dependent manner. ***P < 0.001 vs. control. Data are shown as mean ± SD, n = 5.
tigate the capacity of EPCs to participate in neovascularization, which is the most important activity of EPCs. The responsiveness of tubulogenesis assay to SAs is depicted in Fig. 6. Addition of 30 mg/l SAs increased tubule formation significantly (81 ± 8 vs. control 38 ± 8, P < 0.001).
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Fig. 6. Angiogenic responsiveness of angiogenesis assay. Tubule number increased when exposed to 30 mg/l SAs. ***P < 0.001 vs. control. Data are expressed as mean ± SD, n = 5.
Fig. 7. Effects of SAs on CAM model. SAs showed pro-angiogenic effects exposure at the concentrations of 100 and 300 mg/l. *P < 0.05, **P < 0.01 vs. control. Data are expressed as mean ± SD, n = 8 ∼ 9.
3.6. Effects of SAs on CAM model Having shown the capacity for increasing the number and function promotion of EPCs, we then assessed whether SAs would augment vessel formation in CAM model. To do this, fertile 8-dayold chicken eggs were used to prepare CAM model. After exposure to SAs for 72 h at concentrations of 100 and 300 mg/l, vessel density was increased significantly compared to control (2.98 ± 0.39% and 3.07 ± 0.36%, respectively, vs. 2.47 ± 0.35%) (Fig. 7). 4. Discussion and conclusion
Fig. 5. Incubation of rat EPCs with SAs for 24 h increased the number of adhesiveness cells at 30 min dose-dependently. *P < 0.05, ***P < 0.001 vs. control. Data are expressed as mean ± SD, n = 5.
In the past, the vascularization of adult tissue was considered to occur via angiogenesis, the sprouting of new vessels from existing ones. It relies on the proliferation, migration, and remodeling of fully differentiated endothelial cells (Ausprunk and Folkman, 1977). However, the mature ECs have limited regenerative capability, recent advances in vascular biology have led scientists to revisit this conventional picture of angiogenesis, with the discovery of circulating EPCs having the capacity to home in on sites of endothelial injury (Asahara et al., 1997; Hill et al., 2003), it is now believed that a combination of angiogenesis and a modified type of vasculogenesis contributes to neovascularization. With evidence suggesting EPCs incorporation into the vasculature, EPCsdependent neovascularization in adults may represent a third means of blood vessel formation, paralleling developmental vas-
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culogenesis in the embryo. EPCs may serve as the substrate for new vessel formation. Accumulating evidence suggests that bonemarrow-derived EPCs promote endothelial repair and contribute to ischemia-induced neovascularization. Gurtner’s group demonstrated that postnatal vasculogenesis is initiated primarily as a response to local tissue ischemia. Ischemia release growth factors/cytokines, such as VEGF, which subsequently mobilize EPCs from the bone-marrow. Local tissue hypoxia alters the vascular endothelium in ischemic tissue to arrest EPCs in regions where neovascularization is needed. Adherent EPCs egress into tissue and are themselves exposed to local tissue hypoxia. Over the next few days, these hypoxic conditions stimulate EPCs proliferation and the organization of cell clusters. After 14 days of injury, the increasing pool of EPCs form cordlike vascular structures. Soon after, vascular cords newly formed from bone-marrow-derived cells canalize and connect to existing vasculature (Tepper et al., 2005). CAD and its risk factors, such as diabetes, hypercholesterolemia, hypertension and smoking, are associated with a reduced number and impaired functional activity of circulating EPCs. Moreover, initial data suggest that reduced EPCs levels are associated with endothelial dysfunction and an increased risk of cardiovascular events, compatible with the concept that impaired EPCs-mediated vascular repair promotes progression of vascular disease (Vasa et al., 2001). Therefore, the augmentation of EPCs numbers and functions by pharmacological modulation may be a novel strategy to improve neovascularization after ischemia. In the present experiments, we demonstrated for the first time that SAs could augment EPCs numbers and promote EPCs migration, adhesion and in vitro vasculogenesis capacity. Besides, SAs could also enhance angiogenesis in CAM model. The use of CAM was one of the proposed alternatives of testing models to mimic human tissue with several advantages including simplicity, rapidity, sensitivity, ease of performance, and cost-effectiveness (Saw et al., 2008). Given the well-established role of EPCs participating in neovascularization and reendothelialization, the results from this study thus indicate that SAs may have utility for therapeutic postnatal vasculogenesis of ischemic tissue, limiting neointimal formation and ultimately the occlusion of diseased vessels in part by enhancing the mobilization and homing of EPCs after vascular injury, contribute to the clinical benefit of SM therapy in patient with CAD. However, the mechanisms whereby SAs alters number and function of EPCs will have to be determined in more detail. In particular, the impact of SAs on direct incorporation of EPCs into the endothelial monolayer and the release of paracrine mediators by EPCs remain to be further explored.
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