Protective effects of tanshinone IIA on endothelial progenitor cells injured by tumor necrosis factor-α

Tanshinone IIA (Tan IIA) is a Traditional Chinese Medicine commonly used in Asian and Western countries for the prevention and treatment of cardiovascular disorders, such as atherosclerosis. Endothelial dysfunction and associated inflammatory processes have a critical role in the development of atherosclerosis. Endothelial progenitor cells (EPCs) have been demonstrated to be involved in certain aspects of the endothelial repair process. The present study aimed to investigate the putative protective effects of Tan IIA on EPCs injured by tumor necrosis factor-α (TNF-α). The potential effects of Tan IIA on TNF-α-stimulated EPC proliferation, migration, adhesion, in vitro tube formation ability and paracrine activity were investigated in the current study. The results indicated that TNF-α impaired EPC proliferation, migration, adhesion capacity and vasculogenesis ability in vitro as well as promoted EPC secretion of inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and soluble CD40 ligand (sCD40L). However, Tan IIA was able to reverse these effects. In conclusion, these findings demonstrated that Tan IIA may have the potential to protect EPCs against damage induced by TNF-α. Therefore, these results may provide evidence for the pharmacological basis of Tan IIA and its potential use in the prevention and treatment of early atherosclerosis associated with EPC and endothelial damage.


Introduction
Danshen is a type of Traditional Chinese Medicine, which has been commonly used in Asian and Western countries for the treatment of numerous diseases, including cerebrovascular and coronary artery diseases (1,2). Danshen contains aqueous and lipid soluble fractions. The two active hydrophilic components of Danshen are salvianolic acid B and danshensu, whereas tanshinone ⅡA (Tan ⅡA) and cryptotanshinone are the two lipophilic components (3). Among these components, Tan ⅡA serves as a marker component to exert therapeutic effects. Numerous studies in animal models and human patients have demonstrated that Tan ⅡA may be an effective antioxidant for protecting against atherosclerosis, the pathological basis for most clinical cardiovascular diseases (4,5). Endothelial dysfunction and associated inflammatory processes have been confirmed to have a critical role in the development of atherosclerosis (6). Therefore, maintaining the integrity of the vascular endothelium is essential for the prevention and treatment of early atherosclerosis.
Endothelial progenitor cells (EPCs) comprise a cell population that are able to circulate as well as proliferate and differentiate into mature endothelial cells (ECs). These cells do not express markers which are characteristic of mature endothelial cells and are not involved in lumen formation (7,8). Increasing evidence has suggested that EPCs have a role in the endothelial repair process through differentiation into mature ECs and the release of protective paracrine factors, including vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) (9). Therefore, improving EPC number and function through pharmacological modulation may be a novel strategy for atherosclerosis prevention and treatment.
Li et al reported that incubation of EPCs with salvianolic acids increased EPC number as well as enhanced the migratory, adhesive and vasculogenesis capacities of EPCs in vitro (10). Tan ⅡA is a primary active constituent of Danshen and is involved in mediating the beneficial actions of Danshen. However, there is little evidence for the effect of Tan ⅡA on EPC number and functions. In addition, tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine released in response to pathological conditions, is elevated in atherosclerosis and may promote its pathogenesis (11). TNF-α was demonstrated to reduce the proliferation, migration, adhesion and tube formation capacity of EPCs (12). However, the effect of Tan ⅡA on the functions of EPCs exposed to TNF-α remains to be elucidated. The present study aimed to examine the effects of Tan IIA on TNF-α-treated EPC number, adhesion, migration, tube formation capacity in vitro and paracrine function.

Materials and methods
Isolation and cultivation of EPCs. All experimental procedures involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (Bethesda, MA, USA) and the present study was approved by the Institutional Animal Care Committee of Zhejiang University. A total of 40 male Sprague-Dawley rats (6-7 weeks old; 200 g) were provided by the Laboratory Animal Center of Zhejiang Province (Hangzhou, China), were housed in ten laboratory animal cages and maintained at a controlled temperature (20-22˚C) and humidity (50-60%) with a 12 h light/dark cycle. The animals were provided with a standard diet and water ad libitum.
Expansion of rat bone marrow-derived EPCs was performed in vitro, as previously described (13,14). In brief, EPCs were obtained from the femurs of male Sprague-Dawley rats. Muscles and the connective tissue were detached, and the epiphyses were removed. Bone marrow was harvested by inserting an 18-gauge syringe needle into one end of the bone shaft and flushing the contents into a 60-mm culture dish consisting of endothelial basal medium-2 (EBM-2, Lonza, Basel, Switzerland) supplemented with 5% screened fetal bovine serum (FBS; Gibco Life Technologies, Carlsbad, CA, USA). Density gradient centrifugation for 20 min at 2,000 x g was then used to obtain mononuclear cells (MNCs) fraction. Cells were suspended in EBM-2 supplemented with 10% FBS and plated onto six-well plates (Corning, Inc., Tewksbury, MA, USA). Following 24 h, non-adherent cells were aspirated and transferred to separate plates for a further 24 h, following which the procedure was repeated again in order to rapidly remove adherent mature ECs and hematopoietic cells, which may be present in the aspirate. Non-adherent cells harvested at 48 h were used for subsequent experiments; these cells were cultured in EBM-2 supplemented with microvascular endothelial cell growth medium-2 (EGM-2 MV) single aliquots containing 10% FBS (50 ml), VEGF (50 ml), epidermal growth factor (EGF; 0.5 ml), fibroblast growth factor-2 (2 ml), insulin-like growth factor-1 (0.5 ml) and ascorbic acid (0.5 ml) (Lonza). Following 4 days in culture at 37˚C in 5% humidified CO 2 , non-adherent cells were removed by washing in phosphate-buffered saline (PBS; Keyi, Hangzhou, China) and media was replaced every 3 days for 14 days.
Migration assay. The migration rate of EPCs was determined using a Transwell ® chamber (Greiner; Monroe, NC, USA) with 8-µm pore filters. Trypsin (0.25%) was used to detach isolated cells. Centrifugation for 10 min at 1,000 x g was performed in order to harvest cells, which were then resuspended in 500 µl EBM-2 and counted. Subsequently, 2x10 4 EPCs were placed in the upper Transwell ® chamber. Serum-free EBM-2 with VEGF was placed in the lower compartment of the chamber and the chambers were incubated for 24 h at 37˚C in 5% humidified CO 2 . Following incubation, the lower side of the filter was washed with PBS and a cotton wool swab was used to remove the remaining cells on the upper face. Transwell ® filters were then fixed with 2% paraformaldehyde. EPCs were stained with 0.1% crystal violet solution (Bogoo) and cells migrating into the lower chamber were counted in five randomly selected microscopic fields (magnification, x200; BX51 microscope).
EPC adhesion assay. Following incubation with Tan ⅡA and TNF-α, EPCs were washed with PBS and 0.25% trypsin was used to gently detached cells. Centrifugation for 10 min at 1,000 x g was then performed and cells were resuspended in EBM-2, an equal number of cells were then replated onto fibronectin-coated culture dishes (Corning, Inc.) and incubated at 37˚C in 5% humidified CO 2 for 30 min. Once the non-adherant cells were washed away with PBS, the adherent cells were counted by independent blinded investigators in five randomly selected microscopic fields (magnification, x400; BX51 microscope).
In vitro tube formation assay. A Matrigel ® assay (EMD Millipore, Billerica, MA, USA) was used, according to the manufacturer's instructions, in order to assess endothelial tube formation in vitro. In brief, ECMatrix TM solution (frozen at -20˚C; EMD Millipore) was thawed on ice overnight, mixed with 10X ECMatrix TM diluent (EMD Millipore) and placed into a 96-well plate at 37˚C for 1 h to allow the matrix solution to solidify. Subsequently, 2x10 4 EPCs were harvested and replated onto the solidified matrix solution with 150 µl EBM-2. Cells were incubated for 18 h at 37˚C and then fixed with 2% paraformaldehyde. The lengths of enclosed tubes which formed within the network were measured from five randomly selected microscopic fields (magnification, x200; BX51 microscope). The experiment was repeated five times.
ELISA analyses. Following 7 days of EPC culture, adherent cells were collected, re-inoculated onto six-well culture dishes and cultured for a further 48 h, the supernatant was then discarded through aspiration and supplement-free EBM-2 was added. Tan ⅡA (0, 1, 5, 10 and 20 µM) was then added (200 µl/well) and incubated for 18 h, following which EPCs were treated with 10 ng/ml TNF-α per well and cultured for 6 h. EPCs without treatment served as the control group. The original medium was discarded and cells were re-incubated with fresh EBM-2 with no supplement

Statistical analysis.
Values are presented as the mean ± standard deviation. Differences between group means were assessed by one-way analysis of variance for multiple comparisons followed by the Least Significant Difference post hoc test using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference between values.

Characterization of EPCs.
MNCs isolated from rat bone marrow and cultured for 14 days, resulted in cells with a spindle-shaped, EC-like morphology (Fig. 1A). EPCs were confirmed to be adherent cells due to their double positive staining for DiLDL uptake and lectin binding, as determined using fluorescence microscopy ( Fig. 1B-D).

Effects of Tan ⅡA on proliferation of TNF-α-stimulated EPCs.
The effect of Tan ⅡA on EPC proliferation was assessed using the MTT assay (Fig. 2). TNF significantly reduced the proliferative activity of EPCs (74.59±5.05%) compared with that of the control (P<0.01). By contrast, this effect was reversed by Tan ⅡA in a dose-dependent manner, with significant increases at 10 and 20 µM (87.88±3.58% and 93.09±8.12%, respectively) compared with the TNF-α-treated cells (P<0.05).

Effects of Tan ⅡA on the adhesion of TNF-α-stimulated EPCs.
In order to determine whether Tan ⅡA altered the adhesion of cultured EPCs induced by TNF-α, EPCs were incubated with TNF-α (10 ng/ml) and Tan ⅡA (0, 1, 5, 10 and 20 µM) for 24 h and then replated onto fibronectin-coated dishes. As shown in Fig. 4, EPCs pre-exposed to TNF-α exhibited a

Effects of Tan ⅡA on tube formation of TNF-α-stimulated EPCs in vitro.
EPCs were incubated overnight in starvation medium and then stimulated for 24 h with TNF-α and Tan ⅡA in basal medium. The lengths of enclosed tubes which formed within the network were measured. As shown in Fig. 5, TNF-α significantly impaired EPC vasculogenesis in vitro (38.50±4.48%) compared with the control (P<0.01). Tan ⅡA restored EPC vasculogenesis ability in vitro in a dose-dependent manner following TNF-α treatment, with significant results at 10 and 20 µM Tan IIA (77.86±3.93 and 87.39±3.95%, respectively) compared with the TNF-α-treated cells (P<0.01).

Discussion
The vascular endothelium is located at the interface between blood and tissue and has a crucial role in the maintenance of vessel wall integrity (15). Numerous environmental factors have been reported to damage the structure and function of the endothelium, resulting in the development of various diseases (15). It has been confirmed that endothelial dysfunction has a critical pathogenic role in various diseases, including atherosclerosis, hypertension, diabetes and thrombosis (16). Endothelial dysfunction may induce subsequent pathogenic events, such as inflammation and thrombosis. It was reported that inflammation was involved at all stages of atherosclerosis, including lesion formation and plaque stability (17). Thus, it was suggested that endothelial dysfunction may be a major promoter of atherosclerosis (18). In order to prevent the pathogenesis and development of atherosclerosis, it is essential to repair endothelial dysfunction. Endothelial dysfunction occurs due to an imbalance between the severity of injury and the ability of the endothelium to repair (19). Increasing evidence has suggested that EPCs are involved in certain aspects of this repair process (20)(21)(22). In an animal study, EPCs were demonstrated to contribute to vessel formation through differentiation into mature ECs and incorporation into the vessel wall (20). Other studies have reported that accelerated re-endothelialization by EPCs effectively inhibited smooth muscle cell proliferation, migration and neointima formation, therefore preventing the initiation and development of the early stages of restenosis following vascular injury (21,22). EPCs have an essential role in the prevention of early atherosclerosis and the treatment of restenosis following angioplasty (23).
Tan ⅡA, a major lipid-soluble active compound of Danshen, was demonstrated to have anti-atherosclerotic and anti-inflammatory properties (24). Therefore, Tan ⅡA may be a promising candidate for the development of novel therapeutic strategies for the prevention and treatment of atherosclerosis (25). In vitro studies have indicated that Tan ⅡA may downregulate the expression of intercellular adhesion molecule-1 in TNF-α-induced human umbilical vein endothelial cells as well as inhibit the oxidation of low-density lipoprotein (26,27). Considering the close associations between EPCs and ECs, it was hypothesized that Tan ⅡA may exert anti-atherosclerotic properties through the protection of EPC function. The present study aimed to examine the effect of Tan ⅡA on TNF-α-treated EPC number, adhesion, migration, tube formation capacity in vitro and paracrine activity. To the best of our knowledge, the current study was the first report of the effects of Tan ⅡA on the function of EPCs stimulated by TNF-α.
The present study demonstrated that EPC proliferation, migration, adhesion capacity and vasculogenesis ability in vitro were impaired by 10 ng/ml TNF-α. TNF-α is a pro-inflammatory cytokine that is released in atherosclerosis and has been reported to promote the process of atherosclerosis (11). The results of the present study suggested that TNF-α may participate in the process of atherosclerosis by affecting EPC function; therefore, inhibiting these functions may be a promising therapeutic strategy for the treatment of atherosclerosis. The current study demonstrated that Tan ⅡA was able to significantly improve EPC function, as demonstrated by its capacity to increase EPC proliferation, migration and adhesion. These observations led to the subsequent investigation of the role of Tan ⅡA in promoting angiogenesis. A vasculogenesis assay in vitro demonstrated that Tan ⅡA upregulated vasculogenesis in a dose-dependent manner, with significant results at concentrations of 10 and 20 µM Tan IIA.
Increasing evidence has supported the involvement of inflammation in the pathogenesis and development of atherosclerosis (28,29). It was reported that the inflammatory responses mediated by cytokines, including MCP-1, IL-6 and sCD40L, were important in atherosclerosis (30). In the current study, the effects of Tan ⅡA on EPC secretion of MCP-1, IL-6 and sCD40L following stimulation by TNF-α, were investigated. The results demonstrated that TNF-α enhanced the release of MCP-1, IL-6 and sCD40L by EPCs. However, treatment with Tan ⅡA reduced the levels of IL-6, sCD40L and MCP-1 in the EPC supernatant.
MCP-1 is a pro-inflammatory chemokine, which has been reported to promote vascular inflammation and atherosclerosis (31,32). IL-6 is an inflammatory cytokine of circulation, the levels of which were shown to reactively rise with coronary heart disease; in addition, the IL-6 pathway was suggested to have a role in the process of atherosclerosis (33). CD40 and CD40 ligand activation are known to be important inflammatory signals in atherosclerosis (17). Previous in vivo and in vitro studies have demonstrated that Tan ⅡA exerts protective effects in atherosclerosis through decreasing the expression of CD40 (34,35). In the present study, the release of the pro-atherogenic chemokines MCP-1, IL-6 and sCD40L was markedly reduced by coculturing the cells with Tan ⅡA. Therefore, the present study demonstrated that compared with the control group, Tan ⅡA significantly improved cell function as well as reduced the protein levels of MCP-1, IL-6 and sCD40L in the EPC supernatant. These results suggested that Tan ⅡA may not only be used to reduce the damage of EPCs from TNF-α, but also to significantly enhance the anti-inflammatory ability of EPCs.
Of note, in the present study EPCs were cultured with MNCs derived from healthy rats in order to investigate the protective effects of Tan ⅡA on EPCs in the presence of TNF-α. The findings suggested that Tan ⅡA may exert therapeutic effects on endothelial dysfunction-associated diseases, such as atherosclerosis, through the protection of EPC function. However, in order to further study the anti-atherosclerotic effects of Tan ⅡA, future studies should use MNCs derived from atherosclerotic animals.
In conclusion, the results of the present study demonstrated that TNF-α impaired EPC proliferation, migration, adhesion capacity and vasculogenesis ability in vitro, as well as promoted EPC secretion of inflammatory cytokines, including MCP-1, IL-6 and sCD40L. In addition, it was revealed that Tan ⅡA was able to reverse these effects. Therefore, treatment with Tan ⅡA may have the potential to protect EPCs against damage induced by TNF-α. These results may provide evidence for the pharmacological basis of Traditional Chinese Medicine in the prevention and treatment of early atherosclerosis associated with EPC and endothelial damage.