Enhancing amplification of late‐outgrowth endothelial cells by bilobalide

Abstract Transfusion of autologous late‐outgrowth endothelial cells (OECs) is a promising treatment for restenosis after revascularization. Preparing cells by in vitro amplification is a key step to implement the therapy. This study aimed to demonstrate that bilobalide, a terpenoid, enhances the OEC amplification. Human‐, rabbit‐ and rat OECs and a mouse femoral artery injury model were used. Expanding OECs used endothelial growth medium‐2 as the standard culture medium while exploring the mechanisms used endothelial basal medium‐2. Proliferation assay used MTT method and BrdU method. Migration assay used the modified Boyden chamber. Intracellular nitric oxide, superoxide anion, hydroxyl radical/peroxynitrite and H2O2 were quantified with DAF‐FM DA, dihydroethidium, hydroxyphenyl fluorescein and a H2O2 assay kit, respectively. Activated ERK1/2 and eNOS were tested with the Western blot. Bilobalide concentration‐dependently enhanced OEC number increase in vitro. Transfusion of bilobalide‐based human OECs into femoral injured athymia nude mouse reduced the intimal hyperplasia. Bilobalide promoted OEC proliferation and migration and increased the intracellular nitric oxide level. L‐NAME, a NOS inhibitor, inhibits but not abolishes OEC proliferation, migration and ERK1/2 activation. Bilobalide concentration‐dependently enhanced the eNOS Ser‐1177 phosphorylation and Thr‐495 dephosphorylation in activated OECs. Bilobalide alleviates the increase in hydroxyl radical/peroxynitrite, superoxide anion and H2O2 in proliferating OECs. In conclusion, nitric oxide plays a partial role in OEC proliferation and migration; bilobalide increases OEC nitric oxide production and decreases nitric oxide depletion, promoting the OEC number increase; Bilobalide‐based OECs are active in vivo. The findings may simplify the preparation of OECs, facilitating the implementation of the autologous‐OECs‐transfusion therapy.


| INTRODUCTION
Late-outgrowth endothelial cells (OECs) also known as bone marrow (BM)-derived ECs are a subtype of endothelial progenitor cells. OECs exist in many organs but are rare anywhere. [1][2][3] It is difficult to selectively mobilize sufficient OECs to the circulating blood and the diseased organs. We and other research groups have demonstrated that OECs directly participate in the re-endothelialization and by a paracrine pathway inhibit the migration and proliferation of vascular smooth muscle cells; transfusion of sufficient OECs soon after vascular injury may reduce neointima formation, 4-6 opening a new way to inhibit the restenosis process after revascularization procedures such as percutaneous balloon angioplasty, stent implantation or atherectomy. In the forementioned three studies, we transfused autologous OECs that were prepared by in vitro amplification to the experimental rabbits while the two other research groups delivered allogeneic OECs to the immunodeficient mice. Allogeneic OECs fails to prevent intimal hyperplasia in immunity-intact rats. 7 Expanding OECs is a key step to implement the autologous-OECstherapy. However, as we know, all the current expansion methods [1][2][3][4][5][6][7][8] have low efficiencies, and getting seed OECs demands a large amount of peripheral blood (PB) or BM. Hence, it is useful to enhance the OEC expansion. As OECs have endothelial nitric oxide synthase (eNOS) and can proliferate whereas the early-outgrowth endothelial progenitor cells lack eNOS and do not proliferate, 9,10 we hypothesized that nitric oxide is involved in OEC proliferation, and additives that can modulate the eNOS/nitric oxide pathway may accelerate the OEC amplification.
Bilobalide, a sesquiterpenoid having a 15-carbon skeleton (Fig-ure S1), is originally found in Ginkgo biloba leaves. Ginkgo biloba leaf is widely used in traditional Chinese medicine, and its standardized extract EGb 761 has been used worldwide because of multifaceted pharmacological benefits. 11 Bilobalide accounts for 2.6%-3.2% of EGb 761 and contributes to EGb 761's vasodilating effect. 12 Although there is no report of using bilobalide alone in clinic, animal experiments have attested that bilobalide increases cerebral blood flow 13 and decreases arterial blood pressure, especially the diastolic pressure. 14,15 An in vitro study using aorta ring strips reveals that the vasorelaxation of bilobalide could be reduced significantly by NOS inhibitors. 16 Bilobalide decreases the activity or expression of iNOS in some non-vascular cells or tissues. [17][18][19] Our second hypothesis was that bilobalide modulates eNOS/nitric oxide pathway, promoting the OEC proliferation.
This article reports our verification of the hypotheses. We find that nitric oxide plays a partial role in OEC proliferation and migration; bilobalide promotes eNOS activation and superoxide scavenging to increase intracellular nitric oxide, enhancing the OEC expansion; bilobalide-based OECs are active in vivo.

| Ethics
The procedures of the study received ethics approval from the Ethical Committee for Medical and Biological Research at Xi'an Jiaotong University (No. 2012-0061) on March 10, 2012. Five human blood donators are openly recruited healthy volunteers. They were clearly told the use of their blood samples before signed informed consent forms and drawn 50 mL blood each by a certified medical staff. All efforts were made to reduce animal suffering and the number of animals used. All animals used in the study received care according to the Guide for the Care and Use of Laboratory Animals (Washington DC: National Academy Press, 1996), and was approved by the Animal Administrative Committee of Xi'an Jiaotong University.

| Amplification of OECs
Obtaining rabbit PB and BM and from them isolating mononuclear cells (MNCs) refer to our previous report. 4 Human PB MNCs were isolated in the same way. Rat BM was harvested from the femurs and tibias of male Sprague-Dawley rats (250-280 g, supplied by the Laboratory Animal Center of Xi'an Jiaotong University) killed with inhalation of CO 2 .
MNCs were plated on 100-mm culture dishes that had been coated with human fibronectin (for PB OECs) or rat collagen type I (for BM OECs), and were cultured for over 2 weeks in endothelial growth medium-2 (EGM-2, Lonza-BioWhittaker, Walkersville, MD, USA) at 37°C, letting the included OECs increased while other cell types disappeared.
The medium was changed every other day. Cells were subcultured when they approached 90% confluence. For bilobalide groups, bilobalide (0.1-10 lmol/L; Sigma-Aldrich, St Louis, MO, USA) was added into EGM-2. Bilobalide was dissolved in DMSO and diluted with PBS beforehand. The final concentration of DMSO was below 0.05% (v/v).

| Mouse femoral artery injury and treatment by
OECs or/and bilobalide Male BALB/c nude mice weighing 34-41 g (Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China) were anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg).
Transluminal mechanical injury of the femoral artery was performed according to the literature reports. 5,19,20 Briefly, the left femoral artery and a branch between the rectus femoris and vastus medialis muscles were exposed and looped proximally and distally with silk suture. Transverse arteriotomy was performed in the exposed muscular branch artery, through which a straight spring wire (0.38 mm diameter) was inserted into the femoral artery. The wire was slowly push-and-pulled for 4 times and left in place for 1 minute to denude and dilate the artery, then was removed and the suture looped at the proximal portion of the muscular branch artery was secured.  Related details refer to our previous report. 4
For N G -nitro-L-arginine methyl ester (L-NAME) groups, L-NAME To each well was added 200 lL DMSO, followed by shaking for 10 minutes, and detection of the OD by a microplate spectrophotometer (Bio Rad) set to a wavelength of 490 nm.

| Migration assay
The modified Boyden chamber assay 22 was used. Briefly, transwell inserts (Costar Transwell membrane, 6.5 mm diameter, 12.0 lm pore size) were placed in 24-well dishes. Rabbit PB OECs were plated on the upper wells (5 9 10 4 cells/well) to culture for 24 hours in EBM-2 containing FBS (5%) and stromal cell-derived factor-1a (SDF-1a, 100 ng/ mL; Sigma-Aldrich). Adding bilobalide and L-NAME into the media of related groups refers to the above description. Cells that migrated to the underside of the membrane were Hoechst-stained for counting. Bilobalide did not alter the OEC characteristics. For example, the PB OECs that were amplified in the presence of 10 lmol/L bilobalide have cobblestone shape when they are at adhesion state ( Figure 1A), can take in LDL and bind agglutinin ( Figure 1B Although there is no difference in the intima area and media area between the OECs and OECs + bilobalide groups, the lumen area of OECs + bilobalide group is larger than that of OECs alone group, conforming bilobalide's vasodilating effect.

| Bilobalide promotes OEC proliferation and increases intracellular nitric oxide level
BrdU incorporation assay and cell viability measurement by MTT assay, respectively, are often used direct and indirect methods for attesting cell proliferation. In this study, MTT assay showed that HUVECs and rabbit PB OECs increased their numbers to

| Bilobalide promotes OEC eNOS activation
Late-outgrowth endothelial cells constitutively express eNOS and iNOS. 10 We used siRNA to decrease rat BM OECs expressing eNOS and iNOS; the decrease rates were 62.4% and 67.2%, respectively. 3.6 | Quenching reactive oxygen species contributes to bilobalide enhancing OEC nitric oxide Nitric oxide reacts with O 2 À • forming ONOO À . 23 Bilobalide has antioxidant potential in a variety cell types because of promoting antioxidase expression. 18,19,24,25 We used rabbit PB OECs to dissect whether bilobalide quenching reactive oxygen species (ROS) plays a role in its enhancing nitric oxide level in proliferating OECs. As shown in Figure 8,

| Bilobalide promotes OEC migration
As shown in Figure 9,  Late-outgrowth endothelial cells have eNOS and iNOS. 10 Bilobalide decreases iNOS expression in some cell types. [17][18][19] The effect of bilobalide on eNOS remains unclear so far. Our present data excluded bilobalide increasing OEC's eNOS-and iNOS expression.
The eNOS is regulated activity by phosphorylation at multiple sites.
Two most thoroughly studied sites are activation site Ser-1177 and inhibitory site Thr-495. 32 Multiple stimuli such as growth factors and fluid shear stress activate eNOS by Ser-1177 phosphorylation 33 Bilobalide has antioxidant potential through promoting antioxidase expression in a variety of cell types. 18,19,24,25 We failed to get a special reagent for indication of intracellular ONOO À but HPF, a fluorescence probe reactive to OH • and ONOO À . Our present data showed that bilobalide pre-treatment limited the increase of OH • / ONOO À , O 2 À • and H 2 O 2 in proliferating OECs, implying bilobalide F I G U R E 8 Effect of bilobalide on hydroxyl radical/peroxynitrite, superoxide anion and hydrogen peroxide in proliferating lateoutgrowth endothelial cells (OECs). Rabit PB OECs were stimulated with VEGF to induce proliferating. Data were detected at 5 min after stimulation. Bilobalide treatment means adding bilobalide (1 lmol/L) immediately after VEGF, pre-treatment means adding bilobalide 24 h prior to VEGF. Intracellular hydroxyl radical/peroxynitrite was determined by hydroxyphenyl fluorescein, and superoxide anion by dihydroethidium, hydrogen peroxide by a hydrogen peroxide kit. n = 3 per group. *P < .05 or .01 vs VEGF (À); #P < .01 vs VEGF (+) + bilobalide (À) F I G U R E 9 Effect of bilobalide on migration of rabbit PB OECs. The modified Boyden chamber assay was used. Additions of bilobalide and L-NAME refer to Figure 3. For each sample, migrated cells in 3 randomly chosen visual fields (9100) were counted and averaged. n = 6-7 per group. *P < .05 and **P < .01 vs 0 lmol/L bilobalide; #P < .01 vs none L-NAME scavenges ROS to reduce nitric oxide depletion by O 2 À • in activated OECs.
In summary, the present study demonstrates that nitric oxide plays a role in OEC proliferation and migration; bilobalide increases nitric oxide production and prolongs the lifespan of available nitric oxide to enhance OEC growth. The findings may facilitate in vitro expansion of OECs, which may simplify the preparation of OECs for the autologous-OECs-transfusion therapy.