Activation of ATP‐sensitive potassium channels facilitates the function of human endothelial colony‐forming cells via Ca2+/Akt/eNOS pathway

Abstract Accumulating data, including those from our laboratory, have shown that the opening of ATP‐sensitive potassium channels (KATP) plays a protective role in pulmonary vascular diseases (PVD). As maintainers of the endothelial framework, endothelial colony‐forming cells (ECFCs) are considered excellent candidates for vascular regeneration in cases of PVD. Although KATP openers (KCOs) have been demonstrated to have beneficial effects on endothelial cells, the impact of KATP on ECFC function remains unclear. Herein, this study investigated whether there is a distribution of KATP in ECFCs and what role KATP play in ECFC modulation. By human ECFCs isolated from adult peripheral blood, KATP were confirmed for the first time to express in ECFCs, comprised subunits of Kir (Kir6.1, Kir6.2) and SUR2b. KCOs such as the classical agent nicorandil (Nico) and the novel agent iptakalim (Ipt) notably improved the function of ECFCs, promoting cell proliferation, migration and angiogenesis, which were abolished by a non‐selective KATP blocker glibenclamide (Gli). To determine the underlying mechanisms, we investigated the impacts of KCOs on CaMKII, Akt and endothelial nitric oxide synthase pathways. Enhanced levels were detected by western blotting, which were abrogated by Gli. This suggested an involvement of Ca2+ signalling in the regulation of ECFCs by KATP. Our findings demonstrated for the first time that there is a distribution of KATP in ECFCs and KATP play a vital role in ECFC function. The present work highlighted a novel profile of KATP as a potential target for ECFC modulation, which may hold the key to the treatment of PVD.


Introduction
ATP-sensitive potassium channels (K ATP ), widely expressed in tissues and cells, participate in numerous cellular processes. Mounting evidence has confirmed that K ATP protect myocardium against ischaemia, regulate vascular smooth muscle contraction and are involved in the secretion of insulin from pancreatic b cells [1]. Recently, prevailing attention has been paid to the regulatory role of K ATP in endothelial cells (ECs). As the major components of the pulmonary vasculature, ECs have been widely reported to be impaired during the progression of pulmonary vascular diseases (PVD). Several studies have revealed that K ATP play a critical role in pulmonary ECs to shear stress and arterial vasomotion [2,3]. In mice, knockout of inward rectifier EPCs ameliorated and reversed monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) [7,8]. Endothelial progenitor cell-based cell therapy suppressed the development of atherosclerosis with a down-regulation of pro-inflammatory protein [9]. Although accumulating data have indicated that EPCs may provide a circulating pool of immature ECs for endothelium regeneration, little is known about the role of K ATP in the regulation of EPCs.
Endothelial colony-forming cells (ECFCs), one type of EPC, acquire a completely endothelial phenotype and are thought to be 'true' EPCs [10]. Endothelial colony-forming cells display high proliferative potentials and angiogenic ability both in vitro and in vivo [11,12]. Recently, ECFCs have been used as a therapeutic tool for vascular regeneration to ultimately maintain the integrity of vessels [13][14][15]. Many efforts are made to improve ECFC function, but few have proved effective. Endothelial colony-forming cells injected into peripheral circulation showed low percentage (10%) of cell retention and survival within the injury site [16,17]. Initial attempts with autologous ECFC also failed to achieve prospective outcomes for the deficiency in cell function. Therefore, rescue of ECFC function has been considered as a promising strategy for PVD therapies.
Although the contribution of K ATP on ECs has been confirmed before, it remains unclear whether K ATP is expressed in ECFCs, and what the role K ATP play in ECFC function. This study has determined for the first time that K ATP are expressed in human ECFCs and is composed of Kir6.1/6.2 and sulfonylurea receptor 2b (SUR2b). Furthermore, we also revealed that K ATP openers (KCOs) could promote ECFC proliferation, migration and angiogenesis by Ca 2+ /Akt/endothelial nitric oxide synthase (eNOS) signalling, which will improve our understanding on the respiratory biology of K ATP and open new perspectives for ECFC management.

Materials and methods
Chemicals Iptakalim (Ipt) was a gift from the Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences (Beijing, China). Nicorandil (Nico) and glibenclamide (Gli) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nico and Gli were diluted with dimethyl sulfoxide (DMSO), and Ipt was diluted with double-distilled H 2 O. The concentration of DMSO was controlled below 0.1% (vol/vol), and did not affect the biological viability of the culture cells.

Cell culture
Human ECFCs were cultured using a protocol approved by the First Affiliated Hospital of Nanjing Medical University. Human peripheral bloodderived ECFCs were isolated and cultured as described previously [18,19]. Briefly, human peripheral blood (20 ml) obtained from healthy adult donors with informed consent was fractionated via centrifugation in a Ficoll-Paque (Sigma-Aldrich) gradient. Isolated mononuclear cells were re-suspended in endothelial basal medium-2 (EBM-2) (Lonza, Walkersville, MD, USA), consisting of 10% foetal bovine serum (FBS), human epidermal growth factor (hEGF), human VEGF1, human fibroblast growth factor (hFGF), insulin-like growth factor 1 (IGF-1), ascorbic acid and heparin. Cells were diluted to a final concentration of 7.5 9 10 5 cells per ml and seeded into 2% fibronectin-coated (Sigma-Aldrich) six-well plates with approximately 2 9 10 6 cells per well. Medium was replaced after the first 24 hrs. Under daily observation, colonies of ECFCs were obtained after 14-28 days on average. Cells were used in their fourth to sixth passages.

Reverse transcription polymerase chain reaction
Total mRNA was extracted from ECFCs and HPAECs using the Qiagen RNeasy mini kit (Qiagen, Venlo, The Netherlands), according to the manufacturer's instructions. First-strand cDNA was synthesized using the Prime Script RT reagent kit (TakaraBio, Shiga, Japan) and amplified by Phusion DNA Polymerase (Thermo Fisher Scientific) in a 25 ll reaction mixture. Each cDNA was subjected to PCR amplification using gene-specific primers. The primer sequences for each of the K ATP subtypes and the actin housekeeping genes are listed in Table 1. PCR was conducted using a PCR system Master Cycler. Thermal cycling conditions for all PCR mixtures were as follows: initial denaturation for 30 sec. at 98°C; 40 cycles of 10 sec. at 98°C (denaturation), 30 sec. at 58°C (annealing) and 30 sec. at 72°C (extension); and a final extension for 10 min. at 72°C. PCR mixtures were subsequently separated and analysed on a 1.5% agarose gel. The mRNA expression of actin was utilized as an internal control. HPAECs and mouse brain were used as positive controls. Water was used as a negative control.

Confocal microscopy
In brief, after fixation and blocking, ECFCs were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS, blocked with 5% BSA for 1 hr at room temperature and incubated with antibodies against Kir6.1 (Alomone Labs), Kir6.2 (Abcam), SUR2b (Santa Cruz), CD31 (Santa Cruz) and VE-cadherin (Abcam) at 4°C overnight. After three washes with PBS, ECFCs were incubated with secondary antibodies as stated above for 1 hr at room temperature in the dark. DAPI was used for nuclei staining. Cells were visualized using an LSM 510 confocal microscope (Zeiss).

Cell proliferation assays
The incorporation of 5-ethynyl-2 0 -deoxyuridine (EdU) with the EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China) was used for detecting cell proliferation. All the procedures were performed according to the manufacturer's protocol. In brief, cells were pretreated with Ipt (10 À6 , 10 À5 and 10 À4 M) or Nico (10 À6 , 10 À5 and 10 À4 M) in EBM-2 containing 2% FBS for 24 hrs and then incubated with 50 lM EdU for 4 hrs before fixation, permeabilization and EdU staining. To investigate the interaction between KCOs and K ATP blockers on human ECFCs, Gli (10 À5 M) was added 30 min. prior to the addition of Ipt and Nico. The EdU incorporation rate was determined as the ratio of the EdU-positive cells (red cells) to the total Hoechst 33342-positive cells (blue cells). Five fields were randomly selected from each dish by fluorescence microscopy (DM2500; Leica, Wetzlar, Germany) and at least three dishes were counted per treatment.
Cell counting kit-8 (CCK8) assay was performed to further confirm the effect of KCOs on the proliferation of human ECFCs. Human ECFCs were seeded into a 96-well plate at a density of 10,000/well overnight. Then, cells were treated with Ipt (10 À6 , 10 À5 and 10 À4 M) or Nico (10 À6 , 10 À5 and 10 À4 M) with EBM-2 containing 2% FBS for 24 hrs. Cells were pre-incubated with Gli (10 À5 M) for 30 min. before adding Ipt and Nico. Cell proliferation was analysed with CCK-8 kits (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacture's protocol. Absorbency was measured at 450 nm by a microplate reader (Thermo Scientific, Millipore, Billerica, MA, USA).

Cell migration
We used transwell migration chamber assays to examine ECFC migration. Migration assays were performed using a 24-well Transwell chamber (Corning, Corning, NY, USA). Cells were re-suspended in serum-free EBM-2 at 1 9 10 5 cells/ml and a 100-ll cell suspension was added to the upper chamber. Endothelial basal medium-2 containing 10% FBS in the presence or absence of different concentrations of Ipt or Nico (10 À6 , 10 À5 and 10 À4 M) were added into the lower chamber. Gli (10 À5 M) was added 30 min. before the addition of other drugs. The chamber was incubated for 24 hrs at 37°C. The filter was carefully removed and cells attached on the upper side were wiped off. Cells migrating through the filter and appearing on the lower side were fixed with 4% paraformaldehyde for 20 min. and stained with 5% crystal violet. We randomly selected five vision fields and counted cells using a phase-contrast microscope (Nikon, Tokyo, Japan) for statistical analysis [20].

In vitro tube formation on Matrigel plates
Endothelial colony-forming cell and HPAEC angiogenesis were evaluated by an in vitro tube formation assay. Ten microlitres of Matrigel (Becton-Dickinson, San Diego, CA, USA) basement membrane matrix was added to a u-slide angiogenesis plate (Ibidi, Martinsried, Germany). The slide was incubated at 37°C for 30 min. to allow the matrix solution to solidify. Cells were diluted to a final concentration of 1.5 9 10 5 cells per ml in basal medium containing 2% FBS. Fifty microlitres of cell suspension was seeded onto the surface of the u-slide well pre-coated with 10 ll Matrigel in the presence or absence of different concentrations of Ipt (10 À6 , 10 À5 and 10 À4 M) or Nico (10 À6 , 10 À5 and 10 À4 M). Gli (10 À5 M) was added 30 min. prior to the treatment of Ipt and Nico. After 4 hrs of incubation, we randomly chose four fields from an unbiased examiner in each plate. The length of complete tubes per unit area was calculated using Image-Pro Plus [20].

Statistical analysis
All data were shown as mean AE S.D. Comparisons between controls and samples treated with various drugs were analysed by one-way ANOVA. Least significant difference post hoc tests were conducted when significant effects were found. Statistical significance was defined as P < 0.05.

Results
Characterization of ECFCs derived from human peripheral blood Endothelial colony-forming cells were isolated and expanded ex vivo from adult peripheral blood. Most of the ECFC colonies appeared during 14-28 days. Initially, small colonies were observed; then, they rapidly expanded into large colonies. The cells showed characteristic endothelial 'cobblestone' morphology, providing easy preliminary identification under an optical microscope (Fig. 1A). Several experiments were conducted for further identification. FACS results showed that 99.5% of the isolated cells expressed the endothelial marker VEGFR-2 (CD309); a small percentage of cultured cells expressed CD34 (3%). Nevertheless, CD133, CD15, CD45 and CD115 were almost absent in the obtained cells, which were consistent with previous reports [21] (Fig. 1B). Additionally, the cells displayed an endothelial phenotype: they took up DiI-ac-LDL, bound to FITC-UEA-I (Fig. 1C), and stained positively for the endothelium-specific antigens CD31 and vWF (Fig. 1D).

Determination of K ATP expression in ECFCs
To determine the expression of K ATP in ECFCs, we performed reverse transcription polymerase chain reaction (RT-PCR) and western blotting for the associated mRNA and protein, using HPAECs and mouse brain as positive controls. The mRNA of Kir6.1, 6.2 and SUR2b was detected in human ECFCs, but not SUR1 and SUR2a (Fig. 2A). These subunits were further confirmed in protein level by western blotting. As shown in Figure 2B, the expression of Kir6.1, Kir6.2 and SUR2b was determined.

Subcellular localization of K ATP subunits in ECFCs
Confocal images were gained to identify the distribution of K ATP in ECFCs co-stained with an endothelium-specific marker and showed that Kir6.1 and Kir6.2 were expressed not only in the cell membrane but also in the cytoplasm (Fig. 2C). SUR2b was also detected in the cytoplasm and intracellular patterns of punctuated and filamentous staining were observed (Fig. 2C).

KCOs promoted ECFC proliferation, migration and angiogenesis
As shown in Figures 3-5, activation of K ATP by KCOs significantly promoted ECFC function such as cell proliferation, migration and angiogenesis. The data from EdU incorporation assay showed that the number of EdU+ cells was markedly elevated by Ipt (10 À4 M) and Nico (10 À4 M) (versus control group, P < 0.05) ( Fig. 3A and C). The effects of KCOs on ECFC proliferation was further substantiated by CCK8 assay. The data manifested that cell viability was elevated by Ipt (10 À4 M) and Nico (10 À4 M) (P < 0.05 versus control group) (Fig. 3D). As a novel KCO, the treatment of Ipt in promoting ECFC proliferation presented as good as that of Nico, as there was no significant difference between Ipt and Nico at the concentration of 10 À4 M.
Data from transwell migration assay indicated that KCOs notably improved ECFC migration with more permeation compared with control. Treatment with 10 À5 M Ipt and 10 À4 M Ipt could significantly facilitate the migration of ECFCs, whereas treatment with 10 À4 M Nico could markedly promote the ability of migration (Fig. 4A). Ipt was more effective than Nico that 10 À5 M Ipt had already indicated its work on promoting ECFC migration (Ipt 10 À5 M versus Nico 10 À5 M, P < 0.05); however, there was no difference between Nico and Ipt at the concentration of 10 À4 M.
Moreover, neovascularization is considered as one of the essential functions of ECFCs. Angiogenesis is recognized as the basic step of developing new capillaries in vivo. Using an in vitro model of angiogenesis, we found that KCO treatments facilitated the capacity of ECFCs and HPAECs to form capillary-like structures. Our findings showed that Ipt and Nico markedly promoted ECFC angiogenesis at 10 À4 M (versus control group, P < 0.05) (Fig. 5A), whereas Ipt at 10 À5 M and Nico at 10 À4 M notably improved HPAEC angiogenesis (versus control group, P < 0.05) (Fig. 5C).
Furthermore, ECFCs were pretreated with Gli (10 À5 M), a nonselective K ATP blocker [22], before the administration of KCOs. The results indicated that Gli almost completely abolished the effects of Ipt and Nico, but not affected the basal levels in ECFC proliferation, migration or angiogenesis (Figs 3B-D, 4B and 5B). Moreover, Gli reversed Ipt (10 À5 M) and Nico (10 À4 M) induced angiogenesis of HPAECs (Fig. 5D).

KCOs enhanced the phosphorylation of CaMKII, Akt and eNOS in ECFCs
Western blotting was performed to quantify the phosphorylation of intracellular signal transduction. KCO administration notably increased the phosphorylation of CaMKII, Akt and eNOS in ECFCs (Fig. 6A and  B). Pretreatment with Gil significantly reversed the enhancement elicited by KCOs, while no alteration was observed with Gli alone.

Discussion
Since first being isolated from peripheral blood [6], EPCs have gained increasing attention as a potential reparative cell therapy. Until now, the identification and characterization of EPCs have remained controversial. Based on surface markers and the culture methods used, circulating EPCs have been divided into three groups: colony-forming unit ECs (CFU-ECs), circulating angiogenic cells (CACs) and ECFCs [10,23]. Compared with 'early' EPCs, CFU-ECs and CACs, ECFCs are considered as 'late' EPCs or 'late' outgrowth ECs. Endothelial colony-forming cells are characterized by the absence of hematopoietic and myeloid markers, a hierarchy of proliferative potentials and the capacity for vascular formation [24]. All of these features imply that ECFCs are genuine EPCs. In the current study, we isolated ECFCs from adult peripheral blood. The FACS analysis results showed the expression of EC surface antigen VEGFR-2 but not CD133, CD14, CD45 and CD115 in ECFCs, indicating that no/minimal 'early' EPC contamination. However, only a A B Fig. 4 Effects of Ipt and Nico on ECFC migration. Ipt and Nico increased ECFC migration and Ipt was more effective than Nico at the concentration of 10 À5 M (A). The increase originated from KCOs could be abolished by Gli (B). Data were shown as mean AE S.D. of four independent experiments. Statistically significant differences: *P < 0.05 versus control group; &P < 0.05 Ipt 10 À5 M versus Nico 10 À5 M; #P < 0.05 Ipt group versus Ipt+Gli group; †P < 0.05 Nico group versus Nico+Gli group, scale bar: 50 lm. small percentage of cells expressed CD34. Although some studies have shown that ECFCs from peripheral blood express CD34, there is no evidence suggesting that CD34 is necessary for ECFCs [10].
Studies have identified the expression of potassium channels in ECFCs [25,26]. However, no data are available on whether there is a K ATP distribution on ECFCs and what role K ATP play in the regulation of ECFCs. K ATP were originally detected in the cardiovascular system in 1983 [27]. Serving as a molecular biosensor, K ATP provide a unique connection between cellular metabolic status and membrane excitability [28]. K ATP are a hetero-octameric complex consisting of a Kir6.x (Kir6.1, Kir6.2) pore and a regulatory SUR subunit (SUR1, SUR2a and SUR2b) [29]. K ATP can be assembled by diverse combinations of the Kir6 and SUR subunits, such as the combinations in skeletal muscle cells (SUR2a/Kir6.2), pancreatic b cells (SUR1/Kir6.2) [30] and coronary artery ECs (SUR2/Kir6.1/Kir6.2) [29]. In the present study, the composition of K ATP was shown to include Kir6.1, Kir6.2 and SUR2b. However, neither SUR1 nor SUR2a was detected in ECFCs. The results of confocal imaging revealed that K ATP were distributed in both the cellular membrane and the cytoplasm, which may suggest a contribution of K ATP in the function of subcellular organelles in ECFCs.
Studies have shown that distinct KCOs present diverse subtype selectivity. Classic KCOs include Nico and diazoxide, while Ipt is regarded as one of the novel agents. It has been shown that Nico is a hybrid agent with two pharmacologic actions, the opening of K ATP and the donation of nitrogen oxide, which specifically activates K ATP , consisting of Kir6.1 and SUR2b [31]. Because the expression of K ATP in ECFCs, composed of SUR2b/Kir6.1/Kir6.2, has been confirmed by our results, Ipt and Nico were used to investigate the role of K ATP in the modulation of ECFC function. As a new KCO, Ipt exhibits high selectivity for the SUR2b/Kir6.1 subtype in the previous study [32]. In the current study, both Ipt and Nico significantly increased cellular proliferation, migration and angiogenesis in ECFCs, which are involved in ECFC repair and neovascularization [33]. Moreover, Ipt as well as Nico presented notable superiority in promoting ECFCs proliferation and angiogenesis. However, higher efficiency of Ipt was showed for facilitating ECFC migration at the concentration of 10 À5 M compared with Nico. Although Nico has an additional potential for nitrate properties, few effect of nitrogen oxide was observed in ECFC regulation, specifically the inhibition of KCO effects by the K ATP inhibitor. Taken together, our results suggest that various KCOs exhibit diverse effects on the modulation of ECFCs.
It is well known that KCOs have protective effects on pulmonary vessels in many pathological conditions, such as hypoxia and ischaemia. In vivo studies have shown that KCOs are capable of reversing hypoxia-induced pulmonary arterial remodelling and reducing right ventricular hypertrophy [34]. KCOs have been shown to improve the function of smooth muscle cells (SMCs) by inducing membrane hyperpolarization and relaxing vessels [35]. KCOs were also shown to have beneficial effects in ECs, including decreased hypoxia-induced apoptosis, promotion of nitrogen oxide release and inhibition of endothelin-1 (ET-1) synthesis [36,37]. In our work, we demonstrated  for the first time that KCOs promoted the function of ECFCs, which may uncover a new mechanism involved in the pulmonary vascular protection caused by KCOs.
Accumulating data suggest that ECFCs are a new candidate for cell-based therapies and a new biomarker of disease progression. The number of circulating EPCs is decreased in various disorders, including chronic obstructive pulmonary disease, ischaemic chronic heart failure, coronary heart disease, rheumatoid arthritis and systemic lupus erythematosus, indicating abnormal EPC function [38][39][40][41][42]. Moreover, the excessive mobilization, differentiation and maintenance of mature eosinophil progenitors induced by dysfunctional ECFCs have been shown to result in the development of asthma [43]. Additionally, transplantation of EPCs failed to ameliorate the pathobiological changes in hypoxia-induced PAH, specifically the defective migration and incorporation of EPCs into pulmonary vasculature [44]. These studies have indicated that ECFCs play an integral role in numerous disorders, especially in respiratory diseases. Considering the results of the present study, improving ECFC function using KCOs could be a promising option for PVD management.
Previous studies have demonstrated that the protective effect of KCOs is associated with calcium signalling [36,45]. Ca 2+ is considered a critical node in the signalling network, and it has been implicated in a wide range of cellular processes, including cell proliferation, apoptosis, migration and gene expression [46,47]. In podocytes, excessive intracellular Ca 2+ induced apoptosis and the effacement of foot processes [48]. In ECs, Ca 2+ influx regulated cell migration and the synthesis of vasoactive substances, such as ET-1, nitrogen oxide and prostacyclin [49]. Our previous study found that the decline of intracellular Ca 2+ abolished plateletderived growth factor-BB-induced airway SMCs dysfunction [50]. Ca 2+ influx activates a range of downstream signalling effectors, of which CaMKII is an important molecule. The phosphorylation of CaMKII is an essential early event in Ca 2+ signal transduction that regulates the activation of Akt and eNOS [51][52][53]. Activation of CaMKII is required for H 2 O 2 -induced phosphorylation of Akt in vascular smooth cells. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is considered a regulator of cell proliferation and survival. Numerous studies have confirmed that the active PI3K/Akt pathway enhances the proliferation of numerous cells, including ECFCs [54]. Moreover, Akt is one of the determinants of eNOS phosphorylation at Ser-1177. As a Ca 2+ /CaM-dependent enzyme, the phosphorylation of eNOS can be increased when Ca 2+ binds to calmodulin, which is mediated by CaMKII. It has been shown that insulin upregulates the phosphorylation of eNOS in kidney collecting duct cells, leading to an increased production of nitrogen oxide and reduced hypertension in the insulin-resistant state [55]. The overexpression of eNOS in EPCs that were delivered to patients with PAH could achieve notable hemodynamic improvements, implying a crucial role for eNOS in ECFCs during PVD therapy [56]. These findings indicate that KCOs remarkably improve ECFC function because of the evaluated phosphorylation of CaMKII, Akt and eNOS, which could in turn be inhibited by a K ATP blocker. Altogether, we suggest that Ca 2+ /Akt/eNOS signalling is involved in the regulation of K ATP in ECFCs (Fig. 7).
In conclusion, this study confirmed for the first time that human ECFCs obtained from adult peripheral blood express K ATP , which contains the Kir6.1, Kir6.2 and SUR2b subunits. Additionally, this is the first demonstration that opening K ATP with Ipt and Nico facilitates ECFC proliferation, migration and angiogenesis. Ca 2+ /Akt/eNOS signalling plays a key role in the regulation of K ATP in ECFCs. Because opening K ATP contributes to an improvement in ECFC function, our findings provide an effective target for ECFC regulation, thus constituting a promising therapeutic option for PVD.