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Abstract

Hypoxia-associated pulmonary hypertension is characterized by pulmonary vascular remodeling. Pulmonary arterial endothelial cells dysfunction is considered as the initial event. As precursor of endothelial cells, endothelial colony-forming cells (ECFCs) play significant roles in maintenance of endothelium integrity and restoration of normal endothelial cell function. Accumulating data have indicated that hypoxia leads to a decrease in the number and function of ECFCs with defective capacity of endothelial regeneration. Previous studies have reported that the activation of ATP-sensitive potassium channels (K_ATP) shows therapeutic effects in pulmonary hypertension. However, there have been few reports focusing on the impact of K_ATP on ECFC function under hypoxic condition. Therefore, the aim of this study was to investigate whether the opening of K_ATP could regulate hypoxia-induced ECFC dysfunction. Using ECFCs derived from adult peripheral blood, we observed that Iptakalim (Ipt), a novel K_ATP opener (KCO), significantly promoted ECFC function including cellular viability, proliferation, migration, angiogenesis, and apoptosis compared with ECFCs exposed to hypoxia. Glibenclamide (Gli), a nonselective K_ATP blocker, could eliminate the effects. The protective role of Ipt is attributed to an increased production of nitric oxide (NO), as well as an enhanced activation of angiogenic transduction pathways, containing Akt and endothelial nitric oxide synthase. Our observations demonstrated that K_ATP activation could improve ECFC function in hypoxia via Akt/endothelial nitric oxide synthase pathways, which may constitute increase ECFC therapeutic potential for hypoxia-associated pulmonary hypertension treatment.

Keywords

hypoxia endothelial colony-forming cells [ECFCs]Iptakalim endothelial nitric oxide synthase [eNOS]

Introduction

Pulmonary hypertension (PH) is a progressive and life-threatening disease, defined as elevated pulmonary artery pressure that causes right heart failure and ultimately death.¹ Hypoxia is one of the confirmed factors in the pathogenesis of PH, since numerous hypoxic lung diseases are associated with PH, such as chronic obstructive pulmonary disease (COPD), interstitial lung disease, chronic exposure to high altitude.² Pulmonary vascular remodeling is the basic pathologic features of hypoxia-associated pulmonary hypertension (HPH).³ Acute hypoxia leads to pulmonary vasoconstriction, while chronic hypoxia induces pulmonary artery cell proliferation, hypertrophy, and dysfunction. The injury of pulmonary artery endothelial cells (PAECs) is the initial and central event in the development of PH.^4,5 Mounting studies have indicated that the balance between endothelial injury and repair is a critical component of HPH.^6,7 Recently, endothelial progenitor cells (EPCs), a backup system of ECs, have gained prevailing attention in PH.^8,9

EPCs have been identified originally as circulating cells with pro-angiogenic properties, which can be differentiated into ECs in vitro.¹⁰ These cells consist of a heterogeneous group that can be classified by different expression of surface markers, of which endothelial colony-forming cells (ECFCs) belong to one subtype.¹¹ EPCs have abilities to form blood vessels de novo and proliferative potentials. A number of studies indicated that EPCs could be a promising approach for PH therapy.¹² Growing evidence indicate that hypoxia limits the repair ability of EPCs, since a notable reduction in the number and function of EPCs was demonstrated in patients with chronic hypoxic diseases, such as COPD and idiopathic pulmonary fibrosis (IPF).^13–15 In a chronic hypoxia-induced PH model, hypoxia enhanced the apoptosis and dysfunctions of ECFCs.¹⁶ In vitro, ECFCs displayed decreased capacities in proliferation and migration when exposed to hypoxia.^17,18 Therefore, rescue of ECFC function from hypoxic injury has been regarded as a promising strategy for HPH therapies.

Increasing evidence has suggested that ATP-sensitive potassium channels (K_ATP) could be a new candidate for PH treatment. Iptakalim (Ipt), a lipophilic para-amino compound with a low molecular weight, has been determined as a novel selective K_ATP opener (KCO).¹⁹ Ipt has been shown to reverse pulmonary resistance vascular remodeling, inhibit proliferation of pulmonary arterial smooth muscle cells (PASMCs) and airway smooth muscle cells (ASMCs), as well as protect PAECs from pathological stimulation.^20–23 Our previous study has illustrated the molecular composition of K_ATP in human ECFCs, and the effects of KCOs for promoting ECFC capacity in physiological condition.²⁴ Nevertheless, it remains unclear whether Ipt could regulate ECFC function under hypoxic condition.

In the current study, we determined for the first time that Ipt ameliorated hypoxia-impaired human ECFC viability, proliferation, migration, angiogenesis, and apoptosis. In addition, Ipt regulated ECFC function by increasing the production of nitric oxide (NO) and activating Akt/endothelial nitric oxide synthase (eNOS) signaling. Our data indicated that Ipt can protect ECFCs from hypoxia injury, which may provide a potential target for novel therapeutic strategies in HPH management.

Materials and methods

Chemicals

Iptakalim (Ipt), with a purity of 99.9%, was synthesized and kindly provided from the Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences (Beijing, China). Glibenclamide (Gli) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Isolation and culture of ECFCs

The study was approved by the First Affiliated Hospital of Nanjing Medical University. ECFCs were isolated from adult peripheral blood via density gradient centrifugation as previously stated.^18,24 Blood samples were obtained from healthy donors recruited from the First Affiliated Hospital of Nanjing Medical University. The inclusion criteria were: age between 18 and 60 years old; either sex; clinically healthy; and voluntary consent to participate in the study. Exclusion criteria were: age less than 18 years old or more than 60 years old; clinical evidence of acute or chronic illness; past history of smoking; and refusal to consent to study participation. Ficoll-Paque (Sigma-Aldrich) density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMCs) from 20 mL of peripheral blood. To generate ECFCs, 1 × 10⁶ PBMCs per cm² were seeded on six-well fibronectin-coated tissue culture plates in complete endothelial basal media (EBM-2) medium (Lonza, USA) consisting of 10% fetal bovine serum (FBS), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), recombinant insulin-like growth factor-1 (IGF-1), gentamicin/amphotericin-B, ascorbic acid and heparin. After two days of culture, nonadherent cells were removed. Adherent cells were cultured for 14–28 days to obtain ECFCs. The ECFCs used in the study were less than eighth passages.

Hypoxia experiments

For the hypoxic condition, ECFCs were placed in a modular incubator chamber (YCP, Hua Xi Electronics Technetronic Company, Changsha, China) that maintains a gas mixture within 93% N₂, 5% CO₂, and 2% O₂ at 37℃ in a humidified atmosphere.

Cell counting kit 8 (CCK8) assay

Cell viability was accessed by cell counting kit-8 (Kumamoto, Japan). ECFCs were plated in 96-well at a density of 1 × 10⁴ cell/well in 200 µL of complete EBM-2 medium for 24 h. Then, the cells were exposed to hypoxia for 12 h, 24 h, and 48 h or pre-treated with Ipt (10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M, and 10⁻⁴ M) for 1 h before incubated under the hypoxic condition for 12 h. Gli, as a nonselective K_ATP blocker, were pre-incubated for 30 min at the concentration of 10⁻⁵ M before Ipt. CCK8 was performed according to the manufacture’s protocol. The absorbency at 450 nm was determined using a microplate reader (Thermo Scientific, CA, USA).

EdU proliferation assay

EdU (5-ethynyl-2′-deoxyuridine) incorporation assay (Ribobio, Guangzhou, China) was used to detect ECFC proliferation. Cells were pre-treated with Ipt (10⁻⁵ M) or Gli (10⁻⁵ M) for 2 h and 30 min respectively before exposed to hypoxic condition for 12 h. Then, ECFCs were incubated with 50 µmol/L EdU for 4 h. All of the EdU incorporation experiments were conducted in accordance with the protocol of the kit. The percentage of EdU-positive nuclei to total nuclei was selected randomly as the proliferation rate of ECFCs in five random high-power fields per well. The cells were visualized by fluorescence microscopy (DM2500, Lescia, Wetzlar, Germany).

Cell migration assay

ECFCs were gently detached with 0.25% trypsin, resuspended in serum-free EBM-2. A total of 1 × 10⁵ cells containing Ipt (10⁻⁵ M) were added on the upper chamber of a 24-well Transwell (Corning USA). A total of 600 µL of EBM-2 medium containing 10% FBS was placed in the lower compartment of the chamber. After 24 h incubation at 37℃ under normal or hypoxia conditions, ECFCs attached on the top membrane were wiped off carefully. The cells that migrated onto the lower side of the transwell membrane were fixed with 4% formaldehyde for 20 min. The ECFCs were stained with 5% crystal violet and randomly counted using a phase contrast microscope (Nikon, Tokyo, Japan) in five vision fields. Furthermore, Gli (10⁻⁵ M) was added to find whether the closure of K_ATP could affect ECFC migration.

Tube formation assay

After being placed at 4℃ overnight, 10 µL matrigel (Becton-Dickinson Labware) was added to a u-slide angiogenesis plate (ibidi, German) for pre-incubation at 37℃ for 1 h. Then, 50 µL ECFCs suspension at the concentration of 1.5 × 10⁵ cells per ml by complete EBM-2 medium were seeded on the top of the solidified in the presence or absence of Ipt (10⁻⁵ M) or Gli (10⁻⁵ M). Cells were then incubated in either normal (21% oxygen) or hypoxia (2% oxygen) conditions for 12 h to allow in vitro angiogenesis. Randomly, five images of tube were taken for each well. The length of complete tubes per image was measured to quantify tube formation by Image-Pro Plus.

Cell apoptosis assay

The apoptosis rate was analyzed using flow cytometry. The starved cells were treated with Ipt (10⁻⁵ M) or Gli (10⁻⁵ M) in hypoxia (2% oxygen) for 12 h. Then, the cells were collected and washed with phosphate buffered saline (PBS). After that, cells were incubated in Annexin V FITC and PI according to the protocol. The apoptotic cells were detected by flow cytometer (Becton-Dickinson, CA, USA).

NO measurement

The cultured ECFCs were seeded on 96-well culture plates and incubated overnight. The cells were pretreated with Ipt (10⁻⁵ M) for 1 h before stimulated in hypoxia. The production of NO was analyzed indirectly from ECFC supernatants. Because of the instability of NO in physiological solutions, the majority of the NO is rapidly converted to nitrite (NO²⁻) and further to be broken down into nitrate (NO³⁻). Herein, the levels of NO²⁻/NO³⁻ in the culture medium were determined by a commercially available Nitric Oxide Assay Kit (Beyotime Institute of Biotechnology, Nanjing, China) according to the manufacturer’s instructions. The absorbency was measured at 540 nm with a microplate reader (Thermo Scientific, CA, USA).

Western blot analysis

ECFCs were lysed in radioactive immunoprecipitation assay (RIPA) buffer supplemented with 1% protease inhibitor cocktail (Roche, Basel, Switzerland) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Nantong, China). The lysates were then centrifuged at 12,000 r/min for 15 min at 4℃. BCA Protein Assay kit (Beyotime, Nantong, China) was used to measure protein concentrations. Western blot was conducted according to standard techniques. In brief, the samples were subjected to 10% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE), and transferred onto poly-vinylidene difluoride (PVDF) membranes (Boston, MA, USA). After blocked with 5% bovine serum albumin (BSA) for 1 h, the transferred membranes were incubated with primary antibodies of Akt (pan) (Cell Signaling Technology, USA), phospho-Akt (Ser473) (P-Akt) (Cell Signaling Technology, USA), endothelial nitric oxide synthase (eNOS) (Santa Cruz, USA) and β-actin (Protech, USA). The blots were then incubated with the appropriate horseradish-peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz, CA, USA). The signals were detected via enhanced chemiluminescence (ECL) reagent kit (Boston, MA, USA) by Bio-Rad Gel Doc/Chemi Doc Imaging System and Quantity One software (Hercules, CA, USA).

Statistical analysis

All data were presented as mean ± standard deviation, which were collected from at least three independent experiments. Comparisons between controls and samples treated with various drugs were analyzed by Student’s t-test or one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.

Results

Ipt increases hypoxia-induced loss of human ECFC viability

To determine the effect of hypoxia on human ECFCs, the cells were cultured under hypoxic condition for 0, 12, 24, and 48 h at 1% oxygen or 2% oxygen. As shown in Fig. 1(a), the data from CCK8 assay suggested that hypoxia significantly reduced ECFC viability in a time-dependent manner. In addition, ECFCs cultured at 1% oxygen showed a lower viability than cultured at 2% oxygen. After 12-hour exposure at 2% oxygen, human ECFCs maintained approximately 70% of viability rate compared with the control group. According to the results, the subsequent experiments were conducted at 2% oxygen for 12 h unless otherwise stated. Hypoxia challenge reduced cell viability of ECFCs, which was attenuated by Ipt pretreatment in a dose-dependent manner. Ipt at the indicated concentrations (10⁻⁵ M and 10⁻⁴ M) significantly increased cell viability in hypoxia conditions (Fig. 1(b)). Therefore, Ipt of 10⁻⁵ M was chosen for the subsequent experiments. Moreover, the results indicated that Gli abolished the effects of Ipt (Fig. 1(c)).

Fig.1.

Effects of Ipt on ECFC viability in hypoxia. (a) The results from CCK8 showed that hypoxia inhibited ECFCs viability with the decrease of O₂ level and the elongation of hypoxic time via Student’s t-test. (b) Ipt enhanced the viability of ECFCs compared with hypoxia group in a concentration-dependent way via one-way ANOVA. (c) The increasing effects of Ipt were abolished by Gli. Data represent the mean ± SD of three independent experiments via one-way ANOVA. Statistically significant differences: ^&(p < 0.05) 1% O₂ level 12 h group vs. 2% O₂ level 12 h; ^§(p < 0.05) 1% O₂ level 24 h vs. 2% O₂ level 24 h; (p < 0.05) 1% O₂ level 48 h group vs. 2% O₂ level 48 h group. *(p < 0.05) as compared with the control group; ^#(p < 0.05) as compared with the hypoxia group; ^†(p < 0.05) as compared with the Ipt group.

Ipt improves hypoxia-inhibited human ECFC proliferation

The data from EdU incorporation assay indicated that exposure to hypoxia for 12 h notably inhibited the proliferation of human ECFCs. However, Ipt improved ECFCs proliferation at 2% oxygen in a concentration-dependent manner (Fig. 2(a)). The results suggested that 10⁻⁵ M Ipt markedly increased the number of EdU⁺ cells compared with the hypoxia group, reflecting the increased proliferative capacity of ECFCs. Pretreatment with Gli eliminated the effect (Fig. 2(b)). Combined the data from CCK8 and EdU incorporation assays, we further confirmed that 10⁻⁵ M Ipt was selected for the following study.

Fig. 2.

Effects of Ipt on ECFC proliferation in hypoxia. (a) The results from EdU assay showed that hypoxia inhibited ECFCs proliferation compared with control (C) group. Ipt ameliorated the proliferative ability of ECFCs in a concentration-dependent manner. (b) Gli decreased EdU-positive ECFCs induced by Ipt. Scale bar: 100 µm. Data represent the mean ± SD of three independent experiments. Statistically significant differences analyzed via one-way ANOVA: *(p < 0.05) as compared with the control group; ^#(p < 0.05) as compared with the hypoxia group; ^†(p < 0.05) as compared with the Ipt group.

Ipt attenuates hypoxia-impaired human ECFC migration

A transwell migration chamber was used to determine the function of ECFC migration under hypoxia. As shown in Fig. 3(a) and (d), hypoxia led to a significant decrease in the number of migrated cells compared with the control group, which was notably ameliorated by pre-incubation with Ipt. However, Gli could abolish the protective effects of Ipt on ECFC migration.

Fig. 3.

Effects of Ipt on ECFC migration, angiogenesis and apoptosis in hypoxia. (a and d) Hypoxia impaired ECFC migration, while Ipt increased ECFC migration in hypoxia. (b and e) Hypoxia decreased ECFC angiogenesis, while Ipt increased ECFC angiogenesis. (c and f) Hypoxia induced ECFC apoptosis, while Ipt decreased ECFC apoptosis. Gli abolished the effects of Ipt. The scale bar is 100 µm. Data represent the mean ± SD of three independent experiments. Statistically significant differences analyzed via one-way ANOVA: *(p < 0.05) as compared with the control group; ^#(p < 0.05) as compared with the hypoxia group; ^†(p < 0.05) as compared with the Ipt group.

Ipt reverses hypoxia-injured human ECFC angiogenesis

The ability of angiogenesis is one of the essential functions of ECFCs, which involves a sequence of events such as cell proliferation, migration, differentiation, and tube formation.²⁵ Tube formation is considered as the fundamental step for ECFCs angiogenesis in vivo. The administration of Ipt could rescue the ECFC function of tube formation from hypoxic impairment, which seemed to be abrogated by Gli (Fig. 3(b) and (e)).

Ipt inhibits hypoxia-induced human ECFC apoptosis

Flow cytometry analysis indicated that hypoxia markedly increased apoptosis rate in ECFCs. Pretreatment with Ipt significantly attenuated cell apoptosis, while Gli reversed the effects of Ipt on ECFC apoptosis (Fig. 3(c) and (f)).

Ipt up-regulates the expression of phosphorylated Akt, and eNOS in ECFCs

Western blotting was carried out to quantify the activation of intracellular signal transduction. Ipt administration notably increased the expression of phosphorylated Akt and eNOS in ECFCs, while hypoxia inhibited Akt/eNOS pathways. Pretreatment with Gil blocked the activated signaling induced by Ipt (Fig. 4(a) and (b)).

Fig. 4.

Effects of Ipt on Akt, eNOS, and the release of NO. (a and b) The results demonstrated the down-regulation of phosphorylated Akt and eNOS induced by hypoxia. Ipt promoted the expression of phosphorylated Akt and eNOS. (c) Ipt induced the release of NO in hypoxic condition. Gli reversed the effects of Ipt on protein expression and NO release. At least three independent experiments were conducted. Statistically significant differences analyzed via one-way ANOVA: *(p < 0.05) as compared with the control group; ^#(p < 0.05) as compared with the hypoxia group; ^†(p < 0.05) as compared with the Ipt group.

Ipt promotes NO production of ECFCs under hypoxia

It has been shown that hypoxia may deteriorate the activity of eNOS and NO signaling of ECs.²⁶ We further investigated the effect of hypoxia on NO secretion and the regulatory role of Ipt on the release of NO in ECFCs. The data showed that hypoxia led to a marked down-production of NO compared with the control group, whereas treatment with Ipt increased NO levels under hypoxia. Gli abolished the protective effects of Ipt on regulating NO secretion (Fig. 4(c)).

Discussion

The current study is the first to investigate the regulation of Ipt, a new KCO, on ECFC function under hypoxia as well as the underlying mechanisms. It has been determined that Ipt improved hypoxia-impaired ECFC function, including cellular viability, proliferation, migration, angiogenesis, and apoptosis. Moreover, Gli, a nonselective K_ATP blocker, could eliminate the protective effects of Ipt. The regulation of Ipt were contributed to the increased release of NO, and the enhanced activation of Akt/eNOS signaling. Hence, our findings may reveal a new insight focusing on the cell-based therapy for HPH.

PH is characterized by pathological features in the walls of small pulmonary arteries. Endothelial dysfunction is considered as the initial and central event in vascular remodeling induced by chronic hypoxia, resulting in excessive vascular cell growth, and infiltration of inflammatory mediators. Ultimately, repair of pulmonary endothelium is critical to preventing the development of PH.²⁷ A recent development in the recognition of EPCs has characterized three major types of EPCs, including colony forming unit-ECs (CFU-ECs), circulating angiogenic cells (CACs), and ECFCs.¹¹ A growing body of evidence have confirmed that ECFCs possess the high potentials of proliferation and the ability to form tubes and intact vascular networks.²⁸ ECFCs, also referred as late-outgrowth EPCs, have gained widespread attention as lineage-specific “genuine EPCs”.²⁹ Recently, ECFCs have been considered as one of the ideal candidates for cell therapy in pulmonary diseases, particularly COPD and PH.³⁰ However, previous studies have shown that ECFCs from PH patients are present with impaired ability of angiogenesis.³¹ Additionally, in HPH, the therapeutic potential of EPCs is limited by their poor incorporation rate into pulmonary vascular and impaired function when exposure to hypoxia.^17,32,33 Therefore, it is of the primary concern to improve functions of ECFC under hypoxia as a therapeutic target for HPH.

K_ATP is widely expressed in cells and tissues, providing a unique connection between cellular metabolic status and membrane excitability.³⁴ Ipt, a novel KCO, was primarily used as an antihypertensive agent. Recently, it is reported that Ipt possesses an antidepressant effect in chronic mild stress through the reduced expression of neuro-inflammation and neurogenesis.³⁵ Moreover, Ipt has been shown to prevent monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats with a potent antiproliferative effect on PASMCs and a protective effect on PAECs.^{21–23,35–37} Increasing data have shown that Ipt is a selective KCO, with a high selectivity for cardiac K_ATP (sulfonylurea receptor 2 A (SUR2A)/inward rectifier potassium channel 6.2 (Kir6.2)) and vascular K_ATP (SUR2B/Kir6.1).^38,39 Since our previous study has confirmed the expression of K_ATP in human ECFCs, composed of Kir6.1/Kir6.2 and SUR2B, Ipt was used to further determinate the role of K_ATP in the regulation of ECFC function in hypoxia. In this study, pretreated with Ipt increased the cellular viability and proliferative capacity in a concentration-dependent manner when exposed to hypoxia. Additionally, Ipt ameliorated hypoxia-injured ECFC migration angiogenes is and apoptosis, which have been thought as essential events in the repair and restoration of the endothelium.²⁵ Herein, our findings indicated that administration of Ipt not only up-regulated the function of ECFCs under hypoxia, but may also enhance the therapeutic potential of transplanting ECFCs for endothelial repair in HPH.

NO, a highly reactive molecule has been regarded as a typical marker of endothelial function in various pulmonary disorders. NO is a primary mediator of pulmonary vasodilation both produced and released by the endothelium.⁴⁰ Recent studies have observed that PH patients have lower levels of NO both in pulmonary and total body compared to healthy adults.⁴¹ In the development of PH, the impaired release of NO results in ECs dysfunction, sustained SMCs proliferation and constriction, accompanied by inhibition of platelet aggregation, ultimately vascular remodeling.⁴² Herein, the promotion of endogenous NO production in hypoxia has been considered as a key point for HPH therapies. In our study, pretreatment with Ipt could partially restore the decreased production of NO induced by hypoxia in ECFCs, which may exert a beneficial effect in the prevention of hypoxia-associated pulmonary vascular remodeling.

Accumulating data have indicated that eNOS is the primary enzyme in the pulmonary circulation of three isoforms of nitric oxide synthases.⁴³ eNOS-null mice showed significantly higher pulmonary hypertension when submitted to hypobaric hypoxia compared to controls.⁴⁴ It has been indicated that delivering EPCs, which were modified with overexpressed eNOS gene, could markedly ameliorate acute pulmonary hemodynamics in PH patients, implying increasing activation of eNOS pathway could constitute a promising strategy to facilitating ECFC function.⁴⁵ Furthermore, eNOS has been found to be a vital factor in the protection of KCOs against endothelial dysfunction. Nicorandil, a classic KCO, could attenuate endothelial damage and vascular remodeling mainly through restoration of eNOS expression in MCT-induced PH.⁴⁶ In cultured aortic ECs, opening K_ATP by Ipt could increase NOS activity, enhance NO release, and inhibit endothelin-1 (ET-1) generation, mediating by promotion of Ca²⁺ influx.⁴⁷ Our data suggested that Ipt significantly up-regulated the expression of eNOS, which may contribute to the regulation of Ipt on ECFCs in hypoxia. Moreover, Akt is thought to be a key signaling molecule mediating the activation of eNOS. In ECs, PI3K/Akt pathway contributes to the shear stress-induced activation of eNOS.⁴⁸ Accumulating evidence has implicated that Akt plays a key role in modulating EPC function, including migration and incorporation to ECs.⁴⁹ Our results showed that phosphorylated Akt was significantly increased by Ipt treatment in hypoxia, while the K_ATP blocker suppressed the up-regulated expression of phosphorylated Akt.

However, there are several limitations in the study. A major limitation is the use of normal ECFCs instead of ECFCs from HPH patients. Healthy ECFCs exposed to hypoxia may be an adaptive response, while ECFCs derived from HPH patients suffered from chronic hypoxia and had crosstalk with other types of dysfunctional cells as well as influenced by secreted cytokines in vivo. The effects of Ipt on patients’ ECFCs are needed to be further detected. In addition, the present study was designed to assess the effects of Ipt on ECFC functions in vitro. Although a previous study found that Ipt attenuated HPH in rats, it still remains to be investigated the effects of Ipt on regulating ECFC functions in ameliorating HPH progression in vivo.³⁷ Finally, the CCK8 method was used to evaluate ECFC viability, which was unable to assess cell proliferation, apoptosis, and senescence, respectively.

Footnotes

Acknowledgments

Ipt was kindly provided by Dr H Wang (Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing, China) and Dr G Hu (Department of Pharmacology, Nanjing Medical University, Nanjing, China).

Conflict of interest

The author(s) declare that there is no conflict of interest.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) under Grant 81273571, 81800090, 8187010350, the Jiangsu Clinical Research Center for Respiratory Diseases Project under Grant BL2012012.

References

Yamaji-Kegan

Takimoto

Zhang

, et al. Hypoxia-induced mitogenic factor (FIZZ1/RELMalpha) induces endothelial cell apoptosis and subsequent interleukin-4-dependent pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2014; 306: L1090–1103.

Fadini

Avogaro

Ferraccioli

, et al. Endothelial progenitors in pulmonary hypertension: new pathophysiology and therapeutic implications. Eur Respir J 2010; 35: 418–425.

Hales

. Hypoxia does neither stimulate pulmonary artery endothelial cell proliferation in mice and rats with pulmonary hypertension and vascular remodeling nor in human pulmonary artery endothelial cells. J Vasc Res 2011; 48: 465–475.

Pak

Aldashev

Welsh

, et al. The effects of hypoxia on the cells of the pulmonary vasculature. Eur Respir J 2007; 30: 364–372.

Morrell

Adnot

Archer

, et al. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 2009; 54: S20–31.

Yang

, et al. Targeting mitochondria-associated membranes as a potential therapy against endothelial injury induced by hypoxia. J Cell Biochem 2019.

Stenmark

Fagan

Frid

. Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circ Res 2006; 99: 675–691.

Wang

Hong

Chen

, et al. Modulation of EPC: a new hope for pulmonary artery hypertension treatment. Int J Cardiol 2018; 268: 214.

Guerra

Perrotta

Testa

. Circulating endothelial progenitor cells biology and regenerative medicine in pulmonary vascular diseases. Curr Pharm Biotechnol 2018; 19: 700–707.

10.

Asahara

Murohara

Sullivan

, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: 964–967.

11.

Basile

Yoder

. Circulating and tissue resident endothelial progenitor cells. J Cell Physiol 2014; 229: 10–16.

12.

Vaidya

Gupta

. Novel therapeutic approaches for pulmonary arterial hypertension: unique molecular targets to site-specific drug delivery. J Control Release 2015; 211: 118–133.

13.

Fadini

Schiavon

Cantini

, et al. Circulating progenitor cells are reduced in patients with severe lung disease. Stem Cells 2006; 24: 1806–1813.

14.

Huertas

Palange

. Circulating endothelial progenitor cells and chronic pulmonary diseases. Eur Respir J 2011; 37: 426–431.

15.

Malli

Koutsokera

Paraskeva

, et al. Endothelial progenitor cells in the pathogenesis of idiopathic pulmonary fibrosis: an evolving concept. PloS One 2013; 8: e53658.

16.

Wang

Jia

Luo

, et al. Coordination between NADPH oxidase and vascular peroxidase 1 promotes dysfunctions of endothelial progenitor cells in hypoxia-induced pulmonary hypertensive rats. Eur J Pharmacol 2019; 857: 172459.

17.

Decaris

Lee

Yoder

, et al. Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells. Angiogenesis 2009; 12: 303–311.

18.

Cai

, et al. Hypoxia induces the dysfunction of human endothelial colony-forming cells via HIF-1alpha signaling. Respir Physiol Neurobiol 2018; 247: 87–95.

19.

Costa

. Iptakalim: a new or just another KCO? Cardiovasc Res 2009; 83: 417–418.

20.

Xie

Wang

Ding

, et al. Anti-proliferating effect of iptakalim, a novel KATP channel opener, in cultured rabbit pulmonary arterial smooth muscle cells. Eur J Pharmacol 2005; 511: 81–87.

21.

Zhu

Zhang

Xie

, et al. Iptakalim inhibited endothelin-1-induced proliferation of human pulmonary arterial smooth muscle cells through the activation of K(ATP) channel. Vasc Pharmacol 2008; 48: 92–99.

22.

Zong

Zuo

Wang

, et al. Iptakalim rescues human pulmonary artery endothelial cells from hypoxia-induced nitric oxide system dysfunction. Exp Ther Med 2012; 3: 535–539.

23.

Liu

Kong

Zeng

, et al. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration. Exp Cell Res 2015; 336: 204–210.

24.

, et al. Activation of ATP-sensitive potassium channels facilitates the function of human endothelial colony-forming cells via Ca(2⁺) /Akt/eNOS pathway. J Cell Mol Med 2017; 21: 609–620.

25.

Rosenzweig

. Endothelial progenitor cells. N Engl J Med 2003; 348: 581–582.

26.

Rochette

Lorin

Zeller

, et al.

Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: possible therapeutic targets?

Pharmacol Ther 2013; 140: 239–257.

27.

Farkas

Kolb

. Vascular repair and regeneration as a therapeutic target for pulmonary arterial hypertension. Respiration 2013; 85: 355–364.

28.

Steinmetz

Nickenig

Werner

. Endothelial-regenerating cells: an expanding universe. Hypertension 2010; 55: 593–599.

29.

Alphonse

Vadivel

Zhong

, et al. The isolation and culture of endothelial colony-forming cells from human and rat lungs. Nat Protoc 2015; 10: 1697–1708.

30.

Weiss

. Concise review: current status of stem cells and regenerative medicine in lung biology and diseases. Stem Cells 2014; 32: 16–25.

31.

Toshner

Voswinckel

Southwood

, et al. Evidence of dysfunction of endothelial progenitors in pulmonary arterial hypertension. Am J Respir Crit Care Med 2009; 180: 780–787.

32.

Marsboom

Pokreisz

Gheysens

, et al. Sustained endothelial progenitor cell dysfunction after chronic hypoxia-induced pulmonary hypertension. Stem Cells 2008; 26: 1017–1026.

33.

Liu

Peng

, et al. Non-muscle myosin light chain promotes endothelial progenitor cells senescence and dysfunction in pulmonary hypertensive rats through up-regulation of NADPH oxidase. Eur J Pharmacol 2016; 775: 67–77.

34.

Dzeja

Terzic

. Phosphotransfer reactions in the regulation of ATP-sensitive K+ channels. FASEB J 1998; 12: 523–529.

35.

Yang

Geng

, et al. Iptakalim confers an antidepressant effect in a chronic mild stress model of depression through regulating neuro-inflammation and neurogenesis. Int J Neuropsychopharmacol 2014; 17: 1501–1510.

36.

Long

Cui

, et al. Iptakalim ameliorates monocrotaline-induced pulmonary arterial hypertension in rats. J Cardiovasc Pharmacol Ther 2013; 18: 60–69.

37.

Zhu

, et al. Iptakalim attenuates hypoxia-induced pulmonary arterial hypertension in rats by endothelial function protection. Mol Med Rep 2015; 12: 2945–2952.

38.

Pan

Huang

Cui

, et al. Targeting hypertension with a new adenosine triphosphate-sensitive potassium channel opener iptakalim. J Cardiovasc Pharmacol 2010; 56: 215–228.

39.

Sikka

Kapoor

Bindra

, et al. Iptakalim: a novel multi-utility potassium channel opener. J Pharmacol Pharmacother 2012; 3: 12–14.

40.

Dias-Junior

Cau

Tanus-Santos

. Role of nitric oxide in the control of the pulmonary circulation: physiological, pathophysiological, and therapeutic implications. J Bras Pneumol 2008; 34: 412–419.

41.

Ghosh

Gupta

, et al. Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 2016; 310: L1199–1205.

42.

Crosswhite

Sun

. Nitric oxide, oxidative stress and inflammation in pulmonary arterial hypertension. J Hypertens 2010; 28: 201–212.

43.

Wilkins

Wharton

Grimminger

, et al. Phosphodiesterase inhibitors for the treatment of pulmonary hypertension. Eur Respir J 2008; 32: 198–209.

44.

Kobs

Chesler

. The mechanobiology of pulmonary vascular remodeling in the congenital absence of eNOS. Biomech Model Mechanobiol 2006; 5: 217–225.

45.

Granton

Langleben

Kutryk

, et al. Endothelial NO-synthase gene-enhanced progenitor cell therapy for pulmonary arterial hypertension: the PHACeT trial. Circ Res 2015; 117: 645–654.

46.

Sahara

Sata

Morita

, et al. Nicorandil attenuates monocrotaline-induced vascular endothelial damage and pulmonary arterial hypertension. PloS One 2012; 7: e33367.

47.

Wang

Long

Duan

, et al. A new ATP-sensitive potassium channel opener protects endothelial function in cultured aortic endothelial cells. Cardiovasc Res 2007; 73: 497–503.

48.

Dimmeler

Fleming

Fisslthaler

, et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 1999; 399: 601–605.

49.

Hur

Yoon

Lee

, et al. Akt is a key modulator of endothelial progenitor cell trafficking in ischemic muscle. Stem Cells 2007; 25: 1769–1778.

Iptakalim ameliorates hypoxia-impaired human endothelial colony-forming cells proliferation,migration,and angiogenesis via Akt/eNOS pathways

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Isolation and culture of ECFCs

Hypoxia experiments

Cell counting kit 8 (CCK8) assay

EdU proliferation assay

Cell migration assay

Tube formation assay

Cell apoptosis assay

NO measurement

Western blot analysis

Statistical analysis

Results

Ipt increases hypoxia-induced loss of human ECFC viability

Ipt improves hypoxia-inhibited human ECFC proliferation

Ipt attenuates hypoxia-impaired human ECFC migration

Ipt reverses hypoxia-injured human ECFC angiogenesis

Ipt inhibits hypoxia-induced human ECFC apoptosis

Ipt up-regulates the expression of phosphorylated Akt, and eNOS in ECFCs

Ipt promotes NO production of ECFCs under hypoxia

Discussion

Footnotes

Acknowledgments

Conflict of interest

Funding

References