Slight up‐regulation of Kir2.1 channel promotes endothelial progenitor cells to transdifferentiate into a pericyte phenotype by Akt/mTOR/Snail pathway

Abstract It was shown that endothelial progenitor cells (EPCs) have bidirectional differentiation potential and thus perform different biological functions. The purpose of this study was to investigate the effects of slight up‐regulation of the Kir2.1 channel on EPC transdifferentiation and the potential mechanism on cell function and transformed cell type. First, we found that the slight up‐regulation of Kir2.1 expression promoted the expression of the stem cell stemness factors ZFX and NS and inhibited the expression of senescence‐associated β‐galactosidase. Further studies showed the slightly increased expression of Kir2.1 could also improve the expression of pericyte molecular markers NG2, PDGFRβ and Desmin. Moreover, adenovirus‐mediated Kir2.1 overexpression had an enhanced contractile response to norepinephrine of EPCs. These results suggest that the up‐regulated expression of the Kir2.1 channel promotes EPC transdifferentiation into a pericyte phenotype. Furthermore, the mechanism of EPC transdifferentiation to mesenchymal cells (pericytes) was found to be closely related to the channel functional activity of Kir2.1 and revealed that this channel could promote EPC EndoMT by activating the Akt/mTOR/Snail signalling pathway. Overall, this study suggested that in the early stage of inflammatory response, regulating the Kir2.1 channel expression affects the biological function of EPCs, thereby determining the maturation and stability of neovascularization.

carotid artery injury and hind limb and myocardial ischaemia, paving the way for clinical research. [1][2][3][4] However, an endothelial cell phenotype is not always the final endpoint of the differentiation of EPCs, which is not preset. The recent literature has shown that under certain physiological or pathophysiological conditions, EPCs can undergo an endothelialmesenchymal transition (EndoMT); thus, their function will presumably be altered. 5,6 Unfortunately, the underlying mechanism of this transformation process has not yet been clarified.
According to their functional characteristics and amino acid sequences, channels in the inward rectifier potassium channels (Kir) family can be divided into the following seven subfamilies into four functional groups: Kir1.x, Kir4.x, Kir5.x and Kir7.x (K + transport channels); Kir2.x (classic K + channels); Kir3.x (G-protein-gated K + channels); and Kir6.x (ATP-sensitive K + channels). 7 The channels in this family conduct more inward current under a negative voltage/ balanced potential than under a negative current/outward voltage. Therefore, the Kir channels play an important role in maintaining the resting membrane potential (RMP) in most cells. However, the distributions of Kir subtypes vary due to cell heterogeneity, which also determines the differences in their biological functions. The roles of Kir channels in Andersen's syndrome, cardiac arrhythmias and hypokalaemic periodic paralysis, which have been extensively discussed, involve and affect cell migration, contraction and differentiation. 8 Previous studies have shown that the Kir2.1 channel is the main inward rectifying potassium channel on EPCs. 9 Our recent research also showed that the Kir2.1 channel is closely related to the differentiation of EPCs. 2 Furthermore, blockade of the Kir2.1 channel was found to cause the depolarization of EPCs and accelerate the process of endothelial differentiation, which is related to the formation of autophagy. 2 As shown by our further study, interestingly, we found that the expression of Kir2.1 was increased in EPCs due to oxidative stress caused by hydrogen peroxide. In contrast, inhibiting the channel function with the specific blocker ML133 could promote the occurrence of cell apoptosis ( Figure S1). However, whether alterations in Kir2.1 channel expression affect the differentiation (transdifferentiation) or subsequent cytological functions of EPCs has not been discussed.
Here, we report that the slight up-regulation of Kir2.1 promoted channel hyperpolarization, contributing to EPC mesenchymal transition, and that this process has an essential function in angiogenesis and vascular stability.

| Isolation, extraction, identification and culture of EPCs from rat bone marrow
Sprague Dawley rats weighing 200-300 g (Pengyue Laboratory Animal Breeding Company, Jinan, China) were euthanized by neck dislocation and then soaked in 70-75% alcohol for sterilization. The obtained bone marrow mononuclear cells were inoculated in a T25 culture bottle pre-coated with fibronectin at 5 µg/cm 2 and cultured in EGM-2MV medium. The exact method was carried out by referring to our previous literature. 10

| Cell viability assay
EPC viability assay was performed by the Cell Counting Kit-8 (CCK8) method. Specifically, cells were inoculated in 96-well plates at a density of 5 × 10 3 /well. After 3 days of transfection with the adenovirus vector, the culture medium in each well was exchanged with 10 µl of CCK8 solution in 100 µl of culture solution. After 2 h, the optical density at 450 nm was detected with a microplate spectrophotometer.

| Recombinant adenovirus or siRNA transfection
Adenovirus containing the rat Kir2.1 gene (NM_017296.1, GFP-Ad-Kir2.1 or Ad-Kir2.1) was commercially designed and synthesized by HanBio Company (HanBio). In brief, the cells were washed twice with 1 × PBS, and serum-free medium containing viral particles at different titres (MOIs) was slowly added to each well. Both Stealth RNAi TM siRNA against rat Snail and negative control siRNA were purchased from Invitrogen (Thermal Fisher) The sequence of the siRNA was as follows: 5′-AAUAUUUGCAGUUGAAGGCCUUCCG-3′. Transfection with siRNA or scramble siRNA was carried out using Lipofectamine 3000 according to the manufacturer's recommended instructions.

| SYBR Green-based quantitative RT-PCR
Total mRNA was extracted from samples with TRIzol™ reagent (Invitrogen, Thermal Science). Reverse transcription and quantitative PCR were performed using TaKaRa One-Step TB Green ® PrimeScript™, and the PCR samples were 25 µl. The reaction conditions were as follows: pre-denaturation at 95°C for 30 s followed by 40 cycles of 95°C for 20 s and 60°C for 20 s. The fol-

| Protein extractions and Western blot analysis
Detailed methods are available to our previous literature. 11,12 The protein antibodies used were as follows: antibodies against p53, SIRT1, NG2 and PDGFRβ were purchased from Abcam Company, and antibodies against p-Akt, p-mTOR and Snail were purchased from Cell Signal Company. Protein bands were visualized using a chemiluminescence and spectral fluorescence imaging system (Uvitec alliance Q9). The relative optical densities of the protein bands were analyzed with ImageJ v1.53c software.

| Fluorescence-activated cell sorting analysis
The cells were digested with a trypsin solution (0.25%) to obtain a cell suspension and then centrifuged at 500 × g for 10 min to ob-  The fluorescence intensity of protein expression or the cellular RMP was visualized and quantitatively analyzed by fluorescence microscopy (Olympus IX71, Japan) or confocal laser microscope (Leica TCS SP8, Germany).

| ELISA
The level of TGFβ1 in the supernatant was determined by ELISA according to the manufacturer's instructions (Sangon Biotechnology).
The optical density at 450 nm was measured using Thermo Fisher software (USA).

| Cellular senescence detection (senescenceassociated β-galactosidase staining)
Cell senescence was detected using a senescence-associated βgalactosidase (SAβ-Gal) staining kit (Beyotime, China) following the manufacturer's instructions. In brief, the cell culture solution in the 6-well plate was removed, the cells were washed with PBS three times, and 1 ml of β-Gal fixation solution was added and incubated for 15 min at room temperature to fix the cells. Then, the cells were washed with PBS, and 1 ml of a staining solution containing β-Gal stain A\B\C and X-Gal solution was added to each well. Cells were incubated overnight at 37°C and photographed under an optical microscope the next day. Cells expressing blue β-Gal were positive, and the corresponding senescence rate was calculated.

| In vitro coculture Matrigel angiogenesis assay
First, 100 µl of Matrigel (BD Biosciences, USA) was quickly placed in a 96-well plate on ice and then incubated at 37°C for 10 min.

| Cell contraction assay
Noradrenaline (NE) (0.1 mM, Grand Pharma Company) dissolved in cell culture medium was used as a stimulant. GFP-labelled EPCs were treated for 1 h, and the degree of cell contraction was observed under a fluorescence microscope. Cell morphology was measured before and after NE intervention and compared with that in the control group. The collagen contraction model was performed by the company's kit instructions (Cell BioLabs). In detail, the collagen gel working solution is configured on ice that containing the collagen fluid, 5 × DMEM and neutralization solution. Then, the target cells were digested, and the cell concentration was adjusted to 3 × 10 6 /ml of culture medium.
A total of 0.5 ml of the cell-collagen mixture containing 1 part cell suspension and 4 parts the collagen working solution was added into per well in a 24-well plate and incubated for 1 h in a cell incubator in order to promote the collagen polymerization. At last, 1.0 ml of culture medium was added atop each lattice. After 48 h of incubation, the tensioned collagen matrix was released with 10μl sterile pipette tips before the NE working solution was intervened. The gel area changes were measured at various times with ImageJ v1.53c software.

| Statistical analysis
Experimental data are represented as the mean ± SD. Student's t test was used to analyze data between two groups. Data among the three groups were analyzed by one-way ANOVA (Prism GraphPad 8.2.1).
Differences with p ≤ 0.05 were considered statistically significant.

| The slightly up-regulated expression of Kir2.1 could promote the maintenance of stemness and reduce the senescence of EPCs
In our previous study, we found that blocking the function of Kir2.1 or knocking out its expression could accelerate the differentiation of EPCs into endothelial cells. 2 In addition, the expression of Kir2.1 was found to be slightly increased when EPCs were exposed to temporary oxidative stress (various concentrations of H 2 O 2 stress, Figure S1A), but apoptosis was significantly enhanced by the specific Kir2.1 channel blocker ML133 in a lower concentration of H 2 O 2 ( Figure S1B).
Therefore, EPCs were transfected by adenovirus containing the Kir2.1 gene with different MOI values to promote different degrees of expression. First, the expression of Kir2.1 was slightly increased when adenovirus was transfected at an MOI no higher than 20 ( Figure 1A and B, Figure S2). Second, compared with the control group, the MOI Next, after Kir2.1 transfection, the population of EPCs was performed by FACS as shown in Figure 1E. Compared with the control group, the expression of CD44, CD31 and vWF was decreased, but there was no change in CD29 and CD45 expression. Stem and progenitor cells are characterized by their self-renewal and ability to maintain stemness. In the case of EPCs, their stemness and senescence determine their capacity to accumulate for postnatal vascular repair. Hamid et al. 13 showed that ZFX and NS were marker molecules reflecting the stemness or self-renewal ability of EPCs, while other molecules, such as OCT4 and NANOG, had low or no expression, which is consistent with our results ( Figure S3). In this study, we found that slightly increased Kir2.1 expression was shown to promote the expression of ZFX and NS ( Figure 1F), suggesting that EPC stemness maintenance was enhanced. In addition, increased expression of Kir2.1 reduced the expression of p53 but slightly promoted the expression of SIRT1, which indicates that Kir2.1 can delay or inhibit the ageing of EPCs ( Figure 1G). Furthermore, this effect was confirmed by SAβ-Gal staining ( Figure 1H).

| The slightly up-regulated expression of Kir2.1 could promote EPC transdifferentiation into a pericyte phenotype
Under a moderate oxidative stress environment, neovascularization is not decreased but rather increased. 14,15 Unfortunately, the mechanism of neovascularization is not clear. We found that

| EPC-derived pericytes express the pericyte marker protein Desmin and promote the maturation and stabilization of neovascularization
Desmin is a structural and functional protein of pericytes that is important for the maintenance of vascular stability and pericyte contraction. 18 The results of immunofluorescence assays showed that the protein expression of Desmin was significantly increased when EPCs were transfected with pAV-Kir2.1 ( Figure 3A   Then, as expected, MK2206 was found to significantly inhibit Akt phosphorylation. MK2206 also inhibited mTOR phosphorylation and decreased the expression of Snail. Pretreatment with the mTOR blocker RAD001 reduced expression of the Snail protein. Surprisingly, we found that the mTOR inhibitor RAD001 significantly promoted Akt phosphorylation. However, intervention with Snail siRNA had no significant effect on the phosphorylation of Akt and mTOR ( Figure 5C).
In summary, as shown in Figure 6, these results suggest that slight Kir2.1 overexpression could promote EPC transdifferentiation through the Akt/mTOR/Snail pathway.

| DISCUSS ION
To comprehensively investigate the role of the Kir2.1 channel in the development of EPCs, we first observed the effect of overexpression of Kir2.1 on EPC stemness and senescence, the essential biological characteristics of stem/progenitor cells. In this study, it was surprising to find that the slightly up-regulated expression of Kir2.1, which was achieved by taking advantage of transfection with adenovirus at different MOI values, could promote the maintenance of stemness and reduce the senescence of EPCs. Subsequently, we found that Kir2.1 protein expression was also slightly increased under stimulation with reactive oxygen species (ROS) at a particular concentration. However, when ML133 blocked the function of the Kir2.1 channel, the apoptosis and senescence rate of EPCs was significantly increased in oxidative conditions ( Figure S1 and 5). These data suggest that the Kir2.1 channel plays an essential role in response to changes in the surrounding microenvironment.
More researchers showed that CD44, CD31 and vWF were all related to the differentiation of EPCs into endothelial cells. [20][21][22] Subsequently, we found that the expression of EPC-related marker molecules CD44, CD31 and vWF decreased significantly after promoting the expression of EPC Kir2.1. For example, CD44 was involved in mediating the proliferation and activation of endothelial cells. 20 These data suggest that changes in Kir2.1 expression may influence the differentiation or transdifferentiation process of EPCs.
Traditionally, EPCs residing in the stem cell niche or bone marrow differentiate into vascular ECs, which play a vital role in repairing the endothelium of damaged blood vessels. However, Diez D et al. 23 showed that under the influence of the local environment, especially specific concentrations of TGFβ1, EPCs obtain a mesenchymal phenotype, showing increased expression of the transcription factors slug, Snail, zeb1 and endothelin-1. Therefore, the authors speculated that EPCs transdifferentiate into smooth muscle celllike cells through an EndoMT-like process. In this study, we found no change in the expression of TGFβ1 either in the gene expression of TGFβ1 or the protein level in the supernatant of culture medium after EPCs transfection. Silvia et al. 24 suggested that EPC might be a source of cells with pericyte or perivascular mesenchymal phenotype and function. As reported in the literature, supportive vascular cells, such as pericytes and/or smooth muscle cells, may originate from endothelial cells themselves. 25,26 As reviewed by the first discoverer of EPCs, Asahara, endothelial cells and pericytes are homologous; namely, they are derived from vascular stem cells from the developmental perspective. 27 Therefore, EndoMT may be an essential mechanism to recruit such pericytes during postnatal angiogenesis and neovascularization. 28 Nevertheless, the molecular regulatory mechanisms involved in the process of EndoMT are mostly unknown.
As mentioned above, the Kir2.1 channel likely plays a crucial role in the response of EPCs to changes in the surrounding microenvironment. However, whether changes in Kir2.1 expression determine the process of EPC transdifferentiation is mainly unknown. In this study, we found that slightly elevated Kir2.1 expression inhibited EPC differentiation into ECs; in contrast, expression of the pericyte marker molecules NG2 and PDGFRβ was promoted, suggesting that the Kir2.1 channel contributes to the acquisition of a mesenchymal (pericyte) phenotype by EPCs.
Although NG2 and PDGFRβ are pericyte marker molecules, it must be noted that the levels of these markers in different tissues were highly variable. Therefore, pericyte-related functions were also identified in transdifferentiated EPCs. Our data showed that Further data showed that ML133 attenuated the effect of increased Kir2.1, which promoted the maintenance of EPC stemness and the transition of EPCs into a pericyte phenotype. Therefore, our data suggest that mediation of the ion permeation-independent signalling pathway by the Kir2.1 channel is a key mechanism that enhances the stemness of EPCs and promotes EPC transdifferentiation into a pericyte phenotype. Changes in the RMP, such as hyperpolarization or depolarization, trigger subsequent intracellular signalling pathways that change cellular behaviour. 33 A study by Li et al. 34 showed that enhancing Kir2.1 channel currents, which are related to the regulatory mechanism of the Akt/PI3-kinase activity signalling pathway, rescued acute ischaemic arrhythmia triggered by hypoxia-induced RMP depolarization. Ilaria et al. 30 showed that membrane hyperpolarization inhibited Wnt signalling, thus promoting the development of daughter neurons. Therefore, some researchers have also confirmed that cell membrane hyperpolarization plays a key role through Notch, Ca 2+ sparks, or the PKC signalling pathway and speculated that the specific signalling mechanism is closely related to the factors and type of cells stimulated. [35][36][37] This study found that increasing Kir2.1 expression could promote the phosphorylation of both Akt and mTOR.
It was also found that blocking Akt phosphorylation by MK2206 could effectively reduce the degree of mTOR phosphorylation.
Further study showed that the inhibition of Akt and mTOR phosphorylation by the inhibitors MK2206 and RAD001, respectively, reduced the Kir2.1-induced phenotypic transformation of EPCs to a pericyte phenotype, suggesting that Kir2.1-induced changes in the RMP can affect the development of EPC EndoMT through the Akt/ mTOR pathway. Surprisingly, although RAD001 significantly blocked the mTOR phosphorylation process, it also paradoxically promoted Akt phosphorylation. Some literature confirmed that RAD001induced p-Akt up-regulation was due to the autocrine release of insulin-like growth factor-1 (IGF-1) with the subsequent activation of the IGF-1 receptor (IGF1R) in cell lines. [38][39][40] So we speculated that this RAD001-mediated p-Akt up-regulation was concerned with the IGF-1/IGF-1 receptor autocrine loop: (1) IGF1 and IGF1R are expressed in EPCs derived from rat bone marrow, which according to the microarray data accessed at GEO database with accession number GSE49510 ( Figure S6); (2) it has been confirmed that EPCs can secrete IGF-1 and express IGF1R, which serves the vital cell biological function, [41][42][43] but it requires further exploration.
Some studies suggest that Snail is a pivotal transcription factor involved in EndoMT and EMT. 44,45 In this study, the slight up-regulation of Kir2.1 was found to promote Akt/mTOR phosphorylation, which promoted the expression of Snail in EPCs. As shown in Figure 5, once the expression of Snail was reduced by siRNA interference, the effect of Kir2.1 on promoting EPC EndoMT was weakened, which suggests that the Akt/mTOR/Snail pathway is involved in the process of Kir2.1-mediated EPC transformation to a pericyte phenotype.
In conclusion, combined with our previous studies, our results have demonstrated the following findings (shown in Figure 6). Therefore, we speculate that the Kir2.1 channel is likely a switch molecule by which EPCs perceive the stem niche and thus determine the direction of EPC differentiation or transdifferentiation, and Kir2.1 may be a new therapeutic target for vascular remodelling diseases. F I G U R E 6 Diagrammatic representations of Kir2.1 channelmediated regulation of the direction of EPC differentiation. After the Kir2.1 channel is inhibited, EPC depolarization is induced, and autophagy is then induced, promoting EPC differentiation into ECs. In contrast, if the Kir2.1 channel is agitated, EPC hyperpolarization is promoted, and the Akt/mTOR/Snail signalling pathway is excited, inducing the transdifferentiation of EPCs into mesenchymal cells (pericytes) and moderating/maintaining stem cell stemness