ABSTRACT

   Diabetic wound (DW) represent a common complication of diabetes.
   Despite advances in regenerative repair utilizing endothelial
   progenitor cells (EPCs), challenges such as low survival and impaired
   angiogenic function of EPCs remain. Herein, we explored an effective
   method to induce injury‐induced protection for EPCs and improves their
   function. This was achieved through cell preconditioning under
   conditions of nutrient deprivation and high glucose (NDHG), combined
   with sb431542, a transforming growth factor beta (TGF‐β) signaling
   inhibitor. Specifically, after three generations of cell passage during
   preconditioning, umbilical cord‐derived endothelial cells (ECs)
   exhibited characteristics resembling those of EPCs, with over 80% of
   the cells expressed CD34, a typical marker of EPCs. Notably, these
   preconditioned EPC‐like cells (pEPCs) showed tolerance to pathological
   environment, as evidenced by robust cell viability, improved
   antioxidant capacity, and stable tube‐forming ability under NDHG
   condition. The protective effect of preconditioning in pEPCs is partly
   achieved by activating the PI3K/AKT pathway to upregulate the
   expression of Nrf2 and HIF‐1α. Importantly, pEPCs exhibited therapeutic
   potential in two diabetic mouse models‐limb ischemia and skin wounds by
   enhancing blood vessel formation and facilitating tissue repair.
   Overall, this preconditioning method induced the generation of
   functionally enhanced pEPCs, providing an alternative source of cells
   for treating DWs.

   Keywords: angiogenesis, cell therapy, diabetic wound, endothelial
   progenitor cells, preconditioning, TGF‐β signaling
     __________________________________________________________________

   In this study, we explore a novel and efficient preconditioning method
   that induced EPCs‐like cells from hUVECs. TGF‐β inhibitors–treated
   hUVECs demonstrated significantly enhanced cell activity and
   tube‐forming ability under nutrient‐deficient and
   high‐glucose‐containing diabetic wound simulation conditions.
   Furthermore, they exhibited the ability to repair limb and skin
   injuries in diabetic mice.

   graphic file with name MCO2-6-e70364-g001.jpg

1. Introduction

   The refractory diabetic wound (DW) is a serious complication of
   diabetes mellitus that leads to ulcerations and amputations of the
   lower extremities [[48]1, [49]2]. During the healing of DW, impaired
   angiogenesis is partially responsible for the pathological process
   [[50]3, [51]4, [52]5]. Despite the availability of standard
   revascularization treatments such as angioplasty, arterial bypass, and
   vascular growth factors, a high incidence of amputation remains
   [[53]6]. Therefore, there is an urgent requirement to explore
   alternative therapeutic options for DW.

   Stem cell‐based therapy has emerged as a potential treatment for DW.
   Clinical trials have reported that transplantation of CD34^+
   endothelial progenitor cells (EPCs), derived from bone marrow, can
   result in long‐term improvements of severe limb ischemia for patients
   who are not eligible for standard revascularization procedures [[54]7,
   [55]8]. Nonetheless, the therapeutic efficacy of the transplanted EPCs
   is compromised by their poor survival in the high‐glucose and
   nutrient‐deficient pathological conditions of DW [[56]9, [57]10].
   Although introducing specific genes, such as CXCR7, Nampt, or
   microRNAs, into EPCs has shown promise in enhancing their function
   [[58]9, [59]10, [60]11], the genetic manipulation of cells raises
   safety concerns and is not yet suitable for widespread therapeutic
   application.

   Preconditioning has been shown to improve the resistance of
   transplanted cells to the challenging host microenvironment by
   subjecting them to conditions that mimic the harsh conditions of
   damaged tissues [[61]12, [62]13]. Previous studies have reported
   various preconditioning schemes involving pharmacological and/or
   physical inducers to enhance the reparative potential of EPCs. For
   instance, pinocembrin preconditioning has been found to improve the
   therapeutic efficacy of EPCs in rats with monocrotaline‐induced
   pulmonary hypertension [[63]14]. Ischemic preconditioning of EPCs has
   been shown to attenuate renal ischemia/reperfusion injury [[64]15], and
   EPCs preconditioned with defined small molecules improve their therapy
   against ischemic diseases [[65]16]. However, the optimal
   preconditioning scheme that can increase the proangiogenic responses of
   EPCs and maximize their therapeutic potential for DW remains to be
   elucidated.

   The signaling pathway of transforming growth factor beta (TGF‐β) is
   critical for the function and plasticity regulation of ECs in health
   and diseases [[66]17, [67]18]. Research has indicated that
   hyperglycemia increases TGF‐β signaling in ECs, leading to detrimental
   effects on their viability and cellular function by triggering
   endothelial‐to‐mesenchymal transition and oxidative stress [[68]19].
   Additionally, TGF‐β signaling plays a significant role in the
   development of hyperglycemia‐induced metabolic memory in ECs, resulting
   in long‐lasting damage [[69]20]. These findings highlight the potential
   of inhibiting TGF‐β signaling to reverse the damage to EPCs under the
   pathological conditions of DW. However, many studies have demonstrated
   that TGF‐β inhibitors can facilitate the differentiation of stem cells
   into ECs and enhance the proliferation of induced ECs by inhibiting
   TGF‐β signaling [[70]21]. The potential application of TGF‐β inhibitors
   in the preconditioning of EPCs is not yet fully understood, and the
   underlying mechanism remains uncertain.

   In the present study, we aimed to investigate the optimal components of
   preconditioning conditions and develop an effective scheme for
   preconditioning to obtain EPC‐like cells capable of tolerating the high
   glucose and low nutrient stress in a DW microenvironment. We analyzed
   the biological characteristics of these preconditioning‐induced
   EPC‐like cells (pEPCs), explored the underlying mechanisms, and
   evaluated the therapeutic potential of pEPCs in promoting
   neovascularization and skin regenerative repair in diabetic mice.

2. Results

2.1. Preconditioning Cell With TGF‐β Inhibitor SB431542 Under NDHG Conditions
Enhances Endothelial Viability

   To find effective preconditioning methods that improve EC tolerance to
   harsh environments, we established the nutrient‐deficient and high
   glucose‐containing (NDHG) conditions that mimic the pathological
   condition observed in DW (Figure [71]S1), and the viability of ECs
   decreased with the increasing concentration of d‐glucose. Particularly,
   when the glucose concentration reached 75 mM, an increased number of
   apoptotic cells were observed floating in the culture supernatant.
   These results suggest that NDHG preconditioning alone is ineffective
   for enhancing the survival of ECs.

   To improve the preconditioning conditions, we tested some small
   molecule compounds that have been reported to be beneficial for ECs
   [[72]22, [73]23, [74]24]. Based on the results of cell viability
   (Figure [75]S2), we identified the TGF‐β inhibitor sb431542 and its
   optimal concentration (Figure [76]S3). We found that ECs cultured under
   NDHG conditions with sb431542 (hereafter referred to as sb‐NDHG)
   exhibited high cell viability and retained their cobblestone‐like
   morphology, comparable to parental ECs (Figure [77]1A,B). In our
   supplementation experiments, the activation of the TGF‐β signaling
   pathway in NDHG‐ECs supports the rationale for using sb431542 (Figure
   [78]S4). This result was further confirmed by live/dead cell staining
   and the TUNEL staining (Figure [79]1C–F). Moreover, flow cytometry
   analysis revealed that the number of apoptotic cells in
   sb‐NDHG‐preconditioned ECs (pECs) decreased from nearly 70% to less
   than 5% compared to NDHG‐preconditioned ECs (NDHG‐ECs)
   (Figure [80]1G,H). Our further research demonstrates that the
   combination of NDHG conditions and sb431542 enhances EC survival,
   rather than sb431542 alone (Figure [81]S5).

FIGURE 1.

   FIGURE 1
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   Sb‐NDHG preconditioning restores the viability of ECs under stressful
   conditions. (A) Representative brightfield images of cells cultured in
   different media for two days. (B) Cell viability was evaluated using
   the CCK‐8 assay. (C) Representative brightfield images of live/dead
   fluorescence staining. (D) Quantification of cell viability based on
   the live/dead staining results. (E) Cell apoptosis was tested by TUNEL
   staining. (F) Quantification of cell apoptosis based on the TUNEL
   staining results. (G) Cell apoptosis was assessed by flow cytometric
   analysis. (H) Quantification of cell apoptosis based on the flow
   cytometric analysis results. (I) Immunostaining of JC‐1 fluorescent
   probe (red) and Annexin V (green) in cells from different groups. Data
   are represented as mean ± SEM. **p < 0.01, ***p < 0.001 compared with
   the corresponding control. Scale bars represent 50 µm. ECs, hUVECs,
   NDHG‐ECs), NDHG‐preconditioned ECs; pECs, sb‐NDHG‐preconditioned ECs.

   Changes of mitochondrial membrane potential (MMP) also serve as a
   crucial indicator of cell apoptosis. ECs stained with the MMP
   fluorescent probe (JC‐1) exhibited bright red fluorescence
   (Figure [83]1I). However, in NDHG‐ECs, the fluorescence intensity of
   JC‐1 decreased, while the green fluorescence (annexin V‐FITC+)
   increased. Conversely, the fluorescence intensity of JC‐1 in pECs was
   upregulated, indicating a recovery of cell viability. Collectively,
   these results indicated that preconditioning with sb‐NDHG could confer
   a survival advantage to ECs under stressful conditions.

   While screening small molecular compounds, we also discovered that
   other TGF‐β inhibitors, including SB525334 and A83‐01, enhance EC
   viability under NDHG conditions (Figure [84]S6). In our follow‐up
   study, we chose sb431542 to further investigate whether and how sb‐NDHG
   preconditioning affects ECs.

2.2. Sb‐NDHG Preconditioning Preserves Endothelial‐Specific Characteristics
in pECs Under Stressful Conditions

   EndMT is a complex process in which ECs reduce the expression of
   endothelial markers and undergo trans‐differentiation into
   myofibroblasts or mesenchymal cells involved in pathological fibrosis.
   This process can be robustly induced by hyperglycemia [[85]25].
   Although sb‐NDHG preconditioning has been shown to increase the
   viability of ECs, it remains unclear whether sb‐NDHG preconditioning
   can inhibit EndMT and preserve the original properties of ECs during
   long‐term culture.

   To test this hypothesis, endothelial surface markers were detected
   using flow cytometry (Figure [86]2A). Compared with ECs, pECs expressed
   the same levels of CD31, VE‐cad, KDR, and NRP1, while displaying
   negative expression of CD133 (Figure [87]2B). Notably, the expression
   of CD34, a well‐known marker of EPCs, gradually increased from
   approximately 20% at passage 0 (P0) to nearly 80% at passage 3 (P3) in
   pECs. Immunofluorescent staining of pECs, from P1 to P3 revealed the
   presence of typical endothelial markers, such as CD31, VE‐cad, and vWF.
   Notably, CD34^+ cells, initially scattered among pECs at passage 1
   (P1), gradually increased to became the predominant cell type in pECs
   at passage 3 (P3) (Figure [88]2C).

FIGURE 2.

   FIGURE 2
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   Sb‐NDHG preconditioning contributes to maintaining the
   endothelial‐specific characteristics of ECs. pECs were passaged three
   times and are defined as pECs P1, pECs P2, and pECs P3. (A) Endothelial
   surface markers were detected using flow‐cytometry analysis. (B)
   Quantification of endothelial surface markers based on the results of
   flow cytometry analysis. (C) Immunostaining of endothelial‐specific
   markers. (D) Dil‐acLDL uptake assay in the indicated cells. (E) EndMT
   signature genes were detected by qRT‐PCR. Data are represented as mean
   ± SEM. *p < 0.05, ***p < 0.001 compared with the corresponding control.
   Scale bars represent 50 µm.

   Compared to ECs, pECs showed no difference in the uptake of
   Dil‐acetylated low‐density lipoprotein (ac‐LDL) (Figure [90]2D). In
   addition, EndMT signature genes, including α‐SMA, COLL‐1, Snail, Slug,
   and ZEB, were not significantly upregulated in pECs at P3
   (Figure [91]2E). These results suggest that pECs can stably maintain
   endothelial‐specific properties under stressful conditions.

2.3. Sb‐NDHG Preconditioning Enhanced pECs Angiogenic Activities and
Oxidative Stress Resistance Under Stressful Conditions

   Angiogenesis is a hallmark of endothelial function and is seriously
   impaired in DW [[92]26]. To evaluate the ability of angiogenesis, we
   conducted a Matrigel tube formation assay under NDHG conditions. Over a
   12‐h observation period, ECs and pECs P1 failed to form any tubular
   structures, while only a few tube‐like structures were formed by pECs
   P2 (Figure [93]3A). Notably, pECs P3 exhibited a significant increase
   in both tube length and the number of loops in the tubular structures
   (Figure [94]3B). When cultured in endothelial growth medium (EGM), the
   tubular structures formed by pECs P3 were maintained for at least 44 h,
   which was longer than that of other cells (Figure [95]S7). Based on
   their excellent tube‐formation ability and the high expression of CD34,
   pECs P3 could be considered as preconditioned EPC‐like cells (pEPCs)
   with enhanced endothelial function.

FIGURE 3.

   FIGURE 3
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   Long‐term Sb‐NDHG preconditioning facilitated the generation of pEPCs
   with enhanced endothelial function. (A) The tube formation ability of
   the indicated cells was evaluated under NDHG conditions. (B)
   Quantification of tube lengths and the number of loops was performed.
   (C) Flow cytometry analysis and quantification of ROS production in
   pEPCs were conducted. (D) Immunostaining of JC‐1 fluorescent probe
   (red) and annexin V (green) in pEPCs. (E) The mRNA levels (top) and the
   protein levels (bottom) of Nrf2 and SOD were tested. Data are
   represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared
   with the corresponding control. Scale bars represent 50 µm. pEPCs,
   preconditioned EPC‐like cells

   Hyperglycemia impairs EC function mainly through the accumulation of
   reactive oxygen species (ROS) [[97]27]. The level of ROS in NDHG‐ECs
   was significantly higher than that in ECs (Figure [98]3C). The
   increased accumulation of ROS was linked to mitochondrial oxidative
   damage, which resulted in the loss of MMP in NDHG‐ECs (Figure [99]3D).
   Furthermore, the activity of nuclear factor erythroid 2 like 2 (Nrf2),
   a key transcription factor that regulates hundreds of genes involved in
   oxidative stress, was reduced [[100]28] (Figure [101]3E). Consequently,
   the level of superoxide dismutase (SOD), a potent antioxidant
   transcribed from one of Nrf2's target genes, was also reduced, as
   analyzed by Quantitative Real‐time PCR (qRT‐PCR) and western blot
   (Figure [102]3E). Notably, the elevated ROS levels were significantly
   reduced in pEPCs, accompanied by the restoration of MMP as well as an
   increase in the expression levels of Nrf2 and SOD‐2 at both the mRNA
   and protein levels (Figure [103]3C–E). These findings demonstrate that
   sb‐NDHG preconditioning promotes the generation of pEPCs and enhances
   their endothelial function.

   Two essential factors for cell‐based therapy are the angiogenic
   potential and large‐scale production of cell products. Our results
   indicate that pEPCs can expand in vitro for over five generations while
   maintaining stable biological properties (Figure [104]S8). The pEPCs
   maintained their secretion of pro‐angiogenic cytokines at levels
   comparable to those of normal parental ECs (Figure [105]S9).
   Furthermore, the processes of freezing and thawing did not have a
   detrimental effect on cell proliferation, the expression of cell
   surface markers, or tube formation ability (Figure [106]S10). These
   findings strongly support pEPCs as a promising source of seed cells for
   regenerative repair therapies.

2.4. Protective Effects of sb‐NDHG Preconditioning on pEPCs via Partial
Activation of the PI3K and Nrf2 Signaling Pathway

   To gain insight into the molecular mechanism underlying the protective
   effects of sb‐NDHG preconditioning for pEPCs, we performed whole‐genome
   transcriptome analysis on ECs, NDHG‐ECs, and pEPCs. We identified the
   differentially expressed genes (DEGs) as those with p < 0.05 and a fold
   change > 2. Global gene expression analysis revealed that pEPCs
   clustered more closely with ECs than with NDHG‐ECs, as illustrated by
   hierarchical clustering (Figure [107]S11A). To investigate the altered
   genes in cells, DEGs between pEPCs and NDHG‐ECs were subjected to Gene
   Ontology (GO) analysis. The DEGs were primarily associated with cell
   proliferation, vasculature development, regulation of angiogenesis, and
   tube morphogenesis (Figure [108]4A). To identify the signaling pathways
   associated with these effects, we conducted a Kyoto Encyclopedia of
   Genes and Genomes (KEGG) pathway enrichment analysis on the identified
   DEGs. The PI3K‐AKT signaling pathway was enriched among the cellular
   pathways, The PI3K‐AKT pathway was suppressed in NDHG‐ECs (Figure
   [109]S11B) and activated in pEPCs (Figure [110]4B). This finding was
   further verified by western blot analysis (Figure [111]4C). These
   results indicate that the protective effect of sb‐NDHG preconditioning
   partly relies on the upregulation of the PI3K‐AKT signaling pathway in
   pEPCs.

FIGURE 4.

   FIGURE 4
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    sb‐NDHG preconditioning enhances pEPC resilience through modulation of
   the PI3K‐Nrf2 pathway. (A) GO enrichment analysis of RNA‐seq data of
   DEGs between pEPCs and NDHG‐ECs. (B) KEGG pathway enrichment analysis
   of RNA‐seq data of DEGs between pEPCs and NDHG‐ECs. (C) Western blot
   was conducted to detect the expression of PI3K, P‐PI3K, AKT, and P‐AKT
   in pEPCs and NDHG‐ECs. (D) Cell viability was evaluated using the CCK‐8
   assay. (E) Immunostaining of endothelia markers CD31 and CD34 in
   indicated cells. (F and G) Tube formation assay. Tube lengths and the
   loop number were measured 6 h after seeding under NDHG conditions. (H)
   mRNA levels of Nrf2 and SOD were tested by qRT‐PCR. (I) Protein levels
   of Nrf2, SOD, P‐PI3K, and P‐AKT were determined by western blot
   analysis. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01,
   ***p < 0.001 compared with the corresponding control. Scale bars
   represent 50 µm.

   To determine whether the activation of the PI3K‐AKT signaling pathway
   is essential for the protective effects of sb‐NDHG preconditioning, we
   pretreated ECs with the PI3K inhibitor LY294002 (LY) to block the
   PI3K‐AKT signaling. Following this treatment, the cells underwent
   sb‐NDHG preconditioning and referred to as LY+pECs. Compared to pEPCs
   and ECs, LY+pECs exhibited a reduced proliferation rate, lower CD34
   expression (Figure [113]4D,E), as well as impaired tube formation
   (Figure [114]4F,G). Furthermore, according to the results of qRT‐PCR
   and western blot analysis, the expression levels of Nrf2 and SOD in
   LY+pECs were reduced, similar to those in NDHG‐ECs (Figure [115]4H,I).
   Collectively, these results suggest that sb‐NDHG preconditioning
   promotes the activation of PI3K‐AKT signaling pathway, thereby
   upregulating its downstream signal pathway, which appears to play a
   crucial role in the protective effects for pEPCs mediated by sb‐NDHG
   preconditioning.

2.5. pEPCs Exhibit Longer in Vivo Survival and Retention Than ECs in Diabetic
Mice

   The survival and retention of cells in vivo are important indicators of
   their potential use and effectiveness in cell‐based therapy for a
   variety of refractory diseases, including DW [[116]29, [117]30]. To
   assess the survival and retention of pEPCs in vivo, CM‐Dil‐labeled
   pEPCs were subcutaneously injected into the dorsal skin of diabetic
   mice. Mice injected with CM‐Dil‐labeled ECs served as controls
   (Figure [118]5A). Over an observation period of 24 days, we
   continuously monitored the fluorescence signals at the injection sites
   of the pEPCs. In contrast, the fluorescence signals from the ECs
   decreased dramatically over time and were almost undetectable by the
   19th day (Figure [119]5B). Quantitative analysis of the fluorescence
   signals revealed a trend consistent with these observations
   (Figure [120]5C). Based on these results, we infer that pEPCs maintain
   stability for a longer duration than ECs in the non‐wounded skin of
   diabetic mice following administration.

FIGURE 5.

   FIGURE 5
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   Evaluation of the survival/retention of pEPCs in diabetic mice. (A) A
   description of the experimental scheme. (B) CM‐Dil‐labeled pEPCs or
   CM‐Dil‐labeled ECs were subcutaneously injected into the dorsal skin of
   diabetic mice (n = 6). The fluorescence signal of each recipient mouse
   was measured at the indicated time points. (C) Quantitative analysis of
   the fluorescent signal in each recipient mouse over a 24‐day period.
   (D) CM‐Dil‐labeled pEPCs or CM‐Dil‐labeled ECs were intramuscularly
   injected into the ischemic hind limbs of diabetic mice (n = 6).
   Fluorescence signal of each recipient mouse was measured at the
   indicated times post‐cell injection. (E) Quantitative analysis of
   fluorescence signal in each recipient mouse over a 24‐day period. Data
   are represented as means ± SEM. *p < 0.05; **p < 0.01 compared with the
   corresponding control.

   To further assess whether pEPCs can survive in the areas of lesions,
   CM‐Dil‐labeled pEPCs were injected intramuscularly into the ischemic
   limbs of diabetic mice. CM‐Dil‐labeled ECs as controls
   (Figure [122]5A). The fluorescence signals from ECs remained relatively
   stable for about 7 days after injection, and then began to decrease
   exponentially, reaching baseline levels within 16–20 days. In contrast,
   the fluorescence signals of pEPCs persisted for a longer duration,
   remaining detectable up to 24 days post‐injection (Figure [123]5D,E).
   Biosafety of pEPCs is an important issue that requires attention. To
   assess the safety of pEPCs, pEPCs were transplanted into diabetic model
   mice for a duration of 3 months, in which that levels of alanine
   aminotransferase (ALT), aspartate aminotransferase (AST), and
   immunoglobulin G2 (IgG2) were not significantly different from those in
   normal mice, and no tumor‐like structures were observed in the organs
   of the model mice (Figure [124]S12). Collectively, these findings
   demonstrate that pEPCs exhibit higher levels of survival and retention
   as well as biosafety in vivo.

2.6. pEPC Transplantation Enhances Blood Flow Recovery in Ischemic Limbs of
Diabetic Mice

   To assess the therapeutic efficacy of pEPC transplantation, we
   administered pEPCs or ECs intramuscularly into the ischemic hind limbs
   of diabetic mice that had been subjected to femoral artery ligation.
   Mice injected with an equivalent volume of PBS served as controls, and
   blood flow was monitored using laser Doppler imaging (Figure [125]6A).
   On the zeroth day, the average perfusion rate (PR) of blood flow in all
   mice that were treated by surgery was approximately 20.0%, confirmed
   that severe limb ischemia was successfully induced in the diabetic
   mice. Compared with the PBS‐treated and ECs‐treated mice, the average
   PR in pEPCs‐treated mice gradually increased and peaked 14 days
   post‐injection (Figure [126]6B), indicating that pEPCs significantly
   enhanced the recovery of blood flow in ischemic hindlimbs.

FIGURE 6.

   FIGURE 6
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   pEPCs enhance blood flow recovery in a diabetic mouse model of hindlimb
   ischemia. (A) Representative Doppler images of mice in different groups
   at 0, 7, 14, and 21 days post‐treatment (n = 6). The white square frame
   denotes the ischemic limbs. (B) Perfusion rates of ischemic limbs at
   days 0, 7, 14, and 21 after cell treatment. (C) Immunofluorescence
   staining for endothelial‐specific CD31 in ischemic limbs implanted with
   ECs, pEPCs, or PBS. (D) Quantification of vessel density and the
   percentage of CD31+ vessels. (E) Representative images showing the
   incorporation of pEPCs into the host microvasculature. (F) Expression
   of angiogenesis factors in ischemic muscle tissue was tested by
   qRT‐PCR. Data are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p
   < 0.001 compared with the corresponding control. Scale bars represent
   20 µm.

   The improvement in limb perfusion was further confirmed with
   immunofluorescence quantification of total vascular density using the
   endothelia‐specific CD31 antibody (Figure [128]6C). Quantitative
   analysis showed that mice transplanted with pEPCs had a capillary
   density nearly three times higher than that of mice treated with PBS,
   and approximately 1.5 times higher than that of mice treated with ECs
   (Figure [129]6D). To verify the integration of pEPCs into the host
   vasculature, CM‐Dil‐labeled pEPCs were injected intramuscular into the
   ischemic limbs of diabetic mice. Alexa568‐isolectin‐B4 was perfused
   into the mice 14 days post pEPCs transplantation to identify all blood
   vessels. We observed that CM‐Dil+ human‐derived vessels were
   interspersed among the murine vessels in the ischemic limb. This
   finding indicates that pEPCs have the potential to incorporate with
   host microvasculature (Figure [130]6E).

   Comprehensive expression of angiogenic growth factors is beneficial for
   promoting angiogenesis in ischemic limbs. Seven days after pEPCs
   transplantation, RNA was extracted from the limbs of each group.
   qRT‐PCR analysis revealed that in comparison with PBS‐treated group,
   the expression of the genes VEGF, bFGF, and HGF in the pEPCs‐treated
   group was significantly upregulated (Figure [131]6F). These results
   further confirm the excellent therapeutic effects of pEPCs in the
   treatment of DW.

2.7. Transplantation of pEPCs Enhances Neovascularization and
Re‐Epithelialization in Diabetic Wound Healing

   To assess the therapeutic potential of pEPCs in DW healing, we created
   a 10‐mm full‐thickness skin wound on the dorsum of each diabetic mouse
   and then administered different treatments (Figure [132]7A). First,
   pEPCs and ECs were pre‐planted on an absorbable gelatin sponge (AGS), a
   material widely used in bone regeneration and nerve repair [[133]31].
   After 3–4 days of culture in a shaker, pEPCs proliferated within the
   AGS and formed typical tubular structure (Figure [134]7B). The mice
   successfully modeled were randomly divided into four groups, which
   received (1) pEPCs‐loaded AGS (pEPC‐AGS), (2) ECs‐loaded AGS (EC‐AGS),
   (3) pure AGS, or (4) PBS, respectively. The AGS was placed over the
   wound and affixed to the skin using interrupted 6–0 nylon sutures. The
   wounds were observed with digital photographs (Figure [135]7C, left).
   Quantitative analysis showed that the wound treated with pEPC‐AGS was
   approximately 64% closed after 9 days and nearly fully closed after 12
   days (Figure [136]7C, right), indicating that pEPC‐AGS healed the wound
   much faster than other treatments.

FIGURE 7.

   FIGURE 7
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   pEPCs accelerate wound healing in diabetic mice. (A) Illustration of
   the experimental protocol. (B) Immunostaining of endothelial markers in
   pEPCs cultured in the shaking bed for 3 days. White arrows indicate the
   typical tubular‐like structure formed by pEPCs in AGS. (C)
   Representative wound images at different time points post‐wounding
   (left) and quantification of wound closure rate (right) (n = 6). (D)
   Representative images of capillaries stained with CD31 antibody (red)
   in skin sections. (E) Quantification of the number of capillaries in
   five randomly selected areas of each skin sample. (F) Harvested skin
   tissues (12 days post injury) were cross‐sectioned to assess the degree
   of re‐epithelialization. High‐magnification views of the indicated
   regions are shown (bottom). (G) Quantification of re‐epithelialization
   of skin sections. (H) Evaluation of collagen deposition with Masson's
   trichrome dyes. (I) Quantification of collagen deposition. Data are
   presented as mean ± SEM. *p < 0.05, ***p < 0.001 compared with the
   corresponding control. Scale bars represent 50 µm in (B) and (D),
   500 µm in (F), and 1 mm in (I). EC‐AGS, ECs‐loaded AGS; pEPC‐AGS,
   pEPCs‐loaded AGS.

   Angiogenesis plays a crucial role in wound regeneration. The
   improvement of angiogenesis in DWs was further confirmed 12 days
   post‐wounding with vascular staining with a CD31 antibody
   (Figure [138]7D). Quantitative analysis of capillary density, which is
   defined as the number of CD31‐positive cells per square millimeter,
   showed that wounds transplanted with pEPCs exhibited a higher capillary
   density than other groups (Figure [139]7E).

   H&E staining was performed to evaluate the degree of re‐epithelization
   (Figure [140]7F). Notably, by 12 days post‐wounding, the wounds treated
   with pEPC‐AGS exhibited intact newly formed epidermis and dermis,
   complete with hair follicles. In contrast, wounds treated with either
   PBS or AGS alone did not develop a new epidermis (Figure [141]7G).
   Higher magnification images clearly depicted the extent of
   re‐epithelization across the groups (Figure [142]7F). Masson's
   trichrome staining was utilized to detect collagen deposition,
   revealing a greater quantity of collagen fibers in wounds treated with
   pEPC‐AGS compared to other groups (Figure [143]7H). Quantification of
   the average intensity of Masson‐stained areas further confirmed the
   superior efficacy of pEPC‐AGS in promoting collagen deposition during
   wound healing (Figure [144]7I). These data indicate that the
   combination of pEPC with AGS accelerates cutaneous wound healing in
   diabetic mice.

3. Discussion

   Cell therapy has provided a considerable opportunity for DW treatments,
   for which EPC‐based therapy is uniquely suited to, by allowing a more
   precise route to the area of ischemia, increase in vivo angiogenesis
   and the promotion of tissue repair [[145]32, [146]33]. However, poor
   cell survival and low engraftment of EPCs at the harsh and hostile
   environment after transplantation limit their regeneration potential
   [[147]9, [148]34]. Genetic modification and cellular reprogramming, as
   well as preconditioning, are strategies that could potentially optimize
   cell‐based therapy. Our study demonstrates for the first time that
   preconditioning of ECs under NDHG condition with sb431542 is required
   to induce the conversion of ECs into EPC‐like cells. These pEPCs
   acquire antioxidant capacity, maintain cell viability, and demonstrate
   stable tube formation ability in stressful environments. Importantly,
   pEPCs are more effective for blood vessel formation and tissue repair
   in diabetic mouse models. Collectively, this study provides a simple
   and effective method and can promote pEPC therapeutic functions to help
   to push them toward a more beneficial therapeutic phenotype for the
   treatment of DW.

   Increasing evidence has shown that preconditioning, including exposure
   to hypoxia, pharmacological or chemical agents, growth factors, and
   hormones, can optimize the therapeutic efficacy of transplanted cells.
   Among them, hypoxic‐preconditioning is highly studied. In these
   studies, hypoxic‐preconditioned stem cells showed improved survival and
   enhanced differentiation. Indeed, the ischemic area is strongly
   hypoxic. When EPCs are exposed to hypoxia, cellular adaptation to harsh
   conditions is triggered through the upregulation of HIF‐1α, which would
   activate the HIF‐1a‐VEGF signal pathway and promote the secretion of
   angiogenic factors VEGF and bFGF. This alleviates endothelial injury
   caused by ischemic hypoxia in ischemic conditions, such as acute
   myocardial ischemia. However, in the DW microenvironment, EPCs
   gradually lose their protective regulatory function due to chronic
   hypoxia, resulting in insufficient secretion of angiogenic factors.
   Consequently, after EPCs transplantation, these cells encounter
   survival challenges due to not only elevated glucose levels but also
   reduced growth factors in the pathological microenvironment. To
   simulate the harsh microenvironment of DW, we replaced hypoxia with
   nutrient deprivation and high‐glucose conditions. In this study, ECs
   were preconditioned under sb‐NDHG condition, which promoted cellular
   adaptation to the harsh environment prior to in vivo transplantation by
   amplifying beneficial signaling pathways. The activation of the
   PI3K‐Nrf2/SOD signaling pathway in pEPCs improved stress tolerance and
   enhanced their therapeutic potential in vivo. This method aligns with
   the current popular method of cell preconditioning, also known as
   injury‐induced protection, indicating that NDHG conditions are a
   critical element in this preconditioning method.

   In this study, we also found that inhibition of TGF‐β signaling plays a
   crucial role in this preconditioning method. Several studies have
   provided evidence that inhibition of TGF‐β signaling helps maintain the
   viability and proliferation capacity of ECs. Other studies have
   revealed that the activation of TGF‐β signaling favors
   hyperglycemia‐induced endothelial‐to‐mesenchymal transition as well as
   hyperglycemia‐induced EC metabolic memory [[149]25, [150]35], which
   impedes the proliferation of ECs and abrogates their angiogenic
   function [[151]20]. Our findings indicate that under NDHG conditions,
   TGF‐β signaling in ECs is activated, suggesting a correlation between
   the activation of TGF‐β signaling and the reduced activity of ECs.
   Based on previous research and our experimental results, sb431542, a
   common inhibitor of the TGF‐β signaling pathway, was selected to
   include in our preconditioning protocol. The results indicate that
   sb431542 can enhance the viability of ECs cultivated under NDHG
   conditions. Prolonging the culture time allowed the acclimated ECs to
   gradually increase CD34 expression, stabilize their tube formation
   capability, and transform into enhanced vasculogenic EPC‐like cells.

   Conversely, if NDHG conditions are replaced with EC‐culture medium and
   only sb431542 is added, ECs will not significantly increase the
   expression of CD34 and improve their adaptability to NDHG conditions,
   even with prolonged culture time. The results indicate that the
   compound sb431542 is a key component in this preconditioning protocol
   and works synergistically with the NDHG condition to induce the
   formation of pEPCs.

   In addition, some other small molecule compounds that target other
   signaling pathways, such as AMPK signaling, have been reported to
   protect against high glucose or oxidative stress‐induced endothelial
   dysfunction and are considered drug candidates for cardiovascular
   disorders [[152]36]. In this study, three widely studied small molecule
   compounds, including SRT‐1460, MDL‐28170, and lovastatin, were chosen
   to examine whether they could exert effects similar to those of
   sb431542. Our observations reveal that none of them were effective in
   stabilizing EC viability under NDHG conditions, which makes it even
   less likely that they can enhance EC adaptability to harsh environments
   after preconditioning. These results confirm that the TGF‐β inhibitor
   sb431542 is effective and a vital part of our preconditioning method.

   The primary challenge in cell‐based therapy is the extremely low rate
   of survival and retention of cells in the wound tissue, whereas the
   survival and retention of pEPCs were significantly increased than
   parental ECs, not only in the subcutaneous tissue but also in the
   ischemic limbs of diabetic mice, which is crucial for the success of
   cell therapy [[153]29, [154]30, [155]37]. Moreover, the beneficial
   effects of pEPCs could be in part attributed to their ability to
   increase the paracrine factors in the bloodstream at the wound site,
   such as VEGF and bFGF, and their differentiation into vascular cells
   that integrate into the neovascular system. This study demonstrates
   that the positive effects of pEPCs are likely to be multi‐factorial.

   With advancements in technology, several studies have explored new
   methods to generate autologous ECs from pluripotent stem cells
   [[156]38, [157]39], or reprogrammed non‐endothelial cells [[158]40,
   [159]41, [160]42]. These approaches offer the potential to obtain
   pEPCs. However, there is still a long road ahead, and further research
   is needed to address important aspects such as comparative studies of
   optimal delivery routes, determining the appropriate cell dosage, and
   exploring the combination of pEPCs with biomaterials or growth factors.
   These investigations are crucial to ensure the effectiveness and safety
   of pEPC transplantation in the future.

4. Conclusions

   In summary, the present study provides a simple and effective
   preconditioning protocol for obtaining functionally enhanced pEPCs, and
   they hold promise as an alternative cell source for the treatment of
   diabetic ischemic diseases.

5. Materials and Methods

5.1. Cell Culture and Treatments

   Human umbilical cord was obtained from health donors in West China
   Hospital, Sichuan University, upon consent of its donor according to
   procedures approved by the Medical Ethics Committee, Sichuan
   University. Human umbilical vein endothelial cells (hUVECs, abbreviated
   as ECs) were isolated as described and cultured with EGM, which is
   comprised of endothelial basal medium (EBM, ScienCell, 1001), 5% fetal
   bovine serum (FBS; ScienCell, 0025), and 1% endothelial cell growth
   supplements (ECGS; ScienCell, 1052). The nutrient‐deficient and high
   glucose‐containing (NDHG) conditions was used to simulate the
   pathological condition of DW. Specifically, extra 50 mM d‐glucose was
   added into EBM, which contained only 2% FBS and no ECGS.

   To screen small molecule compounds, ECs were cultured under NDHG
   conditions, each supplemented with a different small molecule. Cell
   viability was evaluated using Cell Counting kit‐8 (CCK8), TUNEL
   staining, flow cytometry, and live/dead staining according to the
   manufacturer's instructions. Important information about small molecule
   compounds is listed in Table [161]S1.

5.2. In Vitro Endothelial Functional Assays

   The uptake of Dil‐acLDL was assessed by incubating cells with 10 µg/mL
   of ac‐LDL (Invitrogen) for 4 h before detection. For Matrigel tube–like
   formation in vitro assay, pECs at passages 1, 2, and 3 were trypsinized
   into single cells. ECs cultured in EGM were used as controls. The
   resuspended cells (5 × 10^4/100 µL) were seeded on top of a thin layer
   of Matrigel with either EGM or NDHG conditions. The formation of
   tube‐like structures was imaged under brightfield microscopy at the
   indicated time points. The total tube length and the number of loops
   were counted and quantified.

5.3. Flow Cytometry Analysis

   To detect cells phenotypes, pECs and ECs were trypsinized into single
   cells and incubated with fluorescence‐labeling mAbs for 30 min on ice
   in the dark. The fluorescence intensity was measured using a flow
   cytometer (BD Biosciences) and analyzed using FlowJo software
   (Treestar, Ashland, OR, USA). The antibodies are listed in Table
   [162]S2.

   To detect the level of intracellular ROS, pECs, ECs, and NDHG‐ECs (ECs
   that were cultured under NDHG conditions) were collected and incubated
   with 10 µM H2DCF‐DA (Beyotime, China) for 20 min at 37°C in dark. After
   washing with cold PBS, the samples were analyzed using a flow
   cytometer. The level of intracellular ROS was calculated based on the
   mean cell fluorescence intensity. To evaluate cellular apoptosis rate,
   the dissociated cells that cultured in different conditions were
   incubated with Annexin V‐FITC apoptosis detection kit (Beyotime, China)
   for 10 min in dark at room temperature (RT), and then analyzed using a
   flow cytometer.

5.4. Immunofluorescence and Histological Analyses

   For immunofluorescence staining, cells fixed with 4% PFA were blocked
   with normal goat serum (NGS, ThermoFisher Scientific) for 30 min at RT,
   and subsequently stained with primary monoclonal antibodies (mAbs) at
   4°C overnight. After thorough washing with PBS, the cells were
   incubated with secondary mAbs for 1 h at RT in the dark. Cellular
   nuclei were stained with DAPI (Roche Basel, Switzerland). For blood
   vessels immunostaining, O.C.T‐embedded tissue was cut into 10‐µm‐thick
   sections. Sections were then fixed with ice‐cold acetone for 10 min
   before being stained with the primary and secondary mAbs. The sections
   were visualized using a laser scanning confocal microscope. The
   antibodies used are listed in Table [163]S2.

5.5. Western Blot Analysis

   Western blot analysis was used to detect the expression of specific
   proteins in cells following different treatments, according to standard
   procedures. Protein concentrations were measured using a BCA protein
   assay kit (Beyotime). A total of 30 µg of protein was separated using a
   12% SDS‐PAGE gel, then transferred to nitrocellulose membranes. After
   blocking with 5% fat‐free milk for 2 h, the membranes were incubated
   with primary antibodies at 4°C overnight, followed by incubation with
   an HRP‐conjugated secondary antibody (GE Healthcare, Buckinghamshire,
   UK). GAPDH mAb was used as an internal control. The primary and
   secondary antibodies are listed in Table [164]S2.

5.6. RNA‐Seq and Quantitative Real‐time PCR

   Total RNAs was extracted from cells and sequenced using the Solexa
   high‐throughput sequencing service (Majorbio, China). The RNA‐seq raw
   expression files and details have been deposited in NCBI GEO under
   accession nos. SRR22198247, SRR22198248, and SRR22198249. The RNA was
   reverse transcribed with M‐MLV Reverse Transcriptase (Takara).
   Quantitative real‐time PCR (qRT‐PCR) was performed using SYBR Green PCR
   Master Mix (Takara). Gene expression was normalized to β‐actin. Fold
   changes in the expression levels of target samples relative to control
   samples were calculated using the 2^−ΔΔCT method. The primers used are
   provided in Table [165]S3.

5.7. Diabetic‐Hindlimb Ischemia Models and Assessments

   Male C57BL/6J mice were purchased from Beijing Huafukang Bioscience.
   Animal experiments were performed in accordance with the protocols
   approved by the Animal Care and Use Committee of Sichuan University.
   Experimental Diabetes Mellitus was induced in 8‐week‐old mice by daily
   injection of streptozotocin (STZ, 70 mg/kg, Amresco, USA) for five
   consecutive days, following a 4‐week high‐fat diet regimen. Blood
   glucose levels were monitored after 4–5 weeks of STZ administration.
   Mice with sustained blood glucose levels above 250 mg/dL were selected
   and defined as STZ‐induced diabetic mice.

   Hind limb ischemia was induced by sequential ligation and excision of
   the femoral arteries, the femoral artery spans from its proximal origin
   at the branch of the external iliac artery to the distal point where it
   bifurcates into the saphenous and popliteal arteries [[166]5]. Right
   after arterial excision, 1.0 × 10^6 ECs or pEPCs (suspended in 150 µL
   PBS) were injected into ischemic limbs at three equally spaced points.
   Cyclosporine A was added to the drinking water at a concentration of
   210 mg/L, during the experimental period. The blood flow was tested
   weekly using the laser Doppler imaging (Laser Doppler Perfusion Imager
   System, Moor Instruments) for 3 weeks. The recovery of the perfusion
   ratio (PR) was calculated as the ratio of ischemic to non‐ischemic hind
   limb blood flow.

   To assess capillary perfusion, tissue sections from mice muscle were
   stained with human/mouse CD31 antibody. The number of capillaries in
   five random microscopic fields was counted and expressed as the number
   of capillaries per square millimeter of tissue. To test whether pEPCs
   could fusion into the host vascular system, the transplanted pEPCs were
   pre‐labeled with cell‐tracker CM‐Dil (Invitrogen, C7000). Fourteen days
   after cell transplantation, the mice were perfused with
   Alexa568‐Isolectin‐B4 to identify functional vessels. The ischemic
   limbs that received cell transplantation were dissected for
   cryosectioning and detected with fluorescence microscopy.

5.8. Diabetic Wound Assay

   A full‐thickness wound, 10 mm in diameter, extending through the
   panniculus carnosus, was created on the dorsum of the diabetic mice
   following an established model [[167]43]. Before transplantation, ECs
   or pEPCs were pre‐seeded onto an AGS and cultured on a shaking bed for
   3–4 days. The mice were randomized into four groups, wounds were
   covered with ECs‐AGS, pEPCs‐AGS, or pure AGS, and then affixed to the
   skin using 6‐0 nylon sutures (Ethicon, Somerville, NJ). Mice received a
   150 µL PBS injection directly into the subcutaneous layer adjacent to
   the wound bed as negative controls. Photographs of the wounds were
   taken during the experimental period. Wound area was quantitated using
   ImageJ software, and wound closure rates were calculated using the
   following formula: wound closure rate (%) = (So − St) × 100%/So. So
   represents the initial wound area, and St represents wound area
   measured at days 0, 4, 8, and 12 post‐treatment.

5.9. In Vivo Imaging

   To assess the survival of pEPCs in diabetic mice, CM‐Dil‐labeled pEPCs
   or CM‐Dil‐labeled ECs were injected into the ischemic limbs or
   non‐wounded dorsum skin of diabetic mice (a single injection of 1 ×
   10^6 cells/mouse). Fluorescent signals were detected at the indicated
   time points using IVIS Spectrum imaging system (Perkin Elmer, Waltham,
   MA). The images were obtained and analyzed, and the signal intensity
   was represented by the radiance unit of photons (p)/s/cm^2/sr.

5.10. Statistical Analysis

   For statistical analysis of our data, GraphPad Prism software (GraphPad
   Software Ltd., San Diego, CA, USA) was used. Each experiment was
   performed a minimum of three times. Results were presented as means ±
   standard errors of the mean. A Student's t test was conducted to detect
   statistical difference between two groups. one‐way ANOVA with
   Bonferroni's post hoc analysis was performed to detect statistical
   difference among three or more groups. The results were considered
   statistically significant when p < 0.05.

Author Contributions

   D. S. S. and F. Y. C. were involved in the conception and design,
   collection and assembly of data, data analysis and interpretation, and
   manuscript writing. Y. Z. performed GO and KEGG analysis. Q. Y. J. and
   F. D. contributed to cell isolation and characterization, provision of
   study material, and assembly of data. C. P., Y. X. Y., and L. Z. were
   involved in the animal experiments. P. S. Z., H. L. W., Q. X., and X.
   L. S. were responsible for manuscript writing and revision of the
   manuscript. H. X. D. were involved in the conception and design,
   manuscript writing, financial support, and final approval of
   manuscript. All authors have read and approved the final manuscript.

Ethics Statement

   Human tissue was obtained according to procedures approved by the
   Medical Ethics Committee, Sichuan University (the registration number
   K‐2018109‐1), and written informed consent was obtained from all
   participants. All animal experiments conducted in our study have been
   approved by the appropriate ethical committee, with the approval number
   2021366A.

Conflicts of Interest

   The authors declare no conflicts of interest.

Supporting information

   Supporting Fig 1: NDHG alone is not a suitable preconditioning
   condition for ECs.

   Supporting Fig 2: Candidate small molecule compounds have limited
   effects on the reduction of cell apoptosis.

   Supporting Fig 3: The optimum working concentration of sb431542 was
   determined.

   Supporting Fig 4: The detection of TGF‐β expression in endothelial
   cells under different conditions.

   Supporting Fig 5: The TGF‐β signaling pathway plays a crucial role in
   EPC preconditioning under NDHG conditions.

   Supporting Fig 6: Other inhibitors targeting TGF‐β pathway can
   effectively reduce cell apoptosis

   Supporting Fig 7: Long‐term preconditioning improves the stability of
   tube‐like structure in pECs

   Supporting Fig 8: pEPCs were expanded in large quantities in vitro with
   stable biological characteristics

   Supporting Fig 9: The detection of pro‐angiogenic cytokines.

   Supporting Fig 10: pEPCs maintain stable biological properties after
   thawing from cryopreservation.

   Supporting Fig 11: Hierarchical clustering and KEGG pathway enrichment
   analysis were performed on the RNA‐seq data for differentially
   expressed genes (DEGs) in the indicated cells.

   Supporting Fig 12: Assessment of the Biosafety of pEPC.

   Supporting Table 1: The list of small 218 molecule compounds.

   Supporting Table 2: The list of antibodies.

   Supporting Table 3: The list of PCR primer sequences.
   [168]MCO2-6-e70364-s001.pdf^ (2MB, pdf)

Acknowledgments