Abstract Endothelial progenitor cells (EPCs) are reduced in number and impaired in function in diabetic patients. Whether and how Nrf2 regulates the function of diabetic EPCs remains unclear. In this study, we found that the expression of Nrf2 and its downstream genes were decreased in EPCs from both diabetic patients and db/db mice. Survival ability and angiogenic function of EPCs from diabetic patients and db/db mice also were impaired. Gain- and loss-of-function studies, respectively, showed that knockdown of Nrf2 increased apoptosis and impaired tube formation in EPCs from healthy donors and wild-type mice, while Nrf2 overexpression decreased apoptosis and rescued tube formation in EPCs from diabetic patients and db/db mice. Additionally, proangiogenic function of Nrf2-manipulated mouse EPCs was validated in db/db mice with hind limb ischemia. Mechanistic studies demonstrated that diabetes induced mitochondrial fragmentation and dysfunction of EPCs by dysregulating the abundance of proteins controlling mitochondrial dynamics; upregulating Nrf2 expression attenuated diabetes-induced mitochondrial fragmentation and dysfunction and rectified the abundance of proteins controlling mitochondrial dynamics. Further RNA-sequencing analysis demonstrated that Nrf2 specifically upregulated the transcription of isocitrate dehydrogenase 2 (IDH2), a key enzyme regulating tricarboxylic acid cycle and mitochondrial function. Overexpression of IDH2 rectified Nrf2 knockdown- or diabetes-induced mitochondrial fragmentation and EPC dysfunction. In a therapeutic approach, supplementation of an Nrf2 activator sulforaphane enhanced angiogenesis and blood perfusion recovery in db/db mice with hind limb ischemia. Collectively, these findings indicate that Nrf2 is a potential therapeutic target for improving diabetic EPC function. Thus, elevating Nrf2 expression enhances EPC resistance to diabetes-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia likely via transcriptional upregulating IDH2 expression and improving mitochondrial function of diabetic EPCs. Keywords: Endothelial progenitor cell, Nuclear factor erythroid 2-related factor 2, Angiogenesis, Hind limb ischemia, Diabetes 1. Introduction Microvascular and macrovascular complications associated with diabetes mellitus are leading causes of morbidity and mortality for diabetic patients. These vascular complications are associated with dysregulation of vascular remodeling and vascular growth, decreased responsiveness to ischemic/hypoxic stimuli, impaired or abnormal neovascularization, and a lack of endothelial regeneration under diabetic conditions, indicating that promoting angiogenesis is a promising strategy for the therapy of diabetic vascular complications [[65]1]. Endothelial progenitor cells (EPCs), precursor of endothelial cells, are considered to play critical roles in physiological angiogenesis and endothelium homeostasis [[66]2]. EPCs facilitate angiogenesis by directly incorporating into ischemic sites to form neo-vessels [[67]3], and/or operate in a paracrine fashion by secreting proangiogenic factors [[68]4]. Local or systemic administration of EPCs from bone marrow (BM) [[69]5], cord blood [[70]6] or peripheral blood [[71]7] can enhance ischemic neovascularization and improve function of ischemic tissues in animals with hind limb ischemia (HLI) or myocardial infarction. However, the number of circulating EPCs is decreased and function is impaired in both type 1 [[72]8] and type 2 diabetes [[73]9], contributing to attenuated angiogenesis and delayed repair of the injured endothelium. Several explanations for diabetic EPC dysfunction have been proposed [[74]10], including excessive generation of reactive oxygen species (ROS) [[75]11], over-production of oxidized low density lipoprotein (LDL) [[76]12], defective nitric oxide pathway [[77]13], impaired EPC mobilization [[78]14], and aggravated inflammation [[79]15]. Although several medications have been tested to promote the function of diabetic EPCs, positive clinic outcomes of these treatments are limited [[80]16]. It is unlikely that EPC dysfunction under diabetic conditions is a consequence of a single independent mechanism, and thus, a more complete understanding of the underlying defects is required in order to ameliorate EPC dysfunction and improve therapeutic strategies for diabetic vascular complications. Our previous study demonstrated that impaired stromal cell-derived factor 1/C-X-C chemokine receptor type 7 (CXCR7) signaling is associated with EPC dysfunction under diabetic conditions. Elevating CXCR7 enhances EPCs resistance to diabetes-induced oxidative injury and improves the therapeutic efficacy of EPCs for diabetic ischemia treatment via activating nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway [[81]3]. Nrf2 is a basic region-leucine zipper-type transcription factor belonging to the Cap ‘n’ collar (CNC) family. It can translocate and accumulate in the nucleus and bind to the antioxidant responsive element (ARE) [[82]17] where it mediates the expression of more than 200 cytoprotective genes, including antioxidant proteins, phase I and II detoxification enzymes, transport proteins, proteasome subunits, chaperones, growth factors and their receptors, and other transcription factors [[83]18]. As a stress-responsive transcription factor, Nrf2 is the primary master of the inducible cell defense system. Whether and how Nrf2 serves a direct role in regulating the survival and function of EPCs under diabetic conditions remains unclear. Mitochondria play an essential role in the maintenance of normal cellular functions. Mitochondrial dysfunction is a key mediator of impaired EPC function under hypertensive conditions [[84]19]. Moreover, mitochondrial dysfunction increases electron leak from the mitochondrial respiratory chain and upregulates mitochondrial ROS (mtROS) production [[85]20], which are well-known inducers of impaired reendothelialization and angiogenic capacity of EPCs under diabetic conditions [[86]21]. To maintain mitochondrial quality and homeostasis, mitochondrial morphologies and metabolic status change rapidly in response to external insults through fusion and fission dynamics [[87]22,[88]23]. Mitochondrial fusion creates large mitochondria, while mitochondrial fission produces small mitochondria. The frequency of mitochondrial fusion and fission is dynamically balanced in healthy cells. However, under pathological conditions, significantly smaller or larger mitochondria have been observed. For example, exposure to excess nutritional environment, such as diabetes or obesity, promotes mitochondrial fission and decreases mitochondrial fusion, resulting in mitochondrial fragmentation and uncoupled respiration [[89]24,[90]25]. It remains largely unknown whether an altered mitochondrial size or imbalanced activities of fission or fusion contributes to EPC dysfunction induced by diabetes. Given that Nrf2 activation is positively associated with mitochondrial biogenesis and mitochondrial quality control [[91]26], we hypothesized that Nrf2 would improve angiogenic function of diabetic EPCs through improvement of mitochondrial dynamics. Thus, the aims of the present study were to investigate whether Nrf2 could rescue EPC dysfunction in diabetes and improve the angiogenic function of EPCs in diabetic limb ischemia and to reveal the possible mechanisms connecting EPC function and mitochondrial dynamics. 2. Materials and methods 2.1. Subject characteristics The patients suffering from type 2 diabetes (n = 8) were recruited from the First Affiliated Hospital of Chengdu Medical College (Chengdu, China). Peripheral blood (20 ml) was obtained from each participant prior to treatment. Written informed consent was obtained from all the study participants. Healthy individuals (n = 8) admitted to the same hospital only for preventive examinations were enrolled as controls. The baseline characteristics were shown in [92]Table S1. The study protocol was approved by the Research Ethics Committee of the First Affiliated Hospital of Chengdu Medical College. 2.2. Animals db/db male mice and their sex-matched littermate wild type (WT) control mice at age of 10–12 weeks were used in this study. All mice were housed in a 12:12-h light-dark cycle and specific pathogen-free facility. All animal procedures were approved by the Animal Policy and Welfare Committee of Chengdu Medical College or the Institutional Animal Care and Use Committee of the University of Louisville, which conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication No. 85–23, revised 2011). 2.3. Isolation and culture of human EPCs from peripheral blood EPCs were isolated from peripheral blood of healthy donors (Healthy-EPC) and diabetic patients (DM-EPC) according to protocols described in our previous studies [[93]3,[94]27,[95]28]. Briefly, blood samples (20 ml) were collected with heparin vacuum blood collection tube (BD Biosciences, San Jose, CA), and mononuclear cells (MNCs) were isolated by density gradient centrifugation using histopaque-1077 (Sigma-Aldrich, St. Louis, MO) within 2 h of sampling. MNCs were suspended in endothelial growth factor-supplemented media (EGM-2 bullet kit, Lonza, Basel, Switzerland) with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA) and seeded into 6-well plates precoated with 50 μg/mL fibronectin (Sigma-Aldrich, St. Louis, MO). Cells were maintained at 37 °C with 5% CO[2] in a humidified incubator. After 3 days of incubation, dead cells were washed away with phosphate buffered saline (PBS) and new medium was added. Medium was changed daily for 7 days, and then every 3 days. After 2 weeks of culture, late EPCs were harvested for identification as described in our previous studies [[96]3,[97]27,[98]28] and used for the following experiments. 2.4. Mouse BM-EPC isolation and culture EPCs were isolated from BM of WT (WT-EPCs) and db/db (db/db-EPCs) mice using density gradient centrifugation according to protocol described in our previous studies [[99]3,[100]29]. Briefly, BM of femurs and tibias were flushed twice using Hank's balanced salt solution (Thermo Fisher Scientific, Waltham, MA). MNCs were collected by density gradient centrifugation using histopaque-1083 (Sigma-Aldrich, St. Louis, MO) and seeded into 6-well plates precoated with 50 μg/mL fibronectin (Sigma-Aldrich, St. Louis, MO). Cells were maintained in EGM-2 bullet kit with 10% FBS at 37 °C with 5% CO[2] in a humidified incubator. After 7 days of culture, early EPCs were characterized as described in our previous studies [[101]3,[102]27] and used for the following experiments. 2.5. Lentiviral vector construction, virus production, and transfection To upregulate Nrf2 expression in EPCs from diabetic patients or db/db mice, the lentivirus vector containing sequences encoding human or mouse Nrf2 gene (Lv-Nrf2) was generated by Genechem (Shanghai, China). In addition, human isocitrate dehydrogenase 2 (IDH2) overexpression lentivirus vector (Lv-IDH2) was constructed to upregulate IDH2 expression in EPCs. The empty lentivirus vector was used as control (Lv-Ctrl). Human or mouse EPCs were transfected with the purified lentivirus carrying recombinant Nrf2, IDH2 or control vector overnight at multiplicity of transfection (MOI) of 50 with 5 μg/mL polybrene (Genechem, shanghai, China), and the medium was replaced with fresh growth medium 24 h after transfection. After transfection for 72 h, the expression of Nrf2 and IDH2 was detected by quantitative real-time PCR (qRT-PCR) and Western blot assay. To knockdown Nrf2 expression in EPCs from healthy subjects or WT mice, EPCs were transfected with the lentiviruses containing shRNA against Nrf2 (Nrf2-shRNA) constructed by Genechem (Shanghai, China). Meanwhile, lentiviruses containing shRNA against IDH2 (IDH2-shRNA) was used to knockdown IDH2 expression in EPCs. The lentiviruses containing nonsense shRNA (Ctrl-shRNA) was used as control. * The target sequences for Nrf2 or IDH2 and nonsense sequence as follows: * Human Nrf2-shRNA: 5′-GTCCAAAGAGCAGTTCAATGA-3′; * Mice Nrf2-shRNA: 5′-GCCTTACTCTCCCAGTGAATA-3′; * Human IDH2-shRNA: 5′-GCTGTACATGAGCACCAAGAA-3′; * Nonsense shRNA: 5′-TTCTCCGAACGTGTCACGT-3′; Transfection was performed following the procedure described above. After transduction for 72 h, the expression of Nrf2 and IDH2 was determined by qRT-PCR and Western blot assay. 2.6. Apoptosis assay EPCs were seeded on 6-well plates (1 × 10^5 cells/well) and maintained under culture conditions detailed in figure legends. The non-adherent cells were removed by washing with PBS. Subsequently, adherent cells were released with 0.25% trypsin without EDTA. EPCs were collected by centrifugation and stained with APC-conjugated Annexin V Apoptosis Detection Kit with propidium iodide (PI), according to the manufacturer's instructions (KeyGen BioTech, Nanjing, China). The apoptotic EPCs were detected by a flow cytometry (BD Accuri C6, San Jose, CA; or Beckman Coulter Cytoflex S, Krefeld, Germany). Early apoptotic cells were defined as AnnexinV^+/PI^−. 2.7. Angiogenesis assay in vitro The in vitro angiogenic capability of EPCs was determined by Matrigel tube formation assay. Briefly, 96-well plates were coated with growth factor-reduced Matrigel (100 μL/per well, BD Biosciences). EPCs were plated (2 × 10^4 cells/well) in 100 μL under culture conditions detailed in figure legends and incubated at 37 °C with 5% CO[2] for 12 h to form tubes. Images of tubes in each well were taken using an inverted microscopy (Nikon Eclipse E600, Nikon, Kanagawa, Japan). The tube lengths were calculated by Image J software (NIH, Bethesda, MD). 2.8. Quantification of mitochondrial morphology EPCs were seeded and grown in glass-bottom dishes. Mitochondrial morphology was visualized by incubating the cells with 200 nmol/L MitoTracker Red CMXRos probe (Life Technologies, Carlsbad, CA) for 30 min at 37 °C. Images were acquired using a confocal microscopy (LSM880NLO FLIM, Zeiss, German). The degree of mitochondrial interconnectivity (perimeter^2/4*π *area) as a measure of both length and degree of branching was calculated using ImageJ software as described in previous reports [[103]30,[104]31]. A degree of interconnectivity of 1 corresponds to a circular and unbranched mitochondrion, whereas a higher degree of interconnectivity indicates a longer and more branched mitochondrion [[105]31]. 2.9. Assay of mitochondrial membrane potential (ΔΨm) The ΔΨm of EPCs was determined by tetramethylrhodamine methyl ester (TMRM, Ex/Em 548/574 nm, Thermo Fisher Scientific, Waltham, MA) according to the instructions. Briefly, EPCs were rinsed twice with PBS and incubated with 100 nmol/L TMRM for 30 min, then cells were washed twice with PBS and detected by BD Accuri C6 flow cytometry. 2.10. mtROS determination mtROS production of EPCs was measured by staining with MitoSOX™ Red mitochondrial superoxide indicator (Ex/Em 510/580 nm, Thermo Fisher Scientific, Waltham, MA). Briefly, EPCs were washed twice with PBS, and then incubated with 5 μmol/L MitoSox for 30 min, after which the cells were washed twice with PBS and analyzed with flow cytometry. 2.11. Determination of oxidative stress To detect the ROS level of EPCs, dihydroethidium (DHE; Molecular Probes, Eugene, OR) probe was used to stain EPCs. DHE is cell permeable and able to react with superoxide to form ethidium, which in turn intercalates with DNA and produces nuclear fluorescence. EPCs were seeded on 24-well plates for 24 h, and then incubated with 5 μmol/L DHE in PBS for 30 min at 37 °C. Nuclear DHE positive staining indicates superoxide generation in cells. The fluorescence intensity was detected by a flow cytometry. 2.12. ATP extraction and quantification ATP content in EPCs was measured by an ATP Measurement Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacture's instruction. In brief, EPCs were plated in a 6-well plate, ATP was extracted with 200 μL ATP lysis buffer and detected by a luminometer. The concentration of ATP was normalized by the protein concentration measured with a BCA protein assay kit (Beyotime Biotechnology). 2.13. qRT-PCR Total RNA was extracted using Trizol reagent (RNA STAT 60, Tel-Test Inc., Austin, TX) and reverse transcribed using a GoScript™ Reverse Transcription System (Promega, Madison, WI) following the manufacturer's protocol. The expression of mRNAs was measured by quantitative real-time polymerase chain reaction (qRT-PCR) using the SYBR-Green method (Go Taq® qPCR, Promega Corporation, USA). All the primers used for qRT-PCR were designed and synthesized by Tsingke Biotechnology Co., Ltd. (Shanghai, China). qRT-PCR was performed in duplicate with a 10 μL reaction system, which contained 5 μL SYBR Green PCR Master Mix, 4 μL cDNA, and 1 μL primer, using the ABI 7500 RT-PCR system (Applied Biosystems, Foster City, CA). The comparative cycle time (Ct) method was used to determine fold differences between samples, and the amount of target genes was normalized to β-actin as an endogenous reference (2^−△△Ct). 2.14. RNA-sequencing (RNA-Seq) assay Total RNA was isolated from EPCs transduced with Lv-Ctrl (n = 3), Lv-Nrf2 (n = 3), Ctrl-shRNA (n = 3), or Nrf2-shRNA (n = 3), respectively, using total RNA isolation kit (Solarbio, Beijing, China). The purity and concentration of isolated RNA were determined by a NanoPhotometer spectrophotometer (IMPLEN, CA), the RNA integrity was assessed by the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA). RNA sequencing libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich) and index codes were added to attribute sequences to each sample. Clusters were generated using TruSeq PE Cluster Kit v3-cBit-HS (Illumina, San Diego, CA), and RNA-seq libraries were sequenced on an Illumina platform at the Novegene Company (Beijing, China). Raw data (raw reads) of fastq format were initially processed through in-house perl scripts. Gene expression was quantified using HTSeq v0.9.1. Differential expression analysis was performed using the DESeqR package (1.18.0). DESeq provided statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. Genes with a P < 0.05 found by DESeq were assigned as differentially expressed. 2.15. Chromatin immunoprecipitation (ChIP) assay ChIP was performed using a Simple ChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA) following the protocol provided by the manufacturer. In brief, EPCs were fixed with formaldehyde, and the chromatin was sheared. Then, the fragmented chromatin was incubated with Nrf2 antibody (Abcam, Cambridge, MA) and protein G magnetic beads. DNA released from the precipitates was analyzed by PCR. IgG was employed as the negative control. The primer sequences specific to the Nrf2 binding region within the IDH2 promoter region were as follows: IDH2 promoter forward: 5′-GACTTGGTGAGGGGAGCTTTGAA-3′ and reverse: 5′-CTGAGGGCTGGGTTTTATTTGGA-3′. As a positive control, the primer sequences specific to the region of NQO1 gene promoter that contains an ARE binding site were as follow: forward primer: 5′-TGCACCCAGGGAAGTGTGTTGTAT-3′ and reverse primer: 5′-CCCTTTTAGCCTTGGCACGAAA-3′. As a negative control, the primer sequences to the region of NQO1 that does not contain an ARE binding site as follow: forward primer: 5′-TCTCAGTTTTTGCCCTTATTTAATC-3′ and reverse primer: 5′-TAAAAAGTAGAGTGGTTGGAGTGATGAC-3′. 2.16. Dual luciferase reporter assay For reporter assay, the human IDH2 promoter sequences -2000 bp ∼ 0 bp and -1000 bp ∼ 0 bp were cloned into the pGL3.0-Basic plasmid (provided by Tsingke Biotechnology) and designated as IDH2-Full-Luc and IDH2-Short-Luc, respectively. Then the putative binding sites (GTGAATTAGCG) of Nrf2 in IDH2-Full-Luc plasmid was mutated to a non-specific sequence (TATCATTAAAG) by using KOD-Plus-Mutagenesis kit (SMK-101, Toyobo, Osaka, Japan) and designated as IDH2-Mutant-Luc. Luciferase reporter assays were performed as described previously [[106]32]. To detect whether knockdown of Nrf2 expression inhibiting IDH2 promoter activity, IDH2-Full-Luc, IDH2-Short-Luc, or IDH2-mutant-Luc reporters were co-transfected with pLKO.1-Ctrl-shRNA or pLKO.1-Nrf2-shRNA vector into HEK-293T cells, respectively. Renilla luciferase reporter plasmid (pRL-TK) was used as an internal control of transfection. In addition, to investigate whether activation of Nrf2 promoting IDH2 promoter activity, we used Nrf2 activator sulforaphane (SFN) to treat HEK-293T cells transfected with IDH2-Full-Luc, IDH2-Short-Luc, or IDH2-mutant-Luc reporters, respectively. Cell lysates were subjected to luciferase assay by using a GloMax 96 microplate luminometer (Promega, Madison, WI) at 48 h post-transfection. For each sample, the pGL3.0-firefly luciferase activity was normalized to Renilla luciferase activity of the pRL-TK control. 2.17. Western blot assay Western blot was performed as described in previous studies [[107]3,[108]27,[109]29]. For cellular protein extraction, EPCs were rinsed twice with PBS and then suspended in ice-cold RIPA lysis buffer (Solarbio Life Sciences, Beijing, China) and incubated for 30 min on ice. Proteins were collected by centrifugation, 12,000 g for 15 min at 4 °C. The protein concentration was determined using a BCA protein assay kit. The proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blocked in tris-buffered saline with 5% non-fat milk and 0.5% BSA for 1 h, and then incubated with primary antibody overnight at 4 °C, followed by incubation with the secondary antibody for 1 h at room temperature after standard washing procedures. The primary antibodies against Nrf2 (1:1000), mitofusin 1 (Mfn1, 1:1000), NAD(P)H dehydrogenase quinone 1 (NQO-1, 1:1000), optic atrophy1 (Opa1, 1:1000) were purchased from Abcam (Cambridge, MA); heme oxygenase-1 (HO-1, 1:1000), mitofusin 2 (Mfn2, 1:1000) and IDH2 (1:1000) were purchased from Cell Signaling Technology; β-actin (1:2000) was purchased from Bioss Biotechnology; dynamic related protein 1 (Drp1, 1:1000) was purchased from Novus Biologicals (Littleton, CO); mitochondrial fission 1 protein (Fis1, 1:1000) was purchased from GeneTex (San Antonio, TX). All horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Bioss Biotechnology (Beijing, China). Blots were visualized with Chemiluminescent HRP Substrate (Millipore, Billerica, MA) and quantified with Quantity 5.2 software System (Bio-Rad). 2.18. HLI model and cell therapy db/db type 2 diabetic mice at the age of 10–12 weeks were used to develop HLI model as reported in our previous studies [[110]3]. Briefly, under sufficient anesthesia with isoflurane (1–3% isoflurane in 100% oxygen at a flowrate of 1 L/min), the right hind limb was shaved and the entire right superficial femoral artery and vein (from just below of deep femoral arteries to popliteal artery and vein) were ligated with 6–0 silk sutures, cut, and excised with an electric coagulator (Fine Science Tools Inc., Foster City, CA), and the skin was closed with 4–0 silk sutures. Real time microcirculation imaging analysis was performed using a Pericam Perfusion Speckle Imager (PSI, Perimed Inc., Kings Park, NY) to evaluate the foot pad blood perfusion ratio [ischemic limb (right)/normal limb (left)] at days 0, 3, 7, 14, 21, and 28 post-ischemia [[111]3]. Then mice were euthanized and gastrocnemius muscle and soleus muscle of the ischemia limb were collected for analysis of capillary density. For cell therapy in db/db mice, 2.5 × 10^6 WT-EPCs or db/db-EPCs were infused into db/db male mice via tail vein within 1 h after HLI surgery. For SFN treatment, db/db mice were pretreated with SFN (1.0 mg/kg body weight, every other day by intraperitoneal injection) [[112]33,[113]34] for 1 week, followed by HLI surgery and continued SFN treatment for an additional 4 weeks until the end of experiment. SFN was dissolved in 1% dimethyl sulfoxide (DMSO) and diluted in PBS; an equivalent concentration of DMSO in PBS was used as vehicle control. To evaluate whether overexpression IDH2 can rescue the angiogenic dysfunction of mouse EPC induced by Nrf2-knockdown, 2.5 × 10^6 WT-EPCs co-transfected with lentivirus carrying Ctrl-shRNA and control vector (Ctrl-shRNA/Lv-Ctrl-EPC), Nrf2-shRNA and control vector (Nrf2-shRNA/Lv-Ctrl-EPC), or Nrf2-shRNA and IDH2 over-expression vector (Nrf2-shRNA/Lv-IDH2-EPC) were infused into db/db male mice via tail vein within 1 h after HLI surgery. Foot pad blood perfusion was evaluated by PSI at days 0, 3, 7, 14, 21, and 28 after surgery. 2.19. Histological assessment Ischemic gastrocnemius muscle and soleus muscle were fixed with 4% paraformaldehyde and embedded with paraffin. Paraffin sections were cut at 5 μm and stained with Alexa Fluor® 594 conjugated isolectin GS-IB4 (Thermo Scientific, Waltham, MA) to evaluate the capillary density. The number of capillaries were calculated in randomly selected fields for a total of 20 different fields ( × 40 magnification) per section and 3 sections per animal. The capillary density was expressed as capillary number per muscle fiber. 2.20. Statistical analysis All data are presented as mean ± SD. Sample size for each study was detailed in figure legends. Statistical analysis was performed using GraphPad Prism 8.0 software with one-way or two-way ANOVA, followed by post-hoc multiple comparisons with the Scheffe’ test. Statistical significance was considered as p < 0.05. 3. Results 3.1. Diabetes attenuates Nrf2 and impairs the survival and angiogenic function of EPCs To investigate the role of Nrf2 in EPCs, the expression of Nrf2 and its downstream genes were detected. The results showed that the expression of Nrf2 and its downstream genes was significantly decreased in DM-EPCs, compared with Healthy-EPCs ([114]Fig. 1A). We further investigated the survival and angiogenic function of Healthy-EPCs and DM-EPCs. Annexin V/PI staining showed significantly higher apoptotic percentage of DM-EPCs than that of Healthy-EPCs ([115]Fig. 1B). In addition, tube formation ability of DM-EPCs was also impaired as indicated by the significantly shorter length of tubes produced by DM-EPCs compared with Healthy-EPCs ([116]Fig. 1C). Similarly in mice, the expression of Nrf2 and its downstream genes in db/db-EPCs were lower than that of WT-EPCs ([117]Fig. S1A), and the survival and angiogenic function of db/db-EPCs were also impaired ([118]Figs. S1B and C). Fig. 1. [119]Fig. 1 [120]Open in a new tab Diabetes attenuates Nrf2 and impairs the survival and angiogenic function of EPCs. EPCs were isolated from healthy donors (Healthy-EPC) or diabetic patients (DM-EPC). (A) The Expression of Nrf2 and its downstream genes were detected by Western blot, and the quantitative data was normalized by the average of Healthy-EPCs for each protein; β-actin was used as a loading control. (B) The survival abilities of EPCs were determined by Annexin V/PI staining, and the quantitative data was expressed as the percentage of AnnexinV^+/PI^– cells. (C) The angiogenic function of EPCs was evaluated by a Matrigel tube formation assay, and the quantitative data was normalized by the average tube length of Healthy-EPCs. n = 8 per group. Data shown in graphs represents the means ± SD. *p < 0.05, vs Healthy-EPC. 3.2. Nrf2 regulates the survival and angiogenic function of EPCs To determine if reduced Nrf2 expression in diabetic EPCs was a cause of angiogenic dysfunction, gain- and loss-of-function studies were performed by Nrf2-shRNA lentivirus to knockdown Nrf2 expression in Healthy-EPCs and WT-EPCs, and Nrf2 overexpression lentivirus to upregulate Nrf2 expression of DM-EPCs and db/db-EPCs, respectively. The expression of Nrf2 and its downstream genes was significantly reduced by the Nrf2-shRNA at mRNA and/or protein levels in Healthy-EPCs ([121]Fig. 2A) and WT-EPCs ([122]Fig. S2A), and this was accompanied by increased apoptosis ([123]Fig. 2C, [124]Fig. S2C) and impaired tube formation ([125]Fig. 2D, [126]Fig. S2D). Conversely, transduction of Lv-Nrf2 into DM-EPCs and db/db-EPCs elevated the expression of Nrf2 and its downstream genes ([127]Fig. 2B, [128]Fig. S2B), diminished cell apoptosis ([129]Fig. 2C, [130]Fig. S2C) and improved tube formation in DM-EPCs and db/db-EPCs ([131]Fig. 2D, [132]Fig. S2D). Fig. 2. [133]Fig. 2 [134]Open in a new tab Downregulation of Nrf2 impairs the survival and anigogenic function of Healthy-EPCs, while upregulation of Nrf2 rescues the survival and angiogenic function of DM-EPCs. Healthy-EPCs were transfected with lentivirus carrying nonsense shRNA (Ctrl-shRNA) or Nrf2 shRNA (Nrf2-shRNA) and DM-EPCs were transfected with lentivirus carrying Nrf2 (Lv-Nrf2) or control vector (Lv-Ctrl). (A-B) The efficiency of Nrf2 knockdown or upregulation was determined by Western blot and qRT-PCR, and the quantitative data was normalized by the average of the respective control group for each protein; β-actin was used as a loading control. (C) The survival abilities were determined by Annexin V/PI staining, and the quantitative data was expressed as the percentage of AnnexinV^+/PI^– cells. (D) Angiogenic function of EPCs was evaluated by a Matrigel tube formation assay, and the quantitative data was normalized by the average tube length of the respective control group. n = 4 per group. Data shown in graphs represents the means ± SD. *p < 0.05, vs Ctrl-shRNA or Lv-Ctrl. To evaluate the role of Nrf2 in EPC-mediated angiogenesis in vivo, we infused mouse EPCs with manipulated Nrf2 expression into db/db mice with HLI that was induced by femoral artery ligation. As shown in [135]Fig. 3A, infusion of WT-EPCs transfected with Nrf2-shRNA lentivirus showed significantly slower blood flow recovery in db/db mice at days 14, 21 and 28 after HLI surgery compared with infusion of WT-EPCs transfected with Ctrl-shRNA lentivirus. However, elevating Nrf2 expression in db/db-EPCs with Nrf2 overexpression lentivirus remarkably promoted the recovery of blood perfusion in db/db mice compared with infusion of db/db-EPCs transfected with control lentivirus. Furthermore, the capillary density in ischemic muscle was measured at 28 days after HLI. The results demonstrated that knockdown of Nrf2 expression in WT-EPCs reduced the capillary density in both gastrocnemius and soleus muscles ([136]Fig. 3B and C). While upregulating Nrf2 expression in db/db-EPCs significantly increased the capillary density in both gastrocnemius muscle and soleus muscle of db/db mice ([137]Fig. 3B and C). These results suggest that diabetic downregulation of Nrf2 plays a causative role in diabetes-induced EPC angiogenic dysfunction, while elevating Nrf2 expression rescued EPC dysfunction induced by diabetes. Fig. 3. [138]Fig. 3 [139]Open in a new tab Nrf2 regulates the angiogenic function of mouse EPCs. WT-EPCs were transfected with lentivirus carrying Ctrl-shRNA or Nrf2-shRNA and db/db-EPCs were transfected with lentivirus carrying Lv-Ctrl or Lv-Nrf2. Within 1 h after hind limb ischemia (HLI) surgery, db/db mice (FVB background) were transplanted with different types of EPCs that have a same genetic background. (A) The time-course of blood perfusion before (BEF) and after HLI surgery was monitored by a Pericam Perfusion Speckle Imager, and the blood perfusion was quantified by Image J and expressed as the percentage of the perfusion relative to the collateral nonischemic limb. Transverse sections of ischemic gastrocnemius (B) and soleus muscle (C) were stained with Alexa Fluor® 594 conjugated isolectin to enumerate isolectin-stained cell number as a proxy of capillary density. Capillary density was expressed as isolectin^+ capillaries per muscle fiber. n = 6 mice per group. Data shown in graphs represents the means ± SD. *p < 0.05, vs Ctrl-shRNA or Lv-Ctrl. 3.3. Diabetes induces mitochondrial dysfunction and ROS overproduction in EPCs Our previous study showed that oxidative stress is the main cause of diabetes-induced EPC dysfunction [[140]3]. Mitochondria are the major source of ROS generation [[141]35]. Mitochondrial fragmentation is an important contributor to ROS overproduction under diabetic conditions, in which it causes deleterious vascular cell signaling and subsequent endothelial dysfunction [[142]36]. Thus, to uncover whether mitochondrial fragmentation is responsible for the dysfunction of diabetic EPCs, the mitochondrial morphology was visualized by mitochondrial fluorescent probe. As shown in [143]Fig. 4A, most mitochondria in Healthy-EPCs exhibited rod and network organization; however, most mitochondria in DM-EPCs were circular and punctate, indicating mitochondrial fragmentation. Fig. 4. [144]Fig. 4 [145]Open in a new tab Diabetes induces mitochondrial fragmentation of EPCs and impairs mitochondrial function. (A) Micrographs of mitochondrial morphology of EPCs were visualized by MitoTracker Red CMXRos probe staining, and the morphological alterations were quantified by Image J and expressed as mitochondrial interconnectivity. (B–C) The expression of mitochondrial fusion and fission related proteins was determined by Western blot, the quantitative data was normalized by the average of Healthy-EPC for each protein, and β-actin was used as a loading control. The levels of (D) intracellular ROS, (E) mitochondrial ROS, and (F) mitochondrial membrane potential were detected by DHE, MitoSOX™, and TMRM staining, respectively, and quantified by a flow cytometry and normalized by the average fluorescence density of Healthy-EPCs. (G) ATP concentration in EPCs was measured by an ATP assay Kit. n = 8 per group. Data shown in graphs represents the means ± SD. *p < 0.05, vs Healthy-EPC. (For interpretation of the references to colour in this figure legend, the