GDF11 Improves Angiogenic Function of EPCs in DiabeticLimb IschemiaJiajia Zhang,1 Yixiang Li,2 Huan Li,1 Biao Zhu,1 Li Wang,1 Bei Guo,1 Lin Xiang,1 Jing Dong,1 Min Liu,1 andGuangda Xiang1

Diabetes 2018;67:2084–2095 | https://doi.org/10.2337/db17-1583

Growth differentiation factor 11 (GDF11) has been shownto promote stem cell activity and rejuvenate the func-tion of multiple organs in old mice, but little is knownabout the functions of GDF11 in the diabetic rat model ofhindlimb ischemia. In this study, we found that systematicreplenishment of GDF11 rescues angiogenic functionof endothelial progenitor cells (EPCs) and subsequentlyimproves vascularization and increases blood flow indiabetic rats with hindlimb ischemia. Conversely, anti-GDF11 monoclonal antibody treatment caused impair-ment of vascularization and thus, decreased blood flow.In vitro treatment of EPCs with recombinant GDF11 at-tenuated EPCdysfunction and apoptosis. Mechanistically,the GDF11-mediated positive effects could be attrib-uted to the activation of the transforming growth factor-b/Smad2/3 and protein kinase B/hypoxia-inducible factor1a pathways. These findings suggest that GDF11 reple-tion may enhance EPC resistance to diabetes-induceddamage, improve angiogenesis, and thus, increase bloodflow. This benefit of GDF11 may lead to a new therapeuticapproach for diabetic hindlimb ischemia.

Diabetes is a chronic metabolic disease with considerablemorbidity and mortality (1–3). One devastating compli-cation of diabetes is peripheral artery disease (PAD).Patients with diabetes with PAD always experience moresevere disease, such as critical limb ischemia (CLI), thanpatients without diabetes. CLI, the most severe form ofPAD, is a leading cause of incurable ulceration, gangrene,and even lower-extremity amputation (4). Many individ-uals with diabetes with PAD are not candidates for cur-rently available surgical or endovascular procedures becauseof diffuse vascular disease (5). Consequently, therapeutic

interventions aimed at enhancing angiogenesis and restor-ing blood flow in diabetic CLI are essential.

Ischemia-induced vascularization is strongly associatedwith endothelial progenitor cells (EPCs), which are mobilizedfrom the bone marrow and then home to sites of vasculardamage where they contribute to neovascularization andrecovery (6). However, the levels and functions of EPCs aredramatically reduced in patients with diabetes (7).

Growth differentiation factor 11 (GDF11) belongs tothe transforming growth factor-b (TGF-b) family and plays anessential role in mammalian development (8). GDF11 signalsthrough the activin type II receptors and activates the ca-nonical Smad2/3 signaling pathway (9). Some reports haverevealed that GDF11 appears to decline with age and thatexogenous administration of GDF11 reverses age-relateddefects in the heart, skeletal muscle, and cerebra (10–12).However, other groups have argued that systemic restorationof GDF11 fails to rejuvenate cardiac pathologies and inhibitsskeletal muscle regeneration (13,14). GDF11 and GDF8 arehighly homologous at the protein level, andmany key reagentsthat recognize GDF11 also recognize GDF8 (14). More re-cently, Poggioli et al. (15) confirmed that exogenous GDF11reduces cardiomyocyte size in mice. Importantly, GDF11exhibits antisenescent effects in lung cells and promotesthe recovery of renal and cardiac function in mice (16–18).

Of note, our early study showed that GDF11 can pro-tect against endothelial cell injury (19). Further researchhas reported that GDF11 improves cardiac function andreduces infarct size in mice after ischemia/reperfusion in-jury (16). However, data regarding the effect of GDF11 onvascularization of EPCs are scarce. In the current study, weaimed to investigate whether GDF11 could rescue EPCs fromdamage induced by diabetes and improve neovascularization

1Department of Endocrinology, Wuhan General Hospital of Guangzhou Command,Wuhan, Hubei, China2Department of Hematology and Medical Oncology, School of Medicine, EmoryUniversity, Atlanta, GA

Corresponding author: Guangda Xiang, guangda64@hotmail.com.

Received 30 December 2017 and accepted 29 June 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1583/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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in diabetic limb ischemia and to reveal the possible mech-anisms involved.

RESEARCH DESIGN AND METHODS

Animals and TreatmentsMale Sprague-Dawley rats weighing 200–220 g were usedin this study. The model of diabetic ischemic limb wasgenerated as described previously (20,21). As shown inSupplementary Fig. 1A, nondiabetic rats served as normalcontrol and further randomized to receive vehicle (citratebuffer) or recombinant GDF11 (rGDF11) (the control andcontrol + rGDF11 groups, respectively; n = 10 per group).Correspondingly, diabetic rats also were randomized toreceive vehicle or rGDF11 (the vehicle and rGDF11 groups,respectively; n = 10 per group).

For rGDF11 treatment, rats were intraperitoneallyinjected daily with 0.1 mg/kg rGDF11 or an equivalentvolume of vehicle for 2 weeks. Two weeks after treatment,the animals were euthanized for blood tests and tissueexaminations. At the beginning and end of the treatments,glucose tolerance tests, insulin tolerance tests, blood pres-sure, and blood biochemical analyses were performed as inour previous studies (19,22).

To determine the optimal dosage for the GDF11 anti-body (Ab) administration, we first performed a dose-response study. The specificity of the Ab was verified inour previous report (22). Fifteen diabetic rats were ran-domized into five groups that received intravenous injec-tions of GDF11 Ab (0, 100, 150, 200, or 250 mg) twicea week or an equivalent volume of IgG Ab for 2 weeks(n = 3 per group). Accordingly, to evaluate the effects ofGDF11 Ab in diabetic rats with hindlimb ischemia, ratswere injected intravenously with GDF11 Ab or IgG Abtwice a week for 2 weeks (n = 10 per group).

To explore the potential mechanism of GDF11, 40diabetic rats were further randomized to four groups:rGDF11 + vehicle (citrate buffer), rGDF11 + SB431542(an inhibitor of TGF-b type I receptor [TbRI]), rGDF11 +Ad-small interfering RNA (siRNA) control (Ad-siCon),and rGDF11 + Ad-siRNA hypoxia-inducible factor 1a(Ad-siHIF1a) (n = 10 per group). To determine the optimaldosage for the SB431542 administration, we performeda dose-response experiment. Twelve diabetic rats wererandomized into four groups that received a single intra-peritoneal injection of rGDF11 + SB431542 (5, 10, or20 mg/kg) or rGDF11 + an equivalent volume of citratebuffer (n = 3 per group). On the next day, the rats wereeuthanized for phosphorylated (p)-SMAD2/3 expressionexamination. For Ad-siHIF1a treatment in vivo, rats re-ceived a single injection of Ad-siHIF1a at a dose of 23 1010

plaque-forming units through the tail vein. To assess trans-fection efficiency, we measured the mRNA and protein levelsof HIF1a by RT-PCR and Western blot.

Construction of Adenovirus VectorsWe synthesized HIF1a-specific siRNA (siHIF1a; GenBankaccession number NM_024359) and scrambled control

siRNA (siCon) oligonucleotides and cloned them intopSUPER vector. Generation, amplification, purification,titer determination, and transduction of adenovirus vec-tors were performed as previously described (23,24).

Blood Flow Analysis and Physical ExaminationBlood flow was scanned using a laser Doppler perfusionimage (LDPI) analyzer (Perimed, Stockholm, Sweden).Perfusion analyses were performed immediately afterfemoral artery excision and at postoperative days 7 and14. The LDPI index was defined as the perfusion ratio ofischemic to nonischemic hindlimb. Ambulatory impair-ment was scored using the criteria described previously(25).

Capillary and Arteriole Density in Diabetic IschemicHindlimbRats were sacrificed 14 days after treatment, and adductormuscle samples were harvested for histological evalua-tion. To visualize capillary and arteriole density, the tis-sue sections were stained with CD31 (Abcam, Cambridge,U.K.) and a-smooth muscle actin (a-SMA; Boster Bio-Engineering) according to the procedures previously pub-lished (26). Capillary or arteriole density was assessed byexpressing the data as CD31+ vessels/mm2 or a-SMA+

vessels/mm2.

Determination of Circulating EPCs and HomingPopulation of EPCs in RatsThe number of circulating EPCs was calculated by flowcytometry as previously described with a little adjustment(27). In brief, total mononuclear cells were isolated fromrat peripheral blood by density gradient centrifugationwith Histopaque-1083 (Sigma-Aldrich, St. Louis, MO) andthen incubated with fluorescein isothiocyanate–labeledkinase insert domain receptor (FITC-KDR; Abcam) andphycoerythrin-labeled CD34 antibodies (PE-CD34; Abcam).To detect the homing population of EPCs, the adductormuscle sections from the ischemic limb were incubatedwith CD34 (R&D Systems) and KDR (R&D Systems) forimmunofluorescent staining.

Bone Marrow EPC Culture and Cell Signaling AnalysesBone marrow EPCs (BM-EPCs) were isolated, cultured, andidentified according to methods described previously (7).For p-SMAD assays, EPCs were fixed and processed aspreviously described (19).

Tube Formation and Migration AssaysA matrigel tube formation and migration assay was per-formed to determine the angiogenic capacity of BM-EPCs(28).

Apoptosis Assay and Western Blot AssayApoptosis was detected using flow cytometry (BD, FranklinLakes, NJ) after double staining with Annexin V-FITC (BDPharmingen) and propidium iodide (22). Western blot wasperformed as previously reported (29). The following anti-bodies were used: Smad2/3, p-Smad2/3, AMPK, p-AMPKa,

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AKT, p-AKT, ERK1/2, p-ERK1/2, HIF1a, vascular endo-thelial growth factor (VEGF), stromal cell-derived factor1a (SDF1a), Bax, Bcl-2, cleaved caspase3 (all from CellSignaling Technology), and b-actin (Boster Bio-Engineering).For real-time PCR, total RNA samples were preparedand measured as previously described (30). The followingoligonucleotides served as primers: b-actin forward: 59-AC-ACTGTGCCCATCTAGGAGG-39, reverse: 59-AGGGGCCGG-ACTCGTCATACT-39; HIF1a forward: 59-AAGTCTAGGGA-TGCAGCAC-39, reverse: 59-CAAGATCACCAGCATCTAG-39.

Statistical AnalysisData are expressed as mean6 SEM. Statistical differenceswere evaluated by Student t test or one-way ANOVA witha least significant difference test. P, 0.05 was consideredsignificant.

RESULTS

GDF11 Restoration Improves MetabolicCharacteristics in Diabetic RatsOur previous report suggested that GDF11 restorationimproves glycolipid homeostasis in diabetic mice (22).Thus, we further confirmed the influence of GDF11 onmetabolic characteristics in diabetic rats. First, circulatingGDF11/8 levels (our assay for serum GDF11 did notdistinguish circulating GDF11 from GDF8) were decreasedin the diabetic rats compared with the nondiabetic rats. Asexpected, rGDF11 treatment elevated GDF11/8 levels inboth diabetic and nondiabetic rats at days 7 and 14 (P ,0.05) (Supplementary Fig. 1B and C). Second, at baseline,no significant differences were observed in glucose orinsulin tolerance among the diabetic groups (Supplemen-tary Fig. 2A–D). Although vehicle-treated diabetic ratsdeveloped severe hyperglycemia, GDF11 restoration at-tenuated the progression of hyperglycemia (Supplemen-tary Fig. 2E). Consistently, the rGDF11 interventionabated HbA1c levels (Supplementary Fig. 2F). After 2 weeks

intervention, rGDF11 treatment attenuated glucose in-tolerance and insulin resistance in diabetic rats (Supple-mentary Fig. 2G–J). Third, after different treatments for2 weeks, administration of rGDF11 improved plasma in-sulin levels and lipid profiles in diabetic rats but not innondiabetic rats. Finally, no significant difference inblood pressure was found among nondiabetic and di-abetic groups (Supplementary Table 1).

GDF11 Restoration Promotes Perfusion Recovery inthe Diabetic Hindlimb Ischemia ModelGDF11 has been shown to protect against ischemia/reperfusion insult in mouse heart (16). Therefore, we in-vestigated the effects of GDF11 on perfusion recovery innondiabetic and diabetic hindlimb ischemia. The resultsshow that GDF11 had no effect on blood flow recovery innondiabetic rats (Supplementary Fig. 3A–C). The perfusionrecovery was worse in diabetic rats than in nondiabeticrats. At 7 and 14 days, recovery of limb perfusion wassignificantly increased in the rGDF11-treated diabetic ratscompared with the vehicle-treated diabetic rats (Fig. 1Aand B). Consequently, the rGDF11 group manifested lessambulatory impairment compared with the vehicle group(Fig. 1C). Overall, these results suggest that GDF11 re-pletion improves blood perfusion in the diabetic hindlimbischemic rats.

GDF11 Restoration Improves Vascularization andIncreases Circulating EPCs and the Homing of EPCsin Diabetic Ischemic HindlimbWe next questioned whether GDF11 has an effect onvascularization. Indeed, significantly increased numbersof CD31+ and a-SMA+ vessels were detected in the ische-mic muscles of the rGDF11 group compared with those inthe vehicle group (Fig. 2). However, GDF11 had no effectson capillary and arteriolar densities in nondiabetic limbischemia (Supplementary Fig. 3D–F). The increased num-bers of CD31+ and a-SMA+ vessels provide a histological

Figure 1—Perfusion recovery and ambulatory impairment of ischemic hindlimb. LDPIs of ischemic hindlimb were taken at days 0, 7, and14 postligation. Blood perfusion is presented as the ratio of blood flow in ischemic limb divided by that in normal hindlimb. Nondiabetic ratsserved as control (control group). A: Representative LDPIs of the time course of ischemic limb perfusion. B: Quantitative analysis of A. TheLDPI index was significantly higher in the rGDF11 group than in the vehicle group (n = 10 per group).C: Ambulatory impairment was scored ateach time point. Data are mean 6 SEM. *P , 0.05, **P , 0.01 vs. control group; †P , 0.05, ††P , 0.01 vs. vehicle group.

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basis for the increased blood flow and tissue viability in thediabetic ischemic limb.

Given that EPCs have been identified as an importantregulator of vascularization, we investigated whether GDF11exerts its neovascularization activities through increasingmobilization and recruitment of EPCs. The results showthat GDF11 had no effect on the number of CD34+/KDR+

cells in nondiabetic rats (Supplementary Fig. 3G). The num-ber of CD34+/KDR+ cells in diabetic rats was less than that innondiabetic rats. However, the rGDF11 group showed in-creased numbers of CD34+/KDR+ cells in diabetic rats (Fig.3A and B). To further confirm these observations, we ex-plored the homing of EPCs to sites of ischemia. GDF11 hadno effect on the number of CD34+/KDR+ cells in ischemicsites in nondiabetic rats (Supplementary Fig. 3H and I).As expected, an approximate twofold increase in the num-ber of CD34+/KDR+ cells was observed in the rGDF11 groupcompared with the corresponding numbers in the vehiclegroup (Fig. 3C and D). These findings suggest that GDF11 isinvolved in EPC mobilization and recruitment in diabeticrats. In addition, the rGDF11 group had higher plasmaVEGF and SDF1a concentrations than the vehicle group.Compared with the IgG Ab group, VEGF and SDF1a levelswere lower in the GDF11Ab group (Supplementary Table 1).These findings further suggest that GDF11 is essential forEPC mobilization and recruitment.

Neutralization of GDF11 Aggravates the Impairment ofHindlimb Ischemia Recovery in Diabetic RatsWe previously demonstrated that administration of GDF11Ab reverses the biological effects of GDF11 (22). Accord-ingly, the effects of GDF11 Ab on the progression ofhindlimb ischemia recovery in diabetic rats were detected

in this work. First, we have previously demonstrated thatthis Ab specifically detects GDF11 (22). Compared withthe control group, the stimulatory effects of rGDF11 onmigration were dose-dependently reversed by GDF11 Abtreatment in vitro (Supplementary Fig. 4A). Second, todetermine the optimal dosage for GDF11 Ab administra-tion, we performed a dose-response study. GDF11 hasbeen shown to alleviate impaired endothelium-dependentrelaxation (19). Therefore, we explored the optimal dosageof GDF11 Ab on the basis of the changes of endothelium-dependent relaxation in diabetic rats. In present study, theresults show that compared with control group, GDF11 Abtreatment decreased vascular endothelium-dependent re-laxation at doses between 200 and 250 mg (SupplementaryFig. 4B); thus, we chose 200mg as the optimum dose in thisanimal study. In addition, vascular endothelium-independentrelaxation in response to sodium nitroprusside did notdiffer among the five groups (Supplementary Fig. 4C).

We next assessed the effects of GDF11 Ab on metaboliccharacteristics in diabetic rats. As expected, glucose in-tolerance and insulin resistance were comparable betweenthe diabetic groups at baseline but were worse in theGDF11 Ab group at the termination of the study (Sup-plementary Fig. 4D–G and J–M). As shown in Supplemen-tary Fig. 4H and I, GDF11 Ab treatment further elevatedthe fasting glucose and HbA1c levels in diabetic rats.Consistently, GDF11 Ab treatment decreased plasma in-sulin levels and exacerbated lipid metabolic disturbance(Supplementary Table 1).

Next, we examined whether neutralization of GDF11reduces blood reperfusion of the ischemic limb. At day 7,the LDPI showed no significant difference between the

Figure 2—Effect of GDF11 on capillary and arteriolar density 14 days after intervention. Ischemic skeletal muscle was collected at 14 dayspostligation and immunostained for CD31 (red) or a-SMA (green). Nondiabetic rats served as control (control group). Representative image(A) and graphical representations (B andC) of the capillary and arteriolar density analysis among the various groups (counts/mm2) (n = 10 pergroup). Significant increases in the capillary and arteriolar density were observed in the rGDF11 group compared with those in the vehiclegroup. Scale bar = 50 mm. Data are mean 6 SEM. *P , 0.05, **P , 0.01 vs. control group; †P , 0.05 vs. vehicle group.

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GDF11 Ab and IgG Ab groups. However, the perfusionrecovery of diabetic hindlimb ischemia in the GDF11 Abgroup was lower than that in the IgG Ab group at day14 (Fig. 4A and B). The GDF11 Ab and IgG Ab groupsdid not significantly differ in ambulatory impairment(Fig. 4C). Further histological analysis demonstrated thatGDF11 Ab treatment results in a notable decrease in vesseldensity compared with IgG Ab treatment (Fig. 4D and E).Likewise, the GDF11 Ab group showed a diminished num-ber of circulating EPCs, suggesting that GDF11 deficiencyimpairs mobilization of EPCs into the circulation in responseto ischemia (Fig. 4F). Moreover, the homing of EPCs to theischemic region was significantly impeded by GDF11 Abtreatment compared with IgG Ab treatment (Fig. 4G and H).

GDF11 Increases EPC Proliferation and AlleviatesApoptosis In VitroSeveral clinical studies have observed that the number ofcirculating EPCs is reduced in individuals with diabetes(31–33). Our animal data suggest that the circulatingnumber of EPCs was decreased in diabetic rats, but GDF11protected against EPC reduction (Fig. 3A and B). There-fore, we next investigated whether rGDF11 pretreat-ment could directly affect EPC proliferation and apoptosisin vitro. BM-EPCs were identified as Dil-ac-LDL and lectindouble-positive cells under fluorescence microscopy (Sup-plementary Fig. 5). First, exposure of EPCs to GDF11promoted EPC proliferation in a dose- and time-dependentmanner (Supplementary Fig. 6A and B). Accordingly, wechose 50 ng/mL rGDF11 and 60 min as the optimumconcentration and time conditions in the in vitro study.Second, flow cytometry analysis indicated that high-glucose (25 mmol/L)/hypoxia/serum-deprivation medium

(HG) greatly increased the percentage of apoptotic EPCs(Supplementary Fig. 6C). However, the effect was atten-uated by rGDF11 pretreatment (Fig. 5A and B). Consis-tently, the increased expression level of the antiapoptoticprotein Bcl-2 and the decreased expression levels of theproapoptotic proteins Bax and cleaved-caspase3 furtherconfirmed the results of the flow cytometry analysis (Fig.5C and D).

GDF11 Improves BM-EPC Angiogenic FunctionsEx Vivo and In VitroThe animal data revealed that increased circulating GDF11levels are associated with increases in vessel density andEPC mobilization. Thus, we next conducted a series ofex vivo or in vitro experiments to determine whether theeffect of GDF11 on angiogenesis and perfusion in diabeticrats with hindlimb ischemia can be attributed, at least inpart, to changes in the angiogenic activity of EPCs. First,the capacity of BM-EPCs for tube formation and migra-tion was assessed by ex vivo experiments. BM-EPCs fromthe rGDF11 group exhibited significant augmentation ofthe tube-like structure formation and migration capacity.Conversely, the angiogenic activities of EPCs from theGDF11 Ab group were impaired compared with those ofEPCs from the IgG Ab group (Fig. 6). Second, angiogenicfunctions were further examined in EPCs from Sprague-Dawley rats in vitro. As shown in Supplementary Fig. 6D–G, EPCs cultured in normal glucose (5 mmol/L)/hypoxia/serum-deprivation medium (NG) exhibited decreasedtube formation and migration capacity compared withthe normal control. Compared with NG treatment, EPCstreated with mannitol/hypoxia/serum-deprivation me-dium, which served as the osmotic control, exhibited

Figure 3—GDF11 enhances the number of circulating EPCs in diabetic rats and increases homing of EPCs. Blood was harvested fromanesthetized rats. Nondiabetic rats served as control. A: After isolation from blood, the number of circulating EPCs were stained with FITC-KDR and PE-CD34 and assessed by flow cytometry (n = 10 per group). B: Quantitative analysis of A. C: Immunolabeling of muscle sectionsfor KDR (red) and CD34 (green) shows EPCs. Scale bars 5 20 mm. D: Quantitative analysis of C. The number of KDR+/CD34+ cells wassignificantly greater in the rGDF11 group than in the vehicle group. Data aremean6 SEM. *P, 0.05, **P, 0.01 vs. control group; ††P, 0.01vs. vehicle group.

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no significant changes in tube formation and migration.However, exposure of cells to HG significantly decreasedboth capacities of tube formation and migration com-pared with NG. As expected, EPCs pretreated withrGDF11 demonstrated increased angiogenic capacitiescompared with EPCs subjected to HG treatment (Fig. 7).These findings indicate that improved tube formationand migration capacities of EPCs may be attributable toGDF11.

TGF-b/Smad and AKT/HIF1a Signals Are Required forthe Effects of GDF11We next explored the possible pathways on which GDF11exerts its positive effects on the recovery of ischemia.GDF11 transmits its signals through dual serine/threoninekinase receptors and transcription factors called Smads(34). We found that rGDF11 upregulates Smad2/3 phos-phorylation in diabetic rats with hindlimb ischemia. How-ever, the GDF11 Ab group exhibited decreased Smad2/3phosphorylation compared with the IgG Ab group (Fig. 8Aand B). We further confirmed the involvement of TGF-bsignaling in vivo by injecting SB431542 intraperitoneally.

We first tested various doses (5, 10, and 20 mg/kg) ofSB431542 and observed that 10 mg/kg was effective atreducing GDF11-induced Smad2/3 phosphorylation (Sup-plementary Fig. 7A and B). Therefore, we chose a dose of10 mg/kg to use in the experiments. As expected, theperfusion recovery of diabetic ischemic hindlimb was par-tially blocked in rats cotreated with rGDF11 and SB431542(Supplementary Fig. 7C and D). In addition, the effects ofrGDF11, including increased circulating EPCs and improvedEPC recruitment, were partially abolished in rats cotreatedwith rGDF11 and SB431542 (Supplementary Fig. 7E–G). Inin vitro experiments, cultured EPCs in HG conditions de-creased Smad2/3 phosphorylation. Of note, treating EPCswith rGDF11 activated the canonical TGF-b/Smad signaling,revealed by an increase in the Smad2/3 phosphorylationcascade, although the phosphorylation could be blockedby coincubation with SB431542 (Fig. 8C and D). Moreover,SB431542 partially abolished the protective effects of GDF11on EPCs, including improved tube formation, migration, andantiapoptosis (Figs. 5 and 7).

We further explored the noncanonical signaling cascadeof GDF11. Activation of HIF1a-related pathways is known

Figure 4—Neutralization of GDF11 aggravates the impairment of hindlimb ischemia recovery in diabetic rats. A: Representative LDPIsdisplay the time course of ischemic limb perfusion (n = 10 per group). B: Quantitative analysis of A. C: Ambulatory impairment was scored. Dand E: Representative image and graphical representation of capillary and arteriolar density analysis among the various groups (counts/mm2)(n = 10 per group). Scale bar = 50 mm. F: Quantitative analysis of the number of circulating EPCs assessed by flow cytometry. G:Immunolabeling of muscle sections for KDR (red) and CD34 (green) shows EPCs. Scale bars = 20 mm. H: Quantitative analysis ofG. Data aremean 6 SEM. *P , 0.05, **P , 0.01.

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to be associated with changes in postischemic neovascu-larization (35). Therefore, we investigated whether ac-tivation of HIF1a-dependent signaling is required inmediating the proangiogenic effects of GDF11. The resultsshow that rGDF11 significantly upregulated HIF1a pro-tein levels in diabetic ischemic rats (Fig. 8E and F). HIF1ais known to regulate angiogenesis through mediators suchas VEGF and SDF1a (36). Consistent with the increase ofHIF1a, rGDF11 elevated the VEGF and SDF1a levels inischemic muscle samples (Fig. 8E and F). However, neu-tralization of GDF11 reduced HIF1a, VEGF, and SDF1aexpression levels (Fig. 8E and F). The activation of HIF1acan be regulated by several kinases (37,38). Thus, weexamined changes in the levels of protein kinase B/AKT,AMPK, and extracellular regulated protein kinases 1/2(ERK1/2). rGDF11 treatments increased AKT phosphory-lation compared with that in the vehicle group, but theydid not influence the expressions of AMPK and extracel-lular regulated protein kinases 1/2 (Supplementary Fig.

8A–D). We next verified this noncanonical signaling incultured EPCs. HG conditions reduced AKT phosphoryla-tion and HIF1a and VEGF expression in EPCs, but pre-incubation with rGDF11 partially rescued these defects(Supplementary Fig. 8E–H). Furthermore, coincubation withan AKT inhibitor exerted a suppressive effect on GDF11-mediated AKT phosphorylation and HIF1a and VEGFexpression (Supplementary Fig. 8E–H).

Importantly, we used Ad-siHIF1a to further confirmthe involvement of HIF1a-related pathways in vivo andin vitro. First, in Ad-siHIF1a–treated cells, we observedsignificant decreases in HIF1a mRNA and protein levelscompared with those in Ad-siCon cells in the hypoxiacondition (Supplementary Fig. 9A–C). Second, the resultsdemonstrate that Ad-siHIF1a partially abolished the pro-tective effects of GDF11 on EPCs, including improvedtube formation and migration, and antiapoptosis (Figs. 5and 7). Next, we evaluated the effect of HIF1a silencingon GDF11-induced neovascularization in vivo. We first

Figure 5—GDF11 alleviates EPC apoptosis in vitro. EPCswere pretreatedwith SB431542 for 30min or transfectedwith Ad-siHIF1a and thenincubated with rGDF11 (50 ng/mL) for 60 min, which was followed by a 24-h incubation with HG. A: After incubation, apoptosis of EPCs wasstained with Annexin V-FITC and propidium iodide and assessed by flow cytometry. B: Quantitative analysis of A. C and D: Representativeimmunoblots and densitometric quantification for the expressions of the proteins Bcl-2, Bax, and cleaved-caspase3. Data are mean6 SEMfor five independent experiments. *P , 0.05, **P , 0.01. MOI, multiplicity of infection.

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evaluated in vivo silencing of endogenous HIF1a. At day2 after interventions, Ad-siHIF1a decreased HIF1amRNAand protein levels in both ischemic and nonischemicmuscle compared with those in the Ad-siCon group (Sup-plementary Fig. 9D–F). As expected, blood perfusion wasreduced in Ad-siHIF1a rats treated with rGDF11 com-pared with that in Ad-siCon rats treated with rGDF11,indicating that silencing of HIF1a partially abrogatesthe effect of rGDF11 (Supplementary Fig. 9G and H). Inaddition, the other positive effects of rGDF11, includingincreased circulating EPCs and improved EPC recruitment,were partially abolished in Ad-siHIF1a rats (Supplemen-tary Fig. 9I–K).

DISCUSSION

The major findings of this study were that 1) GDF11improves vascularization and thus accelerates the recoveryof blood flow in diabetic limb ischemia; 2) GDF11 improvesEPC angiogenic functions, including tube formation andmigration; 3) GDF11 protects against EPC apoptosis; 4)neutralization of GDF11 impairs vascularization in dia-betic hindlimb ischemia; and 5) the molecular mechanisms

underlying these beneficial effects of GDF11 may involvethe activation of the canonical TGF-b/Smad and the non-canonical AKT/HIF1a signaling pathways. The novel find-ings suggest that GDF11 restoration may rescue EPCs fromdamage induced by diabetes and cause greater improvementin vascularization in diabetic hindlimb ischemia.

It is well accepted that the evolution of ischemic damagein patients with diabetes is worsened owing to impairmentof the reparative angiogenesis process (39,40). In thisstudy, we have demonstrated for the first time in ourknowledge that GDF11 treatment significantly increasesvascular density in ischemic tissue in diabetic rats andsubsequently markedly intensifies perfusion of ischemiclimb. However, GDF11 had no angiogenic effect in non-diabetic rats. Conversely, neutralization of GDF11 im-paired neovascularization and alleviated the recovery ofblood flow. In addition, GDF11 replenishment improvedglycolipid metabolism in diabetic rats as previously observed(22), which might partially be of benefit for angiogenesis.Taken together, these data indicate an unequivocal role forGDF11 in revascularization and recovery from hindlimbischemia.

Figure 6—GDF11 improves tube formation and migration of EPCs ex vivo in diabetic rats. A: Representative images of the tube formation ofEPCs from diabetic rats when treated as indicated. Nondiabetic rats served as control. EPC tube formation was measured at 12 h.Photographs were taken with a phase-contrast microscope, and relative tube length was measured with ImageJ software. Scale bar =100 mm. B: Quantitation of A. C: Migration assay photographs of EPCs from the underside of transwell membrane. Scale bars5 200 mm. D:The graph displays the average number of cells that migrated in five independent experiments. Data are mean6 SEM. *P, 0.05, **P, 0.01vs. vehicle group; †P , 0.05 vs. IgG Ab group. hpf, high-power field.

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EPCs are well known to be an important contributor toneovascularization (41). Circulating EPCs are decreased inpatients with diabetes at an early stage and are furtherimpaired in patients with macrovascular complications(32,42), and EPC reduction is related to the worseningof diabetic complications. Our previous studies demon-strated that GDF11 plays a critical role in the regulation ofproliferation and survival of endothelial cells (19). In thecurrent study, we show that GDF11 supplementation leadsto more EPCs homing to sites of ischemia, which conse-quently may participate in the formation of new vessels.Decreased release of VEGF and SDF1a resulted in a defectiverecruitment process (43). In our study, ischemic tissue andplasma levels of VEGF and SDF1a were increased afterGDF11 supplementation, which partially explains the effectof GDF11 on EPC recruitment. With this study, we provideevidence that GDF11 treatment improves impaired EPCfunctions both ex vivo and in vitro, including tube formationand migration. It has been shown that patients with di-abetes with ischemic foot lesions as a result of end-stagePAD have a great reduction of EPCs compared with PAD butwithout foot lesions (32). These data highlight the impor-tance of our finding that GDF11 treatment significantlyincreases the number of EPCs in diabetic rats with CLI.

Next, we explored the underlying pathways that mayexplain the positive effects of GDF11 on vascularizationand recovery of blood flow. It is well documented thatGDF11 activates the canonical Smad signaling pathway (9).

Our data show that GDF11 elevated p-Smad2/3 expres-sion. The TbRI inhibitor partially blocked the ability ofGDF11 to rescue diabetic rats from hindlimb ischemia andEPCs from dysfunction and apoptosis.

In addition, GDF11 can activate several non-Smadsignaling pathways in a context-dependent manner, in-cluding the AKT pathway, which can crosstalk with Smadsignaling (44). Of note, one of the AKT targets that maybe particularly important in the angiogenesis process isHIF1a (38). The HIF1a-related mechanism appears toinvolve an increase in vessel density and limb perfusionand a rise in the number of circulating EPCs (25). The currentdata support the notion that GDF11 exerts its biologicalactivity through the AKT/HIF1a pathway. GDF11 increasedAKT phosphorylation and HIF1a expression both in diabeticrats and in EPCs. Moreover, GDF11-induced neovasculariza-tion was blunted in Ad-siHIF1a rats, and Ad-siHIF1apartially abrogated GDF11-mediated protection againstEPC dysfunction and demise in vitro, indicating thatHIF1a downregulation attenuated the proangiogenic ef-fect of GDF11. Cumulatively, we conclude that the GDF11-mediated beneficial effects may depend on the activationof the Smad and AKT/HIF1a cascades.

Our study had some limitations. First, GDF8 is a closestructural homolog of GDF11, with 90% amino acid se-quence identity shared in its mature active forms. Ourassay for rat serum GDF11 does not distinguish circulatingGDF11 from GDF8, and as a result, we did not accurately

Figure 7—GDF11 improves EPC tube formation and migration in vitro. A: Representative images of tube formation in various treatmentgroups. EPCs were pretreated with SB431542 for 30 min or transfected with Ad-siHIF1a and then incubated with rGDF11 (50 ng/mL) for60 min, which was followed by a 12-h incubation with HG. Scale bars = 100 mm. B: Representative images of migration assay in various treatmentgroups. EPCs were pretreated with SB431542 for 30 min or transfected with Ad-siHIF1a and then incubated with rGDF11 (50 ng/mL) for60min, which was followed by a 24-h incubation with HG. Scale bars5 200 mm.C andD: Quantitative analysis of A andB, respectively. Dataare mean 6 SEM for five independent experiments. *P , 0.05, **P , 0.01, ***P , 0.001. hpf, high-power field; MOI, multiplicity of infection.

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determine the GDF11 concentration in rats. Second, wedid not explore the specific mechanisms of the effect ofGDF11 on metabolism. Third, many other cells, such asmesenchymal stem cells and immunomodulatory cells, areknown to be involved in neovascularization (45). However,we did not further investigate the effect of GDF11 on theother cells associated with vascularization in this study;some additional studies are needed.

In conclusion, the data clearly suggest a critical role forGDF11 in vascularization through increasing EPC numberand improving its functions through the activation of theSmad2/3 and AKT/HIF1a pathways. These results implythe possible translational value of this research in the

clinical care of patients with diabetes in the future. Con-sidering the result that controlled elevation of GDF11 inthe circulation has therapeutic benefits in treating CLI,treatment with GDF11 may be applied in patients withdiabetes with ischemic vascular diseases, including myo-cardial infarction, cerebral stroke, and peripheral arterialocclusive diseases, and especially in patients with diabeticfoot ulcer and gangrene. With regard to the administra-tion routes, we envision that GDF11 treatment couldwork in patients through several approaches, such as inha-lation delivery, wound external application, or intravenous,intramuscular, or subcutaneous injection of exogenousrGDF11 protein and analogs. Long-acting formulation of

Figure 8—GDF11 activates the TGF-b/Smad pathway both in vivo and in vitro and activates HIF1a signaling in vivo. A–D: At the end of thestudy, rats were anesthetized by intraperitoneal administration of pentobarbital sodium (45 mg/kg) and euthanized for muscle isolation.Nondiabetic rats served as control. A: Western blots of total muscle lysates for p-Smad2/3 and Smad2/3. B: The relative expressions of thephosphorylated protein normalized to total protein are shown (n = 3 per group). C: Representative images of p-Smad2/3 in EPC culturestreated with rGDF11 alone or pretreatedwith TbRI inhibitor SB431542 under conditionsmimicking hyperglycemia and ischemia. Scale bars =50 mm. D: Quantitative analysis of C. Each experiment was repeated five times. E: Western blot analysis of p-AKT, AKT, HIF1a, VEGF, andSDF1a normalized to b-actin protein in the ischemic muscle from rats in various treatment groups. F: Quantitative analysis of E. Data aremean 6 SEM. *P , 0.05, **P , 0.01 vs. vehicle group; †P , 0.05 vs. IgG Ab group.

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GDF11 (e.g., microspheres, nanoparticles, fusion proteinthat is based on IgG heavy chain constant regions [IgGFc], adeno-associated virus–mediated stable gene expres-sion) may be developed in the future for the sake of bettermedical compliance. However, there is still a long way togo to get this treatment to market because there are a lotof clinical trials and regulatory affairs to be finished andbecause the cost-effectiveness analysis of this treatmentneeds to be further clarified.

Funding. This work was supported by grants from the National Natural ScienceFoundation of China (81370896, 81570730), National Key Research and De-velopment Program of China (2016YFC1305601), and Research Project of HubeiHealth and Planning Commission (WJ2017H0031).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. J.Z. and L.W. conducted the animal experiments.Y.L., L.X., J.D., and M.L. performed the in vitro experiments. H.L., B.Z., and B.G.analyzed the data and wrote the manuscript. G.X. is the guarantor of this work and,as such, had full access to all the data in the study and takes responsibility for theintegrity of the data and the accuracy of the data analysis.

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