Enhanced angiogenesis by mesenchymal stem cells based on Hyaluronic Acid hydrogel combined with GM-CSF and IL-2 in a rat model of hindlimb ischemia

Enhanced angiogenesis by mesenchymal stem cells based on Hyaluronic Acid hydrogel combined with GM-CSF and IL-2 in a rat model of hindlimb ischemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced angiogenesis by mesenchymal stem cells based on Hyaluronic Acid hydrogel combined with GM-CSF and IL-2 in a rat model of hindlimb ischemia yihang Cai, Junshu Wang, Lin Wu, Xinhuang Hou, Shiping Ji, Jinchi Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5348024/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The clinical application of stem cells in restoring ischemic lower limb perfusion has been hindered by challenges. To promote the directed differentiation of mesenchymal stem cells(MSCs) into endothelial cells(ECs) and enhance the paracrine effect, this paper aim to find a combined therapy for angiogenesis. Methods MSCs based on Hyaluronic Acid(HA) hydrogel combined with GM-CSF and IL-2(HGI-MSCs) were locally injected into the femoral artery ligated ischemia model rat. Recovery of perfusion was assessed by limb temperature and exhaustive distance. Hematoxylin and eosin(H&E) staining, immunohistochemistry and immunofluorescence staining were used to evaluate the quantity of blood vessels, microvessel density(MVD) and the proliferation and maturation of neovascularization in the limb muscle. Migration and tube formation of Human umbilical vein endothelial cells(HUVECs) were evaluated by Transwell and tube formation assays. The expression levels of angiogenesis-related genes, cytokine and proteins were measured by qRT-PCR, ELISA and Western blotting, respectively. Results HGI-MSCs promoted ischemic limb angiogenesis and restored perfusion. Our study showed that HGI-MSCs could promote the proliferation and maturation of neovascularization. Moreover, HGI-MSCs promoted HUVECs migration and tube formation in vitro, up-regulated the expression of VEGF and activated PI3K/Akt signaling pathway. Conclusions HGI-MSCs showed a more robust pro-angiogenic effect than that of pure MSCs in the ischemia limb model rat. It offers novel ideas for the treatment of patients with refractory limb ischemia. Mesenchymal stem cells Human umbilical vein endothelial cells Endothelial cells Hyaluronic Acid hydrogel GM-CSF IL-2 Critical limb ischemia Angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background The prognosis of critical limb ischemia (CLI) is extremely unfavorable, encompassing non-healing ulcers, impaired ambulatory function, limb amputation, and even life-threatening consequences. For the patients with ischemic disease of lower limbs, revascularization is essential in treatment of CLI. However, there are 20–30% of those patients who are not qualifed for endovascular treatment or bypass surgery. In recent years, more experts and clinicians are paying attention on stem cell therapy on ischemic diseases. It has been proven that stem cells exhibit a robust migratory ability, differentiation potential, and secretion of a variety of beneficial cytokinescan. The stem cell therapy can help restore blood perfusion and alleviate secondary injury caused by ischemia [ 1 – 4 ] . However, the application of stem cell therapy is challenged by factors such as impaired multilineage differentiation ability, immune rejection, exposure to ischemia and inflammation, mechanical washout by the vasculature, and leakage of cell suspension from the target injection site, lead to poor efficacy [ 5 ] . Therefore, improving the viability and functionality of stem cells is key to its potential applications.Several approaches,like growth factors, overexpression of stem cell regulatory genes, to improve the tissue-regenetive capacity of stem cells have been investigated. The effect of these approaches on stem cells is related to the activation of phosphoinositol 3-kinase (PI3K)/Akt signaling pathway,which plays a central regulatory role in angiogenesis [ 6 ] . Angiogenesis is a highly regulated process involving the formation of new blood vessels sprouting from preexisting vessels through the activation, migration, and proliferation of vascular endothelial cells(ECs) [ 7 ] . Granulocyte macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that can enhance the functions of various cells that are necessary for inducing endothelial cells to migrate and proliferate [ 8 ] . Interleukin-2(IL-2) plays a major role in the clonal expansion of T lymphocytes(T cells) by interacting with specific cell surface receptor. It binds to the functional receptor and induces activation of PI3K/Akt siganling pathway [ 9 ] , which is well known as a regulator of neovascularization. Hyaluronic acid (HA), the main component of extracellular matrix(ECM) consisting of repeating disaccharide units of b-(1,4)-D-glucuronic acid and b-(1,3)-N-acetyl-D-glucosamine, plays a vital role in cell proliferation and migration during biological processes like tissue regeneration and angiogenesis [ 10 ] . It has been widely utilized as drug-carrying scaffold in regeneration medicine. And numerous studies have demonstrated that all of bioactive factors mentioned above had a certain promoting effect on the secretion of VEGF [ 11 – 15 ] , indicating a synergistic effect on angiogenesis. To our knowledge, relevant research on the combination of MSCs and bioactive cytkine for the treatment of limb ischemia is very limited.Based on the above results, we believe that the application of combined therapy above can improve the efficiency of restoring ischemic limb perfusion. Thus, we developed a rat model of hindlimb ischemia,combined MSCs and bioactive factors(GM-CSF and IL-2) and piggybacked them on hyaluronic acid hydrogels to evaluate the synergistic effect of the combined therapy on enhancing angiogenesis in CLI and study the related mechanism. We found that compared with the treatment of PBS and pure MSCs,the combined therapy enhanced angiogenesis and promoted its maturation. The results revealed the underlying mechanism of HGI-MSCs therapy in promoting angiogenesis and provided strategies for the treatment of limb ischemia in the future. Methods Preparation of cell Four-week-old Sprague Dawley rats(Shanghai SLAC Laboratory Animal Co., Ltd), which were SPF grade, male and weighed ~ 80–120 g, were used. Rats were anaesthetized by intraperitoneal injection of 2% sodium pentobarbital(30mg/kg) in order to minimalize the stress and suffering. 10 min after administration of anaesthetics, animals were euthanized by the disruption of the spinal cord. Next, the bilateral femurs and tibias in rats were removed after decapitation under sterile conditions. Removing the metaphysis, the exposed bone marrow cavity was flushed with Dulbecco’s modified Eagle’s medium(DMEM; Gibco; USA). The rinse solution was collected. Pass the rinsing solution through a 200-mesh screen in order to produce a single cell suspension that was centrifuged 1000 rpm for 15 min. The supernatant was discarded and then PBS was added to blow suspension. BM-MSCs were isolated using the bone marrow adherence method. Cells were cultured in α minimum essential medium (α-MEM, Gibco, USA) supplemented with 10% fetal bovine serum(FBS, cegrogen, Germany) at 37°C in a 5% CO 2 humidified atmosphere. The medium was changed every 2 days. Cells at passages 3 to 5 were used in the experiments. Primary HUVECs were purchased from Zolgene and cultured in endothelial cell growth medium (ECM, ScienCell, USA) containing 5% FBS and supplemented with 100× endothelial cell growth supplement (ECGS, ScienCell, USA). The medium was changed every 2 days. Cells at passages 3 to 5 were used in the experiments. Preparation of the HGI-MSCs-hydrogel HGI-MSCs suspension was formed by mixing 1ml MSCs with 1ml hyaluronic acid (HA) hydrogel(Humedix Co., Ltd.,Korea), rhG-CSF(Hainan Unipul Pharmaceutical Co., LTD., China) and IL-2(Beijing SL Pharmaceutical Co., Ltd.,China) at room temperature. The final concentrations of rhGM-CSF and IL-2 were 150 ng/ml and 1*10^5 U/ml respectively. Animal Model and Treatment The rats(SPF grade, male and weighed ~ 200 ~ 250 g) were weighed and anesthetized under 2% sodium pentobarbital(50mg/kg) for surgical intervention. The surgical intervention was performed to create unilateral hindlimb ischemia model in the rat. Exposure was obtained by performing an incision in the skin overlying the middle portion of the left hindlimb of each rat. After ligating the proximal end of the femoral artery, the distal portion of the saphenous artery was ligated, and the artery and all side-branches were dissected free; the femoral artery and attached side-branches were then excised(Fig. 1 A). The overlying skin was then closed using a surgical stapler. While the corresponding right femoral artery was left unexcised and used as a control. 3 days after the operation, the skin temperature of the dorsum of the foot of both lower limbs were measured every 3 days for 60days. Keep records. After 60 days, all rats were randomly divided into 3 groups of eight each. Firstly, each rat was injected intramuscularly with 0.1ml PBS at 10 different points on the medial and lateral sides of the ischemic limb. It was marked as the PBS group. In the MSCs group, pure MSCs suspension was injected. The third group was injected with HGI-MSCs suspension(composition and concentration have been indicated above), which was marked as the HGI-MSCs group. All injection methods and dosage were the same as the first group [ 16 ] , and they were given for three consecutive days. Skin temperature was measured every 5 days for a period of 30 days subsequent to the injection. The experimental design is shown in Fig. 1 B. The work has been reported in line with the ARRIVE guidelines 2.0. Exhaustive exercise All rats were subjected to forced exhaustive exercise running on treadmill apparatus(KW-PT, KEW, China). The rats were initially familiarized with the treadmill of low intensity running for 3 days. On 1st day of the familiarization protocol, rats placed on the treadmill were given 5 min to acclimate before the treadmill belt was engaged. The belt speed was initially set to 8m/min then increased to 10m/min, then 12m/min for 5 min each, all at an incline of 10%. On day 2, rats were given 2 min to acclimate to the treadmill then exercised at 8m/min, 10m/min, 12m/min and 15m/min for 5 min each. On day 3, rats were placed on the treadmill and immediately exercised at 10m/min for 5 min, then 15m/min and 20m/min for 5 min each.After familiarization, rats were allowed two days to recover before beginning the exhaustive exercise protocol. At the experiment day, all rats were subjected to the following protocol: at a 10% incline, the initial speed was set at 10m/min for 8 min, then increased to 15m/min for 5 min, and then increased by 2.5m/minute every three minutes until reaching a speed of 25m/minute. Rats exercised for 10 min at 25m/min, then speed was then increased by 1m/min every 5 min until exhaustion.Exhaustion was determined when rats failed to re-engage all four paws with the treadmill belt despite negative stimulus[17]. Then, the animals were euthanized. The procedure for euthanasia has been described above. Blood and limb muscle were harvested for further processing.All rats experiments were approved by the Animal Ethics Committee of Fujian Medical University. The work has been reported in line with the ARRIVE guidelines 2.0. Histology and immunohistology analysis Samples harvested from the lower limb muscle were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. Serial 5-mm sections were cut and stained with hematoxylin and eosin (H&E) for observation of the quantity of vessels in the muscle. For immunohistochemical staining, a panel of primary antibodies against rat CD31(1:500, Servicebio, China) were used. The sections were firstly incubated with 3% H2O2 for 10 minutes to deactivate the endogenous peroxidase. To recover antigen, these sections were soaked in 10 mM citrate buffer solution (pH 6.0) and heated twice in the microwave oven. And the slides were then washed thoroughly with PBS(pH 7.4). After blocked with 5% bovine serum albumin in Tris-buffered saline for 20 minutes, the sections were incubated with primary antibodies against CD31 at 4℃ overnight followed by washing with PBS. Afterward, the slides were sequentially incubated with secondary antibody for 1h and streptavidin-horseradish peroxidase for another 20 minutes. The staining was visualized after incubation with a DAB-H2O2 solution. The slides were then counterstained with hematoxylin for 1 minute, dehydrated with ethanol, and sealed in resinene for microscopic observation. Immunofluorescence analysis A double-labeling immunofluorescence technique was applied to analyze the proliferation of endothelial cells using anti-CD31(1:200, Servicebio, China) and anti-Ki67(1:300, Servicebio, China) antibodies. Similarly, the pericyte coverage of microvessels was observed using antiCD31 and anti-α-smooth muscle actin(α-SMA ) (1:200, Abcam, Cambridge, MA) antibodies. The sections were blocked with BSA(5%) for 2 h and incubated with primary antibody at 4℃ overnight. And then, the sections were further incubated with secondary goat anti-rabbit antibodies(Servicebio, 1:400) for 1 h in the dark after washing. Subsequently, the sections were incubated in 4’6-diamidino-2 phenylindole (DAPI) for nuclear staining. Images were obtained with a fluorescence microscope(Carl Zeiss) and were merged using Image-Pro Plus v. 6.0 software. Negative control staining experiments were performed by omission of the primary antibody. Quantitation of MVDs, PCI, and MPI. All sections were assessed for uniformity of staining at low power(c50), and individual microvessel counts were then performed under a ×200 field. Staining of endothelial cells for CD31 was used to express MVD counts. Any CD31 positive endothelial cell clusters clearly separated from each other were considered as single countable microvessels. Microvessel density (MVD) was assessed by light microscopic analysis for areas of the lower limb muscle that contained the most capillaries and small venules (microvessels) (so-called neovascular "hot spots"). Counts were transformed and expressed as the number of microvessels/ mm2(1 HPF = 0.0681mm2). The proliferating capillary index(PCI) was used to assess the proliferating endothelial cells and was quantified by calculating the ratio of the number of microvessels with proliferating endothelial cells(Ki67) to the total number of microvessels(CD31) which was screened for the areas with the highest vessel density under a magnification of ×200. Similarily, the microvessel pericyte coverage index(MPI) is expressed as the α-SMA/CD31 ratio, which is used to assess the maturity of new blood vessels. It was correspondingly established by quantitating the percentage of microvessels that co-localized endothelial cell staining (CD31) and pericyte staining (α-SMA). For all quantification, at least five nonoverlapping microscopic fields per section were independently analyzed under double-blind conditions [ 18 ] . In vitro tube formation assay HUVECs coculture with PBS/MSCs/HGI-MSCs were performed on Basement Membrane Matrigel(MCE, China) in 24-well plates with 100 µL of Matrigel/well. The optimal time for tube formation was determined following an 8-hour incubation at 37℃ with periodic observations. The capillary-like structure was examined under an inverted phase contrast microscope at ×100 magnification. Five fields per test condition were examined. The tube length was measured using ImageJ software. In vitro migration assay HUVECs (4.0 × 10*4 per chamber) were plated into the upperchamber in medium (containing 1% FBS). Meanwhile, the lower chamber was divided into PBS/MSCs/HGI-MSCs group. After 24h of incubation, the cells that had attached to the lower surface were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 15 minutes after removing the paraformaldehyde. The chamber was washed twice with PBS, and the non-migrated cells on the membrane were removed using cotton swabs. Each group was randomly photographed under the microscope(×200) to observe and count the cells in the field of view. Real-Time Polymerase Chain Reaction. Total RNA from muscle samples were isolated using Trizol reagent according to the manufacturer’s instructions. cDNA was synthesized with PrimeScript RT Master Mix kit. Real-time polymerase chain reaction was performed with SYBR green premix in accordance with the manufacturer’s instructions. The following primers were used for cDNA amplification: for VEGF (Norway rat), 5-GCACGTTGGCTCACTTCCAG-3 (forward) and 5-TGGTCGGAACCAGAATCTTTATCTC-3(reverse); for b-actin(Norway rat), 5-GGGAAATCGTGCGTGACATT-3(forward) and 5-GCGGCAGTGGCCATCTC-3(reverse). All of the gene expressions were normalized to that of the internal reference genes, namely b-actin, within the same samples using the 22DDCt method [ 19 ] . Measurement of cytokines Quantification of VEGF in serum was performed using the VEGF ELISA kit specific for mouse(BOSTER, Wuhang) according to the manufacturer’s instructions. The absorbance at 450 nm was read on an automatic ELISA system. Western blot analyze The limb muscle samples stored at -80°C were taken, and weighed for 50 mg, then cut into small pieces. Then, the muscle tissues were homogenized in RIPA buffer(containing RIPA 5×DTT, Protease Inhibitor Cocktail, Phenyl-methysulfonylfluoride(PMSF) and phosphorylase inhibitors cocktail). The homogenization was centrifuged at 12000 rpm for 15 min at 4°C, retaining the supernatant, and the protein concentrations were determined using a bicinchoninic acid(BCA) protein assay kit(Boster, Wuhan, China). The samples were added with 2 × loading buffer, then equal amounts of protein in each sample were separated on 12% SDS polyacrylamide gel electrophoresis(SDS-PAGE) gels and transferred onto polyvinylidene fluoride(PVDF) membrane. The membranes were blocked with 5% bovine serum albumin(BSA; Boster, Wuhan) for 2 h at 4°C, and then incubated with the following primary antibodies: rabbit anti-Akt(1:1000, Cell Signaling Technology), rabbit-anti-phosphoAkt(Ser473) (1:2000, Cell Signaling Technology) and rabbit anti-β-actin(1:5000, Bioworld) overnight at 4°C. Subsequently, washing in Tris-buffered saline plus Tween (TBST) buffer three times for 5 minutes each, membranes were incubated with goat anti-rabbit immunoglobulin (Ig)G-peroxidase conjugated(1:3000, Boster) for 2 h at 37°C. Finally, the protein bands were visualized using an Enhanced Chemiluminescence Detection Kit (Boster, Wuhan, China), captured with iBright 1500(Thermo Fisher Scientific, Waltham, USA), and quantified with Image J software. The protein expression was normalized to β-actin, and the experiment was repeated independently three times. Statistical analysis The values were presented as the mean ± standard deviation (SD). Statistical significance among multiple experimental groups was determined using one-way ANOVA followed by Tukey’s post hoc test with GraphPad Prism 9(GraphPad Software Inc., San Diego, CA). Only dif- ferences with P< 0.05 were considered statistically significant. Results Assessment of ischemia model induced by femoral artery ligation On day 60 after ligation surgery, the skin temperature of the surgical hindlimb, which was significantly lower than that of the normal side,was observed to change(Fig. 2 A). It shows that following ligation of the proximal femoral artery and resection of the distal and all branch arteries and veins, there is a disparity in blood perfusion between the operated and healthy sides(Fig. 2 B), indicating successful establishment of the lower limb ischemia model. With the prolonged ischemia time,refractory wound ulceration and partial gangrene were observed in the paws of rats(Fig. 2 C). Assessment of restored perfussion induced by HGI-MSCs therapy At the 30th day after drug injection, it was observed that the limb ulcers in the PBS group were not significantly improved and healed, while in the MSCs and HGI-MSCs groups, the ulcers were significantly healed, and the skin color of the operated limb was close to the healthy side(Fig. 3 A). There was significant difference in skin temperature among HGI-MSCs/MSCs/PBS group, indicating that the therapeutic efficacy of HGI-MSCs in restoring blood perfusion is superior to that of MSCs group and PBS group. Moreover, the skin temperature of the HGI-MSCs group was close to the healthy side, the difference was not statistically significant(Fig. 3 B). By monitoring the skin temperature of the lower limbs every 5 days, it can be inferred that after 2 months of ligation, the rats began to exhibit a compensatory trend in the lower limbs, which was further enhanced by HGI-MSCs injection therapy. It indicates that while neovascularization in the lower limb of rats has initiated compensation for regional hypoperfusion caused by acute ischemia, HGI-MSCs can expedite neovascularization and enhance restoration of lower limb perfusion(Fig. 3 C). HGI-MSCs induced an increase in exhaustive exercise distance and formed more vessels in ischemic muscles. To determine the enhancement of motor function in rats treated with HGI-MSCs injection therapy, we performed exhaustion experiments(protocol was shown in Fig. 4 A). The results showed that HGI-MSCs injection therapy significantly increased the exhaustive exercise distance of the rats,which provided strong evidence that HGI-MSCs had positive effects on the motor function of ischemia limb rats. Similarly, rats treated with MSCs ran more distance than that with PBS treatment. In the PBS group, several rats became exhausted within just a few minutes on the treadmill due to severe limb ischemia(Fig. 4 B). Histopathological analysis was performed to evaluate the blood perfusion in muscle tissue by HE staining. The findings indicated a high abundance of blood vessels in the HGI-MSCs group, which appeared more perfusion than MSCs group and the PBS group(Fig. 4 C). HGI-MSCs promotes angiogenesis and the proliferation and maturation of endothelial cells The specific markers CD31 were utilized to simultaneously immunostain vascular endothelial cells in order to quantitatively assess the neovasculature in the operated hindlinbs(Fig. 5 A). The results showed that the HGI-MSCs group had much higher average MVD than that in the MSCs group after injection. Meanwhile, we observed the MSCs group had higher average MVD than that in the MSCs group after injection(Fig. 5 B). Meanwhile,to quantify the proliferation of endothelial cell in the muscle of lower limb, we double-stained endothelial cells using CD31 and the cell proliferation marker ki-67 to quantify the proliferation capillary index (PCI), which reflected the percentage of microvessels with Ki67 positive endothelial cell nuclei. We figured that there is significant difference between the three groups. As shown in Fig. 5 C,the PCI value increased significantly in HGI-MSCs group(Fig. 5 D). We evaluated the functional status of neovascularization using double-labeling immunofluorescence techniques with CD31 and α-SMA to detect the colocalization of endothelial cells and pericytes. As expected, it shows that the pericytes of the HGI-MSCs group formed more circles and densely enveloped the endothelial cells than that of the MSCs group and the PBS group(Fig. 5 E).The above trend can be seen through the quantitative analysis of microvessel pericyte coverage index(MPI) (Fig. 5 F). It can be inferred that HGI-MSCs facilitate the advancement of neovascular maturation and functionality. HGI-MSCs enhanced the migration and tube formation of the HUVECs in vitro. In the transwell system, the migration capacity of HUVECs is activated by HGI-MSC. It can be inferred that the number of migrating cells is greater in the HGI-MSCs group than that in the MSCs group and the PBS group(Fig. 6 A&C). In the coculture system, the angiogenesis of HUVECs was observed by tube formation assay. We can detect that HUVECs monoculture formed less lumens or can not form any lumen sometimes. With supplements, HUVECs cocultured with HGI-MSCs formed more capillary-like structures than that of MSCs group(Fig. 6 B&D). HGI-MSCs up-regulated the expressions of VEGF The changes in gene expression of VEGF after drug treatment were quantitatively analyzed by qRT-PCR. The results showed VEGF were significantly higher in the HGI-MSCs group than that in the MSCs group and the PBS group after treatment(Fig. 7 A). Meanwhile, the ELISA analysis of VEGF cytokine in the serum to evaluate the changes in phenotype. The results showed that VEGF elevated in the MSCs group and reached a peak in the HGI-MSCs group compared with that in the PBS group(Fig. 7 B). HGI-MSCs activated PI3K/Akt signaling pathway. Western blot analysis was conducted to evaluate the role of PI3K/Akt pathway in VEGF-induced angiogenesis. The activation of this signaling pathway is controlled by the phosphorylation-induced activation of AKT. As shown in Fig. 7 C, Akt and p-Akt was observed in all groups. However, the expression of p-Akt/Akt in the HGI-MSCs group markedly increased. Discussion In this study, we investigated whether the pro-angiogenic effect of MSCs could be enhanced by combining with GM-CSF and IL-2 on hyaluronic acid-based hydrogels. Our findings revealed that HGI-MSCs was effective in promoting angiogenesis in artery ligated induced hindlimb ishcemia rats to restore perfussion. A significant enhanced effect of HGI-MSCs on proliferation and maturation of endothelial cells was found in double-labeling immunofluorescence. Furthermore, it was demonstrated that HGI-MSCs induced HUVECs migrate and form more lumen by up-regulating VEGF and it might be related with the activation of PI3K/Akt signaling pathway. Importantly, it was found all the effects mentioned above with HGI-MSCs were greater than that with pure MSCs. Taken together, these results provided a novel insights underlying the beneficial effects of HGI-MSCs on pro-angiogenesis of stem cells and offered innovative therapies on ischemic diseases. Researches on lower limb ischemic diseases have shown that the obstruction caused by atherosclerosis and microcirculation dysfunction of the lower extremity leads to hypoperfusion or even no perfusion in part of the blood supply area, which will lead to hypoxia of the limb, and nutrients will not be delivered to the site. MSCs, as far as we know, can be transplanted into tissues ex vivo. In the appropriate microenvironment, MSCs can adhere, proliferate and differentiate into specialized cells of target tissues, thereby playing a role in the regeneration and repair of injured tissues [20] . Despite the existence of strong evidence for the therapeutic effects of MSCs, the mechanisms remain unclear. Current mechanistic studies have revealed two main mechanisms of MSCs: paracrine and multidirectional differentiation [21] . This thesis proposes HGI-MSCs therapy to promote the directional differentiation of MSCs and the proliferation, migration, and maturation of endothelial cells, which might be related to elevated expression of VEGF, eventually form vascular lumen, enhance blood perfusion. Sun et al. have demonstrated that extended ligation of the distal femoral artery will reduce perfusion of lower limbs and lead to ischemia by Laser Doppler perfusion imaging(LDPI) [22] . As expected, the establishment of the lower limb vascular ligation model resulted in a reduction of perfusion in the operated limb of the model rat. This was evidenced by a decrease in temperature of the ischemic limb, as well as the progressive development of ulcers and claudication over time. After HGI-MSCs treatment, the skin temperature of the affected limb increased compared to the PBS group, and ulcer healing was observed. The MSCs group also exhibited a similar trend when compared to the PBS group, suggesting that while pure MSCs therapy has a certain role in enhancing lower limb perfusion, but its efficacy is not as pronounced as that of HGI-MSCs therapy. It is well known that the oxygen and nutrients brought by blood perfusion provide the necessary conditions for excercise. Relevant reports about hypoperfusion resulting in functional decline of exercise have proven that blood perfusion is essential for the preservation of motor function [23] - 24] [25] . Due to the significant ischemia present in lower limbs, the rat in PBS group could merely run on the treadmill for a few minutes, or even less than one minute. This indicates that the blood supply disorder caused by artery ligation will seriously affect the motor function of rat.After injection treatment of HGI-MSCs, the exhaustive exercise distance was significantly increased. Pathological analysis of the lower limb muscles showed that the HGI-MSCs group had more blood vessels than the other two groups. This improvement in motor function was attributed to the enhancement of one type of neovascularization, the apparent improvement in the number of MVD. In this study, immunohistochemical staining of CD31 was used to evaluate changes in the number of MVD. We figure that there would be more neovascularization in the lower limb muscles after treatment with HGI-MSCs than that in the MSCs group and the PBS group in terms of the quantity of MVD. Studies on lower limb ischemic disorders reveal that microcirculation dysfunction of lower limb muscles is the principal cause influencing motor function and wound healing [26] . Therefore, angiogenesis in the lower limbs can increase the motor function of rats, that is, the increase in running distance. MVD was counted in five random fields within the pathological sections of the lower limb muscles of the rats, and the average value was calculated. The correlation between the averaged MVD and the running distance of the rats was analyzed by Pearson correlation analysis, which revealed a linear correlation between the distance and MVD(r=0.8476, p< 0.01).Hence, facilitating angiogenesis constitutes effective therapeutic strategies for restoring perfusion. Previous researches revealed that Hyaluronic acid hydrogels were used as scaffolds on generating of functional vascular networks [27] . And the addition of GM-CSF may stimulate the hematopoietic stem cells (HSCs) in the suspension of bone marrow cells to differentiate into granulocytes and macrophages, participate in the local inflammatory response, and thus stimulate the regeneration of blood vessels [28] . On the other hand, it promotes the proliferation of bone marrow stem cells to maintain their viability. Bae, Jin-Hee et al. also demonstrated endothelial cells express IL-2Rα and β and IL-2 may promote angiogenesis [29] . IL-2 and GM-CSF together regulate the immune function, stimulate the activation of MSCs, make them differentiate into vascular endothelial cells, and secrete VEGF.The generated VEGF can also further stimulate MSCs to differentiate into vascular endothelial cells, thus forming a circulation conducive to angiogenesis. In addition, IL-2 may also stimulate local tissue to secrete VEGF and bFGF through inflammatory response, thus promoting the regeneration of blood vessels.The small molecule hyaluronic acid hydrogel was chosen as the matrix because of its large microchannels and loose ultrastructure. Recent researches have found that capillary-like structures form best in soft matrix [27] . In this study, the proliferation coverage index(PCI) was quantified by co-localized staining for CD31 and Ki67. We found that HGI-MSCs shows obvious advantages in promoting the proliferation of endothelial cells compared with pure MSCs treatment and PBS treatment.Thus, the enhanced effect on promoting the proliferation of endothelial cells may be attributed to HGI-MSCs therapy. Similarly, HGI-MSCs had a positive effect on the maturation of neovascularization. After formation of the original vessels, the stabilization and maturation of the newly formed vessels will become a major concern [30] . Previous studies have shown that insufficient maturity of the vascular system may lead to vascular obstruction, edema, and hemorrhage, affecting the transport of nutrients and oxygen necessary for perfusion restoration. Pericytes are in direct contact with endothelial cells through gap junctions and share the basement membrane with endothelial cells, strategically covering 90% of the capillary surface area,which constitute a vital component of the microvascular wall and are instrumental in regulating microvascular morphology, microcirculation, and permeability [31] . Therefore, pericyte coverage and basement membrane integrity are hallmarks of mature microvessels [32] [33] . When we observed newly formed microvessels at the late stage of ligation, we found not only a higher number of newly formed vessels but also a higher pericyte coverage of microvessels after HGI treatment. Several elongated pericytes encased endothelial cells and bridged adjacent endothelial cells to form tight junctions, indicating that these microvessels were more mature and stable.MPI quantitative analysis showed that MSCs combined with a variety of bioactive factors could promote vascular maturation through immune regulation.Zhao J et al. have demonstrated GM-CSF regulates maturation of microvessels by manipulating the spatial-temporal Ang-1/Ang-2 balance and the phosphorylation of Tie-2 [32] . On the other hand, IL-2 in the microcirculation is readily taken up by the blood vessels [34] . IL-2 has been shown to stimulate glycosaminoglycan synthesis in vascular smooth muscle cells and regulate the responsiveness to angiotensin II, which maintaining vascular wall stability [35] .However,the presence of this IL-2 would have some effect on vascular permeability, which is contrary to our expectation [36] . To gain a deeper understanding of the role of HGI-MSCs in mediating endothelial cell migration and tube formation, we conducted tests on HUVECs at the cellular level and discovered that following treatment with HGI-MSCs, the migration of HUVECs was enhanced and HUVECs can form more vascular network structure.Here, HA hydrogel was used as ECM supplemented with GM-CSF and IL-2 to study vascular morphogenesis at the cellular level.In recent decades, the role of natural ECM, such as collagen and fibrin gels, in vascular morphogenesis has been thoroughly elucidated [37] [38] . In contrast to natural materials, HA hydrogels can be made from well-defined synthetic polymer networks that not only have a high water content to promote cell viability, but also possess biophysical and biochemical properties similar to many tissues which can be tuned to favor vascular morphogenesis [39] [40] . Therefore, HA gel may load bioactive factors and enhance their delivery efficiency, holding great promise for regenerative medicine as a biological scaffold.In the tube formation assay, the combination of GM-CSF and IL-2 promoted tube formation of HUVECs in co-culture system with MSCs [41] [42] . However,Tim D Eubank,Eleni Sakkoula et al. expressed some opposing opinions regarding GM-CSF and IL-2 in angiogenesis when researching oncology [43] [44] . To further explore the above phenomenon,we examined the corresponding expression of mRNA of VEGF, which stimulates the proliferation and survival of endothelial cells and promotes angiogenesis and vascular permeability.In angiogenesis process,cells, including macrophages and fibroblasts, translocate the hypoxia-inducible factor HIF-1α or KRAS to the nucleus in response to hypoxia, thereby activating the expression of target genes including VEGF. VEGFA(but also VEGFE or VEGFC VEGFD) binds to VEGFR2, and upon activation of VEGFR2, several pathways are activated. The family of vascular endothelial growth factors (VEGFs) and their receptor tyrosine kinases (RTKs) are widely recognized as the most extensively studied pathways in developmental angiogenesis [45] , stimulating the PI3K-AKT pathway phosphorylation activation signaling cascade and increasing endothelial cell survival [46] . However, VEGFR1, which is a poor signaling receptor, but very potent VEGF binder [47] . There is a consensus that VEGFR1 is able to regulate VEGF activity on vascular endothelial cells in a negative manner by sequestering and rendering the factor unavailable to VEGFR2 [48] . This may explain the opinions from the above Professor Tim D Eubank and Eleni Sakkoula. We didn’t investigated the subtypes of VEGFR in the combined therapy to angiogenesis,and this question will be addressed in future researches.Back to the signal in the pathway itself, we detected the VEGF levels in serum using ELISA. The results showed that HGI-MSCs treatment may promote the paracrine secretion of VEGF.Subsequently, statistical significance was achieved through Akt and P-Akt Western blot analysis studies. It shows that the activation of PI3K-Akt signaling pathway to promote angiogenesis might be mediated by elevated VEGF in the corresponding tissues. And in term of migration of HUVECs,there were more HUVECs in the lower chamber(supplemented with HGI-MSCs), suggesting that HGI-MSCs activated PI3K/Akt pathway by secreting VEGF, thereby mediating cell migration [49] . In the coculture system of HUVECs and HGI-MSCs, HUVECs can form more vascular network structures, which is also mediated by the activation of VEGF-induced PI3k/Akt pathway [50] [51] . Conclusion Our study demonstrated that mesenchymal stem cells based on hyaluronic acid hydrogel combined with GM-CSF and IL-2 can effectively restore lower limb perfusion and improve motor function in rats. And the effect is more effective than that of pure MSCs therapy. This process is likely to induce the activation of PI3K/Akt pathway through up-regulation of VEGF expression, leading to angiogenesis and maturation of neovacularization. The findings provided a new perspective on the clinical application of MSCs in the field of ischemic diseases. Abbreviations MSC: Mesenchymal stem cell; EC: Endothelial cell; GM-CSF: Granulocyte macrophage colony-stimulating factor; IL-2: Interleukin-2; HA: hyaluronic acid; VEGF: Vascular endothelial growth factor; SD: Sprague-Dawley; PBS: Phosphate-buffered saline; DMEM: Dulbecco’s modified Eagle medium; FBS: Fetal bovine serum; DAB: 3,3′-Diaminobenzidine; DAPI: 6-Diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay; Declarations Competing interest The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate All animal experiments were conducted in accordance with the ARRIVE guidelines 2.0(Animal Research: Reporting of In Vivo Experiments) and approved by the Animal Ethics Committee of Fujian Medical University(Approval No. IACUC FJMU 2022-0709 “Effects of angiogenic therapy on motor function of ischemic hindlimbs in rats.” approved on September 30, 2022). Human umbilical vein endothelial cells were obtained from Zolgene Company (Fuzhou, China). Zolgene Company has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent. HUVECs cultures in this study were reviewed and approved by Ethics Committee of Fujian Medical University(Approval No. IACUC FJMU 2022-0709 “Effects of angiogenic therapy on motor function of ischemic hindlimbs in rats.” approved on September 30, 2022). Funding This study was funded by Natural Science Foundation of Fujian Province(2020J01960) Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.All additional files are included in the manuscript. Authors’contributions YHC and JSW performed the experiments, analyzed the data, and wrote the manuscript; JCZ reviewed and edited the manuscript; and WL,XHH, SPJ researched the data. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5348024","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381945551,"identity":"49c84930-e8e8-4181-9a14-8b2b8c018c64","order_by":0,"name":"yihang Cai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACNmbmAwcSftTwyLM3HyBOCx87W+KBhz3HZAx7jiUQp0WOn8f44AM2ZhuGGzkGxDqMweBAAg8bD+OMnI833jDYyek2ENaScCDBQoaHneftZss5DMnGZgcIazkAsaU9d5s0D8OBxG2EtTA2HEhgYwYqznlGrBagNWAtJ3LYiNXCBlTWc4wHGMjGlnMMiPCLfP/5zx9//KixB0blwxtvKuzkCGpBARI8REYNshZSdYyCUTAKRsGIAAB1jz7qUHNuKAAAAABJRU5ErkJggg==","orcid":"","institution":"Fujian Medical University","correspondingAuthor":true,"prefix":"","firstName":"yihang","middleName":"","lastName":"Cai","suffix":""},{"id":381945552,"identity":"f1bd7aa3-4b7e-4c54-a707-c9ae6d44df22","order_by":1,"name":"Junshu Wang","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Junshu","middleName":"","lastName":"Wang","suffix":""},{"id":381945553,"identity":"f9d560da-8af7-47c1-b7ae-4b5b07cc2222","order_by":2,"name":"Lin Wu","email":"","orcid":"","institution":"Wuzhou Worker Hospital: Guangxi Medical University Seventh Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Wu","suffix":""},{"id":381945554,"identity":"6d6e53ff-60cc-43d7-97a8-68ab32f1eb7a","order_by":3,"name":"Xinhuang Hou","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinhuang","middleName":"","lastName":"Hou","suffix":""},{"id":381945555,"identity":"ed67b755-b826-4563-870d-ea50b87585a3","order_by":4,"name":"Shiping Ji","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shiping","middleName":"","lastName":"Ji","suffix":""},{"id":381945556,"identity":"6cd32b96-7c6b-43a5-80e4-5fcff45a58b5","order_by":5,"name":"Jinchi Zhang","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinchi","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-10-28 14:43:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5348024/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5348024/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71894645,"identity":"c54855d6-72a3-4477-926c-1611cceb2bbe","added_by":"auto","created_at":"2024-12-19 13:35:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":192706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental approach\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eHindlimb ischemia model was created by extended removal of left femoral artery and all attached side-branches. \u003cstrong\u003eB\u003c/strong\u003e. Rats were randomized to receive PBS/MSCs/HGI-MSCs equal volume at 10 different points on the medial and lateral sides of the ischemic limb for three consecutive days, followed by temperature measurement every 5 days for a period of 30 days subsequent to the injection.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/e662980de87f76a40f2cc54c.png"},{"id":71896231,"identity":"cfc81823-95c9-4d03-8c8e-77dfcb5f3447","added_by":"auto","created_at":"2024-12-19 13:44:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":748603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of establishment of ischemia model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eBilateral skin temperature changes in 60days after ischemia model induction. \u003cstrong\u003eB. \u003c/strong\u003eEvaluation of ligation effect. \u003cstrong\u003eC. \u003c/strong\u003eRepresentative image of paws ulcer induced by femoral artery ligation. All data are expressed as mean±SD(n=8). *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, compared with healthy side.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/6f84b74ba8adafdf5aee7869.png"},{"id":71894647,"identity":"2d877223-4c1d-4818-bbec-c6ee250317d3","added_by":"auto","created_at":"2024-12-19 13:36:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":945517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of HGI-MSCs on restoring perfussion of ischemic limb.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative images of ulcer healing analysis in the PBS/MSCs/HGI-MSCs treatment groups in self-controlled rat models at the 30\u003csup\u003eth\u003c/sup\u003e day after the injection event. \u003cstrong\u003eB. \u003c/strong\u003eSkin temperature analysis of operated limb in the PBS/MSCs/HGI-MSCs treatment groups and the healthy limb. \u003cstrong\u003eC. \u003c/strong\u003eThe overall trend of skin temperature changed with time.All data were expressed as mean±SD(n=8). Significance was set to *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, ns: not significant.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/b772e9ad158083eb654bc60c.png"},{"id":71894646,"identity":"b99e8f44-c5c9-4f93-b113-6cd920f719f4","added_by":"auto","created_at":"2024-12-19 13:35:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1378319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of motor function and pathological analysis of lower limb muscle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eRats were placed on the treadmill and conducted exhaustive exercise by stimulus. \u003cstrong\u003eB. \u003c/strong\u003eExhaustive exercise analysis after injection therapy. \u003cstrong\u003eC. \u003c/strong\u003eEffects of PBS/MSCs/HGI-MSCs treatment on angiogenesis in ischemic muscles, as examined using H\u0026amp;E staining. All data were expressed as mean±SD(n= 8). Significance was set to *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/e7f684ab2eb9ac88e5646296.png"},{"id":71894658,"identity":"a16796b9-4fe3-491e-959a-68d97913d3d3","added_by":"auto","created_at":"2024-12-19 13:36:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2673899,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistology and Immunofluorescence analysis of neovascularization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative images of CD31 staining in muscle section from the PBS/MSCs/HGI-MSCs group. Scale bar=100μm. \u003cstrong\u003eB. \u003c/strong\u003eQuantitative analysis of the microvessel density (MVD) in PBS/MSCs/HGI-MSCs group.\u003cstrong\u003eC.\u003c/strong\u003eRepresentative examples of double staining of Ki67/ CD31 (green, CD31; red, Ki67; nucleus, blue) in muscle sections from the PBS/MSCs/HGI-MSCs group. \u003cstrong\u003eD. \u003c/strong\u003eQuantitative analysis of the proliferating capillary index (PCI)in PBS/MSCs/HGI-MSCs group. The PCI was used to assess the percentage of microvessels with proliferating endothelial cells. \u003cstrong\u003eE. \u003c/strong\u003eRepresentative images of double staining of α-SMA/CD31(red, α-SMA; green, CD31; nucleus, blue) in muscle sections from the PBS/MSCs/HGI-MSCs group. \u003cstrong\u003eF. \u003c/strong\u003eQuantitative analysis of the microvessel pericyte coverage index (MPI) in PBS/MSCs/HGI-MSCs group. The microvessel pericyte coverage index (MPI) was quantified by assessing the percentage of microvessels that were associated with α-SMA-positive pericytes. All data are expressed as the mean±SD(n=8).Scale bar=50μm.Significance was set to *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure503.png","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/338158fba80dcc8510848d3f.png"},{"id":71896229,"identity":"960e71a9-ff0e-473f-a5d9-a93cb44e8f47","added_by":"auto","created_at":"2024-12-19 13:44:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2446486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHGI-MSCs promoted migration and angiogenesis of HUVECs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e The transwell system assay was used to test the migration ability of HUVECs cocultured with PBS/MSCs/HGI-MSC. \u003cstrong\u003eB. \u003c/strong\u003eTube formation assay was performed on the Matrigel to test the angiogenic ability of HUVECs cocultured with PBS/MSCs/HGI-MSCs. \u003cstrong\u003eC.\u003c/strong\u003eQuantitative analysis of migrated cells of HUVECs cocultured with PBS/MSCs/HGI-MSCs in vitro. \u003cstrong\u003eD. \u003c/strong\u003eQuantitative analysis of lumen formation of HUVECs cocultured with PBS/MSCs/HGI-MSCs in vitro. All data are expressed as the mean±SD(n=5).Significance was set to *\u003cem\u003ep \u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/ba3c04792d19adf78c8b0899.png"},{"id":71894650,"identity":"c7a21872-6676-4622-aa16-abcab984fb7d","added_by":"auto","created_at":"2024-12-19 13:36:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":185942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of HGI-MSCs on VEGF induced PI3K/Akt signaling pathway activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eVEGF mRNA levels of lower limb muscle treated with PBS/MSCs/HGI-MSCs were assessed by qRT-PCR. \u003cstrong\u003eB. \u003c/strong\u003eQuantitative analysis of VEGF paracrined in serum treated with PBS/MSCs/HGI-MSCs. \u003cstrong\u003eC. \u003c/strong\u003eWestern blotting assay of p-Akt/Akt of lower limb muscle treated with PBS/MSCs/HGI-MSCs.Full-length blots are presented in Supplementary Figure.1. \u003cstrong\u003eD. \u003c/strong\u003eCorrelation between exhausitve distance and microvessel density (MVD) in rats. (Pearson’s r=0.8476, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001). All data are expressed as the mean±SD(n=8). Significance was set to *\u003cem\u003ep \u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/5f54fd7c5d24c3f0a548e964.png"},{"id":76880366,"identity":"7939f5c8-9553-4272-9591-d55e2fa31a4e","added_by":"auto","created_at":"2025-02-21 16:59:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10762248,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/043df0d3-82af-4cdb-b7d1-816aab9d439c.pdf"},{"id":71896737,"identity":"0c40dc17-cc60-4435-af12-09daff9e4bc6","added_by":"auto","created_at":"2024-12-19 13:52:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":225015,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVEChecklistFull.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/76b33489b7ec2132585104a7.pdf"},{"id":71894657,"identity":"2611a51f-1c4a-4396-aba8-ff985a915043","added_by":"auto","created_at":"2024-12-19 13:36:00","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6854851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure.1 Full-length blots are presented.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"renamed5868a.tif","url":"https://assets-eu.researchsquare.com/files/rs-5348024/v1/662312bbe4476c04fe5a6841.tif"}],"financialInterests":"","formattedTitle":"Enhanced angiogenesis by mesenchymal stem cells based on Hyaluronic Acid hydrogel combined with GM-CSF and IL-2 in a rat model of hindlimb ischemia","fulltext":[{"header":"Background","content":"\u003cp\u003eThe prognosis of critical limb ischemia (CLI) is extremely unfavorable, encompassing non-healing ulcers, impaired ambulatory function, limb amputation, and even life-threatening consequences. For the patients with ischemic disease of lower limbs, revascularization is essential in treatment of CLI. However, there are 20\u0026ndash;30% of those patients who are not qualifed for endovascular treatment or bypass surgery. In recent years, more experts and clinicians are paying attention on stem cell therapy on ischemic diseases. It has been proven that stem cells exhibit a robust migratory ability, differentiation potential, and secretion of a variety of beneficial cytokinescan. The stem cell therapy can help restore blood perfusion and alleviate secondary injury caused by ischemia\u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, the application of stem cell therapy is challenged by factors such as impaired multilineage differentiation ability, immune rejection, exposure to ischemia and inflammation, mechanical washout by the vasculature, and leakage of cell suspension from the target injection site, lead to poor efficacy\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, improving the viability and functionality of stem cells is key to its potential applications.Several approaches,like growth factors, overexpression of stem cell regulatory genes, to improve the tissue-regenetive capacity of stem cells have been investigated. The effect of these approaches on stem cells is related to the activation of phosphoinositol 3-kinase (PI3K)/Akt signaling pathway,which plays a central regulatory role in angiogenesis\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Angiogenesis is a highly regulated process involving the formation of new blood vessels sprouting from preexisting vessels through the activation, migration, and proliferation of vascular endothelial cells(ECs)\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that can enhance the functions of various cells that are necessary for inducing endothelial cells to migrate and proliferate\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Interleukin-2(IL-2) plays a major role in the clonal expansion of T lymphocytes(T cells) by interacting with specific cell surface receptor. It binds to the functional receptor and induces activation of PI3K/Akt siganling pathway\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, which is well known as a regulator of neovascularization. Hyaluronic acid (HA), the main component of extracellular matrix(ECM) consisting of repeating disaccharide units of b-(1,4)-D-glucuronic acid and b-(1,3)-N-acetyl-D-glucosamine, plays a vital role in cell proliferation and migration during biological processes like tissue regeneration and angiogenesis\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. It has been widely utilized as drug-carrying scaffold in regeneration medicine. And numerous studies have demonstrated that all of bioactive factors mentioned above had a certain promoting effect on the secretion of VEGF\u003csup\u003e[\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, indicating a synergistic effect on angiogenesis.\u003c/p\u003e \u003cp\u003eTo our knowledge, relevant research on the combination of MSCs and bioactive cytkine for the treatment of limb ischemia is very limited.Based on the above results, we believe that the application of combined therapy above can improve the efficiency of restoring ischemic limb perfusion. Thus, we developed a rat model of hindlimb ischemia,combined MSCs and bioactive factors(GM-CSF and IL-2) and piggybacked them on hyaluronic acid hydrogels to evaluate the synergistic effect of the combined therapy on enhancing angiogenesis in CLI and study the related mechanism. We found that compared with the treatment of PBS and pure MSCs,the combined therapy enhanced angiogenesis and promoted its maturation. The results revealed the underlying mechanism of HGI-MSCs therapy in promoting angiogenesis and provided strategies for the treatment of limb ischemia in the future.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of cell\u003c/h2\u003e \u003cp\u003eFour-week-old Sprague Dawley rats(Shanghai SLAC Laboratory Animal Co., Ltd), which were SPF grade, male and weighed\u0026thinsp;~\u0026thinsp;80\u0026ndash;120 g, were used. Rats were anaesthetized by intraperitoneal injection of 2% sodium pentobarbital(30mg/kg) in order to minimalize the stress and suffering. 10 min after administration of anaesthetics, animals were euthanized by the disruption of the spinal cord. Next, the bilateral femurs and tibias in rats were removed after decapitation under sterile conditions. Removing the metaphysis, the exposed bone marrow cavity was flushed with Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium(DMEM; Gibco; USA). The rinse solution was collected. Pass the rinsing solution through a 200-mesh screen in order to produce a single cell suspension that was centrifuged 1000 rpm for 15 min. The supernatant was discarded and then PBS was added to blow suspension. BM-MSCs were isolated using the bone marrow adherence method. Cells were cultured in α minimum essential medium (α-MEM, Gibco, USA) supplemented with 10% fetal bovine serum(FBS, cegrogen, Germany) at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere. The medium was changed every 2 days. Cells at passages 3 to 5 were used in the experiments. Primary HUVECs were purchased from Zolgene and cultured in endothelial cell growth medium (ECM, ScienCell, USA) containing 5% FBS and supplemented with 100\u0026times; endothelial cell growth supplement (ECGS, ScienCell, USA). The medium was changed every 2 days. Cells at passages 3 to 5 were used in the experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of the HGI-MSCs-hydrogel\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHGI-MSCs suspension was formed by mixing 1ml MSCs with 1ml hyaluronic acid (HA) hydrogel(Humedix Co., Ltd.,Korea), rhG-CSF(Hainan Unipul Pharmaceutical Co., LTD., China) and IL-2(Beijing SL Pharmaceutical Co., Ltd.,China) at room temperature. The final concentrations of rhGM-CSF and IL-2 were 150 ng/ml and 1*10^5 U/ml respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal Model and Treatment\u003c/h3\u003e\n\u003cp\u003eThe rats(SPF grade, male and weighed\u0026thinsp;~\u0026thinsp;200\u0026thinsp;~\u0026thinsp;250 g) were weighed and anesthetized under 2% sodium pentobarbital(50mg/kg) for surgical intervention. The surgical intervention was performed to create unilateral hindlimb ischemia model in the rat. Exposure was obtained by performing an incision in the skin overlying the middle portion of the left hindlimb of each rat. After ligating the proximal end of the femoral artery, the distal portion of the saphenous artery was ligated, and the artery and all side-branches were dissected free; the femoral artery and attached side-branches were then excised(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The overlying skin was then closed using a surgical stapler. While the corresponding right femoral artery was left unexcised and used as a control. 3 days after the operation, the skin temperature of the dorsum of the foot of both lower limbs were measured every 3 days for 60days. Keep records.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 60 days, all rats were randomly divided into 3 groups of eight each. Firstly, each rat was injected intramuscularly with 0.1ml PBS at 10 different points on the medial and lateral sides of the ischemic limb. It was marked as the PBS group. In the MSCs group, pure MSCs suspension was injected. The third group was injected with HGI-MSCs suspension(composition and concentration have been indicated above), which was marked as the HGI-MSCs group. All injection methods and dosage were the same as the first group\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, and they were given for three consecutive days. Skin temperature was measured every 5 days for a period of 30 days subsequent to the injection. The experimental design is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e\n\u003ch3\u003eExhaustive exercise\u003c/h3\u003e\n\u003cp\u003eAll rats were subjected to forced exhaustive exercise running on treadmill apparatus(KW-PT, KEW, China). The rats were initially familiarized with the treadmill of low intensity running for 3 days. On 1st day of the familiarization protocol, rats placed on the treadmill were given 5 min to acclimate before the treadmill belt was engaged. The belt speed was initially set to 8m/min then increased to 10m/min, then 12m/min for 5 min each, all at an incline of 10%. On day 2, rats were given 2 min to acclimate to the treadmill then exercised at 8m/min, 10m/min, 12m/min and 15m/min for 5 min each. On day 3, rats were placed on the treadmill and immediately exercised at 10m/min for 5 min, then 15m/min and 20m/min for 5 min each.After familiarization, rats were allowed two days to recover before beginning the exhaustive exercise protocol. At the experiment day, all rats were subjected to the following protocol: at a 10% incline, the initial speed was set at 10m/min for 8 min, then increased to 15m/min for 5 min, and then increased by 2.5m/minute every three minutes until reaching a speed of 25m/minute. Rats exercised for 10 min at 25m/min, then speed was then increased by 1m/min every 5 min until exhaustion.Exhaustion was determined when rats failed to re-engage all four paws with the treadmill belt despite negative stimulus[17]. Then, the animals were euthanized. The procedure for euthanasia has been described above. Blood and limb muscle were harvested for further processing.All rats experiments were approved by the Animal Ethics Committee of Fujian Medical University. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e\n\u003ch3\u003eHistology and immunohistology analysis\u003c/h3\u003e\n\u003cp\u003eSamples harvested from the lower limb muscle were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. Serial 5-mm sections were cut and stained with hematoxylin and eosin (H\u0026amp;E) for observation of the quantity of vessels in the muscle. For immunohistochemical staining, a panel of primary antibodies against rat CD31(1:500, Servicebio, China) were used. The sections were firstly incubated with 3% H2O2 for 10 minutes to deactivate the endogenous peroxidase. To recover antigen, these sections were soaked in 10 mM citrate buffer solution (pH 6.0) and heated twice in the microwave oven. And the slides were then washed thoroughly with PBS(pH 7.4). After blocked with 5% bovine serum albumin in Tris-buffered saline for 20 minutes, the sections were incubated with primary antibodies against CD31 at 4℃ overnight followed by washing with PBS. Afterward, the slides were sequentially incubated with secondary antibody for 1h and streptavidin-horseradish peroxidase for another 20 minutes. The staining was visualized after incubation with a DAB-H2O2 solution. The slides were then counterstained with hematoxylin for 1 minute, dehydrated with ethanol, and sealed in resinene for microscopic observation.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence analysis\u003c/h3\u003e\n\u003cp\u003eA double-labeling immunofluorescence technique was applied to analyze the proliferation of endothelial cells using anti-CD31(1:200, Servicebio, China) and anti-Ki67(1:300, Servicebio, China) antibodies. Similarly, the pericyte coverage of microvessels was observed using antiCD31 and anti-α-smooth muscle actin(α-SMA\u003c/p\u003e \u003cp\u003e) (1:200, Abcam, Cambridge, MA) antibodies. The sections were blocked with BSA(5%) for 2 h and incubated with primary antibody at 4℃ overnight. And then, the sections were further incubated with secondary goat anti-rabbit antibodies(Servicebio, 1:400) for 1 h in the dark after washing. Subsequently, the sections were incubated in 4\u0026rsquo;6-diamidino-2 phenylindole (DAPI) for nuclear staining. Images were obtained with a fluorescence microscope(Carl Zeiss) and were merged using Image-Pro Plus v. 6.0 software. Negative control staining experiments were performed by omission of the primary antibody.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitation of MVDs, PCI, and MPI.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll sections were assessed for uniformity of staining at low power(c50), and individual microvessel counts were then performed under a \u0026times;200 field. Staining of endothelial cells for CD31 was used to express MVD counts. Any CD31 positive endothelial cell clusters clearly separated from each other were considered as single countable microvessels. Microvessel density (MVD) was assessed by light microscopic analysis for areas of the lower limb muscle that contained the most capillaries and small venules (microvessels) (so-called neovascular \"hot spots\"). Counts were transformed and expressed as the number of microvessels/ mm2(1 HPF\u0026thinsp;=\u0026thinsp;0.0681mm2). The proliferating capillary index(PCI) was used to assess the proliferating endothelial cells and was quantified by calculating the ratio of the number of microvessels with proliferating endothelial cells(Ki67) to the total number of microvessels(CD31) which was screened for the areas with the highest vessel density under a magnification of \u0026times;200. Similarily, the microvessel pericyte coverage index(MPI) is expressed as the α-SMA/CD31 ratio, which is used to assess the maturity of new blood vessels. It was correspondingly established by quantitating the percentage of microvessels that co-localized endothelial cell staining (CD31) and pericyte staining (α-SMA). For all quantification, at least five nonoverlapping microscopic fields per section were independently analyzed under double-blind conditions\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro tube formation assay\u003c/h2\u003e \u003cp\u003eHUVECs coculture with PBS/MSCs/HGI-MSCs were performed on Basement Membrane Matrigel(MCE, China) in 24-well plates with 100 \u0026micro;L of Matrigel/well. The optimal time for tube formation was determined following an 8-hour incubation at 37℃ with periodic observations. The capillary-like structure was examined under an inverted phase contrast microscope at \u0026times;100 magnification. Five fields per test condition were examined. The tube length was measured using ImageJ software.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro migration assay\u003c/h3\u003e\n\u003cp\u003eHUVECs (4.0 \u0026times; 10*4 per chamber) were plated into the upperchamber in medium (containing 1% FBS). Meanwhile, the lower chamber was divided into PBS/MSCs/HGI-MSCs group. After 24h of incubation, the cells that had attached to the lower surface were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 15 minutes after removing the paraformaldehyde. The chamber was washed twice with PBS, and the non-migrated cells on the membrane were removed using cotton swabs. Each group was randomly photographed under the microscope(\u0026times;200) to observe and count the cells in the field of view.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReal-Time Polymerase Chain Reaction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal RNA from muscle samples were isolated using Trizol reagent according to the manufacturer\u0026rsquo;s instructions. cDNA was synthesized with PrimeScript RT Master Mix kit. Real-time polymerase chain reaction was performed with SYBR green premix in accordance with the manufacturer\u0026rsquo;s instructions. The following primers were used for cDNA amplification: for VEGF (Norway rat), 5-GCACGTTGGCTCACTTCCAG-3 (forward) and 5-TGGTCGGAACCAGAATCTTTATCTC-3(reverse); for b-actin(Norway rat), 5-GGGAAATCGTGCGTGACATT-3(forward) and 5-GCGGCAGTGGCCATCTC-3(reverse). All of the gene expressions were normalized to that of the internal reference genes, namely b-actin, within the same samples using the 22DDCt method\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eMeasurement of cytokines\u003c/h3\u003e\n\u003cp\u003eQuantification of VEGF in serum was performed using the VEGF ELISA kit specific for mouse(BOSTER, Wuhang) according to the manufacturer\u0026rsquo;s instructions. The absorbance at 450 nm was read on an automatic ELISA system.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analyze\u003c/h2\u003e \u003cp\u003eThe limb muscle samples stored at -80\u0026deg;C were taken, and weighed for 50 mg, then cut into small pieces. Then, the muscle tissues were homogenized in RIPA buffer(containing RIPA 5\u0026times;DTT, Protease Inhibitor Cocktail, Phenyl-methysulfonylfluoride(PMSF) and phosphorylase inhibitors cocktail). The homogenization was centrifuged at 12000 rpm for 15 min at 4\u0026deg;C, retaining the supernatant, and the protein concentrations were determined using a bicinchoninic acid(BCA) protein assay kit(Boster, Wuhan, China). The samples were added with 2 \u0026times; loading buffer, then equal amounts of protein in each sample were separated on 12% SDS polyacrylamide gel electrophoresis(SDS-PAGE) gels and transferred onto polyvinylidene fluoride(PVDF) membrane. The membranes were blocked with 5% bovine serum albumin(BSA; Boster, Wuhan) for 2 h at 4\u0026deg;C, and then incubated with the following primary antibodies: rabbit anti-Akt(1:1000, Cell Signaling Technology), rabbit-anti-phosphoAkt(Ser473) (1:2000, Cell Signaling Technology) and rabbit anti-β-actin(1:5000, Bioworld) overnight at 4\u0026deg;C. Subsequently, washing in Tris-buffered saline plus Tween (TBST) buffer three times for 5 minutes each, membranes were incubated with goat anti-rabbit immunoglobulin (Ig)G-peroxidase conjugated(1:3000, Boster) for 2 h at 37\u0026deg;C. Finally, the protein bands were visualized using an Enhanced Chemiluminescence Detection Kit (Boster, Wuhan, China), captured with iBright 1500(Thermo Fisher Scientific, Waltham, USA), and quantified with Image J software. The protein expression was normalized to β-actin, and the experiment was repeated independently three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe values were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance among multiple experimental groups was determined using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test with GraphPad Prism 9(GraphPad Software Inc., San Diego, CA). Only dif- ferences with \u003cem\u003eP\u0026lt;\u003c/em\u003e0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of ischemia model induced by femoral artery ligation\u003c/h2\u003e \u003cp\u003eOn day 60 after ligation surgery, the skin temperature of the surgical hindlimb, which was significantly lower than that of the normal side,was observed to change(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). It shows that following ligation of the proximal femoral artery and resection of the distal and all branch arteries and veins, there is a disparity in blood perfusion between the operated and healthy sides(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating successful establishment of the lower limb ischemia model. With the prolonged ischemia time,refractory wound ulceration and partial gangrene were observed in the paws of rats(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of restored perfussion induced by HGI-MSCs therapy\u003c/h2\u003e \u003cp\u003eAt the 30th day after drug injection, it was observed that the limb ulcers in the PBS group were not significantly improved and healed, while in the MSCs and HGI-MSCs groups, the ulcers were significantly healed, and the skin color of the operated limb was close to the healthy side(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There was significant difference in skin temperature among HGI-MSCs/MSCs/PBS group, indicating that the therapeutic efficacy of HGI-MSCs in restoring blood perfusion is superior to that of MSCs group and PBS group. Moreover, the skin temperature of the HGI-MSCs group was close to the healthy side, the difference was not statistically significant(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). By monitoring the skin temperature of the lower limbs every 5 days, it can be inferred that after 2 months of ligation, the rats began to exhibit a compensatory trend in the lower limbs, which was further enhanced by HGI-MSCs injection therapy. It indicates that while neovascularization in the lower limb of rats has initiated compensation for regional hypoperfusion caused by acute ischemia, HGI-MSCs can expedite neovascularization and enhance restoration of lower limb perfusion(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHGI-MSCs induced an increase in exhaustive exercise distance and formed more vessels in ischemic muscles.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine the enhancement of motor function in rats treated with HGI-MSCs injection therapy, we performed exhaustion experiments(protocol was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The results showed that HGI-MSCs injection therapy significantly increased the exhaustive exercise distance of the rats,which provided strong evidence that HGI-MSCs had positive effects on the motor function of ischemia limb rats. Similarly, rats treated with MSCs ran more distance than that with PBS treatment. In the PBS group, several rats became exhausted within just a few minutes on the treadmill due to severe limb ischemia(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Histopathological analysis was performed to evaluate the blood perfusion in muscle tissue by HE staining. The findings indicated a high abundance of blood vessels in the HGI-MSCs group, which appeared more perfusion than MSCs group and the PBS group(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHGI-MSCs promotes angiogenesis and the proliferation and maturation of endothelial cells\u003c/h2\u003e \u003cp\u003eThe specific markers CD31 were utilized to simultaneously immunostain vascular endothelial cells in order to quantitatively assess the neovasculature in the operated hindlinbs(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The results showed that the HGI-MSCs group had much higher average MVD than that in the MSCs group after injection. Meanwhile, we observed the MSCs group had higher average MVD than that in the MSCs group after injection(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Meanwhile,to quantify the proliferation of endothelial cell in the muscle of lower limb, we double-stained endothelial cells using CD31 and the cell proliferation marker ki-67 to quantify the proliferation capillary index (PCI), which reflected the percentage of microvessels with Ki67 positive endothelial cell nuclei. We figured that there is significant difference between the three groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC,the PCI value increased significantly in HGI-MSCs group(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). We evaluated the functional status of neovascularization using double-labeling immunofluorescence techniques with CD31 and α-SMA to detect the colocalization of endothelial cells and pericytes. As expected, it shows that the pericytes of the HGI-MSCs group formed more circles and densely enveloped the endothelial cells than that of the MSCs group and the PBS group(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).The above trend can be seen through the quantitative analysis of microvessel pericyte coverage index(MPI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). It can be inferred that HGI-MSCs facilitate the advancement of neovascular maturation and functionality.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHGI-MSCs enhanced the migration and tube formation of the HUVECs in vitro.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the transwell system, the migration capacity of HUVECs is activated by HGI-MSC. It can be inferred that the number of migrating cells is greater in the HGI-MSCs group than that in the MSCs group and the PBS group(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026amp;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the coculture system, the angiogenesis of HUVECs was observed by tube formation assay. We can detect that HUVECs monoculture formed less lumens or can not form any lumen sometimes. With supplements, HUVECs cocultured with HGI-MSCs formed more capillary-like structures than that of MSCs group(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026amp;D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHGI-MSCs up-regulated the expressions of VEGF\u003c/h2\u003e \u003cp\u003eThe changes in gene expression of VEGF after drug treatment were quantitatively analyzed by qRT-PCR. The results showed VEGF were significantly higher in the HGI-MSCs group than that in the MSCs group and the PBS group after treatment(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Meanwhile, the ELISA analysis of VEGF cytokine in the serum to evaluate the changes in phenotype. The results showed that VEGF elevated in the MSCs group and reached a peak in the HGI-MSCs group compared with that in the PBS group(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHGI-MSCs activated PI3K/Akt signaling pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWestern blot analysis was conducted to evaluate the role of PI3K/Akt pathway in VEGF-induced angiogenesis. The activation of this signaling pathway is controlled by the phosphorylation-induced activation of AKT. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, Akt and p-Akt was observed in all groups. However, the expression of p-Akt/Akt in the HGI-MSCs group markedly increased.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated whether the pro-angiogenic effect of MSCs could be enhanced by combining with GM-CSF and IL-2 on hyaluronic acid-based hydrogels. \u0026nbsp;Our findings revealed that HGI-MSCs was effective in promoting angiogenesis in artery ligated induced hindlimb ishcemia rats to restore perfussion. A significant enhanced effect of HGI-MSCs on proliferation and maturation of endothelial cells was found in double-labeling immunofluorescence. Furthermore, it was demonstrated that HGI-MSCs induced HUVECs migrate and form more lumen by up-regulating VEGF and it might be related with the activation of PI3K/Akt signaling pathway. \u0026nbsp;Importantly, it was found all the effects mentioned above with HGI-MSCs were greater than that with pure MSCs. Taken together, these results provided a novel insights underlying the beneficial effects of HGI-MSCs on pro-angiogenesis of stem cells and offered innovative therapies on ischemic diseases.\u003c/p\u003e\n\u003cp\u003eResearches on lower limb ischemic diseases have shown that the obstruction caused by atherosclerosis and microcirculation dysfunction of the lower extremity leads to hypoperfusion or even no perfusion in part of the blood supply area, which will lead to hypoxia of the limb, and nutrients will not be delivered to the site. MSCs, as far as we know, can be transplanted into tissues ex vivo. In the appropriate microenvironment, MSCs can adhere, proliferate and differentiate into specialized cells of target tissues, thereby playing a role in the regeneration and repair of injured tissues\u003csup\u003e[20]\u003c/sup\u003e. Despite the existence of strong evidence for the therapeutic effects of\u0026nbsp;MSCs, the mechanisms remain unclear. Current mechanistic studies have revealed two main mechanisms\u0026nbsp;of MSCs: paracrine and multidirectional differentiation\u003csup\u003e[21]\u003c/sup\u003e. This thesis proposes\u0026nbsp;HGI-MSCs therapy to promote the directional differentiation\u0026nbsp;of MSCs and the\u0026nbsp;proliferation, migration, and maturation of endothelial cells, which might be related to\u0026nbsp;elevated\u0026nbsp;expression of VEGF, eventually form vascular lumen,\u0026nbsp;enhance blood perfusion.\u003c/p\u003e\n\u003cp\u003eSun et al. have demonstrated that extended ligation of the distal femoral artery will reduce perfusion of lower limbs and lead to ischemia by Laser Doppler perfusion imaging(LDPI)\u003csup\u003e[22]\u003c/sup\u003e. As expected, the establishment of the lower limb vascular ligation model resulted in a reduction of perfusion in the operated limb of the model rat. This was evidenced by a decrease in temperature of the ischemic limb, as well as the progressive development of ulcers and claudication over time. After HGI-MSCs treatment, the skin temperature of the affected limb increased compared to the PBS group, and ulcer healing was observed. The MSCs group also exhibited a similar trend when compared to the PBS group, suggesting that while pure MSCs therapy has a certain role in enhancing lower limb perfusion, but its efficacy is not as pronounced as that of HGI-MSCs therapy.\u003c/p\u003e\n\u003cp\u003eIt is well known that the oxygen and nutrients brought by blood perfusion provide the necessary conditions for excercise. Relevant reports about hypoperfusion resulting in \u0026nbsp;functional decline of exercise have proven that blood perfusion is essential for the preservation of motor function\u003csup\u003e[23]\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e24]\u003c/sup\u003e\u003csup\u003e[25]\u003c/sup\u003e. Due to the significant ischemia present in lower limbs, the rat in PBS group could merely run on the treadmill for a few minutes, or even less than one minute. This indicates that the blood supply disorder caused by artery ligation will seriously affect the motor function of rat.After injection treatment of HGI-MSCs, the exhaustive exercise distance was significantly increased. Pathological analysis of the lower limb muscles showed that the HGI-MSCs group had more blood vessels than the other two groups. This improvement in motor function was attributed to the enhancement of one type of neovascularization, the apparent improvement in the number of MVD. In this study, immunohistochemical staining of CD31 was used to evaluate changes in the number of MVD. We figure that there would be more neovascularization in the lower limb muscles after treatment with HGI-MSCs than that in the MSCs group and the PBS group\u0026nbsp;in terms of the quantity of MVD.\u0026nbsp;Studies on lower limb ischemic disorders reveal that microcirculation dysfunction of lower limb muscles is the principal cause influencing motor function and wound healing\u003csup\u003e[26]\u003c/sup\u003e. Therefore, angiogenesis in the lower limbs can increase the motor function of rats, that is, the increase in running distance. MVD was counted in five random fields within the pathological sections of the lower limb muscles of the rats, and the average value was calculated. The correlation between the averaged MVD and the running distance of the rats was analyzed by Pearson correlation analysis, which revealed a linear correlation between the distance and MVD(r=0.8476,\u003cem\u003ep\u0026lt;\u003c/em\u003e0.01).Hence, facilitating angiogenesis constitutes effective therapeutic strategies for restoring perfusion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious researches revealed that Hyaluronic acid hydrogels were used as scaffolds on generating of functional vascular networks\u003csup\u003e[27]\u003c/sup\u003e. And the addition of GM-CSF may stimulate the hematopoietic stem cells (HSCs) in the suspension of bone marrow cells to differentiate into granulocytes and macrophages, participate in the local inflammatory response, and thus stimulate the regeneration of blood vessels\u003csup\u003e[28]\u003c/sup\u003e. On the other hand, it promotes the proliferation of bone marrow stem cells to maintain their viability. Bae, Jin-Hee et al. also demonstrated endothelial cells express IL-2R\u0026alpha; and \u0026beta; and IL-2 may promote angiogenesis\u003csup\u003e[29]\u003c/sup\u003e. IL-2 and GM-CSF together regulate the immune function, stimulate the activation of MSCs, make them differentiate into vascular endothelial cells, and secrete VEGF.The generated VEGF can also further stimulate MSCs to differentiate into vascular endothelial cells, thus forming a circulation conducive to angiogenesis. In addition, IL-2 may also stimulate local tissue to secrete VEGF and bFGF through inflammatory response, thus promoting the regeneration of blood vessels.The small molecule hyaluronic acid hydrogel was chosen as the matrix because of its large microchannels and loose ultrastructure. Recent researches have found that capillary-like structures form best in soft matrix\u003csup\u003e[27]\u003c/sup\u003e.\u0026nbsp;In this study,\u0026nbsp;the proliferation coverage index(PCI) was quantified by co-localized staining for CD31 and Ki67. We found that HGI-MSCs shows obvious advantages in promoting the proliferation of endothelial cells compared with pure MSCs treatment and PBS treatment.Thus, the enhanced effect on promoting the proliferation of endothelial cells may be attributed to HGI-MSCs therapy.\u003c/p\u003e\n\u003cp\u003eSimilarly, HGI-MSCs had a positive effect on the maturation of neovascularization. After formation of the original vessels, the stabilization and maturation of the newly formed vessels will become a major concern\u003csup\u003e[30]\u003c/sup\u003e. Previous studies have shown that insufficient maturity of the vascular system may lead to vascular obstruction, edema, and hemorrhage, affecting the transport of nutrients and oxygen necessary for perfusion restoration. Pericytes are in direct contact with endothelial cells through gap junctions and share the basement membrane with endothelial cells, strategically covering 90% of the capillary surface area,which constitute a vital component of the microvascular wall and are instrumental in regulating microvascular morphology, microcirculation, and permeability\u003csup\u003e[31]\u003c/sup\u003e. Therefore, pericyte coverage and basement membrane integrity are hallmarks of mature microvessels\u003csup\u003e[32]\u003c/sup\u003e\u003csup\u003e[33]\u003c/sup\u003e. When we observed newly formed microvessels at the late stage of ligation, we found not only a higher number of newly formed vessels but also a higher pericyte coverage of microvessels after HGI treatment. Several elongated pericytes encased endothelial cells and bridged adjacent endothelial cells to form tight junctions, indicating that these microvessels were more mature and stable.MPI quantitative analysis showed that MSCs combined with a variety of bioactive factors could promote vascular maturation through immune regulation.Zhao J et al. have demonstrated GM-CSF regulates maturation of microvessels by manipulating the spatial-temporal Ang-1/Ang-2 balance and the phosphorylation of Tie-2\u003csup\u003e[32]\u003c/sup\u003e. On the other hand, IL-2 in the microcirculation is readily taken up by the blood vessels\u003csup\u003e[34]\u003c/sup\u003e. IL-2 has been shown to stimulate glycosaminoglycan synthesis in vascular smooth muscle cells and regulate the responsiveness to angiotensin II, which maintaining vascular wall stability\u003csup\u003e[35]\u003c/sup\u003e.However,the presence of this IL-2 would have some effect on vascular permeability, which is contrary to our expectation\u003csup\u003e[36]\u003c/sup\u003e. To gain a deeper understanding of the role of HGI-MSCs in mediating endothelial cell migration and tube formation, we conducted tests on HUVECs at the cellular level and discovered that following treatment with HGI-MSCs, the migration of HUVECs was enhanced and HUVECs can form more vascular network structure.Here, HA hydrogel was used as ECM supplemented with GM-CSF and IL-2 to study vascular morphogenesis at the cellular level.In recent decades, the role of natural ECM, such as collagen and fibrin gels, in vascular morphogenesis has been thoroughly elucidated\u003csup\u003e[37]\u003c/sup\u003e\u003csup\u003e[38]\u003c/sup\u003e. In contrast to natural materials, HA hydrogels can be made from well-defined synthetic polymer networks that not only have a high water content to promote cell viability, but also possess biophysical and biochemical properties similar to many tissues which can be tuned to favor vascular morphogenesis\u003csup\u003e[39]\u003c/sup\u003e\u003csup\u003e[40]\u003c/sup\u003e. Therefore, HA gel may load bioactive factors and enhance their delivery efficiency, holding great promise for regenerative medicine as a biological scaffold.In the tube formation assay, the combination of GM-CSF and IL-2 promoted tube formation of HUVECs in co-culture system with MSCs\u003csup\u003e[41]\u003c/sup\u003e\u003csup\u003e[42]\u003c/sup\u003e.\u0026nbsp;However,Tim D Eubank,Eleni Sakkoula et al. expressed some opposing opinions regarding\u0026nbsp;GM-CSF and IL-2\u0026nbsp;in angiogenesis when researching oncology\u003csup\u003e[43]\u003c/sup\u003e\u003csup\u003e[44]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo further explore the above phenomenon,we examined the corresponding expression of mRNA of VEGF, which stimulates the proliferation and survival of endothelial cells and promotes angiogenesis and vascular permeability.In angiogenesis process,cells, including macrophages and fibroblasts, translocate the hypoxia-inducible factor HIF-1\u0026alpha;\u0026nbsp;or KRAS to the nucleus in response to hypoxia, thereby activating the expression of target genes including VEGF. VEGFA(but also VEGFE or VEGFC VEGFD) binds to VEGFR2, and upon activation of VEGFR2, several pathways are activated. The family of vascular endothelial growth factors (VEGFs) and their receptor tyrosine kinases (RTKs) are widely recognized as the most extensively studied pathways in developmental angiogenesis\u003csup\u003e[45]\u003c/sup\u003e, stimulating the PI3K-AKT pathway phosphorylation activation signaling cascade and increasing endothelial cell survival\u003csup\u003e[46]\u003c/sup\u003e. However, VEGFR1, which is a poor signaling receptor, but very potent VEGF binder\u003csup\u003e[47]\u003c/sup\u003e. There is a consensus that VEGFR1 is able to regulate VEGF activity on vascular endothelial cells in a negative manner by sequestering and rendering the factor unavailable to VEGFR2\u003csup\u003e[48]\u003c/sup\u003e. This may explain the opinions from the above Professor\u0026nbsp;Tim D Eubank and Eleni Sakkoula. We didn\u0026rsquo;t investigated the subtypes of VEGFR in the combined therapy to angiogenesis,and this question will be addressed in future researches.Back to the signal in the pathway itself, we detected the VEGF levels in serum using ELISA. The results showed that HGI-MSCs treatment may promote the paracrine secretion of VEGF.Subsequently, statistical significance was achieved through Akt and P-Akt Western blot analysis studies. It shows that the activation of PI3K-Akt signaling pathway to promote angiogenesis might be mediated by elevated VEGF in the corresponding tissues. \u0026nbsp;And in term of migration of HUVECs,there were more HUVECs in the lower chamber(supplemented with HGI-MSCs), suggesting that HGI-MSCs activated PI3K/Akt pathway by secreting VEGF, thereby mediating cell migration\u003csup\u003e[49]\u003c/sup\u003e. In the coculture system of HUVECs and HGI-MSCs, HUVECs can form more vascular network structures, which is also mediated by the activation of VEGF-induced PI3k/Akt pathway\u003csup\u003e[50]\u003c/sup\u003e\u003csup\u003e[51]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study demonstrated that mesenchymal stem cells based on hyaluronic acid hydrogel combined with GM-CSF and IL-2 can effectively restore lower limb perfusion and improve motor function in rats. And the effect is more effective than that of pure MSCs therapy. This process is likely to induce the activation of PI3K/Akt pathway through up-regulation of VEGF expression, leading to angiogenesis and maturation of neovacularization. The findings provided a new perspective on the clinical application of MSCs in the field of ischemic diseases.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMSC: Mesenchymal stem cell; EC: Endothelial cell; GM-CSF: Granulocyte macrophage colony-stimulating factor; IL-2: Interleukin-2; HA: hyaluronic acid; VEGF: Vascular endothelial growth factor; SD: Sprague-Dawley; PBS: Phosphate-buffered saline; DMEM: Dulbecco\u0026rsquo;s modified Eagle medium; FBS: Fetal bovine serum; DAB: 3,3\u0026prime;-Diaminobenzidine; DAPI: 6-Diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the ARRIVE guidelines 2.0(Animal Research: Reporting of In Vivo Experiments) and approved by the Animal Ethics Committee of Fujian Medical University(Approval No. IACUC FJMU 2022-0709 “Effects of angiogenic therapy on motor function of ischemic hindlimbs in rats.” approved on September 30, 2022). Human umbilical vein endothelial cells were obtained from Zolgene Company (Fuzhou, China). Zolgene Company has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent. HUVECs\u0026nbsp;cultures in this study were reviewed and approved by\u0026nbsp;Ethics Committee of Fujian Medical University(Approval No. IACUC FJMU 2022-0709 “Effects of angiogenic therapy on motor function of ischemic hindlimbs in rats.” approved on September 30, 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Natural Science Foundation of Fujian Province(2020J01960)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.All additional files are included in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYHC and JSW performed the experiments, analyzed the data, and wrote the manuscript; JCZ reviewed and edited the manuscript; and WL,XHH, SPJ researched the data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks to the support of Fujian Provincial Institute of Hypertension.The authors declare that they have not use AI-generated work in this manuscript\" in this section.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNakajima, D., Watanabe, Y., Ohsumi, A., Pipkin, M., Chen, M., Mordant, P., ... \u0026amp; Keshavjee, S. (2019). Mesenchymal stromal cell therapy during ex vivo lung perfusion ameliorates ischemia-reperfusion injury in lung transplantation. \u003cem\u003eThe Journal of Heart and Lung Transplantation\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e(11), 1214-1223.\u003c/li\u003e\n\u003cli\u003eSchuleri, K. H., Amado, L. C., Boyle, A. J., Centola, M., Saliaris, A. P., Gutman, M. R., ... \u0026amp; Hare, J. M. (2008). 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PTEN regulate angiogenesis through PI3K/Akt/VEGF signaling pathway in human pancreatic cancer cells. \u003cem\u003eMolecular and cellular biochemistry\u003c/em\u003e, \u003cem\u003e331\u003c/em\u003e, 161-171.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells, Human umbilical vein endothelial cells, Endothelial cells, Hyaluronic Acid hydrogel, GM-CSF, IL-2, Critical limb ischemia, Angiogenesis","lastPublishedDoi":"10.21203/rs.3.rs-5348024/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5348024/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe clinical application of stem cells in restoring ischemic lower limb perfusion has been hindered by challenges. To promote the directed differentiation of mesenchymal stem cells(MSCs) into endothelial cells(ECs) and enhance the paracrine effect, this paper aim to find a combined therapy for angiogenesis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMSCs based on Hyaluronic Acid(HA) hydrogel combined with GM-CSF and IL-2(HGI-MSCs) were locally injected into the femoral artery ligated ischemia model rat. Recovery of perfusion was assessed by limb temperature and exhaustive distance. Hematoxylin and eosin(H\u0026amp;E) staining, immunohistochemistry and immunofluorescence staining were used to evaluate the quantity of blood vessels, microvessel density(MVD) and the proliferation and maturation of neovascularization in the limb muscle. Migration and tube formation of Human umbilical vein endothelial cells(HUVECs) were evaluated by Transwell and tube formation assays. The expression levels of angiogenesis-related genes, cytokine and proteins were measured by qRT-PCR, ELISA and Western blotting, respectively.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHGI-MSCs promoted ischemic limb angiogenesis and restored perfusion. Our study showed that HGI-MSCs could promote the proliferation and maturation of neovascularization. Moreover, HGI-MSCs promoted HUVECs migration and tube formation in vitro, up-regulated the expression of VEGF and activated PI3K/Akt signaling pathway.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eHGI-MSCs showed a more robust pro-angiogenic effect than that of pure MSCs in the ischemia limb model rat. It offers novel ideas for the treatment of patients with refractory limb ischemia.\u003c/p\u003e","manuscriptTitle":"Enhanced angiogenesis by mesenchymal stem cells based on Hyaluronic Acid hydrogel combined with GM-CSF and IL-2 in a rat model of hindlimb ischemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 13:35:54","doi":"10.21203/rs.3.rs-5348024/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"02198f7a-9b07-48de-ac6f-cf51631b0ca8","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-21T16:51:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-19 13:35:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5348024","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5348024","identity":"rs-5348024","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}