Sustained release of vascular endothelial growth factor A and basic fibroblast growth factor from nanofiber membranes reduces oxygen/glucose deprivation-induced injury to neurovascular units

Yifang Wu ,Jun Sun ,Qi Lin ,Dapeng Wang ,Jian Hai

Abstract Upregulation of vascular endothelial growth factor A/basic fibroblast growth factor (VEGFA/bFGF) expression in the penumbra of cerebral ischemia can increase vascular volume,reduce lesion volume,and enhance neural cell proliferation and differentiation,thereby exerting neuroprotective effects.However,the beneficial effects of endogenous VEGFA/bFGF are limited as their expression is only transiently increased.In this study,we generated multilayered nanofiber membranes loaded with VEGFA/bFGF using layer-by-layer self-assembly and electrospinning techniques.We found that a membrane containing 10 layers had an ideal ultrastructure and could efficiently and stably release growth factors for more than 1 month.This 10-layered nanofiber membrane promoted brain microvascular endothelial cell tube formation and proliferation,inhibited neuronal apoptosis,upregulated the expression of tight junction proteins,and improved the viability of various cellular components of neurovascular units under conditions of oxygen/glucose deprivation.Furthermore,this nanofiber membrane decreased the expression of Janus kinase-2/signal transducer and activator of transcription-3 (JAK2/STAT3),Bax/Bcl-2,and cleaved caspase-3.Therefore,this nanofiber membrane exhibits a neuroprotective effect on oxygen/glucose-deprived neurovascular units by inhibiting the JAK2/STAT3 pathway.

Key Words: brain ischemia;brain microvascular endothelial cell;nanofiber membrane;neurovascular unit

Introduction

The incidence of ischemic cerebrovascular diseases,such as severe intracranial arterial stenosis or occlusion,atherosclerosis,moyamoya disease,and cerebral small vessel disease,increases year after year (Sayad et al.,2022).The common pathological feature of these diseases is brain ischemia (Jin et al.,2023).The sudden interruption of local blood flow,resulting in insufficient oxygen and glucose supply,may occur during cerebral ischemia,which leads to a variety of pathologic processes,including neuroinflammation,ischemic neuronal injury,vascular damage,and neurovascular microenvironmental disorders,causing severe brain dysfunction with a poor prognosis (Guse et al.,2022;Liu et al.,2022a).Brain ischemia is a major threat to human health.Efforts to treat brain ischemia by promoting neuron regeneration and repair have largely not been successful,because these approaches do no address the concurrent damage to vascular cells (Candelario-Jalil et al.,2022).Therefore,the mechanism leading to neural and vascular cell injury in brain ischemia,as well as ways to treat the injury as a whole,deserve further investigation.

Neurovascular units (NVUs) consist of neurons,glia,vascular cells,and extracellular matrix (Segarra et al.,2018;Dong et al.,2023).Neuronal architecture and function can be regulated by adjacent astrocytes and brain microvascular endothelial cells (BMECs) (Attwell et al.,2010;Chow et al.,2020).These cells and components are interconnected,interact with each other,and change with the external environment (Castro and Potente,2022).NVUs are crucial for maintaining the homeostasis of the central nervous system microenvironment and responding to the initiation and development of neurologic diseases (Császár et al.,2022;Wu et al.,2023).Owing to this complex interaction between brain cells and the vascular system,there has been a recent shift in understanding the pathophysiology of and designing treatments for brain ischemia,from isolated neuroprotection to NVU protection (Lu et al.,2022).Ischemia and hypoxia can trigger a series of changes in NVU structure and function,resulting in NVU remodeling (Huang et al.,2022).Severe or prolonged cerebral ischemia can lead directly to disruption of NVU structural integrity,which is usually regarded as being responsible for subsequent blood-brain barrier (BBB) leakage and neurologic deficits (Yang et al.,2021);however,the NVU remodeling process is poorly understood.

Nanofiber (NF) materials have gained wide interest among researchers.NF materials have considerable application potential owing to their unique properties,such as high porosity,excellent pore interconnectivity,small pore diameter,and high surface-to-volume ratio (Subramanian et al.,2009;Wu et al.,2022;Zhang et al.,2023).These advantages make NFs promising tools for the treatment of central nervous system diseases,including brain ischemia and infarction (Lu et al.,2019;Liu et al.,2022b).As neural implants,aligned NFs support cell migration and orientation of neurite outgrowth,and are often used to treat peripheral nerve injuries (Bordoni et al.,2020).Randomlyoriented NFs provide high porosity and surface area,which are conducive to cell adhesion and proliferation,and are therefore commonly used in the central nervous system (Wang et al.,2011).NFs can be loaded with therapeutic proteins while maintaining their conformational structure and stability,and a recent study showed that a loaded NF exerted therapeutic effects in an animal model of brain ischemic injury induced by transient middle cerebral artery occlusion (Zhang et al.,2019).Fon et al.(2014) incorporated mimetic brainderived neurotrophic factor into electrospun polycaprolactone (PCL) NFs and found that the loaded NF improved local neuronal plasticity and survival by increasing neurite sprouting at the tissue-implant interface.Moreover,growth factor-loaded NFs not only facilitate angiogenesis and vascular repair,but also enhance nerve regeneration,ultimately promoting functional recovery in rats with sciatic nerve injuries (Rao et al.,2020).Consequently,the application of NFs in brain ischemia warrants investigation.

Vascular endothelial growth factor A (VEGF) is an endogenous angiogenic growth factor that predominantly stimulates vascular endothelial cell proliferation and migration (Ben-Zvi and Liebner,2022).VEGF also acts on neurons,astrocytes,and neural stem cells to regulate neuroprotection and neurogenesis,as well as migration of neural stem cells into the ischemic region of the brain,where they further differentiate into functional neurons (Guo et al.,2016;Ni et al.,2022).Similarly,basic fibroblast growth factor (bFGF) has protects brain neurons from the effects of ischemia and promotes their survival,as well as promoting synapse formation,neurite outgrowth,and neurogenesis (Choi et al.,2021).In addition,bFGF plays a proangiogenic role by activating the caveolin-1/VEGF signaling pathway (Liu et al.,2018).In response to brain ischemia,VEGF and bFGF expression increase in the penumbra,leading to neuroprotection,increased vascular volume,reduced lesion volume,and enhanced neural cell proliferation and differentiation (Geiseler and Morland,2018).These beneficial effects,however,are both limited and temporary,because endogenous VEGF and bFGF expression levels are not elevated sufficiently or for a long enough period of time to ensure complete repair (Xing and Lo,2017).This problem can be addressed by supplementation with exogenous VEGF and bFGF,especially via topical application,which has minor side effects and higher effective targeting compared with systemic administration (Barker et al.,2014).Several studies report that treatment with VEGF leads neovascularization and to the formation of collateral networks,providing a better survival and regeneration environment for neurons in models of global and focal ischemia (Guo et al.,2016;Ghori et al.,2022).Studies have demonstrated that administering intravenous bFGF after the onset of ischemia not only reduces infarct volume,but also enhances recovery of sensorimotor function in the contralateral limbs in a mature rat model of permanent and temporary middle cerebral artery occlusion (Han et al.,2015;Choi et al.,2021).Furthermore,synergistic proangiogenic and neuroprotective effects have been highlighted following combined VEGF and bFGF therapy (Upadhya et al.,2020).Luo et al.(2015) found that activating CD133+ependymal neural stem cells with VEGF and bFGF elicited neural lineage differentiation and migration.We previously designed a PCL NF membrane that mediates sustained release of VEGF and confirmed that it confers synergistic neuroprotective effects against hippocampal neuronal apoptosis in brain ischemia when combined with cell culture medium containing the fatty-acid amide hydrolase inhibitor,URB597 (Wang et al.,2021);however,the neuroprotective effects of this NF membrane loaded with a single growth factor are limited,and its effect on NVUs has not been evaluated in a model of oxygen/glucose deprivation (OGD).In this study,a dual growth factor sustained release delivery system consisting of VEGF-and bFGF-loaded PCL NF membranes was fabricated using layer-bylayer (LBL) self-assembly and electrospinning techniques.The effect of this novel NF membrane on NVUs,as well as the mechanism by which it promotes repair of OGD-induced injury,was investigated.

Methods

Fabrication of electrospun PCL NF membranes loaded with VEGF and bFGF

Randomly-oriented PCL NF membranes were fabricated as previously described (Wang et al.,2021).In brief,a PCL (80 kDa;Sigma-Aldrich,St.Louis,MO,USA) polymer solution dissolved in a mixture of dichloromethane/dimethyl sulfoxide (3:1,w/w;Sigma-Aldrich) at a concentration of 6% (w/w) was electrospun using an applied voltage of 15 kV,a flow rate of 0.5 mL/h,and a needle collector distance of 20 cm.PCL NFs were collected by an aluminum foil-covered cylinder rotating at 100 r/min to yield a randomlyoriented morphology.VEGF (R&D Systems,Minneapolis,MN,USA,Cat# 564-RV) and bFGF (R&D Systems,Cat# 3339-FB) were then loaded onto the PCL NFs using LBL self-assembly technology.In short,poly(sodiumstyrenesulfonate) (PSS;Sigma-Aldrich) and polyallylamine hydrochloride (PAH;Sigma-Aldrich) solutions at a pH of 4.7 and a concentration of 1 mg/mL were prepared.Because VEGF and bFGF are negatively charged,VEGF and bFGF were dissolved in PSS solution.Each LBL cycle consisted of immersing the PCL NF membranes in PAH solution for 1 hour to develop a positively charged precursor,followed by immersing the PCL NF membranes in PSS solution containing dissolved VEGF (1 µg/mL) and bFGF (1 µg/mL) for 5 minutes.The control membrane was fabricated by successively immersing the PCL NFs into the PAH and PSS solutions without VEGF and bFGF.This LBL process was repeated for 5,10,15,and 20 cycles to fabricate VEGF/bFGF PCL NF membranes with 5,10,15,and 20 layers,respectively.

Characterization of PCL NF membranes

Samples were sputter-coated with gold (JEOL JFC-1600 Auto Fine Coater;Tokyo,Japan) and observed by scanning electron microscopy (SEM;S-4800;Hitachi Ltd.,Tokyo,Japan) at 10 kV.The average fiber diameter of each PCL NF membrane was calculated by measuring the width of 100 fibers with ImageJ version 1.53t software (National Institutes of Health,Bethesda,MD,USA) (Schneider et al.,2012).

Membranes from each group were cut into 10 mm×10 mm samples and immersed in 10 mL of phosphate-buffered saline (PBS) at 37°C.At the same time each day for up to 35 days,the medium containing released VEGF and bFGF was collected and replaced with an equal volume of fresh PBS.The amount of VEGF and bFGF released by the membranes with different numbers of layers was analyzed using an enzyme-linked immunosorbent assay kit (Nos.DY564 and MFB00;R&D Systems) according to the manufacturer’s instructions.The assay was performed in triplicate for each time point.Absorbance at 405 nm minus absorbance at 650 nm was determined using a PerkinElmer Victor 3 multilabel reader (Waltham,MA,USA).

Primary BMEC,astrocyte,and hippocampal neuron culture and construction of the NVU model

Sprague-Dawley rats were purchased from Shanghai Slac Laboratory Animal Co.,Ltd.(Shanghai,China;license No.SCXK (Hu) 2017-0005).Rats were anesthetized by isoflurane inhalation and sacrificed by cervical dislocation.This study was approved by the Ethics Committee of Tongji Hospital of Tongji University (No.TJ20191012-A006) on July 16,2019.

Primary BMECs were obtained from the brain cortex of 10,1-day-old neonatal Sprague-Dawley rats,as reported previously (Saleh et al.,2020).The cortical tissue was homogenized,trypsin was added,and the supernatant was collected and centrifuged to obtain mixed cells.All cell types other than BMECs were killed by treatment with 5.5 µM puromycin dihydrochloride (Thermo Fisher Scientific,Waltham,MA,USA).Isolated BMECs were then culturedin vitroat 37°C with 5% CO2in medium containing 20% fetal bovine serum (FBS;Gibco,Waltham,MA,USA).

As previously described (Ouyang et al.,2020),primary astrocytes were prepared from two Sprague-Dawley rats (<2 days old).The cortices were minced in precooled PBS,then treated with pancreatin containing 0.05% ethylene diamine tetraacetic acid at 37°C for 20 minutes.Ten percent FBS medium was added to the suspension,which was then centrifuged at 1000×gfor 5 minutes.The supernatant was discarded,and the astrocytes were resuspended in fresh culture medium,then seeded in culture flasks at a density of 1×105cells/mL.

Primary hippocampal neurons were isolated from 24 embryonic day 18 Sprague-Dawley rats as described previously (Anastasia et al.,2013;Varela et al.,2016).Neurons (1×106/mL) were seeded in plates coated with poly-D-lysine solution.Medium supplemented with 20% FBS was added.After the neurons sprouted axons,NeurobasalA culture medium containing 2% B-27 Supplement and 0.5% GlutaMAX (all from Gibco) was added.Half of the medium was replaced every 2 days.

von Willebrand factor (vWF),glial fibrillary acidic protein (GFAP),and neuron specific enolase (NSE) immunofluorescence assays were performed to confirm the identity and purity of the primary-cultured BMECs,astrocytes,and hippocampal neurons,respectively.

For the NVU model,neurons were planted in the lower chambers of 12-well Transwell plates (Corning Inc.,Corning,NY,USA) at 1×106cells/mL for 48 hours,and astrocytes were planted on the back of Transwell upper chambers at 2×105cells/mL.Astrocytes were inverted in a large glass petri dish and cultured for 4 hours.After astrocytes were attached,the astrocytes were removed from the glass petri dish,and the superior chamber was transferred to the culture plate with neurons.The medium was replaced with neuronal culture medium.After 4 hours of neuron and astrocyte co-culture,BMECs were seeded into the Transwell upper chambers at 5×105cells/mL.The three cells types were then co-cultured for 3-5 days,with the medium changed every day.For neuron-only and BMEC-only culturing,NF membranes were placed on the bottom of all wells prior to cell seeding.For the NVU model,NF membranes were only placed on the bottom of the lower chambers prior to seeding them with neurons,and the loaded growth factors were slowly released into the medium to act on three types of primary cells in the three-dimensional (3D) co-culture system.

OGD model and intervention

The cell experiments were performed separately on primary BMECs,hippocampal neurons,and the NVU model,with the following groups: (1) Con group,cells were incubated in complete medium without OGD;(2) OGD group,cells cultured under OGD conditions without treatment;(3) OGDC group,cells cultured under OGD conditions using the control membrane (without VEGF and bFGF);(4) VEGF group,cells cultured under OGD conditions using PCL NF membranes containing VEGF;(5) bFGF group,cells cultured under OGD conditions using PCL NF membranes containing bFGF;and (6) VEGF+bFGF group,cells cultured under OGD conditions using PCL NF membranes containing VEGF and bFGF (VEGF+bFGF NF membranes).

To induce OGD injury,the medium was completely replaced with glucosefree DMEM (Gibco),and cells were incubated for 4 hours at 37°C in a sealed chamber containing an anaerobic gas mixture (95% N2and 5% CO2) (Xu et al.,2020).After OGD,the medium was replaced with maintenance medium,and the cells were incubated for 72 hours in a regular incubator prior to subsequent experimentation.

Tube formation assay

BMECs in each group were cultured at 1×105cells/mL in 24-well plates coated with Matrigel (Corning Life Sciences,Tewksbury,MA,USA) for 12 hours.Tube length was observed with an Olympus inverted phase contrast microscope (Olympus Corporation,Tokyo,Japan) and calculated from four random fields using ImageJ software.

Immunofluorescence assay

Primary BMECs,astrocytes,and hippocampal neurons were fixed with 4% paraformaldehyde for 30 minutes at room temperature (20°C).Before staining,all samples were permeabilized with PBS-0.2% Triton X-100 (Sigma-Aldrich),followed by blocking with 5% FBS for 30 minutes.Primary antibodies were subsequently applied at 4°C overnight.The cells were incubated with fluorochrome-conjugated secondary antibody at room temperature (20°C) for 1 hour in the dark,then 4′,6-diamidino-2-phenylindole (DAPI;Invitrogen) was added,and the cells were protected from light for 10 minutes.After sealing with anti-fluorescence quenching sealing tablets (Beyotime Institute of Biotechnology,Shanghai,China),samples were observed under a laser confocal microscope (Leica,Wetzlar,Germany).Three random visual fields were photographed per slide,and three slides were analyzed for each experimental group.ImageJ software was used to quantify fluorescence intensity.

Primary antibodies were rabbit anti-Ki67 (1:400,CST,Danvers,MA,USA,Cat# 9129,RRID: AB_2687446),rabbit anti-NSE (1:100,Abcam,Cambridge,MA,USA,Cat# ab180943,RRID: AB_2934109),rabbit anti-vWF (1:100,Abcam,Cat# ab6994,RRID: AB_305689),mouse anti-GFAP (1:50,Abcam,Cat# ab4648,RRID: AB_449329),mouse anti-F-actin (1:100,Abcam,Cat# ab205,RRID: AB_302794),and rabbit anti-zonula occludens-1 (ZO-1;1:100,Invitrogen,Camarillo,CA,USA,Cat# 40-2200,RRID: AB_2533456).Secondary antibodies were donkey anti-rabbit Alexa Fluor® 647 (1:1000,Abcam,Cat# ab150077,RRID: AB_2630356),donkey anti-rabbit Alexa Fluor® 647 (1:1000,Abcam,Cat# ab150075,RRID: AB_2752244),goat anti-mouse Alexa Fluor® 488 (1:1000,Abcam,Cat# ab150113,RRID: AB_2576208),and goat antimouse Alexa Fluor® 488 (1:500,Abcam,Cat# ab150121,RRID: AB_2801490).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Neuron and BMEC viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich).Cells were seeded into 96-well plates (n=6 wells per group).After different treatments,cells were incubated with 20 µL of MTT reagent (5 mg/mL) for 4 hours.MTT solution was then removed,and MTT-formazan crystals were solubilized by adding 100 µL of dimethyl sulfoxide to each well.The optical density (OD) was measured at 490 nm using a plate reader (PerkinElmer,Waltham,MA,USA).

Scanning electron microscopy

Primary hippocampal neuron number and morphology were assessed by SEM,as previously described (Han et al.,2019).After incubation on PCL NF membranes for 72 hours,cells were washed twice with PBS,followed by immersion in PBS containing 1% glutaraldehyde for 4 hours.Samples were then dehydrated using a 20-100% ethanol series,washed,and air-dried at 25°C for 1 hour.The samples were spattered with gold in a vacuum and observed by Scanning electron microscopy (SEM).Three biological repeats were performed for each group,with three random fields of view for each slice photographed.

Flow cytometry assay

Apoptosis of primary hippocampal neurons was detected via flow cytometry using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Biyuntian Biological Co.,Ltd.,Shanghai,China) according to the manufacturer’s instructions.Briefly,cells were resuspended in 100 µL of 1× binding buffer,then incubated with 5 µL of Annexin V-FITC and 10 µL of a PI solution at room temperature (20°C) for 10 minutes in the dark.Flow cytometry data were collected on a FACSCalibur instrument (BD Biosciences,Franklin Lakes,NJ,USA) and analyzed with FlowJo version 10.5.3 software (BD Biosciences).The lower right quadrant (Annexin V-FITC+/PI-) represented early apoptosis,whereas the upper right quadrant (Annexin V-FITC+/PI+) represented late apoptosis.The apoptosis rate was defined as the sum of the lower and upper right quadrants of the flow cytometry images.

TdT-mediated dUTP nick end labeling assay

Neurons were fixed with 4% paraformaldehyde for 25 minutes and permeabilized with 0.2% Triton X-100 for 5 minutes.Samples were incubated with 50 µL of the TdT-mediated dUTP nick end labeling (TUNEL) reagent (Promega,Madison,WI,USA) at 37°C for 1 hour before being counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in the dark.Samples were observed under a fluorescence microscope (Zeiss,Jena,Thuringia,Germany).Three random visual fields were photographed on each slide,and three slides were imaged for each experimental group.The percentage of TUNEL-positive cells was calculated by dividing the number of DAPI/TUNEL double-labeled nuclei by the total number of DAPI-labeled nuclei.

Western blot analysis

Neurons and BMECs were analyzed by western blotting,as previously described (Wang et al.,2022a).Cells were lysed in radioimmunoprecipitation assay buffer (Sigma-Aldrich),and protein concentrations were determined using a bicinchoninic acid kit (Biyuntian Biological Co.,Ltd.).The western blot membranes were incubated with primary antibodies at 4°C overnight and with secondary antibodies at 25°C for 1 hour.The gray values of the protein bands (n=3 per group) were quantified using ImageJ software.The relative gray value was calculated as the ratio of the target band gray value to that of the β-actin band.

Primary antibodies were rabbit anti-claudin-5 (1:50,000,Abcam,Cat# ab172968,RRID: AB_2934110),rabbit anti-occludin (1:1,000,Abcam,Cat# ab216327,RRID: AB_2737295),rabbit anti-ZO-1 (1:1000,Invitrogen,Cat# 40-2200,RRID: AB_2533456),rabbit anti-phospho-Janus kinase-2 (p-JAK2;1:1000,CST,Cat# 3774,RRID: AB_390750),rabbit anti-Janus kinase-2 (JAK2;1:1000,CST,Cat# 3230,RRID: AB_2128522),rabbit anti-phospho-signal transducer and activator of transcription-3 (p-STAT3;1:2000,CST,Cat# 9145,RRID: AB_2491009),rabbit anti-signal transducer and activator of transcription-3 (STAT3;1:2000,CST,Cat# 4904,RRID: AB_331269),rabbit anti-Bax (1:1000,CST,Cat# 2772,RRID: AB_10695870),rabbit anti-Bcl-2 (1:1000,Abcam,Cat# ab196495,RRID: AB_2924862),rabbit anti-cleaved caspase-3 (1:1000,CST,Cat# 9664,RRID: AB_2070042),and mouse anti-β-actin (1:1000,CST,Cat# 3700,RRID: AB_2242334).Secondary antibodies were goat antirabbit H&L horseradish peroxidase (1:2000,Abcam,Cat# ab6721,RRID: AB_955447) and horse anti-mouse H&L horseradish peroxidase (1:2000,CST,Cat# 7076,RRID: AB_330924).

Statistical analysis

SPSS software (SPSS 21.0;IBM,Armonk,NY,USA) was used for statistical testing.Experimental data are presented as the mean ± standard deviation (SD).Multiple comparisons were evaluated by one-way analysis of variance followed by Fisher′s least significance differencepost hoctests.AP-value of <0.05 was considered statistically significant.

Results

Characterization of PCL NF membranes

The microscopic morphology of NF membranes was observed by SEM.As shown inFigure 1A,random PCL NF membranes were successfully prepared by electrospinning.VEGF and bFGF were deposited on the surface of the PCL NF membranes using electrostatic LBL self-assembly technology,and VEGF+bFGF PCL NF membranes with 5,10,15,and 20 layers were fabricated.The SEM results showed that all of the membranes had a clear 3D structure.The fibers on the surface of the untreated PCL NF membranes were smooth,loosely arranged,and randomly oriented.As the number of loaded layers increased,the particles were deposited continuously,the NFs came into closer contact,and the pore size and porosity decreased.SEM revealed a highly uniform and interconnected porous structure of the VEGF+bFGF NF membranes with 5 and 10 layers.In contrast,the presence of an excess amount of particles blocked the micropores of 15-and 20-layer VEGF+bFGF NF membranes,resulting in an unclear fiber structure.In addition,the NF diameter increased as the number of loaded layers increased,and the average diameters of the NFs in the 5-and 10-layer VEGF+bFGF membranes were similar to those in the PCL membrane (Figure 1B).The diameter of the NFs in the membranes with 15 and 20 layers was significantly increased (P<0.05,15 and 20 layersvs.PCL,5 and 10 layers,respectively),but the differences between the 15-and 20-layer membranes was statistically insignificant.

Figure 1｜Characterization of VEGF+bFGF PCL NF membranes.

VEGF and bFGF release by the different membranes was then analyzed using enzyme-linked immunosorbent assay.As displayed inFigure 1CandD,rapid release from all membranes was observed in the first 3 days.Between the 3rdand 10thdays,VEGF and bFGF were released efficiently and steadily.All VEGF+bFGF NF membranes exhibited sustained release for >30 days.The 5-layer membrane exhibited the fastest release and achieved the plateau phase first,followed by the 10-layer membrane.Because high porosity provided enough space for cell growth and adhesion,combined with different sustained release efficiency,we chose 10-layer VEGF+bFGF NF membranes for use in the subsequent experiments.

Establishment of the NVU model and confirmation of the identity of primary BMECs,astrocytes,and hippocampal neurons

A 3D co-culture system that contained three types of primary cells was constructed to simulate the structure of NVUsin vitro(Figure 2A).vWF,GFAP,and NSE immunofluorescence assays confirmed the successful isolation of primary BMECs,astrocytes,and hippocampal neurons,respectively (Figure 2B).

Figure 2｜NVU model and confirmation of the identity of the cellular components.

VEGF+bFGF NF membranes promote BMEC-mediated angiogenesis in response to OGD

We investigated the effects of VEGF+bFGF NF membranes on BMECs subjected to OGD by tube formation assessment,Ki67 immunofluorescence staining,and MTT assay.OGD treatment inhibited tube formation by BMECs (P<0.05,vs.Con group;Figure 3AandB) and decreased the proportion of Ki67-positive cells (P<0.05,vs.Con group;Figure 3CandD).Similar results were observed in the OGDC group.However,the presence of VEGF or bFGF NF membranes relieved OGD-induced inhibition of tube formation (bothP<0.05,vs.OGD group).Furthermore,compared with the VEGF and bFGF groups,the VEGF+bFGF group exhibited longer tube length and higher Ki67 immunofluorescence intensity (bothP<0.05).

Figure 3｜NF membranes loaded with VEGF and bFGF promote BMEC tube formation and proliferation after OGD.

MTT assay was used to measure the relative number and viability of BMECs.As shown inFigure 3E,the OD value in the VEGF group was akin to that in the bFGF group,higher than those in the OGD and OGDC groups,and lower than that in the VEGF+bFGF group.Therefore,VEGF+bFGF NF membranes alleviated OGD-induced inhibition of angiogenesis and promoted BMEC proliferation.

VEGF+bFGF NF membranes promote neuronal survival under OGD conditions

Primary hippocampal neuron number was determined by SEM.We found that there were fewer cells in the OGD and OGDC groups than in the Con group (bothP<0.05;Figure 4AandB).VEGF and bFGF NF membrane treatment distinctly elevated the cell number (bothP<0.05,vs.OGD group),and this increase was even more pronounced with VEGF+bFGF NF membrane treatment (bothP<0.05,vs.VEGF and bFGF groups).

Next,neuronal apoptosis induced by OGD was detected by flow cytometry (Figure 4CandD).The apoptosis rate in the OGD and OGDC groups was markedly increased (bothP<0.05,vs.Con group),while that in the VEGF and bFGF groups was lower than that in the OGD group (bothP<0.05).Compared with the VEGF and bFGF groups,the apoptosis rate was decreased in the VEGF+bFGF group (bothP<0.05).

Consistent with the above observation,OGD increased the percentage of TUNEL-positive cells (P<0.05,vs.Con group;Figure 4EandF).The apoptosis rate did not differ significantly between the OGD and OGDC groups;however,treatment with VEGF and bFGF NF membranes reduced the green fluorescence intensity (bothP<0.05,vs.OGD group).Moreover,VEGF+bFGF NF membrane treatment further decreased the apoptosis rate (bothP<0.05,vs.VEGF and bFGF groups).

MTT assay was performed to quantify neuronal viability (Figure 4G).Similar to the TUNEL results,the VEGF+bFGF group had the highest OD value,followed by the VEGF and bFGF groups,while the OGD and OGDC groups had the lowest values.Collectively,these findings suggest that VEGF+bFGF NF membranes promote neuronal attachment and growth and attenuate OGDinduced apoptosis.

VEGF+bFGF NF membranes promote the repair of NVUs subjected to OGD in vitro

To investigate the protective effect of VEGF+bFGF NF membranes on NVUs subjected to OGDin vitro,we quantified NSE,vWF,and GFAP immunofluorescence staining in the 3D co-culture systems containing primary hippocampal neurons,BMECs,and astrocytes,respectively.The variations in NSE and vWF immunofluorescence intensity in each treatment group were basically the same (Figure 5A,B,D,andE).OGD led to a decline in fluorescence expression compared with the Con group (P<0.05),and OGDC treatment did not improve this negative impact (P>0.05,vs.OGD group).The level of fluorescence expression in the VEGF and bFGF groups increased,but was still significantly lower than that in the VEGF+bFGF group (bothP<0.05).Moreover,the VEGF,bFGF,and VEGF+bFGF groups demonstrated a significant increase in the number of GFAP-positive cells compared with the OGD group (allP<0.05).The VEGF+bFGF NF membranes had the most pronounced effect (bothP<0.05,vs.VEGF and bFGF groups;Figure 5C,andF).In contrast,the OGDC group did not exhibit alleviation of the decrease in the number of GFAP-positive stained astrocytes compared with the OGD group (P>0.05).Taken together,these findings suggest that VEGF+bFGF NF membranes helped improve cellular activity within NVUs and prevented OGDinduced fatal injury.BMECs joined by tight junctions constitute the anatomic basis of the BBB,which is a vital element of the NVU (De Strooper and Karran,2016).The cytoskeletal protein F-actin and the tight junction scaffolding protein ZO-1 were observed in BMECs via immunofluorescence staining to assess BBB disruption.F-actin and ZO-1 expression were reduced in BMECs exposed to OGD (bothP<0.05,vs.Con group;Figure 6A-D).VEGF and bFGF NF membrane treatment partly rescued the expression of both proteins (bothP<0.05,vs.OGD group),and this effect was enhanced by VEGF+bFGF NF membrane treatment (bothP<0.05,vs.VEGF and bFGF groups).To further analyze changes in the expression of tight junction proteins,such as claudin-5,occludin,and ZO-1,western blotting was performed.We found that the expression levels of all of these tight junction proteins were decreased in the OGD and OGDC groups in contrast to the Con group (allP<0.05,Figure 6EH),while VEGF and bFGF NF membrane treatment,especially VEGF+bFGF NF membrane treatment,significantly increased their expression levels (allP<0.05,vs.OGD group).In conclusion,the VEGF+bFGF NF membrane improved neuron,BMEC,and astrocyte activity,repaired BBB dysfunction,and maintained the integrity of functional NVUs subjected to OGD.

Figure 5｜NF membranes loaded with VEGF and bFGF ameliorate OGD-induced injury to cellular components of the NVU.

VEGF+bFGF NF membranes confer neuroprotection via JAK2/STAT3 pathway regulation to NVUs injured by OGD

Previous studies have shown that the JAK2/STAT3 signal transduction pathway,which correlates closely with the regulation of important pathological processes,such as inflammatory responses and neuronal apoptosis,is rapidly activated by ischemic injury (Zhong et al.,2021;Yu et al.,2022).Potential alterations in the expression of JAK2/STAT3 pathway components in neurons in the NVU model were investigated by western blotting.At the outset,OGD treatment increased JAK2 and STAT3 phosphorylation (bothP<0.05,vs.Con group;Figure 7A-C),but this effect was alleviated by treating the neurons with VEGF or bFGF NF membranes (bothP<0.05,vs.OGD group).More importantly,the VEGF+bFGF group exhibited lower JAK2 and STAT3 phosphorylation levels compared with the VEGF and bFGF groups (bothP<0.05),although there was no significant difference between the OGD and OGDC groups (P>0.05).

Figure 7｜NF membranes loaded with VEGF and bFGF inhibit the JAK2/STAT3 pathway,protecting neurons from apoptosis.

We further detected the expression levels of apoptosis-and anti-apoptosisrelated proteins.The Bax/Bcl-2 ratio and the level of cleaved caspase-3 increased after OGD (P<0.05,vs.Con group;Figure 7A,D,andE),and this effect was reversed by VEGF and bFGF NF membrane treatment (bothP<0.05,vs.OGD group).Moreover,VEGF+bFGF NF membrane treatment further reduced the Bax/Bcl-2 ratio and the cleaved caspase-3 expression level (bothP<0.05,vs.VEGF and bFGF groups).In summary,sustained release of both VEGF and bFGF from the NF membrane inhibited apoptosis by suppressing the JAK2/STAT3 pathway,exerting a synergistic neuroprotective effect on NVUs.

Discussion

Biomedical nanotechnology,tissue engineering methods,and electrospinning technology have been used to prepare polymer scaffolds with good electrical,mechanical,and biological properties (Subramanian et al.,2009).Electrospun PCL NFs have a high potential for clinical practice owing to easy fabrication,good mechanical compliance,and biocompatibility (Han et al.,2015).Through the LBL self-assembly technique,oppositely charged polyelectrolytes can be adsorbed onto the fibers of NF membranes.Negatively charged growth factors can be adsorbed by positively charged polyelectrolytes and deposited layer by layer on the surface of NFs.During the degradation of the polyelectrolyte layer,growth factors can be slowly released layer by layer (Zhang et al.,2020).Based on our previous studies,we successfully loaded VEGF and bFGF onto PCL NF membranes by LBL self-assembly technology to generate a membrane that releases VEGF and bFGF continuously and in a controlled fashion.The enzyme-linked immunosorbent assay results suggested that this dual growth factor local sustained-release delivery system released both factors efficiently and stably for >1 month,which provided sufficient concentrations and time for the repair of NVUs injured by ischemia.In addition,as shown by SEM,the VEGF+bFGF PCL NF membranes offered a suitable 3D microenvironment for allowing cell adhesion and maintaining cell viability.Nevertheless,as the number of layers increased,the NF membrane pore size and porosity decreased and the NF diameter increased,resulting in unclear fiber structure and changes in the biological properties of the membranes.Considering both sustained-release efficiency and ultramicroscopic morphology,we found the 10-layered VEGF+bFGF NF membrane to be most suitable for the intended application.The effects of VEGF and bFGF are closely related to route and timing of administration (Xing and Lo,2017).At 0-24 hours after brain ischemia onset,endogenous upregulation or systemic administration of VEGF is likely to cause BBB leakage and subsequent exacerbation of brain edema (Hu et al.,2022).In contrast,treatment with VEGF starting 1 day after an ischemic event decreases ischemia-induced neural damage (Liu et al.,2021).Intracerebroventricular or local application of VEGF at the site of brain injury confers powerful neuroprotection,even when applied early after ischemia (Geiseler and Morland,2018).For bFGF,systemic application may result in severe side effects,such as tumor formation and neovascularization of nontargeted tissues (Liu et al.,2013).Sustained release of VEGF and bFGF from NF membranes at the site of brain ischemic injury harnesses the beneficial effects of these factors,while avoiding their detrimental effects.We found that BMECs treated with VEGF+bFGF sustained-release NF membranes had the longest tube length,as well as the highest MTT OD value and Ki67 immunofluorescence intensity among all groups exposed to OGD.Compared with the groups with the single-factor NF membranes,the dual-factor membrane further decreased the neuronal apoptosis rate,as verified by flow cytometry and TUNEL assay,and increased the number of cells detected by SEM.These results indicate that the VEGF+bFGF NF membranes significantly promoted BMEC proliferation and tube formation,inhibited neuronal apoptosis,and improved cell viability in BMEC and neuron monoculturein vitro.

Brain ischemia disrupts NVUs,involving death of neurons,glia,and BMECs in the core and penumbra regions (Keogh et al.,2007;Lu et al.,2022).Integrated protection targeting the NVU holds immense therapeutic potential for brain ischemia (Wang et al.,2022b).Our study showed that,in a 3D coculture system,the OGD-induced decline in the immunofluorescence intensity of NVU components (NSE and vWF) and the decrease in GFAP-positive stained astrocytes could be reversed by treatment with VEGF+bFGF NF membranes.During the repair period that follows cerebral ischemia,increased numbers of astrocytes contribute to improved communication between components of the NVU,promoting angiogenesis,synapse formation,and neurogenesis (Liu and Chopp,2016).BBB integrity relies not only on cellular components of the NVU (pericytes,astrocytes,and endothelial cells),but also on tight junction proteins (Weber et al.,2020).According to the immunofluorescence staining and western blotting results,VEGF+bFGF NF membranes up-regulated F-actin,claudin-5,occludin,and ZO-1 expression,which could help repair the BBB and maintain the integrity of functional NVUs after OGD-induced injury.Moreover,the combined use of these two growth factors was more effective than their separate use.

Ischemia-induced apoptosis may be regulated by the JAK2/STAT3 signal transduction pathway (Zhong et al.,2021).Some studies have attempted to reduce inflammation and apoptosis and confer neuroprotection by suppressing the JAK2/STAT3 pathway (Zhu et al.,2021;Yu et al.,2022).The JAK-inhibitor ruxolitinib downregulates STAT3 phosphorylation,and consequently the expression of downstream proinflammatory cytokines,which ameliorate cerebral edema and decrease the infarct size in mice subjected to middle cerebral artery occlusion (Zhu et al.,2021).Activation of the JAK2/STAT3 pathway by brain ischemia impairs the recovery of neurologic function by promoting the production and secretion of proinflammatory cytokines,then inducing neuronal loss and apoptosis (Guo et al.,2021).In this study,OGD increased JAK2 and STAT3 phosphorylation upregulated the expression of pro-apoptotic proteins (Bax,cleaved caspase-3),and downregulated the expression of an anti-apoptotic protein (Bcl-2).We previously reported that VEGF protects primary hippocampal neurons against OGD-induced inflammation and apoptosis,thus demonstrating its neuroprotective effects (Wang et al.,2021).The results from the current study show that VEGF and bFGF relieved the OGD-induced increase in JAK2 and STAT3 phosphorylation,especially in combination.Therefore,we speculated that the JAK2/STAT3 pathway may be involved in the synergistic effect that sustained release of both VEGF and bFGF from the NF membrane had on promoting BMEC proliferation and tube formation,inhibiting neuronal apoptosis,upregulating the expression of tight junction proteins,and improving the viability of various cellular components of NVU in anin vitroOGD model.

There were some limitations in this study.Although we investigated the protective effects and potential mechanism of VEGF+bFGF NF membranes on primary BMECs,hippocampal neurons,and an NVU model after OGD,furtherin vivoexperimental animal studies are needed to confirm thein vitrodata,especially using agents that inhibit the JAK2/STAT3 pathway.It has been reported that PCL NFs generally take >2 years to completely degradein vitro(Zhang et al.,2020).Fortunately,the degradation rate is significantly fasterin vivothanin vitro.We plan to conduct animal experiments of longer duration to assess the degradability of VEGF+bFGF NF membranes and to address the other limitations mentioned above.

In summary,we successfully fabricated a dual growth factor sustained-release delivery system composed of electrospun PCL NF the releases VEGF and bFGF continuously and in a controlled fashion.Profiting from the synergism of two growth factors,this novel NF membrane produced directly protected NVUs subjected to OGD by repressing the JAK2/STAT3 pathway.These results may provide a novel therapeutic strategy for ischemic cerebrovascular diseases.

Acknowledgement:We created graphical abstract with Figdraw.

Author contributions:Study design: DW,JH;experimental implementation: JS,YW;data analysis and manuscript draft: JS;manuscript revision: DW,JH,QL.All authors have read and approved the final version of the manuscript.

Conflicts of interest:The authors declare that there is no conflict of interest regarding the publication of this paper.

Data availability statement:No additional data are available.

Open access statement:This is an open access journal,and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License,which allows others to remix,tweak,and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.