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. 2025 May 2;41(9):1522–1536. doi: 10.1007/s12264-025-01403-6

Endothelial Cell Integrin α6 Regulates Vascular Remodeling Through the PI3K/Akt-eNOS-VEGFA Axis After Stroke

Bing-Qiao Wang 1,#, Yang-Ying Duan 2,#, Mao Chen 1,#, Yu-Fan Ma 1, Ru Chen 1, Cheng Huang 1, Fei Gao 1, Rui Xu 1,✉, Chun-Mei Duan 1,✉
PMCID: PMC12433384  PMID: 40316875

Abstract

The angiogenic response is essential for the repair of ischemic brain tissue. Integrin α6 (Itga6) expression has been shown to increase under hypoxic conditions and is expressed exclusively in vascular structures; however, its role in post-ischemic angiogenesis remains poorly understood. In this study, we demonstrate that mice with endothelial cell-specific knockout of Itga6 exhibit reduced neovascularization, reduced pericyte coverage on microvessels, and accelerated breakdown of microvascular integrity in the peri-infarct area. In vitro, endothelial cells with ITGA6 knockdown display reduced proliferation, migration, and tube-formation. Mechanistically, we demonstrated that ITGA6 regulates post-stroke angiogenesis through the PI3K/Akt-eNOS-VEGFA axis. Importantly, the specific overexpression of Itga6 in endothelial cells significantly enhanced neovascularization and enhanced the integrity of microvessels, leading to improved functional recovery. Our results suggest that endothelial cell Itga6 plays a crucial role in key steps of post-stroke angiogenesis, and may represent a promising therapeutic target for promoting recovery after stroke.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12264-025-01403-6.

Keywords: Integrin α6, Ischemia, Angiogenesis

Introduction

Stroke is a prominent cause of long-term disability globally, yet there remains a lack of effective therapies to facilitate recovery from stroke [1–3]. Brain ischemia triggers continuous neurogenesis, prompting neuronal precursor cells to migrate toward ischemic areas to aid in the reconstruction of damaged tissue [4–6]. While several studies suggest that stem cell therapy shows potential to improve functional recovery post-stroke, its efficacy is hindered by the low survival and differentiation rates of transplanted cells due to the inadequate energy and oxygen supply in the ischemic region [6, 7]. Research has demonstrated that brain ischemia triggers vascular remodeling in the peri-infarct region [7, 8]. The newly-formed blood vessels release neurotrophic factors and chemokines, establishing a conducive microenvironment in the injured brain that promotes the survival of emerging neurons [9, 10]. Crucially, newly-formed vessels can provide adequate perfusion in a timely manner, and this can reduce the damage caused by ischemia by mitigating hypoxia and supplying enough energy for the biological process of tissue repair, such as cell proliferation, migration, and differentiation [8]. This heightened vascularization in the peri-infarct zone of stroke patients has been linked to prolonged survival, highlighting the importance of the angiogenic reaction post-stroke in neurological rehabilitation [11]. Therefore, investigating the processes involved in angiogenesis and vascular restoration following brain injury could have significant potential for treating stroke and other degenerative conditions.

Extracellular matrix (ECM) proteins play crucial roles in shaping vascular formation during development and in adulthood [12, 13]. It is essential to determine the specific roles of ECM integrin receptors on the microvascular in regulating vascular remodeling and maintaining blood-brain barrier (BBB) integrity. Integrin α6 (Itga6), a member of the integrin family, is found on brain microvessels and likely plays a significant role in regulating blood vessel remodeling and promoting vascular maturation [14, 15]. Previous studies have shown that the expression of ITGA6 is significantly upregulated in neuroinflammatory [16] and hypoxic conditions [17], but its specific role remains unclear. ITGA6 can interact with integrin β4 or integrin β1 to form functional dimers, which may contribute to maintaining vascular integrity during the neuroinflammation induced by experimental autoimmune encephalomyelitis [14, 18]. BBB maintenance studies have used integrin β4 conditional-knockout mice but did not explore the role of Itga6 [14, 19].

Among the numerous potential regulators of angiogenesis that have been identified [9], vascular endothelial growth factor (VEGF) plays a notable role in this process [20]. It enhances the survival, proliferation, migration, and tube formation of endothelial cells, thereby facilitating the creation of a new vascular network through the interaction between VEGF-A and VEGFR-2 [9]. Previous studies have demonstrated that ITGA6 regulates tumor cell invasion, metastasis, and neurogenesis through the PI3K/Akt pathway [21]. This pathway is also a crucial mediator of angiogenic activity, playing a role in cell migration and proliferation [22, 23]. Sustained activation of Akt in endothelial cells leads to the development of structurally abnormal blood vessels resembling tumor vessels [24]. Inhibition of Akt signaling in endothelial cells can result in the loss of endothelial cell integrity, subsequent thrombosis, and sudden death [25]. Therefore, we hypothesized that ITGA6 regulates post-stroke angiogenesis through the PI3K/Akt-VEGFA (Vascular Endothelial Growth Factor A) axis.

In this study, we examined the role of endothelial cell Itga6 in vascular remodeling following cerebral ischemia. Through the use of inducible genetic elements in mice, we found that deletion of Itga6 in endothelial cells led to decreased neovascularization and compromised vascular integrity after a stroke. Our research delved into the specific mechanism by which ITGA6 in endothelial cells influences cerebral vascular remodeling after stroke, revealing a novel pathway involving activated PI3K/Akt-eNOS-VEGFA signaling. Moreover, the overexpression of Itga6 in endothelial cells was able to reverse the abnormal vascular remodeling after a stroke, suggesting a promising therapeutic approach for addressing compromised vascular function in stroke and other neurodegenerative conditions.

Materials and Methods

Mutant Mice and Inducible Genetic Modifications

All animal procedures were conducted with the approval of the Animal Ethics Committee of the Army Medical University and in accordance with the ARRIVE guidelines (Animal Research: Reporting in vivo Experiments). Itga6flox/flox mice were obtained from GemPharmatech, and Cdh5-P2A-CreERT2 mice were sourced from Shanghai Model Organisms (Stock NM-KI-200173). To generate the CDH5+ cell Itga6 knockout mice, we bred Cdh5-P2A-CreERT2 transgenic mice with Itga6flox/flox mice, resulting in Cdh5-P2A-CreERT2; Itga6flox/flox inducible conditional-knockout (Itga6ECKO) mice.

The Itga6ECKO mice were treated with tamoxifen (MCE, HY-13757A) via intraperitoneal injection at a dose of 1 mg once daily for 10 days, starting at 8 weeks of age, to specifically knock out Itga6 in endothelial cells. Itga6fl/fl littermates served as controls. Pdgfrb-tdTomato mice were generated by crossing Pdgfrb-Cre mice (Biocytogen, 110129) with Ai9 mice (Jackson Lab, 007909). Both male and female mice were used in equal proportions in this study.

Middle Cerebral Artery Occlusion and Reperfusion

Mice were anesthetized via intraperitoneal administration of pentobarbital at a dose of 50 mg/kg body weight. Focal ischemia/reperfusion (I/R) was induced through a transient middle cerebral artery occlusion (MCAO) procedure as previously described [26]. Briefly, following a midline cervical incision, the left common carotid artery, the left external carotid artery (ECA), and the left pterygopalatine artery were isolated and ligated under an operating microscope. The internal carotid artery (ICA) was occluded at the peripheral site of the bifurcation between the ICA and the pterygopalatine artery using a small vascular clip. Subsequently, a 6–0 silicone-coated filament was advanced through the ICA to the bifurcation of the ICA and ECA. The filament was advanced until light resistance was encountered. The filament was removed after a 90-min occlusion. The mice were carefully monitored and left in their cages for the next 24 h. In the sham-operated mice, these arteries were visualized but remained undisturbed.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

We processed the brain tissues at corresponding MCAO time points and obtained lysates. Total RNA was then extracted using an RNA/DNA Isolation Kit (Beyotime, R0017S). RT-qPCR was applied using the PrimeScript RT reagent Kit (Takara, RR047A). All steps were guided by the manufacturer’s instructions. A comparative CT method was used to compare each condition to the control reactions.

The primers were: Itga6 forward: 5’-acctcaatgcagatgggtgg-3’; Itga6 reverse: 5’-taaactgcacccccgacttc-3’; Gapdh forward: 5’-tgctcctccctgttccagag-3’; Gapdh reverse: 5’-cctggaactcacccgttcac-3’.

5-Triphenyltetrazolium Chloride (TTC) Staining

Twenty-four hours after the induction of MCAO, mouse brain tissue was collected and placed at –80℃ for 5 min. The brain tissue was cut into 6 equal parts on ice, stained in 2% TTC solution (Sigma-Aldrich, T8877) in 1× PBS at 37 ℃ for 15–20 min, and then fixed in 4% paraformaldehyde solution (Beyotime, P0099) at 4 ℃ until imaging.

Lateral Scratch Wound Assay

2 × 106 endothelial cells were evenly spread on a 6-well plate. Cells were scratched vertically with a 1 mL pipette tip after they were attached, washed 2–3 times with PBS, and photographed at the location of the scratch at 0 h, 12 h, and 24 h.

Transwell Assay

600 μL endothelial cell medium containing 30% fetal bovine serum was added to a 24-well plate, followed by a transwell chamber (Corning, 3464) to which 5000 endothelial cells re-suspended in 150 μL serum-free medium were added. After 48 h, the images were captured after staining with 0.1% crystal violet.

Cell Viability Assay

800 endothelial cells were added to a 96-well plate. At 12, 24, 36, 48, and 60 h, 10 μL Cell Counting Kit-8 (CCK8) was added to each well, incubated at 37 ℃ for 1 h, and the optical density was determined.

Cell Lines

Mouse brain primary pericytes (MIC-iCell-n003), endothelial cells (MIC-iCell-n001), and astrocytes (MIC-iCell-n009) were obtained from Cellverse Bioscience Technology Co., Ltd. These cells were cultured in their respective media: Pericyte Culture Medium (Cellverse, PriMed-iCell-015), Endothelial Cell Culture Medium (Cellverse, PriMed-iCell-002), and Astrocyte Culture Medium (Cellverse, iCell-007). In addition, human brain primary endothelial cells (HBECs) (ScienCell, 1000) were sourced from ScienCell™ Research Laboratories and were cultured in Endothelial Cell Medium (ScienCell, 1001). All cells were maintained in a humidified atmosphere containing 5% CO2 and 20% O2. The identity of the cell lines was confirmed.

Immunofluorescence Staining

Brain slices and cell samples were washed three times with PBS for 10 min each before being blocked with PBS containing 3% normal goat serum and 0.1% Triton X-100 for 1 h at room temperature. The samples were then incubated with the primary antibody overnight at 4 °C. The following day, after washing three times with PBS for 10 min each, the slices were incubated with a fluorophore-conjugated secondary antibody for 1 h at room temperature. After the final wash, the samples were mounted onto glass slides using a mounting medium. The following primary antibodies were used: anti-CD31 (AF3628, R&D), anti-ITGA6 (710209, Invitrogen), anti-ZO1 (ab221547, Abcam), anti-Occludin (ab216327, Abcam), anti-Claudin5 (ab131259, Abcam), and Alexa FluorTM 488-Phallodin (A12379, Invitrogen).

EdU Administration

To assess the proliferation of endothelial cells, mice were injected intraperitoneally with 5-ethynyl-2'-deoxyuridine (EdU) (50 mg/kg) (MCE, HY-118411) once a day for 7 consecutive days, and were sacrificed after the final EdU injection.

Western Blot Analysis

Equivalent quantities of total protein (20 μg) were resolved by 10% SDS-PAGE and detected by western blotting using the following antibodies: anti-ITGA6 (ab181551; Abcam), anti-ZO1 (ab276131, Abcam), anti-Claudin5 (ab131259, Abcam), Occludin (ab216327, Abcam), anti-PI3K p55 (11889, CST), anti-PI3K p85 (4257, CST), anti-p-PI3K p85 (Tyr458)/p55 (Tyr199), anti-p-Akt (Thr308) (9275, CST), anti-Akt (9272, CST), FAK Antibody Sampler Kit (9330, CST), anti-β-Catenin (9562, CST), anti-SMAD2/3 (3102, CST), anti-p-SMAD2/3 (8828, CST), anti-Erk1/2 (9102, CST), anti-p-Erk1/2 (9101, CST), anti-P38 MAPK (9212, CST), anti-p-P38 MAPK (4511, CST), anti-eNOS (32027, CST), anti-p-eNOS (ab215717, Abcam), anti-VEGFA (ab46154, Abcam), and anti-β-actin (HRP-60008, Proteintech). Proteins were detected using an ECL reagent (Santa Cruz Biotechnology). Western blot signals were quantified using an Amersham Imager 600 (GE Healthcare, Little Chalfont, UK), and band signals are expressed as amounts relative to β-actin.

FITC-dextran Assay

Mice were placed on a warming plate for 15 min to dilate the caudal vein. FITC-dextran (2000-kDa, Sigma-Aldrich) was injected into the tail vein at a dose of 100 mg/kg, using a 30-gauge disposable needle, 3 min before sacrifice. After sacrifice, each animal’s brain was removed and embedded in an OCT medium (Tissue-Tek). After snap-freezing in liquid nitrogen, the brains were sectioned and immunostained.

Overexpression of Itga6 in Endothelial Cells

Adeno-associated virus BI30 (AAV-BI30) vector was purchased from GeneChem (Shanghai, China). The full-length cDNA of Itga6 was inserted into AAV-BI30 to construct an Itga6 overexpression AAV. Subsequently, 8-week-old male Itga6ECKO mice were injected with either AAV-BI30-Itga6 or AAV-BI30-vector in a volume of 100 µL via the tail vein. The viral titer was 2 × 1012 genomes per ml. Three weeks later, these mice underwent MCAO surgery.

Rotarod Test

The rotarod (Unibiolab, Beijing, China) was utilized to assess neuromuscular coordination and balance in mice at 7 days post-MCAO surgery. This assessment was measured by the latency for each mouse to fall from a rotating beam, which started at a speed of 5 r/min and accelerated to 45 r/min during 100 s. Prior to the test, all mice underwent training, consisting of five trials on the day before the actual assessment. Each mouse was placed individually on the beam, and the latency until the mouse slid off was recorded using a stopwatch. The rotarod test was applied three times for each mouse.

Pole-Climbing Test (PCT)

A cork ball 2.5 cm in diameter was affixed to the top of a stick 50 cm long and 1 cm in diameter. Each mouse was placed at the top of the pole in an upright position, and the time taken for the mouse to turn around on the ball, as well as the time taken to climb the entire pole, were recorded. Each mouse underwent five training sessions to familiarize itself with the entire process prior to testing. Each trial was conducted three times, and the average time was calculated. In each trial, the mouse had a maximum of 120 s to complete the task. If a mouse slid down the pole, it received a trial score of 60 s, while a score of 120 s was assigned if the mouse fell. Mice were tested on day 7 after MCAO surgery.

Lentivirus Infection

HBECs were cultured in 6-cm dishes at a density of 1 × 106 cells per dish. After 6 h, lentivirus expressing scrambled (5′-ttctccgaacgtgtcacgt-3′) and shITGA6 purchased from Hanbio (Shanghai, China) were added (MOI = 10). Cells were harvested for assays at the scheduled times.

Statistical Analysis

Data are presented as the mean ± SD. Significant differences between group means were assessed using the unpaired Student’s t-test or one-way ANOVA, conducted with Graph Prism software (version 9.0, GraphPad). For comparisons involving more than two treatment groups, one-way ANOVA was followed by Dunnett’s multiple-comparison test. A P-value <0.05 was considered a statistically significant difference. The number of replicates for each group is indicated in the figure legends. ns: P >0.05, *P <0.05, **P <0.01, ***P <0.001.

Results

Expression of Itga6 After MCAO Surgery

To investigate the potential role of ITGA6 after stroke, we analyzed changes in ITGA6 expression following MCAO surgery. Initially, 8-week-old mice underwent MCAO surgery, and brain tissue from the infarcted area was collected before (day 0) and on days 1, 3, 5, 7, and 14 after surgery. The qPCR analysis revealed an increase in Itga6 mRNA levels on day 1, reaching a peak on day 3, and subsequently decreasing from days 5 to 14 (Fig. 1A). Protein levels exhibited a similar trend, reaching a peak on day 5 and returning to normal by day 14 (Fig. 1B). Subsequently, immunohistochemistry was conducted to assess the expression of ITGA6 in the peri-infarct region (shown in the framed area in Fig. 1C) known for its angiogenic activity following a stroke [27]. Immunofluorescence analysis revealed that the expression of ITGA6 was significantly elevated at day 5 post-stroke compared to the sham group, with localization in CD31+ endothelial cells (Fig. 1D). Furthermore, ITGA6 did not colocalize with pericytes in the brains of Pdgfrb-tdTomato reporter mice, nor with astrocytes in WT mice (Fig. 1E). Western blot analysis confirmed prominent expression of ITGA6 in mouse brain endothelial cells, with limited expression in astrocytes and pericytes (Fig. 1F). These findings collectively highlight the dynamic expression and localization of ITGA6 following a stroke, indicating its crucial involvement in the vascular remodeling response to ischemic injury.

Fig. 1.

Fig. 1

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Dynamic expression of Itga6 after MCAO surgery. A qPCR analysis of Itga6 in brain tissue post-stroke at indicated days (n = 4). B Western blots and analysis of ITGA6 expression post-stroke at indicated days (n = 4). C Schematic of the MCAO model where the shaded area indicates the infarct area. The framed area shows the peri-infarct area which is used for all histological measurements. D Immunostaining of CD31 and ITGA6 depicting the expression of ITGA6 in brain sections at day 5 post-stroke (n = 6; scale bar, 25 μm). E Immunostaining of ITGA6 in Pdgfrb-tdTomato mice, where pericytes are labeled with tdTomato, as well as double-immunostaining of ITGA6 and GFAP in brain sections from wild-type (WT) mice. Arrowheads indicate that ITGA6 does not colocalize with pericytes or astrocytes (n = 3; scale bar, 25 μm). F Western blots showing the ITGA6 expression in astrocytes (AS), pericytes (PC), and endothelial cells (EC) (n = 3). Values are the mean ± SD; unpaired, 2-tailed Student’s t test.

Deletion of Itga6 in Endothelial Cells Results in More Severe Deficiencies in Neural Function

To investigate the impact of endothelial cell Itga6 on neovessel formation following a stroke, we bred Cdh5-CreERT2 transgenic mice, which express tamoxifen-inducible Cre recombinase under the control of the Cdh5 promoter, with Itga6 floxed mice. This breeding approach enabled the generation of Cdh5-CreERT2; Itga6flox/flox (Itga6ECKO) mice with inducible conditional-knockout of Itga6 (Fig. S1A), and Itga6fl/fl littermates served as controls. To validate the endothelial cell-specificity of Cdh5-CreERT2-mediated Itga6 ablation in the mouse brain, both Itga6ECKO mice and Itga6fl/fl mice received intraperitoneal injections of tamoxifen (50 mg/kg once a day for 10 days) (Fig. 2A). Subsequently, brain CD31+ cells were sorted by magnetic cell sorting (MACS), and western blotting analysis were conducted to confirm the absence of Itga6 in endothelial cells (Fig. 2B). Itga6ECKO mice exhibited normal brain vasculature by CD31 immunostaining compared with Itga6fl/fl mice (Supplementary Fig. 2B). In addition, we did not see any abnormal physiological state of Itga6ECKO mice following administration of tamoxifen (Data not shown). We performed MCAO surgery on Itga6fl/fl mice and Itga6ECKO mice 2 days after tamoxifen injection, and conducted functional analysis on day 7 (Fig. 2A). The results displayed a significant increase in infarct size in Itga6ECKO mice compared to Itga6fl/fl mice (Fig. 2C). Moreover, Itga6ECKO mice exhibited more pronounced neurological impairments than Itga6fl/fl mice, as indicated by neurological deficit scoring on day 7 (Fig. 2D). Assessments of motor function, such as the rotarod test and the pole-climbing test (PCT), revealed greater motor deficits in Itga6ECKO mice post-stroke (Fig. 2E–F). In addition, survival rates were notably lower in Itga6ECKO mice following stroke than in Itga6fl/fl mice (Fig. 2G). A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis indicated more apoptotic brain cells in Itga6ECKO mice than in Itga6fl/fl littermates at day 7 post-stroke (Fig. 2H). These findings underscore the critical importance of maintaining neural function after a stroke.

Fig. 2.

Fig. 2

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Deletion of endothelial cell Itga6 impairs neural function after stroke. A Experimental timeline of tamoxifen administration and MCAO surgery, followed by functional analysis. B Western blots and analysis showing the absence of Itga6 in the Itga6ECKO mice after tamoxifen administration (n = 4). C TTC staining and quantification showing the infarct size in Itga6ECKO mice and Itga6fl/fl mice (n = 6). D Neurological deficit scores of Itga6ECKO and Itga6fl/fl mice (n = 8). E Rotarod test results show the latency of Itga6ECKO and Itga6fl/fl mice at day 7 post-stroke to fall from the rotating rod (n = 8). F Pole Climbing Test (PCT) measuring turnaround time and total climbing duration in Itga6ECKO and Itga6fl/fl mice at day 7 post-stroke (n = 8). G Kaplan-Meier survival curves comparing survival rates in Itga6ECKO and Itga6fl/fl mice subjected to MCAO surgery (n = 15). H TUNEL assay showing the apoptotic neural cells in brain sections from Itga6ECKO and Itga6fl/fl mice and quantification at day 7 post-stroke (scale bar, 40 μm). Values are the mean ± SD; unpaired, 2-tailed Student’s t test.

Itga6 Deficiency Leads to Reduced Neovascularization after Stroke

Next, we aimed to investigate the impact of Itga6-deficiency on microvascular remodeling after stroke. Immunofluorescence imaging at various post-stroke time points (Sham, and days 1, 3, 5, 7, and 14 following MCAO surgery) revealed microvascular remodeling in the peri-infarct area of Itga6ECKO and Itga6fl/fl mice. We found that both the vascular length and vascular area were significantly reduced on day 1, followed by a subsequent increase that peaked on day 7 in Itga6fl/fl mice, aligning with the dynamic expression of ITGA6 post-stroke (Fig. 3A–C). In Itga6ECKO mice, both vascular length and vascular area were significantly reduced at the same time point compared to Itga6fl/fl mice (Fig. 3A–C), suggesting that Itga6 plays an important role in post-stroke angiogenesis. Next, we investigated the microvascular phenotypes in Itga6ECKO mice on day 7. The functionality of the vessels was assessed through intravascular injection of tomato-lectin, and this demonstrated a notable reduction in perfused vessels in Itga6ECKO mice (Fig. 3D). We further examined the effect of Itga6 deficiency on endothelial cell proliferation by repeated EdU administration for 7 days and found that the numbers of EdU+/CD31+ endothelial cells were significantly reduced in Itga6ECKO mice after stroke (Fig. 3E). These data suggest that Itga6 plays a modulatory role in neovessel formation by regulating endothelial cell proliferation.

Fig. 3.

Fig. 3

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Deletion of endothelial cell Itga6 reduces the neovascularization after stroke. A Immunostaining of CD31 in brain sections of Itga6ECKO and Itga6fl/fl mice at the indicated days post-stroke (scale bar, 50 μm). B, C Analysis and comparison of vascular length and area between Itga6ECKO and Itga6fl/fl mice at the same time points (n = 6). D Left, lectin perfusion assay showing the microvessel perfusion in the peri-infarct area at day 7 post-stroke; right, quantification of microvessel perfusion in Itga6ECKO and Itga6fl/fl mice (n = 6; scale bar, 50 μm). E Left, EdU and CD31 immunostaining display endothelial cell proliferation at day 7 post-stroke; right, quantification of endothelial cell proliferation in Itga6ECKO and Itga6fl/fl mice (n = 6; scale bar, 25 μm). Values are the mean ± SD; unpaired, 2-tailed Student’s t test.

Endothelial Cell Itga6 Deficiency Impairs Vascular Integrity and Pericyte Coverage

To investigate the impact of endothelial cell-specific Itga6 ablation on cerebrovascular integrity, both Itga6ECKO mice and Itga6fl/fl mice were intravenously administered dextran-FITC (MW, 2000 kDa) on day 7. The extravasation of dextran-FITC dye into the tissue surrounding the cerebral capillaries was notably more pronounced in Itga6ECKO mice than in Itga6fl/fl mice, suggesting a significant increase in cerebrovascular leakage (Fig. 4A). Immunostaining of tight junctions on microvessels revealed a significant reduction in the levels of key proteins, including Claudin5, Occludin, and ZO1, in Itga6ECKO mice compared to Itga6fl/fl mice (Fig. 4B–D). These results were further confirmed by Western blotting analysis (Fig. 4E). Pericytes contribute to the maintenance of microvascular integrity [28]. Using staining for the pericyte markers platelet-derived growth factor receptor-β (PDGFRβ) and CD13, we found that the pericyte coverage was notable less in Itga6ECKO mice than in Itga6fl/fl mice (Fig. 4F–G). These results suggest that deletion of Itga6 in endothelial cells results in disruption of cerebrovascular integrity and reduced pericyte coverage.

Fig. 4.

Fig. 4

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Deletion of Itga6 impairs the microvessel integrity after stroke. A Left, representative images showing dextran-2000 kDa leakage out of the microvessels in brain sections post-stroke; right, quantitation of vessel permeability in Itga6ECKO and Itga6fl/fl mice (n = 6; scale bar, 25 μm). B Left, immunostaining of CD31 and Occludin in brain sections at day 7 post-stroke; right, quantification of Occludin mean fluorescence intensity (MFI) (n = 6; scale bar, 25 μm). C Left, immunostaining of CD31 and Claudin5 in brain sections at day 7 post-stroke; right, quantification of Claudin5 MFI (n = 6; scale bar, 25 μm). D Left, immunostaining of CD31 and ZO1 in brain sections at day 7 post-stroke; right, quantification of ZO1 MFI (n = 6; scale bar, 25 μm). E Left, western blots and analysis (right) demonstrating the tight junction expression (Claudin5, Ocludin, ZO1) on microvessels in the infarct area at day 7 post-stroke (n = 4). F Left, immunostaining of CD31 and PDGFRβ in brain sections at day 7 post-stroke; right, pericyte coverage on the microvessels in Itga6ECKO mice and Itga6fl/fl mice (n = 6; scale bar, 25 μm0. G Left, immunostaining of CD31 and CD13 in brain sections at day 7 post-stroke; right, pericyte coverage on the microvessels in the Itga6ECKO mice and Itga6fl/fl mice (n = 6; scale bar, 25 μm). Values are the mean ± SD; unpaired, 2-tailed Student’s t test.

Itga6 Regulates Brain Endothelial Cell Migration and Proliferation through the PI3K/Akt-eNOS-VEGFA Signaling Pathway

We next assessed the direct effects of ITGA6 knockdown on endothelial cell function and explored the potential downstream signaling pathways associated with ITGA6. Using a shRNA lentivirus, we suppressed ITGA6 expression in HBECs. Phalloidin staining showed that the cytoskeleton of HBECs was reorganized by silencing ITGA6 (Fig. S2A). Both the lateral scratch wound assay and the Transwell assay demonstrated that silencing ITGA6 expression significantly decreased the motility of HBECs (Fig. S2B, C). EdU immunostaining demonstrated that ITGA6 knockdown significantly reduced the proliferation of HBECs, a finding that was further validated by the CCK8 assay (Fig. 5A and S2D). In addition, the tube formation assay revealed that the knockdown of ITGA6 in HBECs inhibited tube formation, as evidenced by a reduction in both vascular length and branch points (Fig. 5B). These results strengthen the assertion that the loss of ITGA6 impairs the migration and proliferation of endothelial cells, thereby inhibiting angiogenesis. To investigate the downstream effector proteins of ITGA6, we applied ELISA to detect various angiogenic cytokines, including VEGFA, angiotensin 2 (Ang-2), fibroblast growth factor 2 (FGF2), matrix metallopeptidase 2 (MMP2), MMP9, MMP14, interleukin-1 beta (IL-1β), and IL-6 [9], in the medium of HBECs following ITGA6 knockdown. Our findings revealed a significant reduction in the level of VEGFA after ITGA6 knockdown, along with slight decreases in Ang-2 and FGF2 levels (Fig. 5C). In contrast, the levels of MMP2, MMP9, MMP14, IL-1β, and IL-6 exhibited no significant differences between the NC and Lv-ITGA6 shRNA groups (Fig. 5C). These findings indicate that ITGA6 primarily regulates endothelial cell function mainly by modulating the expression of VEGFA. Previous studies have established that PI3K/Akt signaling is downstream of ITGA6 [21]. However, it remains unclear whether this axis is involved in the regulation of angiogenesis by ITGA6. We isolated endothelial cells from the infarct area of Itga6fl/fl and Itga6ECKO mice using MACS. Western blot assays revealed that the protein levels of p-PI3K (p85, p55) and p-Akt (Thr308) were significantly decreased in Itga6ECKO mice, while the total levels of Akt and PI3K remained unaffected (Fig. 5D). In addition, the expression of p-eNOS and VEGFA was significantly reduced in the endothelial cells of Itga6ECKO mice (Fig. 5E). Similar results were obtained in cultured ITGA6 knockdown HBECs (Fig. 5F, G). However, we found no changes in the PI3K/Akt-eNOS-VEGF axis proteins under physiological conditions (Fig. S2E), suggesting that ITGA6 modulates this axis during the angiogenesis process, but not under quiescent conditions. Furthermore, we detected other pathways that are well-known for their role in angiogenesis [7, 9, 29]. Our findings indicated that Wnt/β-catenin signaling and TGFβ/Smad2/3 signaling exhibited no differences in the endothelial cells of Itga6ECKO and Itga6fl/fl mice (Supplementary Fig. 2F, I). However, the expression levels of p-FAK (Tyr925), p-FAK (Tyr576/577), and p-Erk1/2 were slightly reduced, while p-FAK (Tyr397) and p-P38 levels remained unchanged in the endothelial cells of Itga6ECKO mice after stroke (Supplementary Fig. 2G, H). These results indicate that the PI3K/Akt-eNOS-VEGFA pathway exhibits the most significant changes following the deletion of Itga6 post-stroke.

Fig. 5.

Fig. 5

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ITGA6 regulates endothelial cell function through the PI3K/Akt-Enos-VEGFA pathway. A Left, EdU immunostaining showing the proliferation of cultured HBECs after ITGA6 knockdown; right, the numbers of EdU+ endothelial cells (n = 4; scale bar, 25 μm). B Left, tube formation assay after ITGA6 knockdown in HBECs; right, vascular length and branch points (n = 4; scale bar, 100 μm). C ELISA analysis of the expression levels of cytokines in the medium of endothelial cells with either ITGA6 knockdown or the NC group (n = 4). D Western blots and analysis showing changes in the PI3K-Akt axis in endothelial cells of Itga6ECKO and Itga6fl/fl mice at day 7 post-stroke (n = 4). E Western blots and analysis showing the expression levels of p-eNOS and VEGFA in endothelial cells of Itga6ECKO and Itga6fl/fl mice (n = 3). F Western blots and analysis showing changes in the PI3K-Akt axis in cultured HBECs after ITGA6 knockdown (n = 4). G Western blots and analysis showing the expression levels of p-eNOS and VEGF in cultured HEBCs after ITGA6 knockdown (n = 3). Values are the mean ± SD; unpaired, 2-tailed Student’s t test. H CCK8 assays demonstrate the viability of HBECs across the Mock, PI3K-IN-1, NC, Lv-ITGA6, and Lv-ITGA6 + PI3K-IN-1 groups (n = 3). Cell viability is compared among the different groups at the same time points. I EdU immunostaining illustrates the proliferation of HBECs in the indicated groups (n = 4; scale bar, 25 μm). J Tube formation assays on HBECs in the indicated groups (n = 4; scale bar, 100 μm). K Western blots and analysis showing the expression levels of p-eNOS and VEGF in the indicated HBEC groups. Values are the mean ± SD; one-way ANOVA.

To further confirm that PI3K/Akt-eNOS-VEGFA is a downstream target of ITGA6 in regulating endothelial cell function, we utilized the PI3K-Akt inhibitor, PI3K-IN-1, along with a lentivirus vector encoding the full-length of ITGA6 to overexpress ITGA6 in HBECs. Phalloidin staining revealed that PI3K-IN-1 disrupted cytoskeletal organization, similar to that in ITGA6 knockdown HBECs (Fig. S2J). Although the overexpression of ITGA6 promoted extensive actin fiber formation, the application of PI3K-IN-1 abolished this effect (Fig. S2J). In addition, consistent with the knockdown of ITGA6, PI3K-IN-1 significantly inhibited the migratory capacity, cell proliferation, and tube formation of HBECs (Figs 5H–J and S2J–L). In contrast, the overexpression of ITGA6 significantly enhanced these cellular activities; however, PI3K-IN-1 negated the effects of ITGA6 overexpression (Fig 5H–J and S2J–L). Furthermore, the overexpression of ITGA6 markedly increased the levels of p-eNOS and VEGFA, while PI3K-IN-1 exhibited contrasting phenotypes and counteracted these effects. Collectively, these results suggest that endothelial cell ITGA6 regulates angiogenesis through PI3K/Akt-eNOS-VEGFA signaling.

Overexpression of Itga6 Rescues the Vascular Remodeling After Stroke

To address the vascular impairments following ischemic stroke, especially in the absence of endothelial cell-specific Itga6, we examined the therapeutic benefits of increasing Itga6 expression. This approach entailed utilizing an adeno-associated virus BI30 (AAV-BI30) vector encoding Itga6 (AAV-BI30-Itga6), which can overexpress Itga6 specifically in endothelial cells [30], with the hypothesis that it could promote angiogenesis and enhance the function of endothelial cells, ultimately restoring cerebrovascular integrity and perfusion in Itga6ECKO mice. The vector was administered via tail vein injection into Itga6ECKO mice at 2 days following tamoxifen administration, followed by MCAO surgery 14 days later and functional analysis 21 days later (Fig. 6A). ITGA6 expression was successfully elevated in the brain endothelial cells of the AAV-BI30-Itga6 group compared with the control group (Fig. 6B). Crucially, the intervention notably reduced the infarct size and improved the neurological impairments in Itga6ECKO mice (Fig. 6C, D). Assessments of motor function showed it was enhanced in the AAV-BI30-Itga6 group, as demonstrated by the rotarod test and PCT analysis (Fig. 6E, F). In addition, overexpression of Itga6 significantly increased survival rates (Fig. 6G). TUNEL assays indicated reduced neuronal death in the peri-infarct area of the AAV-BI30-Itga6 group (Fig. 6H). This improvement suggests that reintroducing ITGA6 in endothelial cells is crucial for neurological recovery after a stroke, potentially through mediating vascular remodeling.

Fig. 6.

Fig. 6

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Rescue of microvascular remodeling via Itga6 overexpression after stroke. A Experimental timeline for tail vein AAV injection for Itga6 overexpression specifically in endothelial cells, followed by MCAO surgery and functional analysis in Itga6ECKO mice. B Western blots and quantification confirm the overexpression of Itga6 in the AAV-BI30-Itga6 group (n = 4). C TTC staining and quantification of the infarct area post-stroke in the AAV-BI30-Itga6 group and control group (n = 5). D Statistical analysis of neurological function score in AAV-BI30-Itga6 and control groups (n = 8). E Rotarod test showing the latency of AAV-BI30-Itga6 and control mice (n = 8). F PCT test showing the iTurn and TD of AAV-BI30-Itga6 and control mice (n = 8). G Kaplan-Meier survival curves for AAV-BI30-Itga6 and control mice subjected to MCAO surgery (n = 15). H TUNEL assays showing the numbers of apoptotic neural cells in brain sections of AAV-BI30-Itga6 and control mice (n = 6; scale bar, 40 μm). I Immunostaining of CD31 and quantification of vascular length and vascular area in brain sections from AAV-BI30-Itga6 and control mice at day 5 post-stroke (n = 6; scale bar, 50 μm). J Lectin perfusion assay and quantification of vascular perfusion in AAV-BI30-Itga6 and control mice (n = 6; scale bar, 25 μm). K Representative images and quantification of EdU+ endothelial cell proliferation post-stroke (n = 6; scale bar, 25 μm). L Representative images and quantification of vessel permeability showing the leakage of dextran-2000 from the microvasculature in AAV-BI30-Itga6 and control mice ( n = 6; scale bar, 25 μm). M Immunostaining of CD31 and PDGFRβ and pericyte coverage in brain sections at day 5 post-stroke in AAV-BI30-Itga6 and control mice (n = 6; scale bar, 25 μm). N Immunostaining of CD31 and CD13 and pericyte coverage in brain sections at day 5 post-stroke in AAV-BI30-Itga6 and control mice (n = 6; scale bar, 25 μm). O Western blots and analysis showing the changes in the PI3K-Akt-VEGFA axis in Itga6-overexpressing endothelial cells versus the control group (n = 4). Values are the mean ± SD; unpaired, 2-tailed Student’s t test.

To assess the impact of Itga6 overexpression in endothelial cells on cerebrovascular remodeling following a stroke, we conducted a series of immunostaining experiments. Our results revealed that the vascular area and length were significantly increased in Itga6ECKO mice upon Itga6 augmentation (Fig. 6I). In addition, the lectin perfusion assay demonstrated a marked increase in perfused microvessels in the AAV-BI30-Itga6 group (Fig. 6J). Further investigation into endothelial cell proliferation following MCAO surgery revealed a significant increase in proliferating endothelial cells in the AAV-BI30-Itga6 group compared to the control group (Fig. 6K). Functional assessments of microvascular integrity were carried out by administering dextran-FITC (MW, 2000 kDa) via tail vein injection. Itga6-overexpressing mice exhibited decreased microvascular permeability, demonstrated by reduced leakage of dextran-FITC in comparison to controls (Fig. 6L). Immunostaining of tight junctions provided additional support for this discovery, demonstrating that the overexpression of Itga6 notably elevated the protein levels of Claudin5, Occludin, and ZO1 on microvessels in the peri-infarct area (Fig. S3A–C). Western blot analysis further validated these results (Fig. S3D). Moreover, mice in the AAV-BI30-Itga6 group exhibited enhanced pericyte coverage, as indicated by PDGFRβ and CD13 immunostaining (Fig. 6M, N). In addition, overexpression of Itga6 significantly increased the levels of p-PI3K (p85, p55), p-Akt (Thr304), p-eNOS, and VEGFA in the endothelial cells sorted from the infarct area (Fig. 6O). These findings collectively show that the overexpression of Itga6 is significantly involved in vascular remodeling and strengthening cerebrovascular impermeability following an ischemic insult. This may provide a potential therapeutic approach for alleviating cerebrovascular dysfunction caused by ischemia.

Discussion

While the role of endothelial cells in cerebrovascular integrity is well documented [31, 32], the molecular mechanisms of angiogenesis remain incompletely understood. Our study focuses on the role of endothelial cell Itga6 in cerebral microvascular remodeling after stroke and demonstrates that endothelial cell Itga6 is crucial for neovascularization following brain ischemia, promoting angiogenesis, maintaining vascular integrity, and ensuring adequate pericyte coverage. In addition, evidence is presented suggesting that ITGA6 regulates vascular remodeling through the intracellular PI3K-Akt signaling pathway. Crucially, this study emphasizes the potential therapeutic importance of targeting Itga6 for the treatment of ischemic stroke and other brain diseases characterized by microvascular dysfunction.

Recuperative therapeutic strategies for stroke are focused on neuroprotection and neuroregeneration, but most of the strategies that have been clinically tested fail to show benefit in stroke patients [33, 34]. Ischemia-induced angiogenesis is an essential event to improve capillary blood flow surrounding an ischemic infarct that promotes regenerative processes and neuroplasticity [29, 35, 36]. Unfortunately, this process is still incompletely understood and therefore not exploited for therapeutic purposes. In this study, we utilized genetical conditional knockout Itga6 mice and an AAV-BI30-Itga6 enabling the specific overexpression of Itga6 in endothelial cells to demonstrate the important role of Itga6 in vascular remodeling, highlighting that Itga6-mediated increased neovascularization improved neural function and enhanced survival rates following MCAO surgery. In addition, complementary in vitro studies using shRNA-mediated ITGA6 knockdown in HBECs revealed significant impairment of proliferative capacity, migratory potential, and tube formation ability- phenotypic alterations that mechanistically mirror the pathological vascular deficits observed in Itga6 ECKO mice. These findings suggest that Itga6 is a novel potential target for ischemic stroke treatment.

The importance of Itga6 in barrier function is best illustrated by the finding that global murine knockout of Itga6 results in perinatal mortality caused by defective epidermal integrity [37]. This manifests as a skin blistering condition that is analogous to the human disease junctional epidermolysis bullosa [37]. In light of the essential role of Itga6 in maintaining epidermal integrity, combined with our results that endothelial cells are the primary sources of ITGA6 in cerebral vessels and a significant increase in ITGA6 expression when exposed to ischemic insult [17], strongly suggest its particular relevance for the regulation of post-stroke microvascular remodeling. Our findings reveal that the inactivation of ITGA6 in endothelial cells affects endothelial cell proliferation, neovessel integrity, and perfusion. Furthermore, pericyte coverage was significantly decreased in Itga6ECKO mice. These findings represent the first evidence of active upregulation of ITGA6 and its involvement in ischemic stroke.

Previous studies have described the regulation of PI3K-Akt signaling by integrin subunits [38–40]. The PI3K/Akt-eNOS-VEGFA axis plays a critical role in regulating vascular remodeling [41–45]. In line with this, Itga6ECKO mice exhibited inactivation of the PI3K/Akt-eNOS-VEGFA signaling pathway, which was also found in ITGA6-knockdown HBECs in vitro. The knockdown of ITGA6 significantly inhibited the migration, proliferation, and tube formation of HBECs, whereas ITGA6 overexpression markedly promoted these processes, accompanied by increased expression of p-eNOS and VEGFA. Notably, the effects of ITGA6 overexpression were abolished by a PI3K-Akt axis antagonist. These results indicated that ITGA6 regulates endothelial cell function through the PI3K/Akt-eNOS-VEGFA axis. In addition, VEGFA not only influences angiogenesis but also affects neurons and neural stem cells to promote recovery from ischemic stroke [46]. Therefore, the role of ITGA6 in the crosstalk between endothelial cells and other types of neural cells warrants further investigation. Although we found significant changes in the ITGA6 knockdown of HBECs, there were some limitations to our study. Specifically, we conducted the in vitro experiments under normal oxygen levels, which may not accurately replicate the conditions associated with post-stroke angiogenesis. Therefore, additional experiments investigating the role of hypoxia in regulating ITGA6 expression, as well as the influence of ITGA6 on HBEC function under hypoxic conditions, would be valuable. Collectively, these results establish a novel connection between endothelial ITGA6, the PI3K/Akt-eNOS-VEGFA signaling pathway, and vascular remodeling in cerebral vessels. These findings also suggest that targeting this pathway could potentially promote neovascularization in patients suffering from cerebral ischemia.

In this study, we found that Itga6ECKO mice exhibited reduced expression of tight junctions, although the underlying mechanism remains unclear. Various studies have reported a major involvement of the PI3K-Akt signaling pathway in the regulation of tight junction expression. Luo et al. demonstrated that triterpene celastrol ameliorates oxygen-glucose deprivation-induced disruption of endothelial barrier assembly by inducing tight junction proteins through activation of the PI3K/Akt/mTOR pathway [47]. Similarly, Hu et al. found that Panax notoginseng saponins protect cerebral microvascular endothelial cells against oxygen-glucose deprivation/reperfusion-induced barrier dysfunction via activation of the PI3K/Akt/Nrf2 signaling pathway [48]. In addition, Zhang et al. reported that DT-13 ameliorates TNF-α-induced vascular endothelial hyperpermeability through the Src/PI3K/Akt signaling pathway and non-muscle myosin IIA [49]. Despite these indications, further investigation is warranted to determine whether Itga6 indeed regulates tight junction expression through PI3K-Akt signaling.

This comprehensive study highlights the significant therapeutic potential of Itga6 in improving recovery from ischemic stroke, especially through targeted overexpression in endothelial cells. It not only improves perfusion and the integrity of the microvasculature in the peri-infarct area but also provides a promising approach to mitigating the effects of stroke, positioning Itga6 as a crucial element in vascular remodeling strategies.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 799 KB) (799.2KB, pdf)

Acknowledgments

This work was supported by grants from the Scientific Research Foundation for Young Doctors of the Second Affiliated Hospital, Army Medical University (2022YQB041), and the Chongqing Natural Science Foundation (CSTB2024NSCQ-MSX0924 and CSTB2024NSCQ-MSX0909).

Conflict of interest

The authors declare that there is no conflict of interest in the research, authorship, or publication of this article.

Footnotes

Bing-Qiao Wang, Yang-Ying Duan, and Mao Chen have contributed equally to this work.

Contributor Information

Rui Xu, Email: xurui007@tmmu.edu.cn.

Chun-Mei Duan, Email: duancm0629@tmmu.edu.cn.

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