Up-regulation of caveolin 1 mediated by chitosan activates Wnt/ β-catenin pathway in chronic refractory wound diabetic rat model.

Diabetes mellitus (DM) can be implicated in the perturbations of vascular integrity and the dysfunction of angiogenesis. Chitosan has the advantage of promoting the vascular endothelial cell proliferation. However, the molecular mechanism of action in the promotion of wound healing by chitosan derivatives is still debated. In the current study, DM with chronic wound (CW) model rats were prepared and treated with chitosan. Vascular endothelial cells isolated from granulation tissues were conducted by RNA sequencing. Two thousand three hundred and sixteen genes were up-regulated, while 1,864 genes were down-regulated after chitosan treatment compared to CW group. Here, we observed that caveolin 1 (CAV1) was highly expressed induced by chitosan. Furthermore, we observed that CAV1 knockdown could compromise the activation of Wnt pathway by reduction of β-catenin in rat aortic endothelial cells (RAOECs) and brain endothelium four cells (RBE4s). Moreover, we determined a direct interaction between CAV1 and β-catenin by IP assay. The C-terminus of CAV1 and β-catenin (24 to 586 amino acids) contributed to the interaction of these two proteins. Finally, the protein docking analysis indicated that the fragments of β-catenin (253-261 'FYAITTLHN' and 292-303 'KFLAITTDCLQI') might have affected the structure by CAV1 and facilitated the resistance to degradation. Taken together, our study demonstrates that chitosan can up-regulate CAV1 expression, and CAV1 can interact with β-catenin for promotion of canonical Wnt signaling pathway activity. Our results deepens the molecular mechanism of the Wnt pathway in vascular endothelial cells and is beneficial to developing new targets to assist in enhancing the pharmacological effect of chitosan on wound healing and angiogenesis against DM.

Introduction

As a chronic condition, diabetes mellitus (DM) has become a major public health problem worldwide. In 2017, the International Diabetes Federation reported that 425 million people suffered from diabetes and 5 million people died of diabetes, with more than 800 million USD for diabetic care [1]. DM2 is associated with numerous comorbidities, including, but not limited to, cardiovascular disease, stroke, chronic renal failure, peripheral neuropathy, and diabetic skin wounds or ulcerations [2].

Angiogenesis is an event induced by almost all injury and repair processes. Angiogenesis in normal wound healing relies on a delicate balance between vessel proliferation, maturation and quiescence, but is perturbed by diabetic disease state. A pro-angiogenic imbalance in this process leads to abnormal growth of new blood vessels. Diabetic hyperglycemia, particularly in DM2, has been implicated in the perturbations of vascular integrity [3]. Endothelial cells exposed to elevated blood glucose for extended periods of time have been shown to become dysfunctional, leading to integrity loss and increased susceptibility to apoptosis, detachment, and circulation into the bloodstream [4]. On the contrary, another foreseen DM complication is the low-grade chronic inflammation involved in multiple organs. Evidence suggests that diabetes is accompanied by a pro-inflammatory state [5]. A large number of inflammatory hallmarks such as high sensitivity C-reactive protein (hsCRP), toll-like receptors (TLRs), oxidative stress and Nuclear Factor-kB (NF-kB), and advanced glycation endproducts (AGEs) and their chief cell-surface receptor, RAGE (receptor for AGEs) produced by different cells compose the angiogenic network, and enhance the pro-angiogenesis response in wound healing [6]. Although the dual effects caused by DM on angiogenesis are complicated, acknowledgment of the coordination of multiple regulatory factors is beneficial to develop more targeted drugs for therapy of wound repair.

Extracellular matrix (ECM) served as a deposit for growth factors, proteases, cytokines and chemokines and plays a crucial role in orchestrating and guiding cell phenotype, adhesion, migration and proliferation among all factors in skin wound. ECM proteins also provide the mechanical support necessary for angiogenesis in the newly formed granulation tissue [7]. Chitosan and its derivatives are a natural cationic polysaccharide consisting of (1-4)-2-amino-2-deoxy-β-d-glucan, is the partially to fully deacetylated form of chitin [8]. Chitosan has attracted great attention owing to the properties beneficial for application to wound healing. Chitosan can enlarge the effect of inflammatory cells, macrophages, and fibroblasts, hence boosting the inflammatory phase and prolonging the proliferative phase in wound healing process [9]. Moreover, chitosan has an ability to regulate granulation tissue formation and angiogenesis, assuring the correct deposition of collagen fibers and further enhancing the correct repair of injured dermal tissue [10]. Meanwhile, the hemostatic and analgesic properties are attributed to the positive charge of chitosan. Nevertheless, the mechanism of action in the promotion of wound healing by chitosan derivatives is still debated but is suggested to depend on the type of functionalization.

In this study, we investigated the molecular role of chitosan in wound healing against DM. RNA sequencing (RNA-seq) was conducted to study RNA profiles of chronic refractory wound DM rat models with chitosan treatment. The expression of the candidate gene FAM83F and activity of the WNT signaling pathway were found to be associated with chitosan. Our results determined a novel therapeutic mechanism of chitosan on promoting wound healing in DM.

Materials and methods

Experimental animals

Adult male Sprague-Dawley rats (200–220 g) acquired from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) were enrolled in this study. The study was approved by the Heilongjiang University of Traditional Chinese Medicine, and all animal protocols were executed following the guidelines accordingly. All rats were housed in a controlled environment of 53% humidity (23 ± 2°C) in a 12/12 h dark/light cycle with free access to a high-calorie diet (60% normal fodder plus 15% animal fat, 20% sucrose and 5% cholesterol), and received intraperitoneal injection with 50 mg/kg streptozotocin (STZ) (EMD Millipore, USA) per month for total 3 months. Blood glucose stably more than 16.7 mmol/L and body weight significantly less than normal control (n = 20) were considered as diabetic model. DM rats were then anesthetized by 10% isopentobarbital sodium 1 mL/100 g and removed the back hair at the area of 4 cm × 4 cm, and cut away the skin to the fascia as chronic wound model. 0.5 g chitosan antibacterial gel (Hubei Puai Medication Co., Ltd, China) was evenly applied to the wound area and changed every 2 days. The wound was covered and bandaged for 2 weeks. After that, wound and granulation tissues were harvested for the consequent experiments.

HE staining

The granulation tissue was fixed in 4% formaldehyde solution, dehydrated with gradient alcohol, paraffin embedded, cut into slices of 5 µm and HE stained. The pathological changes of fibrosis, inflammatory infiltration, vessel angiogenesis and cell regeneration were observed and captured images using Olympus BX-51 light microscope with 200x amplification.

Flow cytometric assay

Isolated of vascular endothelial cells was conducted as previously described [11]. Granulation containing 1 × 107 cells was cut into small pieces and digested by 1% trypsin for 30 min at 37°C, then washed with PBS (pH 7.2) for three times and, respectively, incubated with CD31 (Cat. No. MHCD3101, Thermo Fisher Scientific), CD45 (Cat. No. MHCD4518, Thermo Fisher Scientific) and GP38 (Cat. No. 12–5381-82, eBioscience, USA) in the dark on ice for 2 h, then washed with PBS for three times, and incubated with 1.5 mL PBS containing 100 μg/mL RNase A (Sangon) in the dark on ice for 30 min. The suspension was filtered through a 45 µm mesh and then subjected to FACS Calibur FCM (BD Biosciences, USA). CD45−/CD31+/GP38− cells were sorted for the consequent experiments.

RNA sequencing (RNA-seq)

Wound and granulation tissues containing 1 × 106 cells were stored in 1 mL TRIZOL (Thermo Fisher Scientific, USA) and ground in liquid nitrogen. Next, 200 μL chloroform was added, and the cells were fully mixed and centrifuged at the highest speed at 4°C for 10 min. The supernatant was transferred into a new tube, isopropanol was added to the same volume, and cells were centrifuged at the highest speed at 4°C for 10 min. The precipitate was washed with 75% cold ethanol and dissolved in DEPC water. The concentration and quality of RNA were measured using a Nanodrop 2000 (Thermo Fisher Scientific) and an Agilent Bioanalyzer 2100 (Agilent, USA). A total of 5 μg of RNA from each group was used for library preparation using the NEBNext Ultra-Directional RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s instructions and sequenced on an Illumina HiSeq platform.

The raw data were trimmed for adaptors, and low-quality reads were filtered out using Trimmomatic [12]. The quality of the clean reads was checked using FastQC [13]. Next, clean reads were aligned to the latest rat genome assembly Rnor6.0 using Hisat2 [14]. The transcripts were assembled, and the expression levels were estimated with FPKM values using the StringTie algorithm with default parameters [15]. Differential mRNA and lncRNA expressions among the groups were evaluated using the R package Ballgown [16], and the significance of the differences were computed using the Benjamini & Hochberg (BH) p-value adjustment method. Gene annotation was performed using the Ensembl Genome Browser database (http://www.ensembl.org/index.html). The R package ClusterProfiler was used to annotate the DEGs using Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [17].

Cell culture

Rat aortic endothelial cells (RAOECs) and brain endothelium four cells (RBE4s) purchased from Coweldgen Co., Ltd. (Shanghai, China) were cultured within Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) containing 20% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 100 μg/mL penicillin and streptomycin. CAV1 siRNA (5’-UUGCCUGUGUAAGUUGUACUGU-3’) was synthesized from GenePharma (Shanghai, China), and transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s instructions. Different truncated CAV1 or β-catenin vectors with 3x Flag tag were constructed by GenePharma and transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Immunoprecipitation (IP)

IP protocol was followed as previously described [18]. 1 × 107 cells were disintegrated using Pierce IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) (Thermo Fisher Scientific) containing 1% protease inhibitor cocktail and 1% PMSF on ice for 30 min, then centrifuged to harvest the supernatant, and mixed with 1 μg β-catenin (Cat No. 51067-2-AP, Proteintech Group, USA), CAV1 (Cat No. 16447-1-AP, Proteintech Group), Flag (Cat No. AE063, Abclonal, China) or IgG Rabbit IgG antibody, and 40 μl flurry IgA beads (Thermo Fisher Scientific) for gently rotating overnight at 4°C. Immunoprecipitates were washed with IP buffer (20 mM HEPES [pH 7.9], 350 mM NaCl, 0.1% NP-40, 1 mM DTT, 0.2 mM PMSF, 2 mg/mL leupeptin, and 2 mg/mL aprotinin) for three times and resuspended by 20 μL lysis buffer. The protein lysate was subjected to a Western blot assay.

Western blot (WB) assay

The protein lysate was then analyzed by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, USA). The membrane was blocked with 5% fat-free milk in PBST for 30 min, followed by incubation overnight at 4°C with final dilutions of primary antibodies against β-catenin (Cat No. 17565-1-AP, Proteintech Group, USA), CAV1 (Cat No. A19006, Abclonal), GSK3β (Cat No. 12456, CST, Beverly, USA), p-GSK3β (Cat No. 5558, CST), VEGFB (Cat No. A2132, Abclonal), FGF1 (Cat No. A5900, Abclonal), PDGFB (Cat No. A1195, Abclonal), Flag (Cat No. AE005, Abclonal) or GAPDH (Cat No. A19056, Abclonal). Next, the membrane was washed three times and then incubated with HRP-conjugated secondary antibodies (Proteintech Group). Membranes could be stripped using stripping buffer (Cat No. ab270550, Abcam) at 52°C for 30 min via gently shaking and washed by PBST, then blocked by 5% fat-free milk for 2 h, and re-incubated by antibodies at 4°C overnight. The blotting bands were developed with ECL plus immunoblotting detection reagents (Thermo Fisher Scientific) using UVP Chemstudio Plus System (Analytik Jena, Germany), and captured using Image J.

Immunofluorescence assay

Cells were fixed within 4% solution of paraformaldehyde and washed by phosphate buffer saline (PBS), then permeabilized with 0.1% Triton-X-100 (Sigma) and blocked with 0.5% horse serum in PBS. Immunostainings of samples were performed using β-catenin antibody (1:200) overnight at 4°C. After washing with PBS for 4 times (5 min per time), the donkey anti-rabbit secondary antibodies (1:20000; Jackson ImmunoResearch) were used to incubate for 30 min at room temperature, following further incubation with DAPI for 15 min, washed with PBS for 4 times. After drying in the air, slices were dropped and coated with mounting medium and coated with cover glasses. The positive staining was statistically analyzed using Image J.

Statistical analysis

Data are presented as the mean ± standard deviation for three independent experiments. The differences in values were analyzed using one-way ANOVA. Statistical significance was set at p-value less than 0.05.

Results

To investigate the effects of chitosan on angiogenesis in diabetic wound healing process, we first established the rat model of hyperglycemia and skin injury with chitosan treatment, and performed RNA-seq to study the differential expressed genes in vascular endothelium isolated from granulation, and focused on one candidate up-regulated gene CAV1 as well as the activated Wnt signaling pathway induced by chitosan. Finally, we determined a substantial protein–protein interaction between CAV1 and β-catenin, and concluded that chitosan could enhance the activity of Wnt pathway via CAV1 for angiogenesis against DM.

The therapeutic effect of chitosan on angiogenesis of chronic refractory wound healing against DM

Groups of normal skin (NS), diabetic but not wounded skin (DS), diabetic wound (DW), and diabetic wound treated with chitosan (DWC) (ten rats in each group) were examined the ability of wound self-repair after 2 weeks of model preparation. HE staining indicated a phenotype of numerous infiltrated inflammatory cells, visible necrotic zones, bleeding, sparse fibroblasts, as well as a few narrow blood vessels in DW. While chitosan could enhance the proliferation of fibroblasts in granulation area with a large number of new blood vessels (Figure 1(a)). Moreover, the overall expressions of vascular endothelial growth factor B (VEGFB), fibroblast growth factor 1 (FGF1) and platelet-derived growth factor subunit B (PDGFB) were all elevated in wound area of DWC compared to DW (Figure 1(b)). Taken together, the therapeutic effect of chitosan on angiogenesis was confirmed in our system.

Figure 1.

The therapeutic effect of chitosan on wound healing in chronic wound model of DM rat. (a) HE staining of the wound area in different conditions of animal model. The blue dots represent infiltration of inflammatory cells. Large circles represent the cross section of vessel. Images are captured with 200x amplification. (b) Protein expressions of VEGFB, FGF1 and PDGFB in granulation tissues of different conditions of animal model shown by immunoblotting (left) and statistical graphic (right). NS: normal skin; DS: diabetic but not wounded skin; DW: diabetic wound; DWS: diabetic wound treated with chitosan; VEGFB: vascular endothelial growth factor B; FGF1: fibroblast growth factor 1; PDGFB: platelet derived growth factor subunit B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. The experiments are repeated by three individual experiments. ‘*’ and ‘**’ mean the significant difference with p-value less than 0.05 and 0.01 by the comparison between DW and DWS. normal skin (NS), diabetic but not wounded skin (DS), diabetic wound (DW), and diabetic wound treated with chitosan (DWC.

Chitosan-mediated transcriptome changes in vascular endothelial cells at wound area

To investigate the molecular role of chitosan in proliferation of vascular endothelial cells, CD45−/CD31+/GP38− cells [11] isolated from granulation were conducted RNA-seq to search the potential targets of vascular endothelial cells responding to chitosan (Figure 2(a)). We observed that a total of 722 genes were up-regulated, while 709 genes were down-regulated in DWC compared to DW (log2FC > 1 or < −1, p < 0.05) (Figure 2(b), Table S1). Here, we observed that VEGFB (log2FC = 3.020, p = 0.007), FGF1 (log2FC = 1.467, p = 0.033), PDGFB (log2FC = 2.042, p = 0.019), matrix metallopeptidase 7 (MMP7) (log2FC = 1.852, p = 0.017), caveolin 1 (CAV1) (log2FC = 2.414, p = 0.014), bone morphogenetic protein 4 (BMP4) (log2FC = −3.868, p = 0.043) and SMAD family member 4 (SMAD4) (log2FC = −3.169, p = 0.024) were all significantly changed induced by chitosan (Figure 2(c)). Additionally, GO analysis indicated that multiple functions were connected with smooth muscle cell proliferation, blood vessel endothelial cell migration, inflammatory response and cell apoptosis. The main signaling pathways involved included Wnt/β-catenin, MAPK, and PI3K/Akt signaling pathway was closely associated with chitosan-mediated angiogenesis (Figure 2(d)). Overall, chitosan changed the expression of a large number of target genes in granulation through numerous crucial signaling pathways.

Figure 2.

The transcriptome of vascular endothelial cells affected by chitosan. (a) Flow cytometric assay shows the isolation from cell suspension from granulation tissues by CD45, CD31 and GP38. CD45−/CD31+/GP38− population is considered as vascular endothelial cells. (b) Heatmap shows the differential expressed genes (DEGs) (log2FC > 1 or < −1, p < 0.05) among different conditions of animal model. (c) Scatter plot shows the DEGs between groups with and without chitosan treatment. Dots with different color represent genes that significantly up-regulated in DM+CW+CTS (Red), significantly up-regulated in DM+CW (Blue) and no significance (Grey). (d) Bubble chart shows the enriched functions associated with DEGs between DM+CW and DM+CW+CTS by GO analysis. NC: negative control group; DM: diabetes mellitus group; CW: chronic wound group; CTS: chitosan group; VEGFB: vascular endothelial growth factor B; FGF1: fibroblast growth factor 1; PDGFB: platelet derived growth factor subunit B; MMP7: matrix metallopeptidase 7; CAV1: caveolin 1; BMP4: bone morphogenetic protein 4; SMAD4: SMAD family member 4; GO: gene ontology.

CAV1 is essential for activation of Wnt signaling pathway by chitosan

Previous studies have reported that CAV1 can facilitate the activation of canonical Wnt signaling pathway in a variety of cancers [19,20]. We suspected that CAV1 might bridge between chitosan and Wnt signals activity. To this end, RAOEC and RBE4 cells were treated with 1/10 volume of the chitosan solution (1 mg/mL chitosan in acetic acid) for 24 h [21], and CAV1 mRNA was further interfered using siRNA in RAOEC and RBE4 cells. We observed that the expression (Figure 3(a,b)) and the nucleus-localization of β-catenin (Figure 3(c,d)) could be indeed elevated by chitosan, but obviously compromised by CAV1 knockdown, suggesting that CAV1 might participate in chitosan-mediated activation of Wnt/β-catenin pathway in vascular endothelial cells in vitro. Taken together, we determined that CAV1 played an important role in modulating the effect of chitosan on Wnt/β-catenin pathway in vascular endothelial cells.

Figure 3.

Effect of CAV1 on β-catenin in RAOEC and RBE4 cells. WB assay and analysis show the expression of β-catenin and GSK3β phosphorylation affected by chitosan and CAV-1 knockdown in RAOEC (a) and RBE4 (b) cells. IF assay shows the Subcellular localization of β-catenin affected by chitosan and CAV-1 knockdown in RAOEC (c) and RBE4 (d) cells. RAOEC cells: rat aortic endothelial cells; RBE4 cells: rat brain endothelium 4 cells; NC: negative control group; CTS: chitosan group; KD: knockdown; CAV1: caveolin 1; β-catenin: catenin beta 1; GSK3β: glycogen synthase kinase 3 beta; p: phosphorylated; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. ‘*’ and ‘**’ mean the significant difference with p-value less than 0.05 and 0.01 by the comparison between CTS and NC, ‘#’ and ‘##’ mean the significant difference with p-value less than 0.05 and 0.01 by the comparison between with and without chitosan.

The interaction between CAV1 and β-catenin

Previous study has indicated the interaction between CAV1 and β-catenin in zebra fish [22]. However, whether this effect is also conserved in mammalian cells is unclear. In our results, we noticed that the accumulation of β-catenin could be blocked by CAV1 knockdown, although glycogen synthase kinase 3 beta (GSK3β) was still inactivated, suggesting that CAV1 might play an enhanced role in β-catenin beyond GSK3β. To verify the assumption that CAV1 could directly interact with β-catenin, co-IP was conducted to verify the interaction between CAV1 and β-catenin in RAOEC cells. It was confirmed that the binding affinity between these two proteins was strengthened induced by chitosan treatment compared to control (Figure 4(a,b)). Moreover, IP assay of the interaction between ectopic truncating β-catenin (2–23, 24–234, 235–586, 587–780 respective deficiency) and CAV1 (2–44, 45–142 and 143–177 respective deficiency) with Flag tag suggested that the key binding domains of CAV1 and β-catenin for binding with each other (Figure 4(c,d)). We determined that the C-terminal domain of CAV1 and β-catenin (24 to 586 amino acids) contributed to the interaction of these two proteins. Protein docking analysis between CAV1 (PDB ID: 5IJP_A) and β-catenin (PDB ID: 2Z6G_A) by Z-DOCK system [23] showed that fragments of 253–261 ‘FYAITTLHN’ as well as 292–303 ‘KFLAITTDCLQI’ of β-catenin were two major fulcrums to support the protein binding surface (Figure 4(e)). Our data illustrated the crucial binding domains of the CAV1 and β-catenin.

Figure 4.

The interaction between CAV1 and β-catenin. Co-IP and WB assay shows the interaction between wild type β-catenin and CAV1 via β-catenin pull down (a) and CAV1 pull down in chitosan treated RAOEC cells. (b). (c) Co-IP and WB assay shows the interaction between truncation β-catenin and wild type CAV1 in RAOEC cells treated by chitosan. Δ1 to 4 mean the Flag tag-fused β-catenin with amino acids deletion of 2–23, 24–234, 235–586, 587–780. (d) Co-IP and WB assay shows the interaction between truncation CAV1 and wild type β-catenin in RAOEC cells treated by chitosan. ΔΔ1 to 3 mean the Flag tag-fused CAV1 with amino acids deletion of 2–44, 45–142 and 143–177. Red arrows indicate the difference of binding ability compared between RAOEC cells treated with and without chitosan. (e) Protein docking by Z-DOCK tool shows the core binding domain between β-catenin and CAV1. White: CAV1; Orange: 2–23 of β-catenin; Green: 24–234 of β-catenin; Blue: 235–586 of β-catenin; Yellow: 587–780 of β-catenin; Pink: two core fragments of β-catenin having direct interaction with CAV1.

Discussion

Although chitosan has been acknowledged as a promising substance for governing wound healing, inflammatory response, tissue regeneration and angiogenesis [24] as well as exerting it as nanoparticles for short RNA or drug delivery [25], the global changes of gene expression induced by chitosan are poorly understood. Our transcriptomic study provides a valuable resource for comprehensively understanding the molecular effects of chitosan on vascular endothelial cells particularly. From omics data, a series of novel target genes and signaling pathways responding to chitosan figure that the global gene expression profiles of vascular endothelial cells stimulated by chitosan are largely similar with the normal group (Figure 2(b)). The well-known genes including VEGFB, PDGFB, FGF1, BMP4, SMAD4 and MMP7 all display significant changes (Figure 2(c)) as well as a host of signaling pathways are involved in the process of angiogenesis (Figure 2(d)) compared between vascular endothelial cells with and without chitosan treatment.

CAV1 has been known as a pro-arteriogenic gene [26] and functions through Wnt signaling pathway [19,20] but is never connected with chitosan. However, the cyto-compatibility and cell proliferation enhanced by chitosan have been determined in previous non-vascular studies [27-29]. Therefore, CAV1 is supposed to bridge with chitosan and Wnt signals. Our observations of reduced β-catenin by CAV1 knockdown beyond GSK3β activity in RAOEC and RBE4 cells (Figure 3(a,b)) indicate that β-catenin level controlled by CAV1 in cytoplasm is independent of the phosphorylation-ubiquitination degradation process of β-catenin initiated by GSK3β.

β-Catenin is a typical member protein superfamily having Armadillo (ARM) repeats, which form a superhelix showing a long, positively charged groove arranged in triangular shape ARM repeat via the 42 residues of the central region [30,31]. ARM repeats are involved in strengthening the interaction and avoiding degradation. Multiple binding partners occupy the groove of the central β-catenin region and reject the recruitment of Cadherin 1 (E-cadherin), APC regulator of Wnt signaling pathway (APC) and T-cell factor (TCF)/Lymphoid enhancer-binding factor (Lef) [32]. In our results of co-IP, C-terminus of CAV1 is determined to interact with β-catenin of central region (Figure 4(c,d)). Due to the limitation of protein structural and biochemical data, we can only speculate the potential effect of CAV1 on β-catenin from the perspective of protein docking analysis (Figure 4(e)). Two given protein fragments of β-catenin (253–261 ‘FYAITTLHN’ as well as 292–303 ‘KFLAITTDCLQI’) are consistent with our IP data, and display more precise binding areas. These two areas, respectively, included in two adjacent ARMs (3rd and 4th ARM) appear to fold and approach each other when CAV1 combine to β-catenin. The room at this area likely becomes too narrow to accommodate APC [33]. Of course, this conjecture still needs to be validated in future studies.

Conclusion

In summary, our study demonstrates that chitosan can up-regulate CAV1 expression, and CAV1 can interact with β-catenin to promote the activity of canonical Wnt signaling pathway. The conclusion deepens the molecular mechanism of the Wnt pathway in vascular endothelial cells and is beneficial to developing new targets to assist in enhancing the pharmacological effect of chitosan on wound healing and angiogenesis against DM.

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