Activated mesangial cells induce glomerular endothelial cells proliferation in rat anti‐Thy‐1 nephritis through VEGFA/VEGFR2 and Angpt2/Tie2 pathway

Abstract Objectives We aimed to investigate the underlying mechanism of endothelial cells (ECs) proliferation in anti‐Thy‐1 nephritis. Materials and methods We established anti‐Thy‐1 nephritis and co‐culture system to explore the underlying mechanism of ECs proliferation in vivo and in vitro. EdU assay kit was used for measuring cell proliferation. Immunohistochemical staining and immunofluorescence staining were used to detect protein expression. ELISA was used to measure the concentration of protein in serum and medium. RT‐qPCR and Western blot were used to qualify the mRNA and protein expression. siRNA was used to knock down specific protein expression. Results In anti‐Thy‐1 nephritis, ECs proliferation was associated with mesangial cells (MCs)‐derived vascular endothelial growth factor A (VEGFA) and ECs‐derived angiopoietin2 (Angpt2). In vitro co‐culture system activated MCs‐expressed VEGFA to promote vascular endothelial growth factor receptor2 (VEGFR2) activation, Angpt2 expression and ECs proliferation, but inhibit TEK tyrosine kinase (Tie2) phosphorylation. MCs‐derived VEGFA stimulated Angpt2 expression in ECs, which inhibited Tie2 phosphorylation and promoted ECs proliferation. And decline of Tie2 phosphorylation induced ECs proliferation. In anti‐Thy‐1 nephritis, promoting Tie2 phosphorylation could alleviate ECs proliferation. Conclusions Our study showed that activated MCs promoted ECs proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 pathway in experimental mesangial proliferative glomerulonephritis (MPGN) and in vitro co‐culture system. And enhancing Tie2 phosphorylation could alleviate ECs proliferation, which will provide a new idea for MPGN treatment.

tion. MCs-derived VEGFA stimulated Angpt2 expression in ECs, which inhibited Tie2 phosphorylation and promoted ECs proliferation. And decline of Tie2 phosphorylation induced ECs proliferation. In anti-Thy-1 nephritis, promoting Tie2 phosphorylation could alleviate ECs proliferation.

Conclusions:
Our study showed that activated MCs promoted ECs proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 pathway in experimental mesangial proliferative glomerulonephritis (MPGN) and in vitro co-culture system. And enhancing Tie2 phosphorylation could alleviate ECs proliferation, which will provide a new idea for MPGN treatment.

| INTRODUC TI ON
Mesangial proliferative glomerulonephritis (MPGN) is characterized by mesangial cells (MCs) proliferation and increased extracellular matrix deposition. [1][2][3][4] As the most common pathological change of primary glomerulonephritis worldwide, MPGN leads to glomerular fibrosis and sclerosis and eventually results in end-stage renal disease. 5 Anti-Thy-1 nephritis is the most suitable animal model to study the specific pathogenesis of MPGN. 5,6 It shows that as MCs were damaged, glomerular capillaries proliferated and developed aneurysmal changes, 7 and as MCs continued to return to normal, the "aneurysm" gradually decreased. 8,9 Therefore, there might be cell-cell communication between MCs and ECs, which leads that mesangial areas and the capillaries change synchronously. And strategies to prevent such cell-cell communication would be useful in the resolution of vascular lesions in MPGN.
Vascular endothelial growth factor (VEGF) plays significant role in angiogenesis, tumour growth, development and atherosclerosis, which mainly produced by podocytes, distal tubules and collecting duct epithelial cells in normal human and rat kidneys. 10 As a dimer protein, it composed of 121, 165, 189 or 206 amino acids, respectively. VEGF 165 , VEGFA, is the most common isoform and it has been verified that VEGFA expressed in activated MCs of experimental MPGN. 11 TEK tyrosine kinase (Tie2) is expressed on the surface of ECs and is a tyrosine kinase receptor with two homologous domains, IG and EGF. 12 Angiopoietin1 (Angpt1) and angiopoietin2 (Angpt2) competitively bind to Tie2, which is important in angiogenesis and vascular inflammation. Angpt1 binds to Tie2 to promote Tie2 phosphorylation, thereby maintaining vascular structural integrity, protecting ECs from apoptosis and inhibiting the inflammatory response. [13][14][15] Angpt2 is expressed and stored in the Weibel-Palade body of ECs, interfering with the Angpt1/Tie2 signalling pathway. 16,17 VEGFA and Angpt2 both are considered as pro-angiogenesis factors. [18][19][20] Schmittnaegel et al pointed out that Angpt2 and VEGFA bispecialantibody (A2V) has a better therapeutic effect than single-antibody on metastatic breast cancer, pancreatic neuroendocrine tumour and melanoma. 21 And the phase 2 randomized trial of Faricimab (also a novel bispecific antibody targeting Angpt2 and VEGFA) showed that it could benefit diabetic macular oedema. 22 Even though many studies indicated that VEGFA and Angpt2 had synthetic effects in angiogenesis, 21,23 there are few observations of their roles in glomerular angiogenesis. Therefore, we tried to investigate whether VEGFA and Angpt2 participated in glomerular vascular lesions.
In this study, the rat anti-Thy-1 nephritis model and cell co-culture system were used to explore the specific mechanism of vascular lesions, and we try to alleviate ECs proliferation by blocking the cell-cell communication so as to provide new ideas for disease treatment.

| Animals and biochemical measurements
Wild-type Wistar rats (male, aged 6-8 weeks, 200-250 g) were purchased from the Animal Center of PLA General Hospital. All rats were kept in an environment with constant temperature (20°C), humidity (70%) and alternating day and night cycles. To explore the glomerular endothelial cells (ECs) proliferation in anti-Thy-1 nephritis, the rats were divided into control group (n = 7) and model group (n = 7). Anti-Thy-1 antibody (2.5 mg/kg) was administered by tail vein injection to sober rats in the model group to establish rat anti-Thy-1 nephritis. The control group was injected with saline solution. To evaluate the role of Vasculotide in ECs proliferation, we divided the rats into a control group (n = 7), model group (n = 7) and Vasculotide group (n = 7). The Vasculotide group was repeatedly injected with Vasculotide (3 µg/kg, ip Method and dose of administration reference before 24 ) on the 4th, 5th and 6th days, and model group injected with phosphate buffer saline (PBS). On the 7th day, the rats were sacrificed after sedated by pentobarbital. Glomeruli were isolated from kidney samples as described previously. 25 The experiments were performed according to the Guide for the Care and Use of Laboratory Animals (National Research Council of the USA, 1996).
The blood was collected in vacutainer tubes and centrifugated (100g for 15 minutes at 22°C). Then, the upper serum was left and stored at −80°C until used. Angpt2 in serum was measured by using Angiopoietin-2 Quantikine ELISA Kit (R&D Systems) according to manufacturer's instructions.

| Vasculotide
Tournaire et al 26 were the first to synthesize a short peptide HHHRHSF whose tetramer forming by affinity/biotin could combine with the extracellular part of Tie2 to induce Tie2 phosphorylation.
At present, 4-arm polyethanol glycol (average molecular weight 10 kDa) scaffold is used to replace the avidin/biotin complex. In our study, Vasculotide was synthesized by Shanghai Bootech Bioscience & Technology Co ( Figure S7).

| Periodic Acid-Schiff (PAS) staining
Rat kidneys were fixed in 10% formalin and dehydrated with gradient ethanol. The tissue was embedded in paraffin and sectioned into 2-4 µm slices. Then, the tissue slices were stained with PAS. The mesangial hypercellularity index was used to assess the severity of hypercellular lesions as follows: 0, no hypercellularity, with fewer than three cells per mesangial area; 1, mild focal hypercellularity and <50% of glomeruli with three to five cells per mesangial area; 2, diffuse mild hypercellularity or prominent focal segmental hypercellularity with more than five cells per mesangial area; and 3, prominent, diffuse global hypercellularity. Twenty glomeruli were selected for each section.

| Cell co-culture
Transwells were purchased from Corning and used for co-culturing.
MCs were seeded in transwell inserts and were cultured alone or stimulated with platelet-derived growth factor BB (PDGF-BB, to activate MCs) treatment for 24 hours and were then washed with fresh medium followed by co-culturing with ECs for another 24 hours.

| Real-time quantitative PCR (RT-qPCR)
TRIzol reagent (Invitrogen) was used to lyse the cells and extract the RNA. The ReverTra Ace qPCR RT kit (Toyobo) reverse-transcribed the RNA into cDNA. RT-qPCR was performed using SYBR Select master mix (Life Technologies) and an RT-qPCR detection system (ABI).
The 18s gene was used as an internal control, and the expression of each target gene was calculated by the 2 −ΔΔCT method. All experiments were repeated three times.

| ELISA
The ECs medium was collected after ECs co-cultured with MCs and centrifuged (100 g for 15 minutes at 4°C). Then, the supernatant was left and stored at −80°C until used. Angpt2 in medium was measured by using Angiopoietin-2 Quantikine ELISA Kit (R&D Systems) according to manufacturer's instructions.

| Statistical analysis
All data analyses were performed using GraphPad (version 5.0; GraphPad). The data are expressed as the means ± SD. Comparisons among the groups were conducted using Student t test. A value of P < .05 was considered significant.

| On the 7th day of anti-Thy-1 nephritis, ECs proliferated and the expressions of VEGFA and Angpt2 increased
The rat anti-Thy-1 nephritis model was successfully established, according to increased urine albumin/creatinine ratio (UACR) in model group ( Figure S1A). PAS staining results showed that the number of cells in the glomeruli increased significantly on the 7th day after the model was established ( Figure 1A). Moreover, rat endothelial cells antigen-1 (RECA-1) and proliferative cells nuclear antigen (PCNA) immunohistochemical staining ( Figure 1B,C) and immunofluorescence co-staining ( Figure 1D) results showed that the number of glomerular endothelial cells (ECs) increased. Western blot results showed that expression of CD34 (protein marker of ECs) was also increased significantly in the model group ( Figure S1B). These results indicated that there was abnormal ECs proliferation on the 7th day after the model was established. Western blot and RT-qPCR results showed that the expressions of vascular endothelial growth factor A (VEGFA) and angiopoietin2 (Angpt2) in glomeruli of the model group were increased ( Figure 1E,F). Moreover, serum Angpt2 levels of model group were higher than control ( Figure 1G). VEGFA/Thy-1 (protein marker of MCs) and VEGFA/Wilms tumour protein-1 (WT-1, protein marker of podocyte) immunofluorescence co-staining results showed that the expression of VEGFA apparently increased in the mesangial area of glomerular in the model group ( Figure S1C,D).
Angpt2/RECA-1 (protein marker of ECs) and Angpt2/Thy-1 immunofluorescence co-staining results showed that most of Angpt2 was expressed by ECs ( Figure S1E,F). Therefore, we suspected that MCsderived VEGFA and ECs-derived Angpt2 might play important roles in ECs proliferation of anti-Thy-1 nephritis. MCs was significantly increased (Figure 2A-C). Then, to investigate whether ECs proliferation was stimulated by MCs-derived VEGFA, VEGFA neutralizing antibody was added in the co-culture system ( Figure S2C). EdU assay results showed that adding VEGFA neutralizing antibody in co-culture system could inhibit ECs proliferation ( Figure 2D). Western blot pointed that activated MCs can definitely activate vascular endothelial growth factor receptor 2 (VEGFR2), which was inhibited by VEGFA neutralizing antibody ( Figure 2E).

| MCs promoted ECs proliferation through VEGFA/VEGFR2 pathway
Thus, by demonstrating that blocking VEGFA in co-culture system could inhibit ECs proliferation and VEGFR2 activation, we concluded that MCs generated VEGFA to activate VEGFR2 and promote ECs proliferation.

| MCs-derived VEGFA stimulated Angpt2 expression to promote ECs proliferation
The RT-qPCR, Western blot and Angpt2/CD31 (protein marker of ECs) immunofluorescence co-staining results showed that VEGFA enhanced Angpt2 expression in ECs ( Figure 3A-C). Then, we cocultured ECs with activated MCs and added VEGFA neutralizing antibody in the co-culture system. Western blot and immunofluorescence staining results showed that activated MCs promoted ECs to express Angpt2, which could be abrogated by VEGFA neutralizing antibody ( Figure 3D,G). Moreover, we measured the concentration of Angpt2  Figure 3E). Subsequently, we further explored the effect of Angpt2 on ECs proliferation. Transduction of Angpt2 siRNA in ECs significantly reduced the expression of Angpt2 protein ( Figure S3). EdU results indicated that VEGFA could improve ECs proliferation, and this improvement could be diminished by knockdown of Angpt2 ( Figure 3F,H). It can be seen that VEGFA expressed by MCs stimulated Angpt2 expression of ECs which promoted ECs proliferation.

| MCs-derived VEGFA stimulated Angpt2 expression which inhibited Tie2 phosphorylation on ECs
We continued to co-culture MCs and ECs and add VEGFA neutralizing antibody or Angpt2 neutralizing antibody into the co-culture system to investigate the effects of VEGFA and Angpt2 on Tie2 phosphorylation ( Figure S4). The results of Western blot and p-Tie2/CD31 immunofluorescence co-staining showed that blocking VEGFA or Angpt2 could increase the phosphorylation levels of Tie2 ( Figure 4A

| Enhancing Tie2 phosphorylation can effectively alleviate activated MCs-induced ECs proliferation
Angiopoietin1 (Angpt1) and Angpt2 can bind to the same part of Tie2, but their effects on Tie2 phosphorylation are opposite. Angpt1 promotes Tie2 phosphorylation, which is inhibited by Angpt2. We added Angpt2 or Angpt1 in co-culture system and detected the changes of Tie2 phosphorylation and ECs proliferation under different circumstances, so as to explore the relationship between Tie2 phosphorylation and ECs proliferation ( Figure S5A). The results showed that activated MCs inhibited the phosphorylation of Tie2 on ECs, but Tie2 phosphorylation increased after adding Angpt1 to co-culture system ( Figure 5A). At the same time, Tie2 phosphorylation decreased after the addition of Angpt2 into the co-culture system of static MCs and ECs ( Figure 5A). EdU results showed that activated MCs enhanced ECs proliferation, but ECs proliferation was inhibited after adding Angpt1 to co-culture system ( Figure 5B,D). Moreover, ECs proliferation was boosted after the addition of Angpt2 into the co-culture system of static MCs and ECs ( Figure 5B,D). To further confirm the relationship between ECs proliferation and Tie2, we transfected si-Tie2 to knock down Tie2 expression (inhibited Tie2 phosphorylation) in ECs ( Figure S5B). And EdU results showed that the proliferation ability of ECs was significantly improved by knocking down Tie2 (Figure 5C,E).
In conclusion, the proliferation ability of ECs was correlated with Tie2 phosphorylation levels, and the proliferation ability of ECs increased when Tie2 phosphorylation levels decreased.

| Enhancing Tie2 phosphorylation can effectively alleviate ECs proliferation in anti-Thy-1 nephritis
In vitro experiments, we verified that MCs promoted ECs proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 pathway and the advance RECA-1 and PCNA immunohistochemical staining ( Figure 6A,D,E) and immunofluorescence co-staining ( Figure 6B) results showed that ECs proliferation was alleviated by Vasculotide. Western blot results showed that after Vasculotide treatment, the expression of CD34 was decreased ( Figure S6C), which indicated that Vasculotide could decrease ECs proliferation. Thus, by summarizing experiment results, we concluded that promoting Tie2 phosphorylation by Vasculotide could effectively alleviate ECs proliferation in anti-Thy-1 nephritis.

| D ISCUSS I ON
Mesangial proliferative glomerulonephritis (MPGN) does not represent a specific glomerulopathy. As a pathological description of a class of glomerular diseases with mesangial hyperplasia, it can occur in primary glomerulonephritis (IgA nephropathy, lupus nephritis, infectious glomerulonephritis, IgM nephropathy, etc) and systemic disease (kidney allograft, diabetes, immune complex deposition, etc). 27,28 MPGN is one of the major factors of chronic kidney disease which leads to end-stage renal disease. Therefore, the study on its mechanism can provide new ideas for the treatment of a class of kidney diseases. Studies related to experimental MPGN have shown that when mesangial cells (MCs) damage and F I G U R E 4 MCs-derived VEGFA stimulated Angpt2 expression which inhibited Tie2 phosphorylation on ECs. A, C, p-Tie2 and Tie2 expressions on ECs were detected by Western blot. B, D, p-Tie2 expressions on ECs were detected by p-Tie2/CD31 immunofluorescence co-staining. CD31, red. p-Tie2, green. DAPI, blue. Results are presented as the mean values (±SD), *P < .05, **P < .01. Scale bars, 50 μm. Angpt2, angiopoietin2; ECs, endothelial cells; MCs, mesangial cells; PDGF-BB, platelet-derived growth factor BB, to activate MCs; Tie2, TEK tyrosine kinase. (A, B) Control, ECs co-cultured with inactive MCs. PDGF-BB, ECs co-cultured with PDGF-BB-activated MCs. PDGF-BB+IgG, ECs co-cultured with PDGF-BB-activated MCs and IgG antibody, was added as negative control. PDGF-BB+anti-Angpt2, ECs co-cultured with PDGF-BB-activated MCs and Angpt2 neutralizing antibody, was added. PDGF-BB+anti-VEGFA, ECs co-cultured with PDGF-BB-activated MCs and VEGFA neutralizing antibody, was added. (C, D) Control, inactive ECs. VEGFA, ECs stimulated by VEGFA for 24 h. VEGFA+si-Angpt2, ECs stimulated by VEGFA for 24 h after transfected with si-Angpt2 Angiopoietins belong to a family of growth factors and include Angpt1, Angpt2 and Angpt3. Angpt2 plays an important role in kidney disease, because it was increased in serum of patients with chronic kidney disease, lupus nephritis, thrombotic microangiopathy or antiglomerular basement membrane nephropathy, [39][40][41] and associated with poor prognosis in hypertension and coronary heart disease and also associated with high risk of acute kidney injury in critically ill patients. 42 The expression of Angpt2 of fibrotic kidneys increased, which stimulated the production of chemokines and adhesion factors in aortic, increased the infiltration of Ly6C(Low) macrophages and promoted the expression of fibrotic cytokine TGF-β1. 35 Studies on diabetic nephropathy indicated that Angpt2 promotes autophagy in MCs through miR-33-5p/SOCS5 loop. 36 In our study, we found that Angpt2 expression increased on the 7th day of anti-Thy-1 nephritis. And by co-staining Angpt2 with protein marker of ECs (RECA-1, rat endothelial cells antigen-1) or protein marker of MCs (Thy-1), we verified that Angpt2 was expressed by ECs. Relevant studies have shown that Angpt2 collaborates with VEGFA to promote angiogenesis. 43,44 In breast cancer, non-small cell lung cancer and acute myeloid leukaemia, the expression of VEGFA and Angpt2 increased simultaneously, which promoted tumour growth. High VEGFA expression is considered as the prerequisite for Angpt2-promoted hepatoma carcinoma cells growth. 45 This study confirmed that VEGFA expressed in MCs promoted Angpt2 expression in ECs which promoted ECs proliferation. However, in developing retinas and ischaemic retinal models, it was demonstrated that Angpt2 promotes the effect of VEGFA on retinal angiogenesis. 38 Thus, although VEGFA and Angpt2 have synergistic effects in promoting angiogenesis, the relationship between them is controversial in different organs. we found that the studies on Tie2 activation and angiogenesis are focused on brain, tumour, sepsis and ophthalmic diseases. Among them, most studies have shown that Tie2 activation can reduce permeability and inflammation, stabilize blood vessels and maintain the vascular function (such as stabilizing the blood-brain barrier, 49,50 reducing inflammation and dysregulation of coagulation in sepsis, 51 inhibiting the metastasis of tumour regeneration, 52 and repairing Schlemm's canal integrity and reducing intraocular pressure 53,54 ). However, three studies have suggested that Tie2 activation can promote angiogenesis, two of which mentioned that Tie2 activation promoted vascular repair in diabetes 55 and promoted choroidal angiogenesis to relieve hypoxia. 56 And one mentioned that Tie2 activation promoted angiogenesis to increase treatment resistance of tumour, which could be seen as "tumour self-preservation behavior." Therefore, it is obvious that Tie2 plays different roles in different diseases. We need to intervene Tie2 activation based on the pathological changes of diseases to achieve the effect of alleviating diseases. In this study, we added Angpt1 or Angpt2 to the co-culture system of MCs and ECs in vitro. The results showed that with the decline of Tie2 activation after adding F I G U R E 5 Enhancing Tie2 phosphorylation can effectively alleviate activated MCs-induced ECs proliferation. A, p-Tie2 and Tie2 expressions on ECs were detected by Western blot. B, Qualifications of EdU-positive rates in D. C, Qualifications of EdU-positive rates in E. D, E, EdU was used to detect ECs proliferation. Results are presented as the mean values (±SD), *P < .05, **P < .01. Scale bars, 50 μm. Tie2, TEK tyrosine kinase. MCs, mesangial cells. ECs, endothelial cells. Angpt2, angiopoietin2. Angpt1, angiopoietin. PDGF-BB, platelet-derived growth factor BB, to activate MCs. (A, B, D) Control, ECs co-cultured with inactive MCs. PDGF-BB, ECs co-cultured with PDGF-BB-activated MCs. Angpt2, ECs co-cultured with inactive MCs and Angpt2, was added. PDGF-BB+Angpt1, ECs co-cultured with PDGF-BB-activated MCs and Angpt1, was added. (C, E) Control, normal ECs. NC, ECs transfected with negative control siRNA. si-Tie2, ECs transfected with si-Tie2 F I G U R E 6 Enhancing Tie2 phosphorylation can effectively alleviate ECs proliferation in anti-Thy-1 nephritis. A, PAS staining showed cell number per glomerulus. RECA-1 and PCNA expressions in glomeruli were detected by immunohistochemical staining. B, Locations of RECA-1 and PCNA expressions were showed by immunofluorescence co-staining. White arrows indicated proliferating ECs. RECA-1, red. PCNA, green. DAPI, blue. C, Hypercellularity index per glomerulus was evaluated (n = 7). D, RECA-1 area staining rate per glomerulus was qualified (n = 7). E, PCNA-positive cells rate per glomerulus was qualified (n = 7). Results are presented as the mean values (±SD), *P < .05, **P < .01. Scale bars, 50 μm. ECs, endothelial cells; PCNA, proliferative cells nuclear antigen; RECA-1, rat endothelial cells antigen-1; Tie2, TEK tyrosine kinase. Control, normal rats. Model, anti-Thy-1 nephritis rats. Vasculotide, anti-Thy-1 nephritis rats treated by Vasculotide Angpt2, the proliferation ability of ECs increased. On the contrary, Tie2 phosphorylation increased after adding Angpt1, but the proliferation ability of ECs decreased. In conclusion, Tie2 phosphorylation is negatively correlated with ECs proliferation.
The results showed that VEGFA/VEGFR2 and Angpt2/Tie2 participated in the cell-cell communication between MCs and ECs. Therefore, we can inhibit ECs proliferation by blocking VEGFA or promoting Tie2 phosphorylation. As early as 1999, Tammo Ostendorf et al applied VEGFA-antagonist to anti-Thy-1 nephritis, which though effectively inhibited ECs proliferation, inhibited ECs restoration and increased ECs death at the same time. 38 It seems that alleviating the pathological changes of the glomerulus by blocking VEGFA is not advisable. So, we attempted to inhibit ECs proliferation by promoting Tie2 phosphorylation. A recently developed drug, Vasculotide, enhancing Tie2 phosphorylation, has been applied to a variety of diseases. 24,57,58 It was reported that Vasculotide could alleviate haemorrhagic shock, tumour cells metastasis and acute kidney injury by protecting ECs. 24,26,59 So, we applied Vasculotide to anti-Thy-1 nephritis. And the results showed that the phosphorylation of Tie2 was promoted and ECs proliferation was significantly inhibited, which suggests a possible clinical application for Vasculotide in MPGN.
In summary, the present study found that the VEGFA/VEGFR2 and Angpt2/Tie2 signalling pathway was involved in the cell-cell communication between MCs and ECs. Moreover, in vivo experiments showed that enhancing Tie2 phosphorylation by Vasculotide could alleviate ECs proliferation on the 7th day of anti-Thy-1 nephritis, which provided a new idea and strategy for alleviating the vascular lesions in MPGN.

CO N FLI C T O F I NTE R E S T
All of the authors declared no competing interests.

AUTH O R S ' CO NTR I B UTI O N S
Yinghua Zhao, Lingling Wu and Xiangmei Chen designed research; Yinghua Zhao, Bo Fu and Pu Chen analysed data and performed research; Yinghua Zhao and Lingling Wu wrote the paper; Qinggang Li, Qing Ouyang, Chuyue Zhang, Guangyan Cai and Xiangmei Chen optimized the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data and models generated and used during the study are available from the corresponding author by request.