1. To analyze the expression of ANGPTL7 in RA patients and CIA mice, as well as its correlation with their clinical features.

2. Investigate the effects of ANGPTL7 on pannus formation and bone destruction in RA through in vivo and in vitro experiments.

1. Expression of ANGPTL7 in Serum, Synovial Fluid, Synovial Tissue of RA Patients and Arthritis Animal Models, and Its Association with Clinical Characteristics

(1) We collected serum and synovial fluid samples from RA patients, OA patients, and healthy volunteers. ANGPTL7 levels were measured by ELISA. Correlations with RA clinical and laboratory parameters were assessed using SPSS 25.0; (2) We used immunofluorescence to detect the expression of CD44, CD31, and ANGPTL7 in the synovial tissues of patients with RA, OA, and CIA mice. (3) We employed bioinformatic approaches to analyze the expression levels of ANGPTL7 in synovial tissues from patients with RA, OA and healthy controls.

2. Effects of ANGPTL7 intervention on the phenotype and function of RA-FLSs

We isolated and cultured RA-FLSs from synovial tissues and different concentrations of ANGPTL7 were added to the culture medium for intervention. Using CCK-8 assay, flow cytometry, wound healing assay, and Transwell assay to assessed cell proliferation, apoptosis, migration, and invasion.

3. Effects of ANGPTL7 intervention on the phenotype and function of HUVEC
  Different concentrations of ANGPTL7 were added to the culture medium of HUVEC. Using CCK-8 assay, flow cytometry, wound healing assay, Transwell assay to assessed cell proliferation, apoptosis, migration ,invasion, as well as evaluated tube formation ability using Matrigel tube formation assay.

4. Effects of ANGPTL7 intervention on joint inflammation and bone destruction in CIA mice
  A collagen-induced arthritis (CIA) model was established in DBA mice. After the onset of arthritis, different concentrations of ANGPTL7 were injected into the knee joints. Body weight and clinical manifestations were monitored regularly from the time of model induction, and arthritis scores were recorded for each group. Joint destruction and histopathological changes were comprehensively assessed using Micro-CT, hematoxylin-eosin (HE) staining, and Safranin O-Fast Green staining. We used immunofluorescence staining to detect the expression of the endothelial marker CD31 in the synovial tissues of mice after ANGPTL7 intervention. Furthermore, immunohistochemistry was performed to evaluate the expression of VEGF, MMP1, MMP2, MMP9, and MMP13 in synovial tissues.

1. Expression of ANGPTL7 in Serum, Synovial Fluid, Synovial Tissue of RA Patients and Arthritis Animal Models, and Its Association with Clinical Characteristics

(1) ELISA results demonstrated that serum ANGPTL7 levels were significantly lower in patients with RA than in healthy controls (P<0.001), subgroup analysis showed that RA patients with bone erosion had significantly lower serum ANGPTL7 levels than those without bone erosion (P<0.001). Correlation analysis further demonstrated that serum ANGPTL7 levels in RA were negatively correlated with anti-CCP antibody levels (r=-0.48, P<0.01), Sharp score (r=-0.41, P<0.001), and US28 score (r=-0.51, P<0.05); (2) Immunofluorescence analysis showed that, compared with the control group, the number of CD44+CD31+ANGPTL7+FLSs in the synovial tissues of RA and CIA was markedly reduced; (3) Bioinformatics analysis showed that ANGPTL7 expression in synovial tissues from RA patients was significantly lower than in controls group (P<0.0001).

2. Effects of ANGPTL7 intervention on the phenotype and function of RA-FLSs

(1) CCK-8 proliferation assay showed that, compared with the control group, ANGPTL7 intervention did not significantly affect the proliferative capacity of RA-FLSs (P>0.05); (2) Flow cytometric analysis of apoptosis showed that,compared with the control group, ANGPTL7 intervention did not significantly alter the apoptotic rate of RA-FLSs(P>0.05); (3) Wound healing assay results demonstrated that, compared with the control group, the migration rates of RA-FLSs were significantly reduced in the 10, 100, and 1000 ng/mL ANGPTL7 intervention groups (P<0.05, P<0.001, P<0.0001, respectively). In addition, the migration rates in the 100 and 1000 ng/mL groups were both lower than that in the 10 ng/mL group (P<0.05, P<0.01, respectively); (4) Transwell invasion assay results showed that the numbers of invading RA-FLSs in the 10, 100, and 1000 ng/mL ANGPTL7 intervention groups were significantly lower than that in the control group (P<0.05, P<0.001, P<0.0001, respectively). Moreover, the numbers of invading cells in the 100 and 1000 ng/mL groups were both lower than that in the 10 ng/mL group (both P<0.01).

3. Effects of ANGPTL7 intervention on the phenotype and function of HUVEC

(1) CCK-8 assays showed that, compared with the control group, ANGPTL7 intervention at different concentrations significantly inhibited HUVEC proliferation at 24, 48, and 72 h (all P<0.05); (2) Flow cytometric apoptosis analysis showed that the apoptosis rate of HUVEC in the ANGPTL7 intervention group was significantly higher than that in the control group (P<0.05, P<0.001, P<0.0001, respectively), Moreover, apoptosis rates in the 100 and 1000 ng/mL groups were higher than in the 10ng/mL group (P<0.05, P<0.001, respectively), and were further increased in the 1000 ng/mL group compared with the 100ng/mL group (P<0.05); (3) Wound-healing assay showed that the migration rate of HUVEC was significantly reduced in the ANGPTL7 intervention group compared with the control group (both P<0.001), In addition, migratory capacity in the 100 and 1000 ng/mL groups was lower than that in the 10 ng/mL group (P<0.01, P<0.0001, respectively), and were further reduced in the 1000 ng/mL group compared with the 100 ng/mL group (P<0.05); (4) Transwell invasion assays showed that the number of invading cells were significantly decreased in the 100 and 1000 ng/mL ANGPTL7 groups compared with the control group (P<0.001, P<0.0001, respectively), compared with the 10 ng/mL group, the numbers of invading cells were lower in the 100 and 1000 ng/mL groups (P<0.01, P<0.0001, respectively), and were further decreased in the 1000 ng/mL group compared with the 100 ng/mL group (P<0.05); (5) Tube formation assay showed that the angiogenic capacity of HUVEC in the ANGPTL7 intervention group was significantly impaired, with a statistically significant difference (all P<0.05).

4. Effects of ANGPTL7 intervention on joint inflammation and bone destruction in CIA mice

Compared with the model group, ANGPTL7 intervention group exhibited lower arthritis index scores and reduced radiographic joint surface destruction. Histopathological analysis further showed attenuated synovial inflammation and bone destruction, accompanied by significantly decreased expression of MMP-1, MMP-2, MMP-9, MMP-13,  VEGF and CD31 in synovial tissues (P<0.001).

1. erum ANGPTL7 levels were reduced in patients with RA and were significantly negatively correlated with anti-CCP antibody levels, Sharp scores, and US28 scores. Moreover, RA patients with bone erosion had significantly lower serum ANGPTL7 levels than those without bone erosion. ANGPTL7 expression was also decreased in the synovial tissues of RA patients and CIA mice.

2. ANGPTL7 did not affect the proliferation or apoptosis of RA-FLSs, but inhibited their migration and invasion.

3. ANGPTL7 significantly suppressed the proliferation, migration, invasion, and in vitro tube formation of HUVECs, while promoting apoptosis.

4. ANGPTL7 ameliorated arthritis and bone destruction in CIA mice, exerting anti-inflammatory and bone-protective effects.