Apelin Promotes Endothelial Progenitor Cell Angiogenesis in Rheumatoid Arthritis Disease via the miR-525-5p/Angiopoietin-1 Pathway

Figures

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  Figure 1. APLN increases Ang1 expression in RASFs and promotes EPCs tube formation and migration. (A–C) RASFs were incubated for 24 h with APLN; Ang1 expression was quantified by qPCR, Western blot and ELISA assays. (D, E) RASFs cells were treat with APLN does manner for 24 h and collected conditioned medium (CM) was applied to endothelial progenitor cells (EPCs), then EPC migration and angiogenesis was measured. (F) Matrigel plugs containing CM from APLN-treated RASFs or VEGF (positive control) were applied to 6-day-old fertilized chick embryos for 4 days. CAMs were examined by microscopy and photographed, and vessels counted. *p < 0.05, **p < 0.01 and ***p < 0.001 versus the control group; # p < 0.05 versus the APLN-treated group.

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  Figure 2. PLCγ signaling regulates APLN-induced effects on Ang1 expression and EPC angiogenesis. (A, B) RASFs were left untreated, stimulated with APLN (3 ng/mL) alone for 24 h, or pretreated with U73122 for 30 min, or transfected with PLCγ siRNA for 30 min, prior to 24 h of APLN (3 ng/mL) stimulation. Ang1 expression was examined by qPCR and ELISA assays. (C, D) Collected CM was applied to EPCs, and angiogenesis was determined. (E) RASFs were treated with APLN for varying amounts of time and Western blot (n=3) determined PLCγ phosphorylation. (F) Densitometry analysis of (E). *p < 0.05, **p < 0.01 and ***p < 0.001 versus the control group; # p < 0.05 versus the APLN-treated group.

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  Figure 3. PKCα signaling regulates APLN-induced effects on Ang1 expression and EPC angiogenesis. (A, B) RASFs were pretreated with GF109203X and Go6976 for 30 min, or transfected with PKCα and PKCδ siRNAs for 24 h, then stimulated with APLN for 24 h. Ang1 expression was examined by qPCR and ELISA assays. (C, D) Collected CM was applied to EPCs, and EPC angiogenesis was determined. (E) RASFs were treated with APLN time-manner and Western blot (n=3) determined PKCα phosphorylation. *p < 0.05, **p < 0.01 and ***p < 0.001 versus the control group; # p < 0.05 versus the APLN-treated group.

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  Figure 4. APLN facilitates Ang1 synthesis and EPC angiogenesis by inhibiting miR-525-5p. (A) Open-source software enabled identification of miRNAs that possibly interfere with Ang1 transcription. (B) RASFs were incubated with APLN for 24 h. miR-525-5p expression was determined by the qPCR assay. (C) RASFs were transfected with miR-525-5p mimic for 24 h, then stimulated with APLN for 24 h. Ang1 levels were determined by qPCR. (D, E) Collected CM was applied to EPCs, and EPC angiogenesis was quantified. (F) Schematic 3′UTR representation of human Ang1 containing the miR-525-5p binding site. (G, H) RASFs were transfected with the indicated luciferase plasmid with or without miR-525-5p mimic for 24 h, then stimulated with APLN for 24 h. Relative luciferase activity was determined. (I) RASFs were pretreated with U73122, GF109203X and Go6976 for 30 min, then stimulated with APLN for 24 h. miR-525-5p expression was quantified by qPCR. *p < 0.05, **p < 0.01 and ***p < 0.001 versus the control group; # p < 0.05 versus the APLN-treated group.

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  Figure 5. Inhibition of APLN reduces EPC angiogenesis in vivo. (A, B) RASFs were infected with APLN shRNA for 24 h. APLN and Ang1 expression was examined by Western blot (n=3) and ELISA. (C, D) Collected CM was applied to EPCs, and EPC angiogenesis was quantified. (E) After subjecting RASFs to the treatment conditions as indicated, the harvested CM was applied to 6-day-old fertilized chick embryos for 4 days. CAMs were examined by microscopy and photographed, and vessels were counted manually. (F, G) Matrigel plugs containing the harvested CM were subcutaneously injected into the flanks of nude mice. After 7 days, the plugs were photographed, and hemoglobin levels were quantified. (H) Plug specimens were immunostained with CD31, CD34 and CD133 antibodies. *p < 0.05, **p < 0.01 and ***p < 0.001 versus the control group; # p < 0.05 versus the APLN-treated group.

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  Figure 6. APLN knockdown reduces angiogenesis and the severity of RA in vivo. CIA mice received intra-articular injections of 7.1 × 106 PFU APLN shRNA or control shRNA on day 14 and were euthanized on day 49. (A) In comparisons of digital plethysmometer values for the amounts of hind paw swelling in the CIA and APLN shRNA groups, the statistical comparisons were not significant on days 14 and 21 (p=0.5 and p=0.3, respectively), but they were significant change on days 14, 21, 28,35,42 and 49 (p=0.001, p<0.0001, p<0.0001, and p<0.0001, respectively). (B) Representative micro-CT images of the hind paws taken on day 49. (C–E) Micro-CT SkyScan Software quantified bone mineral density, bone volume and trabecular numbers. (F, G) Histological sections of ankle joints were stained with H&E or Safranin O and immunostained with CD31, CD34 and CD133. *p < 0.05 versus the control group; # p < 0.05 versus the APLN-treated group. *p < 0.05 and **p < 0.01 versus the control group; # p < 0.05 versus the CIA group.

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  Figure 7. The schematic diagram summarizes the mechanisms of APLN-induced Ang1-dependent EPC angiogenesis during RA pathogenesis. APLN induces Ang1 expression in RASFs by suppressing miR-525-5p expression via the PLCγ and PKCα signaling pathways, and promotes EPC angiogenesis in RA.