Fig 2: CCL7 promoted macrophages migration and M1 polarization. (A) Migration of BMDMs in response to rmCCL7(100 ng/ml) stimulation. Scale bars represented 100 µm(n = 3). (B and C), Expression of (B) M1 markers (iNOS, IL-6, IL-12A, IL-12B, TNF-a) and (C) M2 markers (Arg 1, CD206)in response to rmCCL7 in BMDMs examined by qRT-PCR(n = 3). (D) Protein abundance of iNOS and CD206 in abdominal aortas of saline-infused (n = 7) and Ang II-infused (n = 6) mice. (E) Immunofluorescence co-staining of CD68 and iNOS in abdominal aortas of saline-infused (n = 3) and Ang II-infused (n = 3) mice. Values were represented as mean ± SEM; Student's t test was used in (A-D). *P < .05; **P < .01; ***P < .001, respectively

Fig 3: CCL7 blockade attenuated Ang II-induced AAA. (A) Representative images of abdominal aortas measured by ultrasound including maximum abdominal aorta diameter (luminal diameter) and maximum abdominal aorta area (luminal area) of PBS (n = 10), Control IgG (n = 10) and CCL7-nAb group (n = 10). A grid on the scale represented 0.5 mm. (B) Ex vivo maximum abdominal aorta diameter (external diameter) of PBS, Control IgG and CCL7-nAb group. A grid on the scale represented 1 mm. (C) Representative images of M1 macrophage infiltration in abdominal aortas of PBS (n = 10), Control IgG (n = 10) and CCL7-nAb group (n = 10) as revealed by CD68 and iNOS Immunofluorescence co-staining. Scale bars represented 50 µm. Each plot represented 3 independent experiments. Values were represented as mean ±SEM; One Way ANOVA was used for data analysis in (A-B). *P < .05; **P < .01; ***P < .001, respectively. White arrows (C) indicated CD68 and iNOS-positive location

Fig 4: CCL7-CCR1 interaction regulated macrophage phenotype through JAK2 /STAT1 pathway. (A) Protein abundance of JAK2, p-JAK2, STAT1, p-STAT1 and p-JAK2/JAK2, p-STAT1/STAT1 ratio in BMDMs incubated with rmCCL7 (100 ng/ml) and pre-treated 1 hour with CCR1 antagonist-BX471(1 µM and 10 µM). (B) M1 markers (iNOS, IL-6, IL-12A, IL-12B, TNF-a) mRNA level in BMDMs incubated with rmCCL7 alone or in combination with JAK2 inhibitor-Fedratinib (1 µM) and STAT1 inhibitor-Fludarabine (1 µM). (C) Pathological mechanism diagram of CCL7 in Ang II-induced AAA. Values were represented as mean ± SEM; One Way ANOVA was used for data analysis in (A-B) *P < .05; **P < .01; ***P < .001, respectively

Fig 5: Ccl7 is a HIF1a direct target gene.a qPCR analysis of Ccl7 mRNA expression in Hif1afl/fl and Hif1a?SMC VSMCs treated with vehicle, CoCl2, normoxia, or hypoxia (2% O2) for 6, 12, and 24 h. *P < 0.05, **P < 0.01, ***P < 0.001, n = 6 (independent experiments) per group. VSMCs isolated from Hif1afl/fl mice and Hif1a?SMC mice were infected with oxygen-stable HIF1a-expressing lentivirus, and then treated with Ang II for 24 h, b Ccl7 mRNA was measured by qPCR and c CCL7 protein was detected by ELISA. d qPCR analysis of Ccl7 mRNA expression in vehicle or Ang II-treated Hif2afl/fl and Hif2a?SMC VSMCs. e Schematic diagram of the mouse Ccl7 promoter illustrating the HREs in the regulatory region; the upstream regions were numbered in relation to the transcription initiation site. f Luciferase-reporter constructs under the control of the mouse Ccl7 promoter. HEK293T human embryonic kidney cells transiently transfected with the luciferase construct, and cotransfected with empty vector or HIF1a expression plasmids. Standard dual-luciferase assays were performed. EV, empty vector. **P < 0.01, n = 3. g, h ChIP assays of vehicle or Ang II-treated wild-type VSMCs using HIF1a or HIF2a antibodies. Data were normalized to input. *P < 0.05, **P < 0.01, n = 6 per group. i, j ChIP assays of vehicle or Ang II-treated Hif1afl/fl and Hif1a?SMC VSMCs using HIF1a antibody. Data were normalized to input. *P < 0.05, **P < 0.01, n = 6 per group. Statistical significance was determined by one-way ANOVA test followed by the unpaired t-test