Fig 2: The effect of MPO on mRNA expression and collagen production in mammary fibroblasts. Messenger RNA expression of cancer-associated genes (A) and genes involved in extracellular matrix regulation and collagen production (B) measured by RT-PCR in human mammary fibroblasts following treatment with 5 μg/mL of MPO for 72 h. mRNA expression normalised to HPRT1 expression and presented as relative expression where the average for the untreated control is 1. Collagen production was measured using collagen 1 ELISA (C) and insoluble collagen measured by sirius red staining (D). Data presented as mean + SEM with statistical analysis performed using a linear mixed-effects model Tukey’s post hoc comparison. Statistical significance indicated by * when p < 0.05.

Fig 3: The lack of TSP-1 aggravates renal injury in a mouse model of AAV.(A) Experimental setup for murine model of AAV using TSP-1-KO and WT mice. On day 0, mice were immunized with hMPO and 10 days later injected with NTS. Blood was collected on day -7 and day 11; mice were euthanized on day 14. (B) Mice demonstrated significantly increased C5a levels and developed MPO antibodies (C), confirming disease induction. Data were normalized to day –7 (n = 3). (D) Plasma albumin levels showed more severe hypoalbuminemia in TSP-1-KO mice compared to WT mice at day 14. Data were normalized to day –7 (n = 3). (E) Serum creatinine levels were significantly reduced in TSP-1-KO mice compared with controls (n = 3). (F) Representative images of PAS-stained kidney sections from WT and TSP-1-KO mice. Mice developed fibrinoid necrosis and proteinaceous casts (asterisks). Glomeruli show thickening of capillary walls, parietal epithelial cell (PEC) activation (arrowheads), and capillary thrombi (arrows). (G) Quantification of glomeruli with fibrinoid necrosis shows significantly higher injury in TSP-1-KO mice compared with WT (n = 3). (H) TSP-1-KO mice trend toward greater PEC activation, precursor to crescent formation (n = 3). (I) Plasma TSP-1 levels were significantly increased in hMPO/NTS-treated WT mice, similar to AAV patients (n ≥ 2). (J) C3 levels in kidney lysates of hMPO/NTS-treated TSP-1-KO mice were significantly increased compared to WT mice (n ≥ 2). (K) Representative images and analysis of glomerular C3 deposition demonstrating significantly increased C3 deposits in hMPO/NTS-treated TSP-1-KO mice compared to WT mice (n ≥ 2). Scale bar: 40 μm. (L) Citrullinated histone levels were significantly higher in hMPO/NTS-treated TSP-1-KO mice, suggesting enhanced NET formation. Data are shown as mean ± SD. Statistical analysis by Student’s t test, *P < 0.05, **P < 0.01. ND, not detected.