Tesi di specializzazione (4 anni)
Angiotensin II type 2 receptor (AT2R) is over-expressed in synovial OA and RA tissue and increases steadily with inflammatory stimuli: a potential new target for pain and anti-inflammatory therapies.
MEDICINA CLINICA E SPERIMENTALE
Corso di studi
relatore Prof. Matucci Cerinic, Marco
- Rheumatoid arthritis
- new drug
- angiotensin II type 2 receptor
Data inizio appello
Data di rilascio
Background<br><br>Activation of transient potential type I vanilloid receptor (TRPV1) is crucial for pain perception and inflammation. During rheumatoid arthritis (RA) direct stimulation of TRPV1 positive fibroblast-like synoviocites (FLS) promotes secretion of inflammatory citokynes, like IL-6 and IL-8. Despite these data there aren’t TRPV1 antagonist available for human therapies. Recently, a new membrane receptor, the angiotensin II type 2 receptor (AT2R) has been discovered as crucial activator of TRPV1. <br><br>Objective (s)<br>To demonstrate the presence of AT2R in synovial tissues of RA and OA patients.<br>To evaluate expression of AT2R in FLS, macrophages, T and B cells of RA tissue. <br>To evaluate differences of expression of AT2R in cultured healthy FLS (H-FLS), osteoarthritis FLS (OA-FLS), RA-FLS at baseline and after IL1β or TNF-α incubation. <br><br>Materials and method (s) <br>Synovial biopsies were collected from 8 patients with active knee-RA and 8 with OA-knee. For immunohistochemistry, immunocytochemistry and western blotting analysis AT2R antibody were used. For fluorescence immunohistochemistry analysis specimens were also incubated with anti-CD3, anti-CD20, anti-CD68 and anti-vimentin. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). For fluorescence immunocytochemistry and western blotting analysis FLS were serum-starved for 24 hours or incubated with TNF-α, IL-1β alone or in combination. For statistical analysis the Student’s t-test was used (p value < 0.05 was considered statistically significant). <br><br><br>Results <br>AT2R was express from every layers of OA and RA synovial samples (fig 1, A-D). RA lining layer had significantly higher levels of AT2R than OA (p=0,035) (fig 1, A,C,E). Furthermore expression of AT2R was significantly higher in RA sub-lining layer than OA lining layer (p=0,002) (fig 1, B,D,E). Merge immunohistochemistry of RA samples showed that FLS, macrophage and T and B cells strongly expressed AT2R (fig 2, A-D). Immunocytochemistry and western blotting analysis showed that quantitative AT2R baseline expression was significantly higher in OA-FLS than H-FLS (p<0,001), nevertheless RA-FLS showed the higher levels of baseline AT2R (p<0,001)(fig 3). Incubation with TNF-α and IL-1β significantly increased expression of AT2R-FLS baseline levels in all cellular lines (p<0,05)(fig 3). <br><br>Discussion<br>This study demonstrates, for the first time, the presence of AT2R in human synovial specimens. AT2R was found in every RA and OA samples suggesting a possible constitutive role in RA and OA pathogenesis. However RA samples had higher AT2R expression than OA, both in lining and sub-lining layers. Macrophages, FLS, T and B lymphocytes, leading cells in inducing and sustaining RA inflammation, strongly expressed AT2R. Moreover, inflammatory stimuli induce AT2R expression, as demonstrated by levels of AT2R in H-FLS, OA-FLS and RA-FLS at baseline and after incubation with TNF-α and IL-1β. These evidences suggest that AT2R could act as pro-inflammatory receptor in OA and RA. AT2R antagonism could be a potentially new target for OA and RA treatment. <br>
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