Angiotensin converting enzyme in human synovium: increased stromal [I]351A binding in rheumatoid arthritis

Objective—To determine whether tissue angiotensin converting enzyme (ACE) is increased in synovia from patients with rheumatoid arthritis, osteoarthritis or chondromalacia patellae. Methods—Sections of synovia from patients with rheumatoid arthritis (n = 7), osteoarthritis (n = 7) or chondromalacia patellae (n = 6) were tested for immunoreactivity for ACE, and for binding of the ACE inhibitor [I]351A. The amount of ACE was measured with computer assisted image analysis as the proportion of synovial section area occupied by ACEimmunoreactive cells, and the density of [I]351A binding. Results—[I]351A binding sites had characteristics of ACE and colocalised with ACE-like immunoreactivity to microvascular endothelium and fibroblast-like stromal cells in inflamed and noninflamed human synovium. Stromal [I]351A binding densities (Beq) and the fraction of synovial section area occupied by ACE-immunoreactivity (fractional area) were higher in synovia from patients with rheumatoid arthritis (Beq 28 amol/ mm, fractional area 0.21) than from those with osteoarthritis (Beq 9 amol/mm , fractional area 0.10) or chondromalacia patellae (Beq 9 amol/mm , fractional area 0.09)(p < 0.05). Density of [I]351A binding to stroma was similar to that to blood vessels in rheumatoid arthritis, but less dense than vascular binding in chondromalacia patellae and osteoarthritis. Increases in [I]351A binding densities were attributable to increases in the numbers of binding sites, and were consistent with an increase in the density of ACE bearing stromal cells. Conclusion—ACE is upregulated in synovial stroma in rheumatoid arthritis. Increased tissue ACE may result in increased local generation of the vasoconstrictor and mitogenic peptide angiotensin II and thereby potentiate synovial hypoxia and proliferation in rheumatoid arthritis. (Ann Rheum Dis 2000;59:125–131) Angiotensin converting enzyme (ACE, EC 3.4.15.1) is a membrane metallopeptidase that participates in tissue regulatory peptide systems involving angiotensin II (ANG II), bradykinin and substance P. ACE catalyses the formation of ANG II from its inactive precursor, ANG I, which itself is generated by cleavage of angiotensinogen by the protease renin. ANG II exerts its biological eVects via specific, cell surface receptors, of which two major subtypes, named AT1 and AT2 receptors, have been identified in humans. ANG II is a potent vasoconstrictor, and can stimulate angiogenesis, fibroblast proliferation, collagen synthesis and growth factor expression, each mediated by AT1 receptors. 3–7 ACE expression is upregulated by fibroblasts and myofibroblasts after tissue injury. 9 Furthermore, ACE inhibitors and AT1 receptor antagonists can inhibit interstitial hypertrophy, hyperplasia and angiogenesis in a variety of tissues. 10–13 Hypertrophy, hyperplasia and angiogenesis also are characteristic of persistent rheumatoid synovitis. ACE activity is raised in synovial fluids from patients with rheumatoid arthritis compared with those with osteoarthritis, and rheumatoid synovial explants release ACE activity into their culture medium indicating local synthesis. 15 ACE, however, is primarily a membrane bound peptidase, and tissue ACE may be more important than soluble ACE in the local generation of ANG II. In human synovium, ACE-like immunoreactivity (ACELI) is localised both to vascular endothelium, and to fibroblast-like and some macrophagelike cells in the synovial stroma. It is currently unknown, however, whether tissue ACE is increased in either or both of these synovial compartments in rheumatoid arthritis. Increased tissue ACE could potentiate synovial hypertrophy and hyperplasia in rheumatoid arthritis through the increased generation of ANG II. [I]351A, an iodotyrosyl derivative of the ACE inhibitor lisinopril, binds specifically to the active site of ACE, and the density of [I]351A binding correlates closely with enzyme activity. 19 Immunohistochemistry is a sensitive technique for localising ACE immunoreactivity and quantifying the proportion of tissue components expressing ACE. Quantitative in vitro receptor autoradiography permits the density of ACE-like binding sites to be measured and pharmacologically characterised. A combination of the two techniques performed on the same tissue samples provides a powerful tool to localise and quantify ACE at the microscopic level in human synovium. We have now used quantitative in vitro autoradiography with [I]351A and immunohistochemistry to test whether tissue ACE levels are Ann Rheum Dis 2000;59:125–131 125 Academic Rheumatology, University of Nottingham Clinical Sciences Building, City Hospital, Nottingham NG5 1PB