Increased Levels and Activities of Matrix Metalloproteinases in Sickle Cell Disease.

Abstract

Sickle cell disease is characterized by a chronic inflammatory state, however, the mechanism underlying this inflammation is unclear. Elevated basal leukocyte counts, endothelial activation, altered availability of vaso-active factors, increased adhesion molecule expression and cytokines are found in this disease. Matrix metalloproteinases (MMPs) play an important role in physiological and pathological processes, participating in the extracellular matrix turnover as well as tissue degradation, repair mechanisms and cell migration. Since MMPs are important modulators of inflammation and the activity of cytokines and chemokines, our aim was to compare MMP and tissue inhibitor of matrix metalloproteinases (TIMP; inhibitors of MMP) levels and the activities of MMPs in the plasma of healthy subjects and in SCD patients on or off HU therapy (20–30 mg/kg/day, 3 month minimum duration). Gelatin zymography was used to measure MMP-2 and MMP-9 activities and ELISA was used for MMPs and TIMPs determination. After densitometric analysis of zymograms, a significant increase (p=0.03) in the activity of pro-MMP-9 was observed in the plasma of SCD patients (27.09 ± 1.99 average pixel, n=30) compared with the control group (20.96 ± 1.35 average pixel, n=29). Pro-MMP-9 activity in the plasma of SCD patients on HU (SCDHU) was greater (27.2 ± 1.35 average pixel, n=17); however this difference was not quite significant (p=0.07). With regard to MMP-2 activity, no significant difference (P>0.05, data not shown) between groups of patients and control individuals was observed. MMP-9 levels were significantly increased in the plasma of SCD patients (20.99 ± 1.52, n=32) compared to healthy controls (13.96 ± 1.64, n=16, p=0.02), although no effect of HU therapy on these augmented levels was observed (SCDHU; 23.66 ± 2.93, n=22, P>0.05). MMP-9 levels correlated significantly with total WBC counts (r=0.4221, p=0.01) in SCD patients (on and off HU), as well as with neutrophil counts (r=0.4436, p<0.001), but not with mononuclear cell counts. TIMP-1 and TIMP-2 levels were significantly (p<0.0001) higher in SCD patients not on HU therapy (83.69 ± 6.73, n=15; 95.49 ± 4.99, n=14, respectively) compared to control individuals (41.26 ± 3.75, n=11; 62.84 ± 3.43, n=10, respectively). HU therapy had no significant effect on increased TIMP-1 levels (84.14 ± 4.69, n=12, p>0.05) when compared to SCD patients not on HU. TIMP-2 levels were higher in SCD patients treated with HU (87.62 ± 9.39, n=12) than those observed in control subjects, however this difference was not quite significant (p=0.059). In contrast, MMP-2 and MMP-8 plasma concentrations were not significantly different in SCD individuals compared to controls (P>0.05). Data demonstrate, for the first time, that the level and activity of plasma MMP-9 are significantly increased in SCD, and that these levels are not affected by HU therapy. Importantly, MMP-9 levels correlate with leukocyte counts in SCD, indicating that MMP-9 may be a useful marker for inflammation in SCD.