Clinical outcomes of bosentan in pulmonary arterial hypertension do not correlate with levels of TIMPs.

BACKGROUND: Matrix metalloproteinases (MMP) and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), are involved in tissue inflammation and fibrotic processes. Treatment with bosentan has been shown to improve the clinical outcome of patients with pulmonary arterial hypertension (PAH) with and without association with systemic sclerosis (SSc), and also to modulate the serum levels of matrix metalloproteases-9. We measured TIMP-1 and TIMP-2 in the serum of patients with SSc with and without PAH treated with long-term bosentan compared with healthy donors (HD). MATERIALS AND METHODS: Serum samples from HD (n = 16) and patients with SSc (n = 35), including patients with SSc without PAH (n = 23) and patients with PAH (n = 12), were analyzed using enzyme-linked immunosorbent assays (ELISAs) for total TIMP-1 and TIMP-2. RESULTS: Both mean TIMP-1 and TIMP-2 levels were significantly increased in patients with SSc compared with HD, but no differences were observed between patients with SSc with and without PAH. In the eight bosentan-treated patients, TIMP-1 and TIMP-2 levels did not change during 1 year of treatment, while bosentan increased the 6-min walking distance by 136 meters after 1 year, as well as clinical outcomes. CONCLUSIONS: Increased levels of TIMP-1 and TIMP-2 in patients with SSc compared with HD suggest that the inhibition of proteolysis allows the accumulation of ECM proteins. As bosentan does not stimulate TIMPs, it appears to favour proteolytic imbalance and to increase the turnover of ECM proteins.

Infliximab therapy does not modify MMP-2 and MMP-9 serum concentrations in chronic arthritis.

OBJECTIVE: Matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) play a key role in tissue remodelling after processes such as joint destruction in rheumatoid arthritis. Their expression may reflect the disease activity and they could therefore represent a useful marker to assess the efficacy of therapy. In this study MMP-2 and MMP-9 serum were evaluated in patients with chronic arthritis during therapy with the anti-TNFalpha mAb, infliximab. METHODS: Fifty patients with chronic arthritis, 26 with rheumatoid arthritis and 24 with undifferentiated chronic arthritis, were recruited and treated with infliximab (3 mg/kg). Serum concentrations of MMP-2 and MMP-9 were serially measured by gelatine zymography at baseline and after two and fourteen weeks of infliximab therapy. DAS-28 and ACR response criteria were applied to assess disease activity and clinical improvement. Twenty-four healthy donors were included in the study as controls. RESULTS: Although therapy with infliximab induced a statistically significant reduction of the DAS-28 score and improvement of the ACR clinical response, MMP-2 and MMP-9 serum concentrations were not modulated during therapy with infliximab. CONCLUSIONS: Our study provides further evidence that blocking TNFalpha by infliximab is a powerful tool in the management of chronic arthritis. Nevertheless, infliximab does not seem to be able to modify the serum expression of MMP-2 and MMP-9, probably because modification of these enzymes is restricted to the site of joint inflammation and serum detection can not truly mirror the local situation. Additional soluble factors correlating with joint damage should be investigated as possible markers for monitoring anti-TNFalpha therapy.

Matrix metalloproteinase imbalance in muscle disuse atrophy.

Muscle atrophy commonly occurs as a consequence of prolonged muscle inactivity, as observed after cast immobilization, bed rest or space flights. The molecular mechanisms responsible for muscle atrophy are still unknown, but a role has been proposed for altered permeability of the sarcolemma and of the surrounding connective tissue. Matrix metallo-proteinases (MMPs) are a family of enzymes with proteolytic activity toward a number of extracellular matrix (ECM) components; they are inhibited by tissue inhibitors of MMPs (TIMPs). In a rat tail-suspension experimental model, we show that after fourteen days of non-weight bearing there is increased expression of MMP-2 in the atrophic soleus and gastrocnemius and decreased expression of TIMP-2. In the same experimental model the expression of Collagen I and Collagen IV, two main ECM components present in the muscles, was reduced and unevenly distributed in unloaded animals. The difference was more evident in the soleus than in the gastrocnemius muscle. This suggests that muscle disuse induces a proteolytic imbalance, which could be responsible for the breakdown of basal lamina structures such as Collagen I and Collagen IV, and that this leads to an altered permeability with consequent atrophy. In conclusion, an MMP-2/TIMP-2 imbalance could have a role in the mechanism underlying muscle disuse atrophy; more studies are needed to expand our molecular knowledge on this issue and to explore the possibility of targeting the proteolytic imbalance with MMP inhibitors.

MMP-2, MMP-9, TIMP-1 and TIMP-2 levels in patients with rheumatoid arthritis and psoriatic arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) and psoriatic arthritis (PA) are both chronic rheumatic inflammatory diseases characterized by disruption of the extra-cellular matrix (ECM) protein of the cartilage, likely induced by proteolytic enzymes such as matrix metalloproteases (MMPs). The goal of this study was to quantify the expression of MMPs such as MMP-2 and MMP-9, and their physiological tissue inhibitors TIMP-2 and TIMP-1, respectively, in serum and synovial fluid. METHODS: Serum and synovial fluid from 24 RA patients and 17 PA patients were studied to determine the levels of MMP-2 and MMP-9 proteolytic activity using a modified gelatin zymography procedure. TIMP-1 and TIMP-2 were measured by a commercially available ELISA kit. RESULTS: Our results show that MMP-2 was detected in the latent form only, while MMP-9 was present in latent and active form. Both gelatinases were more concentrated in synovial fluid than in serum, and TIMP-1 and TIMP-2 concentrations were also more elevated in synovial fluid than in serum. CONCLUSIONS: To investigate the remodelling of cartilage ECM proteins, the evaluation of synovial fluid concentrations of MMP-2, MMP-9, TIMP-1 and TIMP-2 is more reliable than that determined in serum. In view of these data, MMPs inhibitors might represent a possible target for new therapies delivered directly in the joint space.

Human hepatocellular carcinoma (HCC) cells require both alpha3beta1 integrin and matrix metalloproteinases activity for migration and invasion.

Hepatocellular carcinoma (HCC) is the most frequent malignant tumor of the liver; prognosis depends on the tendency to metastasize. Cancer cell invasion is regulated by proteolytic remodeling of extracellular matrix components and by integrin expression. We have shown that matrix metalloproteinase-2 (MMP-2) and membrane-type-1 matrix metalloproteinase (MT1-MMP) cleave Laminin-5 (Ln-5), stimulating cell migration. Here we report that all HCC cells express MT1-MMP, migrate on Ln-1 and Collagen IV, whereas only HCC cells that express alpha3beta1 integrin secrete detectable levels of gelatinases, migrate on Ln-5, and invade through a reconstituted basement membrane (BM). Migration on Ln-5 is blocked by BB-94, an MMP inhibitor, and by MIG1, a monoclonal antibody that hinders migration on MMP-2-cleaved Ln-5. Invasion through a reconstituted BM is also inhibited by BB-94. HCC alpha3beta1-negative cells migrate on Ln-1 and Collagen IV, but not on Ln-5, and do not invade through a reconstituted BM, although they express MT1-MMP. Anti-alpha3beta1 blocking antibodies inhibit gelatinase activation, cell motility, and cell invasion through MATRIGEL: In vivo, alpha3beta1 integrin and Ln-5 are expressed in HCC tissue but not in normal liver. In conclusion, our data suggest that both alpha3beta1 integrin and gelatinase activity are required for HCC migration and invasion.

Altered expression of integrins and basement membrane proteins in malignant and pre-malignant lesions of oral mucosa.

Integrins are transmembrane receptors that regulate cell-cell and cell-extracellular matrix (ECM) contact. In epithelial tissues, they interact with ECM components of the basement membrane (BM) to maintain the homeostasis and the architecture of the tissue. This interaction controls several cell functions such as adhesion, migration, proliferation, differentiation, and therefore has a key role in cancer development and metastasis. We studied the expression of integrins and ECM components of the BM by immunohistochemistry in frozen specimens of malignant squamous cell carcinoma (SCC), pre-malignant lesions of the oral mucosa (leucoplakia) and oral lichen planus. In invasive SCC, we observed altered polarity and distribution of alpha2beta1, alpha6beta4 and alpha3beta1 integrins, whereas in the in situ carcinoma alpha6beta4 and alpha3beta1 patterns only were altered. Immunostaining for ECM components such as Laminin-1 (Ln-1), Ln-5, and Collagen IV (Coll IV) was discontinuous and interrupted in invasive SCC, whereas it was normal in the in situ carcinoma. In both pre-malignant lesions and lichen planus specimens, integrins were expressed in a polarized manner in the presence of a normal BM, whereas were abnormally distributed in those tissues with altered staining patterns of the ECM components. In conclusion, we suggest that abnormal re-distribution of alpha3beta1 and alpha6beta4 integrins and expression of ECM components such as Ln-5 could play an important role in SCC invasion and metastasis.

Transforming growth factor beta1 and soluble Fas serum levels in hepatocellular carcinoma.

In this study we assessed the usefulness of serum Transforming Growth Factor-beta1 (TGF-beta1) and soluble Fas (sFas) in distinguishing liver cirrhosis (LC) with and without hepatocellular carcinoma (HCC) as compared with alpha-fetoprotein (AFP). Serum TGF-beta1 and sFas levels were measured by ELISA in 51 LC patients, 54 patients with HCC and 30 healthy donors. Considering as a cut-off limit (mean+1SD of controls) 74 pg/ml and 637 pg/ml for TGF-beta1 and sFas, respectively, we computed serum concentrations of TGF-beta1 and sFas as a score (mean+/-SD). The positive frequency of serum TGF-beta1 levels in HCC patients (54%) was greater than in LC patients (26%) and healthy donors (3%). TGF-beta1 levels were higher in HCC (1.6+/-0.5) than in LC (1.1+/-0.2) (P<0.0001) and healthy donors (0.6+/-0.2). Using a cut-off limit of 82 pg/ml (mean+2SD), the positive frequency of TGF-beta1 was 20% in HCC patients. None of the controls and LC patients had TGF-beta1 levels higher than 82 pg/ml. The positive frequency of serum sFas levels was 100% in HCC patients, 98% in LC patients and 3% in healthy controls. Serum sFas levels were higher in HCC (2.5+/-0.7) than in LC (1.9+/-0.5) (P<0. 001) and healthy donors (0.6+/-0.3). No significant change of positive frequency was obtained by setting sFas cut-off at higher levels. sFas values did not correlate with TGF-beta1 levels. No relationship was found between TGF-beta1 amounts and AFP levels. However, in the 23% of HCC patients, with normal AFP values TGF-beta1 levels were higher than the cut off. These findings suggest the potential usefulness for TGF-beta1 assay in AFP-negative HCC.