MT1-MMP evaluation in neointimal hyperplasia in the late follow-up after prosthesis implantation.

Vascular surgical interventions are often burdened with late complications, including thrombosis or restenosis. The latter is generally caused by neointimal hyperplasia. Although extracellular matrix (ECM) remodelling is an important part of neointima formation, this process is not clearly understood. The aim of the study was to assess the content and activity of membrane-type 1 matrix metalloproteinase in human neointima in the late stages of its development. Matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 were also evaluated. The research was performed on neointima samples collected during secondary vascular interventions from patients with chronic limb ischaemia who developed vascular occlusion at 6-18 months after aorto/ilio-femoral bypass grafting. The control material consisted of segments of femoral arteries collected from organ donors. Western blot and/or ELISA were used for the determination of MT1-MMP and TIMP-2 expression. The activity of MT1-MMP was measured by fluorometric assay and that of MMP-2 by zymography. We demonstrated significantly increased MT1-MMP protein content in neointima when compared to normal arteries. However, the activity of MT1-MMP was significantly lower in neointima than in control samples. The decreased MT1-MMP activity was concomitant with reduced activity of MMP-2. The TIMP-2 protein levels in neointima and normal arteries were not significantly different. The results of our study suggest that the reduced activity of MT1-MMP and consequently MMP-2 in human neointima may play a role in decreased degradation of ECM components and thus promote neointimal overgrowth.

Aquaporins in human platelets: intracellular localization and possible role in granule and lysosome secretion.

This study was undertaken to establish the presence and the role of aquaporins (AQPs) in human platelets. Immunodetection with polyclonal antibodies and fluorescent microscopy suggest the presence of AQP isoforms - 0-7 and 9-12 - localized (in resting platelets) in the plasma membrane and in the dense and alpha granules. In thrombin- or monensin-treated platelets, the granules' AQPs become visible in the whole cell body, indicating the granules' swelling. In our studies on the role of AQPs in platelet responses we used tetrachloroauric acid (HAuCl4), a classical water channel blocker. We found that 10-100 µM of Au(III) inhibited the hypotonicity-, monensin (simulating the action of Na+/H+ exchanger)-, and collagen-evoked platelet swelling and reduced tritiated water uptake by platelets treated by collagen or monensin, indicating its ability to block water channels in these cells. HAuCl4, at the concentrations reducing water influx, did not induce cell lysis, alter the plasma membrane shape or the -SH group content. The inhibitor also failed to affect Na+ and Cl--related osmotic gradient formation and protein kinase D2 phosphorylation. In platelets activated by threshold concentrations of collagen, the thrombin receptor activating peptide, ADP, calcium ionophore A23187, phorbol ester and arachidonic acid, HAuCl4 (100 µM) completely inhibited secretion of ATP from dense granules but failed to reduce platelet aggregation. In collagen-stimulated platelets, HAuCl4 (10-100 µM) reduced secretion from dense and alpha granules, as well as lysosomes, in a dose-dependent manner. We conclude that human platelets possess numerous AQPs subtypes localized in the plasma and granule membranes. AQP-mediated water fluxes may be crucial for platelet volume regulation as well as secretion from dense and alpha granules and lysosomes.

Divergent changes in the content and activity of MMP-26 and TIMP-4 in human umbilical cord tissues associated with preeclampsia.

OBJECTIVES: Preeclampsia is the most common disorder associated with pregnancy. Our earlier findings revealed a substantial increase in the amount of matrix metalloproteinase-26 (matrilysin 2; MMP-26) in preeclamptic umbilical cord blood. The role of MMP-26 in preeclamptic umbilical cord tissue has not been fully elucidated. Some reports have indicated that the expression of matrilysin 2 and tissue inhibitor of matrix metalloproteinase 4 (TIMP-4) is coordinately regulated during progression of various diseases. STUDY DESIGN: Therefore, we decided to assess the expression and activity of MMP-26 and TIMP-4 in normal and preeclamptic umbilical cord tissues - umbilical cord arteries (UCA), vein (UCV) and Wharton's jelly (WJ). Tissues obtained from 10 normal (control material) and 10 preeclamptic umbilical cords were assessed using immunoenzymatic assay, Western immunoblotting, reverse transcriptase - polymerase chain reaction and fluorometric determination of the enzyme activity. RESULTS: All umbilical cord tissues, both control and preeclamptic, expressed MMP-26 and TIMP-4 in macromolecular complexes. Preeclampsia induced a significant increase in the content and actual activity of MMP-26 in UCV and WJ, as compared to control. The content of TIMP-4 in preeclamptic UCV and WJ was reduced. The content of MMP-26 mRNA was lower in UCA and UCV, whereas higher in WJ in preeclampsia. CONCLUSIONS: Divergent changes in MMP-26 mRNA and protein expression suggest a difference in the factors controlling the matrilysin synthesis in healthy and preeclamptic subjects. The decrease in TIMP-4 content in preeclamptic UCV might be the main reason for significantly higher actual activity of MMP-26 in that tissue. Only in preeclamptic Wharton's jelly the changes were compatible in terms of the content and activity of MMP-26 and TIMP-4. It cannot be excluded that similar alterations can be observed for the whole vascular system of newborns delivered by mothers with preeclampsia.

Evaluation of Vascular Endothelial Growth Factor and Its Receptors in Human Neointima.

OBJECTIVE: The potential contribution of vascular endothelial growth factor (VEGF) in neointima development has been evaluated in numerous animal studies. However, its role remains controversial. Moreover, little is known about neointima formation in humans. In this study we assessed the expression of VEGF-A and its receptors in the human neointima formed within vascular anastomosis. METHODS: The studied material comprised neointima samples harvested during secondary vascular operations from patients with chronic limb ischemia after aorto-/iliofemoral bypass grafting who developed vascular graft occlusion at 6-18 months after the initial surgical treatment. The control material consisted of segments of femoral arteries without visible macroscopic lesions collected from organ donors. The expression and content of VEGF-A, VEGFR-1 and VEGFR-2 were analyzed with PCR and ELISA methods, respectively. RESULTS: We observed a significantly increased expression of VEGF-A and VEGFR-2 mRNA in neointima compared to the normal aorta. A significantly higher protein content of VEGF-A and VEGFR-2 in neointima samples compared to the controls was also observed. No significant difference of VEGFR-1 content and VEGFR-1 mRNA expression was found in the studied material. CONCLUSION: These results indicate a possible involvement of the VEGF-A and VEGFR-2 system in the pathologic process of human neointima formation after vascular interventions.

Differential effect of flavonoids on glycosaminoglycan content and distribution in skin fibroblasts of patients with type I osteogenesis imperfecta.

We recently reported that, in osteogenesis imperfecta (OI) type I with diminished type I collagen biosynthesis, flavonoids such as apigenin 7-O-glucuronide, apigenin 7-O-methylglucuronide and pectolinarin normalized the level of collagen type I without affecting total protein synthesis. In addition to collagen, glycosaminoglycans (GAGs) play an important role in the formation of a functional supramolecular complex in the extracellular matrix, and any changes in their content and/or composition may be involved in the OI phenotype. We previously detected a marked increase in sulphated GAG content in the OI fibroblasts of more severely affected patients (OI types II and III). These alterations were more pronounced in medium than in cells. Although, in OI type I cells, the increase observed in medium was much smaller (approximately 1.5-fold), it resulted in an increase of approximately 3-fold of the GAG to collagen type I ratio. Therefore, in the potential pharmacotherapy of OI type I with flavonoids, their effect on GAG level may be of importance. In the OI cells, some of the tested flavonoids applied at a concentration of 30 µM affected GAG content in quite the opposite way than type I collagen. Aglicones inhibiting collagen synthesis caused a marked increase in GAG concentration in medium, in contrast to the flavonoid glycosides, which exerted a stimulatory effect on type I collagen synthesis, but had a different effect on GAG content and distribution. Among these, apigenin 7-O-methylglucuronide did not affect GAG level or secretion, and thus may potentially be used in OI type I pharmacotherapy in patients with normal GAG content. However, in patients with increased concentrations of GAG, pectolinarin, which decreases GAG content by approximately 40%, may be more beneficial.

Activity of lysosomal exoglycosidases in serum and synovial fluid in patients with chronic Lyme and rheumatoid arthritis.

Lysosomal exoglycosidases participate in the destruction of the articular cartilage by cleaving glycoside bonds in glycoproteins and proteoglycans. The aim of the study was to determine the activity of exoglycosidases: hexosaminidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase and alpha-fucosidase in serum and synovial fluid of patients with Lyme and rheumatoid arthritis. The study group consisted of 10 patients with chronic Lyme arthritis (age 18 - 74 y), 13 with rheumatoid arthritis (age 32 - 70 y) and 10 with juvenile idiopathic arthritis (age 8 - 17 y). The control group consisted of 9 healthy volunteers (age 24 - 62 y). The activity of the exoglycosidases was determined with the p-nitrophenyl derivatives of sugars as substrates. A significant increase of the activity of all the exoglycosidases in serum and in synovial fluid of the patients with different forms of arthritis was found. The ratio of synovial fluid/serum activity of exoglycosidases was above 2.0 in LA but not in JIA and RA patients. As the main source of exoglycosidases in the joint is the synovial membrane, this result supports the appropriateness of therapeutic synovectomy in chronic Lyme arthritis with knee effusion. The serum activity of hexosaminidase may be used in monitoring the course of Lyme arthritis and the efficiency of treatment.