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.

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.

Distribution of cathepsin L in human umbilical cord tissues.

The extracellular matrix components show specific distribution patterns within various structures of the umbilical cord, among which Wharton's jelly is especially collagen-rich tissue. Cathepsin L is a potent cysteine protease engaged in degradation of extracellular matrix proteins, including collagens. We evaluated the activity and expression of cathepsin L, and the inhibitory effect of cysteine protease inhibitors in the umbilical cord arteries, vein and Wharton's jelly. Cathepsin L activity and anti-papain inhibitory effect of cysteine protease inhibitors were quantified in extracts of separated umbilical cord tissues using fluorogenic substrates. The results were calculated per DNA content. The enzyme expression was assessed by Western immunoblotting. The active cathepsin L activity (without activation by pepsin digestion), its percentage in the total activity (after pepsin activation), and the expression of the mature single-chain enzyme were the lowest in the umbilical cord arteries and the highest in Wharton's jelly. The effect of cysteine protease inhibitors showed similar distribution as in the case of the active enzyme, being the highest in Wharton's jelly. Distribution of the activity and expression of mature cathepsin L within the umbilical cord probably results from distinctions in the proenzyme activation process. Differences in the action of cysteine protease inhibitors can partly restrict divergences in the enzyme activity that could reflect its expression alone. Differential enzyme action seems to contribute to tissue-specific collagen turnover within the umbilical cord cells, especially those of Wharton's jelly.

Cathepsin B in human myometrium and in uterine leiomyomas at various stages of tumour growth.

OBJECTIVE: Cathepsin B is a major cysteine protease involved in the degradation of extracellular matrix proteins, as well as in the activation of precursor forms of other proteases and in release of matrix-bound growth factors. We assessed the expression and activity of cathepsin B, and the inhibitory effect of cysteine protease inhibitors in human myometrium and uterine leiomyomas at various stages of tumour growth. STUDY DESIGN: Studies were performed on human myometrium collected from 12 patients and on uterine leiomyomas of various weights: small (less than or equal to 25 g, taken from 10 patients) and large (more than or equal to 100 g, obtained from 13 patients). Tissue extracts were assayed for cathepsin B activity and for inhibitory effect of cysteine protease inhibitors against papain using fluorogenic substrates, and calculated per DNA content. Statistical analysis was performed by Kruskal-Wallis analysis of variance followed by Dunn's post hoc tests. The enzyme expression was evaluated by SDS/polyacrylamide gel electrophoresis followed by Western immunoblotting. RESULTS: In all the investigated tissues cathepsin B exists mainly in a fully processed double-chain form. The enzyme activity and expression were similar in control myometrium and in small leiomyomas. However, they distinctly increased during tumour growth. The effect of cysteine protease inhibitors was comparable in all the tissues examined. CONCLUSION: These data suggest that the enhanced activity and expression of cathepsin B but not the action of cysteine protease inhibitors contribute to an increased remodelling of extracellular matrix and bioavailability of various growth factors, which favour leiomyoma growth.

Differential distribution of cathepsin B in human umbilical cord tissues.

The extracellular matrix components are differentially distributed among various structures of the umbilical cord. Wharton's jelly is especially rich in collagens and growth factors. Cathepsin B is a major cysteine protease involved in collagen degradation, as well as in the activation of precursor forms of other collagenolytic enzymes and growth factors. We assessed the activity and expression of cathepsin B in the umbilical cord arteries, veins and Wharton's jelly. Extracts of separated umbilical cord components were subjected to an activity assay with the use of specific fluorogenic substrate. The expression of cathepsin B protein was qualitatively evaluated by Western immunoblotting and quantitatively determined with an immunoenzymatic method. The total cathepsin B activity and content calculated per gram of DNA were higher in Wharton's jelly than in the umbilical cord vessels, and the latter parameter was the lowest in the umbilical cord arteries. Moreover, the expression and the activity of latent cathepsin B (following activation by pepsin digestion) calculated per gram of DNA were the highest in Wharton's jelly and the lowest in the umbilical cord arteries. High expression and activity of latent, pepsin-activatable cathepsin B related to DNA content in Wharton's jelly seem to reflect the stimulation of its cells by high amounts of collagen I and growth factors.

Fatty acid composition of triacylglycerols from Wharton's jelly determined by high-performance liquid chromatography.

The improved method for HPLC determination of fatty acids was proposed. The chromatographic separation of p-bromophenacyl derivatives of fatty acids under a gradient elution was achieved at 40 degrees C with an RP-18 LiChroCART 5 column and organic mobile phase containing methanol, acetonitrile, water and TEAP buffer pH 5.6. The quantitative determination of those derivatives was performed at 254 nm. Preeclampsia, the most common pregnancy complication, did not affect triacylglycerol content in the umbilical cord Wharton's jelly in comparison to the control material. However, it changed the composition of fatty acids, bound to that lipid class. The method allows the determination of almost all fatty acids forming the investigated neutral lipid class, contained in a solid tissue sample. The use of TEAP buffer excluded precipitation and flow stoppage in the HPLC system. The method reduced time and costs and might be useful for all other lipid classes and different tissues.

Separation and Determination of Fatty Acids from Lipid Fractions by High-Performance Liquid Chromatography: Cholesterol Esters of Umbilical Cord Arteries.

Preeclampsia is accompanied by an extensive remodeling of the extracellular matrix of umbilical cord. It is associated with an increase in collagen content in the umbilical cord artery. Furthermore, preeclampsia distinctly reduces proteolytic and gelatinolytic activity, especially after activation with various agents.We decided to develop a method for separation and determination of fatty acids from different tissues by high-performance liquid chromatography. That method allowed us to determine cholesteryl ester composition and content in umbilical cord arteries. Studies were performed on the umbilical cord arteries taken from 10 newborns delivered by healthy mothers and 10 newborns delivered by mothers with preeclampsia. Cholesteryl esters were isolated by thin layer chromatography. Fatty acids were liberated by basic hydrolysis and analyzed by HPLC of their p-bromophenacyl derivatives using detection at 254 nm. It was found that saturated fatty acids were the main group of fatty acids incorporated to cholesteryl esters in all control and preeclamptic umbilical cord arteries. Preeclampsia caused a significant increase in cholesteryl ester content in the umbilical cord arteries. An increase of neutral lipid content in vessel walls of newborns delivered by mothers with preeclampsia may be one of the factors that evoke the initiation of hypertension in utero and its amplification throughout childhood and adult life. The described method reduces time and cost consumption and allows us to determine almost all fatty acids forming cholesteryl esters contained in the tissue sample.

Pre-eclampsia-associated alterations in decorin, biglycan and versican of the umbilical cord vein wall.

OBJECTIVE: The role of proteoglycans in the rearrangement of the extracellular matrix of the umbilical cord vein wall in pre-eclampsia is not known. Decorin, biglycan and versican are the main proteoglycans of the umbilical cord vein wall. We decided to test whether the amounts of these proteoglycans alter in pre-eclampsia. STUDY DESIGN: Study was performed on the umbilical cord veins taken from 10 newborns delivered by healthy mothers (control group) and from 10 newborns delivered by mothers with pre-eclampsia. Proteoglycans were extracted in dissociative conditions, purified by Q-Sepharose anion exchange chromatography and lyophilised. Decorin, biglycan and versican were analysed by SDS-PAGE followed by Western blotting before and after treatment with chondroitinase ABC. The amounts of decorin, biglycan and versican core proteins were assessed by ELISA method. RESULTS: We found that both control and pre-eclamptic umbilical cord vein wall contained all the three proteoglycans. ELISA assay showed the amounts of the core proteins of decorin, biglycan and versican were distinctly higher in pre-eclamptic material in comparison to control vessel. Western blotting confirmed that the expression of all these proteoglycan core proteins increased in pre-eclampsia. They featured in the same electrophoretic mobility-45 and 47 kDa for decorin, 45 kDa for biglycan, and 300 and 320 kDa for versican. CONCLUSION: The content of decorin, biglycan and versican in the umbilical cord vein wall is elevated in pre-eclampsia in comparison to the corresponding control vessel. These alterations may affect the mechanical properties of this vessel and disturb foetal blood circulation.

The inhibitory effect of preeclamptic umbilical cord blood serum on matrix metalloproteinase-1 in arterial slices incubated in vitro.

BACKGROUND: Our previous studies demonstrated that preeclampsia is accompanied by significant alterations in the amounts of peptide growth factors in the umbilical cord serum. Some of these factors (especially IGF-1) are known as regulators of collagen metabolism. The umbilical cord arteries (UCAs) of newborns delivered by mothers with preeclampsia contain more than twice the amount of collagen in comparison to newborns delivered by healthy mothers. A significant role in collagen degradation is attributed to matrix metalloproteinase (MMP)-1 (collagenase 1) and tissue inhibitors of metalloproteinases (TIMPs). OBJECTIVE: To compare the effects of umbilical cord (UC) blood serum of control and preeclamptic newborns on the content and activity of MMP-1, TIMP-1 and TIMP-2 in UCA wall slices incubated in vitro. METHODS: Polyacrylamide gel electrophoresis (PAGE) followed by Western immunoblotting allowed to detect MMP-1 as well as TIMP-1 and TIMP-2. The amounts of MMP-1, TIMP-1 and TIMP-2 in UCA slices were measured by immunoenzymatic method (ELISA). MMP-1 activity in the arterial wall was measured using a collagenase-1-specific substrate. RESULTS: Western immunoblot analyses detected MMP-1, TIMP-1 and TIMP-2 in the incubation fluids and in extracts from the UCA wall. Both 43- and 55-kDa (a zymogen) bands of MMP-1 were visible. The control UC serum stimulated both the amount as well as actual and potential activities of MMP-1 in the arterial wall in a time-dependent manner. In contrast to controls, the preeclamptic serum did not exert such an effect. CONCLUSIONS: The small amount and low activity of MMP-1 accompanied by elevated amounts of TIMPs (especially TIMP-1) decelerate the degradation and enhance the accumulation of collagen in the preeclamptic UCA wall.

Pre-eclampsia-associated alterations in Wharton's jelly proteoglycans.

Proteoglycans of Wharton's jelly contain mainly chondroitin/dermatan sulphate chains. The predominant proteoglycan is decorin (core proteins of 45 and 47 kDa), although the core proteins of biglycan (45 kDa), versican (260 kDa) and of other proteoglycans (90, 110, 220 kDa) were also detected (Gogiel et al., 2003). The aim of the present study was to compare the proteoglycan composition of Wharton's jelly of newborns delivered by healthy mothers and those with pre-eclampsia. Proteoglycans from pre-eclamptic Wharton's jelly had a higher sulphated glycosaminoglycan/protein ratio than those of normal tissue. Pre-eclampsia is associated with a lower level of all proteoglycan core proteins, especially those of higher molecular mass (such as versican), although the same set of core proteins were found in normal and pre-eclamptic Wharton's jelly. The alterations in the proteoglycan composition of Wharton's jelly may affect the mechanical properties of the umbilical cord and, in the case of pre-eclampsia, disturb foetal blood circulation.

Preeclampsia-associated reduction of cathepsin D activity in the umbilical cord.

BACKGROUND: Preeclampsia is accompanied by an increase of collagen contents in the umbilical cord (UC) arteries and in Wharton's jelly. Cathepsin D is one of the enzymes which participates in collagen degradation and activates precursor forms of collagenolytic metalloproteinases. It was decided to evaluate the activity of cathepsin D within umbilical cord arteries, veins and Wharton's jelly and its alterations in preeclampsia. MATERIALS AND METHODS: Umbilical cord components were separated and submitted to homogenisation/extraction with 0.05 M Tris-HCl+0.2% Triton X-100, pH 7.5. Proteolytic activities of the extracts were studied with a use of cathepsin D-specific substrate. Western immunoblot technique was employed to detect this enzyme. RESULTS: It was found that human umbilical cord tissues contain both active and inactive forms of cathepsin D. Preeclampsia is associated with a distinct increase in the amount of this enzyme in the umbilical cord, whereas its activity deeply decreased. Activation with trypsin augments cathepsin D activity in preeclamptic umbilical cord to the values observed in control arteries or even exceeds the control values (veins, Wharton's jelly). CONCLUSIONS: Preeclampsia is associated with a reduction in the activity of cathepsin D in human umbilical cord. The low activity of cathepsin D may reduce collagen degradation and enhance its accumulation in the umbilical cord, especially in the arteries. Similar changes in other foetal blood vessels may result in an increase of vascular resistance and hypertension, which may persist after birth.

Proteoglycans of Wharton's jelly.

Wharton's jelly (WJ) is a myxomatous substance surrounding the blood vessels of the umbilical cord. Proteoglycans (PGs) of Wharton's jelly have not been studied to date therefore it was decided to explore proteoglycan composition of this tissue. Proteoglycans were subjected to dissociative extraction with 4M guanidine hydrochloride containing Triton X-100 and protease inhibitors, purified by Q-Sepharose anion-exchange chromatography and lyophilised. They were analysed by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before and after treatment with chondroitinase ABC. It was found that 1g of Wharton's jelly contains 2.43+/-0.63mg (n=10) of sulphated glycosaminoglycans (GAGs), reflecting the presence of proteoglycans. The proteoglycans were mainly substituted with chondroitin/dermatan sulphate (DS) chains. The predominant proteoglycan fraction included small proteoglycans with core proteins of 45 and 47kD, immunologically related to decorin (45 and 47kD) and biglycan (45kD). The expression of decorin core proteins was much higher than that of biglycan. Larger proteoglycans (core proteins of 90, 110, 220 and 260kD) were found in lower amounts. The most abundant of them (core protein of 260kD) was immunologically related to versican. Perlecan was not identified in Wharton's jelly. The study shows that Wharton's jelly contains mainly small chondroitin/dermatan sulphate proteoglycans, with decorin strongly predominating over biglycan. We suggest that an intensive expression of decorin is associated with very high content of its ligand, collagen.