Grade-dependent changes in sphingolipid metabolism in clear cell renal cell carcinoma.

There is a host of evidence for the role of bioactive sphingolipids in cancer biology, and dysregulated sphingolipid metabolism was observed in many malignant tumors. The aim of the present study was to provide more detailed data on sphingolipid metabolism in different stages of clear cell renal cell carcinoma (ccRCC). Samples of the tumor and noncancerous fragments of the same kidney were collected from patients who underwent a radical nephrectomy. The subjects were stratified according to the degree of malignancy of the tumor (n = 14 for G2, 12 for G3, and 9 for G4). The content of bioactive sphingolipids/glycosphingolipids was measured with an HPLC and HPTLC method, and the mRNA and protein expression of sphingolipid transporters and metabolizing enzymes was evaluated using real-time polymerase chain reaction (PCR) and Western blot, respectively. Compared to healthy kidney tissue, ccRCC was characterized by accumulation of sphingosine, sphingosine-1-phosphate (S1P), ceramide, dihydrosphingosine, and dihydroceramide. However, in the case of the latter two, the accumulation was limited to higher malignancy grades. In addition, compared to the healthy tissue, the content of gangliosides in the tumor was increased at the expense of globosides. We also found profound grade-dependent changes in the mRNA level of S1P-metabolizing enzymes, and spinster homolog 2. In general, their expression was much higher in G2 tumors compared to higher malignancy grades. We conclude that ccRCC is characterized by profound and multilevel alterations in sphingolipid metabolism, which to a large extent are grade-dependent. We hypothesize that dysregulation of sphingolipid metabolism contributes to the progression of ccRCC.

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.

Suppressed Expression but Not Activity of Collagenases MMP-1 and MMP-13 in Human Renal Carcinoma.

BACKGROUND: Collagenases are enzymes starting collagen degradation. The role of collagenases in renal carcinoma development is not well understood. OBJECTIVE: Evaluation of collagen content and collagenase expression and activity in human kidney cancers. METHODS: Collagen content was measured by the hydroxyproline assay. The expression and the content of collagenases were evaluated by Western blotting and ELISA. Fluorogenic substrate was used to measure enzyme activity. RESULTS: Collagen content significantly decreases with the progression of kidney cancer. Both collagenases are first present in high molecular complexes in both control and cancer tissue. The healthy part of the kidney contains similar amounts of both collagenases. Collagenase content decreased significantly in tumor tissue with increasing cancer stage. MMP-13 activity is much higher than that of MMP-1 in all tissues investigated. We observed increasing collagenase activity (MMP-1 and MMP-13) with increasing renal cancer grade. CONCLUSIONS: The lower content and higher activity of the collagenases investigated in cancer tissue indicate that most of these enzymes are in active form in renal carcinoma. The lower collagen content in cancer tissue can be explained at least in part by increased collagenase activity.

A New Analytical Method for Determination of Cathepsin L Based on the Surface Plasmon Resonance Imaging Biosensor.

The purpose of this study was to develop a new method for a determination of the cathepsin L-biosensor based on the Surface Plasmon Resonance Imaging technique. The cathepsin L is an endopeptidase, which degrades proteins and plays an important role in various processes occurring in the human body. The detection technique, Surface Plasmon Resonance Imaging, is an optical, label-free technique, which can be used for quantitative determination of the different proteins. In order to bind the enzyme, the cathepsin L inhibitor-RKLLW-NH2 was used. The validation process showed that parameters: precision, accuracy, and selectivity of the method were acceptable. The analytically useful range of the standard curve was 0.50 ng/mL-15.00 ng/mL. The detection and quantification limit of method was 1.67 pg/mL and 5.07 pg/mL, respectively. The usefulness of the developed method was confirmed by the determination of the cathepsin L concentration in the blood plasma of some healthy persons and in the blood plasma of patients. The obtained results were compared with the results obtained by the ELISA. It was found that the correlation between these two methods was very strong, what suggest that the developed method can be used as the competitive method to the ELISA.

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1.

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor-UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5ng/ml. The detection limit is 0.06ng/ml for the biosensor with antibody and 0.08ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20ng/ml for plasma samples and 0.30 to 0.49ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.

Higher Content but No Specific Activity in Gelatinase B (MMP-9) Compared with Gelatinase A (MMP-2) in Human Renal Carcinoma.

Gelatinases belong to a group of enzymes known as matrix metalloproteinases (MMPs). Gelatinases A and B (MMP-2 and MMP-9, respectively) are the enzymes with the highest ability to destroy collagen, primarily type IV collagen, which is an essential component of the base membrane. Hence, it can be assumed that they are involved, among other things, with the metastasis process of cancer. As a result, the objective of this study was to assess the presence, activity, and expression of selected gelatinases in human renal cancer. Healthy (n = 20) and clear-cell kidney cancer tissue samples (G2 n = 10, G3 n = 10) were analyzed. The presence and content of MMPs were measured using the Western blot and ELISA methods, respectively. The activity (actual and specific) was analyzed with a fluorimetric method. The presence of both investigated enzymes was demonstrated in the representative zymogram. MMP-9 showed the most intensive saturation. It has been observed that both gelatinases occur primarily in high molecular complexes in the human kidney, regardless of whether it is a control or tumor tissue. Both gelatinases were present in comparable amounts in healthy tissues of the kidney. MMP-9 showed a higher content than MMP-2 in both renal cancer grades, but we observed the enhanced activity of both gelatinases with an increase in the grade of renal cancer. A higher MMP-9 content and, on the other hand, lower specific activity in the cancer tissue suggest that MMP-9 is predominantly present in an inactive form in renal cancer. The higher activity of MMP-9 demonstrated using the zymography method may be a cause of different values of activity that depend on the phase of the carcinogenic process. The present study revealed changes in the tested gelatinases in healthy and cancerous tissues of renal cell carcinoma. Therefore, it can be concluded that matrix metalloproteinases 2 and 9 are enzymes directly involved in carcinogenesis, and hence, it seems that MMPs may have potential in the diagnosis and treatment of renal carcinoma.

Higher Content but Not Activity of Stromelysin-2 (MMP-10) in Comparison to Stromelysin-1 (MMP-3) in Human Renal Carcinoma.

Stromelysin-1 and stromelysin-2 (matrix metalloproteinase 3; MMP-3 and matrix metalloproteinase 10; MMP-10, respectively) are enzymes that activate other metalloproteinases. Apart from collagen, they also degrade elastin, fibronectin, gelatin and laminin. In carcinogenic processes, they are involved in angiogenesis and metastasis. Therefore, the aim of this study was to evaluate the DNA content, expression and activity of both stromelysines in cancers of human kidney. Renal carcinoma tissue samples were analyzed. Low- and high-grade cancer tissues were collected. Control material was collected from part of the kidney opposite to the tumor. DNA content, stromelysines content and stromelysin-1 and stromelysin-2 activity were measured using ELISA and Western blot methods. A higher content of deoxyribonucleic acid in low- and high-grade cancer tissues in comparison to the respective control tissue was observed. Both stromelysines were presented in control and cancer tissues in high-molecular-weight complexes. The content of MMP-10 was significantly higher in comparison to MMP-3 in all investigated tissues. Moreover, the content of stromelysin-2 was significantly higher in high-grade (G3) tissues compared to grade 2 (G2) kidney cancer. A significant decrease in the actual and specific activities of both stromelysines was observed with the increase in renal cancer grade. The presented results may indicate that the degradation of extracellular matrix increases with a higher grade of cancer. Moreover, the elevated content and decreased specific activity of stromelysin-2 in cancer tissue indicate that MMP-10 is mainly present in an inactive form in renal carcinoma. Detailed knowledge of the mechanism and participation of stromelysines in extracellular matrix degradation may be important in understanding the pathomechanism of renal cancer development. Therefore, the potential application of stromelysines in the monitoring or prognosis of kidney cancer should be discussed.

Enhanced Expression but Decreased Specific Activity of Matrix Metalloproteinase 10 (MMP-10) in Comparison with Matrix Metalloproteinase 3 (MMP-3) in Human Urinary Bladder Carcinoma.

Human urinary bladder cancer is a huge worldwide oncological problem causing many deaths every year. The degradation of extracellular matrix (ECM) induced by molecules such as matrix metalloproteinases (MMPs) is one of the main factors influencing the process of metastasis origination. The MMP expression is tied to tumor aggressiveness, stage, and patient prognosis. The cleavage of constituent proteins is initiated and prolonged by matrix metalloproteinases, such as MMP-3 and MMP-10. The aim of this study was to evaluate the expression and activity of both MMPs in human urinary bladder cancer occurring at various stages of the disease. Tissue samples from patients with urinary bladder cancer were analyzed. Samples were collected from patients with a low- and high-grade cancer. Control tissue was collected from the site opposite to the tumor. DNA content, MMPs content, and activity of MMP-3 and MMP-10 were measured using ELISA and Western blot techniques. MMP-3 and MMP-10 occur in high molecular complexes in human urinary bladder in healthy and cancerous tissues. Particularly, in high-grade tumors, the content of MMP-10 prevails over MMP-3. The actual and specific activities vary in both grades of urinary bladder cancer; however, the highest activity for MMP-3 and MMP-10 was found in low-grade tissues. In conclusion, MMP-10 had a higher content, but a lower activity in all investigated tissues compared to MMP-3. Generally, obtained results demonstrated a contrary participation of MMP-3 and MMP-10 in ECM remodeling what may be crucial in the pathogenesis of human urinary bladder carcinoma.

Lipid compounds of human Wharton's jelly and their alterations in preeclampsia.

Wharton's jelly is a myxomatous substance which surrounds the umbilical cord vessels protecting them against extension, bending, twisting and compression. Very low number of cells in this tissue produce high amounts of extracellular matrix; collagen, hyaluronate and proteoglycans which bind large quantities of peptide growth factors (PGFs). Preeclampsia (the most common pregnancy-associated syndrome) is accompanied by a significant reduction in hyaluronate and a concomitant increase in sulphated glycosaminoglycans/proteoglycans content in Wharton's jelly. Such a phenomenon corresponds to an 'early ageing' of this tissue. We have evaluated the lipid composition of Wharton's jelly and its alteration in preeclampsia. Thin layer chromatography and high-performance liquid chromatography were employed. It was found that Wharton's jelly contains free fatty acids (FFA), mono-, di- and triacylglycerols, free cholesterol and its esters. The characteristic feature is the presence of relatively high amounts of unsaturated fatty acids, including those (C18:2 and C18:3) which are nutritionally essential. Preeclampsia is associated with a slight increase in the total fatty acid content in Wharton's jelly and with marked changes in the proportional relationships between various lipids. A distinct decrease in the amounts of FFA was observed with a concomitant increase in monoacylglycerols and cholesterol esters. At least in some cases the effects exerted by PGFs are mediated by the lipid second messengers. Thus it is possible that alterations in lipid compounds of Wharton's jelly may participate in the deregulation of various cell functions, including overproduction of sulphated glycosaminoglycans or down-regulation of enzymes which participate in their degradation.

Sphingolipids of human umbilical cord vein and their alteration in preeclampsia.

Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by the accumulation of free fatty acids, acylglycerols and cholesterol esters in the umbilical cord vein (UCV). We evaluate the sphingolipid composition of UCV and its alteration in preeclampsia. The veins were taken from 10 newborns delivered by healthy mothers and 10 newborns delivered by mothers with preeclampsia. Thin layer chromatography, solid-phase extraction and high-performance liquid chromatography were employed for sphingolipid analyses. The UCV walls of newborns delivered by healthy mothers are abundant in sphingomyelins and ceramides, whereas the amounts of sphingoid bases are rather low. Preeclampsia is associated with a significant decrease in sphingomyelins and ceramides, whereas the sphingoid bases changed in uncharacteristic manner. The increase in sphinganine and sphingosine 1-phosphate was accompanied with a decrease in sphingosine, hydroxysphinganine and sphinganine 1-phosphate. Stearate is the dominating fatty acid in sphingomyelins and ceramides of both control and preeclamptic veins. Sphingolipids and some sphingoid bases are bioactive molecules which contribute to regulation of signal transduction pathways, protein sorting and mediation of cell-to-cell interactions and recognition. The alteration in sphingolipid content may modify the metabolism of UCV wall resulting in remodelling of its composition.

Altered sphingolipid composition in Wharton's jelly of pre-eclamptic newborns.

OBJECTIVES: Wharton's jelly is a myxomatous substance surrounding the umbilical cord (UC) vessels to protect them against extension, bending, twisting and compression. Pre-eclampsia (hypertension, oedema, proteinuria) is the most common pregnancy-associated pathological syndrome. It is accompanied by significant alterations in UC composition. Here we describe the sphingolipids of Wharton's jelly from 10 newborns delivered by healthy mothers and from 10 babies of pre-eclamptic mothers. METHODS: Thin-layer chromatography, solid-phase extraction and high-performance liquid chromatography were employed. RESULTS: Control tissues were abundant in sphingomyelins (Sms) and ceramides (Cers), whereas the amounts of sphingoid bases were distinctly lower. Pre-eclampsia is associated with a significant increase in Sms, Cers, sphingosine, sphinganine, 4-OH-sphinganine, sphingosine 1-phosphate and glycosylated sphingosine. Furthermore, a decrease in sphinganine 1-phosphate was found. Stearate (C(18:)(0)) is the dominating fatty acid in Sms and Cers of control tissue. In contrast, pre-eclamptic material contained the highest amount of laurate (C(12:)(0)) in Sms and myristoleate (C(14:)(1)) in Cers. CONCLUSIONS: Sphingolipids and some sphingoid bases are bioactive molecules which contribute to the regulation of signal transduction pathways, protein sorting and mediation of cell-to-cell interactions and recognition. The alteration in sphingolipid content may modify the metabolism of Wharton's jelly, resulting in remodelling of its composition.

Dominative role of MMP-14 over MMP-15 in human urinary bladder carcinoma on the basis of its enhanced specific activity.

BACKGROUND: Human urinary bladder cancer is one of the most common cancers worldwide with the mortality rate of approximately 165,000 people annually. The modulation of extracellular matrix is a crucial event in the metastatic spread, among others in angiogenesis. It is initiated and prolonged by the cascade of matrix metalloproteinases. MMP-14 and MMP-15 are associated with a high degree of malignancy, aggressiveness, and survival prognosis by the activation of other matrix metalloproteinases (MMPs). This study was aimed at evaluating the expression and the activity of selected transmembrane metalloproteinases at different stages of human urinary bladder cancer. METHODS: Western blot and enzyme linked immunosorbent assay (ELISA) method were used to evaluate the expression and content of MMPs and TIMP-1. The activity of studied enzymes was determined with fluorometric method. RESULTS: Both transmembrane metalloproteinases are found in healthy or cancerous tissue in high molecular complexes of human urinary bladder. MMP-14 dominates over MMP-15, particularly in high-grade urinary bladder cancer. Their contents significantly change with the grade of bladder tumor. The amount of MMP-14 increases with increasing grade of tumor. MMP-15 content decreases in high-grade bladder cancer. With increasing grade of urinary bladder cancer their actual activity (per kg of total protein content) is varying in different ways. In all examined tissues, the specific activity of MMP-15 (per kg of the enzyme content) is much higher in comparison to MMP-14. Human urinary bladder cancer contains higher TIMP-1 amounts than control tissue but with the decrease with an increase in tumor grade. CONCLUSION: Comparison of investigated enzymes' activity and the inhibitor content suggests it opposite effects, higher suppression of MMP-14 than MMP-15 activity in low-grade bladder cancer and reverse TIMP-1 action in high-grade cancer. The MMP-14 activity determination in urinary bladder cancer tissue may be used as a predictor of a risk of metastasis.

Peptide growth factors and their receptors in the vein wall.

The varicose vein wall remodeling is a very complex process, which is controlled by numerous factors, including peptide growth factors. The aim of the study was to assess a/b FGF, IGF-1, TGF-ß1, VEGF-A and their receptors in the vein wall. Varicose vein samples were taken from 24 patients undergoing varicose vein surgery. The control material consisted of vein specimens collected from 12 patients with chronic limb ischemia. Contents of aFGF, bFGF, IGF-I, TGF-ß1, VEGF, IGF-1R, VEGF R1 and VEGF R2 were assessed with ELISA method. Protein expression of FGF R1 and TGF-ß RII were evaluated with western blot. Increased contents of aFGF, IGF-1 and VEGF-A were found in varicose veins in comparison with normal ones (p<0.05). In contrast, a significant decrease in TGF-ß content was demonstrated in varicose veins (p<0.05). Furthermore, there was no difference in bFGF content in both groups (p>0.05). IGF-1 R content was significantly increased in varicose veins (p<0.05). There was no difference in VEGF R1 content between varicose and normal veins (p>0.05), whereas VEGF R2 content was significantly increased in varicose veins (p<0.05). Western blot demonstrated increased expression of TGF-ß RII in varicose veins (p<0.05) and similar expression of FGF R1 in both groups (p>0.05). Demonstrated changes in peptide growth factors and their receptors may disturb metabolism of extracellular matrix in the varicose vein wall and contribute to the development of the disease to its more advanced stages.

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.

SPR imaging biosensor for the quantitation of fibronectin concentration in blood samples.

The purpose of this study was presentation of a new biosensor capable of determination of fibronectin. This biosensor was based on the specific interaction of anti-fibronectin antibody produced in rabbit with fibronectin. The surface plasmon resonance imaging (SPRI) technique was used as a detecting method. Optimization and characterization properties of the biosensor were studied. The determination of fibronectin concentration in natural samples was done. The results were compared with a reference method (Enzyme-Linked Immunosorbent Assay-ELISA). The analytically useful dynamic response range of biosensor is between 5 and 400ngmL-1. The detection limit is 1.5ngmL-1 and limit quantification is 5ngmL-1. The proposed SPRI biosensor showed good selectivity for potential interferences. It was applied to determine fibronectin concentrations in plasma of healthy donors and of patients after thermal injury. Good correlations between results obtained using the SPRI biosensor and ELISA test (correlation coefficients for healthy donors 0.996, for patients 0.984) were obtained. The average fibronectin concentration of healthy donors was 140.5±24.6µgmL -1 and the average fibronectin concentration of patients was 601.5±72.1µgmL -1, which was in agreement with results obtained by other investigators. The obtained results indicate that the developed biosensor may be a candidate for monitoring fibronectin concentration in blood samples.

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.

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.

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.

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.