Małgorzata Wolańska

Extracellular matrix components in uterine leiomyoma and their alteration during the tumour growth

It was found that both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans. In contrast to many normal and tumour tissues the amount of hyaluronic acid is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. It is of interest that heparan sulphate is the major glycosaminoglycan component both in normal myometrium, and in leiomyoma. The amount of hyaluronic acid in myometrium and in the leiomyoma is very low. No significant change in hyaluronate content was observed during the tumour growth. In contrast to that the amount of some sulphated glycosaminoglycans (heparan sulphate, keratan sulphate, chondroitin sulphates and heparin) distinctly increased. It is suggested that some of the GAGs participate in the creation of a storage depot for biologically active molecules (growth factors, enzymes) which are thereby stabilized and protected. Hydrolytic degradation of some GAGs may result in the release of some cytokines which may promote the tumour growth and stimulate collagen biosynthesis by tumour cells.

Matrix Metalloproteinases of Human Leiomyoma in Various Stages of Tumor Growth

The amounts and activities of matrix metalloproteinases (MMPs) were studied in human myometrium and uterine leiomyomas in various stages of growth. It was found that both myometrium and the investigated tumors contain collagenolytic enzymes. MMP-1, MMP-2, MMP-3 and MMP-9 were found. Gelatinase A (MMP-2) is the most abundant. In control myometrium only 10% of this enzyme exists in an active form, whereas in tumors, especially in large ones, the values reach 30%. It is suggested that the high activity of MMP-2 is responsible for remodelling of extracellular matrix in the growing tumors.

Evaluation of enzymes involved in proteoglycan degradation in the wall of abdominal aortic aneurysms

The abdominal aortic aneurysm (AAA) wall represents an extreme example of arterial remodeling with disturbed elastin, collagen and proteoglycan metabolism. The aim of this study was to evaluate enzymes involved in the degradation of glycosaminoglycan chains and core proteins of proteoglycans in the AAA wall. The study material consisted of wall samples from 10 AAA. Fragments of 5 normal abdominal aortas from organ donors were used as a control. The activity of endoglycosidases, exoglycosidases and sulfatases was measured using colorimetric methods. To assess matrix metalloproteinases (MMPs), Western blot and zymography were performed. The activity of endoglycosidase degrading chondroitin-4-sulfate was lower in the AAA wall. Endoglycosidase degrading heparan sulfate and dermatan sulfate, arylosulfatase B, as well as all the exoglycosidases assessed demonstrated higher activities in the AAA wall. Furthermore, increased expression of MMP1, MMP2 and MMP9 was also shown in the AAA wall. Zymography revealed decreased activity of pro-MMP2 and presence of pro-MMP9 in the AAA wall compared to the wall of normal aorta. Extensive changes in the activity of glycosaminoglycan-degrading enzymes and MMPs may influence the organization of the extracellular matrix network and lead to previously demonstrated changes in the proteoglycan and glycosaminoglycan content in the AAA wall.

The Activity of Collagen-Degrading Enzymes of Wharton’s Jelly in EPH Gestosis (Pre-Eclampsia)

Oedema/proteinuria/hypertension (EPH) gestosis is one of the more common complications observed during pregnancy. Our previous studies demonstrated some qualitative and quantitative changes in the extracellular matrix of Wharton’s jelly in newborns delivered by mothers with EPH gestosis. For this reason it was decided to evaluate the effect of EPH gestosis on the activity of gelatinolytic and proteolytic enzymes which may be involved in collagen degradation in Wharton’s jelly. Zymographic analysis of control and EPH gestosis samples of Wharton’s jelly demonstrates different electrophoretic patterns of gelatinolytic enzymes. The control Wharton’s jelly contains two latent forms of gelatinolytic enzymes: gelatinase A [metalloproteinase (MMP)-2, 72 kD] and gelatinase B (MMP-9, 92 kD). In contrast to control tissue, the main gelatinolytic enzyme of EPH gestosis Wharton’s jelly is gelatinase A (MMP-2). It was found that the proteolytic activity in EPH gestosis Wharton’s jelly differs from control. The decrease in gelatinase activity may be one of the factors which promote the accumulation of collagen in this tissue.

Does vascular endothelial growth factor participate in uterine myoma growth stimulation

OBJECTIVE Peptide growth factors play a role in the rebuilding of extracellular matrix in the course of leiomyoma growth, and exert a regulative effect on the cell only when they bind with a specific membrane receptor and transmit a signal into the cell. A high content of certain peptide growth factors and their receptors in leiomyoma suggests that in the course of the tumour growth hyperstimulation of cells takes place. A combined action of various peptide growth factors causes an amplification of signal paths in cells, inducing gene expression of proteins responsible for cell division and changes of metabolism. We therefore decided to evaluate the amounts and expression of VEGF, their receptor and mRNA levels. STUDY DESIGN Studies were performed on human myometrium and uterine leiomyomas of various weights (small: i.e. less than 10 g, and large: i.e. more than 100 g). Expression and content of VEGF-A, D and VEGF R-1, R-2 were analysed with Western blot and ELISA methods, respectively. The RT-PCR method was used to determine VEGF mRNA levels. RESULTS Our immunoblotting studies and immunoenzymatic assay, as well as RT-PCR technique, did not detect significant differences in the expression of VEGFs and their receptors in control myometrium and in uterine leiomyomas. CONCLUSION The increase in the amount of some peptide growth factors, especially FGFs and IGF-I, in large leiomyomas without any change in VEGF content means a decrease in the proportional relationship of the latter to other growth factors. Stimulation of extracellular matrix formation seems stronger than angiogenesis during myoma growth.

The activities of some glycosaminoglycan-degrading enzymes in uterine leiomyomas

The activities of some glycosaminoglycan-degrading enzymes in uterine leiomyomas. Both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans (GAGs). In contrast to many normal and tumour tissues the amount of hyaluronic acid (HA) is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. We compared the activity of GAG-degrading enzymes in normal myometrium and in uterine leiomyomas. Growth of uterine leiomyomas results in significant reduction in the activities of neutral endoglycosidases degrading most of the sulphated glycosaminoglycans. The activities of acid endoglycosidases also decreased (with the exception of chondroitin-6-sulphate). Thus, the differentiated activity of glycosidases degrading glycosaminoglycans can be a factor modifying the quantity of GAGs.

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 Dunns 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.

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 Whartons 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 Whartons 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 Whartons jelly. The effect of cysteine protease inhibitors showed similar distribution as in the case of the active enzyme, being the highest in Whartons 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 Whartons jelly.

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.

High Molecular Complexes of Insulin-Like Growth Factor-I in Human Saliva

Insulin-like growth factor-I is one of the most important growth factors involved in oral biology. It circulates in plasma in a form of complex with binding proteins—IGFBPs and acid labile subunit—ALS. It was decided to assess the content of IGF-I in human saliva in relation to other proteins, and the expression and content of its binding proteins and ALS of healthy people of different gender and age. Research material was mixed resting saliva obtained from 70 healthy volunteers, which were divided into seven groups, taking into account age and gender. For qualitative and quantitative evaluation of IGF-I complexes with IGFBP-5 and ALS there were used: western immunoblot and ELISA assay. It was shown that human saliva contained IGF-I mainly in the form of macromolecular complexes. Expression and content of IGFBP-5 and ALS were affected by gender and age.