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.

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.

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.

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.

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.

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.

[Tumor necrosis factor in uterine leiomyomas at various stages of tumor growth]. / Czynnik martwicy nowotworów (TNF) w przebiegu wzrostu miesniaków macicy.

OBJECTIVES: The aim of the study was to evaluate the expression of tumor necrosis factors -alpha and beta (TNF), their receptor and content in human uterine leiomyomas at various stages of tumor growth. MATERIAL AND METHODS: Studies were performed on human myometrium and uterine leiomyomas of various weights (small: less than 10 g and large: more than 100 g). Presence of both growth factors and their receptor was detected by Western Immunoblotting technique. The content of TNF-alpha was evaluated by immunoenzymatic method (ELISA). RESULTS: Changes in the expression of tumor necrosis factors and their receptor and difference in content of TNF-alpha during the tumor growth were found. CONCLUSIONS: Myometrium conversion into leiomyoma and an increase in its mass is accompanied by changes in the expression and contents TNF and TNF RI.

[Fibroblast growth factor gene expression in uterine leiomyomas]. / Ekspresja genu czynnika wzrostowego fibroblastów w miesniakach macicy.

OBJECTIVES: The aim of the study was the evaluation of acidic and basic FGF expression, as well as collagenolitic activity in human uterine leiomyomas at various stages of tumour growth. MATERIAL AND METHODS: 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). The RT-PCR method was used to determine the acidic and basic FGF mRNA levels. The content of both FGF was evaluated by immunoenzymatic method (ELISA). The collagenolitic activity was detected by zymography. RESULTS: A distinct increase in the expression of aFGF, the amounts of both FGFs, and collagenolitic activity was observed during the tumour growth. CONCLUSIONS: Myometrium conversion into leiomyoma and an increase in its mass is accompanied by a significant increase in aFGF gene expression. The collagenolitic activity elevation favours a release of both FGF isoform from complexes with extracellular matrix components.

Transforming growth factor beta and platelet-derived growth factor in human myometrium and in uterine leiomyomas at various stages of tumour growth.

OBJECTIVE: Some authors suggest that growth factors are intermediate regulatory elements through which the ovarian hormones exert their growth-stimulatory effects on uterine leiomyomas. STUDY DESIGN: It was decided to compare the amounts of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) in myometrium and in uterine leiomyomas of various weights (small: less than 10 g and large: more than 100 g). The tissues were homogenised and extracted with 1M acetic acid or with 0.05 M Tris-HCl, pH 7.6. The extracts were assayed for TGF-beta and PDGF with the use of the ELISA technique. RESULTS: The Tris-HCl was more efficient at extracting solvent than 1M of acetic acid. Both myometrium and leiomyomas contained nanogram amounts of extractable TGF-beta and picogram amounts of PDGF. Western immunoblotting demonstrated that both factors exist as stable complexes, probably with extracellular matrix components. The PDGF/TGF-beta ratio in Tris-HCl extracts was higher in leiomyomas than in myometrium and it increased during tumour growth. CONCLUSION: It is known that low concentrations of TGF-beta induce proliferation of cells by stimulating autocrine PDGF secretion. Higher concentrations of TGF-beta1 evoke a reverse effect by the down-regulation of the PDGF receptor and by direct growth inhibition. The increase in the PDGF/TGF-beta ratio during tumour growth seems be important in tumour biology. The low amount of TGF-beta eliminates the inhibitory effect of this factor on cell proliferation and stimulates both autocrine PDGF secretion and promotes the synthesis of PDGF receptors. It is thus possible to bind more PDGF by myometrial cells resulting in a hyperplasia of myometrium and enhancement of extracellular matrix synthesis.

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.

[Epidermal growth factor (EGF) in uterine leiomyoma during tumour growth]. / Czynnik wzrostu naskórka (EGF) w przebiegu wzrostu miesniaków macicy.

OBJECTIVES: Extracellular matrix is a place where various growth factors are bound and immobilised. It is expected that leiomyoma-associated remodelling of extracellular matrix in the uterus may be evoked by changes in the content of some growth factors. DESIGN: The amount and distribution of EGF in the normal myometrium and in the leiomyoma during various growth stages were investigated. MATERIAL AND METHODS: The assay of EGF was carried out with the use of ELISA commercial kit. SDS/polyacrylamide gel electrophoresis of tissue extracts followed by Western immunoblot was performed. RESULTS AND CONCLUSIONS: It was found that all investigated tissues contained endogenous EGE Extractability of EGF depended on type of extracting solvent. Only slight amount of EGF could be extracted by 1 M acetic acid. Much more EGF could be solubilized in 0.05M Tris/HCl, pH 7.6. Our results showed that EGF bound to the uterus (normal and leiomyoma) components of different molecular mass. It is of interest that some components of both, acidic and neutral extracts (myometrium and leiomyomas) were not able to bind exogenous 125I-labelled EGF.

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.

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.

[Basic fibroblast growth factor in leiomyoma]. / Zasadowy czynnik wzrostu fibroblastów w miesniakach macicy.

Fibroblast growth factor (FGF) is a potent stimulator of collagen and glycosaminoglycan biosynthesis and may play an important role in extracellular matrix (ECM) remodelling. Heparin sulphate was shown to be the major proteoglycan molecule in ECM of leiomyoma. It shows ability to bind some growth factors, including FGF. It was decided to evaluate bFGF presence and binding in leiomyoma tissues. Our results show that bFGF binds to the leiomyoma components of different molecular mass. Most of bFGF was identified in large leiomyoma. We suggest that the level of bFGF in leiomyoma tissue may reflect the intensity of tumour growth.

An accumulation of insulin-like growth factor I (IGF-I) in human myometrium and uterine leiomyomas in various stages of tumour growth.

It is commonly thought that uterine leiomyomas result from hyperstimulation of myometrium by ovarian hormones. Some observations suggest that cytokines and growth factors are intermediate elements through which the ovarian hormones may exert their growth-stimulatory effects on leiomyomas. Human myometrium and uterine leiomyomas of various weights were homogenised and extracted with 1 M acetic acid or with 0.05 M Tris/HCl, pH 7.6. The extracts were assayed for IGF-I using the ELISA technique. It was found that 0.05 M Tris/HCl extracts contained several times more IGF-I than the 1 M acetic acid extracts. Nanogram amounts of IGF-I were found in both control myometrium and in leiomyomas. It was found that the amounts of IGF-I extracted from leiomyomas were distinctly higher in comparison to control myometrium and they increased as a function of tumour growth. Polyacrylamide gel electrophoresis, followed by Western immunoblotting, demonstrated that IGF-I in acidic and alkaline extracts exists as stable complexes, probably with extracellular matrix components. No free IGF-I was detected. Furthermore, it was found that some components of both the acidic and alkaline extracts were able to bind exogenous (125)I-labeled IGF-I. It is suggested that IGF-I plays an important role both in myometrium biology and in the growth of uterine leiomyomas.

[Extracellular matrix of the umbilical cord vein in EPH gestosis and fibroblast growth factor]. / Skladniki macierzy pozakomórkowej zyly pepowinowej w przebiegu gestozy EPH a czynnik wzrostu fibroblastów (FGF).

It was found in our previous studies that EPH gestosis is accompanied by an extensive remodeling of the extracellular matrix of the umbilical cord. Studies were performed on the umbilical cord veins of 10 control and 10 newborns delivered by mothers with EPH gestosis. It was decided to determine umbilical cord vein ability to bind of labeled (125J)-basic fibroblast growth factor (bFGF), and FGF content by Western immunoblot and ELISA methods. Our experiments indicated that the extracts of umbilical cord vein contain endogenous bFGF and several soluble FGF-binding compounds of different molecular weight. It is of interest that EPH gestosis is associated with a decrease in bFGF content. It seems be possible that the decrease of bFGF amount may be one of the factors, which constrain the biosynthesis of collagen in EPH gestosis umbilical cord vein wall.

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.

[Gelatinolitic and proteolytic activity of Wharton's jelly in EPH-gestosis (preeclampsia)]. / Aktywnosc enzymów zelatynolitycznych i proteolitycznych galarety Whartona w przebiegu gestozy EPH (preeclampsia).

EPH-gestosis is accompanied by an extensive remodeling of the extracellular matrix of Wharton's jelly. The gelatinolytic and proteolytic activities were measured. A decrease in gelatinolityc activity and an increase in proteolytic activity in EPH-gestosis were observed. The decrease in gelatinase activity in EPH-gestosis may be one of factors involved in extracellular matrix rebuilding of Wharton's jelly.

Glycosaminoglycan-degrading enzymes in the skin of fasted rats.

During fasting of animals, there is decreased content of skin glycosaminoglycans (GAGs) accompanied by decrease in their biosynthesis. Since tissue GAG content depends on both synthesis and degradation of these molecules, we asked whether fasting affects the activity of several tissue glycosidases. Therefore we measured the activity of skin neutral and acidic endoglycosidases, some exoglycosidases: beta-N-acetylhexosaminidase [EC 3.2.1.30], beta-galactosidase [EC 2.1.23], beta-glucuronidase [EC 3.2.1.31], alpha-iduronidase [EC 3.2.1.76], and two sulfatases: arylsulfatase B [EC 3.1.6.1] and 6-sulfatase [EC 3.1.6.14] in the skin of control and fasted rats. Although fasting was accompanied by distinct decrease in the activity of most neutral endoglycosidases, no characteristic changes in the activity of exoglycosidases were found. In contrast, we found that fasting is associated with increase in the activity of acidic endoglycosidases (of lysosomal origin) which degraded hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate and heparin. The same GAGs were decreased in the skin of fasted rats. Our data suggest that the phenomenon is a result of increased intracellular degradation of these molecules. Therefore, not only decreased biosynthesis of GAGs during fasting, but also increased their intracellular degradation may contribute to decrease in GAG skin content.