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.

Identification and localization of S100A6 in human umbilical cord.

S100A6, a calcium-binding protein also known as calcyclin, was detected in human umbilical cord by immunoblotting. Immunohistochemical studies showed an intensive reaction for S100A6 in the walls of vessels and Wharton's jelly. In the latter, S100A6 was found not only in the myofibroblasts but also in the ECM (extracellular matrix) surrounding these cells. Affinity chromatography of S100A6 resin indicated that Wharton's jelly contains some proteins that could bind to S100A6. Thus these novel results show the presence of S100A6 in umbilical cord and suggest the involvement of this protein in intra- and extra-cellular signalling pathways in this tissue.

Glucose deficiency reduces collagen synthesis in breast cancer MCF7 cells.

We decided to study the effect of glucose deprivation on collagen metabolism in MCF7 cells. The incorporation of [3H]-proline into collagenase-sensitive and hydroxyproline-containing proteins was used as an index of collagen synthesis, whereas pulse-chase technique was employed to evaluate the degradation of newly synthesized proteins. The MCF7 cells incubated in high glucose medium synthesized detectable amounts of collagenous proteins. Most of them were found in the cell layer. The shortage of glucose resulted in about 30% reduction in collagen synthesis. The pulse-chase experiments demonstrated that proportionally less collagen was degraded in cultures incubated in low-glucose than in high-glucose media.

Matrix metalloproteinases, MMP-7 and MMP-26, in plasma and serum of control and preeclamptic umbilical cord blood.

OBJECTIVE: Our previous paper demonstrated that preeclampsia-associated accumulation of collagen and proteoglycans in the umbilical cord tissues is a result of increased biosynthesis and decreased degradation of these components. Metalloproteinases (MMPs) are enzymes engaged in degradation of collagen and protein cores of proteoglycans, including those which bind peptide growth factors. Some MMPs, among them matrilysins MMP-7 and MMP-26, participate in activation other members of the MMP family. STUDY DESIGN: Studies were performed on the umbilical cord blood taken from 10 control (healthy) newborns and 10 newborns of preeclamptic women. We used Western immunoblotting, immunoenzymatic assay (ELISA) and zymography techniques for detection of matrilysins. The results were submitted to Student's t-test and Mann-Whitney test. RESULTS: Umbilical cord blood plasma and serum of control and preeclamptic newborns contained MMP-7 and MMP-26. Both enzymes existed in the form of complexes with other extracellular matrix components and/or their tissue inhibitors in control and preeclamptic subjects. Free latent forms of both matrilysins were observed after the action of reducing agent. Furthermore, we found a distinct increase in the amount of MMP-26 in preeclamptic umbilical cord (UC) blood. No significant differences in MMP-7 content and activity in control and preeclamptic UC blood were observed. CONCLUSIONS: MMP-7 and MMP-26 could activate MMP-9 by cleavage of some sites in pro-MMP-9. Our results suggest that the high activity of MMP-9 participates in a proteolytic release of peptide growth factors from their complexes with extracellular matrix components, which facilitate their interaction with membrane receptors and stimulate cell division and extracellular matrix synthesis in these cells. It may be one of the mechanisms of extracellular matrix remodelling in the umbilical cord of preeclamptic newborns.

Glucose deficiency inhibits glycosaminoglycans synthesis in fibroblast cultures.

We decided to study the effect of glucose deprivation on glycosaminoglycan (GAG) synthesis and degradation in fibroblast cultures, vitality of these cells and a correlation of these processes with the expression of oxygen/glucose-regulated proteins (ORP150/GRP170). The incorporation of [(3)H]-glucosamine into both newly synthesised hyaluronic acid and sulphated GAGs and [(35)S]-sulphate into GAGs was used as an index of glycosaminoglycan synthesis. Quantitative evaluation of newly synthesised GAGs degradation was determined by pulse-chase experiments. We demonstrated that fibroblasts incubated in high glucose medium synthesised significant amounts of GAGs. Most of them were secreted into the culture medium. The shortage of glucose resulted in about 40% reduction in synthesis of GAGs, both those secreted into culture medium and remaining in the cell layer. The pulse-chase experiments demonstrated that the reduced amount of newly synthesised glycosaminoglycans was protected against intracellular degradation. Proportionally less GAGs were degraded in cultures incubated in low glucose than in high glucose media. These phenomena were accompanied by an increase in the expression of chaperon--ORP150 in cultures growing in low glucose medium. We suggest that the increased expression of ORP150 is a factor which prolongs the cell vitality and protects glycosaminoglycans against intracellular degradation induced by glucose deprivation.

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.

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.

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.

Preeclampsia-associated alterations in sphingolipid composition of the umbilical cord artery.

OBJECTIVES: Preeclampsia, the most common pregnancy-associated pathological syndrome, is accompanied by significant remodelling of the extracellular matrix and alteration in lipid composition of the umbilical cord artery (UCA). DESIGN AND METHODS: We evaluate the sphingolipid composition of UCA and its alteration in preeclampsia. Thin layer chromatography, solid-phase extraction and high-performance liquid chromatography were employed for these analyses. RESULTS: The UCA wall is abundant in sphingomyelins and ceramides, whereas the amounts of sphingoid bases are rather low. Preeclampsia is associated with significant increases in sphingomyelins and sphinganine with a decrease in ceramides and other sphingoid bases. CONCLUSIONS: Sphingoids, as secondary messengers, may evoke preeclampsia-associated decrease in hyaluronate and accumulation of collagen, sulphated glycosaminoglycans and cholesterol in the UCA wall. This we propose corresponds to an "early ageing" of the umbilical cord and may be a potential mechanism by which preeclampsia evokes an initiation of hypertension in utero and its amplification through childhood and adult life.

The effect of glucose deprivation on collagen synthesis in fibroblast cultures.

It was decided to study the effect of glucose deprivation on collagen synthesis and degradation in fibroblast cultures and a correlation of these processes with the expression of oxygen/glucose regulated proteins (ORP150/GRP170). The incorporation of radiolabeled proline into collagenase-sensitive and hydroxyproline-containing proteins was used as an index of collagen synthesis, whereas pulse-chase technique was employed to evaluate the degradation of newly synthesised proteins. We demonstrated that fibroblasts incubated in high-glucose medium synthesised detectable amounts of collagenous proteins. Most of them were secreted into the culture medium. The shortage of glucose resulted in about 30% reduction in synthesis of collagenous proteins, both those secreted into culture medium and remaining in the cell layer. The pulse-chase experiments demonstrated that the reduced amount of newly synthesised collagen was protected against intracellular degradation. Proportionally less collagen was degraded in cultures incubated in low-glucose than in high-glucose media. These phenomena were accompanied by an increase in the expression of chaperon-ORP150 in cultures growing in low-glucose medium. We suggest that the increased expression of ORP150 is a factor which protects collagen against intracellular degradation induced by glucose deprivation.

Lipid compounds of the umbilical cord vein and their alterations in preeclampsia.

The lipid composition of vascular walls changes during development, ageing and pathological processes. Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by significant remodelling of the extracellular matrix, both in the umbilical cord vessels and in the surrounding Wharton's jelly. Lipids of the umbilical cord have not been extensively studied. Here we evaluate the lipid composition of the umbilical cord vein and its alteration in preeclampsia. Thin layer chromatography and high-performance liquid chromatography were employed for these analyses. It was found that the umbilical cord vein wall, as with most human tissues, contains free fatty acids, mono-, di- and triacylglycerols, free cholesterol and its esters. The characteristic feature is the presence of high amounts of monounsaturated fatty acids, mainly myristoleic acid (C14:1) and oleic acid (C18:1), and polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), which are rather minor lipid components of most human tissues. They exist both in a free form and in a form of acylglycerols and cholesterol esters. Preeclampsia is associated with an increase in the accumulation of free fatty acids, acylglycerols and cholesterol esters in the umbilical cord vein wall, with a proportional reduction in unsaturated fatty acid contents in all the investigated lipid fractions. Total amount of myristoleate was similar to control values. It is suggested that stimulation of lipolysis in maternal tissues increases supply of free fatty acids to foetal blood and promotes the accumulation fatty acids and their esters in some foetal vascular walls.

Lipid compounds of the umbilical cord artery and their alterations in preeclampsia.

The lipid composition of arterial walls changes during development, ageing and pathological processes. Preeclampsia is the most common pregnancy-associated pathological syndrome. It is accompanied by significant remodelling of the extracellular matrix, both in the umbilical cord vessels and in the surrounding Wharton's jelly. Lipids of the umbilical cord have not been extensively studied. Here we evaluate the lipid composition of the umbilical cord artery (UCA) and its alteration in preeclampsia. Thin layer chromatography and high-performance liquid chromatography were employed for these analyses. It was found that the UCA wall, as with most human tissues, contains free fatty acids, mono-, di- and triacylglycerols, free cholesterol and its esters. The characteristic feature of the UCA wall is the presence of high amounts of long chain polyunsaturated fatty acids (PUFA), including eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), which are rather minor lipid components of most human tissues. They exist both in a free form and in a form of acylglycerols and cholesterol esters. Preeclampsia is associated with a marked decrease in most free fatty acids and acylglycerols. The total amount of long chain PUFA: C18:2, C:18:3, C20:4, C20:5 and C22:6 in these lipid fractions is decreased by half, with a concomitant increase in free cholesterol and its esters. We propose that these lower levels of PUFA may reduce prostaglandin synthesis in the arterial wall and thereby impair blood flow in the foetal vascular system, leading to preeclamptic symptoms.

Gelatinase matrix metalloproteinase (MMP)-2 and MMP-9 of the umbilical cord blood in preeclampsia.

BACKGROUND: Preeclampsia is associated with accumulation of collagen and proteoglycans in the umbilical cord tissues as a result of increased biosynthesis and decreased degradation of these components. Matrix metalloproteinases (MMPs) are enzymes engaged in the degradation of collagen and the protein core structures of proteoglycans, including those which bind peptide growth factors. METHODS: We used Western immunoblots, immunoenzymatic assay (ELISA) and zymography techniques for the detection of gelatinases and their inhibitors. RESULTS: We found that both umbilical cord blood plasma and serum of controls and preeclamptic newborns contained MMP-2 and MMP-9, as well tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2. The umbilical cord plasma of preeclamptic subjects contained large amounts of MMP-9 in a form of complexes with other plasma components, and zymographic analysis demonstrated increased gelatinolytic activity at a position corresponding to MMP-9, compared to control samples. By contrast, MMP-2, TIMP-1 and TIMP-2 data showed no significant differences between preeclamptic and control samples. CONCLUSIONS: The high activity of MMP-9 in preeclamptic plasma suggests its participation in the proteolytic release of peptide growth factors from their complexes with other matrix components, with subsequent stimulation of cell division and matrix biosynthesis. We suggest this might represent one of the mechanisms for matrix remodeling in the umbilical cord of preeclamptic newborns.

TGF-beta binding in human Wharton's jelly.

Our previous study reported that TGF-beta may be isolated from human Wharton's jelly (WJ) in a form of soluble, high molecular complex(es). We decided to study the effect of extracellular matrix degradation and reduction of disulphide bridges reduction on the release of TGF-beta from WJ. The WJ prepared from the umbilical cords of newborns delivered at term by healthy mothers was homogenised and treated with hyaluronidase, collagenase, heparinase, chondroitinase and beta-mercaptoethanol, the resulting extracts were then submitted to TGF-beta immunoassay and SDS/PAGE followed by Western immunoblotting. The effect of metalloproteinase activation on TGF-beta was also studied. Pre-treatment of WJ homogenates with hyaluronidase or collagenase markedly increased the extractability of TGF-beta, but did not dissociate the complexes. In contrast, the action of beta-mercaptoethanol resulted in the release of free TGF-beta; but activation of metalloproteinases resulted in the disappearance of this factor. We conclude that TGF-beta1 is bound through disulphide bonds to an extracellular matrix component of WJ. The large amount of collagen fibrils and hyaluronate molecules which surround the cells scattered in WJ may prevent the access of extracting solution to TGF-beta causing a low extractability of this factor. Although hyaluronate and collagen do not bind TGF-beta directly, they may present a barrier that prevents the diffusion of TGF-beta in WJ and results in its concentration around the cells thereby facilitating its interaction with membrane receptors and subsequent stimulation of cell division and synthesis of extracellular matrix components.

Glucose-depleted medium reduces the collagen content of human skin fibroblast cultures.

Glucose deprivation appeared to be a factor which induces oxygen regulated protein (ORP) 150 expression in the human skin fibroblasts cultures. Whereas glucose deprivation resulted in a slight (statistically insignificant) decrease of protein content in these cultures, a marked decrease of collagen content was observed, resulting in a distinct reduction of hydroxyproline: protein ratio. Furthermore, the appearance of ORP150 in glucose-deprived cultures coexisted with an increase of gelatinolytic activity and slight reduction in the expression of insulin-like growth factor-I (IGF-I) receptor. Since IGF-I is a main stimulator of collagen synthesis, the reduction in the expression of IGF-I receptor may result in a decrease of collagen synthesis. It is suggested that ORP 150 is a chaperon, which protects intracellular proteins against proteolytic effects exerted by hypoxia or glucose shortage. Since the total amount of protein in fibroblast cultures did not change much, it appears that collagen (in contrast to other proteins) was not efficiently protected. The decrease in collagen synthesis and the enhancement of collagen degradation by gelatinases may result in distinct reduction of collagen content in glucose-deprived fibroblast cultures.

FGF binding by extracellular matrix components of Wharton's jelly.

Our earlier paper has reported that Wharton's jelly is a reservoir of several peptide growth factors, including acidic and basic fibroblast growth factors (aFGF and bFGF, respectively). Both can be extracted by buffered salts solutions in the form of high molecular mass complexes, probably with a component(s) of the extracellular matrix. Both aFGF and bFGF from such extracts hardly penetrate 10% polyacrylamide gels during electrophoresis. Pre-treatment of Wharton's jelly with hyaluronidase slightly increased the extractability of aFGF, but did not affect the extractability of bFGF. In contrast, the pre-treatment of tissue homogenate with bacterial collagenase (2000 U/ml, 37 degrees C, 18 h) increased the extractability of bFGF. The presence of beta-mercaptoethanol in the extracting solutions increased the extractability of both FGFs, but did not release FGFs in their free form, despite reducing the molecular mass of the FGF-containing complexes. We conclude that both aFGF and bFGF are bound through disulphide bonds to a protein component of Wharton's jelly. We propose that ground substance composed mainly of collagen fibrils and hyaluronate molecules, which surrounds the cells of Wharton's jelly, prevents the access of the extracting solution to aFGF and bFGF. Although hyaluronate and collagen do not bind aFGF or bFGF directly, they may constitute a barrier which prevents the dispersion of FGFs in Wharton's jelly. Thus, the high concentration of FGFs around the cells of Wharton's jelly may facilitate the interaction of these factors with membrane receptors, thereby resulting in stimulation of cell division and differentiation, as well as of the synthesis of 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.

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 effect of hypoxia on the expression of 150 kDa oxygen-regulated protein (ORP 150) in HeLa cells.

Correct protein folding is an important factor, for the translocation of newly synthesised proteins to specific subcellular compartments, extracellular matrix or to biological fluids. This process is regulated by a group of specific proteins, referred to as chaperones. Many stress conditions, such as oxygen or glucose deprivation, slow down the folding process and cause accumulation of unfolded/misfolded proteins in the cell. Molecular chaperones are induced in these conditions; with some named as oxygen-regulated proteins (ORPs). These bind to unfolded / misfolded proteins to facilitate correct assembly. ORP 150 is the subject of this study. Hypoxia results in an enhancement of ORP 150 expression in several tumour cell lines cultured in vitro. HeLa cells grown in hypoxic conditions (despite an intensive expression of ORP 150) demonstrate higher rates of apoptosis in comparison to those cultured in normoxic conditions. Furthermore, the inhibition of ORP 150 synthesis by transfection of these cells with a specific siRNA resulted in an intensification of apoptosis, as indicated by specific markers of this process; the enhancement of poly ADP-ribose protein cleavage and the increase in Bim protein expression. We conclude from our study that the increase in ORP 150 synthesis protects the cells against the proapoptotic effect of hypoxia.

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.