Edward Bańkowski

Collagen and Glycosaminoglycans of Wharton’s Jelly

In this study, we report quantity, solubility and molecular polymorphism of collagen, proportional relationships between various types of collagen, ultrastructure of collagen fibres, the amounts of various glycosaminoglycans (GAGs) and proportional relationships between them in Whartons jelly of normal umbilical cords. We compare the extracellular matrix components in Whartons jelly with those in the umbilical cord artery. Collagen of the Whartons jelly demonstrates some specific features. It is very insoluble in neutral salt and in a slightly acidic solution and appears to be resistant to the action of depolymerizing agent (EDTA-Na2). Only 50% of total collagen may be solubilized by pepsin digestion and fractionated by differential salt precipitation. Four collagen fractions were obtained. Three of them were identified by polyacrylamide gel electrophoresis as type I, type III, and type V collagen and proportional relationship between them was calculated. Hyaluronic acid is the most abundant component of GAGs contained in Whartons jelly. The amounts of sulphated GAGs-keratan sulphate, heparan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate and heparin-are distinctly lower. Each of them constitutes only a few percent of total GAGs.

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.

Collagen of umbilical cord arteries and its alterations in EPH-gestosis

We decided to investigate the EPH-gestosis--connected alterations in collagen of the umbilical cord arteries. The samples of arterial walls were submitted to histological and biochemical studies. It was found that umbilical cord arteries taken from newborns of mothers with EPH-gestosis contain more than twice the amount of collagen in comparison to corresponding arteries of newborns from normal pregnancies. An increase of collagen content in these vessels in accompanied by a slight decrease of its solubility. Types I, III, IV, V and VI collagens were found both in normal umbilical cord arteries and in those of newborns delivered by mothers with EPH-gestosis but their proportional relationships were different. EPH-gestosis is accompanied by an increase of a proportional amount of type III-collagen and a decrease of type I collagen in umbilical cord arteries. It seems that these changes in the umbilical cord arteries may be responsible for the decrease of blood flow in fetus of woman with EPH-gestosis.

Elastin of the Umbilical Cord Arteries and Its Alterations in EPH Gestosis (Preeclampsia)

Edema, proteinuria, hypertension (EPH) gestosis is accompanied by an increase of collagen content and premature replacement of hyaluronic acid by sulfated glycosaminoglycans both in the umbilical cord arteries and in Wharton’s jelly. The effect of EPH gestosis on elastin content and metabolism in the umbilical cord arterial wall was the aim of this work. Studies were performed on normal umbilical cord arteries and those taken from newborns of mothers with EPH gestosis. Elastin was isolated from the arterial wall and quantified by a dye-binding method. Biosynthesis and degradation of this protein was evaluated by a pulse-chase experiment with the use of 14C-proline. It was found that EPH gestosis is associated with a significant reduction of elastin content in the umbilical cord arteries as a result of decrease in elastin biosynthesis rate and accelerated degradation of this protein. The replacement of elastin by collagen, and hyaluronate by sulfated glycosaminoglycans, may decrease the hydration of arterial wall and reduce its elasticity. Such rearrangement of extracellular matrix of the umbilical cord arteries may affect mechanical properties of these vessels and disturb fetal blood circulation.

Proteoglycans of Wharton’s jelly

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

Pre-eclampsia (EPH-gestosis)-induced decrease of MMP-s content in the umbilical cord artery.

BACKGROUND It was found in our previous paper that pre-eclampsia-associated accumulation of collagen in the umbilical cord artery (UCA) is a result of increased biosynthesis and decreased degradation of this protein. It is known that the activity of collagenolytic enzymes is a main factor regulating collagen degradation rate in various tissues. METHODS For this reason it was decided to evaluate the effect of pre-eclampsia on the content and activity of metalloproteinases by immunoenzymatic method (ELISA), zymographic technique and with the use of specific substrates. RESULTS A low amount of MMP-1 (collagenase 1), MMP-9 (gelatinase B) and MMP-3 (stromelysin 1) was detected in the extracts from the wall of the umbilical cord artery. MMP-2 (gelatinase A) is the main collagenolytic enzyme in UCA wall (both latent and active form). Pre-eclampsia is associated with a distinct reduction in those metalloproteinases content in comparison to control UCAs. It can be concluded from zymography that MMP-2 (gelatinase A) of the umbilical cord artery forms an inactive complex with a tissue inhibitor of metalloproteinases (TIMP). Such a complex dissociates under the action of p-aminophenylmercuric acetate (APMA) or sodium dodecyl sulphate. CONCLUSIONS The decrease of metalloproteinases content and activity in the umbilical cord artery may be a factor that reduces the breakdown of collagen in the arterial wall and promotes the accumulation of this protein. The accumulation of collagen with simultaneous reduction in elastin content in the UCA may be the factor that reduces the elasticity of arterial wall and decreases the blood flow in the fetus of women with pre-eclampsia.

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.

Decrease in the glycosaminoglycan content in the skin of diabetic rats. The role of IGF-I, IGF-binding proteins and proteolytic activity

The results of our previous studies demonstrated that acute streptozotocin-induced diabetes in rats evoked a decrease in skin collagen content with little effect on glycosaminoglycans (GAG) content. In our present study we employed the model of chronic diabetes in order to check its effect on skin GAG content.It was found that the skin of diabetic rats showed a significant decrease in almost all the investigated GAGs by 50-70%. The decrease in heparan sulfate content was slight and statistically insignificant. We sought to determine whether the insulin-like growth factor-I (IGF-1) and IGF-binding proteins (IGF-BPs) levels are altered in animals with experimental diabetes and might contribute to the decrease in tissue GAG content. Circulating IGF-I level was found to be reduced in animals with diabetes and significant changes in serum IGF-BPs were observed. The amount of high molecular weight binding proteins (HMW-BPs) was decreased and the content of low molecular weight binding proteins (LMW-BPs), known as IGF-I inactivating substances, markedly increased. Furthermore, diabetic rats demonstrated an increase of skin proteolytic activity. We conclude that the decrease of GAG content in the skin of diabetic rats is a result of three co-existing phenomena: decreased circulating IGFI level, increased plasma content of LMW BPs and increased proteolytic activity of the skin.

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

Abstract 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. Clin Chem Lab Med 2008;46:517–22.

An accumulation of IGF-1 and IGF-binding proteins in human umbilical cord

It is known that extracellular matrix components (ECM) may serve as a storage site to concentrate and stabilize growth factors in the vicinity of cells. IGF-I is expressed in most fetal tissues and it is involved in anabolic effects on protein and sulphated glycosaminoglycans biosynthesis, cell proliferation and differentiation. We demonstrated that human umbilical cord (UC) tissues contain large amounts of IGF-I and IGF-I-binding proteins (BP-3 and BP-1). Particularly Whartons jelly appears to be an abundant reservoir of IGF-I and BPs. Relatively low amount of cells and large amounts of collagen and glycosaminoglycans in UC tissues (especially in Whartons jelly) suggest that IGF-I may play a major role in stimulation of these cells to produce ECM components. The specific BPs in these tissues may be important modulators of IGF-I action during fetal development.