Krzysztof Sobolewski

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.

Extracellular matrix components of the wall of umbilical cord vein and their alterations in pre-eclampsia

Abstract Pre-eclampsia — edema, proteinuria, hypertension (EPH-gestosis) is one of the more common complications observed during pregnancy. The umbilical cord vein walls were taken from newborns delivered by healthy mothers (control material) and by mothers with polysymptomatic pre-eclampsia (investigated material). Normal saphenous vein walls were collected from adult subjects undergoing varicose vein surgery. The collagen content was measured by the assay of hydroxyproline. Elastin was determined according to Fastin Elastin Assay and gravimetrically. Glycosaminoglycans content was determined by uronic acids assay. The collagen content decreased in the pre-eclampsia material. The amount of soluble elastin increased in the investigated material. The insoluble elastin content decreased in the umbilical cord veins of newborns delivered by mothers with pre-eclampsia. Reconstructing the umbilical cord vein wall may disturb fetal blood flow and affect the vascular system in adulthood.

Deregulation of Collagen Metabolism in Human Stomach Cancer

Objective: The defects of collagen metabolism are responsible for the disorganization of extracellular matrix in stomach cancer. Collagen through interaction with integrin receptors regulates the cellular growth, differentiation, gene expression, prolidase and gelatinase activity and plays an important role in tumorigenesis and invasiveness. Although extracellular metalloproteinases initiate the breakdown of collagen in tissues, the final step of its degradation is mediated by prolidase. Therefore, we decided to compare the degradation of collagen in control tissues to gastric cancer tissues. Methods: We investigated the collagen content (hydroxyproline assay), expressions of β1 integrin, prolidase and gelatinases A and B (Western immunoblot) as well activities of prolidase (colorimetric assay) and gelatinases (zymography) in stomach cancer tissue (n = 10). The results were compared with corresponding data obtained for control tissues (n = 10). Results: No differences in the collagen content were found between the studied tissue samples. However, an increase in free proline pool, enhanced gelatinase expression and elevated gelatinolytic activity were found in the tumor tissue. These phenomena were accompanied by a significant elevation in prolidase activity and an increase in β1 integrin expression in stomach cancer, compared to control tissue. Conclusion: The data presented suggest an enhancement of collagen turnover in stomach cancer. It may be suggested that the increased degradation of collagen by gelatinase in cancer tissue is balanced by an increased biosynthesis of this protein.

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.

CCN2 protein is an announcing marker for cardiac remodeling following STZ-induced moderate hyperglycemia in mice

Diabetes causes changes in the myocardium, which are often called diabetic cardiomyopathy. This condition has been extensively investigated in animal models with high glucose levels. Nevertheless, it has not been investigated whether moderate hyperglycemia, in the absence of other features of metabolic syndrome, may also cause similar changes in the heart. The aim of the study was to assess changes in the myocardium in an animal model of mild type 1 diabetes. Moderate hyperglycemia was induced in 8- to 10-week-old male C57BL6J mice by 5 intraperitoneal injections of streptozotocin (40 mg/kg). After 16 weeks, they were sacrificed, and left ventricle (LV) dimensions and extent of cardiac fibrosis were assessed by morphometry. The abundance of CCN proteins in LVsamples was assessed using western blotting, while activity of metalloproteinase 2 was established in zymography. Real time PCR was used to investigate the expression of transforming growth factor beta1 (TGFbeta1) and atrial natriuretic peptide. Mice with moderate hyperglycemia presented comparable cardiac dimensions with fibrosis and hypertrophy parameters as the non-diabetic controls. However, the abundance of profibrotic CCN2 protein was significantly increased in hyperglycemic animals (1.67 +/- 0.28 vs. 1 +/- 0.47, p < 0.05). Interestingly, this change was independent from the TGFbeta1 expression, as its RNA abundance was similar in both groups. Moderate hyperglycemia also caused an increase in the activity of the metalloproteinase 2 (1.21 +/- 0.17 vs. 1 +/- 0.07, p < 0.05). Despite diabetes, no profound changes in cardiac morphology were found. In our animal model, moderate hyperglycemia caused activation of a profibrotic gene expression program, which was counterbalanced by the increase of metalloproteinase activity.

Evaluation of Transforming Growth Factor-β Signaling Pathway in the Wall of Normal and Varicose Veins

Objective(s): Extracellular matrix remodeling in the vein wall is involved in varicose vein pathogenesis, with transforming growth factor β1 (TGF-β1) playing a potential role. The aim of the study was to assess the TGF-β signaling pathway including its receptor (TGF-β RII) and phosphorylated receptor-regulated Smads (p-Smad2/3) in varicose veins. Methods: Varicose veins from patients undergoing varicose vein surgery were the studied material, whereas normal greater saphenous veins from patients undergoing infrainguinal arterial bypass surgery were the control material. Expression of TGF-β RII mRNA was assessed with RT-PCR, whereas expression of TGF-β RII and p-Smad2/3 proteins was assessed with Western blot. Results: A significantly increased TGF-β RII mRNA level was found in varicose veins (287 ± 24%), when compared with normal veins (100 ± 26%). The receptor protein expression reflected a changed mRNA level with significantly increased TGF-β RII protein in varicose veins (290 ± 21%), when compared with controls (100 ± 16%). Enhanced TGF-β RII expression was accompanied by increased p-Smad2/3 protein expression in varicose veins (257 ± 19%) in comparison with normal veins (100 ± 9%). Conclusion(s): Increased TGF-β RII expression and activation in the wall of varicose veins may be involved in extracellular matrix remodeling related to TGF-β1 and supports its role in the disease pathogenesis.

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.

Evaluation of aFGF/bFGF and FGF Signaling Pathway in the Wall of Varicose Veins

BACKGROUND Extensive extracellular matrix remodeling of the vein wall is involved in varicose veins pathogenesis. The process is controlled by numerous factors, including peptide growth factors. The aim of the study was to evaluate acidic (aFGF) and basic (bFGF) fibroblast growth factors, their receptor (FGFR) and the MAP kinase pathway (ERK 1/2) in the wall of varicose and varicose veins complicated by thrombophlebitis, when compared to normal ones. METHODS Segments of normal, varicose, and varicose veins complicated by thrombophlebitis were collected during varicose veins surgery in 17 patients. Expression and content of aFGF and bFGF were evaluated with Western blot and enzyme-linked immunosorbent assay (ELISA) methods, respectively, whereas RT-PCR was employed to assess mRNA level of growth factors. Expression of FGFR and ERK 1/2 was examined with Western blot method. RESULTS Increased aFGF expression and content were accompanied by increased aFGF mRNA level in the wall of varicose veins. Furthermore, alternatively spliced aFGF mRNA was shown in varicose veins complicated by thrombophlebitis. Expression, content, and mRNA level of bFGF were comparable in the investigated material. FGFR and ERK 1/2 expression was demonstrated in the wall of diseased veins, however, without any significant differences in comparison with the wall of normal veins. CONCLUSIONS Overexpressed aFGF in the wall of varicose veins via FGFR and the MAP kinase pathway may influence expression of enzymes involved in extracellular matrix metabolism and play a role in vein wall remodeling, as well as in the disease pathogenesis.

Decreased expression of the insulin-like growth factor-I-binding protein-1 (IGFBP-1) phosphoisoform in pre-eclamptic Wharton's jelly and its role in the regulation of collagen biosynthesis.

Abstract Insulin-like growth factor-I (IGF-I) is known as an important stimulator of collagen and glycosaminoglycans (GAGs) biosynthesis in tissues. IGF-I activity is under control of IGF-I-binding proteins (IGFBPs) with different IGF-I-binding affinity. IGFBP-1 is known as an inhibitor of IGF-dependent functions. Some IGFBPs (e.g., IGFBP-1) may undergo phosphorylation that dramatically increases IGFBP affinity for IGF. Whartons jelly represents a reservoir of IGF-I and its binding proteins (BPs). Pre-eclampsia, the most common, pregnancy-associated pathological syndrome, contributes to a significant decrease in IGF-I and IGFBP-1 content in Whartons jelly, although it does not affect collagen content in this tissue. In the present study we show that control Whartons jelly contains phosphorylated forms of IGFBP-1 that are dramatically dephosphorylated during pre-eclampsia. A dramatic decrease in IGF-I binding to immunoprecipitated IGFBP-1 from pre-eclamptic Whartons jelly compared to the control was observed. Western immunoblot analysis with anti-phosphothreonine antibodies for immunoprecipitated IGFBP-1 from control and pre-eclamptic Whartons jelly revealed that both tissues contain phosphorylated forms of IGFBP-1. However, a distinct decrease in the expression of phosphorylated IGFBP-1 from pre-eclamptic Whartons jelly was observed. The functional significance of the phenomenon was found in cultured fibroblasts treated with IGFBP isolated from Whartons jelly extracts. A significant decrease in collagen biosynthesis was found in the cells treated with IGFBP of control Whartons jelly, while in the presence of IGFBP from pre-eclamptic Whartons jelly, the rate of collagen biosynthesis was similar to that in the control cells. The result was corroborated by data showing increase in expression of IGF-I receptor and phosphorylated MAP kinases (ERK1 and ERK2) in fibroblasts cultured in the presence of IGFBP from pre-eclamptic Whartons jelly, compared to control. The data suggest that the decrease in phosphorylated IGFBP-1 in pre-eclamptic Whartons jelly may decrease IGF-I-binding affinity for IGF and increase the bioavailability of IGF-I for receptor interaction. This mechanism may facilitate IGF-I-dependent stimulation of fibroblasts to produce extracellular matrix (ECM) components even at a low IGF-I tissue level. Therefore, IGFBP-1 phosphoisoforms in Whartons jelly may play an important role in the regulation of IGF-I-dependent functions during pre-eclampsia.