Lech Romanowicz

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.

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.

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.

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.

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1.

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor-UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5ng/ml. The detection limit is 0.06ng/ml for the biosensor with antibody and 0.08ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20ng/ml for plasma samples and 0.30 to 0.49ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.

Glycosaminoglycan-biosynthesis in the wall of the umbilical cord artery and its alteration in EPH-gestosis.

The mechanism of edema, proteinuria, hypertension (EPH)-gestosis-associated premature replacement of hyaluronic acid by sulphated glycosaminoglycans (GAGs) in the umbilical cord arteries is not known. It may result from altered biosynthesis, a different degradation rate or a combination of both phenomena. In order to solve this problem, it was decided to evaluate the biosynthesis and degradation of newly synthesized GAGs in the umbilical cord arteries of control newborns and those delivered by mothers with EPH-gestosis. Incorporation of radioactive precursors ([14C]glucosamine and [35S]sulphate) into GAGs and degradation of newly synthesized GAGs using the pulse-chase experiment were evaluated. We found that the investigated tissue slices incorporated distinctly less [14C]glucosamine into hyaluronic acid in comparison to controls. In contrast to that, the biosynthesis of sulphated GAGs did not change significantly. However, the degradation of newly synthesized sulphated GAGs was distinctly slower than in control tissues. It may be concluded that an EPH-gestosis associated decrease in hyaluronic acid content in the umbilical cord artery is a result of decreased biosynthesis of this substance, whereas an increase in sulphated GAGs-content is rather a result of slower degradation of newly synthesized GAGs.

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 Students 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.

Extracellular Matrix Remodeling of the Umbilical Cord in Pre-eclampsia as a Risk Factor for Fetal Hypertension

The human umbilical cord forms a connection between the placenta and the foetus. It is composed of two arteries and one vein surrounded by Whartons jelly. Pre-eclampsia is accompanied by extensive remodeling of extracellular matrix of umbilical cord. Matrix metalloproteinases (MMPs) are engaged in degradation of extracellular matrix proteins and activation/inactivation of certain cytokines and enzymes. These enzymes will probably play a central role in the release of matrix-embedded cytokines and growth factors. MMP-2 (gelatinase A) is the main collagenolytic enzyme of both umbilical artery and vein. Other metalloproteinases are present in several times lower amounts. Reduced activity of collagen-degrading enzymes may be a factor, which enhances the accumulation of collagen and some other proteins in the pre-eclamptic umbilical cord tissues. It seems to be possible that similar alterations occur in other fetal blood vessels. It may result in an increase in peripheral resistance as well as an increase in the blood pressure in the fetal vascular system. Some observations suggest that the raised pressure may persist after birth. Pre-eclampsia may be a factor that evokes an initiation of hypertension in utero and its amplification through childhood and adulthood.

EPH-gestosis (pre-eclampsia)-induced decrease of gelatinase activity may promote an accumulation of collagen in the umbilical cord artery

It was found in our previous paper that edema, proteinuria, hypertension (EPH)-gestosis-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. For this reason it was decided to evaluate the effect of EPH-gestosis on the activity of proteolytic enzymes which may be involved in collagen degradation in the UCA wall. Proteolytic activity against bovine serum albumin, reconstituted collagen fibres and gelatin were evaluated. Latent forms of proteolytic enzymes were activated by the action of trypsin, p-chloromercuric benzoate (PCMB) and p-aminophenylmercuric acetate (APMA). A low activity of gelatinase (type IV collagenase) was detected in the extracts from the wall of the umbilical cord artery. This enzyme increased its activity several times after the action of trypsin, PCMB and APMA. EPH-gestosis results in a distinct reduction in gelatinase activity. Despite the action of activating agents the gelatinase from EPH-gestosis UCAs was considerably lower in comparison to control UCAs. It can be concluded that gelatinase of the umbilical cord artery forms an inactive complex with a tissue inhibitor of metalloproteinases. Such a complex dissociates under the action of trypsin, PCMB or APMA or sodium dodecyl sulphate. The decrease of gelatinolytic activity in the umbilical cord artery may be a factor that reduces the breakdown of collagen in the arterial wall and promotes an accumulation of this protein. The accumulation of collagen with simultaneous reduction in elastin content in the UCA may be the factors which reduce the elasticity of arterial wall and decrease the blood flow in the fetus of woman with EPH-gestosis.

Preeclampsia-associated decrease of potential collagenolytic and gelatinolytic activities in the wall of the umbilical cord vein

Preeclampsia is the most common pathological syndrome associated with pregnancy. It is accompanied by remodelling of the extracellular matrix of the umbilical cord. A decrease of collagen content in the umbilical cord vein was described. This decrease may result from reduced collagen biosynthesis or enhanced collagen degradation. It was decided to evaluate whether or not this phenomenon is associated with alterations in the activities of collagenolytic, gelatinolytic and non-specific proteolytic enzymes that may be involved in collagen degradation, as well as the activity of prolidase which provides proline as a substrate for collagen biosynthesis. Studies were performed on the umbilical cord veins of newborns delivered by healthy mothers and those with preeclampsia. The control vein extract, activated with trypsin, degraded reconstituted collagen fibres (64.4+/-2.9 nmol Hyp x mg(-1) protein), whereas the preeclamptic material demonstrated only a trace activity. The venous wall extract contained a latent form of gelatinase that might have been activated by trypsin and 4-aminophenylmercuric acetate. A decrease in the gelatinolytic and proteolytic activities of preeclamptic vein extract at neutral pH was found. Prolidase activity was almost 3-fold lower in the preeclamptic extract (240.6+/-29.3 nmol Pro x min(-1) x mg(-1) protein) in comparison to the control (608.2+/-63.7 nmol Pro x min(-1) x mg(-1)protein). It was concluded that the umbilical cord vein contains a latent form of gelatinase A. The decrease in prolidase activity may reduce collagen biosynthesis, resulting in a decrease of this protein in the preeclamptic umbilical cord vein.