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E Kaczmarek

Publications and source records attributed to E Kaczmarek.

At least 37 records · Page 2Linked to original sources
PubMed2000

CD39 modulates IL-1 release from activated endothelial cells.

The activation of endothelial cells (EC) and monocyte-macrophages (Mφ) by lipopolysaccharide (LPS) is considered an important element of the vascular injury observed in endotoxemia. Interleukin-1 (IL-1) beta release from Mφ in response to LPS, appears to be mediated by the autocrine/paracrine release of ATP via P2X7 receptor activation. In EC, similar nucleotide-mediated signaling pathways may be influenced by high levels of expression of CD39, the vascular nucleoside triphosphate diphosphohydrolase (NTPDase; ENTPD I). To determine whether CD39 modulates ATP-mediated release of IL-1 from EC, we stimulated human EC with LPS and measured levels of ATP secretion and IL-1 release. LPS triggered ATP secretion from EC that was soon followed by IL-1alpha release. Overexpression of CD39 following infection with recombinant CD39 adenoviral vectors (AdCD39) abrogated the initial phase of ATP secretion and inhibited IL-1alpha release; comparable results were obtained with soluble NTPDase. These data demonstrate that CD39/NTPDase modulates IL-1alpha release from LPS stimulated human EC.

Adenosine Triphosphatases↗
PubMed2000

Palmitoylation targets CD39/endothelial ATP diphosphohydrolase to caveolae.

Ectonucleotidases influence purinergic receptor function by the hydrolysis of extracellular nucleotides. CD39 is an integral membrane protein that is a prototype member of the nucleoside 5'-triphosphate diphosphohydrolase family. The native CD39 protein has two intracytoplasmic and two transmembrane domains. There is a large extracellular domain that undergoes extensive glycosylation and can be post-translationally modified by limited proteolysis. We have identified a potential thioester linkage site for S-acylation within the N-terminal region of CD39 and demonstrate that this region undergoes palmitoylation in a constitutive manner. The covalent lipid modification of this region of the protein appears to be important both in plasma membrane association and in targeting CD39 to caveolae. These specialized plasmalemmal domains are enriched in G protein-coupled receptors and appear to integrate cellular activation events. We suggest that palmitoylation could modulate the function of CD39 in regulating cellular signal transduction pathways.

Adenosine Triphosphatases↗
PubMed2000

CD39 modulates endothelial cell activation and apoptosis.

BACKGROUND: CD39 is the dominant vascular nucleoside triphosphate diphosphohydrolase (NTPDase) that exerts major effects on platelet reactivity by the regulated hydrolysis of extracellular adenine nucleotides. The effects of NTPDases on endothelial cell (EC) activation and apoptosis remain unexplored. MATERIAL AND METHODS: Recombinant replication-deficient adenoviruses were constructed with human CD39 cDNA (rAdCD39) or the bacterial beta-galactosidase (rAdbetagal). RESULTS: Intact human umbilical vein EC cultures infected with rAdCD39 had substantial and stable increases in NTPDase biochemical activity (14.50 +/- 3.50 Pi nmole/well/min), when contrasted with noninfected cells (0.95 +/- 0.002) and rAdbetagal infected cells (1.01 +/- 0.02; p<0.005). Increased NTPDase activity efficiently inhibited immediate type 2Y purinergic receptor (P2Y)-mediated EC activation responses viz. von Willebrand factor secretion in response to extracellular ATP. In addition, CD39 up-regulation blocked ATP-induced translocation of the transcription nuclear factor (NF)-kappaB to the cell nucleus, and abrogated transcription of mRNA encoding E-selectin, and consequent protein synthesis. CD39 also decreased the extent of apoptosis triggered by putative type-2X purinergic (P2X7) receptors in response to high concentrations of extracellular ATP in vitro. CONCLUSION: These properties of CD39 indicate primary vascular protective effects with potential therapeutic applications.

Adenosine Triphosphatases↗
PubMed1999

Suppression of ATP diphosphohydrolase/CD39 in human vascular endothelial cells.

Vascular ATP diphosphohydrolase/CD39 is an endothelial cell membrane protein with both ecto-ATPase and ecto-ADPase activities. Suppression of constitutive CD39 expression may result in elevated concentrations of ATP and ADP at the vascular interface that could predispose to thrombosis and inflammation. To study the effects of suppression of CD39 synthesis, stable 25-base antisense chimeric oligonucleotides targeting sequences at the 5' region of CD39 were designed. Transfection of these stable oligomers into cultured human endothelial cells resulted in dramatic decreases in levels of CD39 mRNA transcripts. Following transfection with antisense oligonucleotides, total ADPase activity fell from 26.0 +/- 3.1 in control cultures to 9.5 +/- 3.4 nmol of P(i) min(-1) (mg of protein)(-1) (p < 0.005); suppression of CD39 protein expression was also observed by Western blotting. Decreases in ATP diphosphohydrolase activity were associated with increases in concentrations of extracellular purine nucleotides released following stimulation of endothelial cells. Rates of initial hydrolysis of extracellular ATP released from purinergic agonist-stimulated endothelial cells decreased from 17.9 +/- 5.0 to 4.8 +/- 0.5 pmol min(-1) per 10(6) cells (p < 0.005) in antisense transfected cells. Therefore, CD39 regulates extracellular ATP concentrations and may be an important modulator of purinergic receptor activity in vascular endothelial cells.

Adenosine Diphosphate↗
PubMed1999

CD39 as a caveolar-associated ectonucleotidase.

CD39 is a human lymphoid cell activation antigen, (also referred to E-ATPDase or apyrase) that hydrolyzes extracellular ATP and ADP. Although it has been widely studied, its physiological role, however, still remains unclear. This ectonucleotidase generally is said to be evenly distributed in the membrane of the cells. However, we observed that in cell types which possess caveolae, specialised membrane invaginations involved in signalling, CD39 is preferentially targeted to these membrane microdomains. Since all molecules involved in signalling (eNOS, G-proteins, receptors) which are targeted to the caveolae undergo posttranslational modifications (e.g., palmitoylation) we hypothesize the same to be the case for CD39. Furthermore, its presence in the caveolae supports its participation in signalling events.

Adenosine Triphosphatases↗
PubMed1999

Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity.

CD39, the mammalian ATP diphosphohydrolase (ATPDase), is thought to contain two transmembrane domains and five "apyrase conserved regions" (ACR) within a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by sequential deletions at both termini. ATPDase activity was well preserved with FLAG tagging, followed by the removal of either of the demonstrated C- or N-transmembrane regions. However, deletions within ACR-1 (aa 54-61) or -4 (aa 212-220), as well as truncation mutants that included ACR-1, -4, or -5 (aa 447-454), resulted in substantive loss of biochemical activity. Intact ACR-1, -4, and -5 within CD39 are therefore required for maintenance of biochemical activity. Native and mutant forms of CD39 lacking TMR were observed to undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two noncovalently membrane-associated fragments (56 and 27 kDa) that substantially augmented ATPDase activity. Glycosylation variation accounted for minor heterogeneity in native and mutant forms of CD39 but did not influence ATPDase function. Enzymatic activity of ATPDase may be influenced by certain posttranslational modifications that are relevant to vascular inflammation.

Adenosine Triphosphatases↗
PubMed1999

Visualisation and modelling of renal capillaries from confocal images.

A computer aided design was developed to support three-dimensional visualisation and modelling of vascular networks. Volume data comprised a series of images obtained using a Zeiss confocal laser scanning microscope. The profiles of vessels were automatically segmented using two-dimensional morphological filters. Segmented contours of the vessels were used to form a spatial model of the network. The centre points of segmented contours were used to derive a three-dimensional graph representing the vascular network. The proposed method was applied to renal capillary networks of normal rats, and showed well the lobular structure of glomeruli. The average length of renal capillary networks was 6.09 mm. Three-dimensional models based on confocal data require much less effort than reconstructions based on serial sections, and can be adapted for any vascular patterns.

Animals↗
PubMed1999

Analysis of CD39/ATP diphosphohydrolase (ATPDase) expression in endothelial cells, platelets and leukocytes.

Purinergic signaling may influence hemostasis, inflammatory responses and apoptosis. Therefore, hydrolysis of extracellular ATP and ADP by the ATP diphosphohydrolase (ATPDase) could regulate these processes. We have previously demonstrated the identity between the vascular ATPDase and CD39. Here we show that levels of CD39 expression correlate with ATPDase activity in human endothelial cells (EC), platelets and selected monocyte, NK, and megakaryocyte cell lines. Western blotting revealed one to three isoforms of CD39/ATPDase: mobility variations of major protein resulted from post-translational modifications. Northern blotting and primer extension indicated two major mRNA transcripts and one transcription start point, respectively. In addition, mRNAs specific for purinergic P2 receptors were detected in all of the investigated cells, suggesting that the coexpressed CD39/ATPDase may regulate purinergic signaling. Thrombotic and inflammatory responses may be modulated by the expression of CD39/ATPDase.

Adenosine Diphosphate↗
PubMed1999

Clinical and morphological (including morphometric) aspects of minimal change disease and mesangial glomerulonephritis with unfavourable course in children.

Clinical and morphological analysis was made to assess 9 cases of minimal change disease (MCD) and 30 cases of mesangial glomerulonephritis (GNMES) recognized by light microscopy with unfavourable course. Case selection was based exclusively on the clinical course suggesting a possibility of early sclerosis (long-term steroid resistance, frequent recurrences, rare short-lasting remissions, hypertension, renal failure). It was found that the unfavourable clinical course was clearly more frequently associated with electron microscopic than light microscopic changes. Marked increase of the matrix was observed also in those glomeruli in which light microscopy did not reveal any changes or only the signs of immaturity. It was also noticed that in those cases in which the assessment of mesangial matrix increase (which means the onset of sclerosis) is not certain, it is useful to make a morphometric analysis of electron microscopic material.

Adolescent↗
PubMed1999

[Occurrence of Escherichia coli O157 in feces of healthy persons, from selected groups, in the country].

The purpose of the study was determination of the occurrence of E. coli O157 in faeces samples of healthy subjects and characterization of the isolated strains with respect to their potential pathogenicity. The study was carried out in two stages. In the first one in 5 sanitary-epidemiological stations samples were tested from healthy subjects after inoculation onto McConkey (MC) or/and McConkey with sorbitol (SMC) media and isolating from each culture 10 lactose-positive (on MC medium) or sorbitol-negative (on SMC) colonies. Then latex test was done with each isolate for E. coli O157 presence. In all, 1005 samples were studied, including 260 taken from children aged 0-2 years, 180 samples from children aged 3-10 years, and 565 samples from older children and adults. E. coli O157 rods were cultured from 6 adults (0.6%). In the second stage carried out at the Laboratory of Enterobacteriaceae, Bacteriology Department, National Institute of Hygiene strains obtained from territorial laboratories were studied determining their phenotypic and genotypic traits regarded as virulence markers of verotoxic E. coli O157 strains, such as inability to ferment sorbitol and MUG breakdown, and production of verotoxins and enterohaemolysin. By the PCR method fragments were sought of genes coding for production of verotoxins, intimin and enterohaemolysin. The results showed that no E. coli O157 strain obtained from healthy individuals produced verotoxins, but three studied strains contained the eae gene determining intimin production and they were regarded as enteropathogenic.

Adult↗
PubMed1998

Adenovirus-mediated expression of a dominant negative mutant of p65/RelA inhibits proinflammatory gene expression in endothelial cells without sensitizing to apoptosis.

We hypothesized that blocking the induction of proinflammatory genes associated with endothelial cell (EC) activation, by inhibiting the transcription factor nuclear factor kappaB (NF-kappaB), would prolong survival of vascularized xenografts. Our previous studies have shown that inhibition of NF-kappaB by adenovirus-mediated overexpression of I kappaB alpha suppresses the induction of proinflammatory genes in EC. However, I kappaB alpha sensitizes EC to TNF-alpha-mediated apoptosis, presumably by suppressing the induction of the NF-kappaB-dependent anti-apoptotic genes A20, A1, manganese superoxide dismutase (MnSOD), and cellular inhibitor of apoptosis 2. We report here that adenovirus mediated expression of a dominant negative C-terminal truncation mutant of p65/RelA (p65RHD) inhibits the induction of proinflammatory genes, such as E-selectin, ICAM-1, VCAM-1, IL-8, and inducible nitric oxide synthase, in EC as efficiently as does I kappaB alpha. However, contrary to I kappaB alpha, p65RHD does not sensitize EC to TNF-alpha-mediated apoptosis although both inhibitors suppressed the induction of the anti-apoptotic genes A20, A1, and MnSOD equally well. We present evidence that this difference in sensitization of EC to apoptosis is due to the ability of p65RHD, but not I kappaB alpha, to inhibit the constitutive expression of c-myc, a gene involved in the regulation of TNF-alpha-mediated apoptosis. These data demonstrate that it is possible to block the expression of proinflammatory genes during EC activation by targeting NF-kappaB, without sensitizing EC to apoptosis and establishes the role of c-myc in controlling induction of apoptosis during EC activation. Finally, these data provide the basis for a potential approach to suppress EC activation in vivo in transgenic pigs to be used as donors for xenotransplantation.

Adenoviridae↗
PubMed1998

Extracellular ATP and ADP activate transcription factor NF-kappa B and induce endothelial cell apoptosis.

Inflammation within the vasculature is associated with endothelial cell (EC) perturbation, loss of vascular ATP-diphosphohydrolase activity, and platelet microthrombus formation with release of ATP and ADP into the micro-environment. The nature and effects of purinergic stimulation of EC under these circumstances remain largely undetermined. ATP and ADP activated EC transcribed mRNA from certain transcription factor NF-kappa B target genes and expressed E-selectin protein on cell membranes. Band shift analysis and reporter assays confirmed the activation of NF-kappa B in response to both ATP and ADP. Apoptosis was shown to occur in response to purinergic signaling, potentially through the activation of P2z/P2x7 receptors. Induction of EC activation responses and apoptosis in response to stimulation with ATP and ADP is associated with activation of NF-kappa B.

Adenosine Diphosphate↗
PubMed1998

Regulation of monocyte tissue factor activity by allogeneic and xenogeneic endothelial cells.

The regulation of tissue factor (TF) activity by the cell associated tissue factor pathway inhibitor (TFPI) during monocyte (Mo) and endothelial cell (EC) interactions is not fully understood. This report describes co-ordinate induction of TF antigen (TF-Ag) and membrane-associated TFPI-Ag on human Mo following coculture with human aortic (HAEC) or porcine aortic EC (PAEC) or after stimulation with TNFalpha. We show that both allo- and xenogeneic EC induce human Mo-TF antigen in short-term culture. However, the TF activity of TNFalpha-primed Mo is suppressed when these cells are cocultured with HAEC [by 40.3 +/- 6.3% (p<0.02)] or PAEC [by 50.5 +/- 10.6% (p<0.001)]. Antibody (Ab) blocking studies confirm that TFPI is the principal anticoagulant associated with this suppression of TF-activity. Our data indicate that anti-TF activity originates, at least in part, from the activated human Mo in the coculture; additionally, specific generation of TFPI by Mo is observed under the xenogeneic culture conditions. As Mo associated TF, induced by allo- or xenogeneic EC interactions, is regulated by cell-associated TFPI, we propose that infiltrating Mo may modulate the thrombotic process at sites of vascular injury in association with both allo- and xenograft rejection.

Animals↗
PubMed1998

Is vacuolization of podocytes and glomerular endothelial cells of prognostic value with respect to FSGS?

Focal segmental glomerulosclerosis (FSGS) poses a major problem both from the clinical and pathomorphological viewpoint. The diagnosis of FSGS in its early stage is vital mainly because of rapidly developing therapeutic modalities. In the literature various changes are discussed which may be of prognostic value (may predict the development of FSGS). One the these changes in vacuolization, mainly of podocytes and less frequently of endothelial cells. The purpose of the present study was to analyse biopsy specimens to find out to what extent vacuolization of podocytes and endothelial cells is associated with FSGS. We compared vacuolization in minimal change disease (MCD), mesangial glomerulonephritis (GNMES) and FSGS. A similar analysis was made also with respect to those cases of MCD and GNMES, in which electron microscopy suggested an early stage of FSGS. In each group electron micrographs obtained from 15 children were analysed. Electron micrographs (12-15 on average) were obtained most frequently from 3 glomeruli. Each case electron micrographs contained 90-100 podocytes. Based upon the same electron micrographs we counted capillary lumina and defined the percentage of those which contained vacuolized endothelia (we counted the capillary lumina, and not the cells, because it is most frequently impossible to identify the border of vacuolized endothelial cells). The number of capillary cross-sections was 60 on average. The results of the analysis were compared with the clinical data. This comparison did not confirm the hypothesis of other investigators that vacuolization is of a prognostic value. Additionally we evaluated the character of vacuoles. Within podocytes the vacuoles were varying in shape. Surrounded by a single membranous layer most frequently they contained material corresponding to proteins or proteoglycans, rarely to lipids. Sometimes the vacuoles were autophagosomal, occasionally they consisted of the dilated rough endoplasmic reticulum. Vacuole-like changes within the capillary lumina were related to the swelling of endothelial cytoplasm or mesangial processes. The reasons for a discrepancy between our results and those reported by other investigators necessitate further studies.

Child↗
PubMed1997

Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors.

BACKGROUND: Delayed xenograft rejection (DXR) is characterized by inflammation and vascular thrombosis. Activation of coagulation may occur as a result of tissue factor (TF) expression on both activated donor endothelial cells (EC) and recipient infiltrating monocytes (Mo). In addition, natural anticoagulants associated with porcine endothelial cells may not function adequately across species. METHODS: In the present study, we examined the interaction of the TF pathway of coagulation with the natural anticoagulant TF pathway inhibitor, in xenogeneic leukocyte-EC cultures in vitro, and during rejection of discordant xenografts in vivo. RESULTS: Coculture of human Mo with pig aortic EC (PAEC) resulted in 1.7-fold and 2-fold higher induction of Mo TF and Mo intercellular adhesion molecule-1, respectively, when compared with coculture with human aortic endothelial cells (HAEC). In addition, TF-dependent and -independent activation of coagulation factor X was higher on PAEC than on HAEC. Low levels of mRNA for tissue factor pathway inhibitor (TFPI) and its variant, TFPI-2, in resting PAEC were up-regulated by stimulation with tumor necrosis factor alpha. Procoagulant activity of recombinant human TF complexed to activated factor VII was inhibited by PAEC and HAEC-associated TFPI by 22% and 56%, respectively. In contrast, human activated factor X (factor Xa) activity was inhibited by human, but not porcine, EC-associated TFPI, suggesting functional incompatibility of PAEC for human factor Xa. Endothelial TFPI was detected in pig control organs and after hyperacute rejection, but was lost from the vasculature during DXR. CONCLUSIONS: Lack of appropriate human factor Xa inhibition by porcine EC during hyperacute rejection and loss of porcine EC TFPI during DXR could promote the development of a procoagulant environment leading to xenograft rejection.

Amino Acid Sequence↗
PubMed1997

Loss of ATP diphosphohydrolase activity with endothelial cell activation.

Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.

Adenosine Diphosphate↗
PubMed1997

Three-dimensional modeling of renal glomerular capillary networks.

OBJECTIVE: To quantitatively characterize three-dimensional (3D) vascular patterns based on the path length distribution in networks. STUDY DESIGN: Volume data were stacks of confocal fluorescence images of renal glomeruli, obtained using a confocal laser scanning microscope and spanning 60-130 microns in the z axis. After manual editing to remove nonglomerular components, transverse sections of glomerular capillary lumens were segmented automatically using two-dimensional morphologic filters. The center points and the overlap (between adjacent sections) of lumens segmented in the x vs. y plane were used to derive a graph (i.e., a multiply connected network) for each glomerulus. The average degree and the distance between connected nodes were used to derive the number of graph edges and the overall length of the capillary network. RESULTS: Renderings of the 3D reconstructions demonstrated well the lobular structure of the glomerular tufts. Mean capillary length ranged from 53 to 180 microns in 10 glomeruli. Total capillary length ranged from 3,500 to 9,500 microns (mean 5,833). CONCLUSION: Structural measurements based on confocal data require less effort than do measurements based on serial sections and make detailed study of diseased glomerular populations practical.

Animals↗