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Prostaglandyny są hormonami występującymi niemal we wszystkich ssaczych tkankach. Jako cząsteczki sygnałowe odgrywają one kluczową rolę w regulacji wielu procesów fizjologicznych, m. in. cyklu wzrostu włosa. W artykule opisano historię odkrycia prostaglandyn, w tym prace profesora Ryszarda Gryglewskiego - odkrywcy prostacykliny. Szczególną uwagę zwrócono na syntetyczny analog prostaglandyny F2α - latanoprost. Jest to lek wskazany w leczeniu jaskry, którego działaniem ubocznym jest nadmierny wzrost rzęs. Jako prolek, latanoprost ulega przekształceniu do aktywnego metabolitu - kwasu latanoprostowego. Dzięki ostatnim badaniom wiadomo, że kwas latanoprostowy ma szanse stać się skuteczną alternatywą dla minoksydylu i finasterydu - jedynych leków zarejestrowanych obecnie do leczenia łysienia androgenowego. Wprowadzenie na rynek leków przeciwko łysieniu zawierających pochodne prostaglandyn, w tym kwas latanoprostowy będzie procesem znacznie szybszym w porównaniu do tradycyjnej ścieżki rozwoju produktu, opartego o nowy związek chemiczny.

Background: The tobacco use is one of the biggest public health threats worldwide. Cigarette smoke contains over 7000 chemicals among other aldehydes, regarded as priority toxicants. β-escin (a mixture of triterpenoid saponins extracted from the Aesculus hippocastanum. L) is a potent activator of aldehyde dehydrogenase (ALDH) - an enzyme catalyzing oxidation of aldehydes to non-toxic carboxylic acids., Purpose: The aim of this study was to evaluate the effect of β-escin on ALDH activity, ALDH isoforms mRNA expression and cytotoxicity in nasal epithelial cells exposed to cigarette smoke extract (CSE)., Methods: Nasal epithelial cells from healthy non-smokers were treated with β-escin (1 µM) and exposed to 5% CSE. After 6- or 24-hours of stimulation cell viability, DNA damage, ALDH activity and mRNA expression of ALDH isoforms were examined., Results: 24 h β-escin stimulation revised CSE induced cytotoxicity and DNA damage. Cells cultured with β-escin or exposed to CSE responded with strong increase in ALDH activity. This effect was more pronounced in cultures treated with combination of β-escin and CSE. The strongest stimulatory effect on ALDH isoform mRNA expression was observed in cells cultured simultaneously with β-escin and CSE: at 6 h for ALDH1A1 and ALDH3A1, and at 24 h for ALDH1A3, ALDH3A2, ALDH3B1, and ALDH18A1. Combined β-escin and CSE treatment prevented the CSE-induced inhibition of ALDH2 expression at 24 h., Conclusions: β-escin is an effective ALDH stimulatory and cytoprotective agent and might be useful in the prevention or supportive treatment of tobacco smoke-related diseases., Competing Interests: Declaration of Competing Interest K.K., M.D. and O.Z.-S. are inventors on patents related to β-escin and its derivatives. The other authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Chronic overproduction of IL-15 contributes to the pathogenesis of numerous inflammatory and autoimmune disorders. Experimental methods used to reduce the cytokine activity show promise as potential therapeutic approaches to modify IL-15 signaling and alleviate the development and progression of IL-15-related diseases. We previously demonstrated that an efficient reduction of IL-15 activity can be obtained by selective blocking of the specific, high affinity subunit alpha of the IL-15 receptor (IL-15Rα) with small-molecule inhibitors. In this study, we determined the structure-activity relationship of currently known IL-15Rα inhibitors in order to define the critical structural features required for their activity. To validate our predictions, we designed, analyzed in silico, and assessed in vitro function of 16 new potential IL-15Rα inhibitors. All newly synthesized molecules were benzoic acid derivatives with favorable ADME properties and they efficiently reduced IL-15 dependent peripheral blood mononuclear cells (PBMCs) proliferation, as well as TNF-α and IL-17 secretion. The rational design of IL-15 inhibitors may propel the identification of potential lead molecules for the development of safe and effective therapeutic agents.

Background: Recent advancements in understanding β-escin action provide basis for new therapeutic claims for the drug. β-escin-evoked attenuation of NF-κB-dependent signaling, increase in MMP-14 and decrease in COUP-TFII content and a rise in cholesterol biosynthesis could be beneficial in alleviating muscle-damaging processes., Purpose: The aim of this study was to investigate the effect of β-escin on skeletal muscle regeneration., Methods: Rat model of cardiotoxin-induced injury of fast-twich extensor digitorum longus (EDL) and slow-twich soleus (SOL) muscles and C2C12 myoblast cells were used in the study. We evaluated muscles obtained on day 3 and 14 post-injury by histological analyses of muscle fibers, connective tissue, and mononuclear infiltrate, by immunolocalization of macrophages and by qPCR to quantify the expression of muscle regeneration-related genes. Mechanism of drug action was investigated in vitro by assessing cell viability, NF-κB activation, MMP-2 and MMP-9 secretion, and ALDH activity., Results: In rat model, β-escin rescues regenerating muscles from atrophy. The drug reduces inflammatory infiltration, increases the number of muscle fibers and decreases fibrosis. β-escin reduces macrophage infiltration into injured muscles and promotes their M2 polarization. It also alters transcription of muscle regeneration-related genes: Myf5, Myh2, Myh3, Myh8, Myod1, Pax3 and Pax7, and Pcna. In C2C12 myoblasts in vitro, β-escin inhibits TNF-α-induced activation of NF-κB, reduces secretion of MMP-9 and increases ALDH activity., Conclusions: The data reveal beneficial role of β-escin in muscle regeneration, particularly in poorly regenerating slow-twitch muscles. The findings provide rationale for further studies on β-escin repositioning into conditions associated with muscle damage such as strenuous exercise, drug-induced myotoxicity or age-related disuse atrophy., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

A continuing quest for specific inhibitors of proinflammatory cytokines brings promise for effective therapies designed for inflammatory and autoimmune disorders. Cefazolin, a safe, first-generation cephalosporin antibiotic, has been recently shown to specifically interact with interleukin 15 (IL-15) receptor subunit α (IL-15Rα) and to inhibit IL-15-dependent TNF-α and IL-17 synthesis. The aim of this study was to elucidate cefazolin activity against IL-2, IL-4, IL-15 and IL-21, i.e. four cytokines sharing the common cytokine receptor γ chain (γ c ). In silico, molecular docking unveiled two potential cefazolin binding sites within the IL-2/IL-15Rβ subunit and two within the γ c subunit. In vitro, cefazolin decreased proliferation of PBMC (peripheral blood mononuclear cells) following IL-2, IL-4 and IL-15 stimulation, reduced production of IFN-γ, IL-17 and TNF-α in IL-2- and IL-15-treated PBMC and in IL-15 stimulated natural killer (NK) cells, attenuated IL-4-dependent expression of CD11c in monocyte-derived dendritic cells and suppressed phosphorylation of JAK3 in response to IL-2 and IL-15 in PBMC, to IL-4 in TF-1 (erythroleukemic cell line) and to IL-21 in NK-92 (NK cell line). The results of the study suggest that cefazolin may exert inhibitory activity against all of the γ c receptor-dependent cytokines, i.e. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

Upregulation of interleukin 15 (IL-15) contributes directly i.a. to the development of inflammatory and autoimmune diseases. Selective blockade of IL-15 aimed to treat rheumatoid arthritis, psoriasis and other IL-15-related disorders has been recognized as an efficient therapeutic method. The aim of the study was to identify small molecules which would interact with IL-15 or its receptor IL-15Rα and inhibit the cytokine's activity. Based on the crystal structure of IL-15Rα·IL-15, we created pharmacophore models to screen the ZINC database of chemical compounds for potential IL-15 and IL-15Rα inhibitors. Twenty compounds with the highest predicted binding affinities were subjected to in vitro analysis using human peripheral blood mononuclear cells to validate in silico data. Twelve molecules efficiently reduced IL-15-dependent TNF-α and IL-17 synthesis. Among these, cefazolin - a safe first-generation cephalosporin antibiotic - holds the highest promise for IL-15-directed therapeutic applications., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Interleukin 15 is a pleiotropic cytokine of the four α helix bundle family. Binding to a heterotrimeric receptor complex, which consists of a unique, high affinity IL‑15Rα‑chain and IL-2/IL-15Rβ and IL‑2Rγ chains, IL‑15 activates signaling pathways leading to activation and proliferation of T and B cells, as well as natural killer cells. At the same time, IL‑15 protects effector cells from T regulatory cells and does not induce immune tolerance. The significant regulatory action of IL‑15 on the immune system provides new opportunities for development of anti‑cancer therapies. As documented in many experiments using different tumor models, IL‑15 enhances antitumor effects. To improve the efficiency of IL‑15, several strategies, including combination with other anti‑cancer therapies such as chemotherapy, additional use of antibodies (anti‑PD‑L1, anti‑CTLA‑4, anti‑CD40), or other cytokines, have been evaluated. Increased anti‑tumor activity can also be obtained by using IL‑15 agonists. However, acting as a growth factor for immune cells but also for tumor cells, IL‑15 may promote their proliferation, survival and dissemination. Of significance seems the role of IL‑15 in the pathogenesis of hematological malignancies, which is due to the involvement in the proliferation and differentiation of NK, T and B cells. Currently, several experimental strategies are available to block biological activity of IL‑15. Among compounds inhibiting the activity of IL‑15 are not only monoclonal antibodies interacting directly with the cytokine or with IL‑15R subunits, but also mutant forms of IL‑15 and protein constructs.

β-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds (Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of β-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of β-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of β-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells β-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-α-induced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of β-escin beyond the current vascular indications., Competing Interests: DD, OZ-S, DM, MK and KK have a potential financial competing interest related to a patent application EP15001035.3 “New therapy for Alzheimer’s disease. OZ-S, IG, DM, MK and KK have a potential financial competing interest related to a patent application PCT/IB2016/000187 “Protoescigenin derivative, process of its preparation, use of said compound and pharmaceutical composition comprising that compound. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Coronary artery bypass grafting (CABG) triggers oxidative stress and platelet activation. High acetylsalicylic acid (ASA) dose might mitigate the transient proinflammatory state. We compared the effect of three ASA dosages on post-CABG platelet reactivity, oxidative stress, and serum CD39 and CD73 levels. Thirty-six consecutive patients undergoing elective off-pump CABG, pre-treated with ASA 1 × 75 mg for ≥7 days, were randomized to continue the prior treatment regimen, switch to ASA 1 × 150 mg, or ASA 2 × 75 mg. Blood was collected on admission, 7 days, 1 month, and 3 months after CABG. Platelet reactivity was assessed using impedance aggregometry. Platelet oxidative stress was measured as platelet mitochondria extracellular oxygen consumption rate and oxidatively damaged whole-blood DNA cleavage. Serum CD39 and CD73 levels were determined using ELISA. Platelet reactivity and oxidative stress parameters were comparable in all groups. Patients treated with ASA 2 × 75 mg had higher CD39 levels at 7 days and 1 month ( p  = .049, p  = .033), compared to the control group. ASA 2 × 75 mg was associated a beneficial effect on serum CD39 levels after off-pump CABG, without a significant effect on oxidative stress parameters.

The Parkes Weber syndrome is a congenital vascular malformation, characterized by varicose veins, arterio-venous fistulas and overgrown limbs. No broadly accepted animal model of Parkes Weber syndrome has been described. We created side-to-side arterio-venous fistula between common femoral vessels with proximal non-absorbable ligature on common femoral vein limiting the enlargement of the vein diameter in Wistar rats. Contralateral limb was sham operated. Invasive blood pressure measurements in both iliac and inferior cava veins were performed in rats 30 days after fistula creation. Tight circumference and femoral bone length were measured. Histopathology and morphology of soleus muscle, extensor digitorum longus muscle, and the common femoral vessel were analyzed. 30 days following arterio-venous fistula creation, a statistically significant elevation of blood pressure in common iliac vein and limb overgrowth was observed. Limb enlargement was caused by muscle overgrowth, varicose veins formation and bone elongation. Arterio-venous fistula with proximal outflow limitation led to significant increase of femoral vein circumference and venous wall thickness. Our study indicates that the described rat model mimics major clinical features characteristic for the human Parkes Weber syndrome: presence of arterio-venous fistula, venous hypertension and dilatation, varicose veins formation, and the limb hypertrophy. We reveal that limb overgrowth is caused by bone elongation, muscle hypertrophy, and venous dilatation. The newly established model will permit detailed studies on the mechanisms underlying the disease and on the efficacy of novel therapeutic strategies for the Parkes Weber syndrome treatment.

A two-step chemical process for controlled degradation of escin, affording a mixture of olean-12-ene sapogenins, was elaborated and scaled up. The main component of the mixture--protoescigenin--was isolated and purified, in the form of its corresponding monohydrate, without resource to chromatographic methods. This material was further converted into the high purity 3,24;16,22-di-O,O-isopropylidene derivative in a validated large scale laboratory process.

Creating a functional vascularized bone tissue remains one of the main goals of bone tissue engineering. Recently, a growing interest in the crosstalk between endothelial cells (EC) and osteoblasts (OB), the two main players in a new bone formation, has been observed. However, only a few reports have addressed a mutual influence of OB and EC on cell proliferation. Our study focuses on this issue by investigating cocultures of human bone-derived cells (HBDC) and human umbilical vein endothelial cells (HUVEC). Three various proportions of cells have been used that is, HBDC:HUVEC 1:1, 1:4, and 4:1 and the cocultures were investigated on day 1, 4, and 7, while HUVEC and HBDC monocultures served as reference. We have detected enhanced alkaline phosphatase (ALP) activity in a direct HBDC-HUVEC coculture. This effect was not observed when cells were separated by an insert, which is consistent with other reports on various OB-EC lineages. The appearance of gap-junctions in coculture was confirmed by a positive staining for connexin 43. The number of cells of both phenotypes has been determined by flow cytometry: CD-31-positive cells have been considered EC, while CD-31-negative have been counted as OB. We have observed an over 14-fold increase in OB number after a week in the 1:4 HBDC:HUVEC coculture as compared with less than fourfold in monoculture. The increase in HBDC number in 1:1 coculture has been less pronounced and has reached the value of about sevenfold. These results correspond well with the cell proliferation rate, which has been measured by 5-bromo-2'-deoxyuridine incorporation. Moreover, at day 7 EC have been still present in the coculture, which is inconsistent with some other reports. Real-time polymerase chain reaction analysis has revealed the upregulation of ALP and collagen type I genes, but not osteocalcin gene, in all the cocultures grown without pro-osteogenic additives. Our study indicates that HUVEC significantly promote HBDC expansion and upregulate collagen I gene expression in these cells. We believe that these findings have application potency in bone tissue engineering.

Statins, HMG-CoA reductase inhibitors, are used in the prevention and treatment of cardiovascular diseases owing to their lipid-lowering effects. Previous studies revealed that, by modulating membrane cholesterol content, statins could induce conformational changes in cluster of differentiation 20 (CD20) tetraspanin. The aim of the presented study was to investigate the influence of statins on glucose transporter 1 (GLUT1)-mediated glucose uptake in tumor cells. We observed a significant concentration- and time-dependent decrease in glucose analogs' uptake in several tumor cell lines incubated with statins. This effect was reversible with restitution of cholesterol synthesis pathway with mevalonic acid as well as with supplementation of plasma membrane with exogenous cholesterol. Statins did not change overall GLUT1 expression at neither transcriptional nor protein levels. An exploratory clinical trial revealed that statin treatment decreased glucose uptake in peripheral blood leukocytes and lowered (18)F-fluorodeoxyglucose ((18)F-FDG) uptake by tumor masses in a mantle cell lymphoma patient. A bioinformatics analysis was used to predict the structure of human GLUT1 and to identify putative cholesterol-binding motifs in its juxtamembrane fragment. Altogether, the influence of statins on glucose uptake seems to be of clinical significance. By inhibiting (18)F-FDG uptake, statins can negatively affect the sensitivity of positron emission tomography, a diagnostic procedure frequently used in oncology.

Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-α. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that β-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model.

Well-established evidence links extracellular nucleotides to numerous vascular pathologies, including restenosis associated with angioplasty, atherosclerosis and transplant arteriosclerosis. Through activation of purinergic P2 receptors, extracellular nucleotides contribute to the pathogenesis of occlusive vascular diseases by mediating thrombosis, and vascular smooth muscle proliferation and migration. Therefore, there is a growing interest in the enzymes that hydrolyze nucleotides for their capability to modulate nucleotide-triggered pathologies. In this review, we present the current data addressing the therapeutic potential of nucleoside triphosphate diphosphohydrolases (NTPDases) to prevent intimal hyperplasia and treat vascular intimal disease. In addition, we discuss the mechanisms by which NTPDases exert protective effects in vascular function.

Indomethacin is a nonsteroidal anti-inflammatory drug used frequently to control chronic or temporary pain. In the kidney, indomethacin decreases medullary and cortical perfusion, resulting in hypoxia. Kidney hypoxia has many effects, including changes in gene expression, and is a strong stimulus for angiogenesis. Other angiogenic factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2), transforming growth factor beta 1 (TGFbeta1), and platelet-derived growth factor (PDGF). Our goal was to examine the influence of indomethacin on mRNA expression of these factors and their selected receptors in the renal cortex of healthy rats. Groups of 8 healthy, male, six-week-old Wistar rats received either indomethacin (5 mg/kg/day) or placebo orally for three months. RNA from renal cortex biopsies was analyzed by real-time polymerase chain reaction to quantify the mRNA levels of each cytokine. We observed significantly higher mRNA levels for VEGF (1.73-fold), FGF-2 (5.6-fold) and TGFbeta receptor III (2.93-fold), PDGF receptor alpha (2.93-fold) and receptor beta (2.91-fold) in rats receiving indomethacin compared to rats given placebo (p < 0.05). Amounts of mRNA for TGFbeta1, PDGF, FGF receptors 1 and 2 and TGFbeta receptor I did not differ between analysed groups. Our data indicates that indomethacin may regulate the expression of potent angiogenic factors VEGF and FGF-2.

Extracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here, it is shown that stimulation of human umbilical vein endothelial cells (HUVEC) with extracellular ATP or UTP increased intracellular free calcium ion concentrations ([Ca(2+)](i)), induced phosphorylation of focal adhesion kinase (FAK), p130(cas) and paxillin, and caused cytoskeletal rearrangements with consequent cell migration. Furthermore, UTP increased migration of HUVEC in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. BAPTA or thapsigargin inhibited the extracellular nucleotide-induced increase in [Ca(2+)](i), a response crucial for both FAK phosphorylation and cell migration. Furthermore, long-term exposure of HUVEC to ATP and UTP, agonists of the G protein-coupled P2Y2 and P2Y4 receptor subtypes, caused upregulation of alpha(v) integrin expression, a cell adhesion molecule known to directly interact with P2Y2 receptors. Our results suggest that extracellular nucleotides modulate signaling pathways in HUVEC influencing cell functions, such as cytoskeletal changes, cellular adhesion and motility, typically associated with integrin-activation and the action of growth factors. We propose that P2Y2 and possibly P2Y4 receptors mediate those responses that are important in vascular inflammation, atherosclerosis and angiogenesis.

Purpose: The aim of the present study was to potentiate the antitumor effectiveness of photodynamic therapy (PDT). A cDNA microarray analysis was used to evaluate the gene expression pattern after Photofrin-mediated PDT to find more effective combination treatment with PDT and inhibitor(s) of the identified gene product(s) overexpressed in tumor cells., Experimental Design: Atlas Mouse Stress Array was used to compare the expression profile of control and PDT-treated C-26 cells. The microarray results have been confirmed using Western blotting. Cytostatic/cytotoxic in vitro assay as well as in vivo tumor models were used to investigate the antitumor effectiveness of PDT in combination with cyclooxygenase (COX) 2 inhibitors., Results: PDT induced the expression of 5 of 140 stress-related genes. One of these genes encodes for COX-2, an enzyme important in the tumor progression. Inhibition of COX-2 in vitro with NS-398, rofecoxib, or nimesulide, or before PDT with nimesulide did not influence the therapeutic efficacy of the treatment. Administration of a selective COX-2 inhibitor after PDT produced potentiated antitumor effects leading to complete responses in the majority of treated animals., Conclusions: COX-2 inhibitors do not sensitize tumor cells to PDT-mediated killing. However, these drugs can be used to potentiate the antitumor effectiveness of this treatment regimen when administered after tumor illumination.

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.

In this study we examined the effect of experimentally induced asynchrony between male and female pronuclei on male pronucleus formation and developmental potential of the resulting mouse embryos. We demonstrate that when the interval between oocyte activation and sperm incorporation is up to 1.5-2 hr, the spermatozoa transform into normal pronuclei. These male pronuclei can replicate their chromosomes during the first embryonic cell cycle and are transcriptionally competent. During the first cleavage these "delayed" male pronuclei condense into discrete mitotic chromosomes and when resulting embryos are transplanted into oviducts of pregnant females at least some of them can develop to term. In contrast, when sperm nuclei are introduced into parthenogenetic eggs 3 hr or more after activation, their transformation into pronuclei is significantly impaired, and they neither replicate nor transcribe. During the first mitosis they form a group of condensed chromatin, which is displaced into one of the resulting blastomeres leading to formation of haploid/diploid mosaic embryos. These mosaic embryos have poor developmental potential: only a few can reach blastocyst stage in vitro and no full-term development of such embryos was observed after transfer into pregnant females. We conclude that the cytoplasmic factors that make possible the transformation of a sperm nucleus into a functional male pronucleus exhaust within 1.5-2 hr after fertilization and that the male genome which had skipped the first cell cycle cannot become a functional partner in the embryonic genome., (Copyright 1999 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

Erythrocytosis (i.e., elevation in red cell mass) frequently develops after renal transplantation and is associated with increased risk of thromboembolic incidents and hypertension. Because it has been reported that enalapril may induce anemia in renal allograft recipients, we have undertaken a prospective study to estimate the efficacy and safety of enalapril therapy for erythrocytosis and to establish the mechanism by which enalapril reduces red cell mass. Seventeen (12 male and 5 female) long-term renal allograft recipients with increased hematocrit value (> 55% for male and > 50% for female) and elevated red cell mass as determined with 51Cr-labeled autologous erythrocytes were treated with enalapril. After 3 months of therapy, enalapril was withdrawn and patients were observed in order to differentiate spontaneous remission of erythrocytosis from effects of enalapril therapy. After 3 months of the treatment, mean hematocrit decreased from 51.1% (range 47-56%) to 42.9% (range 37-51%; P < 0.01). Red cell mass significantly decreased during this period (from 46.7 ml/kg, range 32.5-60.7 ml/kg, to 32.9 ml/kg, range 20.1-60.1 ml/kg; P < 0.01). Serum erythropoietin levels also changed from 12.2 mIU/ml (range 1.0-33.0 mIU/ml) at baseline to 5.4 mIU/ml (range 0.7-24.2 mIU/ml; P < 0.05). During the following 3 months without enalapril treatment, an increase in hematocrit was noted, reaching 51.7% (range 46-58%; P < 0.05). No serious side effects of enalapril were observed during the study, but there was a need to reduce other hypotensive drugs in some patients. Serum creatinine did not change significantly during enalapril therapy (1.49 mg/dl, range 0.9-2.3 mg/dl, and 1.55 mg/dl, range 1.0-2.3 mg/dl; before and after 3 months of therapy, respectively). Our study proves that enalapril can be safely and effectively used to treat posttransplant erythrocytosis. The effect of enalapril on red cell mass results from reducing erythropoietin production.

The role of protein kinase C (PKC) in the successive steps of mouse oocyte meiotic process was investigated. We have used either OAG, an analog of diacylglycerol, or mezerein, a nonphorbol ester diterpene, less tumor promoting than phorbol esters, as PKC activators, and staurosporine as PKC inhibitor. Cumulus-free oocytes were cultured in minimum essential medium with each of these PKC modulators and maturation stages were screened every two hours until the end of the process. Both PKC activators prevented GVBD at each tested dose for 4 hr (OAG) and 8 hr (mezerein), and decreased the frequencies of PB oocytes. The inhibitory effects of both activators were dose dependent and reversible. The addition of OAG to the culture medium after GVBD occurrence (i.e., after 4 hrs) did not affect PB extrusion whereas similar addition of mezerein significantly decreased the frequency of PB oocytes. Inhibition of PKC by staurosporine accelerated GVBD and increased the frequency of PB extrusion. When staurosporine was added after GVBD, PB extrusion occurred earlier but PB oocyte frequency was not increased. Fertilizability was not affected when oocyte maturation occurred in the presence of any of these substances despite the delay in maturation process. These results clearly indicate that the PKC pathway is involved in mouse oocyte meiotic process: activation of the enzyme would arrest meiotic process whereas its inhibition would participate in meiosis induction.