Protective effect of the multitarget compound DPH-4 on human SSAO/VAP-1-expressing hCMEC/D3 cells under oxygen-glucose deprivation conditions: an in vitro experimental model of cerebral ischaemia.

BACKGROUND AND PURPOSE: Stroke and Alzheimer's disease (AD) are related pathologies in which the cerebrovascular system is involved. Plasma levels of semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1, also known as Primary Amine Oxidase -PrAO) are increased in both stroke and AD patients and contribute to the vascular damage. During inflammation, its enzymatic activity mediates leukocyte recruitment to the injured tissue, inducing damage in the blood-brain barrier (BBB) and neuronal tissue. We hypothesized that by altering cerebrovascular function, SSAO/VAP-1 might play a role in the stroke-AD transition. Therefore, we evaluated the protective effect of the novel multitarget-directed ligand DPH-4, initially designed for AD therapy, on the BBB. EXPERIMENTAL APPROACH: A human microvascular brain endothelial cell line expressing human SSAO/VAP-1 was generated, as the expression of SSAO/VAP-1 is lost in cultured cells. To simulate ischaemic damage, these cells were subjected to oxygen and glucose deprivation (OGD) and re-oxygenation conditions. The protective role of DPH-4 was then evaluated in the presence of methylamine, an SSAO substrate, and/or ß-amyloid (Aß). KEY RESULTS: Under our conditions, DPH-4 protected brain endothelial cells from OGD and re-oxygenation-induced damage, and also decreased SSAO-dependent leukocyte adhesion. DPH-4 was also effective at preventing the damage induced by OGD and re-oxygenation in the presence of Aß as a model of AD pathology. CONCLUSIONS AND IMPLICATIONS: From these results, we concluded that the multitarget compound DPH-4 might be of therapeutic benefit to delay the onset and/or progression of the neurological pathologies associated with stroke and AD, which appear to be linked.

BBB on chip: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function.

The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm(2) (± 0.9 Ω.cm(2)) and 28.2 Ω.cm(2) (± 1.3 Ω.cm(2)) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm(2). Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm(2). This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.

Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-beta variants in cells composing the cerebral vessel walls.

Cerebral amyloid angiopathy (CAA) is an age-associated condition and a common finding in Alzheimer's disease in which amyloid-beta (Abeta) vascular deposits are featured in >80% of the cases. Familial Abeta variants bearing substitutions at positions 21-23 are primarily associated with CAA, although they manifest with strikingly different clinical phenotypes: cerebral hemorrhage or dementia. The recently reported Piedmont L34V Abeta mutant, located outside the hot spot 21-23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. We monitored the apoptotic events occurring after stimulation of human brain microvascular endothelial and smooth muscle cells with nonfibrillar structures of both variants and wild-type Abeta40. Induction of analogous caspase-mediated mitochondrial pathways was elicited by all peptides, although within different time frames and intensity. Activated pathways were susceptible to pharmacological modulation either through direct inhibition of mitochondrial cytochrome c release or by the action of pan- and pathway-specific caspase inhibitors, giving a clear indication of the independent or synergistic engagement of both extrinsic and intrinsic mechanisms. Structural analyses of the Abeta peptides showed that apoptosis preceded fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA.

Review article: the pathophysiology of thrombocytopenia in hepatitis C virus infection and chronic liver disease.

BACKGROUND: The pathophystology of thrombocytopenia in patients with chronic liver disease resulting from hepatitis C virus (HCV) infection is complex and involves several complementary mechanisms that likely act in concert. AIM: To summarize the available data on the etiology of thrombocytopenia in patients with chronic liver disease. RESULTS: In patients with untreated hepatitis C, both prevalence and severity of thrombocytopenia increase in parallel with the extent of disease, usually becoming clinically relevant when patients develop extensive fibrosis and/or cirrhosis. Pathogenetic mechanisms include hypersptenism secondary to portal hypertension, bone marrow suppression resulting from either HCV itself or interferon treatment, aberrations of the immune system resulting in the formation of anti-platelet antibodies and/or immune-complexes that bind to platelets and facilitate their premature clearance, development of immunologically-mediated extrahepatic manifestations including mixed cryoglobulinemia with or without associated joint, renal, or cutaneous involvement, and thrombopoietin (TPO) deficiency secondary to liver dysfunction. In chronic liver disease, the natural inverse relationship between TPO and platelet levels is not maintained; therefore, blood TPO levels fail to have clinical relevance or predictive value in assessing the thrombocytopenic status of a given patient. CONCLUSIONS: The development of thrombocytopenisa in patients with chronic liver disease is complex and multifactorial.

Blood-brain barrier-specific properties of a human adult brain endothelial cell line.

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Rituximab for autoimmune haemophilia: a proposed treatment algorithm.

We previously reported durable complete responses following brief courses of rituximab and prednisone with or without cyclophosphamide in four patients with autoimmune haemophilia and inhibitor titres of 5-60 BU. We report here responses to this monoclonal anti-CD20 antibody in four additional patients, including two patients with inhibitor titres >200 BU. Factor VIII levels became normal 2-35weeks after 4 or 8 weekly doses of rituximab, brief courses of prednisone and in one patient immunoglobulin. Complete responses are ongoing at 10 months in two patients. Two patients relapsed: a patient whose initial inhibitor titre was 525 BU relapsed at 3.5 months and a long-term prednisone-dependent patient at 8.5 months. Both responded to second courses of rituximab and prednisone and are in remission. Our experience suggests that rituximab is a safe and effective addition to immunosuppression with prednisone and cyclophosphamide to treat autoimmune haemophilia, and may permit early discontinuation or even avoidance of these potentially toxic agents. High-titre inhibitor patients, however, may require multiple courses of rituximab or the addition of cyclophosphamide. Pending randomized studies, we propose an algorithm based on our experience and other reports for incorporating rituximab in the treatment of this rare disorder.

Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor signals through multiple pathways in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) encodes a chemokine-like G protein-coupled receptor (KSHV-GPCR) that is implicated in the pathogenesis of Kaposi's sarcoma (KS). Since endothelial cells appear to be targets for the virus, we developed an in vitro mouse lung endothelial cell model in which KSHV-GPCR is stably expressed and KSHV-GPCR signaling was studied. In mouse lung endothelial cells: 1) KSHV-GPCR does not exhibit basal signaling through the phosphoinositide-specific phospholipase C pathway but inositol phosphate production is stimulated by growth-related oncogene alpha (Gro-alpha) via a pertussis toxin (PTX)-insensitive pathway; 2) KSHV-GPCR signals basally through a PTX-sensitive pathway leading to a lowering of intracellular cAMP level that can be lowered further by Gro alpha and increased by interferon gamma-inducible protein 10; 3) KSHV-GPCR stimulates phosphatidylinositol 3-kinase via a PTX-insensitive mechanism; and 4) KSHV-GPCR activates nuclear factor-kappa B (NF-kappa B) by a PTX-sensitive G beta gamma subunit-mediated pathway. These data show that KSHV-GPCR couples to at least two G proteins and initiates signaling via at least three cascades in endothelial cells thereby increasing the complexity of regulation of endothelial cell function by KSHV-GPCR that may occur during viral infection.

[Arachidonic acid and prostaglandins, inflammation and oncology]. / Acide arachidonique et prostaglandines, inflammation et oncologie.

CYCLOOXYGENASE PATHWAY: Among the 3 metabolic pathways leading to oxidation of arachidonic acid, the cyclooxygenase (COX) pathway produces prostaglandin G2 that is rapidly transformed into prostaglandin H2. PROSTAGLANDINS: Prostaglandins are inflammation mediators that are strongly implicated in tumorgenesis. They participate in tumor initiation, promotion and growth. INFLAMMATION AND EPITHELIAL CANCER: Chronic inflammation is a risk factor for epithelial cancer. It induces prostaglandin synthesis via activation of COX-2. There is a cause and effect relationship between chronic inflammation and carcinogeneis via COX-2 expression. It has been demonstrated that COX-2 favors tumor invasion and inhibits apoptosis. Tumor growth is favored by PGE2-induced reduction in immunity; COX-2 inhibitors reinforce the immune response. Finally COX-2 is expressed in tumor neovessels and plays a role in angiogenesis.

[Cyclooxygenases]. / Les cyclooxygénases.

TWO ISOFORMS: There are two isoforms of cyclooxygenase (COX) with similar structure and metabolic activity. COX-1 is a constitutional enzyme. COX-2 is an inductible form. CYCLOOXYGENASE-1: COX-1 is present in most tissues, particularly in the kidney, the digestive tract mucosa, platelets, brain, liver and spleen. CYCLOOXYGENASE-2: COX-2 expression can be induced by an inflammatory process. Implicated in the development of certain cancers, COX-2 is expressed in numerous tumor cell lines and in most epithelial carcinomas. COX-2 favors tumor invasion and inhibits apoptosis. When it is absent, tumor growth is slower or stopped.

[Cyclooxygenase inhibitors]. / Les inhibiteurs des cyclooxygénases.

DISTINCT EFFECTS: Since the discovery of the two isoforms of COX, the therapeutic effects of nonsteroidal antiinflammatory drugs (NSAID) can be distinguished from their adverse effects linked to the inhibition of the constitutional form via selective inhibition of the inducible form. Non-selective NSAID that inhibit both COX isoforms are difficult to use for long-term regimens. NSAID AND CANCER PREVENTION: Epidemiology studies and animal and in vitro studies have demonstrated that regular use of NSAID reduced the incidence of colorectal cancer and certain precancer lesions. PROMISING PERSPECTIVES: COX-2 specific inhibitors, for example cerecoxib or rofecoxib, are potentially interesting for human therapy for chemoprevention of epithelial cancer or as medical treatment, alone or in combination with other anticancer treatments.

Genetic hypofibrinolysis in complicated pregnancies.

OBJECTIVE: To assess the hypofibrinolytic 4G/4G mutation of the plasminogen activator inhibitor (PAI-1) gene as a possible factor contributing to severe preeclampsia, abruptio placentae, fetal growth restriction, and stillbirth. METHODS: We compared 94 women from a previous report who had obstetric complications to 95 controls with normal pregnancies matched for ethnic background and age. We collected blood and extracted DNA after delivery. All subjects had been tested for thrombophilic mutations factor V Leiden, C677T mutation in the methylenetetrahydrofolate reductase gene, and the G20210A mutation in the prothrombin gene. In the present study we tested for the hypofibrinolytic 4G/4G mutation in the PAI-1 gene. RESULTS: Women who had obstetric complications were more likely than controls to be 4G/4G homozygotes, 32% (30 of 94) women versus 19% (18 of 95) controls, odds ratio (OR) and 95% confidence intervals (CI) 2.0 (1.02, 3.9). Mutations in the PAI-1 gene were independently associated with obstetric complications (OR 1.56, 95% CI 1.005, 2.43). Heterozygosity for the factor V Leiden mutation was more common in the 30 women who had PAI-1 4G/4G than in the 18 4G/4G controls (33% versus 0%, Fisher P =.008). Seventy-six percent of women had some form of thrombophilia or hypofibrinolysis compared with 37% of controls (Fisher P <.001). CONCLUSIONS: Women with severe preeclampsia, abruptio placentae, fetal growth restriction, and stillbirth had increased incidence of the hypofibrinolytic 4G/4G mutation of the PAI-1 gene that is frequently associated with the thrombophilic factor V Leiden mutation, further predisposing them to thrombosis.

Cyclo-oxygenase 2: a pharmacological target for the prevention of cancer.

Understanding the mechanisms underlying carcinogenesis provides insights that are necessary for the development of therapeutic strategies to prevent cancer. Chemoprevention--the use of drugs or natural substances to inhibit carcinogenesis - is an important and rapidly evolving aspect of cancer research. We discuss evidence that cyclooxygenase 2 (COX 2), an inducible form of the enzyme, is a potential pharmacological target to prevent cancer. Key data implicating a causal relation between increased activity of COX 2 and carcinogenesis and possible mechanisms of action of COX 2 in this context are covered. Importantly, selective COX 2 inhibitors appear to be safe enough in human beings to allow large-scale clinical testing in healthy people. Several chemoprevention trials using selective COX 2 inhibitors are underway.

Antiplatelet agents in stroke prevention. combination therapy: present and future.

Platelets contribute to arterial thrombosis by multiple mechanisms that promote blood clotting, favor vasoconstriction, activate the procoagulant capacity of endothelium, and stimulate inflammation. These activities are augmented by turbulent blood flow. Classic antiplatelet therapy with aspirin to prevent occlusive stroke offers significant clinical benefit (20-25% risk reduction), yet is less effective than in prevention of coronary artery occlusion (up to 50% risk reduction of myocardial infarction in unstable angina). Since aspirin's antiplatelet effects are limited to blocking a single metabolic pathway - namely inhibition of thromboxane A(2) formation -, and aspirin fails to alter platelet adhesion, other antiplatelet agents that target ADP receptors, platelet surface glycoproteins (such as the GPIIb/IIIa complex), or platelet-dependent thrombin generation offer additional clinical benefits by blocking additional separate pathways or the final common pathway of platelet activation. Combinations of antiplatelet agents, such as aspirin/dipyridamole, aspirin/clopidogrel, or aspirin/GPIIb/IIIa inhibitors, have recently been tested for improved efficacy in clinical trials. Soluble recombinant CD39, an ecto-ADPase, protects against stroke in animal models by metabolizing released ADP/ATP to antiplatelet derivatives. In general, combinations of antiplatelet agents promise greater efficacy than single drugs in preventing stroke, since interactions among different antiplatelet mechanisms can be synergistic. However, such combinations may also increase the risk of bleeding, so that precise understanding of risk/benefit ratios that address the possibility of intracranial as well as gastrointestinal bleeding will require careful monitoring in large clinical trials of patients at risk of stroke, with particular attention to the elderly.

Annexin 1 binds to U937 monocytic cells and inhibits their adhesion to microvascular endothelium: involvement of the alpha 4 beta 1 integrin.

Annexin 1 (ANX1), a calcium-binding protein, participates in the regulation of early inflammatory responses. Whereas some of its effects depend on intracellular interactions, a growing number of observations indicate that ANX1 may also act via autocrine/paracrine functions following externalization to the outer side of the plasma membrane. We studied the effects of ANX1 on leukocyte adhesion to endothelial cells using as a model system the monocytic cell line U937 and human bone marrow microvascular endothelial cells. Exogenous rANX1, as well as endogenous ANX1 externalized by U937 differentiated in vitro, inhibited monocyte firm adhesion to vascular endothelium. Both binding of ANX1 to U937 cells and ANX1-mediated inhibition of cell adhesion involved the short N-terminal domain of the ANX1 molecule. Under experimental conditions in which ANX1 inhibited U937 adhesion to human bone marrow microvascular endothelial cells, this protein specifically colocalized with the alpha 4 integrin, and a direct interaction between ANX1 and the alpha 4 integrin could be documented by immunoprecipitation experiments. Moreover, ANX1 competed with the endothelial integrin counterreceptor, VCAM-1, for binding to alpha 4 integrin. These results indicate that ANX1 plays an important physiological role in modulating monocyte firm adhesion to the endothelium.

Benzo[a]pyrene induces the transcription of cyclooxygenase-2 in vascular smooth muscle cells. Evidence for the involvement of extracellular signal-regulated kinase and NF-kappaB.

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) present in tobacco smoke and tar, have been implicated in the development of atherosclerosis as well as cancer. Increased expression of cyclooxygenase-2 (COX-2) has been detected both in atherosclerotic lesions and in epithelial cancers. To determine whether polycyclic aromatic hydrocarbons might directly affect COX expression in vascular cells, we investigated the effects of B[a]P on COX-2 expression in human and rat arterial smooth muscle cells (SMC). Treatment with B[a]P increased levels of COX-2 protein and mRNA and enhanced prostaglandin synthesis. Nuclear runoff assays and transient transfections revealed increased COX-2 gene transcription after treatment with B[a]P. Experiments were done to define the signaling mechanism by which B[a]P induced COX-2. B[a]P caused a rapid increase in phosphorylation of extracellular signal-regulated kinase (ERK); pharmacologic inhibition of mitogen-activated protein kinase kinase blocked B[a]P-mediated induction of COX-2. Depletion of the intracellular antioxidant, glutathione, with buthionine sulfoximine significantly increased B[a]P-mediated induction of COX-2 while exposure to N-acetylcysteine, a precursor of glutathione, suppressed the induction of COX-2 by B[a]P. Several lines of evidence suggest that the induction of COX-2 by B[a]P is mediated, at least in part, by NF-kappaB. Treatment with B[a]P increased binding of NF-kappaB to DNA. Moreover, B[a]P-mediated stimulation of COX-2 promoter activity was blocked when a construct containing a mutagenized NF-kappaB site was used. Pharmacological inhibitors of NF-kappaB blocked the induction of COX-2 protein and the stimulation of COX-2 promoter activity by B[a]P. Taken together, these data are likely to be important for understanding the atherogenic effects of tobacco smoke.

Immunohistochemical detection of polycyclic aromatic hydrocarbon-DNA damage in human blood vessels of smokers and non-smokers.

The presence of cigarette smoking-related polycyclic aromatic hydrocarbon (PAH)-DNA adducts was investigated in human arterial tissue using an immunoperoxidase staining method with a monoclonal antibody that recognizes benzo(a)pyrene and structurally related PAH diol epoxide-DNA adducts. This is the first time that immunohistochemical methods for detection of PAH-DNA damage have been applied to human endothelial and smooth muscle cells of blood vessels. Internal mammary artery specimens from a total of 37 smokers and non-smokers were tested. Positive nuclear staining was observed in both endothelial and smooth muscle cells, with higher staining in the endothelium. The correlation between smoking status, available for 33 subjects, and detectable PAH-DNA adducts in endothelium did not reach statistical significance (odds ratio = 3.38, 95% confidence interval is 0.47-27.60) in this small series. While no causal role can be inferred from our results, they support the theory that endothelial injury caused by cigarette smoking and other environmental exposures may be an early event in the process of atherosclerosis.

Profibrinolytic properties characterize a stably transformed human endothelial cell line.

A stable immortalized venous endothelial cell (IVEC) line, obtained by transfection of human umbilical vein endothelial cells (HUVEC), retains many normal differentiated endothelial characteristics. We compared the fibrinolytic activities of IVEC and HUVEC, and observed that IVEC express a more profibrinolytic phenotype than HUVEC, since they bind and activate plasminogen more efficiently, produce more tissue plasminogen activator and urokinase-type plasminogen activator antigens, and secrete less plasminogen activator inhibitor-1 antigen both under basal conditions and after stimulation with lipopolysaccharide, phorbol ester and tumor necrosis factor. Moreover, immunostaining and Western blotting of IVEC for the plasminogen/tissue plasminogen activator receptor annexin II, as well as Northern blotting of annexin II mRNA, revealed similar patterns of surface expression in IVEC and HUVEC. Plasminogen activator inhibitor-2 is expressed similarly in both cell types. IVEC may be a useful human model for functional and pharmacological explorations and modulations of fibrinolytic system components.

Genomic instability in the type II TGF-beta1 receptor gene in atherosclerotic and restenotic vascular cells.

Cells proliferating from human atherosclerotic lesions are resistant to the antiproliferative effect of TGF-beta1, a key factor in wound repair. DNA from human atherosclerotic and restenotic lesions was used to test the hypothesis that microsatellite instability leads to specific loss of the Type II receptor for TGF-beta1 (TbetaR-II), causing acquired resistance to TGF-beta1. High fidelity PCR and restriction analysis was adapted to analyze deletions in an A10 microsatellite within TbetaR-II. DNA from lesions, and cells grown from lesions, showed acquired 1 and 2 bp deletions in TbetaR-II, while microsatellites in the hMSH3 and hMSH6 genes, and hypermutable regions of p53 were unaffected. Sequencing confirmed that these deletions occurred principally in the replication error-prone A10 microsatellite region, though nonmicrosatellite mutations were observed. The mutations could be identified within specific patches of the lesion, while the surrounding tissue, or unaffected arteries, exhibited the wild-type genotype. This microsatellite deletion causes frameshift loss of receptor function, and thus, resistance to the antiproliferative and apoptotic effects of TGF-beta1. We propose that microsatellite instability in TbetaR-II disables growth inhibitory pathways, allowing monoclonal selection of a disease-prone cell type within some vascular lesions.

Characterization of a newly established human bone marrow endothelial cell line: distinct adhesive properties for hematopoietic progenitors compared with human umbilical vein endothelial cells.

Human bone marrow endothelial cells (HBMEC) are intimately involved in the homing of hematopoietic progenitor cells (HPC) to the bone marrow and in the regulation of proliferation and differentiation of these cells. Because availability of primary HBMEC and their capacity to be cultured in vitro are limited, we used isolated HBMEC to establish a cloned cell line by microinjection of a recombinant plasmid expressing simian virus 40 early genes under the control of a deletion mutant of the human vimentin promoter. Serum requirements for growth of a transformed HBMEC line (TrHBMEC) were markedly decreased compared with those of primary cells, and added growth factors were not required for proliferation. Cells took up acetylated low-density lipoprotein normally, bound to Ulex europaeus lectin, and stained positively for von Willebrand factor, P-selectin, CD31, CD34, CD44, very late antigen-5, and intercellular adhesion molecule-2 (ICAM-2). After treatment with TNF-alpha or lipopolysaccharide, TrHBMEC increased surface expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 in a manner similar to primary HBMEC. In contrast, IL-1 beta elicited much less up-regulation of these adhesion molecules than in primary cells. In previous work, we reported that, in a flow adhesion model, rolling of peripheral blood CD34+ cells on primary HBMEC was E-selectin-dependent, whereas VCAM-1 and ICAM-1 contributed to firm adhesion. In the present study, we show that HPC adhere in a similar way to TrHBMEC. A less-pronounced role for VCAM-1 and ICAM-1 was found in the adhesion of HPC to human umbilical vein endothelial cells. Furthermore, significantly more CD34+ cells adhered to TNF-alpha-stimulated HBMEC and TrHBMEC than to similarly stimulated human umbilical vein endothelial cells. These data emphasize the importance of using microvessel HBMEC for studying the homing of HPC to the bone marrow, and indicate the usefulness of the above-described bone marrow endothelial cell line.

Inhibition of cyclooxygenase: a novel approach to cancer prevention.

An expanding body of evidence indicates that downregulation of the cyclooxygenases (Cox-1 and Cox-2) will be an important strategy for preventing cancer because cyclooxygenases catalyze the formation of prostaglandins (PGs), and PGs have multiple effects that favor tumorigenesis. PGs also are more abundant in cancers than in the normal tissues from which cancers arise. Overexpression of Cox-2 in epithelial cells inhibits apoptosis and increases the invasiveness of tumor cells; inhibitors of Cox (e.g., NSAIDS) are chemopreventive; and tumorigenesis is inhibited in Cox-2 knockout mice. We focus in this review on strategies to selectively inhibit and downregulate the Cox-2 isoform. This is important because simultaneous inhibition of Cox-1 (constitutively expressed) and Cox-2 (inducible isoform), which is achieved with classical NSAIDs, interferes with the housekeeping functions of Cox-1 and thereby causes serious side effects, such as peptic ulcer disease. Simultaneous inhibition of Cox-1 and Cox-2 hence is not a realistic approach for chemoprevention in individuals at low to moderate risk for cancer. On the other hand, it appears possible to avoid many NSAID-dependent side effects by selective inhibition of Cox-2, which is also the isoform that is upregulated in benign and malignant tumors. Through understanding the biochemistry of these enzymes and the regulation of Cox-1 and Cox-2 gene expression, we review how Cox-2 can be regulated selectively as a target for chemopreventive therapy. We also discuss the potential importance and advantages of a multifaceted approach to diminishing the function of Cox-2 (i.e., combining inhibitors of enzyme function with inhibitors of gene expression).