Non-invasive diagnosis of intracranial aneurysms.

Patients need to be examined for intracranial aneurysms if they have had a subarachnoid hemorrhage. The preferred technique in this situation is CT angiography. Screening can be done for familial forms or for elastic tissue disorders, for which the first line investigation is magnetic resonance angiography. These non-invasive methods have now taken over from conventional angiography that was reserved for the pretreatment phase. A good technical knowledge of these imaging methods, their artifacts and misleading images enables reliable detection of intracranial aneurysms and for an accurate report to be returned to clinicians.

Imaging of cervical artery dissection.

Cervical artery dissection (CAD) may affect the internal carotid and/or the vertebral arteries. CAD is the leading cause of ischemic stroke in patients younger than 45 years. Specific treatment (aspirin or anticoagulants) can be implemented once the diagnosis of CAD has been confirmed. This diagnosis is based on detection of a mural haematoma on ultrasound or on MRI. The diagnosis can be suspected on contrast-enhanced MRA (magnetic resonance angiography) or CT angiography, in case of long stenosis, sparing the internal carotid bulb, or suspended, at the junction of V2 and V3 segments of the vertebral artery, in patients with no signs of atheroma of the cervical arteries. MRI is recommended as the first line imaging screening tool, including a fat suppressed T1 weighted sequence, acquired in the axial or oblique plane at 1.5T, or 3D at 3T. Complete resolution of the lumen abnormality occurred in 80% of cases, and CAD recurrence is rare, encountered in less than 5% of cases. Interventional neuroradiology (angioplasty and/or stenting of the dissected vessel) may be envisaged in rare cases of haemodynamic effects with recurring clinical infarctions in the short-term.

Targeting cancer stem cells expressing an embryonic signature with anti-proteases to decrease their tumor potential.

Cancer stem cells (CSCs) are a specific subset of cancer cells that sustain tumor growth and dissemination. They might represent a significant treatment target to reduce malignant progression and prevent tumor recurrence. In solid tumors, several hierarchically organized CSC clones coexist, even within a single tumor. Among them, CSCs displaying an embryonic stem cell 'stemness' signature, based on the expression of Oct-4, Nanog and Sox2, are present in distinct high-grade tumor types associated with poor prognosis. We previously designed a model to isolate pure populations of these CSCs from distinct solid tumors and used it to screen for molecules showing selective toxicity for this type of CSC. Here we show that human immunodeficiency virus (HIV)-protease inhibitors (HIV-PIs) specifically target CSCs expressing an embryonic signature derived from tumors with distinct origins. They reduced proliferation in a dose-dependent manner with a higher specificity as compared with the total population of cancer cells and/or healthy stem cells, and they were efficient in inducing cell death. Lopinavir was the most effective HIV-PI among those tested. It reduced self-renewal and induced apoptosis of CSCs, subsequently impairing in vivo CSC-induced allograft formation. Two key pharmacophores in the LPV structure were also identified. They are responsible for the specificity of CSC targeting and also for the overall antitumoral activity. These results contribute to the identification of molecules presenting selective toxicity for CSCs expressing an embryonic stemness signature. This paves the way to promising therapeutic opportunities for patients suffering from solid cancer tumors of poor prognosis.

Self-renewal gene tracking to identify tumour-initiating cells associated with metastatic potential.

Tumour-initiating cells (TICs) are rare cancer cells isolated from tumours of different origins including high-grade tumours that sustain neoplasic progression and development of metastatic disease. They harbour deregulated stem cells pathways and exhibit an unchecked ability to self-renew, a property essential for tumour progression. Among the essential factors maintaining embryonic stem (ES) cells properties, OCT-4 (also known as POU5F1) has been detected in tumours of different origins. Although ectopic expression results in dysplasic growth restricted to epithelial tissues, overexpression expands the proportion of immature cells in teratomas. However, OCT-4-expressing cells have not been purified from spontaneously occurring tumours, thus information concerning their properties is rather scant. Here, using p53-/- mice expressing green fluorescent protein and the puromycin resistance gene under the control of the Oct-4 promoter, we show that OCT-4 is expressed in 5% onwards of the undifferentiated tumour cell populations derived from different organs. OCT-4 expression was low as compared with ES cells, but was associated with a 'stemness' signature and expression of the chemokine receptor CXCR4. These cells displayed cancer stem cell features, including increased self-renewal and differentiation ability in vitro and in vivo. They not only formed allografts containing immature bone regions but also disseminated into different organs, including lung, liver and bone. Experiments based on RNA interference revealed that Oct-4 expression drives both their engraftment and metastasis formation. This work points out the crucial contribution of Oct-4-expressing TICs in the hierarchical organization of the malignant potential, leading to metastasis formation. Consequently, it provides an appropriate model to develop novel therapies aiming to strike down TICs by targeting self-renewal genes, therefore efficient to reduce tumour growth and metastatic disease.

Cloning of hOST-PTP: the only example of a protein-tyrosine-phosphatase the function of which has been lost between rodent and human.

Protein-tyrosine-phosphatases (PTP-ases), in concert with protein tyrosine kinases, control various biological activities such as cell growth and differentiation. In rodents, around 40 PTP-ases have been described. Functional orthologue for each of these PTP-ases have been identified in human, except for OST-PTP. OST-PTP is a transmembrane PTP-ase with a restricted tissue distribution. In silico analysis on public sequence databases reveals a human OST-PTP gene orthologue that encompasses 21 kb on chromosome 1q32.1. Using RT-PCR we isolated a 4 kb hOST-PTP transcript. hOST-PTP cDNA sequence exhibits numerous disablements indicating that it does not code for a PTP-ase but is rather a pseudogene with unique features. Indeed, (i) it has no "functional" parent in the human genome, (ii) it has retained an "intron-exon" structure, and (iii) it is transcribed in a regulated manner. Interestingly, we found two ESTs, from domesticated pig and from cow that exhibit ORF that would predict a functional OST-PTP orthologue in Artiodactyls. Taken together, these results indicate that OST-PTP is the only PTP-ase the function of which has been lost during the evolution process between rodents and human.

Coordinated Up-regulation by hypoxia of adrenomedullin and one of its putative receptors (RDC-1) in cells of the rat blood-brain barrier.

Adrenomedullin (ADM) is a potent hypotensive peptide, which is produced during sepsis and ischemia. We demonstrate here that hypoxia induced a time-dependent increase of both ADM mRNA and protein expressions in cultured astrocytes and endothelial cells from rat brain microvessels. Gene reporter analyses showed a 2-fold increase in ADM gene transcription which was suppressed when the ADM promoter was deleted of its hypoxia responsive element. Hypoxia increased 7-fold the stability of pre-formed ADM mRNAs. Rat brain microvessels expressed mRNAs coding for the different putative ADM receptors but they did not respond to exogenous ADM and calcitonin gene-related peptide by the formation of cAMP. In contrast, ADM and calcitonin gene-related peptide increased the formation of cAMP in astrocytes and their actions were potentiated about 2-fold after hypoxia. Messenger RNA species coding for three putative ADM receptors (the L1 orphan receptor, RDC-1, and calcitonin receptor-like receptor) and accessory proteins (receptor-activity modifying proteins) were present in astrocytes. Hypoxia selectively up-regulated expression of RDC-1 receptor mRNAs. The results indicate that ADM and RDC-1 are hypoxia-sensitive genes and that RDC-1 receptors may mediate some actions of ADM in hypoxic astrocytes.

Vascular endothelial growth factors and angiogenesis.

VEGF was discovered in 1989. Research -conducted over the past 10 years has -demonstrated that VEGF is a major regulator of angiogenesis and vasculogenesis. This paper reviews the molecular data on the multiple forms of VEGF, their signalling and accessory receptors. Five genes encoding VEGF-like proteins have been identified; the different isoforms of each VEGF molecule are generated by alternative splicing mechanisms. The different VEGF's recognize signalling tyrosine kinase receptors (Flt-1, Flk-1 and Flt-4) and accessory receptors. VEGF expression is stimulated by hypoxia-dependent and -independent mechanisms. Hypoxic responses are mediated by specific transcription factors that are expressed in a tissue dependent fashion and that are developmentally regulated. VEGF is thought to play a role in tumor angiogenesis and may contribute to cardioprotection in ischemic heart -diseases. Its role in pulmonary hypertension induced by chronic hypoxia is discussed. This review also stresses the difficulty of applying results from in vitro -studies to in vivo situations.

Regulation of vascular endothelial growth factor expression in human keratinocytes by retinoids.

Vascular endothelial growth factor (VEGF) is overexpressed in hyperproliferative diseases, such as psoriasis and cancers, which are characterized by increased angiogenesis. Experimentally, VEGF overexpression can be induced by the treatment of cell cultures and biological tissues with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Using normal human keratinocytes in conventional cultures and skin grafted onto nude mice in vivo, we show that retinoids can inhibit TPA-mediated VEGF gene induction at the transcriptional level. Because retinoids are biologically active either by interacting with the nuclear retinoic acid receptors or by interfering with the activator protein 1 (AP1) transcription factor, we studied the effect of the retinoic acid derivative CD 2409, which exhibits strong anti-AP1 activity but does not bind to the known retinoic acid receptors in vitro. The results demonstrate that the inhibition of VEGF expression by retinoids only depends on their anti-AP1 activity and does not require gene transactivation via retinoic acid response elements. Because the VEGF promoter contains four potential AP1 binding sites, we used different promoter constructs to identify the functional site responsible for TPA induction and retinoid inhibition. This site turned out to be localized at position -621 of the 5' flanking region of the VEGF gene.

Hypoxia-induced transcriptional activation of vascular endothelial growth factor is inhibited by serum.

Expression of vascular endothelial growth factor (VEGF) by cultured vascular smooth muscle cells was analyzed. Serum and hypoxia had nearly additive actions on VEGF mRNA expression. The function of the VEGF promoter in smooth muscle cells was analyzed using transient luciferase reporter assays. Serum and hypoxia stimulated expression of luciferase. The presence of hypoxia response element (HRE) was necessary for the hypoxic induction. AP-1 sequences located upstream of HRE and AP-2/Sp-1 sequences located downstream of HRE are not necessary. Hypoxic responses were best observed in serum-deprived cells. They were largely absent in serum-stimulated cells. Serum did not suppress the hypoxic response by interfering with the hypoxia sensor mechanism or with the signaling cascade that leads to the activation of HIF-1. It is concluded that growth-promoting cytokines regulate hypoxic gene induction in smooth muscle cells.

Expression of vascular endothelial growth factor-b in human astrocytoma.

Growth of human malignant gliomas is stringently dependent on an angiogenic process that probably involves vascular endothelial growth factor (VEGF). Expressions of mRNA coding for the different forms of VEGF were analyzed in surgical specimens from human astrocytomas. Low levels of placental growth factor (PGF) and VEGFC mRNA were observed in polymerase chain reaction, but not in Northern blot experiments. VEGF mRNA was found in some but not all grade and grade IV astrocytomas. VEGFB mRNA was observed in all tissue samples analyzed irrespective of the tumor grade. A new splice variant of VEGFB (VEGFB155) that lacks exons 5 and 6 is described. Expressions of VEGF mRNA in cultured glioblastomas cells were upregulated by hypoxia, but the sensitivity of the cells to hypoxia was reduced as compared with normal rat astrocytes. VEGF expression was depressed by dexamethasone. Expressions of VEGFB mRNA were affected neither by hypoxia nor by dexamethasone. The results indicate a coexpression of VEGF mRNA and VEGFB mRNA in human astrocytomas. Expression of VEGFB is markedly different from that of VEGF. Possible roles of VEGFB as a cofactor for hypoxia-induced angiogenesis in human astrocytomas are discussed.

P2X7 receptor activation-induced contraction and lysis in human saphenous vein smooth muscle.

In cutaneous veins where purinergic neurotransmission is more prominent compared with in deep vessels, physiological and pathological roles of nerve-released ATP have been described. Neuronally released ATP has been reported to act through activation of unidentified ionotropic P2X receptor(s). This study analyzed P2X receptor subtypes expressed in human saphenous vein smooth muscle and their physiological functions. Transcripts for both hP2X1 receptors, already identified in other smooth muscles, and, surprisingly, hP2X7 receptors known to be responsible for the cytotoxic effect of ATP in macrophages were detected by Northern blot analysis in total RNA from saphenous vein smooth muscle. ATP and other P2X receptor agonists [alphabeta-methylene-ATP, 2-methylthio-ATP, and 2',3'-(4-benzoyl)benzoyl-ATP] dose-dependently contracted venous rings, but the contraction induced by 2-methylthio-ATP was more transient than that evoked by the other P2X agonists. The effect of hP2X1 agonists involved the activation of a rapidly desensitizing cation current recorded in freshly isolated myocytes. The action of hP2X7 receptor agonists was related to a maintained nondesensitizing cation current. In addition, hP2X7 receptor activation formed membrane pores that were permeable to large molecules. hP2X1 and hP2X7 receptors coexpressed in COS cells did not associate to form heteromultimers. Our data indicate that both hP2X1 and hP2X7 receptors are expressed as 2 separated populations of channels in human saphenous vein myocytes and are involved in ATP-induced tension. We suggest that cell lysis consequent to hP2X7 receptor-induced pore formation contributes to the disorganization and decrease in the amount of contractile myocytes in the media of varicose veins.

Cardiac expressions of HIF-1 alpha and HLF/EPAS, two basic loop helix/PAS domain transcription factors involved in adaptative responses to hypoxic stresses.

Expression of many mammalian genes is regulated by oxygen tension. HIF-1 alpha and HLF/EPAS are two basic helix-loop-helix/PAS domain transcription factors that bind to hypoxia sensitive elements in the promoters /enhancers of hypoxia sensitive genes such as vascular endothelial growth factor (VEGF). This paper describes the structure of rat HIF-1 alpha and analyses expressions HIF-1 alpha and of HLF/EPAS mRNAs in lung and cardiac tissues from the rat. HLF/EPAS mRNAs appear at birth in the two tissues and are maintained at high levels throughout adult life. HIF-1 alpha mRNAs are expressed at a constant level during lung development. Their abundance increase transiently at birth in cardiac tissues. Cultured cardiomyocytes from new born rats only express HIF-1 alpha mRNAs. HIF-1 alpha mRNA expression is increased by phorbol myristate acetate but not by anoxia or cobalt. The results indicate (i) that HIF-1 alpha and HLF/EPAS are expressed in a cell specific manner and (ii) that the hypoxic induction of VEGF mRNA expression by isolated cardiomyocytes is independent of HLF/EPAS. Finally, they suggest that protein kinase C may prime hypoxia induced gene regulation by inducing expression of HIF-1 alpha mRNAs.

Differential regulation of cardiac heme oxygenase-1 and vascular endothelial growth factor mRNA expressions by hemin, heavy metals, heat shock and anoxia.

Increasing attention has been paid to the effects of hypoxia, heavy metals and heat shocks on gene expression and to the similarities in their actions. This paper compares mRNA levels of two putative hypoxia, heavy metal and heat shock sensitive genes: heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) in myocyte-enriched cultures of neonatal rat heart cells. HO-1 mRNA expression is stimulated by hemin, Cd2+, Co2+ and heat shocks but not by Ni2+ or Mn2+. It is stimulated by long (13h) but not by short (4h) periods of anoxia. Conversely, VEGF mRNA expression is stimulated by short as well as long periods of anoxia, by Cd2+, Co2+, Ni2+ and Mn2+ but not by hemin or heat shocks. The results suggest that heavy metals, anoxia and heat shocks exert their effects on VEGF and HO-1 mRNA expression through separate though potentially overlapping mechanisms. Increased expressions of HO-1 and VEGF may be both cardioprotective under hypoxic/ischemic conditions.

Contributions of NO synthase and heme oxygenase to cGMP formation by cytokine and hemin treated brain capillary endothelial cells.

Two mechanisms contribute to cGMP formation by soluble guanylyl cyclase (i) NO production by NO synthase and (ii) CO production by heme oxygenase. We analyze here the contributions of these two pathways to IL1, TNF, lipopolysaccharide and hemin treated brain capillary endothelial cells. Cytokines and LPS induced cGMP formation in manners that were completely prevented by LY 83,583, methylene blue and by cyclosporin A. They were partially inhibited by inhibitor of NO synthase. Cyclosporin A acts by a posttranscriptional mechanism. Cells constitutively expressed mRNAs for heme oxygenase-1. Expression was enhanced by hemin but not by IL1 or lipopolysaccharide. Induction of heme oxygenase-1 and its inhibition by Sn protoporphyrin IX had no effect on cGMP levels.

Cobalt stimulates the expression of vascular endothelial growth factor mRNA in rat cardiac cells.

We have previously demonstrated that an exposure of rat cardiomyocytes to anoxia induces the expression of mRNAs coding for vascular endothelial growth factor (VEGF). The action of anoxia was mimicked by Co, Ni and Mn. The actions of Co and of anoxia were not additive and did not involve AP-1 binding sites. Experiments using actinomycin D and cycloheximide indicated that VEGF mRNA levels in cardiac cells are regulated both at transcriptional and post transcriptional levels. It is concluded that an oxygen sensing mechanism is present in cardiac cells and controls the expression of VEGF mRNAs. It may be important for the neovascularization of ischemic myocardium.

Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells. A 3.9 kb VEGF transcript is expressed by all cardiac tissues from rat, mouse and guinea pig examined. VEGF expression was not developmentally regulated. The major form of VEGF mRNAs expressed by cardiac tissues coded for VEGF188. Myocyte enriched and fibroblast enriched cultures of new born rat heart cells also expressed VEGF transcripts but the major mRNA found coded for VEGF164. The expression of VEGF mRNA in myocyte enriched cultures of new born rat ventricles was increased 2 fold by serum, 5 fold by phorbol myristate acetate and 7 fold by hypoxic conditions. We conclude that hypoxic conditions may promote cardiac capillary cell growth by inducing VEGF expression.

Expression of vascular endothelial growth factor by cultured endothelial cells from brain microvessels.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells that may be involved in tumor angiogenesis. Cultured capillary endothelial cells from rat brain (BCEC) express transcripts for VEGF as assayed by Northern blots and polymerase chain reaction analysis. The three forms of VEGF (VEGF120, VEGF164 and VEGF188) are produced, VEGF188 being detected in lower amounts. The sequence of rat VEGF188 was determined. Rat and human exons 6 differ at only one position (human Tyr134-->rat Phe133). Transcripts for VEGF were observed in different clones of rat BCEC, in bovine BCEC but not in bovine aortic endothelial cells.

Function of vasoactive factors in the cerebral microcirculation.

The properties of brain capillary endothelial cells (BCECs) have been analyzed. BCECs express two types of receptor sites for endothelins (ETs), and ETA-like receptor, and an ETB-like receptor that is not coupled to phospholipase C but whose occupancy activates Na+/H+ exchange activity. The ETA receptor is positively coupled to phospholipase C and negatively coupled to adenylate cyclase. BCECs, unlike aortic endothelial cells, express high-affinity receptor sites for C-type natriuretic peptide. They respond to exogenous nitric oxide (NO) and to NO donor molecules by large activations of soluble guanylate cyclase. They produce little cGMP in response to A23187 or to agonists of phospholipase C but do so after an exposure to interleukin-1. The physiological consequence of the high reactivity of BCECs to vasoactive factors is discussed.

Endothelins inhibit adenylate cyclase in brain capillary endothelial cells.

The action of endothelins (Et) on cAMP formation was studied in endothelial cells from rat brain microvessels. Et-1 and Et-3 had no action by themselves. They both inhibited cholera toxin stimulated adenylate cyclase by about 50%. K0.5 values were observed at 2 nM and 40 nM for Et-1 and Et-3 respectively, indicating an involvement of a low affinity Et-3 receptor. Coupling to adenylate cyclase was achieved by a pertussis toxin sensitive mechanism. Another action of endothelins in brain capillary endothelial cells was to stimulate phospholipase C. This action involved a low affinity Et-3 receptor and a pertussis toxin insensitive mechanism. It is concluded that in brain capillary endothelial cells, ETA like receptors are coupled to phospholipase C and to adenylate cyclase via two different mechanisms.