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Biomedical subjects

I Laurendeau

Publications and source records attributed to I Laurendeau.

At least 19 recordsLinked to original sources
PubMed2006

The angiopoietin pathway is modulated by PAR-1 activation on human endothelial progenitor cells.

OBJECTIVES: The importance of protease-activated receptor-1 (PAR-1) in blood vessel development has been shown in knock-out mice. As endothelial progenitor cells (EPCs) express functional PAR-1, we examined whether PAR-1 stimulation by the peptide SFLLRN interfered with the angiopoietin pathway, that is EPC commitment, proliferation and migration. METHODS AND RESULTS: Given the strong PAR-1 expression on CD34+ cells, we tested the effect of SFLLRN 75 micromol L(-1) on the emergence of EPCs from cord blood. PAR-1 activation did not modify the number of colonies or the day of emergence, in keeping with the lack of induction of angiopoietin 1 gene expression. Conversely, SFLLRN treatment of EPCs induced angiopoietin 2 gene expression and protein synthesis. Experiments with polyclonal blocking antibodies showed that angiopoietin 2 was involved in the proliferative effect of PAR-1 activation. PAR-1 activation also enhanced migration toward angiopoietin 1 in a Boyden chamber assay. CONCLUSIONS: Our study demonstrates that PAR-1-induced proliferation of EPCs involves angiopoietin 2. PAR-1 also enhances EPC migration toward angiopoietin 1. These findings might explain the role of thrombin in neovascularization via the angiopoietin pathway.

Angiopoietin-1↗
PubMed2006

Human chorionic gonadotropin expression in human trophoblasts from early placenta: comparative study between villous and extravillous trophoblastic cells.

Human trophoblast differentiates into two pathways: extravillous cytotrophoblasts (EVCT) that invade the uterus wall and villous cytotrophoblasts (VCT) that fuse to form the syncytiotrophoblast (ST) involved in placental exchanges and endocrine function. It is established that hCG is produced and secreted by the ST into the maternal compartment where it plays a key endocrine role and stimulates ST formation in an autocrine manner. Herein, we investigated hCG expression in early placentas by immunohistochemistry using different antibodies. We then compared hCG secretion by primary cultures of VCT and EVCT isolated from the same first trimester human chorionic villi. In situ hCG was immunodetected in EVCT all along their invasive differentiating pathway except in cells near the stromal core of the proximal column. hCG expression was confirmed in vitro by immunocytochemistry and hCG secretion quantified in cell supernatants. Interestingly, whereas hCG secretion increased during VCT differentiation into ST (from 60 to 350UI/L/microg DNA), EVCT secretion remained constant and at a high level during the same culture period (160UI/L/microg DNA). Our data demonstrated that in addition to the ST, invasive EVCT also expressed and secreted high levels of hCG, suggesting a specific paracrine and/or autocrine role for hCG from EVCT origin.

Animals↗
PubMed2006

Modulation of PAPP-A expression by PPARgamma in human first trimester trophoblast.

Pregnancy-associated plasma protein-A (PAPP-A) is a metzincin metalloproteinase that cleaves the insulin-like growth factor (IGF)-dependent binding protein-4 and increases in maternal serum during pregnancy. In human placenta PAPP-A is expressed both in villous cytotrophoblasts (VCT) that cover the chorionic villi and in extravillous cytotrophoblasts (EVCT) of the anchoring villi. Due to the key role of PPARgamma in human trophoblast differentiation such as syncytiotrophoblast formation and EVCT invasion, we studied the effect of PPARgamma activation on PAPP-A expression using our in vitro model of EVCT and VCT primary cultures isolated from the same first trimester chorionic villi. First, we demonstrated that invasive EVCT expressed and secreted 10 times more PAPP-A than VCT did. Then, we showed that activation of PPARgamma inhibited PAPP-A gene expression and secretion in EVCT, whereas it had no effect in VCT. Since we have previously shown that PPARgamma agonist inhibits EVCT invasion in vitro, we suggest that PPARgamma-mediated inhibition of PAPP-A might decrease the amount of bioactive IGFII, a factor known to promote trophoblast invasion.

Chorionic Villi↗
PubMed2005

Quantitative gene expression in Budd-Chiari syndrome: a molecular approach to the pathogenesis of the disease.

BACKGROUND: Budd-Chiari syndrome (BCS) is associated with parenchymal changes leading to major architecture remodelling. In order to gain further insight into the pathogenesis of BCS, we investigated expression of a set of genes involved in the course of chronic liver diseases. METHODS: Quantitative expression of 35 selected genes involved in extracellular matrix regulation, growth factors, and angiogenesis was investigated in 13 cases of BCS and compared with 10 normal livers and 13 cirrhosis cases by real time reverse transcription-polymerase chain reaction. Differential gene expression was considered significant for genes showing at least a twofold variation, with p < 0.05. RESULTS: Expression of 14 genes was significantly increased in BCS versus normal liver, with the highest increase in superior cervical ganglion 10 (SCG10) gene. BCS cases were classified according to their evolution and morphological pattern as either acute or chronic in six and seven cases, respectively. Unsupervised hierarchical clustering of acute and chronic BCS cases on the basis of similarity in gene expression pattern led to distinction between the two groups. Expression of three genes was significantly different in acute versus chronic BCS (increase in matrix metalloproteinase 7 and SCG10, decrease in thrombospondin-1 for chronic BCS). Seventeen and 10 genes, mainly involved in extracellular matrix and vascular remodelling, were significantly deregulated in acute BCS versus normal liver and cirrhosis, respectively. CONCLUSION: These results show that BCS cases display a specific gene expression profile that is different from that of normal liver and cirrhosis; the molecular configuration of BCS can be readily distinguished by its evolution and morphological pattern.

Acute Disease↗
PubMed2004

Large variability of trophoblast gene expression within and between human normal term placentae.

Human placenta extracts are widely used in clinical and fundamental research, particularly to study the hormonal and exchange functions of the placenta. However, very little is known about the distribution of the main hormone mRNAs in the placenta as a whole. Total placenta extracts are heterogeneous in their cellular components, as they contain material of both fetal and maternal origin, and in their structure. Results vary greatly depending upon the location of the biopsy and the number of biopsies performed. We used real-time quantitative RT-PCR to determine whether transcripts corresponding to the main hormones secreted by the human placenta (e.g. hCG, HPL and PGH) are equally distributed within and between term placentae. We also measured cytokeratin 7 transcripts, which are specifically expressed in the trophoblast, and transcripts corresponding to nuclear receptors PPARgamma and RXRalpha. A comparison of the results obtained with 12 different samples from each of four normal term placentae revealed that the amounts of transcripts differ considerably within and between each placenta. This emphasizes the need to study large numbers of samples when looking for significant differences in gene expression.

Analysis of Variance↗
PubMed2004

A role for thrombin in liver fibrosis.

BACKGROUND/AIM: Several lines of evidence incriminate the serine proteinase thrombin in liver fibrogenesis either through its procoagulant function or its signaling via cell-surface receptors. The aim of this study was therefore to evaluate the effect of thrombin inhibition on experimental liver fibrosis. METHODS: Fibrosis was induced in rats by administration of CCl4 for either three or seven weeks. Oral administration of the thrombin antagonist SSR182289 started one week after the start of CCl4 intoxication. Fibrosis and the area occupied by alpha smooth muscle actin (ASMA) positive cells were quantified with histomorphometry. Expression of fibrosis related genes was measured by real time RT-PCR. RESULTS: After three weeks of CCl4, treatment with SSR182289 did not significantly decrease the area of fibrosis but significantly decreased the area of ASMA positive cells by 22% (p = 0.03) and the expression of TIMP-1 mRNA by 52% (p = 0.02). There was no effect on gene expression of collagen I, MMP-2, or TIMP-2. After seven weeks of CCl4, treatment with SSR182289 resulted in a significant decrease in fibrosis (-30%, p = 0.04) and ASMA positive areas (-35%, p = 0.05). SSR182289 alone had no effect on the measured parameters. Additionally, it did not alleviate the acute toxicity of CCl4 as shown by measuring levels of serum aminotransferases and the area of necrosis. CONCLUSIONS: These data provide evidence that thrombin antagonism can reduce liver fibrogenesis. The early effect of SSR182289 on ASMA and TIMP-1 expression suggests that it is beneficial in reducing fibrogenic cell activation.

Actins↗
PubMed2001

Quantification of estrogen receptor alpha and beta expression in sporadic breast cancer.

The recent cloning of a second estrogen receptor (ER), designated ERbeta, has prompted a reevaluation of the role of ERs in breast cancer. We have developed and validated a real-time RT-PCR assay to quantify ERalpha and ERbeta gene expression at the mRNA level in a series of 131 patients with unilateral invasive primary breast cancer. Although ERbeta expression showed wide variations in tumor tissues, its range (nearly three orders of magnitude) was smaller than that of ERalpha (nearly four orders of magnitude), suggesting that ERbeta is more tightly controlled than ERalpha. We observed a negative correlation between ERalpha and ERbeta expression. 'ERalpha-negative' tumors (containing very low ERalpha mRNA levels) were associated with SBR histopathological grade III, RB1 underexpression and ERBB2 overexpression, confirming that ERalpha negativity delineates poorly differentiated tumors. The amount of ERalpha mRNA (but not that of ERbeta mRNA) increased with age and was consequently higher in postmenopausal patients' tumors. Expression of ERalpha (but not that of ERbeta) also correlated strongly with progesterone receptor (PR) and PS2 expression, suggesting that ERalpha has stronger transcriptional activity than ERbeta towards genes containing an ERE (estrogen response element) in their promoters. Interestingly, we found a negative correlation between the expression of ERbeta (but not ERalpha) and CCND1, which contains an AP1 element but not an ERE in its promoter. Taken together, these data confirm that ERalpha and ERbeta play different roles in breast cancer, partly by mediating the transcription of various genes via different types of DNA enhancer. PR and PS2 seem to be mainly ERalpha-responsive genes, whereas CCND1 may be mainly ERbeta-responsive. Our findings also underline the need for a reliable method, providing full range of quantitative values, to determine ERalpha and ERbeta status in the clinical setting.

Breast Neoplasms↗
PubMed2001

Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development.

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29-1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

Amino Acid Sequence↗
PubMed2000

Pleiotropic syndrome of dehydrated hereditary stomatocytosis, pseudohyperkalemia, and perinatal edema maps to 16q23-q24.

Dehydrated hereditary stomatocytosis (DHS) is a rare genetic disorder of red cell permeability to cations, leading to a well-compensated hemolytic anemia. DHS was shown previously to be associated in some families with a particular form of perinatal edema, which resolves in the weeks following birth and, in addition, with pseudohyperkalemia in one kindred. The latter condition was hitherto regarded as the separate entity, "familial pseudohyperkalemia." DHS and familial pseudohyperkalemia are thought to stem from the same gene, mapping to 16q23-q24. This study screened 8 French and 2 American families with DHS. DHS appeared to be part of a pleiotropic syndrome in some families: DHS + perinatal edema, DHS + pseudohyperkalemia, or DHS + perinatal edema + pseudohyperkalemia. If adequately attended to, the perinatal edema resolved spontaneously after birth. Logistic regression showed that increased mean corpuscular volume and mean corpuscular hemoglobin concentration were the parameters best related to DHS. In patients in whom cation fluxes were investigated, the temperature dependence of the monovalent cation leak exhibited comparable curves. Specific recombination events consistently suggested that the responsible gene lies between markers D16S402 and D16S3037 (16q23-q24). The 95% confidence limits (Z(max) >/= 3.02) spanned almost the complete 9-cM interval between these 2 markers.

Adolescent↗
PubMed2000

Somatic mosaicism and compound heterozygosity in female hemophilia B.

Sequencing the complete factor IX gene of 2 sisters with hemophilia B with different phenotypes and no family history of hemorrhagic diathesis revealed a common 5' splice site mutation in intron 3 (T6704C) in both and an additional missense mutation (I344T) in one. The presence of dysfunctional antigen in the latter strongly suggested that these mutations are in trans. Neither mutation was found in leukocyte DNA from the asymptomatic parents, but the mother was in somatic mosaicism for the shared splice site mutation. This case illustrates the importance of defining the phenotype and considering somatic mosaicism in sporadic cases. It underlines the limitations of complete gene sequencing for the detection of mosaicism and has implication for genetic counseling. (Blood. 2000;96:1585-1587)

Adolescent↗
PubMed2000

Retinoids stimulate leptin synthesis and secretion in human syncytiotrophoblast.

The syncytiotrophoblast (ST), which forms the outer layer of the chorionic villi, is the endocrine unit of the human placenta. Bathing in the maternal blood of the intervillous space, the ST secretes its hormonal products directly into the maternal circulation. Leptin is expressed in the ST and is secreted into the maternal circulation. However, its regulation and physiological role during pregnancy remain poorly known. In the present work we used the in vitro model of human cytotrophoblast differentiation into ST to study the effect of physiological and synthetic retinoids on leptin synthesis and secretion. Using specific antibodies we first illustrated by immunocytochemistry the expression of retinoic acid (RA) receptor alpha and retinoid X receptor alpha (RXRalpha) in ST. We then observed that leptin messenger ribonucleic acid and protein expression increased with in vitro ST formation. The 9-cis isomer of RA and the synthetic retinoid specific for RXRs (BMS 649) stimulated leptin messenger ribonucleic acid expression and secretion. In contrast, all-trans-RA and a RA alpha-specific ligand had no effect. These results suggest that retinoids regulate leptin expression and highlight a role for RXRalpha in this process.

Adult↗
PubMed1999

Quantitation of MYC gene expression in sporadic breast tumors with a real-time reverse transcription-PCR assay.

MYC gene overexpression was identified recently as a downstream step at the end of the Wnt/APC/beta-catenin pathway dysregulation observed in colorectal cancer (T-C. He et al., Science (Washington DC), 281: 1509-1512, 1998). It thus appears that an excess of c-myc protein is a primary cause of numerous cancers. In breast cancer, MYC has been studied mostly at the DNA level because of the poor quality of available antibodies against the protein product. The renewed interest in MYC calls for a sensitive and accurate method for analyzing MYC overexpression in breast tumors. We have developed a real-time quantitative reverse transcription-PCR assay based on TaqMan fluorescence methodology to quantify the MYC mRNA copy number. We validated the method on a large series of breast tumors. MYC gene overexpression was observed in 29 of 134 (22%) breast tumor RNAs, ranging from 3.2 to 19 times the level in normal breast tissues. These data imply that dysregulated MYC gene expression is potentially involved in the pathogenesis of breast cancer, especially by favoring local cell proliferation. We also found that MYC gene overexpression was rarely due to an increased MYC gene copy number in breast cancer. This new, simple, rapid, and semiautomated method will be useful for screening cancer patients for MYC overexpression and will prove a powerful tool in large, randomized, prospective, cooperative group trials and in the MYC-based therapy project.

Adult↗
PubMed1999

Expression of the RNA component of human telomerase (hTR) in prostate cancer, prostatic intraepithelial neoplasia, and normal prostate tissue.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)-based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non-radioactive in situ hybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin-embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situ hybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate.

Aged↗
PubMed1999

Prion protein and neuronal differentiation: quantitative analysis of prnp gene expression in a murine inducible neuroectodermal progenitor.

The biological function of the cellular prion protein, PrP(c), is currently unknown. The presence of PrP(c) transcripts in the developing neural tube from embryonic day 13.5 and the predominant expression of PrP(c) in the adult brain is suggestive of a role in the onset and/or modulation of neuronal functions. We took advantage of the bipotential neuroectodermal 1C11 cell line to monitor PrP(c) expression during its bioaminergic differentiations. The F9-derived 1C11 precursor cell line displays a stable and immature phenotype in the absence of extracellular signal and, upon induction, has the capacity to acquire a complete serotonergic or noradrenergic phenotype, the two pathways being mutually exclusive. A real-time quantitative PCR assay was developed to assess PrP(c) gene expression at definite times of the two programs that correspond to sequential acquisition of neurotransmitter-specific functions. 1C11 cells and their differentiated progenies express significant amounts of PrP transcripts and of the corresponding protein. A unique decrease in prnp gene expression is observed upon entry into the serotonergic pathway, correlating with a downregulation at the protein level. Moreover, nerve growth factor (NGF) is shown to induce a decrease in the level of prnp gene expression along the serotonergic - but not the noradrenergic - pathway. Our study accurately establishes that prnp gene expression (i) is strongly upregulated concomitantly with cell fate restriction of multipotential cells towards the neural lineage; (ii) is differentially regulated along the serotonergic versus noradrenergic differentiation program of a unique neuroectodermal progenitor. The 1C11 cell line may provide a new tool for studying prion infectivity in a well-defined neuronal context.

Amyloid↗
PubMed1999

TaqMan PCR-based gene dosage assay for predictive testing in individuals from a cancer family with INK4 locus haploinsufficiency.

BACKGROUND: A genetic syndrome of cutaneous malignant melanoma and nervous system tumors recently has been characterized and shown to be linked to the INK4 locus in the 9p21 region. Hemizygosity at adjacent physically mapped microsatellite markers indicated deletion of p16, p19, and p15 clustered tumor suppressors. Because individuals from this family could benefit from predictive testing in terms of cancer prevention, we developed a direct test without need to analyze parental DNAs to comply with the rules of individual consent and secrecy. METHODS: We developed an assay using TaqManTM real-time quantitative PCR, with p15 as the test sequence and albumin (ALB) as the reference gene. The normalized ratio of p15/ALB is expected to yield a value of approximately 1 in individuals without the deletion, whereas a ratio of approximately 0.5, indicating p15 haploinsufficiency, is expected in predisposed individuals. RESULTS: All patients harboring the previously defined at-risk haplotype were correctly identified using this approach. In six individuals with deletions, the p15/ALB ratios were 0.472-0.556 (SD, 0.013-0.078). In the five individuals without deletions, the ratios were 0.919-1.019 (SD, 0.006-0.075). CONCLUSIONS: This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion. This approach, not limited by marker informativeness or the need for harvesting live cells, can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.

Carrier Proteins↗
PubMed1999

Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications.

BACKGROUND: Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations. METHODS: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA. RESULTS: The dynamic range was >1000-fold. The relationship between C(t) and log starting concentration was linear (r(2) >/=0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (>5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio (<5) in breast cancers without gene amplification. CONCLUSIONS: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.

Adult↗