CX3CL1/Fractalkine as a biomarker for early pregnancy prediction of preterm premature rupture of membranes.

OBJECTIVES: The objective of our study was to evaluate serum CX3CL1/Fractalkine, a monocyte/macrophage chemoattractant expressed in cytotrophoblasts and decidual cells, as a predictive biomarker for the occurrence of preterm premature rupture of membranes (PPROM). METHODS: A case-control study of 438 pregnancies including 82 PPROM cases and 64 preterm labor with intact membranes cases with blood samples collected at first trimester, second trimester and delivery was conducted. The predictive ability of CX3CL1 and maternal risk factors for the occurrence of PPROM was assessed by receiver operating characteristic curve analysis. A second, independent cohort was prospectively constituted to confirm the case-control study results. RESULTS: First trimester CX3CL1 was significantly increased in PPROM cases when compared to matched controls. Multivariate regression analysis highlighted a significant difference for CX3CL1 measured during the first trimester (p<0.001). Alone, CX3CL1 predicts PPROM with a 90 % sensitivity and a specificity around 40 %. The area under the receiver operating characteristic curve for PPROM prediction were 0.64 (95% confidence interval: 0.57-0.71) for first trimester CX3CL1, and 0.61 (95% confidence interval: 0.54-0.68) for maternal risk factors (body mass index<18.5 kg/m2, nulliparity, tobacco use and the absence of high school diploma). The combination of CX3CL1 and maternal risk factors significantly improved the area under the curve: 0.72 (95% confidence interval: 0.66-0.79) (p<0.001). The results were confirmed on a second independent cohort. CONCLUSIONS: CX3CL1 is a promising blood biomarker in the early (first trimester) prediction of PPROM.

Effects of Totum-63 on glucose homeostasis and postprandial glycemia: a translational study.

Global prevalence of type 2 diabetes (T2D) is rising and may affect 700 million people by 2045. Totum-63 is a polyphenol-rich natural composition developed to reduce the risk of T2D. We first investigated the effects of Totum-63 supplementation in high-fat diet (HFD)-fed mice for up to 16 wk and thereafter assessed its safety and efficacy (2.5 g or 5 g per day) in 14 overweight men [mean age 51.5 yr, body mass index (BMI) 27.6 kg·m-2] for 4 wk. In HFD-fed mice, Totum-63 reduced body weight and fat mass gain, whereas lean mass was unchanged. Moreover, fecal energy excretion was higher in Totum-63-supplemented mice, suggesting a reduction of calorie absorption in the digestive tract. In the gut, metagenomic analyses of fecal microbiota revealed a partial restoration of HFD-induced microbial imbalance, as shown by principal coordinate analysis of microbiota composition. HFD-induced increase in HOMA-IR score was delayed in supplemented mice, and insulin response to an oral glucose tolerance test was significantly reduced, suggesting that Totum-63 may prevent HFD-related impairments in glucose homeostasis. Interestingly, these improvements could be linked to restored insulin signaling in subcutaneous adipose tissue and soleus muscle. In the liver, HFD-induced steatosis was reduced by 40% (as shown by triglyceride content). In the subsequent study in men, Totum-63 (5 g·day-1) improved glucose and insulin responses to a high-carbohydrate breakfast test (84% kcal carbohydrates). It was well tolerated, with no clinically significant adverse events reported. Collectively, these data suggest that Totum-63 could improve glucose homeostasis in both HFD-fed mice and overweight individuals, presumably through a multitargeted action on different metabolic organs.NEW & NOTEWORTHY Totum-63 is a novel polyphenol-rich natural composition developed to reduce the risk of T2D. Totum-63 showed beneficial effects on glucose homeostasis in HFD-fed mice, presumably through a multitargeted action on different metabolic organs. Totum-63 was well tolerated in humans and improved postprandial glucose and insulin responses to a high-carbohydrate breakfast test.

Hormonal Status and Cognitivo-Emotional Profile in Real-Life Patients With Neuropathic Pain: A Case Control Study.

BACKGROUND: The specific impact of neuropathic pain and recommended neuropathic pain treatments on the hormonal and immune status of patients has been so far poorly explored. This study aimed at studying, in real life, the hypothalamic-pituitary-adrenal axis and the cytokine profile of patients with neuropathic pain. It also explored their links with cognition, emotion, quality of life, and drug treatment. METHODS: This prospective study (clinicaltrials.gov NCT01543425) included 60 patients with neuropathic pain and 60 age- and gender-matched healthy volunteers after obtaining signatures of informed consent. A number of parameters were measured: adrenocorticotropic hormone, cortisol, cortisol awakening response, dehydroepiandrosterone sulphate, sex hormone binding globulin, testosterone, 17-ß-estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, cytokines, brain-derived neurotrophic factor, and vitamin D. Psychological parameters were assessed by questionnaires. RESULTS: Patients with neuropathic pain had lower levels of adrenocorticotropic hormone (P = 0.009) and dehydroepiandrosterone sulphate (P < 0.001) than controls, and the cortisol awakening response was impaired. Patients were more depressed and anxious (P < 0.001) and had a diminished quality of life (P < 0.001), which was influenced by cytokines (P = 0.0067) and testosterone (P = 0.028). Antidepressants and antiepileptics appeared to interfere with testosterone and cognitivo-emotional domains. CONCLUSION: An impairment of the hormonal status and of the immune system was observed in patients. It identified testosterone as a potential pivotal mediator between antidepressants/antiepileptics and quality of life. Further studies must address the exact impact of different types of drugs on central effects, of gender differences, and of the immune system of neuropathic pain.

Ethanolic extract of Algerian propolis decreases androgen receptor transcriptional activity in cultured LNCaP cells.

Antiandrogens have a peculiar place in the treatment of metastatic prostate cancer by blocking the androgen receptor (AR). Unfortunately, aggressive tumors could rapidly develop into a castration resistant state. It is therefore essential to look for new molecules that are more effective, affecting not only the androgen signaling and with minimum undesirable effects. Natural products are an interesting source of new therapeutics, especially for cancer therapy as 70% of them have botanical origin. Based on an ethnobotany screening, we evaluated the effects of ethanolic extract of propolis (EEP) from Algeria on LNCaP cells. Results pointed out that EEP reduces the survival of LNCaP cells with an IC50 of 0.04 mg/ml, induces the apoptosis and blocks the cell cycle at G0/G1 phase. Interestingly, EEP decreased the accumulation of AR suggesting some anti-androgen activity. Indeed, secreted amount of the androgen target protein PSA was decreased when LNCaP cells were incubated with EEP, starting after 4 h of treatment. This anti-androgen activity was also shown on the androgen target genes Fkbp5 and Sgk1. Finally, the capacity of EEP to block AR functioning was demonstrated in transient transfections with human AR and the reporter gene ARE-tk-Luc. Propolis antagonizes the induction of the luciferase activity induced by the natural androgen DHT (10-8M) or the synthetic AR agonist R1881 (10-7M). Altogether, these results highlight the potential pharmacological effects of EEP in future treatments of prostate cancer.

Retinoic acid and tracheal occlusion for diaphragmatic hernia treatment in rabbit fetuses.

INTRODUCTION: Lung hypoplasia and pulmonary arterial hypertension in congenital diaphragmatic hernia lead to a high perinatal mortality. Although sustained fetoscopic tracheal occlusion (TO) improves lung development, a major side effect is abnormal pneumocyte differentiation. This study evaluated the potential ability of intratracheal retinoic acid (RA) administration to reduce adverse effects of sustained TO in a rabbit model of diaphragmatic hernia. METHODS: A left diaphragmatic defect was created on day 23 in time-dated pregnant rabbits. On day 28, the same rabbits underwent sham surgery or TO, with an injection of empty or RA-loaded liposomes. On day 30, the fetuses were harvested, and the lungs were processed for histology, immunohistochemistry, and gene expression quantification. RESULTS: A tracheal RA injection at the time of TO had no effect on the lung-to-body-weight ratio, radial alveolar count or lung connective tissue composition. Retinoic acid plus TO had synergic effects on vascular measurements, proportional medial thickness, and endothelin-1 receptor type-A gene expression. The most noticeable effect was recovery of normal pneumocyte differentiation. CONCLUSION: Retinoic acid plus TO prevented abnormal pneumocyte differentiation and seemed to have a beneficial effect on pulmonary vascularization.

Different dose rate-dependent responses of human melanoma cells and fibroblasts to low dose fast neutrons.

PURPOSE: To analyze the dose rate influence in hyper-radiosensitivity (HRS) of human melanoma cells to very low doses of fast neutrons and to compare to the behaviour of normal human skin fibroblasts. MATERIALS AND METHODS: We explored different neutron dose rates as well as possible implication of DNA double-strand breaks (DSB), apoptosis, and energy-provider adenosine-triphosphate (ATP) levels during HRS. RESULTS: HRS in melanoma cells appears only at a very low dose rate (VLDR), while a high dose rate (HDR) induces an initial cell-radioresistance (ICRR). HRS does not seem to be due either to DSB or to apoptosis. Both phenomena (HRS and ICRR) appear to be related to ATP availability for triggering cell repair. Fibroblast survival after neutron irradiation is also dose rate-dependent but without HRS. CONCLUSIONS: Melanoma cells or fibroblasts exert their own survival behaviour at very low doses of neutrons, suggesting that in some cases there is a differential between cancer and normal cells radiation responses. Only the survival of fibroblasts at HDR fits the linear no-threshold model. This new insight into human cell responses to very low doses of neutrons, concerns natural radiations, surroundings of accelerators, proton-therapy devices, flights at high altitude. Furthermore, ATP inhibitors could increase HRS during high-linear energy transfer (high-LET) irradiation.

Energy expenditure, spontaneous physical activity and with weight gain in kidney transplant recipients.

BACKGROUND & AIMS: Alterations in energy metabolism could trigger weight gain after renal transplantation. METHODS: Nineteen transplanted non-diabetic men, 53 ± 1.6 years old, receiving calcineurin inhibitors but no corticosteroids were studied. They were compared with nine healthy men matched for height, age and lean body mass. Daily energy expenditure and its components (sleeping, basal and absorptive metabolic rates) were analyzed for 24 h in calorimetric chambers and for 4 days in free living conditions using calibrated accelerometry. Other variables known to influence energy expenditure were assessed: body composition, physical activity, 4-day food intake, drug consumption, serum C-reactive protein, interleukin-6, thyroid and parathyroid hormones, and epinephrine. Transplant recipients who gained more than 5% body weight after transplantation (n = 11, +11.0 ± 1.5 kg) were compared with those who did not (n = 8) and with the controls. RESULTS: Weight gain compared with non-weight gain patients and controls exhibited higher fat mass without change in lean body mass. Daily, sleeping and resting energy expenditure adjusted for lean body mass was significantly higher in non-weight gain (167.1 ± 4.2 kJ/kg/lean body mass/24 h, P < 0.05) compared with weight gain patients (147.4 ± 3.6) and controls (146.1 ± 4.6). Weight gain compared with controls and non-weight gain subjects had lower free living physical activity and a higher consumption of antihypertensive drugs and ß-blockers. CONCLUSIONS: After kidney transplantation, weight gain patients were characterized by lower adjusted energy expenditure, reduced spontaneous physical activity but a more sedentary life style and a trend toward a higher energy intake explaining the reason they gained weight. The nWG KTR had increased resting and sleeping EE which protected them from weight gain. Such hypermetabolism was also observed in 24-h EE measurements. By comparison with the nWG patients, the WG transplant recipients were characterized by higher ß-blocker consumption. These data could be helpful in the prevention of weight gain in kidney transplant recipients.

BRCA1 induces major energetic metabolism reprogramming in breast cancer cells.

The hypermetabolic nature of cancer cells and their increased reliance on "aerobic glycolysis", as originally described by Otto Warburg and colleagues, are considered metabolic hallmarks of cancer cells. BRCA1 is a major tumor suppressor in breast cancer and it was implicated in numerous pathways resulting in anticarcinogenic functions. The objective of our study was to address specific contributions of BRCA1 to the metabolic features of cancer cells, including the so-called "Warburg effect". To get a comprehensive approach of the role of BRCA1 in tumor cell metabolism, we performed a global transcriptional and metabolite profiling in a BRCA1-mutated breast cancer cell line transfected or not by wild-type BRCA1. This study revealed that BRCA1 induced numerous modifications of metabolism, including strong inhibition of glycolysis while TCA cycle and oxidative phosphorylation tended to be activated. Regulation of AKT by BRCA1 in both our cell model and BRCA1-mutated breast tumors was suggested to participate in the effect of BRCA1 on glycolysis. We could also show that BRCA1 induced a decrease of ketone bodies and free fatty acids, maybe consumed to supply Acetyl-CoA for TCA cycle. Finally increased activity of antioxidation pathways was observed in BRCA1-transfected cells, that could be a consequence of ROS production by activated oxidative phosphorylation. Our study suggests a new function for BRCA1 in cell metabolic regulation, globally resulting in reversion of the Warburg effect. This could represent a new mechanism by which BRCA1 may exert tumor suppressor function.

Levels of liver X receptors in testicular biopsies of patients with azoospermia.

OBJECTIVE: To determine whether the transcription factors liver X receptors (LXRs) and their downstream genes, which are involved in the regulation of several testicular functions in mouse models, are differentially expressed in testes of men with nonobstructive azoospermia (NOA) or obstructive azoospermia (OA). DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with various types of NOA (n=22) and with OA (n=5). INTERVENTION(S): Human testicular biopsies. MAIN OUTCOME MEASURE(S): Transcript levels were measured in testicular biopsies with the use of quantitative polymerase chain reaction. Correlations of LXR mRNA levels with the number of germ cells, the expression of proliferation and apoptosis markers, and the amount of intratesticular lipids and testosterone were evaluated. The localization of LXRα was analyzed by immunofluorescence. RESULT(S): LXR mRNA levels were decreased by 49%-98% in NOA specimens and positively correlated with germ cell number. Accumulations of IDOL and SREBP1c (LXR targets involved in lipid homeostasis) were 1.8-2.1 times lower in NOA samples and mRNA levels of the SREBP1c target gene ELOVL6 were increased 1.9-2.4-fold. Interestingly, the amount of triglycerides and free fatty acids were higher in NOA testes (3.4-12.2-fold). LXRα was present in Leydig cells. Accumulations of LXR downstream genes encoding the steroidogenic proteins StAR and 3ßHSD2 were higher in NOA testes (5.9-12.8-fold). CONCLUSION(S): Knowledge of changes in the transcript levels of LXRs and some of their downstream genes during altered spermatogenesis may help us to better understand the physiopathology of testicular failure in azoospermic patients.

Cushing's syndrome and fetal features resurgence in adrenal cortex-specific Prkar1a knockout mice.

Carney complex (CNC) is an inherited neoplasia syndrome with endocrine overactivity. Its most frequent endocrine manifestation is primary pigmented nodular adrenocortical disease (PPNAD), a bilateral adrenocortical hyperplasia causing pituitary-independent Cushing's syndrome. Inactivating mutations in PRKAR1A, a gene encoding the type 1 alpha-regulatory subunit (R1alpha) of the cAMP-dependent protein kinase (PKA) have been found in 80% of CNC patients with Cushing's syndrome. To demonstrate the implication of R1alpha loss in the initiation and development of PPNAD, we generated mice lacking Prkar1a specifically in the adrenal cortex (AdKO). AdKO mice develop pituitary-independent Cushing's syndrome with increased PKA activity. This leads to autonomous steroidogenic genes expression and deregulated adreno-cortical cells differentiation, increased proliferation and resistance to apoptosis. Unexpectedly, R1alpha loss results in improper maintenance and centrifugal expansion of cortisol-producing fetal adrenocortical cells with concomitant regression of adult cortex. Our data provide the first in vivo evidence that loss of R1alpha is sufficient to induce autonomous adrenal hyper-activity and bilateral hyperplasia, both observed in human PPNAD. Furthermore, this model demonstrates that deregulated PKA activity favors the emergence of a new cell population potentially arising from the fetal adrenal, giving new insight into the mechanisms leading to PPNAD.

Increased expression of the oncogenic KLF6-SV1 transcript in human glioblastoma.

BACKGROUND: Gliomas constitute the vast majority of primary central nervous system tumors in adults. Glioblastoma multiforme (GBM) is the most aggressive form of these primary brain tumors. There is a need to define diagnostic and prognostic markers that may help to distinguish GBM from non-GBM tumors. The Krüppel-like factor 6 (KLF6) gene has recently emerged as a promising candidate. The goal of our study was to determine if there is a link between KLF6 splice variants expression and different grades of gliomas. METHODS: Fifty-three primary gliomas tumor samples were analyzed using quantitative real-time PCR for the total KLF6, wild-type and alternatively spliced (SV1) KLF6 mRNA. RESULTS: Compared to the non-GBM group, the GBM group had a 2.2-fold increase in the mean level of total KLF6 mRNA expression. GBM showed a 2.1-fold increase in the KLF6 splicing ratio. In addition, KLF6-SV1 mRNA expression levels were also 2.2-fold higher in the GBM group, suggesting that the increase in the KLF6 splicing ratio was due to increased expression of the KLF6-SV1 oncogenic splice variant. CONCLUSIONS: Our study demonstrates that quantification of total and spliced forms of KLF6 may provide a new and useful supplementary molecular tool for grading glioma.

Absence of nuclear receptors for oxysterols liver X receptor induces ovarian hyperstimulation syndrome in mice.

Ovarian hyperstimulation syndrome is a frequent complication occurring during in vitro fertilization cycles. It is characterized by a massive ovarian enlargement associated with an accumulation of extra vascular fluid. Here we show that liver X receptor (LXR)-alpha and LXR-beta deficient mice present many clinical and biological signs of ovarian hyperstimulation syndrome: ovarian enlargement, hemorrhagic corpora lutea, increased ovarian vascular permeability, and elevated estradiol. Ovulation stimulation resulted in excessive ovarian response to exogenous gonadotropins because follicle number and estradiol production were higher in transgenic mice. LXR deficiency also leads to perturbations in general inflammatory status, associated with ovarian il-6 deregulation. Upon treatment with the synthetic LXR agonist T09101317, serum estradiol and expression of star and cyp11a1 genes were markedly increased in wild-type mice, showing that LXRs are key regulators of ovarian steroidogenesis. These results suggest that LXRs control the ovulation by regulating endocrine and vascular processes.

Effects of maternal retinoic acid administration in a congenital diaphragmatic hernia rabbit model.

Maternal retinoid administration has beneficial effects on lung development in the nitrofen rodent toxic model of congenital diaphragmatic hernia (DH). We wanted to investigate the effects in a surgical model, where the retinoid signaling pathway is not primarily disrupted by the toxic agent. We created DH in fetal rabbits at day 23 of gestation, administrated to the does all trans-retinoic acid (ATRA) or vehicle (VHC) intramuscularly for 8 consecutive days and harvested normal and operated (DH) fetuses at 31 d (n = 7 in each group). Normal lungs exposed to ATRA had increased surfactant protein mRNA levels without change in type II pneumocyte density. There was no measurable effect on lung-to-body weight ratio and airway morphometry by ATRA. In DH lungs (DH/VHC) surfactant protein mRNA levels were increased, as well as the density of type II pneumocytes. When supplemented with ATRA (DH/ATRA) these parameters returned to normal (VHC). Cell proliferation or apoptosis were not influenced by ATRA supplementation. In conclusion, maternal ATRA supplementation does not affect gross anatomic, morphologic or proliferation indices in hypoplastic lungs related to surgically induced DH in rabbit. However, ATRA lowers surfactant protein expression and normalizes type I/II pneumocyte ratio to what is observed in normal lungs.

Endoglin (CD105) expression is regulated by the liver X receptor alpha (NR1H3) in human trophoblast cell line JAR.

Human implantation involves invasion of the uterine wall and remodeling of uterine arteries by extravillous cytotrophoblasts. Defects in these early steps of placental development lead to poor placentation and are often associated with preeclampsia, a frequent complication of human pregnancy. One of the complex mechanisms controlling trophoblast invasion involves the activation of the liver X receptor beta (or NR1H2, more commonly known as LXRbeta) by oxysterols known as potent LXR activators. This activation of LXRbeta leads to a decrease of trophoblast invasion. The identification of new target genes of LXR in the placenta could aid in the understanding of their physiological roles in trophoblast invasion. In the present study, we show that the endoglin (ENG) gene is a direct target of the liver X receptor alpha (NR1H3, also known as LXRalpha). ENG, whose gene is highly expressed in syncytiotrophoblasts, is part of the transforming growth factor (TGF) receptor complex that binds several members of the TGFbeta superfamily. In the human placenta, ENG has been shown to be involved in the inhibition of trophoblast invasion. Treatment of human choriocarcinoma JAR cells with T0901317, a synthetic LXR-selective agonist, leads to a significant increase in ENG mRNA and protein levels. Using transfection and electrophoretic mobility shift assays, we demonstrate that LXR (as a heterodimer with the retinoid X receptor) is able to bind the ENG promoter on an LXR response element and mediates the activation of ENG gene expression by LXRalpha in JAR cells. This study suggests a novel mechanism by which LXR may regulate trophoblast invasion in pathological pregnancy such as preeclampsia.

Molecular and metabolic retinoid pathways in the human ocular surface.

PURPOSE: To maintain its integrity, the human ocular surface (cornea and conjunctiva) has an absolute requirement for vitamin A and its active derivatives, the retinoic acids. These retinoids regulate transcriptional levels of target genes through the activation of members of a super-family of ligand-dependant nuclear receptors that feature retinoic acid receptors (RAR) alpha, beta, and gamma as well as retinoid X receptors (RXR) alpha, beta, and gamma. The expression patterns of these receptors have been partial characterized in rabbit, mouse, and human cornea and conjunctiva, but systematic tissue and cellular expression of the three RARs and three RXRs had to be completed at the adult human ocular surface. The first objective of our work was to define their expression patterns in term of genes and proteins for total human conjunctiva, cornea, and the major cell types comprising the adult human ocular surface. The second objective was to demonstrate the presence of different enzymes transforming vitamin A to retinoic acid and the functionality of this metabolic pathway in the corneal epithelium. METHODS: Total mRNA was extracted from human total cornea, conjunctiva, corneal epithelial cells (primary culture and established cell line), corneal keratocytes (primary culture), corneal endothelial cells (established cell line), and conjunctival epithelial cells (established cell line) and was submitted to reverse transcription-polymerase chain reaction (RT-PCR) analysis to determine the expression patterns of the RARs and RXRs using specific primers. Immunological staining (via histochemistry and cellular chemistry) experiments were performed to better localize RAR and RXR proteins in the ocular surface at tissue and cellular levels. We also checked mRNA expression of cellular retinol binding proteins (CRBPs) and cellular retinoic acid binding proteins (CRABPs) with the enzymes involved in retinoic acid generation, i.e., alcohol dehydrogenases (ADHs) and retinal dehydrogenases (RALDHs) or degradation (Cyp26 family members). The enzymatic generation of functional retinoids was confirmed using epithelial corneal cells treated with specific inhibitors of retinol metabolism. RESULTS: RAR alpha, RAR gamma, and RXR alpha are expressed in the cornea, conjunctiva, and all of their constitutive cells, whereas RXR gamma and RXR beta were never detected in the cornea or conjunctiva. RAR beta was absent in primary cultures of corneal keratinocytes. ADH3, ADH4, dehydrogenase/reductase (SDR family) 4 (DHRS4), dehydrogenase/reductase (SDR family) 9 (DHRS9), RALDH1, and RALDH3 are expressed in the ocular surface, as were the retinoid-binding proteins CRBP1, CRABP1, and CRABP2. Retinol dehydrogenase 4 (RODH4) was only detected in the conjunctiva. Corneal epithelial cells convert retinol into retinoic acid using an enzymatic pathway. CONCLUSIONS: For the first time, we have established an exhaustive description of the expressions patterns of RARs, RXRs, ADHs, RALDHs, CRBP, and CRABPs in the human ocular surface. Our results for the human ocular surface demonstrated the presence of all the metabolic and molecular actors of the retinoic acid signaling pathway. We also demonstrated the enzymatic conversion of retinol into active retinoids in the corneal environment.

The changes in angiogenic gene expression in recurrent multiple chorioangiomas.

OBJECTIVES: To assess the messenger ribonucleic acid expression in placental tissue of growth factors, cytokines, angiogenic and anti-angiogenic factors in one case of recurrent multiple chorioangiomas. METHODS: Complementary deoxyribonucleic acid array analysis was performed on the affected placentae and on normal placentaes (controls) to compare messenger ribonucleic acid levels of 96 genes involved in angiogenesis. RESULTS: Eleven genes presented more than two-fold alteration in expression levels: undetectable (angiopoietin 1, osteonectin, tyrosine kinase endothelial, neuropilin 1), decreased (transcription growth factor beta receptor 3, tissue inhibitor of metalloproteinase type 2, EGF receptor, integrin-alpha V, tyrosine kinase vascular endothelial growth factor receptor 2), increased (angiopoietin 2, osteopontin). CONCLUSIONS: We illustrated the complexity of angiogenic disruption in recurrent multiple chorioangiomas leading to the difficulty to propose a single candidate gene to explain this pathology.

Kruppel-like factor 6 (KLF6) affects the promoter activity of the alpha1-proteinase inhibitor gene.

PURPOSE: Keratoconus is a progressive disease that thins and scars the cornea. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of inhibitors alpha1-proteinase inhibitor (alpha1-PI) and alpha2-macroglobulin (alpha2-M) are reduced. The present study explored the possible involvement in keratoconus of Krüppel-like factor 6 (KLF6), a transcription factor previously described to be essential for the integrity of the corneal epithelium. The transcript and proteins level of KLF6 and its action in regulating the genes affected in keratoconus were examined in this study. METHODS: Semiquantitative RT-PCR, Western blot analysis, immunofluorescence and in situ hybridization were used to investigate the expression of KLF6 mRNA and protein in normal and keratoconus corneas. Modulation by KLF6 of the promoter activity of alpha1-PI, LAP, cathepsin B, and alpha2-M genes was studied after transient transfection of KLF6 expression plasmid into corneal epithelial cells using promoter-reporter gene assays. Chromatin immunoprecipitation (ChIP) assays were performed to confirm the interactions between KLF6 and promoters of the genes affected in keratoconus. RESULTS: A global increased expression of the transcription factor KLF6 in terms of mRNAs and proteins was observed in total cornea and/or the epithelium in a substantial number of the keratoconus specimens. The promoter activity of the human alpha1-PI gene was suppressed by expression of KLF6 in corneal epithelial cells. The ChIP assay confirmed a physical interaction between KLF6 and the alpha1-PI promoter. CONCLUSIONS: Transcription factor KLF6 downregulates the alpha1-PI gene in corneal epithelial cells and may thereby be involved in keratoconus.

Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae.

Recently, we and others have identified two human endogenous retroviruses that entered the primate lineage 25-40 million years ago and that encode highly fusogenic retroviral envelope proteins (syncytin-1 and -2), possibly involved in the formation of the placenta syncytiotrophoblast layer generated by trophoblast cell fusion at the materno-fetal interface. A systematic in silico search throughout mouse genome databases presently identifies two fully coding envelope genes, present as unique copies and unrelated to any known murine endogenous retrovirus, that we named syncytin-A and -B. Quantitative RT-PCR demonstrates placenta-specific expression for both genes, with increasing transcript levels in this organ from 9.5 to 14.5 days postcoitum. In situ hybridization of placenta cryosections further localizes these transcripts in the syncytiotrophoblast-containing labyrinthine zona. Consistently, we show that both genes can trigger cell-cell fusion in ex vivo transfection assays, with distinct cell type specificities suggesting different receptor usage. Genes orthologous to syncytin-A and -B and disclosing a striking conservation of their coding status are found in all Muridae tested (mouse, rat, gerbil, vole, and hamster), dating their entry into the rodent lineage approximately 20 million years ago. Together, these data strongly argue for a critical role of syncytin-A and -B in murine syncytiotrophoblast formation, thus unraveling a rather unique situation where two pairs of endogenous retroviruses, independently acquired by the primate and rodent lineages, would have been positively selected for a convergent physiological role.