Carmen Gagnon

Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells.

Nonstop proliferation and vigorous neovascularization are two prominent characteristics of cancer. Antiangiogenic therapy has emerged as an important modality in treatment of solid tumors. Our previous work demonstrated that microparticles derived from apoptotic T-lymphocytes (LMPs) not only reduced the viabilities of high-proliferating cells, but also exhibited potent anti-angiogenic effects through inhibition of the vascular endothelial growth factor (VEGF)/VEGF receptor 2 signalling pathway. In the present study, we extended these studies to explore the anticancer potential of LMPs using a murine model of Lewis lung carcinoma (LLC). Results show that intratumoral injection of LMPs (2.5 mg/kg) decreased tumor size by more than 50% relative to control. Tumor microvessel density and VEGF-A levels were also markedly reduced upon LMPs treatment. To elucidate the underlying mechanisms of LMPs mediated antitumor activity, LLC cells were utilized in in vitro experiments. LMPs suppressed VEGF-A protein levels in LLC cells and led to inhibition of LLC cell viability and proliferation. In addition, knockdown of the low density lipoprotein receptor (LDLR) expression reduced the uptake of LMPs into LLC cells and attenuated the inhibitory effects of LMPs on cell growth and VEGF-A expression. Our findings demonstrate that LMPs exert antiangiogenic and proapoptotic effects that lead to inhibition of lung carcinoma by reducing VEGF-A levels, and LDLR mediates the anti-VEGF effect of LMPs through translocating LMPs into LLC cells. These results suggest that LMPs are promising anti-angiogenic therapeutic agent and represent a new therapeutic strategy for treating lung carcinomas.

Photoprotection prevents TPN-induced lung procollagen mRNA in newborn guinea pigs.

BACKGROUND Photo-exposed intravenous multivitamin solutions (MVP) carry a peroxide load. Peroxidation induces gene expression of procollagen. We hypothesized that photo exposure of the MVP solution might promote pulmonary fibrosis. The aim of the study was to assess the potential for MVP to increase procollagen mRNA. METHODS Three day old guinea pigs were assigned to the following intravenous regimens, either: Control (C): 5% dextrose + 0.45% NaCl; C + 200 or 500 microM H(2)O(2); C + 500 microM H(2)O(2) + 10 microM GSSG; [C + 1% MVP +/- [amino acids + lipids]] +/- photoprotected. After 4 d, levels of pulmonary alpha1(I) procollagen mRNA and glutathione were determined. Results were compared by ANOVA. RESULTS Photoprotection of MVP or TPN prevents light induction of procollagen mRNA. The effect of MVP + light was associated with a peroxide load coupled with a low glutathione level. This was also observed with the 500 microM H(2)O(2) group. The addition of GSSG prevented the increase of procollagen mRNA caused by H(2)O(2). CONCLUSION An oxidant stress caused by the infusion of peroxides in an organism with a weak antiperoxide capacity induces the transcription of the gene encoding for procollagen alpha1(I). The results confirm the antiperoxide activity of lung glutathione. Parenteral nutrition could be a clinical condition favoring the initiation of lung fibrosis, especially in premature newborn infants who have low glutathione levels.

The Role of Lysophosphatidic Acid Receptor (LPA1) in the Oxygen-Induced Retinal Ganglion Cell Degeneration

PURPOSE Although previous studies have demonstrated that hypoxia induces retinal ganglion cell (RGC) apoptosis and that transient retinal ischemia upregulates the expression of lysophosphatidic acid (LPA) receptors, it remains to be determined whether LPA(1) receptor mediates RGC degeneration during retinopathy of prematurity (ROP). By using an immortalized RGC line (RGC-5), primary neonatal RGC cultures, and oxygen-induced retinopathy (OIR) to model ROP, the authors explored whether LPA(1) receptor induces RGC degeneration and the potential mechanisms thereof. METHODS OIR was induced by exposing rat pups to alternating cycles of hyperoxia/hypoxia from postnatal day (P) 0 to P14. RGC viability was evaluated by Fluorogold labeling. Effects of hyperoxia or hypoxia on LPA(1) expression were determined in the RGC-5 line by Western blot. Roles of hypoxia, LPA(1) receptor (with agonist, stearoyl-LPA; antagonist, THG1603; LPA(1) knock-down, shRNA-LPA(1)), and Rho kinase (with inhibitor Y-27632) in mediating RGC survival and neurite outgrowth were assessed by MTT assay and phase-contrast microscopy, respectively. Expression of GFP-LPA(1) in RGC-5 under hypoxia was examined by confocal microscopy. RESULTS OIR caused pronounced RGC loss in the retina. LPA(1) receptor was expressed by RGCs in retinal tissue, whereas oxygen stress induced its expression in RGC-5. Exposure to stearoyl-LPA or hypoxia substantially reduced the viability of RGCs; this was abrogated by THG1603 and shRNA-LPA(1). THG1603 and Y-27632 treatment also attenuated the adverse effects of hypoxia on RGC-5 neurite outgrowth, and their intravitreal administration prevented OIR-induced RGC loss. Interestingly, overexpression of LPA(1) increased RGC-5 susceptibility to hypoxia-induced cell loss. CONCLUSIONS Current data strongly support a critical role for LPA(1) receptor in mediating RGC degeneration during OIR.

Association of -158(C |[rarr]| T) (XmnI) DNA Polymorphism inG|[gamma]|-Globin Promoter with Delayed Switchover from Fetal to Adult Hemoglobin Synthesis

In this study, the effect of -158(C → T) (XmnI) polymorphism on the synthesis of fetal Hb and its Gγ component during the switchover from fetal to adult Hb was examined using cord blood samples from normal Caucasian term infants. The presence of -158(C→T) mutation was determined by amplification of Gγ- and Aγ-globin gene promoter fragments from the DNA isolated from cord blood samples, followed by XmnI restriction enzyme digestion. The syntheses of fetal and adult Hb in cord blood were measured by [3H]leucine incorporation in globin synthesis, separation of the globin polypeptides by HPLC, and scintillation counting of the fractions. The presence of -158(C→ T) substitution in the Gγ-globin promoter region was positively correlated with elevated synthesis of fetal Hb and itsGγ-globin component in term newborn infants and is associated with delayed switchover from fetal to adult Hb. In addition, analysis of cord blood samples from 100 normal Caucasian French Canadian term infants revealed that the frequency of -158(C → T) substitution inGγ-promoter was 0.32.

The Reactivation of Fetal Hemoglobin Synthesis during Anemia of Prematurity

ABSTRACT: Increased fetal Hb (HbF) synthesis has been shown to occur during fetal hypoxemia and severe anemia. To determine whether increased HbF synthesis occurs during anemia of prematurity, the levels of HbF synthesis were correlated with the degree of anemia and plasma erythropoietin levels. Thirteen newborn infants born at 29.2 ± 1.7 wk of gestation were studied at a postconceptional age 36.0 ± 1.1 wk. Hb levels ranged from 65 to 78 g/L. Blood samples were incubated in an amino acid mixture containing [3H]leucine and chromatographed allowing the separation and quantitation of the α, β, and ν (AνT, Gν AνI) chains. Erythropoietin was determined by RIA. The mean HbF synthesis was 77.9 ± 8.9% of total Hb synthesis (range: 61 to 91%). Plasma erythropoietin concentrations were 21.4 ± 6.4 mU/mL. There was no correlation between the total Hb or HbF synthesis and the level of erythropoietin. There was, however, a significant inverse correlation between the Hb level and HbF synthesis (p < 0.01). Nine infants who had received transfusions during the first few days of life had a mean HbF that was 53.5 ± 15.2% of total Hb, whereas their HbF synthesis was 78.4 ± 7.6%. Four of the infants never received transfusions; the total circulating HbF and HbF synthesis in these infants were 87.7 ± 7.7% and 76.8 ± 12.7%, respectively. This study shows that there can be a reactivation of HbF synthesis during severe anemia of prematurity.

The Proportions of G|[gamma]|- and A|[gamma]|-Globins in the Fetal Hemoglobin Synthesized in Preterm and Term Infants

ABSTRACT: The change in Gγ to Aγ ratio in relation to the switchover of fetal HB (HbF) to adult Hb (α2β2) synthesis has not been well defined. The γ-globins of HbF (α2γ2) have either glycine (Gγ) or alanine (Aγ) at position 136. During fetal life the Gγ makes up 70% of the γ-globins, although they are 40% of the small amounts of HbF in the adult. To further the understanding of this switchover, globin chain synthesis was determined sequentially in eight preterm and 20 full-term infants postnatally. To complete the study, single analysis was also carried out in eight term infants at birth and six preterm infants at the postconceptional age equivalent to term. Blood samples were incubated in an amino acid mixture containing 3H-leucine and subjected to reversed-phase liquid chromatography. The results demonstrated that the fetal proportions of Gγ to Aγ are rigidly controlled according to postconceptional age and not affected by postnatal age after preterm birth. During the early postconceptional age switchover from HbF to adult Hb synthesis, an equal repression of the Gγ and Aγ chains was found. However, based on the values obtained from the term infants, after a postconceptional age of 44 wk, the levels of Gγ to total γ synthesis begin to decrease and become more variable.

Hypoxemia and increased fetal hemoglobin synthesis

Fetal hemoglobin (HbF) synthesis in children with congenital cyanotic heart disease was compared that in normal children. Children with hypoxemia had higher levels of hemoglobin, total HbF, and HbF synthesis. In these children there was also an inverse correlation between HbF synthesis and oxygen content, as well as between HbF synthesis and hemoglobin concentration. Thus hypoxemia increases HbF synthesis.

Quantitative correlation between globin mRNAs and synthesis of fetal and adult hemoglobins during hemoglobin switchover in the perinatal period

To determine whether a quantitative relationship existed between globin mRNAs and their translation products during the period of switchover, the relative amounts of the mRNAs of α-, β-, and γ-globins and their protein synthesis in cord blood samples were measured and compared. The synthesis of globins in immature red cells was measured by the incorporation of [3H]leucine followed by separation and quantitation of the polypeptides on a C4-reverse phase HPLC. The relative proportions of the mRNAs of globins were determined by RNase protection assay. A comparison of cord blood samples from 45 newborn infants of different gestational ages(25-41 wk; birth weight, 850-4695 g) revealed a very significant correlation(r2 = 0.924) between the ratio of globin mRNAs encoding HbF([γ/(γ + β)] mRNAs) and HbA ([β/(γ + β)] mRNAs) and the ratio of de novo synthesis of HbF [γ/(γ +β)] and HbA [β/(γ + β)]. There was a linear relationship between the proportions of globin mRNAs encoding HbF with the proportional synthesis of HbF throughout the developmental stage studied. The ratio ofα2/α1-globin mRNAs increased from 2.0 ± 0.2 between 24 and 36 wk of gestation to 2.3 ± 0.4 (p = 0.02) during 37-41 wk of gestation. These results of the complementary changes atα- and β-loci during fetal development may further the understanding of the coordinated regulation of globin gene expression.

Lymphocyte-derived microparticles induce bronchial epithelial cells' pro-inflammatory cytokine production and apoptosis.

OBJECTIVE The aim of this study was to determine if human CEM (human lymphoblastoma) T cell-derived microparticles (LMPs) could directly induce human bronchial epithelial cells (BECs) apoptosis and cytokine production. We also tested if LMPs phagocytosis by BECs played a role in mediating these effects. METHODS We generated LMPs from CEM (human lymphoblastoma) T cells to investigate their effects on a human BEC cell line (16HBE) in vitro. RESULTS BECs (16HBE cells) incubation with LMPs resulted in significant production of inflammation-associated cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8, in a dose- and time-dependent manner. LMPs also induced increased activities of caspase-3, caspase-8, and caspase-9 in BECS, which resulted in increased BECs apoptosis as assessed by flow cytometry (Annexin V and propidium iodide staining) and transmission electronic microscopy (TEM). Interestingly, LMPs effects on BECS were inhibited by the phagocytosis inhibitors cytochalasin D and chloroquine. CONCLUSIONS These results suggest that phagocytosis plays an important role in mediating the effects of LMPs on BECs. Thus, increased LMP concentrations may contribute to increased respiratory inflammatory responses and innate immune response maintenance in airway epithelium after LMPs engulfment by endothelial cells.

Decreasing oxygen saturation in very early preterm newborn infants after transfusion.

Very early preterm infants are born at a time when more than 90% of their red blood cells contain fetal haemoglobin (HbF)1 and therefore their blood has a high affinity for oxygen.2 Because of blood sampling, early preterm newborns often receive transfusions for blood volume replacement. These transfusions are carried out with adult red blood cells containing adult haemoglobin (HbA) and decrease the HbO2 affinity. The change in P50 after transfusion has not previously been published in human preterm …