Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation.

Mammalian blastocyst formation is dependent on establishment of trophectoderm (TE) ion and fluid transport mechanisms. We have examined the expression and function of aquaporin (AQP) water channels during murine preimplantation development. AQP 3, 8, and 9 proteins demonstrated cell margin-associated staining starting at the 8-cell (AQP 9) or compacted morula (AQP 3 and 8) stages. In blastocysts, AQP 3 and 8 were detected in the basolateral membrane domains of the trophectoderm, while AQP3 was also observed in cell margins of all inner cell mass (ICM) cells. In contrast, AQP 9 was predominantly observed within the apical membrane domains of the TE. Murine blastocysts exposed to hyperosmotic culture media (1800 mOsm; 10% glycerol) demonstrated a rapid volume decrease followed by recovery to approximately 80% of initial volume over 5 min. Treatment of blastocysts with p-chloromercuriphenylsulfonic acid (pCMPS, \u3e or =100 microM) for 5 min significantly impaired (P \u3c 0.05) volume recovery, indicating the involvement of AQPs in fluid transport across the TE. Blastocysts exposure to an 1800-mOsm sucrose/KSOMaa solution did not demonstrate volume recovery as observed following treatment with glycerol containing medium, indicating glycerol permeability via AQPs 3 and 9. These findings support the hypothesis that aquaporins mediate trans-trophectodermal water movements during cavitation

Long-Term Safety of PEGylated Coagulation Factor VIII in the Immune-Deficient Rowett Nude Rat

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26- and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ~75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immune-deficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals

Long-Term Safety of PEGylated Coagulation Factor VIII in the Immune-Deficient Rowett Nude Rat

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26-and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ∼75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immunedeficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals

Plasminogen Activator Inhibitor 1 Protects Fibrosarcoma Cells from Etoposide-Induced Apoptosis through Activation of the PI3K/Akt Cell Survival Pathway1

High levels of plasminogen activator inhibitor (PAI-1) in tumors are associated with poor prognosis in several cancer types, and the reason for this association is not fully understood. Plasminogen activator inhibitor 1 has been suggested to contribute to tumor growth by protecting cancer cells from apoptosis, and we have previously shown that wild type murine fibrosarcoma cells are significantly more resistant to apoptosis induced by chemotherapy than PAI-1-deficient fibrosarcoma cells. Here, we further investigated the molecular mechanisms underlying the antiapoptotic function of PAI-1 focusing on the phosphatidylinositol 3-phosphate kinase (PI3K)/Akt cell survival pathway. We demonstrate that the activation level of the Akt cell survival pathway is reduced in PAI-1-deficient cells. Inhibition of either PI3K or Akt by synthetic inhibitors sensitized the wild type but not the PAI-1-deficient cells to etoposide-induced cell death. More importantly, reintroduction of PAI-1 expression in PAI-1-deficient cells induced an increase in Akt activity and protection against etoposide-induced apoptosis. Concordantly, silencing of PAI-1 by RNA interference in wild type fibrosarcoma cells decreased the level of active Akt, and this was accompanied by a sensitization of the cells to etoposide-induced cell death. Altogether, our data suggest that PAI-1 influences sensitivity to etoposide-induced apoptosis through the PI3K/Akt cell survival pathway by acting upstream of PI3K and Akt. This points to PAI-1 as a possible therapeutic target in cancer diseases where PAI-1 inhibits chemotherapy-induced apoptosis