Cardiomyocyte Specific Ablation of p53 Is Not Sufficient to Block Doxorubicin Induced Cardiac Fibrosis and Associated Cytoskeletal Changes

Doxorubicin (Dox) is an anthracycline used to effectively treat several forms of cancer. Unfortunately, the use of Dox is limited due to its association with cardiovascular complications which are manifested as acute and chronic cardiotoxicity. The pathophysiological mechanism of Dox induced cardiotoxicity appears to involve increased expression of the tumor suppressor protein p53 in cardiomyocytes, followed by cellular apoptosis. It is not known whether downregulation of p53 expression in cardiomyocytes would result in decreased rates of myocardial fibrosis which occurs in response to cardiomyocyte loss. Further, it is not known whether Dox can induce perivascular necrosis and associated fibrosis in the heart. In this study we measured the effects of acute Dox treatment on myocardial and perivascular apoptosis and fibrosis in a conditional knockout (CKO) mouse model system which harbours inactive p53 alleles specifically in cardiomyocytes. CKO mice treated with a single dose of Dox (20 mg/kg), did not display lower levels of myocardial apoptosis or reactive oxygen and nitrogen species (ROS/RNS) compared to control mice with intact p53 alleles. Interestingly, CKO mice also displayed higher levels of interstitial and perivascular fibrosis compared to controls 3 or 7 days after Dox treatment. Additionally, the decrease in levels of the microtubule protein α-tubulin, which occurs in response to Dox treatment, was not prevented in CKO mice. Overall, these results indicate that selective loss of p53 in cardiomyocytes is not sufficient to prevent Dox induced myocardial ROS/RNS generation, apoptosis, interstitial fibrosis and perivascular fibrosis. Further, these results support a role for p53 independent apoptotic pathways leading to Dox induced myocardial damage and highlight the importance of vascular lesions in Dox induced cardiotoxicity

Expression in Trichoplusia ni (High Five) insect cells and partial purification of a chimeric version of active human factor VIII

Introduction/Objectives: Purification of recombinant Factor VIII from mammalian cells poses remarkable challenge, because of its suboptimal yield and significant1y high cost. Bacalovirus derived expression in insect cells is an appropiate system to overcome these limitations. However, several reports have concluded that Sf9 cells are not suitable for expression of structurally correct factor VIII (fVIII). We have developed a cDNA construct encoding chimeric, full-length human fVIII in which dispensable B-domain has been replaced with significantly shorter B-domain derived from its ortholog in Fugu rubripe. This cDNA was successfully applied for expression and purificacion of active fVIII protein in Trichoplusia ni insect cells, known as High Five (Invitrogen)

Expression in Trichoplusia ni (High Five) insect cells and partial purification of a chimeric version of active human factor VIII

Introduction/Objectives: Purification of recombinant Factor VIII from mammalian cells poses remarkable challenge, because of its suboptimal yield and significant1y high cost. Bacalovirus derived expression in insect cells is an appropiate system to overcome these limitations. However, several reports have concluded that Sf9 cells are not suitable for expression of structurally correct factor VIII (fVIII). We have developed a cDNA construct encoding chimeric, full-length human fVIII in which dispensable B-domain has been replaced with significantly shorter B-domain derived from its ortholog in Fugu rubripe. This cDNA was successfully applied for expression and purificacion of active fVIII protein in Trichoplusia ni insect cells, known as High Five (Invitrogen)