Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo.

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-beta superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.

Combination bcl-2 antisense and radiation therapy for nasopharyngeal cancer.

PURPOSE: A wide variety of tumors depend on the dysregulation of Bcl-2 family proteins for survival. The resulting apoptotic block can often provide a mechanism for resistance to anticancer treatments, such as chemotherapy and radiation. This current study evaluates the efficacy of combining systemically delivered Bcl-2 phosphorothioate antisense (Bcl-2 ASO) and radiation for nasopharyngeal cancer therapy. RESULTS: Antisense uptake was unaffected by 0, 3, or 6 Gy radiation. Radiation decreased the fraction of viable C666-1 cells to 60%, with a further decrease to 40% in combination with Bcl-2 ASO. Despite a modest in vitro effect, Bcl-2 ASO alone caused the regression of established xenograft tumors in mice, extending survival by 15 days in a C666-1 and by 6 days in a C15 model. The survival times for mice treated with both Bcl-2 ASO and radiation increased by 52 days in C666-1 and by 20 days in C15 tumors. This combination resulted in a more-than-additive effect in C666-1 tumors. Less impressive gains observed in C15 tumors might be attributable to higher expression of antiapoptotic Bcl-2 family proteins and limited drug distribution in the tumor. Retreatment of C666-1 tumors with the Bcl-2 ASO-radiation combination, however, was effective, resulting in mice surviving for >80 days relative to untreated controls. CONCLUSIONS: Our results show that the Bcl-2 ASO and radiation combination is a highly potent therapy for nasopharyngeal cancer. Further examination of combination therapy with radiation and other Bcl-2 family-targeted anticancer agents in both preclinical and clinical settings is definitely warranted.

Local radiotherapy induces homing of hematopoietic stem cells to the irradiated bone marrow.

Local breast radiation therapy (RT) is associated with a 3-fold increased risk of secondary acute myeloid leukemia. As a first step in determining the mechanism(s) underlying this observation, we investigated the role of RT in mediating the active recruitment of hematopoietic stem cells (HSC) to the site of RT. Our results show in a mouse model that local RT delivered to the left leg causes preferential accumulation of bone marrow mononuclear cells to the irradiated site, with maximum signal intensity observed at 7 days post-RT. This is associated with a 4-fold higher number of donor-derived HSC present in the left leg, demonstrating recruitment of HSC to the site of RT. SDF-1, matrix metalloproteinase 2 (MMP-2), and MMP-9 expression is significantly increased in the irradiated bone marrow, and their inhibition significantly reduced HSC recruitment to the irradiated bone marrow. Our data show that local RT has significant systemic effects by recruiting HSC to the irradiated bone marrow site, a process mediated by SDF-1, MMP-2, and MMP-9. These results raise the possibility that the exposure of increased numbers of HSC at a local site to fractionated irradiation may increase the risk of leukemogenesis. Our data also suggest some opportunities for leukemia prevention in breast cancer patients undergoing RT.

Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.

Successful therapy for many inherited disorders could be improved if the intervention were initiated early. This is especially true for lysosomal storage disorders. Earlier intervention may allow metabolic correction to occur before lipid buildup has irreversible consequences and/or before the immune system mounts limiting responses. We have been developing gene therapy to treat lysosomal storage disorders, especially Fabry disease. We describe studies directed toward metabolic correction in neonatal animals mediated by recombinant lentiviral vectors. To develop this method, we first injected a marking lentiviral vector that engineers expression of luciferase into the temporal vein of recipient neonatal animals. The use of a cooled charged-coupled device camera allowed us to track transgene expression over time in live animals. We observed intense luciferase expression in many tissues, including the brain, that did not diminish over 24 weeks. Next, we injected neonatal Fabry mice a single time with a therapeutic lentiviral vector engineered to express human alpha-galactosidase A. The injection procedure was well tolerated. We observed increased plasma levels of alpha-galactosidase A activity starting at our first plasma collection point (4 weeks). Levels of alpha-galactosidase A activity were found to be significantly elevated in many tissues even after 28 weeks. No immune response was observed against the corrective transgene product. Increased levels of enzyme activity also led to significant reduction of globotriaosylceramide in the liver, spleen, and heart. This approach provides a method to treat lysosomal storage disorders and other disorders before destructive manifestations occur.