Faculty Bibliography

Recent experiments show that hematopoietic progenitor cell populations contain endothelial precursor cells. We have isolated a population of CD34(+) cells that expresses fibroblast growth factor receptor-1 (FGFR-1) and that differentiates into endothelial cells in vitro. We find that 4.5% +/- 2.1% of CD34(+) cells isolated from bone marrow, cord blood, and mobilized peripheral blood express FGFR-1 and that viable CD34(+)FGFR(+) cells are small, with little granularity, and express both primitive hematopoietic and endothelial markers on their surface. The primitive hematopoietic markers AC133, c-kit, and Thy-1 are coexpressed by 75%, 85%, and 64% of CD34(+)FGFR(+) cells, respectively. Most of the CD34(+)FGFR(+) cells also express antigens found on endothelial cells, such as CD31, vascular endothelial growth factor receptor-2, and the endothelial-specific cell surface marker, vascular endothelial cadherin (VE-cadherin), whereas 56% to 60% of the cells express Tie, Tek, and the endothelial-specific marker, P1H12. The CD34(+)FGFR(+) population is enriched in cells expressing endothelial-specific antigens compared with the CD34(+) population. Isolated CD34(+)FGFR(+) cells grow slowly in culture, are stimulated by fibroblast growth factor-2 and vascular endothelial growth factor, and give rise to cells that express von Willebrand factor and VE-cadherin and that incorporate acetylated low-density lipoprotein. These experiments show that FGFR-1 is expressed by a subpopulation of CD34(+) cells that give rise to endothelial cells in vitro, indicating that this population contains endothelial stem/progenitor cells

PURPOSE: The majority of the research on the biologic effects of ionizing radiation has focused on the impact of radiation on cells in terms of gene expression, DNA damage, and cytotoxicity. In comparison, little information is available concerning the direct effects of radiation on the extracellular microenvironment, specifically the extracellular matrix and its main component, collagen. We have developed a series of monoclonal antibodies that bind to cryptic epitopes of collagen Type IV that are differentially exposed during matrix remodeling and are key mediators of angiogenesis. We have hypothesized that ionizing radiation might affect the process of angiogenesis through a direct effect on the extracellular matrix and specifically on collagen Type IV. METHODS AND MATERIALS: Angiogenesis was induced in a chick chorioallantoic membrane (CAM) model; 24 h later, a single-dose treatment with ionizing radiation (0.5, 5, and 20 cGy) was administered. Angiogenesis was assessed, and the exposure of two cryptic regulatory epitopes within collagen Type IV (HUI77 and HUIV26) was studied in vitro by solid-phase ELISA and in vivo by immunofluorescence staining. RESULTS: A dose-dependent reduction of angiogenesis with maximum inhibition (85%-90%) occurring at 20 cGy was demonstrated in the CAM model. Exposure of the cryptic HUIV26 site, an angiogenesis control element, was inhibited both in vitro and in vivo by the same radiation dose, whereas little if any change was observed for the HUI77 cryptic epitope. CONCLUSIONS: A dose-dependent alteration of the functional exposure of the HUIV26 cryptic epitope is induced by radiation in vitro and in the CAM model in vivo. This radiation-induced change in protein structure and function may contribute to the inhibitory effects of ionizing radiation on new blood vessel growth and warrants further studies in other models

The Sonic hedgehog (Shh) signaling pathway plays a critical role in normal cerebellar development and has been implicated in medulloblastomas, common malignant childhood tumors of the cerebellum. To test whether Shh mis-expression is sufficient for medulloblastoma formation, we used ultrasound biomicroscopy-guided in utero injection of a Shh-expressing retrovirus into the cerebellum of 13.5-day mouse embryos to show that direct activation of the Shh pathway can lead to tumor formation. Significantly, medulloblastomas were observed in 76% of the mice infected with Shh-expressing retrovirus. Furthermore, contrary to recent suggestions that the Shh transcriptional target Gli1 plays a critical role in Shh-induced tumorigenesis, we found that medulloblastomas form in Gli1 null mutant mice. We have developed an efficient mouse model of medulloblastoma and shown that Gli1 is not required for tumorigenesis when Shh signaling is activated upstream in the pathway

We have generated an animal model for mitochondrial myopathy by disrupting the gene for mitochondrial transcription factor A (Tfam) in skeletal muscle of the mouse. The knockout animals developed a myopathy with ragged-red muscle fibers, accumulation of abnormally appearing mitochondria, and progressively deteriorating respiratory chain function in skeletal muscle. Enzyme histochemistry, electron micrographs, and citrate synthase activity revealed a substantial increase in mitochondrial mass in skeletal muscle of the myopathy mice. Biochemical assays demonstrated that the increased mitochondrial mass partly compensated for the reduced function of the respiratory chain by maintaining overall ATP production in skeletal muscle. The increased mitochondrial mass thus was induced by the respiratory chain deficiency and may be beneficial by improving the energy homeostasis in the affected tissue. Surprisingly, in vitro experiments to assess muscle function demonstrated that fatigue development did not occur more rapidly in myopathy mice, suggesting that overall ATP production is sufficient. However, there were lower absolute muscle forces in the myopathy mice, especially at low stimulation frequencies. This reduction in muscle force is likely caused by deficient formation of force-generating actin-myosin cross bridges and/or disregulation of Ca(2+) homeostasis. Thus, both biochemical measurements of ATP-production rate and in vitro physiological studies suggest that reduced mitochondrial ATP production might not be as critical for the pathophysiology of mitochondrial myopathy as thought previously

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies

The discovery of small molecules that act as agonists and antagonists of the Hedgehog-Gli signaling pathway, which plays important roles in the embryo and adult, opens a new avenue for the treatment of diseases caused by aberrant suppression or activation of this complex pathway

Children and adolescents are increasingly exposed to psychostimulants, either illicitly or for the treatment of common neuropsychiatric conditions, such as attention deficit disorder with and without hyperactivity. Despite the widespread use of psychomotor stimulants in younger age groups, little is known regarding the chronic molecular neuroadaptive responses to these agents in the immature brain. Here we demonstrate that, after chronic administration of the psychostimulants cocaine and amphetamine, the transcription factor DeltaFosB is upregulated in the nucleus accumbens of periadolescent mice but not in post-weanling or adult mice. Induction of DeltaFosB also occurs exclusively in the caudate putamen of periadolescent mice after amphetamine administration. These results demonstrate the unique plasticity in the adolescent brain of a critical molecule that regulates psychostimulant action and suggest that these neuroadaptive changes may be involved in the mediation of enhanced addictive tendencies in the adolescent relative to the adult

Mice with fibroblast-specific expression of TAP-1 were generated by expressing the TAP-1 transgene under the control of the fibroblast-specific protein (FSP) 1 promoter/enhancer on TAP-1-deficient background. MHC class I expression in primary fibroblast cultures isolated from the resulting strain mimicked that of wild-type counterparts. MHC class I was detected in both types of fibroblasts following treatment with IFN-alphabeta. Positive selection of CD4(-)CD8(+) thymocytes was observed in neither adult nor fetal/neonatal thymus of transgenic mice. IFN-alphabeta-induced expression of MHC class I rescued positive selection of CD4(-)CD8(+) T cells in fetal thymic organ cultures, but not in adult mice. Contrary to previous suggestions, our results indicate a limited role of fibroblasts in promoting positive selection. In addition, the results suggest that positive selection may occur by a different mechanism in fetal vs adult thymus