Browsing by Subject "Progenitor cells"

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    Vascular Remodeling of the Blood Brain Barrier
    (2011-01) Johnson, Arial Raina Larson
    The human brain contains a vast network of blood vessels, capillaries, and microvessels. The blood brain barrier (BBB) is made up of three main components: resident endothelial cells (ECs), tight junctions, and a basement membrane. This barrier is impermeable to most solutes, bacteria, antibodies, chemicals, and drugs. It does, however, allow for the transport and diffusion of substances that are metabolically necessary in the brain such as glucose and oxygen. The transport of glucose is facilitated by Glut-1, a brain endothelial cell specific glucose transporter. The loss or deficiency of Glut-1 in the BBB has been clinically diagnosed in humans. Glut-1 deficiency syndrome is characterized by a haploinsufficiency of the wild type Glut-1; the dominant non-functional mutation causes the clinical manifestations related to the syndrome. The manifestations begin in infancy and, if undiagnosed, may cause serious developmental delay, acquired microcephaly, seizures, ataxia, and spasticity. The only known treatment is a ketogenic diet which eliminates the brain’s need for glucose in metabolism. Incorporation of genetically engineered ECs or endothelial progenitor cells (EPCs) that contain the gene for the wild type Glut-1 into the brain vasculature would correct this syndrome in addition to opening the door for treating other CNS diseases. The proposed mechanism for incorporating new cells into the BBB is via postnatal neovasculogenesis. Postnatal neovasculogenesis occurs in ischemia, hypoxia, and tumor growth. There are two modes of postnatal neovasculogenesis: angiogenesis and vasculogenesis. Angiogenesis is the process by which the resident ECs proliferate when the signal for growth of new vessels is received. Theoretically, during the process of vasculogenesis EPCs are recruited from the bone marrow, differentiate, proliferate, and migrate to the signaling tissue and incorporate into the new vessel. My research project focused on method development to incorporate cells into the brain neovasculature. I focused this development further to incorporation of cultured and bone marrow-derived ECs and EPCs into the neovasculature through hypoxia-mediated outgrowth or BBB disruption. We have now developed a method for investigating the effects of hypoxia on vascular remodeling and EPC and EC recruitment into the neovasculature. In this method we utilized two models; direct injection of cultured ECs and EPCs into the brain followed by hypoxia, or osmotic disruption of the BBB followed by injection of ECs and EPCs. Cultured ECs were isolated from brain microvessels. Cultured human EPCs were isolated from peripheral blood. ECs and EPCs display different antigens that allow for immunohistochemical detection. The different combinations of antigens elucidate the different cell types. ECs display antigens for Glut-1, CD31, and von Willebrand Factor. Bone marrow-derived EPCs display antigens for CD31 and Tie2, but do not display antigens for von Willebrand Factor. Immunohistochemistry was used to characterize the cells as EPCs or ECs prior to injection and determine location of the cells in the brain after animals are exposed to hypoxia using the specific antigens for ECs and EPCs.