Two membrane glycoproteins acting as energy‐dependent efflux pumps, mdr‐encoded P‐glycoprotein (P‐gp) and the more recently described multidrug resistance‐associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P‐gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood‐brain barrier, but localization of MRP has not been well characterized yet. Using RT‐PCR and immunoblot analysis, we have compared the expression of P‐gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P‐gp‐encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P‐gp, is not predominantly expressed in the blood‐brain barrier endothelial cells and that Mrp1 and the mdr1b P‐gp isoform may be present in other cerebral cells.

Hematopoietic stem cells (HSCs) arise first in the third week of human ontogeny inside yolk sac developing blood vessels, and independently, from the wall of the embryonic aorta and vitelline arteries one week later. HSCs produced in the yolk sac and in the embryonic truncal arteries migrate to and transiently colonize the embryonic liver (EL), and thereafter the bone marrow (BM), their permanent site of residence. At the moment, the origin of human HSCs is still controversial; one of the main hypotheses being that they are generated by hemogenic endothelial cells (ECs). To proove definitively the endothelial origin of HSCs that arise within the human embryo, we previously purified ECs from either the yolk sac or the truncal arteries and reported that they were able to produce blood cells in vitro. We then found that some of the HSCs present in the human EL were co-expressing vascular endothelial (VE)-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker, and demonstrated that these HSCs bearing a dual hemato-endothelial phenotype were endowed with remarkably high self renewal and proliferative potentials. Furthermore, a transgenic mouse model based on the VE-cadherin cis-regulating elements that we engineered to trace the fate of the first VE-cadherin expressing cells allowed us to clearly demonstrate that a majority of adult BM HSCs derived from a VE-cadherin ancestor. Altogether our studies strongly suggest that at least a part of both the human and the murine hematopoietic systems arise from an endotheliumlike ancestor.

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The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes.