Loss of endothelial-ARNT in adult mice contributes to dampened circulating proangiogenic cells and delayed wound healing.

The recruitment and homing of circulating bone marrow-derived cells include endothelial progenitor cells (EPCs) that are critical to neovascularization and tissue regeneration of various vascular pathologies. We report here that conditional inactivation of hypoxia-inducible factor's (HIF) transcriptional activity in the endothelium of adult mice (Arnt(ΔiEC) mice) results in a disturbance of infiltrating cells, a hallmark of neoangiogenesis, during the early phases of wound healing. Cutaneous biopsy punches show distinct migration of CD31(+) cells into wounds of control mice by 36 hours. However, a significant decline in numbers of infiltrating cells with immature vascular markers, as well as decreased transcript levels of genes associated with their expression and recruitment, were identified in wounds of Arnt(ΔiEC) mice. Matrigel plug assays further confirmed neoangiogenic deficiencies alongside a reduction in numbers of proangiogenic progenitor cells from bone marrow and peripheral blood samples of recombinant vascular endothelial growth factor-treated Arnt(ΔiEC) mice. In addition to HIF's autocrine requirements in endothelial cells, our data implicate that extrinsic microenvironmental cues provided by endothelial HIF are pivotal for early migration of proangiogenic cells, including those involved in wound healing.

Hypoxia influences the vascular expansion and differentiation of embryonic stem cell cultures through the temporal expression of vascular endothelial growth factor receptors in an ARNT-dependent manner.

Adaptive responses to low oxygen (O(2)) tension (hypoxia) are mediated by the heterodimeric transcription factor hypoxia inducible factor (HIF). When stabilized by hypoxia, bHLH-PAS alpha- and beta- (HIF-1beta or ARNT) HIF complex regulate the expression of multiple genes, including vascular endothelial growth factor (VEGF). To investigate the mechanism(s) through which hypoxia contributes to blood vessel development, we used embryonic stem cell (ESC) differentiation cultures that develop into embryoid bodies (EBs) mimicking early embryonic development. Significantly, low O(2) levels promote vascular development and maturation in wild-type (WT) ESC cultures measured by an increase in the numbers of CD31(+) endothelial cells (ECs) and sprouting angiogenic EBs, but refractory in Arnt(-/-) and Vegf(-/-) ESC cultures. Thus, we propose that hypoxia promotes the production of ECs and contributes to the development and maturation of vessels. Our findings further demonstrate that hypoxia alters the temporal expression of VEGF receptors Flk-1 (VEGFR-2) and the membrane and soluble forms of the antagonistic receptor Flt-1 (VEGFR-1). Moreover, these receptors are distinctly expressed in differentiating Arnt(-/-) and Vegf(-/-) EBs. These results support existing models in which VEGF signaling is tightly regulated during specific biologic events, but also provide important novel evidence that, in response to physiologic hypoxia, HIF mediates a distinct stoichiometric pattern of VEGF receptors throughout EB differentiation analogous to the formation of vascular networks during embryogenesis.

Effects of green tea polyphenols on murine transplant-reactive T cell immunity.

Green tea polyphenols (GrTP), the active ingredient of green tea, may have immunosuppressive properties, but whether and how GrTP affect transplant-reactive T cells is unknown. To address this, we tested the effects of GrTP on in vitro and in vivo transplant-reactive T cell immunity. GrTP inhibited IFNgamma secretion by cultured monoclonal T cells and by alloreactive T cells in mixed lymphocyte reactions. Oral GrTP significantly prolonged minor antigen-disparate skin graft survival and decreased the frequency of donor-reactive interferon gamma-producing T cells in recipient secondary lymphoid organs compared to controls. In contrast to other hypothesized actions, oral GrTP did not alter dendritic cell trafficking to lymph nodes or affect metalloproteinase activity in the graft. This is the first report of an immunosuppressive effect of GrTP on transplant-reactive T cell immunity. The results suggest that oral intake of green tea could act as an adjunctive therapy for prevention of transplant rejection in humans.

Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter.

Sodium-bicarbonate cotransporters are homologous membrane proteins mediating the electrogenic or electroneutral transport of sodium and bicarbonate. Of the functionally characterized sodium-bicarbonate cotransporters (NBC), NBC1 proteins are known to be electrogenic. Here we report the cloning and functional characterization of NBC4c, a new splice variant of the NBC4 gene. At the amino acid level, NBC4c is 56% identical to NBC1 protein variants and 40% identical to electroneutral NBC3. When expressed in mammalian cells, NBC4c mediates electrogenic sodium-bicarbonate cotransport. The transport of sodium and bicarbonate is chloride independent and is completely inhibited by DIDS. NBC4c transcripts were detected in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes. The data indicate that NBC4c is an electrogenic sodium-bicarbonate cotransporter. The finding that both NBC1 and NBC4c proteins function as electrogenic sodium-bicarbonate cotransporters will aid in determining the structural motifs responsible for this unique functional property, which distinguishes these transporters from other members of the bicarbonate transporter superfamily.