Vascular endothelial growth factor-A (VEGF-A) is required for vascular development throughout the embryo and has been proposed to play an important role in pulmonary vascular patterning. Expressed by the embryonic respiratory epithelium, VEGF-A signals endothelial cells within the splanchnic mesenchyme. To refine understanding of the spatial and temporal role of VEGF-A in lung morphogenesis, isoform VEGF164 was expressed under conditional control in distal and proximal airway epithelial cells. Unexpectedly, increased expression of VEGF164 in distal lung disrupted peripheral vascular net assembly and arrested branching of airways tubules without altering endothelial cell proliferation or apoptosis. Peripheral airway branching and vascular smooth muscle patterning were also altered. In contrast, expression of VEGF164 by epithelial cells of the conducting airways caused atypical evaginations of small capillary-like vessels into large airways but did not alter peripheral vascular net assembly or branching morphogenesis. These data demonstrate that the differential response of endothelial cells in distal vascular beds and large central blood vessels is established early in lung development. Precise temporal and spatial expression of VEGF-A is required for vascular patterning during lung morphogenesis. Disruption of pulmonary vascular assembly perturbs reciprocal interactions with epithelium leading to altered airway branching morphogenesis.

Distribution of vascular endothelial cell growth factor A (VEGF-A) as a gradient determines microvascular endothelial cell (EC) fate during organogenesis. While much is understood about mechanisms of differential distribution, less is known about how EC perceive and interpret a graded VEGF-A signal to generate positional target gene activation. Using microvascular EC, we analyzed the effect of time and graded VEGF-A input on VEGFR2 autophosphorylation, signal kinase activation and induction of immediate-early genes. The threshold and time to peak activation of VEGFR2 were dependent on signal strength over a 50-fold range in concentration with 3-fold concentration differences readily distinguished. Longer duration of exposure did not compensate for low concentration of VEGF-A, suggesting intensity and duration of signal were not interpreted equivalently. With the same conditions graded and time-sensitive information was transduced through the PLCγ/p44/p42MAPK signal pathway but not the parallel AKT pathway. Analysis of MAPK-induced angiogenic immediate-early genes determined EGR-1, EGR-3, and NR4A1 were dependent on graded input while NR4A2 and DSCR1 were independent with 'switch-like' induction. These data demonstrate rapid, linear integration of VEGF-A levels but independent interpretation of duration of signal and identify potential nodes for segregation of gradient-dependent andindependent responses. These results describe how microvascular EC fate decisions can be determined by comparatively moderate changes in VEGF signal strength, resulting in combinatorial changes in the repertoire of immediate-early genes for transcription effectors. Keywords morphogen; microvascular; organogenesis; cell fate During organogenesis, important cell fate decisions are dependent upon positional information provided by graded distribution of morphogens. The impact of morphogens during development is well characterized in Drosophila and Xenopus (reviews Ashe and Briscoe, 2006;Gurdon and Bourillot, 2001). In these systems, morphogens are critical for the formation of sharp borders of distinct cell types from common progenitors during tissue and organ development. It is proposed that localized expression of vascular endothelial growth factor A (VEGF-A) provides positional information that determines endothelial cell patterning during

The temperature dependence of lipid-depleted beef heart cytochrome c oxidase activity was studied in a series of chemically homogeneous detergents. The detergents that were tested included C10 to C18 maltosides, C8 to C12 glucosides, C8 to C16 Zwittergents, and C12 poly(oxyethylene) ethers. The observed rates of electron transport were dependent upon the structure of the polar head group and the length of the hydrocarbon tail. Of the detergents tested, the alkyl maltosides were the best in terms of both high rates of electron transport and superior enzyme stability. With the maltosides, changing the length of the alkyl tail affected the activity of cytochrome c oxidase in a manner quite similar to that reported with synthetic phosphatidylcholines and phosphatidylethanolamines [Vik, S. B., & Capaldi, R. A. (1977) Biochemistry 16, 5755-5759], suggesting that the alkyl maltosides can mimic some of the features of the membrane environment. In each of the detergents, the activation enthalpy (determined from the slope of an Arrhenius plot) was nearly identical, suggesting that the same electron-transfer step within cytochrome c oxidase is rate limiting. This result has been interpreted as evidence for the existence of two or more conformers of cytochrome c oxidase, one of which is significantly more active than the other(s). The enzyme turnover number, which changes by 2 orders of magnitude depending upon the structure of the bound detergent, may reflect the ability of each detergent to alter the equilibrium between the active and nearly inactive conformers.