Biomedical subjects

L M DeFouw

Publications and source records attributed to L M DeFouw.

5 recordsLinked to original sources

PubMed2001

Differential phosphodiesterase activity contributes to restrictive endothelial barrier function during angiogenesis.

Angiogenic endothelial hyperpermeability is abruptly diminished between days 4.5 and 5.0 of the 18-day lifespan of the chick chorioallantoic membrane. Here, we evaluated phosphodiesterase (PDE) activity during the differentiation of barrier function. At day 4.5, rolipram-mediated inhibition of cAMP-specific PDE IV reduced FITC-dextran extravasation. Moreover, inhibition of PDE III by HL 725, but not PDE I by 8-IBMX, decreased the temporal angiogenic endothelial hyperpermeability. Reduced FITC-dextran was also observed at day 4.5 after application of KT 5823, a selective inhibitor of cGMP-specific protein kinase G (PKG), LY 83583, an inhibitor of soluble guanylate cyclase, or LNMMA, an inhibitor of nitric oxide synthase. At day 5.0, Rp-cAMPS-mediated inhibition of cAMP-specific protein kinase A (PKA) diminished barrier function and interstitial accumulation of FITC-dextran was increased. In all cases, the mean widths of interendothelial separation remained uniform. Together, the results support the concept that differentiation of restrictive angiogenic endothelial barrier function in vivo includes inactivation of PDE III and PDE IV with consequent up-regulation of cAMP/PKA signaling and down-regulation of the cGMP/PKG pathway.

Allantois↗

PubMed2000

Restrictive endothelial barrier function during normal angiogenesis in vivo: partial dependence on tyrosine dephosphorylation of beta-catenin.

Differentiation of a restrictive endothelial barrier in the chick chorioallantoic membrane (CAM) occurs between Day 4.5 and Day 5.0 of the normal 21-day gestation. Whether molecular changes in the endothelial cell-cell junctional protein complex contribute to the ontogeny of barrier function represents the principal focus of this study. VE-cadherin has been shown to contribute to the regulation of endothelial cell monolayer permeability in vitro. Accordingly, VE-cadherin is complexed to the cytosolic catenins, and changes in monolayer permeability have been linked to alterations of the cadherin/catenin complex. Currently, a CAM endothelial VE-cadherin/beta-catenin complex was identified, and phosphotyrosine labeling of beta-catenin was decreased concurrently with the abrupt increase in CAM endothelial selectivity between Day 4.5 and Day 5.0. Further, inhibition of protein tyrosine phosphatases impeded regular tyrosine dephosphorylation of beta-catenin at Day 5.0 and this served to partially restore macromolecular extravasation to elevated levels normally present at Day 4.5. Thus, differentiation of selective barrier function in the angiogenic CAM endothelium in vivo is dependent, in part, on tyrosine dephosphorylation of beta-catenin.

Allantois↗

PubMed2000

Vascular endothelial growth factor fails to acutely modulate endothelial permeability during early angiogenesis in the chick chorioallantoic membrane.

The angiogenic endothelium of the chorioallantoic membrane (CAM) offers minimal restriction to macromolecular efflux at Day 4.5 of the normal 21-day chick gestation. Vascular endothelial growth factor (VEGF)-specific Flk-1 and Flt-1 tyrosine phosphorylation was observed at Day 4.5 by receptor immunoprecipitation and requisite immunoblotting. Further, general inhibition of tyrosine phosphorylation by either genistein or tyrphostin (10(-4) M) served to reduce FITC-Dextran 40 extravasation at Day 4.5. Likewise, anti-VEGF, but not anti-FGF-2 mAb, abolished the temporal endothelial hyperpermeability. These results are consistent with the established permeability-enhancing function of VEGF. Normal differentiation of the restrictive CAM endothelial barrier at Day 5. 0 was associated with reduced Flk-1 and Flt-1 expression, but sustained tyrosine phosphorylation of the residual RTKs. Moreover, inhibition of VEGF/RTK activity by anti-VEGF mAb at Day 5.0 did not enhance normal endothelial barrier function. Likewise, neither VEGF (5 x 10(-4) to 10(-15) M) nor PlGF (10(-6) to 10(-8) M), which selectively binds Flt-1, served to increase FITC-Dextran 40 efflux at Day 5.0. Together, these results are consistent with the suggestion that down-regulation of the permeability-related VEGF signal correlates temporally with the ontogeny of restrictive endothelial barrier function during angiogenesis in vivo.

Allantois↗

PubMed2000

Differentiation of endothelial barrier function during normal angiogenesis requires homotypic VE-cadherin adhesion.

Endothelial cells express two principal cadherins: VE-cadherin and N-cadherin. We established previously that only VE-cadherin expression was increased during differentiation of barrier function by angiogenic endothelium of the chick chorioallantoic membrane (CAM). Presently anti-VE-cadherin mAb, applied to the CAM at day 4.5 of gestation, served to inhibit the abrupt reduction of macromolecular extravasation that occurs normally at day 5.0. Neither anti-N-cadherin nor nonimmune IgG, on the other hand, prevented this temporal decrease of endothelial permeability. Despite the differential permeability responses, morphometric evaluations defined a reduction of mean paracellular cleft width after the application of either anti-VE-cadherin or anti-N-cadherin. Hence, alteration of molecular sieving characteristics within the junctional clefts, rather than modification of cleft dimensions; likely served as the principal modulator of macromolecular extravasation after inhibition of homotypic VE-cadherin adhesion. These results provide support to the concept that VE-cadherin contributes to the normal differentiation of endothelial barrier function during CAM angiogenesis in vivo.

Animals↗

PubMed1999

Modulation of angiogenic endothelial permselectivity by the cAMP pathway.

The chorioallantoic membrane (CAM) of the chick embryo provides an accessible model of normal angiogenesis in vivo. Previously, we reported a rapid reduction in CAM microvascular permeability to macromolecules between Days 4.5 and 5.0 of the normal 21-day gestation (V. Rizzo et al., 1995, Microvasc. Res. 49, 49-63). Here, we tested the hypothesis that activation of the cAMP signaling pathway at Day 4.5 would acutely increase permselectivity prior to normal differentiation of CAM endothelial barrier properties at Day 5.0. Changes in interstitial optical intensities due to extravasation of a graded series of FITC-dextrans (20, 40, and 70 kDa) were evaluated by computer-assisted image analysis, and endothelial ultrastructure was monitored by transmission electron microscopy. The cAMP analogue 8-bromo-cAMP (10(-4) and 10(-3) M) and forskolin (10(-5) and 10(-4) M), an adenylyl cyclase activator, acutely decreased permeability of the graded FITC-dextran series in a dose-dependent fashion. In addition, the nonspecific phosphodiesterase inhibitor IBMX (10(-4) M) served to increase basal restriction of the 20- and 40-kDa tracers. Further, Rp-cAMPS (10(-4) M), a cAMP antagonist for cAMP-dependent protein kinase, abolished the effects of both 8-bromo-cAMP (10(-3) M) and forskolin (10(-4) M) on FITC-Dextran 40 restriction. In all cases, ultrastructural presentation of both the endothelial cell junctions and the vesicles remained unchanged. The present results are consistent with the concept that exogenous cAMP activation decreased permeability of the angiogenic CAM endothelium at Day 4.5 without concomitant ultrastructural changes in the transendothelial macromolecular exchange pathways. Whether endogenous activity of cAMP contributes to normal differentiation of CAM endothelial barrier properties between Days 4.5 and 5.0 remains to be tested.

1-Methyl-3-isobutylxanthine↗