Factores que Influyen en el Rendimiento Escolar de un Niño

The finding of the novel role of ephrinB2 to mediate internalization of VEGFR2 together with the reported function of VEGFR2 in control of tip cell sprouting activity urge an appealing assumption that the regulation of filopodial extension by ephrinB2 might involve its capability to trigger VEGFR2 endocytosis and downstream signaling. To test this, a short- term culture system of explanted retinas was developed. Briefly, retinas were dissected from P4-5 pups and flat-mounted onto hydrophilic membrane filter with the nerve fiber layer facing the membrane. Cultured medium was layered under the filter. Retina explants were incubated for 2-4 h for recovery. Stimulation of acute tip cell responses was carried out for 4h by addition of exogenous stimulators and/or inhibitor into the medium.

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6.3.1. VEGF-induced extension of endothelial tip cell filopodia requires

receptor endocytosis

Explanted retinas robustly responded to stimulation with VEGF as evidenced by the drastic increase in number of filopodial extensions at the sprouting front of vascular bed (Fig. 6-23). This observation confirms the cellular function of VEGFR2 as a pivotal regulator of actin dynamic and guided migration of endothelial tip cells. Simultaneous treatment of explanted retinas with VEGF and dynasore dampened the stimulatory effect of VEGF and caused the reduction in number of filopodial extensions to almost the basal level when no stimulant was presented (Fig. 6-23), demonstrating the crucial requirement of endocytosis for VEGFR2 function at the endothelial tip cell.

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Figure 6-23 Acute stimulation of explanted retina with VEGF induces tip cell filopodial extension in an endocytosis-dependent manner. a, P4 retina explants were stimulated with VEGF or simultaneously treated with VEGF and Dynasore for 4 h. Vessels were stained with isolectin-B4 and filopodial extension along the length of the sprouting front was analyzed. In the lower panels are high magnification pictures of the same vascular beds shown in the upper panels. Green dots indicate filopodia and red lines show the length of vascular front. Scale bars represent 25 µm. b, Quantification of filopodial extension per vessel length revealed an average of 18.21±1.34 filopodia per 100 µm of vessel length in the untreated control which was increased up to 37.39±1.6334 filopodia per 100 µm of vessels after VEGF-stimulation. In the presence of Dynasore, VEGF-stimulation failed to promote filopodial extension. Only 20.99±2.69 filopodia per 100 µm vessel length (in average) was observed following the co-treatment of Dynasore and VEGF. Error bars represent SEM. Two-tailed t-test, n=10-12 retinas in each condition, *** P<0.001.

6.3.2. Activation of ephrinB2 signaling is sufficient to rescue compromised

filopodial extension caused by VEGF deprivation

In agreement with the evidences presented above that point out to a crucial requirement of ephrinB2 PDZ-interactions for VEGFR2 endocytosis and activation of its downstream signaling, stimulation of ephrinB2 reverse signaling in explanted retinas with EphB4-Fc resulted in a significant increase in the extension of filopodial structures at the leading front of vascular network (Fig. 6-24). The effect following EphB4-Fc stimulation was similar to the cellular response observed upon VEGF-stimulation (Fig. 6-23). To further verify the entwined relation between VEGFR2 and ephrinB2 signaling cascades and the dependency of the former on the latter to exert its function particularly in the endothelial tip cells, the ability of ephrinB2 to trigger activation of VEGFR2 signaling and consequently rescue the cellular outcome of inactivated VEGFR2 was examined.

Figure 6-24. Activation of ephrinB2 induces tip cell filopodial extension in explanted retinas. a, P4 retina explants were stimulated with pre-clustered EphB4-Fc or hFc for 4h. Vessels were stained with isolectin-B4 and filopodial extension at the sprouting front was analyzed. In the lower panel are high magnification pictures of the upper panel. Green dots indicate filopodia and red lines show the length of vascular front. Scale bars represent 25 µm. b, In the control condition in which only Fc was presented, retina explants exhibited 19.27±3.13 filopodia per 100 µm of vessel length (in average). Upon stimulation with EphB4-Fc, filopodia extension was increased up to an average of 29.83±1.81 per 100 µm of vessels. Error bars represent SEM. Two-tailed t-test, n=10-12 retinas in each conditions, ** P<0.001.

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Soluble VEGFR1 extracellular domain-Fc fusion protein (sFlt1) has previously been shown to efficiently inhibit VEGFR2 activation. The mode of action of this inhibitor is based on its higher VEGF-binding affinity as compared with VEGFR2, which therefore make it an efficient competitor to bind and seclude VEGF from VEGFR2. Intraoccular injection of sFlt1 has previously been reported to cause a complete retraction of endothelial tip cell filopodia at the sprouting front of retinal vasculature (Gerhardt et al., 2003). Similarly, deprivation of VEGF and thus inhibition of VEGFR2 activation by sFlt1 treatment abrogated extension of filopodia in explanted retina vessels (Fig. 6-25). Importantly, co-treatment of sFlt1 with EphB4-Fc to activate ephrinB2 reverse signaling was able to diminish the inhibitory effect of sFlt1 and rescue filopodial extension in the explants (Fig. 6-25), underscore the credible role of ephrinB2 as a prime regulator of VEGFR2 internalization and activation of its downstream signaling to control the active sprouting activity of endothelial tip cells.

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Figure 6-25 EphrinB2 activation rescues tip cell filopodial dynamics following VEGF sequestering. a, P4 retina explants were simultaneously treated with soluble Flt-1 (sFlt1) and hFc or sFlt1 and EphB4-Fc for 4h. Vessels were visualized by isolectin-B2 staining and filopodial extension at the sprouting front was analyzed. In the lower panels are high magnification pictures of the same vascular beds shown in upper panels. Green dots indicate filopodia and red lines show the length of vascular front. Scale bars represent 25 µm. b, Quantification of number of filopodial processes per vessel length is shown in average values. Retinal explants treated with sFlt1 and hFc showed 13.01±1.33 filopodia per 100 µm of vessels. In the presence of EphB4-Fc number of filopodia was increased to 25.46±1.92 per 100 µm of vessels. Error bars represent SEM. Two-tailed t-test, n=10-12 retinas in each conditions, *** P<0.0001.

6.4. EphrinB2 PDZ-signaling in endothelial cells influences tumor growth

In document Estudio sobre la disfuncionalidad familiar y su incidencia en el aprendizaje de los niños y niñas de primero y segundo año de educación primaria de la escuela Sagrado Corazón de Jesús de Tulcán (página 40-44)