The mechanism by which angiogenic endothelial cells break the physical barrier of the vascular basement membrane and consequently sprout to form new vessels in mature tissues is unclear. Here, we show that the angiogenic endothelium is characterized by the presence of functional podosome rosettes. These extracellular-matrix-degrading and adhesive structures are precursors of de novo branching points and represent a key feature in the formation of new blood vessels. VEGF-A stimulation induces the formation of endothelial podosome rosettes by upregulating integrin α6β1. In contrast, the binding of α6β1 integrin to the laminin of the vascular basement membrane impairs the formation of podosome rosettes by restricting α6β1 integrin to focal adhesions and hampering its translocation to podosomes. Using an ex vivo sprouting angiogenesis assay, transgenic and knockout mouse models and human tumour sample analysis, we provide evidence that endothelial podosome rosettes control blood vessel branching and are critical regulators of pathological angiogenesis.

Thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP-1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor-A (VEGF-A). To demonstrate that the TSP-1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36-deficient endothelial cells. Both TSP-1 and the agonist anti-CD36 mAb SMO, which mimics TSP-1 activity, reduced the VEGF-A165-induced migration and sprouting of CD36-ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C-terminal cytoplasmic tail by site-directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP-1. In contrast, point mutation of C466 did not alter TSP-1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP-1 down-modulates the VEGF receptor-2 (VEGFR-2) and p38 mitogen-associated protein kinase phosphorylation induced by VEGF-A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C-terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP-1 and extend our knowledge of regulation of VEGFR-2-mediated biological activities on ECs.

Somatic activating mutations within the PIK3CA gene have been recently detected in sporadic lymphatic and venous malformations, and in vascular malformations (VM) associated to overgrowth syndromes, such as CLOVES and Klippel–Trenaunay syndrome. Although VM are often limited to specific tissue areas and can be well treated, in extended or recurrent lesions novel therapeutic approaches are needed. We generated a mouse model of VM by local expression of PIK3CA-activating mutation in endothelial cells. PIK3CA-driven lesions are characterized by large areas of hemorrhage, hyperplastic vessels, infiltrates of inflammatory cells, and elevated endothelial cell density. Such vascular lesions are ameliorated by administration of dual PI3K/mTOR inhibitor, BEZ235, and mTOR inhibitor, Everolimus. Unexpectedly, the expression of PIK3CA-activating mutations in human endothelial cells results in both increased proliferation rates and senescence. Moreover, active forms of PIK3CA strongly promote the angiogenic sprouting. Treatment with PI3K/mTOR inhibitors restores normal endothelial cell proliferation rate and reduces the amount of senescent cells, whereas treatment with Akt inhibitor is less effective. Our findings reveal that PIK3CA mutations have a key role in the pathogenesis of VM and PIK3CA-driven experimental lesions can be effectively treated by PI3K/mTOR inhibitors.

The serine/threonine protein kinase phosphoinositide-dependent kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases, including PKB/Akt. We now present evidence showing that PDK1 is essential for the motility of vascular endothelial cells (ECs) and that it is involved in the regulation of their chemotaxis. ECs differentiated from mouse embryonic stem cells lacking PDK1 completely lost their ability to migrate in vitro in response to vascular endothelial growth factor-A (VEGF-A). In addition, PDK1−/− embryoid bodies exhibit evident developmental and vascular defects that can be attributed to a reduced cell migration. Moreover, the overexpression of PDK1 increased the EC migration induced by VEGF-A. We propose a model of spatial distribution of PDK1 and Akt in which the synthesis of phosphatidylinositol 3,4,5 triphosphate at plasma membrane by activation of phosphoinositide 3-kinase recruits both proteins at the leading edge of the polarized ECs and promotes cell chemotaxis. These findings establish a mechanism for the spatial localization of PDK1 and its substrate Akt to regulate directional migration.

Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels. Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85β). Down-regulation of miR-126 and over-expression of p85β in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.

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MRCKα is activated by PDK1 through a PIP3-dependent, kinase-independent mechanism that drives the relocation of both proteins to lamellipodia and regulates lamellipodial retraction and directional migration.

Hydrogels are promising materials in regenerative medicine applications due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. Hydrogel preparation process was optimized to allow VEGF functional incorporation, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay whereas released VEGF bioactivity was validated in Human Umbilical Vein Endothelial Cells (HUVEC) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF releasing hydrogels displayed increased neurite outgrowth proving confirmation that released VEGF maintained its effect, as also confirmed in tubulogenesis assay. In conclusion, a gelatin based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering.