PKCδ Regulates Force Signaling during VEGF/CXCL4 Induced Dissociation of Endothelial Tubes

Abstract

Wound healing requires the vasculature to re-establish itself from the severed ends; endothelial cells within capillaries must detach from neighboring cells before they can migrate into the nascent wound bed to initiate angiogenesis. The dissociation of these endothelial capillaries is driven partially by platelets' release of growth factors and cytokines, particularly the chemokine CXCL4/platelet factor-4 (PF4) that increases cell-cell de-adherence. As this retraction is partly mediated by increased transcellular contractility, the protein kinase c-δ/myosin light chain-2 (PKCδ/MLC-2) signaling axis becomes a candidate mechanism to drive endothelial dissociation. We hypothesize that PKCδ activation induces contractility through MLC-2 to promote dissociation of endothelial cords after exposure to platelet-released CXCL4 and VEGF. To investigate this mechanism of contractility, endothelial cells were allowed to form cords following CXCL4 addition to perpetuate cord dissociation. In this study, CXCL4-induced dissociation was reduced by a VEGFR inhibitor (sunitinib malate) and/or PKCδ inhibition. During combined CXCL4+VEGF treatment, increased contractility mediated by MLC-2 that is dependent on PKCδ regulation. As cellular force is transmitted to focal adhesions, zyxin, a focal adhesion protein that is mechano-responsive, was upregulated after PKCδ inhibition. This study suggests that growth factor regulation of PKCδ may be involved in CXCL4-mediated dissociation of endothelial cords.

The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control

“…The finding that the negative correlation observed between fibrin matrix mass and releasate-induced tube formation was, at least partly, mediated through PF4 strongly suggests that the matrix can exercise biochemical control on angiogenesis by balancing the relative concentrations of pro- and anti-angiogenic factors. Importantly, while our data confirm that fibrin matrix releasates can support EC sprouting, they also point to a more complex dependence of this process on PF4, since presence of the factor at low concentrations might be required for EC detachment from pre-existing vasculature [ 32 ], while higher concentrations inhibit cell proliferation and tubulogenesis [ 33 – 36 ]. We do note that in HCM experiments the fibrinogen concentration used to prepare fibrin matrices was deliberately higher than that normally encountered in plasma, due to the low response threshold of in vitro angiogenesis assays, which made observation of the postulated inhibition more challenging.…”

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

“…More studies are still necessary to address the role played by platelet exhaustion in severity of dengue. Because of its roles in endothelial dissociation and angiogenesis [ 35 ], PF4 is potentially involved in vasculopathy of dengue syndromes.…”

Retinoblastoma Binding Protein 2 (RBP2) Promotes HIF-1α–VEGF-Induced Angiogenesis of Non-Small Cell Lung Cancer via the Akt Pathway

“…HUVECs (1×10 4 cells) were seeded in wells with different conditioned media from RBP2-siRNA1 H1975 cells, RBP2-siRNA2 H1975 cells and control-siRNA H1975 cells. A VEGFR inhibitor (sunitinib malate, 2.5 µM) [32] was added to the conditioned medium of the control siRNA H1975 cells and recombinant human VEGF-165 (rhVEGF165, Millipore, Billerica, MA, USA, 2 ng/ml) was added to the conditioned medium of the RBP2-siRNA2 H1975 cells. The 96-well plates were incubated for 6 h, and tube formation was then photographed under an inverted microscope.…”