Cardiovascular Functions of Ena/VASP Proteins: Past, Present and Beyond.

Actin binding proteins are of crucial importance for the spatiotemporal regulation of actin cytoskeletal dynamics, thereby mediating a tremendous range of cellular processes. Since their initial discovery more than 30 years ago, the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family has evolved as one of the most fascinating and versatile family of actin regulating proteins. The proteins directly enhance actin filament assembly, but they also organize higher order actin networks and link kinase signaling pathways to actin filament assembly. Thereby, Ena/VASP proteins regulate dynamic cellular processes ranging from membrane protrusions and trafficking, and cell-cell and cell-matrix adhesions, to the generation of mechanical tension and contractile force. Important insights have been gained into the physiological functions of Ena/VASP proteins in platelets, leukocytes, endothelial cells, smooth muscle cells and cardiomyocytes. In this review, we summarize the unique and redundant functions of Ena/VASP proteins in cardiovascular cells and discuss the underlying molecular mechanisms.

EVL regulates VEGF receptor-2 internalization and signaling in developmental angiogenesis.

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.

AKAP12 deficiency impairs VEGF-induced endothelial cell migration and sprouting.

AIM: Protein kinase (PK) A anchoring protein (AKAP) 12 is a scaffolding protein that anchors PKA to compartmentalize cyclic AMP signalling. This study assessed the consequences of the downregulation or deletion of AKAP12 on endothelial cell migration and angiogenesis. METHODS: The consequences of siRNA-mediated downregulation AKAP12 were studied in primary cultures of human endothelial cells as well as in endothelial cells and retinas from wild-type versus AKAP12-/- mice. Molecular interactions were investigated using a combination of immunoprecipitation and mass spectrometry. RESULTS: AKAP12 was expressed at low levels in confluent endothelial cells but its expression was increased in actively migrating cells, where it localized to lamellipodia. In the postnatal retina, AKAP12 was expressed by actively migrating tip cells at the angiogenic front, and its deletion resulted in defective extension of the vascular plexus. In migrating endothelial cells, AKAP12 was co-localized with the PKA type II-α regulatory subunit as well as multiple key regulators of actin dynamics and actin filament-based movement; including components of the Arp2/3 complex and the vasodilator-stimulated phosphoprotein (VASP). Fitting with the evidence of a physical VASP/AKAP12/PKA complex, it was possible to demonstrate that the VEGF-stimulated and PKA-dependent phosphorylation of VASP was dependent on AKAP12. Indeed, AKAP12 colocalized with phospho-Ser157 VASP at the leading edge of migrating endothelial cells. CONCLUSION: The results suggest that compartmentalized AKAP12/PKA signalling mediates VASP phosphorylation at the leading edge of migrating endothelial cells to translate angiogenic stimuli into altered actin dynamics and cell movement.

VASP regulates leukocyte infiltration, polarization, and vascular repair after ischemia.

In ischemic vascular diseases, leukocyte recruitment and polarization are crucial for revascularization and tissue repair. We investigated the role of vasodilator-stimulated phosphoprotein (VASP) in vascular repair. After hindlimb ischemia induction, blood flow recovery, angiogenesis, arteriogenesis, and leukocyte infiltration into ischemic muscles in VASP-/- mice were accelerated. VASP deficiency also elevated the polarization of the macrophages through increased signal transducer and activator of transcription (STAT) signaling, which augmented the release of chemokines, cytokines, and growth factors to promote leukocyte recruitment and vascular repair. Importantly, VASP deletion in bone marrow-derived cells was sufficient to mimic the increased blood flow recovery of global VASP-/- mice. In chemotaxis experiments, VASP-/- neutrophils/monocytes were significantly more responsive to M1-related chemokines than wild-type controls. Mechanistically, VASP formed complexes with the chemokine receptor CCR2 and ß-arrestin-2, and CCR2 receptor internalization was significantly reduced in VASP-/- leukocytes. Our data indicate that VASP is a major regulator of leukocyte recruitment and polarization in postischemic revascularization and support a novel role of VASP in chemokine receptor trafficking.

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets.

BACKGROUND: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbß3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo. METHODS: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins. RESULTS: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl. CONCLUSIONS: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Exposure to Carbon Ions Triggers Proinflammatory Signals and Changes in Homeostasis and Epidermal Tissue Organization to a Similar Extent as Photons.

The increasing application of charged particles in radiotherapy requires a deeper understanding of early and late side effects occurring in skin, which is exposed in all radiation treatments. We measured cellular and molecular changes related to the early inflammatory response of human skin irradiated with carbon ions, in particular cell death induction and changes in differentiation and proliferation of epidermal cells during the first days after exposure. Model systems for human skin from healthy donors of different complexity, i.e., keratinocytes, coculture of skin cells, 3D skin equivalents, and skin explants, were used to investigate the alterations induced by carbon ions (spread-out Bragg peak, dose-averaged LET 100 keV/µm) in comparison to X-ray and UV-B exposure. After exposure to ionizing radiation, in none of the model systems, apoptosis/necrosis was observed. Carbon ions triggered inflammatory signaling and accelerated differentiation of keratinocytes to a similar extent as X-rays at the same doses. High doses of carbon ions were more effective than X-rays in reducing proliferation and inducing abnormal differentiation. In contrast, changes identified following low-dose exposure (≤0.5 Gy) were induced more effectively after X-ray exposure, i.e., enhanced proliferation and change in the polarity of basal cells.