Biology subjects

Benz, P. M.

Publications and source records attributed to Benz, P. M..

5 recordsLinked to original sources

bioRxiv2023

Mena and VASP are required for vascular smooth muscle relaxation

Enabled/vasodilator stimulated phosphoproteins (Ena/VASP) proteins are important regulators of the cytoskeleton, linking kinase signaling pathways to actin assembly. In mammals, the Ena/VASP family of proteins consists of mammalian enabled (Mena), VASP, and Ena-VASP-like protein (EVL). The proteins are well known targets of cAMP- and cGMP-dependent protein kinases, PKA and PKG, respectively. Given the importance of cyclic nucleotide signaling in mediating vasodilation, we investigated the role of Ena/VASP protein in vascular smooth muscle relaxation. Whereas VASP and Mena were strongly expressed in vascular smooth muscle cells, EVL was undetectable in the arterial wall and EVL-deficiency had no impact on agonist-induced smooth muscle relaxation. VASP deletion impaired the acetylcholine (ACh)- and nitric oxide (NO)-induced relaxation murine mesenteric arteries ex vivo. Similarly, the ACh-induced and NO-dependent relaxation of aorta from 7-month-old but not 3- month-old VASP-/- mice was also reduced. Aortas from animals lacking VASP and expressing only minimal amounts of Mena displayed significantly impaired relaxations in response to NO, cAMP and cGMP stimulation. These results suggest that Mena and VASP play an important role in agonist induced smooth muscle relaxation and functionally compensate for each other.

physiology↗

bioRxiv2023

Ena/VASP proteins mediate endothelial cell repulsion from ephrin ligands

The interaction of Eph receptor tyrosine kinases with their transmembrane ligands; the ephrins, is important for the regulation of cell-cell communication. Ephrin-Eph signaling is probably best known for the discrimination of arterial and venous territories by repulsion of venous endothelial cells away from those with an arterial fate. Ultimately, cell repulsion is mediated by initiating the collapse of the actin cytoskeleton in membrane protrusions. Here, we investigated the role of the Ena/VASP family of actin binding proteins in endothelial cell repulsion initiated by ephrin ligands. Human endothelial cells dynamically extended sheet-like lamellipodia over ephrin-B2 coated surfaces. While lamellipodia of control siRNA transfected cells rapidly collapsed, resulting in a pronounced cell repulsion from the ephrin-B2 surfaces, the knockdown of Ena/VASP proteins impaired the cytoskeletal collapse of membrane protrusions and the cells no longer avoided the repulsive surfaces. Mechanistically, ephrin-B2 stimulation elicited the EphB-mediated tyrosine phosphorylation of VASP, which abrogated its interaction with the focal adhesion protein Zyxin. Nck2 was identified as a novel VASP binding protein, which only interacted with the tyrosine phosphorylated VASP protein. Nck links Eph-receptors to the actin cytoskeleton. Therefore, we hypothesize that Nck-Ena/VASP complex formation is required for actin reorganization and/or Eph receptor internalization downstream of ephrin-Eph interaction in endothelial cells, with implications for endothelial navigation and pathfinding.

cell biology↗

bioRxiv2023

Impaired H19 lncRNA expression contributes to the compromised developmental angiogenesis in EVL-deficient mice

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. Endothelial-specific deletion of EVL, a member of the mammalian Ena/VASP protein family, reduced the expression of the tip cell marker protein endothelial cell specific molecule-1 (Esm1) and compromised the radial sprouting of the vascular plexus in the postnatal mouse retina. The latter effects could at least partly be attributed to reduced VEGF receptor 2 (VEGFR2) internalization and signaling but the underlying mechanisms(s) are not fully understood. In the present study, we revealed that the expression of the long non-coding RNA H19 was significantly reduced in endothelial cells from postnatal EVL-/- mice and in siRNA-transfected human endothelial cells under hypoxic conditions. H19 was recently shown to promote VEGF expression and bioavailability via Esm1 and hypoxia inducible factor 1 (HIF-1). Similar to EVL-/- mice, the radial outgrowth of the vascular plexus was significantly delayed in the postnatal retina of H19-/- mice. In summary, our data suggests that loss of EVL not only impairs VEGFR2 internalization and downstream signaling, but also impairs VEGF expression and bioavailability in the hypoxic retina via downregulation of lncRNA H19.

cell biology↗

bioRxiv2023

Impaired AMPK activity contributes to the inflammatory phenotype and the reduced phagocytosis capacity of VASP-deficient macrophages

Macrophage polarization plays an important role in tissue regeneration. Numerous factors and signaling molecules affect polarization processes. Here we investigated the consequences of the genetic deletion of vasodilator-stimulated phosphoprotein (VASP), which increases macrophage M1 polarization through augmented signal transducer and activator of transcription 1 (STAT1) signaling, and AMP-activated protein kinase (AMPK), which attenuates inflammation by inhibiting STAT1 expression and signaling. While a basal activity of AMPK (phosphorylation on Thr172) was detected in macrophages from wild-type mice, AMPK phosphorylation was significantly reduced in VASP-deficient M1 macrophages in vitro and the expression of the pro-inflammatory cytokines TNF and IL-1{beta} was increased in these cells. Consistent with the role of AMPK in macrophage phagocytosis, VASP-/- macrophage phagocytosis was also significantly impaired. Interestingly, impaired phagocytosis could be rescued by exogenous activation of AMPK. Mechanistically, we found that VASP binds directly to protein phosphatase 1 regulatory subunit 6 (PP1-R6) and we hypothesize that VASP-binding to PP1- R6/PP1 limits the PP1-dependent de-phosphorylation of AMPK in wild-type cells. Conversely, AMPK dephosphorylation by the PP1-R6/PP1 complex is enhanced in the absence of VASP. In summary, we have identified a link between VASP and AMP-activated protein kinase (AMPK) activity, which may contribute to the pro-inflammatory phenotype of VASP-deficient macrophages.

cell biology↗

bioRxiv2023

The vasodilator-stimulated phosphoprotein (VASP) supports the conduction of vasodilator signals and NO-induced arteriolar dilations in murine arterioles in vivo

VASP is a member of the Enabled/VASP protein family that is involved in cortical actin dynamics and may also contribute to the formation of gap junctions. In vessels, gap junctional coupling allows the transfer of signals along the vessel wall and coordinates vascular behavior. Moreover, VASP is reportedly a mediator of NO-induced inhibition of platelet aggregation. Therefore, we hypothesized that VASP exerts also important physiologic functions in arterioles. We examined the spread of vasodilations enabled by gap junctional coupling in endothelial cells as well as NO-induced arteriolar dilations in VASP-deficient mice by intravital microscopy of the microcirculation in a skeletal muscle in anesthetized mice. Conducted dilations were initiated by brief, locally confined stimulation of the arterioles with acetylcholine. The maximal diameters of the arterioles under study ranged from 30 to 40 m. Brief stimulation with acetylcholine induced a short dilation at the local site that was also observed at remote, upstream sites without an attenuation of the amplitude up to a distance of 1.2 mm in control animals (wild-type). In contrast, remote dilations were reduced in VASP-deficient mice despite a similar local dilation indicating an impairment of conducted dilations. Superfusion of NOdonors induced a concentration-dependent dilation in wild-type mice. However, these dilations were slightly reduced in VASP-deficient animals. In contrast, dilations induced by the endothelial stimulator acetylcholine were fully preserved in VASP-deficient mice. In summary, this study suggests that VASP exerts critical functions in arteriolar diameter control. It is crucial for the conduction of dilator signals along the endothelial cell layer. The impairment possibly reflects a perturbed formation of gap junctions in the endothelial cell membrane. VASP also participates in the full dilatory potential of NOdonors although the effect of its deficiency is only subtle. In contrast, VASP is not required for dilations initiated by endothelial stimulation which are mediated in the murine microcirculation by an EDH-mechanism.

physiology↗