Understanding the regulation of endothelial cell migration in response to chemokines has proven to be a daunting task that requires the investigation of a manifold of factors known to affect chemotaxis (e.g., chemokine sensing by receptor-mediated signaling, cellular locomotion by actin-based molecular motors, and even the cell's energy status). Previous studies from our laboratory on CD34+
EPCs showed that diabetic EPCs have reduced intracellular NO concentration as well as a concomitantly reduced migratory capability. We found that, when pretreated with an NO donor, cell migration can be restored (13
), and we further demonstrated that this exogenous NO exposure resulted in enhanced diabetic cell migration, with attendant changes in phosphorylation of the actin cytoskeletal protein VASP (13
Given the robust nature of the vasodilator gases CO and NO in modulating vascular function, we focused on the following questions: Can CO suffice in place of NO in promoting vascular cell migration? If so, do NO and CO exert their signaling effects on the same downstream phosphorylation target(s)? In this study, we demonstrate that CO can regulate VASP phosphorylation and in turn alter cell migration. We also directly compare the CO effects to those of NO in EPCs, platelets, and microvascular endothelial cells. Our studies demonstrate that both vasoactive gases promote cytoskeletal changes through site- and cell type–specific VASP phosphorylation; however, these responses to NO and CO are blunted in diabetes and may be responsible for reduced vascular repair and tissue perfusion (24
The actoclampins are the force-generating motors responsible for actin-based cell crawling and vesicle motility (14
). Each of these membrane surface–bound molecular motors consists of an actin filament plus-end tracking protein called a clampin (e.g., VASP, N-WASP [neural Wiskott–Aldrich syndrome protein], formins, etc.) and its actin filament partner. The energy for force production appears to be derived from nucleotide hydrolysis at the filament's penultimate actin-ATP subunits, thereby promoting clampin release, translocation, and rebinding to terminal actin-ATP subunits. Before the active motor complex is assembled, clampins must be recruited to the membrane surface, where they dock at motility sites at the tips of filopodia and lamellipodia as well as in the focal adherens complex. Rottner et al. (23
), for example, showed that VASP not only colocalizes to adhesion sites with the adaptor proteins vinculin and zyxin, but is also recruited to the tips of lamellipodia in amounts that are directly proportional to the rate of lamellipodial protrusion. Tokuo and Ikebe (26
) further showed that myosin X specifically transports VASP and other members of the Ena/VASP clampin protein family to the leading edge, where VASP then binds to a membrane docking protein such as vinculin, zyxin, or migfilin (27
). Only then can the actoclampin motor assemble and generate the forces needed for cell migration. When viewed from this perspective, VASP phosphorylation may affect the recruitment by myosin X, VASP docking with membrane components, and/or VASP-mediated formation of an active motor that can propel cell crawling. Moreover, as cells stop moving, VASP is known to redistribute to other intracellular sites. Benz et al. (29
), for example, showed that VASP interacts with αII-spectrin in endothelial cells and that PKA-mediated VASP phosphorylation at Ser-157 inhibits this binding interaction. They also showed that VASP is dephosphorylated upon formation of cell-cell contacts and that, in confluent cells, αII-spectrin colocalizes with nonphosphorylated VASP at cell-cell junctions (29
). The exact details of how VASP phosphorylation at Ser-157 or Ser-239 controls one or more of these steps remain to be worked out. Even so, the observation that different cell types contain various clampins and their respective membrane-docking proteins is likely to explain why VASP phosphorylation can either stimulate or suppress cell crawling and actin-based cell shape changes in a manner depending on cell type and/or culture conditions.
VASP is also the most abundant platelet and endothelial protein phosphorylated by PKG in NO signaling pathways, and, as noted above, this cytoskeletal adaptor protein is also a PKA substrate. Three serine/threonine phosphorylation sites within VASP play roles resulting in the inhibition of platelet aggregation and focal adhesion assembly (30
). In this study, we demonstrated that CO can also regulate VASP phosphorylation, and we directly compare CO and NO effects in EPCs, platelets, and microvascular endothelial cells. We found that CO pretreatment can similarly stimulate EPC migration () and phosphorylation of VASP's Ser-157, whereas NO exposure results in Ser-239 phosphorylation.
While we did not perform direct measurements of CO levels in our experiments, we did use CO concentrations that are achievable in vivo in physiological conditions and previously used by investigators (32
). Endogenous CO has been reported to be generated in many cell types, and the amount of CO released via the heme oxygenase reaction can reach up to 12 ml/day (~16 μmol · l−1
). Previous studies have reported that tissues can produce 0.1–100 μmol/l CO in vivo from the HO reaction (33
); it is therefore quite reasonable to believe that sufficient levels of HO-1 are present in cells to provide sufficient levels of CO, explaining our observed changes in VASP phosphorylation.
Moreover, in response to incubation of microvascular endothelial cells with NO or CO donors, VASP was readily redistributed to the peripheral membrane and filipodia (). Thus, with regard to VASP localization, our data suggest that CO and NO may both have critical roles in vascular cell dynamics, and CO may have important contributions at low NO bioavailability, a condition already known to occur in diabetes. Our studies clearly indicate that both vasoactive gases promote cytoskeletal changes through site- and cell type-specific VASP phosphorylation and that responses to NO and CO are blunted in diabetes. While the migratory deficiency seen in diabetic EPCs could be overcome by exogenous NO (13
), exogenous NO did not result in phosphorylation and redistribution of VASP in mature endothelial cells cultured in high glucose conditions. This defect can be viewed as “diabetes-induced NO resistance.”
What also becomes apparent from the present study is that exposure of cells to NO or CO can greatly alter VASP recruitment to the leading edge with consequential effects on cell motility. Perhaps even more significant, in the context of diabetes, is that culturing endothelial cells at high glucose conditions that mimic diabetes results in motility defects that can be traced, at least in part, to altered VASP phosphorylation. We therefore postulate that these defects contribute to reduced vascular repair and tissue perfusion.
Both heme oxygenases generate CO, but they do so with very different kinetics (34
); HO-1 is induced by oxidants such as hydrogen peroxide, UV radiation, and proinflammatory cytokines and by growth factors, hemodynamic or shear stress, heat shock, and even by NO (3
). Endothelial cells derived from either the micro- or macrovasculature (36
) responded equally to CO and NO, reflecting the key roles of both HO-1 and endothelial NO synthase throughout in the entire vasculature. In contrast, platelets do not respond to CO, perhaps reflective of the low levels of heme oxygenase in adult platelets (37
). By altering HO-1 and NOS gene expression, hypoxia potentially modulates the availability of these gaseous second messengers. Thus, fluxes in the CO and NO generation during hypoxic stress are likely to have dramatic consequences on the regulation of vascular functions such as dilation, expression of vasomodulators, inhibition of platelet aggregation, and smooth muscle cell proliferation (38
). Although the findings in this report support a role for CO sufficing for NO in promoting vascular cell migration, the signaling action of CO results in the phosphorylation of different VASP sites than NO. Furthermore, we demonstrate that, while both vasoactive gases promote cytoskeletal changes through site- and cell type–specific VASP phosphorylation, these responses are blunted in diabetes and may be responsible for the altered vascular repair and tissue perfusion associated with diabetic vascular complications.