Abstract-We studied the association between the production of reactive oxygen species, actin organization, and cellular motility. We have used an endothelial cell monolayer-wounding assay to demonstrate that the cells at the margin of the wound thus created produced significantly more free radicals than did cells in distant rows. The rate of incorporation of actin monomers into filaments was fastest at the wound margin, where heightened production of free radicals was detected. We have tested the effect of decreasing reactive oxygen species production on the migration of endothelial cells and on actin polymerization. The NADPH inhibitor diphenylene iodonium and the superoxide dismutase mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) virtually abolished cytochalasin D-inhibitable actin monomer incorporation at the fast-growing barbed ends of filaments. Moreover, endothelial cell migration within the wound was significantly retarded in the presence of both diphenylene iodonium and MnTMPyP. We conclude that migration of endothelial cells in response to loss of confluence includes the intracellular production of reactive oxygen species, which contribute to the actin cytoskeleton reorganization required for the migratory behavior of endothelial cells. (Circ Res. 2000;86:549-557.)

Despite its small size, profilin is an amazingly diverse and sophisticated protein whose precise role in cells continues to elude the understanding of researchers 15 years after its discovery. Its ubiquity, abundance and necessity for life in more evolved organisms certainly speaks for its extreme importance in cell function. So far, three ligands for profilin have been well-characterized in vitro: actin monomers, membrane polyphosphoinositides and poly-L-proline. In the years following its discovery, profilin's role in vivo progressed from that of a simple actin-binding protein which inhibits actin polymerization, to one which, as an important regulator of the cytoskeleton, can even promote actin polymerization under the appropriate circumstances. In addition, interactions with components of the phosphatidylinositol cycle and the RAS pathway in yeast implicate profilin as an important link through which the actin cytoskeleton is able to communicate with major signaling pathways.

Profilin is a small 12-15-kDa actin-binding protein, which in eukaryotic organisms is ubiquitous and necessary for normal cell growth and function. Although profilin's interactions with its three known ligands (actin monomers, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), and poly-L-proline (PLP)) have been well characterized in vitro, its precise role in cells remains largely unknown. By binding to clusters of PIP 2 , profilin is able to inhibit the hydrolysis of PIP 2 by phospholipase C␥1 (PLC␥1). This ability is the result of profilin's affinity for PIP 2 , but the specific residues of profilin's amino acid sequence involved in the binding of PIP 2 are not known. Using site-directed mutagenesis, we sought to localize regions of profilin important for this interaction by generating the following mutants of human profilin (named according to the wild-type amino acid altered, its position, and the amino acid substituted in its place): Y6F, D8A, L10R, K25Q, K53I, R74L, R88L, R88L/K90E, H119D, G121D, and K125Q. With the exception of L10R, all of the mutants were successfully expressed in Escherichia coli and purified by affinity chromatography on PLP-Sepharose. Only Y6F and K25Q demonstrated moderately less stringent binding to PLP, indicating that most of the mutations did not induce marked alterations of profilin's structure. When tested for their relative abilities to inhibit the hydrolysis of PIP 2 by PLC␥1, most of the mutants were indistinguishable from wild-type profilin. Exceptions included D8A, which demonstrated increased inhibition of PLC␥1, and R88L, which demonstrated decreased inhibition of PLC␥1. To assess the importance of the region surrounding residue 88 of human profilin, three synthetic decapeptides selected to correspond to non-overlapping stretches of the human profilin sequence were tested for their abilities to inhibit PLC␥1. We found that only the decapeptide that matched the peptide stretch centered around residue 88 was able to inhibit PLC␥1 activity substantially and was able to do so at nearly wild-type profilin levels. Taken together with the finding that mutating residue 88 resulted in decreased inhibition of PLC␥1 activity, these data provide strong evidence that this region of human profilin represents an important binding site for PIP 2 .Since the molecule's discovery 20 years ago (1), profilin's interactions with its three ligands (actin monomers, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), 1 and PLP) have been well established through in vitro studies (for review, see Ref.2). For example, by binding to actin monomers in a 1:1 complex, profilin decreases the critical concentration of monomeric actin in the presence of thymosin ␤4 (3), inhibits the spontaneous nucleation of actin filaments (4), and catalyzes the exchange of adenosine nucleotides bound to actin monomers (5). By binding to PIP 2 and to a lesser degree its precursor PIP (6), profilin prevents PLC␥1 from hydrolyzing PIP 2 (7). However, when PLC␥1 is phosphorylated on specific tyrosine residues, such as it occurs...

Inflammation and thrombosis are increasingly recognized as interrelated biologic processes. Endothelial cell expression of thrombomodulin (TM), a key component of the anticoagulant protein C pathway, is potently inhibited by inflammatory cytokines. Because the mechanism underlying this effect is largely unknown, we investigated a potential role for the inflammatory transcription factor nuclear factor-kappa B (NF-B). Blocking NF-B activation effectively prevented cytokineinduced down-regulation of TM, both in vitro and in a mouse model of tumor necrosis factor-␣ (TNF-␣)-mediated lung injury. Although the TM promoter lacks a classic NF-B consensus site, it does contain tandem Ets transcription factor binding sites previously shown to be important for both constitutive TM gene expression and cytokine-induced repression. Using electrophoretic mobility shift assay and chromatin immunoprecipitation, we found that multiple Ets species bind to the TNF-␣ response element within the TM promoter. Although cytokine exposure did not alter Ets factor binding, it did reduce binding of p300, a coactivator required by Ets for full transcriptional activity. Overexpression of p300 also prevented TM repression by cytokines. We conclude that NF-B is a critical mediator of TM repression by cytokines. Further evidence suggests a mechanism involving competition by NF-B for limited pools of the transcriptional coactivator p300 necessary for TM gene expression. IntroductionSystemic inflammatory conditions, such as bacterial sepsis and vasculitis, are frequently complicated by the development of pathologic thrombosis. Inflammatory processes can shift the hemostatic balance toward thrombus formation not only by stimulating tissue factor-dependent coagulation but also by inhibiting anticoagulant and fibrinolytic pathways. 1 Thrombomodulin (TM), a 100-kDa transmembrane protein expressed in abundance by vascular endothelial cells, is a critical component of the anticoagulant protein C pathway. 2 TM binds thrombin and alters its active site specificity to facilitate proteolytic activation of circulating protein C. In concert with its cofactor protein S, activated protein C (APC) enzymatically degrades factors Va and VIIIa of the clotting cascade, thereby suppressing further thrombin generation. Growing clinical evidence suggests that dysfunction of the TM-APC pathway caused by inflammation compromises vascular thromboresistance. For example, TM expression is markedly reduced in skin biopsy specimens taken from patients with severe bacterial sepsis complicated by microvascular thrombosis and associated with abnormally low circulating levels of APC. 3 Impaired TM activity provides the rationale for administering recombinant APC to patients with severe sepsis, a therapy that has been shown to reduce the risk of death in such patients by nearly 20%. 4,5 In vitro studies have demonstrated that endothelial TM expression is potently inhibited by inflammatory mediators such as bacterial endotoxin and several inflammatory cytokines. [6][7][8] Of these...

Abstract-Thrombosis is the major cause of early vein graft failure. Our aim was to determine whether alterations in the expression of the anticoagulant proteins, thrombomodulin (TM) and the endothelial cell protein C receptor (EPCR), impair endothelial thromboresistance that may contribute to vein graft failure. Immunohistochemical staining of autologous rabbit vein graft sections revealed that the expression of TM, but not EPCR, was reduced significantly early after graft implantation. Western blot analysis revealed that TM expression was reduced by Ͼ95% during the first 2 weeks after implantation, with gradual but incomplete recovery by 42 days. This resulted in up to a 95% reduction in the capacity of the grafts to activate protein C and was associated with an increase in bound thrombin activity, which peaked on day 7 at 28.7Ϯ3.8 mU/cm 2 and remained elevated for more than 14 days. Restoration of TM expression using adenovirus vector-mediated gene transfer significantly enhanced the capacity of grafts to activate protein C and reduced bound thrombin activity on day 7 to levels comparable to that of normal veins (5.7Ϯ0.4 versus 5.2Ϯ1.1 mU/cm 2 , respectively, Pϭ0.74). Surprisingly, neointima formation was not affected by this inhibition of local thrombin activity. These data suggest that the early loss of TM expression significantly impairs vein graft thromboresistance and results in enhanced local thrombin generation. Although enhanced local thrombin generation may predispose to early vein graft failure due to thrombosis, it does not seem to contribute significantly to late vein graft failure due to neointimal hyperplasia.

We conclude that an important role for profilin in mammalian cells may be its contribution to the formation of focal contacts, particularly those involving the fibronectin receptor.

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Objective: The aim is to evaluate the feasibility of using mechanical circulatory support, specifically the Impella device, in spontaneous coronary artery dissection (SCAD) patients with cardiogenic shock. Background: The therapeutic options for managing SCAD complicated by cardiogenic shock are limited. Risky revascularization procedures are often necessary. Methods: This was a multicenter case series in the United States. Approximately 20 cases of Impella implantation in patients with SCAD are known. The implanting physician for each of these cases was contacted and de-identified records were requested. The records were analyzed for Impella indications, outcomes, and complications. Results: Records from four cases were received. All patients survived to hospital discharge and no major complications were observed. In two cases, cardiogenic shock developed in the absence of ongoing ischemia, suggesting a Takotsubo-like cardiomyopathy. In these cases, the Impella provided hemodynamic support until the patient's cardiac function recovered. Conclusion: Although a small case series, given the scarcity of SCAD cases complicated by cardiogenic shock and the limited therapies available to treat these patients, these data are of clinical value in highlighting the feasibility of Impella use in SCAD. The Impella can be valuable for procedural support and in cardiogenic shock, especially in cases without evidence of ongoing ischemia.