Anthony W. Ashton

Inhibition of Endothelial Cell Migration, Intercellular Communication, and Vascular Tube Formation by Thromboxane A2

The eicosanoid thromboxane A2 (TXA2) is released by activated platelets, monocytes, and the vessel wall and interacts with high affinity receptors expressed in several tissues including endothelium. Whether TXA2 might alter endothelial migration and tube formation, two determinants of angiogenesis, is unknown. Thus, we investigated the effect of the TXA2 mimetic [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5′-heptenoic acid (IBOP) on human endothelial cell (HEC) migration and angiogenesis in vitro. IBOP stimulation inhibited HEC migration by 50% and in vitro capillary formation by 75%. These effects of IBOP were time- and concentration-dependent with an IC50 of 25 nm. IBOP did not affect integrin expression or cytoskeletal morphology of HEC. Since gap junction-mediated intercellular communication increases in migrating HEC, we determined whether IBOP might inhibit coupling or connexin expression in HEC. IBOP reduced the passage of microinjected dyes between HEC by 50%, and the effects of IBOP on migration and tube formation were mimicked by the gap junction inhibitor 18β-glycyrrhetinic acid (1 μm) with a similar time course and efficacy. IBOP (24 h) did not affect the expression or phosphorylation of connexin 43 in whole HEC lysates. Immunohistologic examination of HEC suggested that IBOP may impair functional coupling by altering the cellular distribution of gap junctions, leading to increased connexin 43 internalization. Thus, this finding that TXA2 mimetics can prevent HEC migration and tube formation, possibly by impairing intercellular communication, suggests that antagonizing TXA2 signaling might enhance vascularization of ischemic tissue.

Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug

Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled AnxA5 and also imaged with atomic force microscopy. Immunoglobulin G levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells and a syncytialized trophoblast cell line. AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by atomic force microscopy. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of human umbilical vein endothelial cells and syncytialized trophoblast cell lines, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell-surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs and cultured cells, and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.

Regulation of Connexin43 Protein Complexes by Intracellular Acidification

Abstract— Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K+ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury.

Caveolin-1 Knockout Mice Show an Impaired Angiogenic Response to Exogenous Stimuli

Recent studies have shown that caveolin-1 (Cav-1) plays an important role as a regulator of angiogenesis in vitro. Here, we use Cav-1 knockout (KO) mice as a model system to examine the in vivo relevance of these findings. A primary mediator of angiogenesis is basic fibroblast growth factor (bFGF). Thus, we studied bFGF-induced angiogenesis in Cav-1 KO mice using a reconstituted basement membrane system, ie, Matrigel plugs, supplemented with bFGF. In Cav-1 KO mice, implanted Matrigel plugs showed a dramatic reduction in both vessel infiltration and density, as compared with identical plugs implanted in wild-type control mice. We also examined the necessity of Cav-1 to support the angiogenic response of an exogenous tumor by subcutaneously injecting Cav-1 KO mice with the melanoma cell line, B16-F10. We show that tumor weight, volume, and vessel density are all reduced in Cav-1 KO mice, consistent with diminished angiogenesis. Ultrastructural analysis of newly formed capillaries within the exogenous tumors reveals a lack of endothelial caveolae and incomplete capillary formation in Cav-1 KO mice. These results provide novel evidence that Cav-1 and caveolae play an important positive role in the process of pathological angiogenesis in vivo.

Endothelial Cell Activation in Patients With Decompensated Heart Failure

Background—Vascular endothelial functions, other than nitric oxide (NO)–mediated control of vasomotor tone, are poorly characterized in patients with chronic heart failure (CHF). Veins and arteries are exposed to the same circulating proinflammatory mediators in patients with CHF. The present study tested whether endothelial cell activation occurs in veins of patients with decompensated CHF and whether activation, if present, subsides with return to a clinically compensated state. Methods and Results—Fifteen patients with decompensated CHF requiring transient inotropic support and 6 age-matched, healthy controls were studied. Endothelial cells and blood were collected from a forearm vein, and brachial artery flow–mediated dilation (FMD) was measured before and 24 hours after discontinuation of short-term inotropic therapy, when patients had returned to a steady compensated state. Nitrotyrosine immunoreactivity (an intracellular marker of oxidative stress), cyclooxygenase-2 (COX-2), and inducible NO synthase (iNOS) expression were significantly higher in venous endothelial cells of patients in clinical decompensation when compared with healthy subjects. Return to a compensated state resulted in a significant reduction in nitrotyrosine immunoreactivity, COX-2, and iNOS expression. Concomitantly, a significant increase in FMD and a decline in plasma total 8-isoprostane and bicycloprostaglandin E2 levels were observed. Venous endothelial NOS expression was unaffected by clinical decompensation. Conclusions—Clinical decompensation in CHF is associated with activation of the venous endothelium. Return to a compensated state after short-term inotropic therapy results in a significant reduction in endothelial nitrotyrosine formation, COX-2, and iNOS expression.

Thromboxane A2 Receptor Signaling Inhibits Vascular Endothelial Growth Factor–Induced Endothelial Cell Differentiation and Migration

Vascular endothelial growth factor (VEGF) is an important patho-physiological mediator of angiogenesis. VEGF-induced endothelial cell (EC) migration and angiogenesis often occur in complicated environments containing multiple agents capable of modifying the response. Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of pathogenesis of many vascular diseases. Human EC express both TXA2 receptor (TP) isoforms; however, the effects of individual TP isoforms on VEGF-induced EC migration and angogenesis are unknown. We report here that the TXA2 mimetic [1S-(1&agr;, 2&bgr;(5Z), 3&agr;(1E, 3R), 4&agr;]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5′-heptenoic acid (IBOP) (100 nmol/L) is a potent antagonist (IC50 30 nmol/L) of VEGF-induced EC migration and differentiation. TP&bgr;, but not TP&agr;, expression is required for the inhibition of VEGF-induced migration and angiogenesis. IBOP costimulation suppressed nitric oxide (NO) release from VEGF-treated EC through decreased activation of Akt, eNOS, and PDK1. TP&bgr; costimulation also ablated the increase in focal adhesion formation in response to VEGF. This mechanism was characterized by decreased recruitment of focal adhesion kinase (FAK) and vinculin to the &agr;v&bgr;3 integrin and reduced FAK and Src activation in response to VEGF. Addition of NO donors together with transfection of a constitutively active Src construct could circumvent the blockade of VEGF-induced migration by TP; however, neither intervention alone was sufficient. Thus, TP stimulation appears to limit angiogenesis, at least in part, by inhibiting the pro-angiogenic cytokine VEGF. These data further support a role for antagonism of TP activation in enhancing the angiogenic response in tissues exposed to elevated TXA2 levels in which revascularization is important.

Regulation of p53 tetramerization and nuclear export by ARC

Inactivation of the transcription factor p53 is central to carcinogenesis. Yet only approximately one-half of cancers have p53 loss-of-function mutations. Here, we demonstrate a mechanism for p53 inactivation by apoptosis repressor with caspase recruitment domain (ARC), a protein induced in multiple cancer cells. The direct binding in the nucleus of ARC to the p53 tetramerization domain inhibits p53 tetramerization. This exposes a nuclear export signal in p53, triggering Crm1-dependent relocation of p53 to the cytoplasm. Knockdown of endogenous ARC in breast cancer cells results in spontaneous tetramerization of endogenous p53, accumulation of p53 in the nucleus, and activation of endogenous p53 target genes. In primary human breast cancers with nuclear ARC, p53 is almost always WT. Conversely, nearly all breast cancers with mutant p53 lack nuclear ARC. We conclude that nuclear ARC is induced in cancer cells and negatively regulates p53.

Reversal of Angiogenesis In Vitro, Induction of Apoptosis, and Inhibition of Akt Phosphorylation in Endothelial Cells by Thromboxane A2

Thromboxane A(2) (TxA(2)) causes platelet aggregation, vasoconstriction, and inhibition of endothelial cell (EC) migration and prevents vascular tube formation via its specific receptors (TP), of which there are two isoforms (TPalpha and TPbeta), both expressed in human ECs. In this study, we demonstrate that the TxA(2) mimetic IBOP increases apoptosis of human ECs and inhibits the phosphorylation of Akt kinase, an intracellular mediator required for cell survival. Treatment with IBOP destroyed EC networks formed on a basement membrane matrix in vitro. To distinguish the role of the TP isoforms, each isoform was expressed in TP-null ECs to create TPalpha and TPbeta ECs. IBOP induced apoptosis and inhibited phosphorylation of Akt kinase in both TPalpha and TPbeta. IBOP increased cAMP levels in TPalpha but not in TPbeta. Apoptosis induced by IBOP in TPalpha was not affected by either the adenylyl cyclase activator forskolin or the protein kinase A inhibitor 14-22 amide or H-89, whereas that in TPbeta was suppressed by forskolin and enhanced by the protein kinase A inhibitor 14-22 amide or H-89, suggesting that the TP isoforms differ in their signal pathways in mediating apoptosis. In conclusion, apoptosis may be the mechanism by which TxA(2)-mediated destruction of vascular structures in ECs occurs; although both TP isoforms induce apoptosis, possibly via inhibiting Akt phosphorylation, the signaling differs in each isoform, in that activation of the adenylyl cyclase pathway prevents apoptosis caused by TPbeta, but not by TPalpha, stimulation.

Anthrax Lethal Toxin Kills Macrophages in a Strain-Specific Manner by Apoptosis or Caspase-1-Mediated Necrosis

Murine macrophages have been classified as either susceptible or nonsusceptible to killing by anthrax lethal toxin (LT) depending upon genetic background. While considered resistant to LT killing, we found that bone marrow-derived macrophages (BMMs) from DBA/2, AKR, and C57BL/6 mice were slowly killed by apoptosis following LT exposure. LT killing was not restricted to in vitro assays, as splenic macrophages were also depleted in LT-injected C57BL/6 mice. Human macrophages, also considered LT resistant, similarly underwent slow apoptosis in response to LT challenge. In contrast, LT triggered rapid necrosis and a broad protein release in BMMs derived from BALB/c and C3H/HeJ, but not C57BL/6 mice. Released proteins included processed interleukin-18, confirming reports of inflammasome and caspase-1 activation in LT-mediated necrosis in macrophages. Complete inhibition of caspase-1 activity was required to block LT-mediated necrosis. Strikingly, minimal residual caspase-1 activity was sufficient to trigger significant necrosis in LT-treated macrophages, indicating the toxicity of caspase-1 in this process. IL-18 release does not trigger cytolysis, as IL-18 is released late and only from LT-treated macrophages undergoing membrane perturbation. We propose that caspase-1-mediated macrophage necrosis is the source of the cytokine storm and rapid disease progression reported in LT-treated BALB/c mice.

Protein Kinase Cδ Inhibition of S-Phase Transition in Capillary Endothelial Cells Involves the Cyclin-dependent Kinase Inhibitor p27Kip1

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Overexpression of PKCα has little effect on proliferation, whereas PKCδ slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKCα (PKCαEC) or PKCδ (PKCδEC) to determine the role of specific cell cycle regulatory proteins in the PKCδ-induced cell cycle arrest. Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKCδEC line in association with S-phase arrest. However, the protein levels for cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 protein in response to serum was also delayed in PKCδEC. In the PKCδEC line, serum induced p27Kip1 but not p16Ink4a or p21Cip1. Serum did not affect p27Kip1 levels in the control vascular endothelial cell line. Immunoprecipitation-Western blotting analysis of p27Kip1 showed serum stimulation of the vascular endothelial cell line resulted in increased amounts of cyclin D1 bound to p27Kip1. In the PKCδEC line, serum did not increase the amount of cyclin D1 bound to p27Kip1. Transfection of full-length p27Kip1 antisense into the PCKδEC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27Kip1 in the PKCδ-mediated S-phase arrest.