Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2-O-methyladenosine-3, 5-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.

Production of thromboxane (TX) A 2 and PG I 2 /prostacyclin (PGI 2 ) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE -/-TP -/-mice exhibited a significant delay in atherogenesis, and apoE -/-IP -/-mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE -/-IP -/-mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE -/-TP -/-mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE -/-IP -/-and apoE -/-TP -/-mice, respectively, than in those of apoE -/-mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE -/-IP -/-mice than in either apoE -/-TP -/-or apoE -/-mice. We conclude that TXA 2 promotes and PGI 2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

Production of thromboxane (TX) A 2 and PG I 2 /prostacyclin (PGI 2 ) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE -/-TP -/-mice exhibited a significant delay in atherogenesis, and apoE -/-IP -/-mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE -/-IP -/-mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE -/-TP -/-mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE -/-IP -/-and apoE -/-TP -/-mice, respectively, than in those of apoE -/-mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE -/-IP -/-mice than in either apoE -/-TP -/-or apoE -/-mice. We conclude that TXA 2 promotes and PGI 2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type-specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitortreated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represents a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.

Background-The vascular smooth muscle cell (VSMC) is the central cell component involved in the fibroproliferative response in atherogenesis. As the lesion advances, VSMCs migrate from the media into the subendothelial space, thereby forming fibrous plaque lesions. Platelet-derived growth factor (PDGF) has been known to be a potent chemoattractant and mitogen for SMCs, but the pathophysiological role of the 2 PDGF receptors, receptor-␣ (PDGFR-␣) and receptor-␤ (PDGFR-␤) in atherogenesis is poorly understood. To clarify this problem, we prepared antagonistic rat monoclonal antibodies, APA5 and APB5, against murine PDGFR-␣ and PDGFR-␤, respectively. Methods and Results-Apolipoprotein E-deficient mice were fed a high-fat diet containing 0.3% cholesterol from 6 weeks of age and subjected to injection with 1 mg/d IP of either antibody from 12 to 18 weeks every other day. In the mice injected with APB5, the aortic atherosclerotic lesion size and the number of intimal VSMCs were reduced by 67% and 80%, respectively, compared with the control mice injected with irrelevant rat IgG. In contrast, the mice that received APA5 showed only minimal reduction of lesion size, and a large number of VSMCs were observed in the intima. In the intima of advanced lesions, APB5 immunolabeled VSMCs, whereas APA5 could detect VSMCs mainly in the media. Conclusions-These

A critical event in the early stages of atherosclerosis is the focal accumulation of lipid‐laden foam cells derived from macrophages. In various cholesterol‐fed animal models of atherosclerosis, localized attachment of circulating monocytes to arterial endothelial cells appeared to precede the formation of foam cells. It is suggested that monocyte recruitment into early lesions depends on the endothelial adhesiveness for monocytes and lymphocytes. In vivo and in vitro experiments have identified molecules, such as ICAM‐1, VCAM‐1, and P‐selectin, that can support the adhesion of monocytes and lymphocytes. Moreover, oxidized LDL, lysophosphatidyl‐choline, and oxidized fatty acids induce the expression not only of these adhesion molecules but also of scavenger receptors, such as CD‐36, SR‐A, and LOX‐1. Recently, we isolated and characterized the novel receptors for oxidized LDL, namely, LOX‐1 and SR‐PSOX. Expression of LOX‐1 is found on endothelial cells, smooth muscle cells, and macrophages, whereas SR‐PSOX is expressed on macrophages. In this paper the significance of oxidized LDL and its receptors, LOX‐1 and SR‐PSOX, in terms of atherogenesis is discussed.

Snail family proteins are zinc finger transcriptional regulators first identified in Drosophila which play critical roles in cell fate determination. We identified a novel Snail -related gene from murine skeletalmusclecells designated Smuc. Northern blot analysis showed that Smuc was highly expressed in skeletal muscle and thymus. Smuc contains five putative DNA-binding zinc finger domains in its C-terminal half. In electrophoretic mobility shift assays, recombinant zinc finger domains of Smuc specifically bound to CAGGTG and CACCTG E-box motifs (CANNTG). Because basic helix-loop-helix transcription factors (bHLH) bind to the same E-box sequences, we examined whether Smuc competes with the myogenic bHLH factor MyoD for DNA binding. Smuc inhibited the binding of a MyoD-E12 complex to the CACCTG E-box sequence in a dose-dependent manner and suppressed the transcriptional activity of MyoD-E12. When heterologously targeted to the thymidine kinase promoter as fusion proteins with the GAL4 DNA-binding domain, the non-zinc finger domain of Smuc acted as a transcriptional repressor. Furthermore, overexpression of Smuc in myoblasts repressed transactivation of muscle differentiation marker Troponin T. Thus, Smuc might regulate bHLH transcription factors by zinc finger domains competing for E-box binding, and non-zinc finger repressor domains might also confer transcriptional repression to control differentiation processes.

Background-Atherosclerosis results from complex inflammatory-fibroproliferative responses. To elucidate the central role of macrophage and macrophage-colony stimulating factor (M-CSF) during atherogenesis, we used a new strategy to administer to adult apolipoprotein E (apoE)-deficient mice a monoclonal antibody (AFS98) raised against c-fms, the receptor of M-CSF. Methods and Results-When 6-week-old apoE-deficient mice were fed a high-fat diet and injected with 2 mg of AFS98 intraperitoneally on alternate days for 6 weeks, accumulation of macrophage-derived foam cells in the aortic root was suppressed by 70% compared with that in controls. This preventive effect was associated with neither remarkable decrease of the number of circulating monocytes nor systemic growth retardation. In contrast, when apoE-deficient mice that had been fed a high-fat diet from 6 weeks of age were given AFS98 from 12 to 18 weeks of age, a minimal protective effect on lesion size was observed. Conclusions-These results suggest that (1)