Endothelial dysfunction or activation elicited by oxidatively modified low-density lipoprotein (Ox-LDL) has been implicated in the pathogenesis of atherosclerosis, characterized by intimal thickening and lipid deposition in the arteries. Ox-LDL and its lipid constituents impair endothelial production of nitric oxide, and induce the endothelial expression of leukocyte adhesion molecules and smooth-muscle growth factors, which may be involved in atherogenesis. Vascular endothelial cells in culture and in vivo internalize and degrade Ox-LDL through a putative receptor-mediated pathway that does not involve macrophage scavenger receptors. Here we report the molecular cloning, using expression cloning strategy, of an Ox-LDL receptor from vascular endothelial cells. The cloned receptor is a membrane protein that belongs structurally to the C-type lectin family, and is expressed in vivo in vascular endothelium and vascular-rich organs.

Acute activation of the serine-threonine kinase Akt is cardioprotective and reduces both infarction and dysfunction after ischemia/reperfusion injury (IRI). However, less is known about the chronic effects of Akt activation in the heart, and, paradoxically, Akt is activated in samples from patients with chronic heart failure. We generated Tg mice with cardiac-specific expression of either activated (

Endothelial dysfunction, or activation, elicited by oxidized LDL (Ox-LDL) or its lipid constituent, has been implicated in the initiation and progression of atherosclerosis. We have recently identified a C-type lectin-like molecule, designated lectin-like Ox-LDL receptor-1 (LOX-1), which acts as a cell-surface receptor for Ox-LDL in cultured vascular endothelial cells. In this study, we provide evidence that LOX-1 expression can be upregulated by tumor necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA) in cultured bovine aortic endothelial cells. TNF-alpha and PMA upregulated LOX-1 protein and mRNA in a time- and dose-dependent manner. Nuclear runoff assay revealed that TNF-alpha stimulates transcription of the LOX-1 gene. Chinese hamster ovary K1 cells stably expressing LOX-1 internalized 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled Ox-LDL but did not significantly internalize acetylated LDL (Ac-LDL), which was effectively suppressed by excess amounts of unlabeled Ox-LDL but not by Ac-LDL. Upregulated expression of LOX-1 by TNF-alpha and PMA was associated with increased uptake of DiI-Ox-LDL that cannot be blocked by excess amounts of unlabeled Ac-LDL. Taken together, LOX-1 is a receptor specific for Ox-LDL, and enhanced uptake of Ox-LDL via this novel receptor on vascular endothelial cells may play an important role in endothelial activation in atherogenesis.

Background-The presence of apoptotic myocytes has been reported in human hearts with dilated cardiomyopathy (DCM) on the basis of a positive finding of DNA in situ nick end-labeling (TUNEL). However, ultrastructural evidence of myocyte apoptosis has not been obtained. Methods and Results-A total of 80 endomyocardial biopsies were obtained from right and left ventricles of 20 patients with DCM and 20 normal control subjects. TUNEL-positive myocytes were found by light microscope in 15% of DCM specimens (controls, 0%, PϽ0.05), and the percentage of TUNEL-positive myocytes per section in DCM was 1.0Ϯ2.7% (meanϮSD). According to TUNEL at the electron microscopic level (EM-TUNEL), immunogold particles, which label DNA breaks with 3Ј-OH terminals, were markedly accumulated in the bizarre-shaped nuclei, with widespread clumping of chromatin (so-called "hypertrophied nuclei") of the myocytes obtained from DCM. Their ultrastructure was neither apoptotic nor necrotic but rather that of living cells. Taq polymerase-based DNA in situ ligation assay, which detects double-stranded DNA fragments more specifically than TUNEL, did not detect a positive reaction in any case. In mirror sections, all of the TUNEL-positive myocytes in DCM simultaneously expressed proliferating cell nuclear antigen, which is required for both DNA replication and repair, but Ki-67, a replication-associated antigen, was completely negative in all cases, which appeared to rule out cell proliferation activity. Conclusions-Most

Endothelial dysfunction, or activation, elicited by oxidized low density lipoprotein (Ox-LDL) and its lipid constituents has been shown to play a key role in the pathogenesis of atherosclerosis. We recently have identified a novel receptor for Ox-LDL-designated lectin-like Ox-LDL receptor (LOX-1) in vascular endothelial cells. To examine ligand specificity of LOX-1, we established CHO cell lines stably expressing both human and bovine LOX-1 (LOX-1-CHO). LOX-1-CHO bound and degraded 125I-labeled Ox-LDL but did not significantly degrade 125I-labeled acetylated LDL (Ac-LDL). Fucoidin and maleylated BSA (M-BSA), which inhibit 125I-Ox-LDL binding to class A scavenger receptors, did not inhibit 125I-Ox-LDL binding or degradation in LOX-1-CHO. Polyinosinic acid and carrageenan, in contrast, significantly reduced 125I-Ox-LDL binding to LOX-1-CHO by 62% and 60%, respectively. Delipidated and untreated 125I-Ox-LDL were bound and degraded equally in LOX-1-CHO; furthermore, excess amounts of unlabeled, delipidated Ox-LDL inhibited binding and degradation of untreated 125I-Ox-LDL. Taken together, LOX-1 is a receptor for Ox-LDL but not for Ac-LDL. LOX-1 recognizes protein moiety of Ox-LDL, and its ligand specificity is distinct from other receptors for Ox-LDL, including class A and B scavenger receptors.

Background— Serum- and glucocorticoid-responsive kinase-1 (SGK1), a serine-threonine kinase that is highly expressed in the heart, has been previously reported to regulate sodium channels. Because SGK1 is a PI 3-kinase–dependent kinase with structural homology to Akt, we examined its regulation in the heart and its effects on cardiomyocyte (CM) apoptosis and hypertrophy in vitro. Methods and Results— Rats were subjected to aortic banding, and expression of total and phosphorylated SGK1 was examined. Both phospho- and total SGK1 increased 2 to 7 days after banding. Phospho-SGK1 was also upregulated in CMs stimulated in vitro with IGF-I or phenylephrine. Infection of CMs with an adenoviral vector encoding constitutively active SGK1 (Ad.SGK1.CA) inhibited apoptosis after serum-deprivation or hypoxia ( P <0.05), whereas expression of kinase-dead SGK1 (Ad.SGK1.KD) increased it and partially mitigated the protective effects of IGF-I ( P <0.05). SGK1 activation was also sufficient to increase cell size, protein synthesis, sarcomere organization, and ANF expression both at baseline and in response to phenylephrine but was not necessary for the hypertrophic response to phenylephrine. Evaluation of potential downstream signaling pathways demonstrated that SGK1 induces phosphorylation of tuberin, p70s6kinase, and GSK3β in CMs, which may contribute to its effects. Conclusions— SGK1 is dynamically regulated during acute biomechanical stress in the heart and inhibits CM apoptosis while enhancing the hypertrophic response.

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A novel receptor for oxidized low-density lipoprotein (OxLDL), lectin-like OxLDL receptor (LOX-1), was cloned from endothelial cells. Since OxLDL is taken up by vascular smooth muscle cells (VSMC) in atheroma, we analyzed the inducible expression of LOX-1 in VSMC in the present study. Incubation of cultured bovine VSMC with lysophosphatidylcholine (LPC), an atherogenic component of OxLDL, increased the level of mRNA for LOX-1 in a dose-and time-dependent manner. Since LPC did not significantly change the half-life of LOX-1 mRNA, the induction seemed to occur at the transcriptional level. The induction accompanied an increase in the protein level of LOX-1 and activity of OxLDL uptake. Blocking antibody against LOX-1 significantly suppressed the enhanced uptake of OxLDL. Thus, LOX-1 is a major receptor for OxLDL in VSMC as in endothelial cells. The enhanced expression of LOX-1 by LPC suggests that OxLDL and LPC would progressively change the function of VSMC and accelerate atherogenesis in vivo.z 2000 Federation of European Biochemical Societies.