Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. While numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here, we describe the endothelial-derived secreted molecule, developmental endothelial locus-1 (Del-1), as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1-deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/-mice displayed significantly higher neutrophil accumulation in LPS-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1-double deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.Leukocyte extravasation is integral to the response to infection or injury and to inflammation and autoimmunity. Leukocyte recruitment comprises a well coordinated cascade of adhesive events including selectin-mediated rolling, firm adhesion of leukocytes to endothelial cells and & This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS. †To whom correspondence should be addressed chavakist@mail.nih.gov. * EYC and EC contributed equally # MAC and HL contributed equally NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript their subsequent transendothelial migration. The interaction between LFA-1 (αLβ2, CD11a/ CD18) and endothelial ICAM-1 is crucial during firm endothelial adhesion of leukocytes (1-5). Whereas numerous adhesion receptors promoting inflammatory cell recruitment have been identified, very little information exists about endogenous inhibitors of the leukocyte adhesion cascade (1-7). Developmental endothelial locus-1 (Del-1) is a glycoprotein that is secreted by endothelial cells and can associate with the endothelial cell surface and the extracellular matrix (8-10). Del-1 is regulated upon hypoxia or vascular injury and has been implicated in vascular remodelling during angiogenesis (10-12). Here, we sought to determine whether endothelial-derived Del-1 participates in leukocyte-endothelial interactions. RT-PCR analysis revealed Del-1 mRNA predominantly in the brain and lung, with no expression in liver, spleen, or whole blood (Fig. 1A and fig. S1A). Del-1 was expressed in WT but not in Del-1-/-murine lung endothelial cells (Fig. 1B, 9). Immunohistochemistry of lung tissues demonstrated the presence of Del-1 in vessels, as observed by co-staining with the endothelial marker PECAM-1 ( fig. S1B).To determine whether Del-1 participates in leukocyte recruitmen...

Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II-mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II-mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.

Apelin reduces left ventricular preload and afterload and increases contractile reserve without evidence of hypertrophy. These results associate apelin with a positive hemodynamic profile and suggest it as an attractive target for pharmacotherapy in the setting of heart failure.

The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelin's effects, apelin-13 was also delivered to cultured C 2C12 myotubes. 2-[ 3 H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulininduced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C 2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPK␣1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelin's effects on glucose uptake and Akt phosphorylation are in part mediated by a G i and AMPK-dependent pathway. insulin resistance; obesity; diabetes; hormones INSULIN RESISTANCE, defined as a diminution in a cell, tissue, or organism's ability to take up glucose in response to insulin, is the pathophysiological hallmark of type 2 diabetes mellitus. Although insulin resistance is typically asymptomatic, it is independently and strongly associated with an increased risk of coronary disease (22), heart failure (15), and mortality (19). Insulin resistance is thus rapidly gaining in importance as a disease entity in the Western world. Unfortunately, despite the clear need for novel therapies for insulin resistance, our understanding of its pathogenesis and mechanisms remains incomplete.Apelin is a peptide hormone recently identified as an endogenous ligand (37) for the G i protein-coupled, angiotensin receptor-like receptor APJ (26, 31). The human preproapelin gene, located on chromosome Xq25-26.1, encodes a 77-amino acid preproprotein (20) that is cleaved to active forms that are 36, 17, 13, and 12 residues in length (37). Of these, the 36-amino acid isoform is the most widely expressed, although the shorter isoforms are more potent and more abundant in the circulation (38). Apelin has gained significant attention in recent years because it has been found to possess numerous di...

Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA-26a for additional studies. Inhibition of miRNA-26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA-26a blunted differentiation. As a potential mechanism, we investigated whether miRNA-26a influences TGF-β-pathway signaling. Dual-luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA-26a inhibition, and the opposite effect with miRNA-26a overexpression in transfected human cells. Furthermore, inhibition of miRNA-26a increased gene expression of SMAD-1 and SMAD-4, while overexpression inhibited SMAD-1. MicroRNA-26a was also found to be downregulated in two mouse models of AAA formation (2.5- to 3.8-fold decrease, P < 0.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA-26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF-β pathway signaling. MicroRNA-26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease.

Objective The G-protein–coupled receptor APJ and its ligand apelin are highly expressed in the pulmonary vasculature, but their function in this vascular bed is unclear. We hypothesized that disruption of apelin signaling would lead to worsening of the vascular remodeling associated with pulmonary hypertension (PH). Methods and Results We found that apelin-null mice developed more severe PH compared with wild-type mice when exposed to chronic hypoxia. Microcomputed tomography of the pulmonary arteries demonstrated significant pruning of the microvasculature in the apelin-null mice. Apelin-null mice had a significant reduction of serum nitrate levels. This was secondary to downregulation of endothelial nitric oxide synthase (eNOS), which was associated with reduced expression of Kruppel-like factor 2, a known regulator of eNOS expression. In vitro knockdown studies targeting apelin in human pulmonary artery endothelial cells demonstrated decreased eNOS and Kruppel-like factor 2 expression, as well as impaired phosphorylation of AMP-activated kinase and eNOS. Moreover, serum apelin levels of patients with PH were significantly lower than those of controls. Conclusion These data demonstrate that disruption of apelin signaling can exacerbate PH mediated by decreased activation of AMP-activated kinase and eNOS, and they identify this pathway as a potentially important therapeutic target for treatment of this refractory human disease.

A new member of the lipase gene family, initially termed endothelial lipase (gene nomenclature, LIPG; protein, EL), is expressed in a variety of different tissues, suggesting a general role in lipid metabolism. To assess the hypothesis that EL plays a physiological role in lipoprotein metabolism in vivo, we have used gene targeting of the native murine locus and transgenic introduction of the human LIPG locus in mice to modulate the level of EL expression. Evaluation of these alleles in a C57Bl/6 background revealed an inverse relationship between HDL cholesterol level and EL expression. Fasting plasma HDL cholesterol was increased by 57% in LIPG–/– mice and 25% in LIPG+/– mice and was decreased by 19% in LIPG transgenic mice as compared with syngeneic controls. Detailed analysis of lipoprotein particle composition indicated that this increase was due primarily to an increased number of HDL particles. Phospholipase assays indicated that EL is a primary contributor to phospholipase activity in mouse. These data indicate that expression levels of this novel lipase have a significant effect on lipoprotein metabolism

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.