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Rationale: Sprouting angiogenesis is governed by the concept of tip/stalk cells that guides our understanding of the transition from vascular sprouting to maturation and ultimately quiescence. The VEGF and Notch signaling pathways have been extensively described in regulating the discrimination between these two cell populations. However, several additional tip and stalk cell specific genes have been identified. To date, unresolved questions remain, and our understanding of the mechanisms by which these signaling processes are integrated is incomplete. Objective: We set out to investigate novel mechanisms by which signaling pathways involving two G protein coupled receptors (GPCRs), expressed in a mutually exclusive fashion in the tip/stalk cell populations, are intricately linked in vascular development. Methods/Results: Using a combination of in vivo and in vitro techniques, we demonstrate the critical role of crosstalk between APLNR and CXCR4 in vascular maturation. We show robust flow induced expression of the stalk cell specific APLNR, that leads to marked suppression of CXCR4 expression, a mechanism to achieve tip cell restricted expression of the latter. Retinas from Apln (ligand), Aplnr (receptor) and endothelial specific Aplnr deleted mice show retarded vascular expansion, reduced vascularized area and fewer vascular branch points. These phenotypes are in part due to increased expression of Cxcr4 in Apln-/- and Aplnr-/- retinal vessels as Cxcr4 inhibition through a selective inhibitor can ameliorate the Aplnr phenotype. The crosstalk between the two GPCRs was found to involve a key shear responsive microRNA, miR-139-5p, which is upregulated by APLN/APLNR signaling and directly targets CXCR4 in endothelial cells. In accordance, Apln-/- and Aplnr-/- retinal endothelial cells showed depleted levels of miR-139-5p. Lastly, we demonstrate that atorvastatin, an HMG-CoA reductase inhibitor shown to enhance APLNR signaling, can induce miR-139-5p expression and rescue the vascular phenotypes associated with APLN/APLNR deficiency. Conclusions: These findings provide key mechanistic insights into a critical microRNA based crosstalk between two GPCR signaling cascades, which regulates important steps in vascular maturation.

To investigate the hypothesis that Apelin treatment would attenuate aortic injury in a murine model of elastase-induced abdominal aortic aneurysm (AAA) disease, by limiting disease-related vascular wall inflammatory. Male C57BL/6 mice were implanted with osmotic mini-pumps filled with Apelin (2 mg/kg/day) or saline (Control), and subsequently treated with intra-aortic porcine pancreatic elastase to create infrarenal AAA’s. Mice were sacrificed at day 3 for aortic PCR analysis or followed ultrasonographically until day 7 and then sacrificed for histological analysis (Elastic Masson, VCAM, ICAM-1, E-Selectin, and Mac-2 semi-quantitative analysis). The cellular expression of inflammatory cytokines and chemokines in response to Apelin was assessed by TaqMan analysis of both macrophages (J774, RAW, and harvested intraperitoneal cells) and rat aortic smooth muscle cells (A7R5) and compared to other stimuli. Apelin treatment resulted in diminished AAA formation, with a 30% reduction in in vivo aortic diameter by ultrasound (1.04 mm vs 1.48 mm, p < 0.05), and a 59% reduction in maximal aortic cross-sectional area by histology (0.59 mm2 vs 1.43 mm2, p < 0.05) relative to the saline-treated group. The macrophage infiltrate also was significantly reduced in the Apelin-treated mice compared to the Control group (68.6 cells/hpf vs 285.4 cells/hpf, p < 0.001). Apelin treatment was associated with reduced mean aortic MCP-1, MIP 1-alpha, IL-6 and TNF-alpha mRNA levels, although these did not reach statistical significance. Apelin stimulation of cultured macrophage models significantly reduced MCP-1 and TNF-alpha mRNA levels compared to basal conditions (−1.12 and −1.49 fold expression change, p < 0.05, respectively). No difference in endothelial cell adhesion molecule expression or smooth muscle cell cytokine production was found in response to Apelin. Apelin significantly reduces AAA formation in this accepted model of human AAA disease. The mechanism appears to be decreased macrophage infiltration, perhaps related to an apelin-mediated decrease in pro-inflammatory cytokine and chemokine activation. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).

The metabolic syndrome includes high triglyceride (TG) and low HDL-cholesterol (HDL-C) levels in the plasma, and often accompanies steatosis in the liver. Endothelial lipase (EL) is a phospholipase that regulates HDL metabolism. EL is expressed by hepatocytes, while the function of EL in the liver has not been identified. Here, we examined the role of EL in the liver using a mouse model of metabolic syndrome. The EL expression in the liver was analyzed by real-time PCR. It revealed that liver EL expression was significantly increased in obese and diabetic db/db mice compared to that of control mice. To examine the significance of the EL upregulation in the liver, we injected the recombinant adenovirus encoding human EL into mice. The EL overexpression in the liver resulted in a significant decrease in plasma HDL-C, TG, and free fatty acid levels. Interestingly, the EL overexpression in the liver increased liver weight and liver TG content both in wild-type and db/db mice. In db/db mice, particularly, EL overexression accelerated the formation of steatosis by increasing the mRNA level of fatty acid synthase. These findings indicate that EL expression is increased in the liver in the metabolic syndrome. The upregulation of EL promotes the uptake of plasma lipids by hepatocytes, and accelerates the progression of steatosis in db/db mice. Thus EL may play a role in the genesis of steatosis as well as dyslipidemia in the metabolic syndrome.