SUMMARY Impaired angiogenesis has been implicated in adipose tissue dysfunction and the development of obesity and associated metabolic disorders. Here, we report the unexpected finding that vascular endothelial growth factor B (VEGFB) gene transduction into mice inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition. Mechanistically, the binding of VEGFB to VEGF receptor 1 (VEGFR1, also known as Flt1) activated the VEGF/VEGFR2 pathway and increased capillary density, tissue perfusion, and insulin supply, signaling, and function in adipose tissue. Furthermore, endothelial Flt1 gene deletion enhanced the effect of VEGFB, activating the thermogenic program in subcutaneous adipose tissue, which increased the basal metabolic rate, thus preventing diet-induced obesity and related metabolic complications. In obese and insulin-resistant mice, Vegfb gene transfer, together with endothelial Flt1 gene deletion, induced weight loss and mitigated the metabolic complications, demonstrating the therapeutic potential of the VEGFB/VEGFR1 pathway.

lesterol transport during the acute phase response but not group IIA secretory phospholipase A 2 . J. Lipid Res . 2010. 51: 743-754. Supplementary key words feces • infl ammation • sepsis • atherosclerosis • miceEpidemiological studies established a strong inverse association between plasma HDL cholesterol levels and the risk of atherosclerotic cardiovascular disease (CVD) ( 1, 2 ). A major anti-atherogenic activity of HDL is regarded to be reverse cholesterol transport (RCT), a process comprising removal of excess cholesterol from peripheral cells, most importantly macrophage foam cells in atherosclerotic lesions, and transport back to the liver for subsequent excretion into bile and feces ( 2, 3 ). Understanding the pathophysiological factors regulating RCT is therefore of prime importance.Infl ammation is strongly linked to atherosclerosis ( 4-6 ). The atherosclerotic plaque itself is increasingly considered a site of chronic infl ammation within the vessel wall ( 5, 7 ). In addition, markers of infl ammation are elevated in plasma of patients with established atherosclerotic CVD, and circulating levels of several acute phase proteins Abstract Atherosclerosis is linked to infl ammation. HDL protects against atherosclerotic cardiovascular disease, mainly by mediating cholesterol effl ux and reverse cholesterol transport (RCT). The present study aimed to test the impact of acute infl ammation as well as selected acute phase proteins on RCT with a macrophage-to-feces in vivo RCT assay using intraperitoneal administration of [ 3 H]cholesterol-labeled macrophage foam cells. In patients with acute sepsis, cholesterol effl ux toward plasma and HDL were signifi cantly decreased ( P < 0.001). In mice, acute infl ammation (75 µg/mouse lipopolysaccharide) decreased [ 3 H] cholesterol appearance in plasma ( P < 0.05) and tracer excretion into feces both within bile acids ( ؊ 84%) and neutral sterols ( ؊ 79%, each P < 0.001). In the absence of systemic infl ammation, overexpression of serum amyloid A (SAA, adenovirus) reduced overall RCT ( P < 0.05), whereas secretory phospholipase A 2 (sPLA 2 , transgenic mice) had no effect. Myeloperoxidase injection reduced tracer appearance in plasma ( P < 0.05) as well as RCT ( ؊ 36%, P < 0.05). Hepatic expression of bile acid synthesis genes ( P < 0.01) and transporters mediating biliary sterol excretion ( P < 0.01) was decreased by infl ammation. In conclusion, our data demonstrate that acute infl ammation impairs cholesterol effl ux in patients and macrophage-to-feces RCT in vivo in mice. Myeloperoxidase and SAA contribute to a certain extent to reduced RCT during infl ammation but not sPLA 2 . However, reduced bile acid formation and decreased biliary sterol excretion might represent major contributing factors to decreased RCT in infl ammation. -Annema, W., N. Nijstad, M. Tölle, J. F. de Boer, R. V. C. Buijs, P. Heeringa, M. van der Giet, and U. J. F. Tietge. Myeloperoxidase and serum amyloid A contribute to impaired in vivo reverse cho-

Except for conversion to bile salts, there is no major cholesterol degradation pathway in mammals. Efficient excretion from the body is therefore a crucial element in cholesterol homeostasis. Yet, the existence and importance of cholesterol degradation pathways in humans is a matter of debate. We quantified cholesterol fluxes in 15 male volunteers using a cholesterol balance approach. Ten participants repeated the protocol after 4 weeks of treatment with ezetimibe, an inhibitor of intestinal and biliary cholesterol absorption. Under basal conditions, about 65% of daily fecal neutral sterol excretion was bile derived, with the remainder being contributed by direct transintestinal cholesterol excretion (TICE). Surprisingly, ezetimibe induced a 4-fold increase in cholesterol elimination via TICE. Mouse studies revealed that most of ezetimibe-induced TICE flux is mediated by the cholesterol transporter Abcg5/Abcg8. In conclusion, TICE is active in humans and may serve as a novel target to stimulate cholesterol elimination in patients at risk for cardiovascular disease.

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Bile acids (BAs) facilitate intestinal absorption of lipid-soluble nutrients and modulate various metabolic pathways through the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5. These receptors are targets for therapy in cholestatic and metabolic diseases. However, dissimilarities in BA metabolism between humans and mice complicate translation of preclinical data. Cytochrome P450 family 2 subfamily c polypeptide 70 (CYP2C70) was recently proposed to catalyze the formation of rodent-specific muricholic acids (MCAs). With CRISPR/Cas9-mediated somatic genome editing, we generated an acute hepatic Cyp2c70 knockout mouse model (Cyp2c70ako) to clarify the role of CYP2C70 in BA metabolism in vivo and evaluate whether its activity modulates effects of pharmacologic FXR activation on cholesterol homeostasis. In Cyp2c70ako mice, chenodeoxycholic acid (CDCA) increased at the expense of βMCA, resulting in a more hydrophobic human-like BA pool. Tracer studies demonstrated that, in vivo, CYP2C70 catalyzes the formation of βMCA primarily by sequential 6β-hydroxylation and C7-epimerization of CDCA, generating αMCA as an intermediate metabolite. Physiologically, the humanized BA composition in Cyp2c70ako mice blunted the stimulation of fecal cholesterol disposal in response to FXR activation compared with WT mice, predominantly due to reduced stimulation of transintestinal cholesterol excretion. Thus, deletion of hepatic Cyp2c70 in adult mice translates into a human-like BA pool composition and impacts the response to pharmacologic FXR activation. This Cyp2c70ako mouse model may be a useful tool for future studies of BA signaling and metabolism that informs human disease development and treatment.

Background and Aims Bile acids (BAs) aid intestinal fat absorption and exert systemic actions by receptor-mediated signaling. BA receptors have been identified as drug targets for liver diseases. Yet, differences in BA metabolism between humans and mice hamper translation of pre-clinical outcomes. Cyp2c70 -ablation in mice prevents synthesis of mouse/rat-specific muricholic acids (MCAs), but potential (patho)physiological consequences of their absence are unknown. We therefore assessed age- and gender-dependent effects of Cyp2c70 -deficiency in mice. Methods The consequences of Cyp2c70 -deficiency were assessed in male and female mice at different ages. Results Cyp2c70 -/- mice were devoid of MCAs and showed high abundances of chenodeoxycholic and lithocholic acids. Cyp2c70 -deficiency profoundly impacted microbiome composition. Bile flow and biliary BA secretion were normal in Cyp2c70 -/- mice of both sexes. Yet, the pathophysiological consequences of Cyp2c70 -deficiency differed considerably between sexes. Three-week old male Cyp2c70 -/- mice showed high plasma BAs and transaminases, which spontaneously decreased thereafter to near-normal levels. Only mild ductular reactions were observed in male Cyp2c70 -/- mice up to 8 months of age. In female Cyp2c70 -/- mice, plasma BAs and transaminases remained substantially elevated with age, gut barrier function was impaired and bridging fibrosis was observed at advanced age. Addition of 0.1% ursodeoxycholic acid to the diet fully normalized hepatic and intestinal functions in female Cyp2c70 -/- mice. Conclusion Cyp2c70 -/- mice show transient neonatal cholestasis and develop cholangiopathic features that progress to bridging fibrosis in females only. These consequences of Cyp2c70 -deficiency are restored by treatment with UDCA, indicating a role of BA hydrophobicity in disease development.

High-density lipoprotein (HDL) particles are involved in the protection against cardiovascular disease by promoting cholesterol efflux, in which accumulated cholesterol is removed from macrophage foam cells. We investigated whether HDL cholesterol efflux capacity is associated with cardiovascular mortality, all-cause mortality, and graft failure in a cohort of renal transplant recipients (n=495, median follow-up 7.0 years). Cholesterol efflux capacity at baseline was quantified using incubation of human macrophage foam cells with apolipoprotein B-depleted plasma. Baseline efflux capacity was not different in deceased patients and survivors (P=0.60 or P=0.50 for cardiovascular or all-cause mortality, respectively), whereas recipients developing graft failure had lower efflux capacity than those with functioning grafts (P,0.001). Kaplan-Meier analysis demonstrated a lower risk for graft failure (P=0.004) but not cardiovascular (P=0.30) or all-cause mortality (P=0.31) with increasing gender-stratified tertiles of efflux capacity. Cox regression analyses adjusted for age and gender showed that efflux capacity was not associated with cardiovascular mortality (hazard ratio [HR], 0.89; 95% confidence interval [95% CI], 0.67 to 1.19; P=0.43). Furthermore, the association between efflux capacity and all-cause mortality (HR, .79; 95% CI, 0.63 to 0.98; P=0.031) disappeared after further adjustment for potential confounders. However, efflux capacity at baseline significantly predicted graft failure (HR, 0.43; 95% CI, 0.29 to 0.64; P,0.001) independent of apolipoprotein A-I, HDL cholesterol, or creatinine clearance. In conclusion, this prospective study shows that cholesterol efflux capacity from macrophage foam cells is not associated with cardiovascular or all-cause mortality but is a strong predictor of graft failure independent of plasma HDL cholesterol levels in renal transplant recipients.