Publications

Placental growth factor (PlGF)-deficient (PlGF−/−) and wild-type mice were kept on a standard-fat or high-fat diet for 15 weeks. With the standard-fat diet, the body weights of PlGF−/− and wild-type mice were comparable, whereas the combined weight of subcutaneous and gonadal adipose tissues was lower in PlGF−/− mice (P = 0.02). With the high-fat diet, PlGF−/− mice had a lower body weight (P < 0.05) and less total subcutaneous plus gonadal adipose tissue (P < 0.0001). Blood vessel size was lower in gonadal adipose tissue of PlGF−/− mice with both the standard-fat and high-fat diet (P < 0.05). Blood vessel density, normalized to adipocyte number, was significantly lower in subcutaneous adipose tissue of PlGF−/− mice fed the high-fat diet (P < 0.01). De novo adipose tissue development in nude mice injected with 3T3-F442A preadipocytes was reduced (P < 0.005) by administration of a PlGF-neutralizing antibody. Bone marrow transplantation from wild-type or PlGF−/− mice to wild-type or PlGF−/− recipient mice revealed significantly lower blood vessel density in PlGF−/− recipient mice without an effect on adipose tissue growth. Thus, in murine models of diet-induced obesity, inactivation of PlGF impairs adipose tissue development, at least in part as a result of reduced angiogenesis.

Objective: Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown. Approach and Results: Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Krüppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with 13 C-labeled glucose. Conclusions: These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis.

Summary The pathogenesis of vascular tumors such as angiosarcomas is poorly understood. Cadherin expression inversely correlates with tumor malignancy and the endothelial specific VE-cadherin is low or absent in angiosarcomas, suggesting an inhibitory role for this protein in tumor progression. In this paper we report that Pmy T VE-cadherin null (VEC null) endothelial cells form larger vascular tumors in nude mice when injected subcutaneously as compared to isogenic VE-cadherin positive (VEC pos) cells. This effect requires the association of β-catenin to VE-cadherin, since a VE-cadherin mutant lacking the domain responsible for β-catenin binding (Δβcat) cannot rescue the phenotype. In VEC null cells β-catenin is phosphorylated and partly degraded. N-cadherin is increased and detected at junctions. VEC null cells also present an altered fibrinolytic activity with increases in tPA, uPA, uPAR and a strong reduction in PAI-1, which may be correlated to the high incidence of abrupt hemorrhages in VEC null tumors. Overall, these data strongly suggest that downregulation of VE-cadherin in endothelial tumors may have important consequences for tumor growth and bleeding complications.

Variations in the CLL DNA methylome reflect modifications that occur during normal B cell maturation, along with IGHV mutated (M-CLL) and unmutated CLL (U-CLL), retaining an imprint of the DNA methylation signature of memory (m-CLL) and naive B cells (n-CLL), respectively, with a third intermediate epigenetic subgroup (i-CLL) (1-3). To further test the clinical utility of DNA methylation signatures, we performed the first analysis of patients entering clinical trials; we tested treatment-naive CLL patients [n=605] randomized to CLL4 (chemotherapy, CT) (4), ARCTIC and ADMIRE (both chemo-immunotherapy, CIT) (5, 6). We identified n-, i- and m-CLL in 49.3% (n=299), 32.0% (n=195) and 18.5% (n=112) of our patients, respectively. Fewer m-CLL patients were identified in our study compared to published data reflecting the progressive nature of our cohort, with 80% (n=245/305, P<0.001) of U-CLL cases exhibiting the n-CLL signature (i-CLL: 17% and m-CLL: 3%). For M-CLL cases, 9%, 50% and 41% exhibited the n-, i- and m-CLL epigenetic signature, respectively. 68% (80/117, p<0.001) of cases with del(11q), 77% (41/53, p<0.001) with trisomy 12, and 68% (38/56, p=0.03) with TP53 lesions were n-CLL. Cases with NOTCH1 (p=0.01) and SF3B1 (p=0.02) mutations were also enriched in n-CLL. Next, we investigated the impact of methylation signatures on progression-free (PFS) and overall survival (OS). In CT patients, n-, i- and m-CLL patients exhibited a median PFS of 23, 34 and 35 months, and OS of 63, 66 and 106 months, respectively. n-CLL showed significantly shorter PFS than i-CLL (HR 0.64, p<0.001) and m-CLL (HR 0.52, p<0.001), and had the shortest OS, again compared to i-CLL (HR 0.73, p=0.01) and m-CLL (HR 0.33, p<0.001). Ten-year OS differed according to epigenetic signature (P<0.001) and was reached by only 14% of n-CLL patients. Multivariate Cox proportional analysis, controlling for confounding variables (incl. clinical features, IGHV status, TP53, NOTCH1 and SF3B1) in 278 patients, showed that m-CLL was an independent prognostic factor for OS (HR 0.46, p<0.01). In 247 CIT patients, univariate analysis showed that the i- (HR:0.57, p=0.05) and m-CLL (HR:0.3, p=0.002) subgroups displayed longer PFS. In a multivariate model, including TP53 lesions and IGHV status (239 patients), the m-CLL subgroup retained independent prognostic significance (HR:0.25, p<0.001). In conclusion, we provide important evidence that DNA methylation analysis may aid in the identification of patients destined to demonstrate durable remissions when treated with these agents.

Background Vascular endothelial growth factor (VEGF) is critical for angiogenesis, vasculogenesis and alveolar type II cell differentiation. We aimed to study the functional pulmonary effects of antenatal intra-amniotic VEGF administration in a preterm rabbit model. Methods At 26 d gestational age (GA; term = 31–32 d), 60 μg recombinant rat VEGF164 in 250 μL vehicle or 250 μL placebo were injected intra-amniotically. Unexposed littermates served as internal controls At 28 d GA, fetuses were harvested (11 VEGF, 10 placebo, 10 control) for morphologic assessment or ventilated (11VEGF, 9 placebo, 8 control) with a Flexivent (Scireq, Montreal) ventilator allowing measurement of total lung capacity, resistance, compliance. Morphologic assessment includes airway and vascular morphometry and immunohistochemistry for Sp B, Flk 1, apoptotic (caspase 3) and proliferative markers (PCNA). Results Lung mechanics as well as number of Flk-1 positive cells were higher in treated pups than in placebo and control animals (ANOVA p Conclusion Antenatal intra-amniotic injection of VEGF during the canalicular phase results in a significant improvement in lung mechanics and a higher number of Flk-1 positive cells at 28 d (saccular stage). This may be a new approach to improve lung maturation.

Abstract The importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevis tadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of xVEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of xVEGF-D with xVEGF-C at suboptimal morpholino concentrations resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or xVEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.

Liver cirrhosis is a very complex disease in which several pathological processes such as inflammation, fibrosis, and pathological angiogenesis are closely integrated. We hypothesized that treatment with pharmacological agents with multiple mechanisms of action will produce superior results to those achieved by only targeting individual mechanisms. This study thus evaluates the therapeutic use of the multitargeted receptor tyrosine kinase inhibitor Sunitinib (SU11248). The in vitro effects of SU11248 were evaluated in the human hepatic stellate cell line LX-2 by measuring cell viability. The in vivo effects of SU11248 treatment were monitored in the livers of cirrhotic rats by measuring angiogenesis, inflammatory infiltrate, fibrosis, α-smooth muscle actin (α-SMA) accumulation, differential gene expression by microarrays, and portal pressure. Cirrhosis progression was associated with a significant enhancement of vascular density and expression of vascular endothelial growth factor-A, angiopoietin-1, angiopoietin-2, and placental growth factor in cirrhotic livers. The newly formed hepatic vasculature expressed vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1. Interestingly, the expression of these adhesion molecules was adjacent to areas of local inflammatory infiltration. SU11248 treatment resulted in a significant decrease in hepatic vascular density, inflammatory infiltrate, α-SMA abundance, LX-2 viability, collagen expression, and portal pressure. Conclusion: These results suggest that multitargeted therapies against angiogenesis, inflammation, and fibrosis merit consideration in the treatment of cirrhosis. (Hepatology 2007.)