Publications

Abstract Placental growth factor (PlGF) is associated selectively with pathological angiogenesis, and PlGF blockade does not affect the healthy vasculature. Anti-PlGF is therefore currently being clinically evaluated for the treatment of cancer patients. In cirrhosis, hepatic fibrogenesis is accompanied by extensive angiogenesis. In this paper, we evaluated the pathophysiological role of PlGF and the therapeutic potential of anti-PlGF in liver cirrhosis. PlGF was significantly up-regulated in the CCl4-induced rodent model of liver cirrhosis as well as in cirrhotic patients. Compared with wild-type animals, cirrhotic PlGF−/− mice showed a significant reduction in angiogenesis, arteriogenesis, inflammation, fibrosis, and portal hypertension. Importantly, pharmacological inhibition with anti-PlGF antibodies yielded similar results as genetic loss of PlGF. Notably, PlGF treatment of activated hepatic stellate cells induced sustained extracellular signal-regulated kinase 1/2 phosphorylation, as well as chemotaxis and proliferation, indicating a previously unrecognized profibrogenic role of PlGF. Conclusion: PlGF is a disease-candidate gene in liver cirrhosis, and inhibition of PlGF offers a therapeutic alternative with an attractive safety profile. (Hepatology 2011;)

Objective— To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Methods and Results— Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain–containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. Conclusion— Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell–expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

2056 Background: Medulloblastoma survival rates have significantly improved over the last decades due to surgery and chemoradiation regimens. However, pediatric patients with high-risk disease and those with recurrence often succumb to their disease. The majority of children suffer from severe adverse effects of intense therapy regimens. We recently demonstrated the role of the Placental Growth Factor (PlGF)/Nrp-1 signaling pathway in medulloblastoma progression in preclinical models (Snuderl et al., Cell 2013). The aim of this study was to evaluate the role of Nrp-1 as a biomarker and therapeutic target in medulloblastoma patients. Methods: Thirty-two surgical samples of pediatric medulloblastoma were analyzed for expression of Nrp-1 and its ligand PlGF by immunohistochemistry, array-comparative genomic hybridization and deep gene sequencing. On an independent, clinically annotated cohort of 42 medulloblastoma samples, we evaluated the correlation between Nrp-1 expression and 5-year overall survival. The therapeutic relevance of Nrp-1 signaling was tested in a D283 orthotopic human xenograft mouse model using shRNA and a monoclonal antibody against Nrp-1. Results: Nrp-1 expression was detectable in 100% and PlGF in 90% of all medulloblastoma samples. No significant differences in expression could be detected between known histological or molecular medulloblastoma subtypes. Patients with high Nrp-1 expression had a significantly worse 5-year overall survival compared to patients with a low or moderate expression (40% vs. 86%, p = 0.0058). Treatment with an antibody against Nrp-1 or genetic silencing of Nrp-1 with shRNA significantly prolonged survival in a D283 orthotopic human xenograft mouse model (for both experiments: p < 0.01). Conclusions: Nrp-1 is widely expressed across all molecular subgroups of medulloblastoma and its expression levels are inversely associated with survival of patients. Targeting Nrp-1 leads to a significant survival benefit in an orthotopic human xenograft mouse model. This data support the hypothesis that targeting the PlGF/Nrp-1 signaling pathway may be a novel therapy approach for pediatric medulloblastoma.

Several lines of indirect evidence suggest that plasminogen activation plays a crucial role in degradation of the follicular wall during ovulation. However, single-deficient mice lacking tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), or PA inhibitor type 1(PAI-1) gene function were recently found to have normal reproduction, although mice with a combined deficiency of tPA and uPA were significantly less fertile. To investigate whether the reduced fertility of mice lacking PA gene function is due to a reduced ovulation mechanism, we have determined the ovulation efficiency in 25-day-old mice during gonadotropin-induced ovulation. Our results reveal that ovulation efficiency is normal in mice with a single deficiency of tPA or uPA but reduced by 26% in mice lacking both physiological PAs. This result suggests that plasminogen activation plays a role in ovulatory response, although neither tPA nor uPA individually or in combination is obligatory for ovulation. The loss of an individual PA seems to be functionally complemented by the remaining PA but this compensation does not appear to involve any compensatory up-regulation. Our data imply that a functionally redundant mechanism for plasmin formation operates during gonadotropin-induced ovulation and that PAs together with other proteases generate the proteolytic activity required for follicular wall degradation.

<b>Purpose:</b> MMP-9 is the prominent isozyme of matrix metalloproteinases (MMP) known to be highly expressed in human atherosclerotic plaques. One potent inhibitor of MMP-9 production is the transforming growth factor beta (TGF-β). Similar to this, we also observed inhibitory effects with sphingosine 1-phosphate (S1P), accumulation in high density lipoprotein (HDL), which presumably suppresses MMP-9 production. The aim of this study was to reveal the inhibitory influence of HDL on MMP-9 production and the molecular mechanisms behind these processes. <b>Methods:</b> Rat vascular smooth muscle cells (VSMC) were stimulated with interleukin-1β (IL-1β). The following MMP-9 expression was quantified by real-time PCR and activity by zymography. For receptor knockdown studies, cells were transfected with siRNA against S1P3 receptor. <b>Results:</b> TGF-β significantly down-regulated IL-1β stimulated MMP-9 expression (78.6% ± 5.4 decrease; p < 0.05, n = 10). This inhibition could be significantly blocked with the TGF-β receptor inhibitor R1 (36.7% ± 2.9 vs. 78.6% ± 5.4 decrease; p < 0.05; n = 9). Like S1P, HDL also significantly suppressed the IL-1β stimulated MMP-9 expression (S1P: 42.2% ± 8.3 decrease n = 5; HDL: 44.3% ± 9.8 decrease; n = 10; p < 0.05). To reveal if there is any cross-communication between the TGF-β and S1P receptors, we also tested the TGF-β receptor inhibitor in the IL-1β/HDL and IL-1β/S1P costimulation, which significantly reversed the effect of HDL (12.2 % ± 4.6 vs. 44.3 % ± 2.4 decrease; p < 0.05; n = 7) and S1P (42.2% ± 3.5 vs. 72.3% ± 8.3 decrease; p < 0.05; n = 7). Investigation of the MMP-9 activity via zymography showed corresponding results after TGF-β and S1P treatment in the absence and presence of the inhibitor. The specific downregulation of the S1P3 receptor by siRNA abolished the HDL/S1P induced TGF-β signaling. <b>Conclusions:</b> HDL, S1P and TGF-β all down-regulate MMP-9 expression. The HDL and S1P mediated inhibition of MMP-9 expression could be diminished by a TGF-β receptor inhibitor and by S1P3 receptor knockdown. Therefore, HDL and its component S1P could cross activate the TGF-β signaltransduction pathway via the S1P3 receptor.