Victor L. Thijssen

17β-Estradiol Antagonizes Cardiomyocyte Hypertrophy by Autocrine/Paracrine Stimulation of a Guanylyl Cyclase A Receptor-Cyclic Guanosine Monophosphate-Dependent Protein Kinase Pathway

Background—Significant gender-related differences exist in the development of left ventricular hypertrophy (LVH). In addition, administration of 17&bgr;-estradiol (E2) to ovariectomized female mice attenuates the development of LVH, demonstrating an antagonistic role for E2 in this process, although no molecular mechanism has been proposed for this phenomenon. Methods and Results—E2 attenuated phenylephrine and endothelin-1 induced hypertrophy in neonatal cardiomyocytes, and E2 directly induced atrial natriuretic factor (ANF) expression as assessed by Northern blot, immunocytochemical analyses, and transient transfection assays using ANF promoter deletion fragments. Both the antihypertrophic effects and ANF induction could be blocked by the estrogen receptor antagonist ICI 182,780, which demonstrates a genomic, estrogen receptor-dependent pathway. To mimic E2-induced autocrine/paracrine effects through stimulation of the guanylyl cyclase A receptor (ANF receptor), cardiomyocytes were stimulated with phenylephrine or endothelin-1 in the presence of exogenous ANF or 8-bromo-cyclic guanosine monophosphate (cGMP), both of which attenuated agonist-induced hypertrophy. Both estrogen and ANF increased cGMP activity. The antihypertrophic effect of ANF could be reduced with extracellular ANF antibodies in a dose-dependent manner. cGMP-dependent protein kinase mediates the antihypertrophic effects of E2, so cardiomyocytes were agonist stimulated in the presence of the cGMP-dependent protein kinase blocker KT-5823. KT-5823 not only reversed the antihypertrophic properties of E2, ANF, or 8-bromo-cGMP, but also evoked potentiation of hypertrophy. Conclusions—E2-mediated induction of ANF in cardiac hypertrophy contributes to its antagonistic effects in LVH.

MicroRNAs in the tumor endothelium: novel controls on the angioregulatory switchboard.

Tumor angiogenesis facilitates tumor metastasis and allows malignant tissues to grow beyond a diffusion limited size. It is a complex process that requires endothelial cells to execute specific steps during different phases. miRNAs are small non-coding RNAs that act as molecular switches to redirect the expression profile of a cell. Evidence is emerging that miRNAs are important players in endothelial cell biology and tumor angiogenesis. In this review we summarize the available data of miRNA expression in the endothelium. In addition, we describe the current knowledge regarding the function of miRNAs in endothelial cell biology. Finally, we discuss the potential applications of miRNA based treatment strategies in angiostatic cancer therapy.

Interfering with Gal-1–mediated angiogenesis contributes to the pathogenesis of preeclampsia

Preeclampsia (PE) is a pregnancy-specific disorder characterized by sudden onset of hypertension and proteinuria in the second half of pregnancy (>20 wk). PE is strongly associated with abnormal placentation and an excessive maternal inflammatory response. Galectin-1 (Gal-1), a member of a family of carbohydrate-binding proteins, has been shown to modulate several processes associated with placentation and to promote maternal tolerance toward fetal antigens. Here, we show that Gal-1 exhibits proangiogenic functions during early stages of pregnancy, promoting decidual vascular expansion through VEGF receptor 2 signaling. Blocking Gal-1–mediated angiogenesis or lectin, galactoside-binding, soluble, 1 deficiency results in a spontaneous PE-like syndrome in mice, mainly by deregulating processes associated with good placentation and maternal spiral artery remodeling. Consistent with these findings, we observed a down-regulation of Gal-1 in patients suffering from early onset PE. Collectively, these results strengthen the notion that Gal-1 is required for healthy gestation and highlight Gal-1 as a valuable biomarker for early PE diagnosis.

Myocyte Enhancer Factor 2 and Class II Histone Deacetylases Control a Gender-Specific Pathway of Cardioprotection Mediated by the Estrogen Receptor

Rationale: Gender differences in cardiovascular disease have long been recognized and attributed to beneficial cardiovascular actions of estrogen. Class II histone deacetylases (HDACs) act as key modulators of heart disease by repressing the activity of the myocyte enhancer factor (MEF)2 transcription factor, which promotes pathological cardiac remodeling in response to stress. Although it is proposed that HDACs additionally influence nuclear receptor signaling, the effect of class II HDACs on gender differences in cardiovascular disease remains unstudied. Objective: We aimed to examine the effect of class II HDACs on post–myocardial infarction remodeling in male and female mice. Methods and Results: Here we show that the absence of HDAC5 or -9 in female mice protects against maladaptive remodeling following myocardial infarction, during which there is an upregulation of estrogen-responsive genes in the heart. This genetic reprogramming coincides with a pronounced increase in expression of the estrogen receptor (ER)&agr; gene, which we show to be a direct MEF2 target gene. ER&agr; also directly interacts with class II HDACs. Cardioprotection resulting from the absence of HDAC5 or -9 in female mice can be attributed, at least in part, to enhanced neoangiogenesis in the infarcted region via upregulation of the ER target gene vascular endothelial growth factor-a. Conclusions: Our results reveal a novel gender-specific pathway of cardioprotection mediated by ER&agr; and its regulation by MEF2 and class II HDACs.

Endothelial LGALS9 splice variant expression in endothelial cell biology and angiogenesis

Galectins are carbohydrate binding proteins with versatile functions in tumor progression. Galectin-9, encoded by LGALS9, has been associated with metastasis and immunosuppression. We previously reported on regulation of LGALS9 expression during endothelial cell activation. Here, we show increased galectin-9 protein levels in the endothelium of different tumors, including carcinomas of the lung, liver, breast and kidney. Endothelial cells were found to express five LGALS9 splice variants, two of which have not been reported before. Splicing was found to be confined to exons 5, 6 and 10. Transfection of human microvascular endothelial cells (HMEC) with galectin-9∆5, a specific LGALS9 splice variant, induced a small but significant increase of proliferation, while migration was not affected by any LGALS9 splice variant. Application of recombinant galectin-9∆5 protein dose-dependently reduced proliferation and migration of HMEC as well as human umbilical vein endothelial cells in vitro. Enhanced sprouting and migration of human umbilical vein endothelial cell (HUVEC) towards a galectin-9∆5 gradient were observed. Interestingly, galectin-9∆5 was found to induce a small inhibitory effect on angiogenesis in vivo. Collectively, these data show that endothelial cells regulate the expression and splicing of LGALS9 during angiogenesis. The function of the dominant splice variant, i.e. galectin-9∆5, in endothelial cell biology depends on the concentration and environmental context in which it is presented to the cells.

Profiling Lgals9 Splice Variant Expression at the Fetal-Maternal Interface: Implications in Normal and Pathological Human Pregnancy

ABSTRACT Disruption of fetal-maternal tolerance mechanisms can contribute to pregnancy complications, including spontaneous abortion. Galectin-9 (LGALS9), a tandem repeat lectin associated with immune modulation, is expressed in the endometrium during the mid and late secretory phases and in decidua during human early pregnancy. However, the role of LGALS9 during pregnancy remains poorly understood. We used real-time PCR and immunohistochemical staining to analyze the expression of Lgals9/LGALS9 during mouse gestation as well as in human tissues obtained from normal pregnancy and spontaneous abortions. In mice, three Lgals9 splice variants were detected, the expression of which was differentially regulated during gestation. Furthermore, decidual Lgals9 expression was deregulated in a mouse model of spontaneous abortion, whereas placental levels did not change. We further found that the LGALS9 D5 isoform suppresses interferon gamma production by decidual natural killer cells. In human patients, six Lgals9 splice variants were detected, and a decrease in Lgals9 D5/10 was associated with spontaneous abortion. Altogether, these results show a differential regulation of Lgals9 isoform expression during normal and pathological pregnancies and designate Lgals9 as a potential marker for adverse pregnancy outcomes.

The clinical application of angiostatic therapy in combination with radiotherapy: past, present, future

Although monotherapy with angiostatic drugs is still far from effective, there is abundant evidence that angiostatic therapy can improve the efficacy of conventional treatments like radiotherapy. This has instigated numerous efforts to optimize and clinically implement the combination of angiostatic drugs with radiation treatment. The results from past and present clinical trials that explored this combination therapy indeed show encouraging results. However, current findings also show that the combination has variable efficacy and is associated with increased toxicity. This indicates that combining radiotherapy with angiostatic drugs not only holds opportunities but also provides several challenges. In the current review, we provide an update of the most recent insights from clinical trials that evaluated the combination of angiostatic drugs with radiation treatment. In addition, we discuss the outstanding questions for future studies in order to improve the clinical benefit of combining angiostatic therapy with radiation therapy.

Abstract 797: Kinase activity profiling of tumor tissues of different origin

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnIntroduction:nnMany new anti-cancer drugs target kinase activity. Unfortunately, methods to monitor the drug effects at the enzymatic level in patient derived tumor tissue are lacking. Here, a novel molecular profiling method for application in biomarker discovery is presented. This method is based on measuring kinase activities in tumor tissue extracts, and involves assessment of inhibition by a drug of interest. This ex vivo activity-based approach is enabled by dynamic peptide microarrays. These arrays comprise of peptides, which are known substrates for phosphorylation by protein kinases. Here we investigated the applicability of this approach in the classification of multiple tumors of different origin.nnMethod:nnPatient-derived fresh frozen tissues of different tumor types (24 breast cancer (BC), 14 non-small cell lung cancer (NSCLC), 12 renal cell carcinomas (RCC)) were used for extraction of total protein in lysis buffer. Dependent on the tissue source, 6 slices of 10 µm were sufficient for 20-100 microarray analyses. Equal amounts of total protein were analyzed for kinase activity on dynamic PamChip peptide microarrays. These arrays comprised 144 or 256 different peptides. A PamChip96 plate format was used, enabling 96 microarray incubations per run. Protein amounts ranging from 1 − 5 µg were used per microarray analysis. Tumor extracts were analysed in the presence and absence of kinase inhibitor drugs (RCC: sorafenib and sunitinib, NSCLC: gefitinib).nnResults:nnWe show here that from different tumors kinase activity profiles could be generated reproducibly (10-15% CV for selected peptides). Control experiments showed protein concentration and ATP dependency. In the RCC experiments different phosphorylation profiles were obtained when the non-tumor tissue was compared to tumor tissue derived from the same patient, showing higher kinase activities in the latter. In addition, inhibitor dependent modulation of the peptide phosphorylations was observed (sorafenib vs. sunitinib). Furthermore, differential kinase activity profiles were obtained that could be correlated to different patient subgroups (BC) or clinical data (long vs. short term survivors; NSCLC).nnConclusion:nnA novel molecular profiling approach was successfully applied for classification of different tumors. This approach is based on detection of kinase activities as well as inhibition of kinase activity in tumor tissues. Application of this method in the discovery of biomarkers for diagnosis, prognosis or prediction of drug response is foreseen.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 797.