To provide an investigative tool for the study of osteosarcoma (OSA) biology we have developed a syngeneic (balb/c) murine model of OSA, using cell lines derived from a spontaneously occurring murine OSA (Schmidt et al. Differentiation 1988; 39: 151-60). This model is characterized by orthotopic primary tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and clonally related variants (K7M2 and K12) that differ in pulmonary metastatic potential. Primary tumor and pulmonary metastasis histology was consistent with OSA in human patients. Expression of bone sialoprotein, biglyan, decorrin, and osteopontin was suggestive of bone lineage cells. The development and use of a more aggressive OSA cell line (K7M2) resulted in spontaneous metastasis to the lungs in over 90% of mice, whereas metastases were seen in only 33% of mice when a less aggressive OSA cell line (K12; Schmidt et al. Differentiation 1988; 39: 151-60) was used. Death from metastasis occurred at a median of 76 days using K7M2 whereas no median was achieved after 140 days using K12. Angiogenic potential, characterized by CD31 and factor VIII staining of primary tumors and pulmonary metastases, was greater in the K7M2 model compared to the K12 model. No significant differences in the in vitro or in vivo expression of angiogenesis associated genes (flt1, flt4, TIE1, TIE2, and VEGF) was found between K7M2 and K12. This well characterized and relevant model of OSA will be a valuable resource to improve our understanding of the biology and treatment of metastasis in OSA.

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh(-/-) mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and alpha-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh(-/-) lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or SMO: Whereas Ihh(-/-) yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo(-/-) yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh(-/-) yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.

Preeclampsia is a systemic syndrome of pregnancy that originates in the placenta and is characterized by widespread maternal endothelial dysfunction. Until recently, the molecular pathogenesis of preeclampsia was largely unknown, but recent work suggests a key role for altered expression of placental antiangiogenic factors. Soluble Flt1 and soluble endoglin, secreted by the placenta, are increased in the maternal circulation weeks before the onset of preeclampsia. These antiangiogenic factors produce systemic endothelial dysfunction, resulting in hypertension, proteinuria, and the other systemic manifestations of preeclampsia. The molecular basis for placental dysregulation of these pathogenic factors remains unknown, and the role of angiogenic proteins in early placental vascular development is just beginning to be explored. These discoveries have exciting clinical implications and are likely to transform the detection and treatment of preeclampsia in the future.

Although the systemic administration of a number of different gene products has been shown to result in the inhibition of angiogenesis and tumor growth in different animal tumor models, the relative potency of those gene products has not been studied rigorously. To address this issue, recombinant adenoviruses encoding angiostatin, endostatin, and the ligand-binding ectodomains of the vascular endothelial growth factor receptors Flk1, Flt1, and neuropilin were generated and used to systemically deliver the different gene products in several different preexisting murine tumor models. Single i.v. injections of viruses encoding soluble forms of Flk1 or Flt1 resulted in approximately 80% inhibition of preexisting tumor growth in murine models involving both murine (Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic carcinoma) tumors. In contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. A strong correlation was observed between the effects of the different viruses on tumor growth and the activity of the viruses in the inhibition of corneal micropocket angiogenesis. These data underscore the need for comparative analyses of different therapeutic approaches that target tumor angiogenesis and provide a rationale for the selection of specific antiangiogenic gene products as lead candidates for use in gene therapy approaches aimed at the treatment of malignant and ocular disorders.

The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney.

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies.

Preeclampsia, a hypertensive disorder peculiar to pregnancy, is a systemic syndrome that appears to originate in the placenta and is characterized by widespread maternal endothelial dysfunction. Until recently, the molecular pathogenesis of phenotypic preeclampsia was largely unknown, but recent observations support the hypothesis that altered expression of placental anti-angiogenic factors are responsible for the clinical manifestations of the disease. Soluble Flt1 and soluble endoglin, secreted by the placenta, are increased in the maternal circulation weeks before the onset of preeclampsia. These anti-angiogenic factors produce systemic endothelial dysfunction, resulting in hypertension, proteinuria, and the other systemic manifestations of preeclampsia. The molecular basis for placental dysregulation of these pathogenic factors remains unknown, and as of 2011 the role of angiogenic proteins in early placental vascular development was starting to be explored. The data linking angiogenic factors to preeclampsia have exciting clinical implications, and likely will transform the detection and treatment of preeclampsia.

The microRNA-200 (miR-200) family is part of a gene expression signature that predicts poor prognosis in lung cancer patients. In a mouse model of K-ras/p53-mutant lung adenocarcinoma, miR-200 levels are suppressed in metastasis-prone tumor cells, and forced miR-200 expression inhibits tumor growth and metastasis, but the miR-200 target genes that drive lung tumorigenesis have not been fully elucidated. Here, we scanned the genome for putative miR-200 binding sites and found them in the 3'-untranslated region (3'-UTR) of 35 genes that are amplified in human cancer. Mining of a database of resected human lung adenocarcinomas revealed that the levels of one of these genes, Flt1/VEGFR1, correlate inversely with duration of survival. Forced miR-200 expression suppressed Flt1 levels in metastasis-prone lung adenocarcinoma cells derived from K-ras/p53-mutant mice, and negatively regulated the Flt1 3'-UTR in reporter assays. Cancer-associated fibroblasts (CAFs) isolated from murine lung adenocarcinomas secreted abundant VEGF and enhanced tumor cell invasion in coculture studies. CAF-induced tumor cell invasion was abrogated by VEGF neutralization or Flt1 knockdown in tumor cells. Flt1 knockdown decreased the growth and metastasis of tumor cells in syngeneic mice. We conclude that miR-200 suppresses lung tumorigenesis by targeting Flt1.

Nonalcoholic fatty liver disease (NAFLD) is an emerging disease with a broad spectrum of liver conditions. The complex molecular pathogenesis of NAFLD is still unclear. In this study, we conducted an analysis of microRNA (miRNA) expression profiles in liver of rats made NAFLD by different diets. To this aim, Sprague-Dawley rats were fed ad libitum for 3 months with different diets: standard diet (SD), diet enriched in fats and low in carbohydrates (HFD), SD with high fructose (SD-HF) and diet with high levels of fats and fructose (HFD-HF). Our results demonstrated that the treatment with different dietetic regimens caused a significant increase of the body weight and the alteration of some metabolic parameters compared with control animals, as well as various liver injuries. The miRNAs analysis showed the significant downregulation of three miRNAs (miR-122, miR-451 and miR-27) and the upregulation of miR-200a, miR-200b and miR-429 in HFD, SD-HF and HFD-HF rats. Besides, miR-21 expression was significantly decreased only in fructose-enriched diets. These miRNAs target molecules involved in the control of lipid and carbohydrate metabolism, signal transduction, cytokine and chemokine-mediated signaling pathway and apoptosis. Western blot analysis of PKCδ, LITAF, ALDOLASE-A, p38MAPK, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 showed a diet-induced deregulation of all these proteins. Interestingly, the expression pattern of LITAF, PTEN, LIPIN1, EPHRIN-A1, EPHA2 and FLT1 might be well explained by the trend of their specific mRNAs, by potentially regulatory miRNAs, or both. In conclusion, we highlight for the first time the potential involvement of novel determinants (miRNAs and proteins) in the molecular pathogenesis of diet-induced NAFLD.

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.

CD34 is frequently used as a marker of adipose-derived stem/stromal/progenitor cells (ASCs). However, CD34 expression in human ASCs (hASCs) decreases over time in culture, and the implications of this remain unclear. In this study, we sorted shortly-cultured hASCs into CD34+ and CD34- fractions and compared their biological functions. Results indicated that CD34+ hASCs were more proliferative and had a greater ability to form colonies. In contrast, CD34- cells showed a greater ability for differentiation into adipogenic and osteogenic lineages. Both CD34+ and CD34- cells showed similar abilities in migration and capillary formation in response to growth factors. Expression levels of endothelial progenitor markers (Flk-1, FLT1, and Tie-2) were higher in CD34+ cells, whereas those of pericyte markers (CD146, NG2, and alpha-smooth muscle actin) were higher in CD34- cells. Both CD34+ and CD34- cells showed similar expression levels of vascular endothelial growth factor and hepatocyte growth factor, although hypoxia affected the expression levels. Together these results suggest that CD34 expression in hASCs may correlate with replicative capacity, differentiation potentials, expression profiles of angiogenesis-related genes, and immaturity or stemness of the cells. Loss of CD34 expression may be related to the physiological process of commitment and/or differentiation from immature status into specific lineages such as adipose, bone, or smooth muscle.

Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment in vivo, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.

Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). Endothelial-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of vascular endothelial growth factor (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, we identified two allelic zebrafish mutants in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression was severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis was unchanged. Overexpression of vegf(121+165) led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. We demonstrate that flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos. However, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis.

A CpG island methylator phenotype (CIMP) is displayed by a distinct subset of colorectal cancers with a high frequency of DNA hypermethylation in a specific group of CpG islands. Recent studies have shown that an activating mutation of BRAF (BRAF(V600E)) is tightly associated with CIMP, raising the question of whether BRAF(V600E) plays a causal role in the development of CIMP or whether CIMP provides a favorable environment for the acquisition of BRAF(V600E). We employed Illumina GoldenGate DNA methylation technology, which interrogates 1,505 CpG sites in 807 different genes, to further study this association. We first examined whether expression of BRAF(V600E) causes DNA hypermethylation by stably expressing BRAF(V600E) in the CIMP-negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We determined 100 CIMP-associated CpG sites and examined changes in DNA methylation in eight stably transfected clones over multiple passages. We found that BRAF(V600E) is not sufficient to induce CIMP in our system. Secondly, considering the alternative possibility, we identified genes whose DNA hypermethylation was closely linked to BRAF(V600E) and CIMP in 235 primary colorectal tumors. Interestingly, genes that showed the most significant link include those that mediate various signaling pathways implicated in colorectal tumorigenesis, such as BMP3 and BMP6 (BMP signaling), EPHA3, KIT, and FLT1 (receptor tyrosine kinases) and SMO (Hedgehog signaling). Furthermore, we identified CIMP-dependent DNA hypermethylation of IGFBP7, which has been shown to mediate BRAF(V600E)-induced cellular senescence and apoptosis. Promoter DNA hypermethylation of IGFBP7 was associated with silencing of the gene. CIMP-specific inactivation of BRAF(V600E)-induced senescence and apoptosis pathways by IGFBP7 DNA hypermethylation might create a favorable context for the acquisition of BRAF(V600E) in CIMP+ colorectal cancer. Our data will be useful for future investigations toward understanding CIMP in colorectal cancer and gaining insights into the role of aberrant DNA hypermethylation in colorectal tumorigenesis.

Mucoepidermoid carcinomas (MECs) of the salivary and bronchial glands are characterized by a recurrent t(11;19)(q21;p13) translocation resulting in a MECT1-MAML2 fusion in which the CREB-binding domain of the CREB coactivator MECT1 (also known as CRTC1, TORC1 or WAMTP1) is fused to the transactivation domain of the Notch coactivator MAML2. To gain further insights into the molecular pathogenesis of MECs, we cytogenetically and molecularly characterized a series of 29 MECs. A t(11;19) and/or an MECT1-MAML2 fusion was detected in more than 55% of the tumors. Several cases with cryptic rearrangements that resulted in gene fusions were detected. In fusion-negative MECs, the most common aberration was a single or multiple trisomies. Western blot and immunohistochemical studies demonstrated that the MECT1-MAML2 fusion protein was expressed in all MEC-specific cell types. In addition, cotransfection experiments showed that the fusion protein colocalized with CREB in homogeneously distributed nuclear granules. Analyses of potential downstream targets of the fusion revealed differential expression of the cAMP/CREB (FLT1 and NR4A2) and Notch (HES1 and HES5) target genes in fusion-positive and fusion-negative MECs. Moreover, clinical follow-up studies revealed that fusion-positive patients had a significantly lower risk of local recurrence, metastases, or tumor-related death compared to fusion-negative patients (P = 0.0012). When considering tumor-related deaths only, the estimated median survival for fusion-positive patients was greater than 10 years compared to 1.6 years for fusion-negative patients. These findings suggest that molecularly classifying MECs on the basis of an MECT1-MAML2 fusion is histopathologically and clinically relevant and that the fusion is a useful marker in predicting the biological behavior of MECs.

Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.

Sprouting angiogenesis expands the embryonic vasculature enabling survival and homeostasis. Yet how the angiogenic capacity to form sprouts is allocated among endothelial cells (ECs) to guarantee the reproducible anatomy of stereotypical vascular beds remains unclear. Here we show that Sema-PlxnD1 signaling, previously implicated in sprout guidance, represses angiogenic potential to ensure the proper abundance and stereotypical distribution of the trunk's segmental arteries (SeAs). We find that Sema-PlxnD1 signaling exerts this effect by antagonizing the proangiogenic activity of vascular endothelial growth factor (VEGF). Specifically, Sema-PlxnD1 signaling ensures the proper endothelial abundance of soluble flt1 (sflt1), an alternatively spliced form of the VEGF receptor Flt1 encoding a potent secreted decoy. Hence, Sema-PlxnD1 signaling regulates distinct but related aspects of angiogenesis: the spatial allocation of angiogenic capacity within a primary vessel and sprout guidance.

A human-specific splicing variant of vascular endothelial growth factor (VEGF) receptor 1 (Flt1) was discovered, producing a soluble receptor (designated sFlt1-14) that is qualitatively different from the previously described soluble receptor (sFlt1) and functioning as a potent VEGF inhibitor. sFlt1-14 is generated in a cell type-specific fashion, primarily in nonendothelial cells. Notably, in vascular smooth muscle cells, all Flt1 messenger RNA is converted to sFlt1-14, whereas endothelial cells of the same human vessel express sFlt1. sFlt1-14 expression by vascular smooth muscle cells is dynamically regulated as evidenced by its upregulation on coculture with endothelial cells or by direct exposure to VEGF. Increased production of soluble VEGF receptors during pregnancy is entirely attributable to induced expression of placental sFlt1-14 starting by the end of the first trimester. Expression is dramatically elevated in the placenta of women with preeclampsia, specifically induced in abnormal clusters of degenerative syncytiotrophoblasts known as syncytial knots, where it may undergo further messenger RNA editing. sFlt1-14 is the predominant VEGF-inhibiting protein produced by the preeclamptic placenta, accumulates in the circulation, and hence is capable of neutralizing VEGF in distant organs affected in preeclampsia. Together, these findings revealed a new natural VEGF inhibitor that has evolved in humans, possibly to protect nonendothelial cells from adverse VEGF signaling. Furthermore, the study uncovered the identity of a VEGF-blocking protein implicated in preeclampsia.

BACKGROUND

No biomarkers that could guide patient selection for treatment with the anti-VEGF monoclonal antibody bevacizumab have been identified. We assessed whether genetic variants in the VEGF pathway could act as biomarkers for bevacizumab treatment outcome.

METHODS

We investigated DNA from white patients from two phase 3 randomised studies. In AViTA, patients with metastatic pancreatic adenocarcinoma were randomly assigned to receive gemcitabine and erlotinib plus either bevacizumab or placebo. In AVOREN, patients with metastatic renal-cell carcinoma were randomly assigned to receive interferon alfa-2a plus either bevacizumab or placebo. We assessed the correlation of 138 SNPs in the VEGF pathway with progression-free survival and overall survival in a subpopulation of patients from AViTA. Significant findings were confirmed in a subpopulation of patients from AVOREN and functionally studied at the molecular level.

RESULTS

We investigated DNA of 154 patients from AViTA, of whom 77 received bevacizumab, and 110 patients from AVOREN, of whom 59 received bevacizumab. Only rs9582036, a SNP in VEGF receptor 1 (VEGFR1 or FLT1), was significantly associated with overall survival in the bevacizumab group of AViTA after correction for multiplicity (per-allele hazard ratio [HR] 2·1, 95% CI 1·45-3·06, p=0·00014). This SNP was also associated with progression-free survival (per-allele HR 1·89, 1·31-2·71, p=0·00081) in bevacizumab-treated patients from AViTA. AC and CC carriers of this SNP exhibited HRs for overall survival of 2·0 (1·19-3·36; p=0·0091) and 4·72 (2·08-10·68; p=0·0002) relative to AA carriers. No effects were seen in placebo-treated patients and a significant genotype by treatment interaction (p=0·041) was recorded, indicating that the VEGFR1 locus containing this SNP serves as a predictive marker for bevacizumab treatment outcome in AViTA. Fine-mapping experiments of this locus identified rs7993418, a synonymous SNP affecting tyrosine 1213 in the VEGFR1 tyrosine-kinase domain, as the functional variant underlying the association. This SNP causes a shift in codon usage, leading to increased VEGFR1 expression and downstream VEGFR1 signalling. This VEGFR1 locus correlated significantly with progression-free survival (HR 1·81, 1·08-3·05; p=0·033) but not overall survival (HR 0·91, 0·45-1·82, p=0·78) in the bevacizumab group in AVOREN.

CONCLUSIONS

A locus in VEGFR1 correlates with increased VEGFR1 expression and poor outcome of bevacizumab treatment. Prospective assessment is underway to validate the predictive value of this novel biomarker.

BACKGROUND

F Hoffmann-La Roche.

MicroRNAs comprise a group of non-coding small RNAs (17-25 nt) involved in post-transcriptional regulation that have been identified in various plants and animals. Studies have demonstrated that miRNAs are associated with stem cell self-renewal and differentiation and play a key role in controlling stem cell activities. However, the identification of specific miRNAs and their regulatory roles in the differentiation of multipotent mesenchymal stromal cells (MSCs) have so far been poorly defined. We isolated and cultured human MSCs and osteo-differentiated MSCs from four individual donors. miRNA expression in MSCs and osteo-differentiated MSCs was investigated using miRNA microarrays. miRNAs that were commonly expressed in all three MSC preparations and miRNAs that were differentially expressed between MSCs and osteo-differentiated MSCs were identified. Four underexpressed (hsa-miR-31, hsa-miR-106a, hsa-miR-148a, and hsa-miR-424) and three novel overexpressed miRNAs (hsa-miR-30c, hsa-miR-15b, and hsa-miR-130b) in osteo-differentiated MSCs were selected and their expression were verified in samples from the fourth individual donors. The putative targets of the miRNAs were predicted using bioinformatic analysis. The four miRNAs that were underexpressed in osteo-differentiated MSCs were predicted to target RUNX2, CBFB, and BMPs, which are involved in bone formation; while putative targets for miRNAs overexpressed in osteo-differentiated MSCs were MSC maker (e.g., CD44, ITGB1, and FLT1), stemness-maintaining factor (e.g., FGF2 and CXCL12), and genes related to cell differentiation (e.g., BMPER, CAMTA1, and GDF6). Finally, hsa-miR-31 was selected for target verification and function analysis. The results of this study provide an experimental basis for further research on miRNA functions during osteogenic differentiation of human MSCs.

OBJECTIVE

In the era of DNA-guided personalized cancer treatment, it is essential to conduct predictive analysis on the tissue that matters. Here, we analyzed genetic differences between primary colorectal adenocarcinomas (CRC) and their respective hepatic metastasis.

METHODS

The primary CRC and the subsequent hepatic metastasis of 21 patients with CRC were analyzed using targeted deep-sequencing of DNA isolated from formalin-fixed, paraffin-embedded archived material.

RESULTS

We have interrogated the genetic constitution of a designed "Cancer Mini-Genome" consisting of all exons of 1,264 genes associated with pathways relevant to cancer. In total, 6,696 known and 1,305 novel variations were identified in 1,174 and 667 genes, respectively, including 817 variants that potentially altered protein function. On average, 83 (SD = 69) potentially function-impairing variations were gained in the metastasis and 70 (SD = 48) variations were lost, showing that the primary tumor and hepatic metastasis are genetically significantly different. Besides novel and known variations in genes such as KRAS, BRAF, KDR, FLT1, PTEN, and PI3KCA, aberrations in the up/downstream genes of EGFR/PI3K/VEGF-pathways and other pathways (mTOR, TGFβ, etc.) were also detected, potentially influencing therapeutic responsiveness. Chemotherapy between removal of the primary tumor and the metastasis (N = 11) did not further increase the amount of genetic variation.

CONCLUSIONS

Our study indicates that the genetic characteristics of the hepatic metastases are different from those of the primary CRC tumor. As a consequence, the choice of treatment in studies investigating targeted therapies should ideally be based on the genetic properties of the metastasis rather than on those of the primary tumor.

OBJECTIVE

We investigated whether vascular endothelial growth factor (VEGF) was associated with the angiogenic and therapeutic effects induced after transplantation of skeletal muscle-derived stem cells (MDSCs) into a myocardial infarction (MI).

BACKGROUND

Because very few MDSCs were found to differentiate into new blood vessels when injected into the heart, the mechanism underlying the occurrence of angiogenesis after MDSC transplantation is currently unknown. In the present study, we used a gain- or loss-of-VEGF function approach with skeletal MDSCs engineered to express VEGF or soluble Flt1, a VEGF-specific antagonist, to identify the involvement of VEGF in MDSC transplantation-induced neoangiogenesis.

METHODS

Vascular endothelial growth factor- and soluble Flt1-engineered MDSCs were injected into an acute MI. Angiogenesis and cardiac function were evaluated by immunohistochemistry and echocardiography.

RESULTS

Both control and VEGF-overexpressing MDSCs induced angiogenesis, prevented adverse cardiac remodeling, and improved function compared with saline-injected hearts. However, these therapeutic effects were diminished in hearts transplanted with MDSCs expressing soluble Flt1 despite successful cell engraftment. In vitro experiments demonstrated that MDSCs increased secretion of VEGF in response to hypoxia and cyclic stretch (likely conditions in ischemic hearts), suggesting that transplanted MDSCs release VEGF in vivo.

CONCLUSIONS

Our findings suggest that VEGF is essential for the induction of angiogenesis and functional improvements observed after MDSC transplantation for infarct repair.

Pre-eclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset pre-eclampsia (EOPET) is a severe form of pre-eclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. This case-control study focused on DNA methylation and gene expression of whole chorionic villi samples from 20 EOPET placentas and 20 gestational age-matched controls from pre-term births. DNA methylation was also assessed in placentas affected by late-onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR). The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation at >480 000 cytosine-guanine dinucleotide (CpG) sites. The Illumina HT-12v4 Expression BeadChip was used to assess gene expression of >45 000 transcripts in a subset of cases and controls. DNA methylation analysis by pyrosequencing was used to follow-up the initial findings in four genes with a larger cohort of cases and controls, including nIUGR and LOPET placentas. Bioinformatic analysis was used to identify overrepresentation of gene ontology categories and transcription factor binding motifs. We identified 38 840 CpG sites with significant (false discovery rate <0.01) DNA methylation alterations in EOPET, of which 282 had >12.5% methylation difference compared with the controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. EOPET, but not associated clinical features, such as intrauterine growth restriction (IUGR), presented a distinct DNA methylation profile. CpG sites from four genes relevant to pre-eclampsia (INHBA, BHLHE40, SLC2A1 and ADAM12) showed different extent of changes in LOPET and nIUGR. Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Results could be confounded by altered cell populations in abnormal placentas. Larger sample sizes are needed to fully address the possibility of sub-profiles of methylation within the EOPET cohort. Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. This study identifies DNA methylation changes at many loci previously reported to have altered gene expression in EOPET placentas, as well as in novel biologically relevant genes we confirmed to be differentially expressed. These results may be useful for DNA- methylation-based non-invasive prenatal diagnosis of at-risk pregnancies.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine involved in angiogenesis, inflammation, and wound healing. It is secreted by a variety of tumor cell lines, including hematopoietic lines. Therefore, we investigated expression of VEGF and its receptors on fresh leukemic blasts. VEGF-specific transcripts were detected by polymerase chain reaction (PCR) in 20 of 28 patients with de novo acute myeloid leukemia (AML) and in 3 of 5 patients with secondary AML. Using immunocytochemistry, we found VEGF protein in 2 leukemic cell lines and in 8 AML patients, in concordance with PCR results. Supernatants of fresh leukemic cells from 24 AML patients contained significantly more VEGF than supernatants from bone marrow cells of 9 normal donors or of CD34-enriched cells from 3 normal volunteer donors as determined by an enzyme-linked immunosorbent assay. VEGF possesses two high-affinity receptors, KDR and FLT1. Using a sensitive nested PCR assay, we detected expression of FLT1 in 10 of 20 patients with de novo AML and 3 of 5 patients with secondary AML. KDR was expressed in 4 of 22 patients with de novo AML and 1 of 4 with secondary AML. To study possible paracrine growth stimulation of AML blasts, endothelial cells from human umbilical cords were incubated with increasing concentrations of VEGF. A dose-dependent increase of granulocyte-macrophage colony-stimulating factor secretion from endothelial cells was identified.