Tumor Vascular Morphology Undergoes Dramatic Changes during Outgrowth of B16 Melanoma While Proangiogenic Gene Expression Remains Unchanged

In established tumors, angiogenic endothelial cells (ECs) coexist next to “quiescent” EC in matured vessels. We hypothesized that angio-gene expression of B16.F10 melanoma would differ depending on the growth stage. Unraveling the spatiotemporal nature thereof is essential for drug regimen design aimed to affect multiple neovascularization stages. We determined the angiogenic phenotype—represented by 52 angio-genes—and vascular morphology of small, intermediate, and large s.c. growing mouse B16.F10 tumors and demonstrated that expression of these genes did not differ between the different growth stages. Yet vascular morphology changed dramatically from small vessels without lumen in small to larger vessels with increased lumen size in intermediate/large tumors. Separate analysis of these vascular morphologies revealed a significant difference in αSMA expression in relation to vessel morphology, while no relation with VEGF, HIF-1α, nor Dll4 expression levels was observed. We conclude that the tumor vasculature remains actively engaged in angiogenesis during B16.F10 melanoma outgrowth and that the major change in tumor vascular morphology does not follow molecular concepts generated in other angiogenesis models

Molecular characterization of tumor vascular phenotype and pharmacology of antiangiogenic therapy

Kanker vormt al jaren één van de belangrijkste doodsoorzaken. Wereldwijd stierven er in 2004 zo’n 7,4 miljoen mensen aan kanker. Ondanks grote vooruitgang in de ontwikkeling van verschillende therapieën voor kanker, verwacht men dat dit aantal zal stijgen tot 12 miljoen in 2030. Deze cijfers van de World Health Organization geven aan dat verbetering van de huidige kankertherapieën nog steeds noodzakelijk is. Naast de conventionele therapeutische strategieën, zoals het chirurgisch verwijderen van de tumor, chemotherapie en bestraling, die zich vooral richten op de tumorcellen zelf, zijn er nieuwe therapieën in ontwikkeling die zich richten op de bloedvaten van een tumor. De groei van tumoren is in zeer grote mate afhankelijk van de (nieuw)vorming van bloedvaten, een proces dat we angiogenese noemen. Immers, zonder goede bloedvoorziening, dus zonder aanvoer van zuurstof en voedingsstoffen, en zonder afvoer van afvalstoffen kunnen cellen niet overleven. Bovendien spelen bloedvaten een belangrijke rol bij het ontstaan van uitzaaiingen, waarbij tumorcellen via de bloedbaan getransporteerd worden naar andere organen. Al deze aspecten maken het proces van tumorbloedvatvorming een aantrekkelijk doelwit voor therapie. Het vormen van een bloedvat is een complex proces. Een scala aan groeifactoren, receptoren, adhesiemoleculen, proteases en moleculen die zorgen voor de transductie van signalen van de ene cel naar de andere en ook binnen een cel, leiden het proces van nieuw-vorming van bloedvaten in de juiste banen. Het proces van angiogenese omvat verschillende stadia. Allereerst worden de cellen die de bloedvatwand bekleden, de endotheelcellen, geactiveerd door groeifactoren. Proteases zorgen ervoor dat het weefsel om de bloedvaten heen afgebroken wordt. De endotheelcellen krijgen daardoor de ruimte om te delen en te migreren in de richting waar het nieuwe zijtakje van het bloedvat gevormd gaat worden. De nieuwgevormde endotheelcellen moeten dan een buisachtige structuur vormen die aansluiting vindt op een bestaand bloedvat, zodat het onderdeel wordt van de bloedcirculatie. Tenslotte worden er pericyten, spiercelachtige steuncellen, gerekruteerd die het bloedvat ondersteunen. De endotheelcellen komen hierdoor weer terug in hun rusttoestand. Dit laatste noemen we vasculaire stabilisatie. Fysiologische angiogenese leidt tot de vorming van functionele bloedvaten, echter, pathologische angiogenese, zoals dat voorkomt in tumoren, kan leiden tot abnormale, slecht functionerende bloedvaten. In tegenstelling tot gezonde bloedvaten, worden tumorbloedvaten gekenmerkt door hun verwijde en bochtige karakter, sterke variaties in diameter, onvolledige bedekking met pericyten en grotere permeabiliteit, wat kan leiden tot chaotische bloeddoorstroming.

Microvascular endothelial cell heterogeneity:general concepts and pharmacological consequences for anti-angiogenic therapy of cancer

Microvascular endothelial cells display a large degree of heterogeneity in function depending on their location in the vascular tree. The existence of organ-specific, microvascular-bed-specific, and even intravascular variations in endothelial cell gene expression emphasizes their high cell-to-cell variability, which is furthermore extremely adaptable to altering conditions. The ability of microvascular endothelial cells to respond dynamically to pathology-related microenvironmental changes is particularly apparent in tumor-growth-associated angiogenesis. An understanding of how they behave, how their behavior varies between and within tumors, and how their behavior is related to responsiveness to drugs is critical for the development of effective anti-angiogenic treatment strategies. In this review, we introduce some general issues concerning organ-imprinted microvascular heterogeneity and highlight the importance of studying microvascular endothelial cell behavior in an in vivo context. This is followed by an overview of state-of-the-art knowledge regarding the nature of the variation in microenvironmental conditions in pre-clinical and clinical tumors and their consequences for tumor endothelial behavior. We provide recent insights into the in vivo molecular activation status of the endothelium and, finally, outline our current understanding of the way that anti-angiogenic drugs affect tumor endothelial cells in relation to their anti-tumor effects

Innovations in studying in vivo cell behavior and pharmacology in complex tissues - microvascular endothelial cells in the spotlight

<p>Many studies on the molecular control underlying normal cell behavior and cellular responses to disease stimuli and pharmacological intervention are conducted in single-cell culture systems, while the read-out of cellular engagement in disease and responsiveness to drugs in vivo is often based on overall tissue responses. As the majority of drugs under development aim to specifically interact with molecular targets in subsets of cells in complex tissues, this approach poses a major experimental discrepancy that prevents successful development of new therapeutics. In this review, we address the shortcomings of the use of artificial (single) cell systems and of whole tissue analyses in creating a better understanding of cell engagement in disease and of the true effects of drugs. We focus on microvascular endothelial cells that actively engage in a wide range of physiological and pathological processes. We propose a new strategy in which in vivo molecular control of cells is studied directly in the diseased endothelium instead of at a (far) distance from the site where drugs have to act, thereby accounting for tissue-controlled cell responses. The strategy uses laser microdissection-based enrichment of microvascular endothelium which, when combined with transcriptome and (phospho)proteome analyses, provides a factual view on their status in their complex microenvironment. Combining this with miniaturized sample handling using microfluidic devices enables handling the minute sample input that results from this strategy. The multidisciplinary approach proposed will enable compartmentalized analysis of cell behavior and drug effects in complex tissue to become widely implemented in daily biomedical research and drug development practice.</p>

Identification of epigenetically silenced genes in tumor endothelial cells

Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated by DNMTs and HDAC are involved in regulation of endothelial cell gene expression during tumor angiogenesis. To understand the mechanisms behind the epigenetic regulation of tumor angiogenesis, we used microarray analysis to perform a comprehensive screen to identify genes down- regulated in tumor-conditioned versus quiescent endothelial cells, and reexpressed by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA). Among the 81 genes identified, 77% harbored a promoter CpG island. Validation of mRNA levels of a subset of genes confirmed significant down-regulation in tumor-conditioned endothelial cells and reactivation by treatment with a combination of DAC and TSA, as well as by both compounds separately. Silencing of these genes in tumor-conditioned endothelial cells correlated with promoter histone H3 deacetylation and loss of H3 lysine 4 methylation, but did not involve DNA methylation of promoter CpG islands. For six genes, down-regulation in microdissected human tumor endothelium was confirmed. Functional validation by RNA interference revealed that clusterin, fibrillin 1, and quiescin Q6 are negative regulators of endothelial cell growth and angiogenesis. In summary, our data identify novel angiogenesis-suppressing genes that become silenced in tumor-conditioned endothelial cells in association with promoter histone modifications and reactivated by DNMT and HDAC inhibitors through reversal of these epigenetic modifications, providing a mechanism for epigenetic regulation of tumor angiogenesis

Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGF beta 2 in vascular abnormalization

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGF beta 2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGF beta signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGF beta 2 may represent a new target for vascular normalization therapy. Copyright (c) 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd