Kaposi sarcoma herpesvirus (KSHV) induces transcriptional reprogramming of endothelial cells. In particular, KSHV-infected lymphatic endothelial cells (LECs) show an up-regulation of genes associated with blood vessel endothelial cells (BECs). Consequently, KSHV-infected tumor cells in Kaposi sarcoma are poorly differentiated endothelial cells, expressing markers of both LECs and BECs. MicroRNAs (miRNAs) are short noncoding RNA molecules that act post-transcriptionally to negatively regulate gene expression. Here we validate expression of the KSHV-encoded miRNAs in Kaposi sarcoma lesions and demonstrate that these miRNAs contribute to viral-induced reprogramming by silencing the cellular transcription factor MAF (musculoaponeurotic fibrosarcoma oncogene homolog). MAF is expressed in LECs but not in BECs. We identify a novel role for MAF as a transcriptional repressor, preventing expression of BEC-specific genes, thereby maintaining the differentiation status of LECs. These findings demonstrate that viral miRNAs could influence the differentiation status of infected cells, and thereby contribute to KSHV-induced oncogenesis.

The angiogenic activity of peptide adrenomedullin (AM) was first shown in 1998 . Since then, a number of reports have confirmed the ability of AM to induce the growth and migration of isolated vascular endothelial and smooth muscle cells in vitro and to promote angiogenesis in xenografted tumours in vivo. In addition, knockout murine models point to an essential role for AM in embryonic vasculogenesis and ischaemic revascularisation. AM expression is upregulated by hypoxia (a typical feature of solid tumours) and a potential role as a regulator of carcinogenesis and tumour progression has been proposed based on studies in vitro and in animal models. Nevertheless, translational research on AM, and in particular, confirmation of its importance in the vascularisation of human tumours has lagged behind. In this commentary, we review current progress and potential directions for future research into the role of AM in tumour angiogenesis.

Kaposi's sarcoma (KS) is caused by Kaposi's sarcoma–associated herpesvirus (KSHV) and consists of proliferating spindle cells, which are related to lymphatic endothelial cells (LEC). Angiopoietin-2 (Ang2) is a secreted proangiogenic and lymphangiogenic molecule. Here, we show the expression of Ang2 protein in KS and confirm that KSHV infection up-regulates Ang2 in LEC. We show that a paracrine mechanism contributes to this up-regulation. A lentiviral library of individual KSHV-encoding genes, comprising the majority of known latent genes and a selection of lytic viral genes, was constructed to investigate the underlying mechanism of this up-regulation. Two lytic genes, viral interleukin-6 (vIL6) and viral G-protein–coupled receptor (vGPCR), up-regulated Ang2 expression in LEC. Both vIL6 and vGPCR are expressed in KSHV-infected LEC and caused up-regulation of Ang2 in a paracrine manner. KSHV, vIL6, and vGPCR up-regulated Ang2 through the mitogen-activated protein kinase (MAPK) pathway. Gene expression microarray analysis identified several other angiogenic molecules affected by KSHV, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis, which is also affected by vIL6 and vGPCR in LEC, and matrix metalloproteinases, which could act in concert with Ang2 to contribute to KS development. These findings support the paracrine and autocrine roles of the lytic KSHV-encoded proteins, vIL6 and vGPCR, in KS pathogenesis and identify Ang2 as a potential therapeutic target for this neoplasm. [Cancer Res 2007;67(9):4042–50]

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.

Human endometrium is a mucosa served by a microvascular blood supply that involves benign angiogenesis under the control of ovarian steroids throughout reproductive life. Adrenomedullin is a multifunctional 52-amino acid peptide involved in numerous physiological and pathological processes, including angiogenesis, growth regulation, differentiation, vasodilation and smooth muscle relaxation. We have previously shown that adrenomedullin is present in the human uterus. To investigate further the role of adrenomedullin in human endometrial angiogenesis, a method for the isolation and culture of non-pregnant endometrial endothelium was developed. Enzymatic dispersion and `Percoll' gradient centrifugation, followed by positive selection using Ulex europaeus agglutinin-coated immunomagnetic beads, yielded pure isolates of endothelium. The cells formed a typical `cobblestone' monolayer within 5–7 days and expressed the classic endothelial markers, CD31 and von Willebrand factor. The presence of adrenomedullin immunoreactivity in endometrial endothelial cells was shown by immunohistochemistry both in vitro and in vivo. Adrenomedullin promotes growth of endothelial cells as measured by [methyl-³H] thymidine uptake. Adrenomedullin also induced cyclic AMP in endometrial endothelial cells. These results demonstrate, for the first time, that adrenomedullin is an autocrine growth factor for human endometrial endothelial cells and is thus involved in endometrial angiogenesis.

Adrenomedullin is a 52 amino acid peptide that shows a remarkable range of effects on the vasculature that include inter alia , vasodilatation, regulation of permeability, inhibition of endothelial cell apoptosis, and promotion of angiogenesis. Recently the G‐protein coupled receptor (GPCR) calcitonin receptor‐like receptor (CRLR), and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signaling system. However, mechanisms of regulation of CRLR expression are still largely unknown. This is in part due to lack of information on the gene promoter. In this study we have determined the transcriptional start of human CRLR cDNA by 5′‐RACE and cloned the proximal 5′‐flanking region of the gene by PCR. The 2318 bp genomic fragment contains the basal promoter of human CRLR, including potential TATA‐boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp‐1, Pit‐1, glucocorticoid receptor, and hypoxia‐inducible factor‐1 α (HIF‐1α) were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5′‐flanking region isolated by PCR contains the gene promoter of human CRLR. Of significance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site‐directed mutagenesis of the consensus hypoxia‐response element (HRE) in the 5′‐flanking region abolished such a response. We also demonstrated by semi‐quantitative RT‐PCR that transcription of the gene is activated by hypoxia in microvascular endothelial cells. In contrast, expression of RAMPs 1, 2, and 3 was unaffected by low oxygen tension. We conclude that simultaneous transcriptional up‐regulation of CRLR and its ligand adrenomedullin in endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia.