The biology of Kaposi sarcoma is poorly understood because the dominant cell type in Kaposi sarcoma lesions is not known 1-4. We show by gene expression microarrays that neoplastic cells of Kaposi sarcoma are closely related to lymphatic endothelial cells (LECs) and that Kaposi sarcoma herpesvirus (KSHV) 5,6 infects both LECs and blood vascular endothelial cells (BECs) in vitro. The gene expression microarray profiles of infected LECs and BECs show that KSHV induces transcriptional reprogramming of both cell types. The lymphangiogenic molecules VEGF-D and angiopoietin-2 were elevated in the plasma of individuals with acquired immune deficiency syndrome and Kaposi sarcoma. These data show that the gene expression profile of Kaposi sarcoma resembles that of LECs, that KSHV induces a transcriptional drift in both LECs and BECs and that lymphangiogenic molecules are involved in the pathogenesis of Kaposi sarcoma. The cellular origin of the spindle cells of Kaposi sarcoma lesions is poorly defined 3,7. Kaposi sarcoma spindle cells express endothelial cell markers but also have features of other cell lineages, including fibroblasts, macrophages and smooth muscle cells 1-3. Kaposi sarcoma could be a tumor originating from LECs, as spindle cells ubiquitously express VEGFR-3 and podoplanin and stain with the antibody D2-40 recognizing LECs 4,8. But these markers can also be expressed on angiogenic blood vessels, or on other cell types 9. Furthermore, some BEC markers (e.g., CD34) are expressed in all Kaposi sarcoma spindle cells 1. KSHV is the infectious cause of Kaposi sarcoma 5,6. In vitro, KSHV can infect both micro-and macrovascular endothelial cells, and these cells are useful to study the role of KSHV in the pathogenesis of Kaposi sarcoma 10-12 .

MicroRNAs are small, non-coding RNAs that negatively regulate gene expression. It has been proposed that microRNAs could function in the regulation of innate immunity, but this has not been demonstrated for viral infection. Here we test this hypothesis using the human pathogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) and one of its putative natural cellular targets, primary lymphatic endothelial cells (LECs). We show that an early antiviral microRNA response (6 h post-infection) includes expression of microRNAs that enhance viral gene expression. In particular, the CREB-induced miR-132 microRNA is highly upregulated after infection and has a negative effect on the expression of interferon-stimulated genes, facilitating viral replication. We show a similar function for miR-132 during infection of monocytes with herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV). miR-132 regulates innate antiviral immunity by inhibiting expression of the p300 transcriptional co-activator. p300 is downregulated early after KSHV infection, and inhibition of miR-132 induction restores p300 expression. Furthermore, p300 regulates miR-132 levels, revealing a dynamic equilibrium between miR-132 and p300. By targeting p300, rather than a transcription factor or signalling protein, miR-132 has a broad role in the regulation of antiviral immunity.

IntroductionDLL4 plays a fundamental role in vascular development and angiogenesis. 1,2 DLL4 haploinsufficiency results in extensive arterial defects and embryonic lethality, 3 indicating that the developing vasculature is sensitive to minor alterations in DLL4 dosage. DLL4 expression is mainly restricted to the endothelium of nascent vessels, particularly the tip cells, where it maintains stalk cell identity in neighboring cells, thereby regulating vessel sprouting and branching in response to angiogenic stimuli. 4 The importance of optimal DLL4 expression in physiologic angiogenesis is illustrated through its regulation of intersegmental vessel (ISV) development in zebrafish. Morpholino (MO) knockdown of dll4 in zebrafish results in an increased number of endothelial cells within the ISVs and ectopic ISV branching from the dorsal aorta (DA) because of overactivation of Vegfa signaling. 5,6 DLL4 is relevant in pathologic angiogenesis and is overexpressed in human tumors, often in association with markers of inflammation, hypoxia and angiogenesis. [7][8][9] Inhibition of DLL4 suppresses experimental tumor growth by inducing nonproductive, deregulated angiogenesis. 10,11 We and others have shown that DLL4 expression is up-regulated in lymphatic endothelial cells (LECs) after infection by Kaposi sarcoma herpesvirus (KSHV), 12,13 an oncogenic ␥-herpesvirus that is the etiologic agent of Kaposi sarcoma (KS). KS is an angioproliferative neoplasm composed of cells of endothelial origin. 14 Although accurate regulation of DLL4 levels is a hallmark of angiogenesis, the mechanisms that finely regulate DLL4 expression are not completely defined. Therefore we hypothesized that, in addition to well-known transcriptional mechanisms that affect DLL4 expression, DLL4 is regulated at the posttranscriptional level.MicroRNAs (miRNAs) are small, noncoding RNAs that influence target gene expression through mRNA degradation and translation inhibition. 15 Implicated in key cellular processes, miRNAs play a role in angiogenesis and cancer. 16,17 miR-27b is the only miRNA thus far implicated in DLL4 regulation 18 ; however, this miRNA also regulates sprouty homologue 2 (SPRY2) and semaphorin 6A (SEMA6A), and it is unclear whether its proposed suppression of DLL4 specifically leads to vascular defects. 18,19 We previously described the miRNA signature in KSHV-infected LECs (KLECs). 20 These data indicated significant down-regulation of members of the miR-30 miRNA family postinfection (PI). Encoded by 6 genes and expressed from 4 distinct transcripts across the human genome, the members of the miR-30 family share an identical seed sequence and hence have common predicted targets. 21 Here we show that miR-30b and miR-30c target DLL4 in vitro and in vivo, and that the miR-30 family regulates angiogenesis. Methods Cell cultureLECs were purchased from Promocell and grown in endothelial growth medium MV (Promocell) supplemented with 10 ng/mL VEGF-C (R&D Systems). HUVECs were purchased from Promocell and grown in endothelial growth medium MV2 ...