Alan R. Brooks

Background: The Hedgehog (Hh) signaling pathway regulates a variety of developmental processes, including vasculogenesis, and can also induce the expression of pro-angiogenic factors in fibroblasts postnatally. Misregulation of the Hh pathway has been implicated in a variety of different types of cancer, including pancreatic and small-cell lung cancer. Recently a putative antagonist of the pathway, Hedgehog-interacting protein (HIP), was identified as a Hh binding protein that is also a target of Hh signaling. We sought to clarify possible roles for HIP in angiogenesis and cancer. Methods: Inhibition of Hh signaling by HIP was assayed by measuring the induction of Ptc-1 mRNA in TM3 cells treated with conditioned medium containing Sonic hedgehog (Shh). Angiogenesis was assayed in vitro by EC tube formation on Matrigel. Expression of HIP mRNA was assayed in cells and tissues by Q-RT-PCR and Western blot. HIP expression in human tumors or mouse xenograft tumors compared to normal tissues was assayed by Q-RT-PCR or hybridization of RNA probes to a cancer profiling array. Results: We show that Hedgehog-interacting protein (HIP) is abundantly expressed in vascular endothelial cells (EC) but at low or undetectable levels in other cell types. Expression of HIP in mouse epithelial cells attenuated their response to Shh, demonstrating that HIP can antagonize Hh signaling when expressed in the responding cell, and supporting the hypothesis that HIP blocks Hh signaling in EC. HIP expression was significantly reduced in tissues undergoing angiogenesis, including PC3 human prostate cancer and A549 human lung cancer xenograft tumors, as well as in EC undergoing tube formation on Matrigel. HIP expression was also decreased in several human tumors of the liver, lung, stomach, colon and rectum when compared to the corresponding normal tissue. Conclusion: These results suggest that reduced expression of HIP, a naturally occurring Hh pathway antagonist, in tumor neo-vasculature may contribute to increased Hh signaling within the tumor and possibly promote angiogenesis.

Background: Although the transient nature of transgene expression using first‐generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain.Methods: Rats were given intramuscular (i.m.) injections of a first‐generation Ad containing the human fibroblast growth factor 4 (hFGF‐4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV‐PE). The copy number of hFGF‐4 mRNA and viral DNA was measured in the same muscles by quantitative RT‐PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF‐4 transcription unit DNA was also performed, and the methylation status of the CMV‐PE DNA in the muscle was determined using bisulfite sequencing.Results: The copy number of hFGF‐4 mRNA peaked at 6 h then decreased 56‐fold by 24 h, and a further 240‐fold between days 3 and 28. Although the viral DNA copy number also decreased 23‐fold between 6 h and 28 days, the ratio of copies of hFGF‐4 mRNA per copy of viral DNA decreased 385‐fold over this period. Methylation of the CMV‐PE DNA in the muscle at both CpG and non‐CpG sites was observed 24 h after virus administration and had increased at day 7.Conclusions: Decreased transcription associated with extensive methylation of the CMV‐PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors. Copyright © 2004 John Wiley & Sons, Ltd.

Gene expression profiling has revealed that cultured vascular endothelial cells (EC) respond to fluid mechanical forces by modulating the mRNA level of a large number of genes. However, differences between the gene arrays and the experimental conditions employed by different researchers make comparison between data sets difficult, and limit the interpretation of the results. Despite these problems, analysis of recent data indicates that the transcriptional response of cultured EC to low-shear disturbed flow conditions similar to those at atherosclerosis-prone areas is distinct from that elicited by atheroprotective high shear laminar flow, providing a molecular basis for the focal nature of atherosclerosis. Many of the genes altered by disturbed flow are involved in key biological processes relevant to atherosclerosis such as inflammation, cell cycle control, apoptosis, thrombosis and oxidative stress. Overall, the gene expression profiling data are consistent with the hypothesis of the hemodynamic etiology of atherosclerotic predilection, vizthat at predilected areas in vivothe presence of low shear, non-laminar flow is sufficient to induce a gene expression profile that pre-disposes the endothelium to the initiation and development of atherosclerotic lesions.

Subtraction cloning and cDNA arrays were used to compare steady-state mRNA levels in cultured human aortic endothelial cells (HAEC) exposed for up to 24 h to either high-shear (13 dyn/cm²) steady laminar flow (LF), an established representation of “atheroprotective” flow conditions, or low-shear (2), pulsatile, nonsteady, non-unidirectional flow (disturbed flow, DF) that simulates conditions in the atherosclerosis-prone areas of the arterial circulation. More than 100 genes not previously known to be flow regulated were identified. Analysis of selected genes by quantitative RT-PCR confirmed the results obtained from the microarrays. These data demonstrate that DF is not simply the absence of LF but in fact represents a distinct biomechanical stimulus that has a profound impact upon the gene expression profile of HAEC in culture. In line with previous studies, many of the changes in mRNA levels induced by LF are atheroprotective. In contrast, DF upregulated the mRNA levels of a plethora of proatherosclerotic genes including proinflammatory, proapoptotic, and procoagulant molecules. For some of the genes whose expression was altered by DF and LF, corresponding changes in EC function (proliferation and monocyte adhesion) could be demonstrated. Specifically, the sustained upregulation of VCAM-1 and increased monocyte adhesion to EC exposed to DF was similar to that found in EC in vivo at atherosclerosis-prone regions, confirming the relevance of our model system for in vivo conditions. Distinct differences in the cellular response induced by TNFα and DF suggest that the effects of DF are not mediated entirely by the same signaling pathways that activate NF-κB. These studies demonstrate extensive and pathophysiologically relevant changes in sustained gene expression patterns in aortic EC exposed to DF compared with LF which are predicted to induce a proatherogenic EC phenotype.