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.

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/cm(2)) steady laminar flow (LF), an established representation of "atheroprotective" flow conditions, or low-shear (<1 dyn/cm(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 TNFalpha and DF suggest that the effects of DF are not mediated entirely by the same signaling pathways that activate NF-kappaB. 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.

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, Hedgehoginteracting 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.

The D-type cyclins promote progression through the G 1 phase of the cell cycle and may provide a link between growth factors and the cell cycle machinery. We determined the nucleotide sequence of the 5-flanking region of the human cyclin D2 and cyclin D3 genes and identified the transcription start sites. Analysis of the upstream sequences required for transcription of the cyclin D2 and cyclin D3 genes in continuously dividing cells revealed marked differences in their regulatory elements. In the cyclin D2 gene positive elements were localized between positions ؊306 and ؊114 relative to the ATG codon at ؉1. Additional positive elements were localized between ؊444 and ؊345, whereas sequences that reduced transcription were identified between nucleotides ؊1624 and ؊892. In the cyclin D3 gene all of the positive elements required for maximal transcription were localized between nucleotides ؊366 and ؊167, and no negative elements were found. The activities of a reporter gene linked to the upstream regulatory sequences of the cyclin D2 gene but not the cyclin D3 gene were induced when starved cells were serum stimulated. This suggests that although the abundance of both the cyclin D2 and cyclin D3 mRNAs is increased by serum stimulation, only the cyclin D2 gene is up-regulated at the transcriptional level. Sequences between nucleotides ؊306 and ؊1624 of the cyclin D2 gene were necessary for serum inducibility.

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, viz that at predilected areas in vivo the 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.