Hepatocyte clearance and pharmacokinetics of recombinant factor IX glycosylation variants.

Addition of N-linked glycosylation sites has been shown to increase serum half-life and decrease clearance for proteins such as recombinant erythropoietin (EPO). However, factor IX (FIX) variants with additional N-linked glycans ("HG" variants) that were expressed in HKB11 cells showed increased clearance in rat in vivo pharmacokinetic studies relative to FIX variants with no additional glycans. Variants with multiple additional glycans were the most rapidly cleared. A rat hepatocyte clearance assay was developed to measure intrinsic clearance of these FIX variants in vitro. The rank order of clearance of the variants was the same both in vivo and in the in vitro hepatocyte assay. In the in vitro assay, heparin, galactose, and asialo-orosomucoid inhibited clearance of a FIX HG variant by hepatocytes, and asialo-FIX was rapidly cleared, suggesting roles for the asialoglycoprotein receptor (ASGPR) and cell surface proteoglycans in FIX clearance. Thus the in vitro hepatocyte intrinsic clearance assay is both useful and predictive for identifying rapidly cleared recombinant proteins and for helping to identify receptors involved in clearance of proteins by the liver.

Knockdown of endothelial NOS by lentivirus-mediated short hairpin RNA in hemangioendothelioma cells increases proliferation and tumor formation.

Endothelial nitric oxide synthase (ecNOS) derived nitric oxide (NO) is a key contributor to the angiogenic process. By augmenting angiogenesis NO could potentially promote tumor progression. The object of this study was to determine how knockdown of ecNOS affects endothelial NO production and the angiogenic response in endothelial cells. EOMA cells derived from a spontaneously arising murine hemangioendothelioma were genetically manipulated to stably express siRNA targeting ecNOS. Knockdown of ecNOS in different stably transfected EOMA cell lines was demonstrated by quantitative RT-PCR, Western blot and ecNOS specific ELISA. An EOMA cell line with near complete knockdown of ecNOS exhibited dramatically altered morphology and changes in the expression of mRNAs encoding proteins involved in angiogenesis. This cell line exhibited a 4-fold increase in proliferation in vitro, altered tube formation in matrigel and formed tumors in mice more rapidly than the parental cells. In contrast, a cell line in which ecNOS protein levels were reduced only 5-fold did not show changes in proliferation rate, tube formation or tumor growth. These results suggest that ecNOS derived nitric oxide reduces the growth of hemangioendothelioma derived tumors, and underscore the importance of careful consideration of the tumor type when selecting modulation of nitric oxide signaling as a treatment strategy.

Hedgehog-interacting protein is highly expressed in endothelial cells but down-regulated during angiogenesis and in several human tumors.

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.

Gene expression profiling of vascular endothelial cells exposed to fluid mechanical forces: relevance for focal susceptibility to atherosclerosis.

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.

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle.

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.

Gene expression profiling of human aortic endothelial cells exposed to disturbed flow and steady laminar flow.

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.