Quantitative CT analysis using functional imaging is superior in describing disease progression in idiopathic pulmonary fibrosis compared to forced vital capacity.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is chronic fibrosing pneumonia with an unpredictable natural disease history. Functional respiratory imaging (FRI) has potential to better characterize this disease. The aim of this study was to identify FRI parameters, which predict FVC decline in patients with IPF. METHODS: An IPF-cohort (treated with pamrevlumab for 48 weeks) was retrospectively studied using FRI. Serial CT's were compared from 66 subjects. Post-hoc analysis was performed using FRI, FVC and mixed effects models. RESULTS: Lung volumes, determined by FRI, correlated with FVC (lower lung volumes with lower FVC) (R2 = 0.61, p < 0.001). A negative correlation was observed between specific image based airway radius (siRADaw) at total lung capacity (TLC) and FVC (R2 = 0.18, p < 0.001). Changes in FVC correlated significantly with changes in lung volumes (R2 = 0.18, p < 0.001) and siRADaw (R2 = 0.15, p = 0.002) at week 24 and 48, with siRADaw being more sensitive to change than FVC. Loss in lobe volumes (R2 = 0.33, p < 0.001), increasing fibrotic tissue (R2 = 0.33, p < 0.001) and airway radius (R2 = 0.28, p < 0.001) at TLC correlated with changes in FVC but these changes already occur in the lower lobes when FVC is still considered normal. CONCLUSION: This study indicates that FRI is a superior tool than FVC in capturing of early and clinically relevant, disease progression in a regional manner.

Connective tissue growth factor promotes fibrosis downstream of TGFbeta and IL-6 in chronic cardiac allograft rejection.

Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long-term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGFbeta, IL-6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGFbeta transduction. Further, intragraft CTGF expression was inhibited by IL-6 neutralization whereas TGFbeta expression remained unchanged, indicating that IL-6 effects may potentiate TGFbeta-mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGFbeta and IL-6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR.

Transcriptional and posttranscriptional regulation of the proliferating cell nuclear antigen gene.

The steady-state mRNA levels of the proliferating cell nuclear antigen (PCNA) gene are growth regulated. In a previous paper (L. Ottavio, C.-D. Chang, M. G. Rizzo, S. Travali, C. Casadevall, and R. Baserga, Mol. Cell. Biol. 10:303-309, 1990), we reported that introns (especially intron 4) participate in growth regulation of the PCNA gene. We have now investigated the role of the 5'-flanking sequence of the human PCNA gene stably transfected into BALB/c 3T3 cells. Promoters of different lengths (from -2856 to -45 upstream of the cap site) were tested. All promoters except the AatII promoter (-45), including a short HpaII promoter (-210), were sufficient for a response to serum, platelet-derived growth factor, and to a lesser extent epidermal growth factor. No construct responded to insulin or platelet-poor plasma. The AatII promoter had little detectable activity. Transcriptional activity was also determined in BALB/c 3T3 cells carrying various constructs of the human PCNA gene by two methods: run-on transcription and reverse transcription-polymerase chain reaction (the latter measuring the heterogeneous nuclear RNA [hnRNA] steady-state levels). There was very little difference in the rate of transcription of the PCNA gene between G0 cells and serum-stimulated cells, although the levels of hnRNA were much higher after stimulation. In G0 cells carrying a human PCNA gene without introns 4 and 5, both transcription rate and hnRNA levels were high. Together with data on the mRNA half-life, these results suggest a posttranscriptional component in the regulation of PCNA mRNA levels after serum stimulation but a transcriptional regulation by intron 4.

Role of the promoter in the regulation of the thymidine kinase gene.

To identify the regulatory elements of the human thymidine kinase (TK) gene, we have established stable cell lines carrying different chimeric constructs of the TK gene. Our results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G0 cells are stimulated by growth factors), TK mRNA levels are higher in G1-arrested cells than in proliferating cells; (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. We conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.

Weekly dosing with the platelet-derived growth factor receptor tyrosine kinase inhibitor SU9518 significantly inhibits arterial stenosis.

The platelet-derived growth factor (PDGF) ligands and their receptors have been implicated as critical regulators of the formation of arterial lesions after tissue injury. SU9518 (3[5-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrol-3-yl]propionic acid) is a novel synthetic indolinone that potently and selectively inhibits the cellular PDGF receptor kinase and PDGF receptor-induced cell proliferation. Inhibition of PDGF receptor phosphorylation in cell-based assays occurs within 5 minutes after drug exposure and persists for >6 hours after drug removal. The pharmacokinetics indicate plasma levels that exceeded the effective concentration required to inhibit the PDGF receptor in cells for up to 8 hours or 7 days after a single oral or subcutaneous administration, respectively. In the rat balloon arterial injury-induced stenosis model, once-daily oral or once-weekly subcutaneous administration of SU9518 reduced intimal thickening of the carotid artery (ratio of neointimal to medial area, 1.94+/-0.38 versus 1.03+/-0.29 [P<0.01] 2.21+/-0.32 versus 1.34+/-0.45 [P<0.01], respectively). These studies provide the rationale to evaluate PDGF receptor tyrosine kinase inhibitors, including inhibitors related to the indolinone, SU9518, for the treatment of arterial restenosis.

A novel cdk2-selective inhibitor, SU9516, induces apoptosis in colon carcinoma cells.

Recent studies have indicated that the development of cyclin-dependent kinase (cdk)2 inhibitors that deregulate E2F are a plausible pharmacological strategy for novel antineoplastic agents. We show here that 3-[1-(3H-Imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), a novel 3-substituted indolinone compound, binds to and selectively inhibits the activity of cdk2. This inhibition results in a time-dependent decrease (4-64%) in the phosphorylation of the retinoblastoma protein pRb, an increase in caspase-3 activation (5-84%), and alterations in cell cycle resulting in either a G(0)-G(1) or a G(2)-M block. We also report here cell line differences in the cdk-dependent phosphorylation of pRb. These findings demonstrate that SU9516 is a selective cdk2 inhibitor and support the theory that compounds that inhibit cdk2 are viable resources in the development of new antineoplastic agents.

Indolinone tyrosine kinase inhibitors block Kit activation and growth of small cell lung cancer cells.

Six indolinone tyrosine kinase inhibitors were characterized for their ability to inhibit Kit kinase and for their effects on the growth of small cell lung cancer (SCLC) cell lines. All of the six compounds were potent inhibitors of Kit kinase in a biochemical assay. A homology model of compound binding to the ATP binding site could account for the increased potency observed with the addition of a propionate moiety to the indolinone core but not the increase observed with addition of a chloride moiety. Although all of the compounds tested were potent in the biochemical assay, several exhibited significantly less potency in cellular kinase assays. Their effects on stem cell factor (SCF)-dependent Kit autophosphorylation and SCLC cell growth were also examined. Inhibition of SCF-stimulated Kit activation and cell growth in the H526 cell line was dose-dependent. At concentrations that inhibited SCF-stimulated H526 cell growth, there was little effect on insulin-like growth factor-1-stimulated growth, suggesting that these compounds exhibit reasonable selectivity for inhibition of Kit-mediated proliferation. Higher doses of the compounds were needed to inhibit serum-stimulated growth. Of the six compounds examined, SU5416 and SU6597 demonstrated the best cellular potency and, therefore, their effect on the growth of multiple SCLC cell lines in serum-containing media was examined. In addition to inhibiting proliferation, these compounds also induced significant cell death of several SCLC cell lines, but not of normal human diploid fibroblasts, in complete media. These observations suggest that Kit kinase inhibitors such as these may offer a new approach for inhibiting Kit-mediated proliferation of tumors such as SCLC, gastrointestinal stromal tumors, seminomas, and leukemias.

A potential role for c-jun in cell cycle progression through late G1 and S.

When the level of c-jun mRNA was analyzed in WI-38 human fibroblasts exciting short- and long-term quiescence, two peaks of c-jun mRNA accumulation were found. The first occurred one hour after stimulation and lasted three to five hours, whereas the second occurred at the G1/S border and was coupled to the time of entry to S phase rather than to the time of stimulation. The early peak is well documented and in agreement with the proposed role of c-Jun/AP-1 in mediating cellular responses to receptor-generated signals. The later peak, however, has not been previously reported. Additional follow-up studies showed that late G1/S expression was not solely a phenomenon of cells existing a quiescent state, nor was it restricted only to human cells. Gel retardation studies confirmed that there is AP-1 specific DNA binding activity in nuclear extracts isolated in late G1 and S phase, and that this AP-1 binding activity is due to the presence of Jun protein. An anti-Fos antibody was able to significantly decrease AB-1 binding activity in early G1 extracts, but had no effect on extracts isolated in late G1 and S phase, indicating that the complexes observed in late G1 and S phase are clearly different from those seen in early G1. These studies are among the first to suggest a functional dissociation of c-Jun from c-Fos. Our results identify a new, previously unreported role for c-Jun/AP-1 in regulation of cell cycle progression and mammalian cell growth.

Identification of substituted 3-[(4,5,6, 7-tetrahydro-1H-indol-2-yl)methylene]-1,3-dihydroindol-2-ones as growth factor receptor inhibitors for VEGF-R2 (Flk-1/KDR), FGF-R1, and PDGF-Rbeta tyrosine kinases.

A series of new 3-substituted indolin-2-ones containing a tetrahydroindole moiety was developed as specific inhibitors of receptor tyrosine kinases associated with VEGF-R, FGF-R, and PDGF-R growth factor receptors. These compounds were evaluated for their inhibitory properties toward VEGF-R2 (Flk-1/KDR), FGF-R1, PDGF-Rbeta, p60(c)()(-)()(Src)(), and EGF-R tyrosine kinases and their ability to inhibit growth factor-dependent cell proliferation. Structure-activity relationships of this new pharmacophore have been determined at the level of kinase inhibition. Compounds containing a propionic acid moiety at the C-3' position of the tetrahydroindole ring represented the most potent indolin-2-ones to inactivate the VEGF, FGF, and PDGF receptor kinases. The inhibitory activities of 9d against VEGF-R2 (Flk-1), 9h against FGF-R1, and 9b against PDGF-Rbeta were 4, 80, and 4 nM, respectively. However, all of these compounds were inactive when tested against the EGF-R tyrosine kinase. Compounds 9a and 9b represented the most potent inhibitors of these classes to inhibit both biochemical kinase and growth factor-dependent cell proliferation for these three targets. In addition, compound 9a was cocrystallized with the catalytic domain of FGF-R1 providing evidence to explain the structure-activity relationship results. This study has provided evidence to support the potential of these new tyrosine kinase inhibitors for the treatment of angiogenesis and other growth factor-related diseases including human cancers.

Carboxy-substituted cinnamides: a novel series of potent, orally active LTB4 receptor antagonists.

A series of carboxy-substituted cinnamides were investigated as antagonists of the human cell surface leukotriene B4 (LTB4) receptor. Binding was determined through measurement of [3H]LTB4 displacement from human neutrophils. Receptor antagonism was confirmed through a functional assay, which measures inhibition of Ca2+ release in human neutrophils. Potent antagonists were discovered through optimization of a random screening hit, a p-(alpha-methylbenzyloxy)cinnamide, having low-micromolar activity. Substantial improvement of in vitro potency was realized by the attachment of a carboxylic acid moiety to the cinnamide phenyl ring through a flexible tether, leading to identification of compounds with low-nanomolar potency. Modification of the benzyloxy substituent, either through ortho-substitution on the benzyloxy phenyl group or through replacement of the ether oxygen with a methylene or sulfur atom, produced achiral antagonists of equal or greater potency. The most potent compounds in vitro were assayed for oral activity using the arachidonic acid-induced mouse ear edema model of inflammation. Several compounds in this series were found to significantly inhibit edema formation and myeloperoxidase activity in this model up to 17 h after oral administration. Representatives of this series have been shown to be potent and long-acting orally active inhibitors of the LTB4 receptor.

Merged screening for human immunodeficiency virus Tat and Rev inhibitors.

In addition to "conventional" drug discovery targets used in modern strategies, mainly focusing on proteins, recent insights into gene regulation as a novel drug concept have begun to invite the targeting of biomolecular interactions between proteins and RNA. Because two protein-RNA interactions (Tat and trans-activation-responsive element, Rev and Rev-responsive element) are essential for any productive replication of human immunodeficiency virus, this important human pathogen was used as a model system for our studies. The design of a fluorescence-based high throughput assay, in which both targets were presented in the same vessel, enabled us to simultaneously interrogate two characteristics of a potential inhibitor: potency of interference and selectivity toward each of the interactions. Although related systems have been reported for several DNA binders, an extension into interference with transcription events would open a new dimension of cellular regulation. Here we describe the setup of the screening assay for over 110,000 compounds as well as a primary characterization of identified hits. The assay's characteristics demonstrate that a microwell-based dual screening system for RNA binders may add a powerful tool to modern drug discovery.

Hepatocyte growth factor expression in human cancer and therapy with specific inhibitors.

Hepatocyte growth factor/Scatter Factor (HGF/SF) mediated stimulation of the Met receptor tyrosine kinase results in pleiotropic cellular effects including proliferation, morphogenesis, motility and invasion. In vivo, HGF/SF-Met activation has been shown to participate in tumorigenesis, angiogenesis and metastasis. Coupled with accumulating evidence that aberrant HGF/SF-Met expression is frequently observed in a variety of human tumors, often in association with progressive disease, these data present HGF/SF-Met as an attractive target for therapeutic intervention in human cancer. In this review, we will present the most compelling evidence suggesting a key role for HGF/SF-Met signaling in tumorigenesis, and discuss several possible therapeutic strategies.

Isolation of the North American strain of viral hemorrhagic septicemia virus (VHSV) associated with epizootic mortality in two new host species of Alaskan marine fish.

Thousands of dead Pacific herring Clupea pallasi, Pacific hake Merluccius productus and walleye pollock Theragra chalcogramma were reported in Lisianski Inlet near Pelican, Alaska, USA, on August 1, 1998. The Pacific hake and pollock continued to die through the end of September. Virological examinations of dead fish identified the North American strain of viral hemorrhagic septicemia virus (VHSV) from all 3 species of fish as well as associated high virus titers and possible histopathological lesions. No other primary fish pathogens were detected and there were no apparent environmental causes for fish mortality. This is the first report of VHSV in 2 new Alaskan fish host species and of a natural epizootic associated with VHSV in which progressive mass mortality was observed simultaneously in herring and 2 other species of free-ranging marine fish.

Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater.

Soluble antigen of Renibacterium salmoninarum (Rs) was detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) at significantly higher prevalences in adult chinook salmon Oncorhynchus tshawytscha that matured in freshwater than in the same cohort of fish spawned after maturation in seawater. The cumulative results were consistent during 4 yr of comparison at the Little Port Walter Hatchery on Baranof Island, Alaska, USA. Possible causes for this difference are discussed. Maturation of chinook salmon broodstock in seawater has become a practical strategy at this hatchery to reduce the prevalence of Rs-positive parent fish and the numbers of culled eggs.

Sequence analysis of the human thymidine kinase gene promoter: comparison with the human PCNA promoter.

We report the sequence of 4264 nucleotides of 5' flanking sequence of the human thymidine kinase gene, a gene that is maximally expressed at the G1/S boundary of the cell cycle. The position of nucleotide sequences which can act as binding sites for trans-acting factors, Sp-1, AP-1/jun, AP-2, OTF-1 and CAAT box factors as well as other potential cis-acting sequences have been mapped. The organization of these cis-acting sequences in the promoter of the human PCNA gene (another gene that is maximally expressed at the G1/S boundary) are shown for comparison. The potential role that these sequences may play in the transcriptional regulation of these genes is discussed.

Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe.

We have used the technique of reverse transcription coupled to the polymerase chain reaction to detect mRNA precursors [heterogeneous nuclear RNA (hnRNA)] transcribed from the thymidine kinase (TK) gene of human diploid fibroblasts. With this method, the amplification products of both hnRNA (containing the introns) and mature mRNA can be detected on Southern blots with appropriate hybridization probes. With the experimental conditions used, the sensitivity of the technique is such that TK mRNA can be detected in as few as 20 S-phase cells. TK hnRNA is maximally expressed early in the S phase of the cell cycle after quiescent human fibroblasts are stimulated to proliferate. At this point, the ratio of TK hnRNA to TK mRNA is 1:155. A small amount of TK hnRNA can be detected in populations of cells that appear to be quiescent. However, the presence of the precursor in these populations correlates with the number of cells still cycling. No TK hnRNA can be detected in truly quiescent human diploid fibroblasts, suggesting that in these cells, the TK gene is not transcribed in G0.

Two zinc-dependent steps during G1 to S phase transition.

The influence of zinc (Zn) availability on thymidine kinase mRNA concentration has been investigated in cells in which production of the mRNA was regulated by either truncated thymidine kinase promoters or by the SV40 early promoter. Thymidine kinase mRNA concentrations were decreased by low Zn availability even when the promoter was truncated to 80 bp but not when it was replaced by the SV40 promoter. However, thymidine incorporation by the SV40 cells was still sensitive to lack of Zn, suggesting a second Zn-sensitive process involved in commitment to S phase. The increase in histone H3 mRNA production prior to S phase was not inhibited by lack of Zn leading to a preferential increase in this mRNA in exponentially growing cells deprived of Zn.

Effect of angiogenesis inhibitors on oestrogen-mediated endometrial endothelial cell proliferation in the ovariectomized mouse.

It has been suggested that endometrial angiogenesis in response to the sex steroids oestrogen and progesterone is mediated at a local level via compounds such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), acting through their respective tyrosine kinase receptors. The aim of the present study was to use SUGEN tyrosine kinase receptor angiogenic inhibitor compounds SU5416, SU5402, SU11652 and SU11685, to determine whether VEGF, FGF or PDGF play a role in mediating endometrial endothelial cell proliferation after administration of oestrogen and progesterone. Endometrial endothelial cell proliferation was induced in adult ovariectomized mice by either oestrogen alone for 24 h (E1), or a regimen using oestrogen alone, then progesterone with low dose oestrogen, followed by progesterone with high-dose oestrogen (PE) over a total of 7 days. Each angiogenesis inhibitor compound was injected daily for 4 days (100 mg kg(-1) day(-1), s.c.) before endometrial tissue collection at either the E1 or PE stage. This study also evaluated the effect of VEGF antiserum (0.2 ml, i.p.) on endothelial cell proliferation at the E1 stage. All four angiogenic inhibitor compounds significantly reduced endothelial cell proliferation activity at the E1 and PE stages. The greatest reduction in the endothelial cell proliferative index was at the E1 stage in the group treated with the VEGF receptor inhibitor SU5416 (2.5 +/- 0.7% versus 27.9 +/- 1.1%, P < 0.001), with a reduction of similar magnitude in the group treated with anti-VEGF antibody. At the PE stage, all four inhibitors significantly reduced endothelial cell proliferation to a similar extent, indicating that VEGF, FGF and PDGF are all involved. These results demonstrate that endometrial angiogenesis after acute oestrogen treatment is primarily mediated by VEGF, but that under the influence of combined oestrogen and progesterone, FGF and PDGF are also probably involved.

Structure and proteolysis of the growth hormone receptor on rat hepatocytes.

125I-Labeled human growth hormone is isolated in high molecular weight (Mr) (300,000, 220,000, and 130,000) and low molecular weight complexes on rat hepatocytes after affinity labeling. The time-dependent formation of low molecular weight complexes occurred at the expense of the higher molecular weight species and was inhibited by low temperature or inhibitors of serine proteinases. Exposure to reducing conditions induced loss of Mr 300,000 and 220,000 species and augmented the amount of Mr 130,000 complexes. The molecular weight of growth hormone (22,000) suggests that binding had occurred with species of Mr 280,000, 200,000, and 100,000. Two-dimensional gel electrophoresis demonstrated that the 100,000-dalton receptor subunit is contained in both the 280,000- and 200,000-dalton species. Reduction of interchain disulfide bonds in the growth hormone receptor did not alter its elution from gel filtration columns, but intact, high molecular weight receptor constituents were separated from lower molecular weight degradation products. Digestion of affinity-labeled growth hormone-receptor complexes with neuraminidase increased the mobility of receptor constituents on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These observations show that the growth hormone receptor is degraded by hepatic serine proteinases to low molecular weight degradation products which can be separated from intact receptor by gel filtration. Intact hormone-receptor complexes are aggregates of 100,000-dalton sialoglycoprotein subunits held together by interchain disulfide bonds and by noncovalent forces.