Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension.

Our understanding of the pathobiology of severe pulmonary hypertension, usually a fatal disease, has been hampered by the lack of information of its natural history. We have demonstrated that, in human severe pulmonary hypertension, the precapillary pulmonary arteries show occlusion by proliferated endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR-2) are involved in proper maintenance, differentiation, and function of endothelial cells. We demonstrate here that VEGFR-2 blockade with SU5416 in combination with chronic hypobaric hypoxia causes severe pulmonary hypertension associated with precapillary arterial occlusion by proliferating endothelial cells. Prior to and concomitant with the development of severe pulmonary hypertension, lungs of chronically hypoxic SU5416-treated rats show significant pulmonary endothelial cell death, as demonstrated by activated caspase 3 immunostaining and TUNEL. The broad caspase inhibitor Z-Asp-CH2-DCB prevents the development of intravascular pulmonary endothelial cell growth and severe pulmonary hypertension caused by the combination of SU5416 and chronic hypoxia.

Inhibition of VEGF receptors causes lung cell apoptosis and emphysema.

Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH(2)-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema.

Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular receptor tyrosine kinases.

3-Substituted indolin-2-ones have been designed and synthesized as a novel class of tyrosine kinase inhibitors which exhibit selectivity toward different receptor tyrosine kinases (RTKs). These compounds have been evaluated for their relative inhibitory properties against a panel of RTKs in intact cells. By modifying the 3-substituted indolin-2-ones, we have identified compounds which showed selective inhibition of the ligand-dependent autophosphorylation of various RTKs at submicromolar levels in cells. Structure-activity analysis for these compounds and their relative potency and selectivity to inhibit particular RTKs has determined that (1) 3-[(five-membered heteroaryl ring)methylidenyl]indolin-2-ones are highly specific against the VEGF (Flk-1) RTK activity, (2) 3-(substituted benzylidenyl)indolin-2-ones containing bulky group(s) in the phenyl ring at the C-3 position of indolin-2-ones showed high selectivity toward the EGF and Her-2 RTKs, and (3) the compound containing an extended side chain at the C-3 position of the indolin-2-one (16) exhibited high potency and selectivity when tested against the PDGF and VEGF (Flk-1) RTKs. Recent published crystallographic data for two of these 3-substituted indolin-2-ones provides a rationale to suggest that these compounds may bind in the ATP binding pocket of RTKs. The structure-activity analysis supports the use of subsets of these compounds as specific chemical leads for the development of RTK-specific drugs with broad application for the treatment of human diseases.

Inhibition of platelet-derived growth factor and epidermal growth factor receptor signaling events after treatment of cells with specific synthetic inhibitors of tyrosine kinase phosphorylation.

The receptor kinase activity associated with the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor plays an important role in ligand-induced signaling events. The effect of specific, synthetic chemical inhibitors of PDGF- and EGF-mediated receptor tyrosine autophosphorylation on receptor signaling were examined in NIH 3T3 cells overexpressing PDGF or EGF receptors. Specific inhibition of ligand-dependent receptor autophosphorylation, PI3K activation, mitogen-activated protein kinase (MAPK) activation, cyclin E-associated kinase activity and cell proliferation was measured after treatment of cells with these inhibitors. A synthetic PDGF receptor kinase inhibitor exhibited specific inhibitory properties when tested for PDGF-induced receptor autophosphorylation, MAPK activity, PI3K activation, entry into S phase and cyclin E-associated kinase activity. A synthetic EGF receptor kinase inhibitor showed selective inhibitor properties when tested for EGF-induced receptor autophosphorylation, MAPK activation, PI3K activation, entry into S phase and cyclin E-associated kinase activity. In both cases, these compounds were found to be effective as inducers of growth arrest and accumulation of cells in the G1 phase of the cell cycle after ligand treatment. However, at high concentrations, the EGF receptor kinase inhibitor was observed to exhibit some nonspecific effects as demonstrated by attenuation of PDGF-induced receptor autophosphorylation and cell cycle progression. This demonstrates that it is critical to use the lowest concentration of such an inhibitor that will alter the response under investigation, to have confidence that the conclusions derived from the use of such inhibitor are valid. We conclude that these experimental parameters signify useful end points to measure the relative selectivity of tyrosine kinase inhibitors that affect receptor-mediated signal transduction.

Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors.

A new class of protein tyrosine kinase inhibitors was identified that is based on an oxindole core (indolinones). Two compounds from this class inhibited the kinase activity of fibroblast growth factor receptor 1 (FGFR1) and showed differential specificity toward other receptor tyrosine kinases. Crystal structures of the tyrosine kinase domain of FGFR1 in complex with the two compounds were determined. The oxindole occupies the site in which the adenine of adenosine triphosphate binds, whereas the moieties that extend from the oxindole contact residues in the hinge region between the two kinase lobes. The more specific inhibitor of FGFR1 induces a conformational change in the nucleotide-binding loop. This structural information will facilitate the design of new inhibitors for use in the treatment of cancer and other diseases in which cell signaling by tyrosine kinases plays a crucial role in disease pathogenesis.

Tyrphostins IV--highly potent inhibitors of EGF receptor kinase. Structure-activity relationship study of 4-anilidoquinazolines.

Potent 4-anilido-substituted quinazolines which potently inhibit epidermal growth factor receptor (EGFR) kinase were prepared. Structure-activity relationship studies reveal high sensitivity to substitution at the aniline ring.

The metabolism of recombinant erythropoietin in the isolated perfused rat liver.

Indirect evidence points to extrarenal organs, presumably the liver, as the site of degradation of erythropoietin (EPO). The metabolism of both fully glycosylated and desialated intrinsically labelled 35S-Cysteine recombinant human erythropoietin (rhEPO) was therefore studied in isolated Wistar rat livers perfused in a recirculating mode for 180 min with a hemoglobin-free medium containing rhEPO. Perfusate and bile levels of rhEPO were measured by RIA. Total 35S-radioactivity in liver, bile and perfusate as well as non-acid precipitable radioactivity in perfusate were determined. In addition, detection of 35S-radioactivity was performed after subcellular fractionation of rat livers perfused with desialo-35S-Cysteine rhEPO. While concentrations of fully glycosylated 35S-Cysteine rhEPO did not exhibit any detectable decrease during perfusion, desialo-35S-Cysteine rhEPO was rapidly cleared from the perfusate. After 60 min of perfusion, only 32% of the initial levels of both immunoreactive rhEPO and total radioactivity remained in the perfusate. Quantitative hepatic accumulation of desialated tracer was demonstrated. Subcellular fractionation showed extensive hepatic degradation of the desialated tracer. Furthermore, during perfusion progressively larger amounts of small molecular weight degradation products of the tracer were found in the perfusate. Bile excretion of both fully glycosylated and desialated tracer was negligible. The significance of hepatic metabolism of desialo-35S-Cysteine rhEPO was supported by reduced removal of desialo-35S-Cysteine rhEPO from plasma in hepatectomized rats. It is hypothesized that continuous in vivo desialation is a crucial rate-limiting step in the degradation of circulating EPO.

Evaluation of the stability of human erythropoietin in samples for radioimmunoassay.

Radioimmunoassays for erythropoietin are limited so far to a few specialized laboratories and this requires transport and storage of samples. We therefore tested the stability of immunoreactive erythropoietin in serum and plasma samples obtained from a uremic and a nonuremic anemic patient. No significant change in the concentration of immunoreactive erythropoietin was found in either serum or plasma samples for up to 14 days of storage. This type of stability was observed no matter whether the samples were stored at room temperature, 4 degrees C, or -20 degrees C. There was no difference between the estimates of erythropoietin in serum and heparinized plasma. Validity of the radioimmunoassay used in this study was demonstrated by parallelism of dilution curves of test specimens and the 2nd International Reference Preparation for erythropoietin and by a close correlation between the immunoreactivity and the bioactivity of the hormone, as assessed in the same samples by the exhypoxic polycythemic mouse bioassay. In conclusion the data obtained clearly indicate that the necessity of storage and transport of clinical samples does not limit the practicability of the radioimmunoassay for erythropoietin.

Probing an adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein.

Expression of developmentally regulated membrane proteins of aggregating cells of Dictyostelium discoideum is subject to several control mechanisms. One of them involves periodic cyclic-AMP pulses as signals for gene expression. To increase the probability of selecting mutants specifically defective in the contact site A (csA) glycoprotein, one of the characteristic proteins of aggregating cells, we have bypassed the requirement for both cyclic-AMP pulses and another control element by two runs of mutagenesis. A ;double bypass' mutant, HG592, was obtained which aggregated in nutrient medium where wild-type did not develop. Mutants defective in expression of the csA-glycoprotein were selected from HG592 by fluorescence-activated cell sorting and colony immunoblotting using a monoclonal antibody specific for that protein. One among 51 csA-negative mutants, HG693, specifically lacked the capability of forming EDTA-stable intercellular contacts. It acquired chemotactic responsiveness and developed into fruiting bodies. Expression of the transcripts for eight developmentally regulated proteins was determined in HG693. Seven of the RNA species were normally expressed; they were recognized by cDNA clones which had been produced from poly(A) RNA isolated from membrane-bound polysomes. The single RNA species which was not substantially expressed in HG693 was recognized by a cDNA clone that was obtained by screening a lambdagt11 library with an antibody specific for the csA-glycoprotein. When probing RNA from wild-type cells, this clone hybridized with a single developmentally regulated RNA species of 1.9 kb whose expression was strongly enhanced by cyclic-AMP pulses. Appearance of this RNA coincided with the expression of the csA-glycoprotein.