<em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) and parathyroid hormone blood levels rise following progressive loss of renal function. Here we measured parameters of phosphate metabolism in 100 patients with autosomal dominant polycystic kidney disease (ADPKD) in stage 1 or 2 of chronic kidney disease, <em>20</em> patients with non-diabetic chronic kidney disease, and 26 with type 2 diabetes. Twenty healthy volunteers served as controls. The mean levels of FGF23 were significantly (4-fold) higher in ADPKD compared to non-diabetic and diabetic patients, and healthy volunteers. Mean serum phosphate levels were significantly lower in ADPKD patients compared to non-diabetic and diabetic patients, and the healthy volunteers. The prevalence of hypophosphatemia was 38, 25, 27, and 5% in ADPKD, non-diabetic and diabetic patients, and healthy volunteers, respectively. The tubular maximum of phosphate reabsorption per glomerular filtration rate was lowest in ADPKD patients with a significantly high positive correlation with serum phosphate levels. Estimated glomerular filtration rates were approximately 100 ml/min per 1.73 m² in all groups and parathyroid hormone and vitamin D metabolite levels were in the normal range. Thus, FGF23 was substantially elevated in ADPKD patients compared to other CKD patients matched for glomerular filtration rate, and was associated with increased renal phosphate excretion. The mechanism for this anomaly will require further study.

In many cell types including myoblasts, <em>growth</em> <em>factors</em> control proliferation and differentiation, in part, via the mitogen-activated protein kinase (MAPK) pathway (also known as the extracellular regulated kinase (Erk) pathway). In C2C12 myoblast cells, insulin-like <em>growth</em> <em>factor</em>-1 and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) activate MAPK/Erk, and both <em>growth</em> <em>factors</em> promote myoblast proliferation. However, these <em>factors</em> have opposing roles with respect to differentiation; insulin-like <em>growth</em> <em>factor</em>-1 enhances muscle cell differentiation, whereas bFGF inhibits the expression of the muscle-specific transcription <em>factors</em> MyoD and myogenin. Cells treated with bFGF and PD98059, a specific inhibitor of the MAPK pathway, show enhanced expression of the muscle-specific transcription <em>factors</em> MyoD and myogenin as compared with cells not exposed to this inhibitor. Inhibiting MAPK activity also enhances myoblast fusion and the expression of the late differentiation marker myosin heavy chain. Basic FGF mediated repression of muscle-specific genes does not result from continued cell proliferation, since bFGF-treated cells progress through only one round of cell division. We have identified a critical boundary 16 to <em>20</em> h after plating during which bFGF induced MAPK activity is able to repress myogenic gene expression and differentiation. Thus, the targets of MAPK that regulate myogenesis are functional at this time and their identification is in progress.

The expression of platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but <em>fibroblast</em>-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately <em>20</em>% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that <em>fibroblasts</em> and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, <em>fibroblasts</em> and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell <em>growth</em> in vivo is dependent on <em>factors</em> regulating expression of the receptor.

With use of single-channel patch-clamp recording, we found five distinct types of stretch-activated ion channels (SACs) in tissue-cultured embryonic chick cardiac myocytes. With 140 mM K+ saline in the pipette, four channels had linear conductances of approximately equal to 25, 50, 100, and <em>20</em>0 pS and other channel was an inward rectifier of approximately equal to 25 pS at 0 mV membrane potential. The 100- and <em>20</em>0-pS channels were K+ selective, whereas the others passed alkali cations and Ca2+. From reversal potentials, the permeability ratio of K+/Na+, PK/PNa, was 3-7 for nonselective channels and 7-16 for K(+)-selective channels. Channel density was approximately equal to 0.3/microns2 for linear conductances and approximately equal to 0.1/microns2 for inward rectifier. Open-channel noise was a function of pipette filling solution with root-mean-square (RMS) noise increasing in the order K+ < isosmotic sucrose (plus trace ions) < Na+, probably reflecting short-lived block by extracellular ions. All channels were blocked by <em>20</em> microM Gd3+. The 25-pS linear channel was also blocked by 12.5 microM tetrodotoxin and 10 microM diltiazem, but the others were insensitive at these concentrations. Extracellular Cs+ and tetraethylammonium chloride did not block any channels. We saw no SAC activity in cells grown without embryo extract (EE), which demonstrates that channel expression, or some necessary co<em>factor</em>, is under control of <em>growth</em> <em>factors</em>. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) could replace EE in supporting channel expression. The presence of SACs capable of generating inward currents might explain how stretch increases automaticity in the heart. Because some SACs were permeable to Ca2+, they could contribute to the Starling curve and perhaps to initiating stretch-induced hypertrophy.

Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive <em>factors</em> that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and <em>growth</em> <em>factors</em> on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of <em>growth</em> <em>factors</em> including hepatocyte <em>growth</em> <em>factor</em> (HGF), bone morphogenetic protein 4, <em>fibroblast</em> <em>growth</em> <em>factor</em> 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and <em>20</em> ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.

Two endothelial cell <em>growth</em> <em>factors</em> (ECGF) have been purified from bovine brain and termed alpha- and beta-ECGF [Burgess, W. H., Mehlman, T., Friesel, R., Johnson, W. V. & Maciag, T. (1985) J. Biol. Chem. 260, 11389-11392]. Amino acid sequence analysis indicates that beta-ECGF represents a <em>20</em> amino acid amino-terminal extension of alpha-ECGF and a 14 amino acid amino-terminal extension of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>. These data indicate that both alpha-ECGF and acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> may be derived from beta-ECGF by posttranslational processing. Analysis of the amino-terminal 14 residues of beta-ECGF by fast-atom-bombardment mass spectrometry established the amino acid sequence of this region and the identity of the blocking group at the amino terminus (acetyl).

OBJECTIVE

Hemangiomas are benign tumors occurring in 10% of infants. A small percentage are complicated by blockage of vital structures, consumptive coagulopathy, or heart failure, resulting in a mortality of -<em>20</em>% of patients with complications. Here, we describe four infants with complicated hemangiomas responding to interferon-alpha-2b therapy.

METHODS

Four children with hemangiomas were treated with interferon-alpha-2b for complicating heart failure (1), visual impairment (2), or coagulopathy (1). Patients received interferon-alpha-2b alone or in conjunction with corticosteroid therapy over 2 to 9 months. Imaging studies and urinary basic fibroblast growth factor (bFGF) levels were used to monitor treatment response.

RESULTS

Three of four patients demonstrated involution of the hemangiomas with improvement in their coagulopathy or visual impairment. The fourth patient expired due to cardiac complications despite radiologic evidence of hemangioma involution. Side effects associated with interferon-alpha-2b treatment included elevated transaminases (2) and leukocytosis (2), which resolved upon completion of therapy. One patient developed mild gross motor delay (1), which improved after cessation of therapy. Decreased urinary bFGF levels correlated with hemangioma involution.

CONCLUSIONS

Interferon-alpha-2b therapy is an effective, well-tolerated treatment for complicated hemangiomas. Measurement of urinary bFGF levels may provide an objective method for monitoring treatment response.

The identity and functional potential of dopamine neurons derived in vitro from embryonic stem cells are critical for the development of a stem cell-based replacement therapy for Parkinson's disease. Using a parthenogenetic primate embryonic stem cell line, we have generated dopamine neurons that display persistent expression of midbrain regional and cell-specific transcription <em>factors</em>, which establish their proper identity and allow for their survival. We show here that transplantation of parthenogenetic dopamine neurons restores motor function in hemi-parkinsonian, 6-hydroxy-dopamine-lesioned rats. Exposure to Wnt5a and <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) <em>20</em> and 2 at the final stage of in vitro differentiation enhanced the survival of dopamine neurons and, correspondingly, the extent of motor recovery of transplanted animals. Importantly for future development of clinical applications, dopamine neurons were post-mitotic at the time of transplantation and there was no tumour formation. These data provide proof for the concept that parthenogenetic stem cells are a suitable source of functional neurons for therapeutic applications.

OBJECTIVE

To examine for the expression of 15-lipoxygenase 1 (15-LOX1) and 15-LOX2 in human retinal microvascular endothelial cells (HRMVECs) and study the role of arachidonic acid metabolites of these enzymes in angiogenesis.

METHODS

Quantitative RT-PCR and reverse-phase HPLC analyses were used to determine 15-LOX1/2 expression and their arachidonic acid metabolites in HRMVECs. The role of MEK1 in 15(S)-HETE-induced angiogenesis was studied using HRMVEC migration, tube formation, and basement membrane matrix plug angiogenesis.

RESULTS

HRMVECs expressed both 15-LOX1 and 15-LOX2. Hypoxia induced the expression of 15-LOX1 and the production of its arachidonic acid metabolites 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 15(S)-HETE stimulated HRMVEC migration and tube formation as potently as <em>20</em> ng/mL <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2). In addition, 15(S)-HETE stimulated the phosphorylation of ERK1/2, JNK1, p38 MAPK, and MEK1 in a time-dependent manner in these cells. Inhibition of MEK1 by pharmacologic and dominant-negative mutant approaches attenuated 15(S)-HETE-induced phosphorylation of ERK1/2 and JNK1 but not p38 MAPK. Blockade of ERK1/2 and JNK1 activation suppressed 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. Inhibition of p38 MAPK attenuated 15(S)-HETE-induced HRMVEC migration only. Inhibition of MEK1 also blocked 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis.

CONCLUSIONS

These results suggest that hypoxia, through the induction of 15-LOX1 expression, leads to the production of 15(S)-HETE in HRMVECs. In addition, 15(S)-HETE, through MEK1-dependent activation of ERK1/2 and JNK1, stimulates the angiogenic differentiation of HRMVECs and basement membrane matrix plug angiogenesis.

BACKGROUND

Epidermal growth factor (EGF)-related proteins, such as transforming growth factor-alpha (TGF-alpha), control cancer cell growth through hormonal pathways (i.e., autocrine [hormone acts on cell that produces it] and paracrine [hormone acts on nearby cells] pathways). Overexpression of TGF-alpha and/or its receptor (EGFR) has been detected in human cancers. The blockade of EGFR activation by the use of anti-EGFR monoclonal antibodies (MAbs) has been proposed as a potential anticancer therapy. The type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKAI) is generally overexpressed in human cancer cells and is involved in neoplastic transformation. Inhibition of PKAI by selective cAMP analogues, such as 8-chloro-cAMP (8-CI-cAMP), induces growth inhibition in various human cancer cell lines.

OBJECTIVE

On the basis of our previous observations of a cooperative anti-proliferative effect of anti-EGFR Mab 528 and 8-Cl-cAMP in human cancer cell lines in vitro, we evaluated the anticancer activity in vivo of the combination of an anti-EGFR MAb (MAb C225) and 8-Cl-cAMP.

METHODS

Athymic mice were injected subcutaneously with 10(7) human colon carcinoma GEO cells. After 7 days, when established tumor xenografts of 0.30-0.35 cm3 were detectable, 10-15 mice per group were treated intraperitoneally twice weekly with different doses of 8-Cl-cAMP and/or MAb C225. Cancer cell expression of various growth factors was evaluated by immunohistochemical analysis in tumors obtained from control and treated mice. Data were evaluated for statistical significance using the Student's t test and the Mantel-Cox logrank test. All P values represent two-sided tests of statistical significance.

RESULTS

A 5-week treatment with low doses of 8-Cl-cAMP (0.5 mg/dose) and MAb C225 (0.25 mg/dose) blocked GEO tumor growth (compared with that in control mice; P < .00001) and suppressed cancer cell production of autocrine growth factors, such as TGF-alpha, amphiregulin, and CRIPTO, and of angiogenic (promotes new blood vessel formation) factors, such as vascular endothelial growth factor and basic fibroblast growth factor, with no signs of toxicity. Control and 8-Cl-cAMP (0.5 mg/dose)-treated mice died within 9-10 weeks after tumor cell injection. In MAb C225 (0.25 mg/dose)-treated mice, GEO tumors resumed a growth rate comparable to that in control animals within 3 weeks following the end of treatment and the mice died between 11 and 20 weeks after tumor cell injection. GEO tumor growth was significantly delayed in the MAb C225 plus 8-Cl-cAMP treatment group (P < .00001) and was accompanied by a prolonged survival of mice (P < .00001) as compared with the control group.

CONCLUSIONS

Long-term treatment with a combination of agents that selectively inhibit two intracellular signal-transduction enzymes, such as the PKAI serine-threonine kinase and the EGFR tyrosine kinase, has anticancer activity in vivo, reflected by suppression of tumor proliferation and angiogenesis, with no signs of toxicity.

CONCLUSIONS

Since these inhibitors of intracellular mitogenic (growth-stimulating) signaling have a different mechanism(s) of action and do not antagonize the effects of cytotoxic therapy, a combination of anti-EGFR MAb C225 and 8-Cl-cAMP should be investigated as a nontoxic, long-term treatment for cancer patients following chemotherapy.

OBJECTIVE

Bile acids may play a role in the pathogenesis of IBS. We investigated the potential effects of bile acids entering the colon and its role in the symptom pattern in IBS.

METHODS

We measured 75Se-labelled homocholic acid-taurine (75SeHCAT) retention, and serum levels of 7α-hydroxy-4-cholesten-3-one (C4) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 19 in patients with IBS (n=141) and control subjects (75SeHCAT n=29; C4 and FGF19 n=435). In patients with IBS stool frequency and form, as well as GI symptom severity were registered, and in a proportion of patients colonic transit time and rectal sensitivity were measured (n=66). An 8-week open-label treatment with colestipol was offered to patients with 75SeHCAT (<em>20</em>%, and the effect of treatment was evaluated with IBS severity scoring system and adequate relief of IBS symptoms.

RESULTS

Compared with controls, patients with IBS had lower 75SeHCAT values (p=0.005), higher C4c levels (C4 corrected for cholesterol) (p<0.001), but similar FGF19 levels. Abnormal 75SeHCAT retention (<10%) was seen in 18% of patients, whereas 23% had elevated C4c levels. Patients with IBS with 75SeHCAT retention <10% had more frequent stools, accelerated colonic transit time, rectal hyposensitivity, a higher body mass index, higher C4c and lower FGF19 levels. Colestipol treatment improved IBS symptoms (IBS severity scoring system 2<em>20</em>±109 vs. 277±106; p<0.01), and 15/27 patients fulfilled criteria for treatment response (adequate relief ≥50% of weeks 5-8).

CONCLUSIONS

Increased colonic bile acid exposure influences bowel habit and colonic transit time in patients with IBS. A high response rate to open label treatment with colestipol supports this, but placebo-controlled studies are warranted.

OBJECTIVE

To document the expression patterns of various matrixins, cytokines and angiogenic factors in plasma to assess their involvement in the pathogenesis of moyamoya disease (MMD).

METHODS

This study included plasma samples from 20 MMD patients and nine healthy individuals. The plasma concentration of five matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, MMP-12), monocyte chemoattractant protein-1 (MCP-1), resistin, three interleukins (IL-1beta, IL-6, IL-8), tumour necrosis factor-alpha, vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB) and basic fibroblast growth factor was determined using multianalyte profiling systems. The concentration of the tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) was measured using ELISA. Gelatin zymography for MMP-2 and MMP-9 was also performed.

RESULTS

MMD patients exhibited significantly higher plasma concentrations of MMP-9, MCP-1, IL-1beta, VEGF and PDGF-BB, and lower plasma concentrations of MMP-3, TIMP-1 and TIMP-2 compared with healthy controls. Significant correlations were found among MMP-9, MCP-1, VEGF, PDGF-BB and TIMP-2 in MMD patients.

CONCLUSIONS

There were distinctive expression patterns of matrixins, cytokines and angiogenic factors in MMD patients, which seemed to correlate with disease pathogenesis. The balance between MMPs and TIMPs was disrupted in MMD and correlated with disease pathogenesis. Increased plasma levels of MCP-1 and VEGF in MMD patients may play a role in the recruitment of vascular progenitor cells and in the formation of collateral vessels.

Platelet concentrates (PCs) constitute new biological mediators used in osseous reconstructive surgery. In this study, we assessed (i) the effects of various concentrations of calcium and thrombin on the kinetics of platelet-derived <em>growth</em> <em>factor</em> (PDGF-BB), transforming <em>growth</em> <em>factor</em>-beta1(TGF-beta 1), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) release by PCs and (ii) the contribution of PC supernatants to endothelial cell proliferation. Our results indicate that high concentrations of calcium (Ca) and thrombin (Thr) trigger an immediate and significant increase in bFGF, TGF-beta 1 and PDGF-BB concentrations. Thereafter, PDGF-BB, VEGF and TGF-beta 1 levels remained generally constant over a 6-day period while a decrease in bFGF concentrations was noted after 24h. Lower Ca and Thr concentrations tended to reduce and delay <em>growth</em> <em>factors</em> release from PCs. Endothelial cell proliferation was greatly enhanced with PC supernatants (mean: <em>20</em>-fold increase). This was especially evident when endothelial cells were treated with supernatants harvested early after PC treatment with high concentrations of Ca and Thr or later after PC treatment with low Ca and Thr concentrations. Additional research aiming to measure the effects of Ca and Thr on bone formation in vivo is needed.

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) resulted within 30s in an insoluble, flexible hydrogel. About <em>20</em>% of the FGF-2molecules were released from the FGF-2-incorporated chitosan hydrogel into phosphate buffered saline (PBS) within 1 day, after which no further significant release occurred under in vitro non-degradation conditions of the hydrogel. The FGF-2molecules retained in the chitosan hydrogel remained biologically active, and were released from the chitosan hydrogel upon the in vivo biodegradation of the hydrogel. In order to evaluate its accelerating effect on wound healing, full thickness skin incisions were made on the back of healing-impaired diabetic (db/db) mice and their normal (db/+) littermates. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure in both db/db and db/+ mice. However, the addition of FGF-2 in the chitosan hydrogel further accelerated wound closure in db/db mice, although not in db/+ mice. Histological examination also has demonstrated an advanced granulation tissue formation, capillary formation and epithelialization in wounds treated with FGF-2-incorporated chitosan hydrogels in db/db mice.

Posthemorrhagic hydrocephalus remains a complication of preterm birth for which we lack a clear understanding and a curative therapy. Transforming <em>growth</em> <em>factor</em> beta (TGF-beta) is a cytokine that upregulates the production by <em>fibroblasts</em> of extracellular matrix proteins. We hypothesized that TGF-beta might be released into cerebrospinal fluid (CSF) after intraventricular hemorrhage and play a role in posthemorrhagic hydrocephalus. Total TGF-beta1 and TGF-beta2 were measured by immunoassay in CSF samples from 12 normal preterm infants, nine preterm infants with transient posthemorrhagic ventricular dilation, and 10 infants who subsequently developed permanent hydrocephalus. Five infants received intraventricular tissue plasminogen activator, and two infants were treated by drainage irrigation and fibrinolytic therapy. Median TGF-beta1 in normal CSF was 0.495 ng/mL. In infants with transient posthemorrhagic ventricular dilation, median initial CSF TGF-beta1 was 2.1 ng/mL. Infants who subsequently had permanent hydrocephalus had median initial CSF TGF-beta1, 9.7 ng/mL (differences between groups p < 0.01). Intraventricular recombinant tissue plasminogen activator was followed by a rise in CSF TGF-beta1 (p = 0.0007). Drainage irrigation and fibrinolytic therapy was followed by a fall in CSF TGF-beta1. TGF-beta2 was detected in CSF and showed similar trends, but the CSF concentration of TGF-beta1 was more than <em>20</em> times higher. These findings support the hypothesis that TGF-beta1 is released into CSF after intraventricular hemorrhage and may play an important part in hydrocephalus. The results help to explain the failure of intraventricular fibrinolytic therapy.

BACKGROUND

Transforming growth factor-alpha and epidermal growth factor (EGF), the ligands for EGF receptor (EGFR), stimulate fibroblast proliferation and play an important role in the pathogenesis of pulmonary fibrosis. Therefore, inhibition of the EGFR signal by an EGFR tyrosine kinase inhibitor (EGFR-TKI) may prevent pulmonary fibrosis. However, there is a possibility that blocking the EGFR signal may inhibit epithelial cell repair, thereby exaggerating lung fibrosis.

OBJECTIVE

To investigate the effect of EGFR-TK inhibition on lung fibrosis.

METHODS

We looked at the effects of the EGFR-TKIs gefitinib (20, 90, 200 mg/kg) and AG1478 (12 mg/kg) on a bleomycin-induced lung fibrosis model in mice.

RESULTS

Gefitinib prevented lung fibrosis at all three doses. Furthermore, in those mice that did not receive bleomycin treatment, gefitinib at 200 mg/kg did not induce lung fibrosis. Immunohistochemistry revealed that phosphorylation of EGFR in lung mesenchymal cells induced by bleomycin was inhibited by gefitinib. AG1478 also attenuated the lung fibrosis. In vitro studies further demonstrated that the addition of gefitinib or AG1478 suppressed the EGFR ligand-induced proliferation of lung fibroblasts.

CONCLUSIONS

These findings suggest that, in the preclinical setting, EGFR-TKIs may have a protective effect on lung fibrosis induced by bleomycin. Because these molecular targeted drugs may have differing effects depending on species and individuals, a cautious interpretation is warranted.

We investigated potential mechanisms by which lymphocytes infiltrating rheumatoid synovium become immunosuppressed. In <em>20</em> of 22 synovial fluids and 12 of 13 synovial tissue culture supernatants, no IL-1 bioactivity could be detected in the thymocyte proliferation assay. These same preparations could, however, support proliferation of <em>fibroblast</em> monolayers, consistent with the presence of IL-1 and/or other <em>fibroblast</em> <em>growth</em> <em>factors</em>. Addition of either rheumatoid synovial fluids or synovial culture supernatants to exogenous IL-1 in the IL-1 bioassay caused marked inhibition of the assay indicative of an IL-1 inhibitor. This inhibition of IL-1 could be reversed by treating the effusions or supernatants with a neutralizing antibody to transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). Furthermore, monocyte-macrophages isolated from rheumatoid synovial fluid constitutively released both latent and active TGF-beta in culture at levels sufficient to completely block the biologic activity of 100 U/ml IL-1. The production of substantial levels of TGF-beta by synovial macrophages, as well as the apparent ability of these inflammatory macrophages to activate latent TGF-beta, implicates TGF-beta not only as an important inhibitor of IL-1-induced lymphocyte proliferation, but also as a key cytokine in promoting synovial <em>fibroblast</em> hyperplasia and pathology.

The mandibular processes are specified as at least two independent functional regions: two large lateral regions where morphogenesis is dependent on <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-8 signaling, and a small medial region where morphogenesis is independent of FGF-8 signaling. To gain insight into signaling pathways that may be involved in morphogenesis of the medial region, we have examined the roles of pathways regulated by FGFs and bone morphogenetic proteins (BMPs) in morphogenesis of the medial and lateral regions of the developing chick mandible. Our results show that, unlike in the lateral region, the proliferation and <em>growth</em> of the mesenchyme in the medial region is dependent on signals derived from the overlying epithelium. We also show that medial and lateral mandibular mesenchyme respond differently to exogenous FGFs and BMPs. FGF-2 and FGF-4 can mimic many of the effects of mandibular epithelium from the medial region, including supporting the expression of Msx genes, out<em>growth</em> of the mandibular processes and elongation of Meckel's cartilage. On the other hand, laterally placed FGF beads did not induce ectopic expression of Msx genes and did not affect the <em>growth</em> of the mandibular processes. These functional studies, together with our tissue distribution studies, suggest that FGF-mediated signaling (other than FGF-8), through interactions with FGF receptor-2 and downstream target genes including Msx genes, is part of the signaling pathway that mediates the <em>growth</em>-promoting interactions in the medial region of the developing mandible. Our observations also suggest that BMPs play multiple stage- and region-specific roles in mandibular morphogenesis. In this study, we show that exogenous BMP-7 applied to the lateral region at early stages of development (stage <em>20</em>) caused apoptosis, ectopic expression of Msx genes, and inhibited out<em>growth</em> of the mandibular processes and the formation of Meckel's cartilage. Our additional experiments suggest that the differences between the effects of BMP-7 on lateral mandibular mesenchyme at stage <em>20</em> and previously reported results at stage 23 (Wang et al., [1999] Dev. Dyn. 216:3<em>20</em>-335) are related to differences in stages of differentiation in that BMP-7 promotes apoptosis in undifferentiated lateral mandibular mesenchyme, whereas it promotes chondrogenesis at later stages of development. We also showed that, unlike mandibular epithelium and medially placed FGF beads, medially placed BMP-7 did not support out<em>growth</em> of the isolated mesenchyme and at stage <em>20</em> induced the formation of a duplicated rod of cartilage extending from the body of Meckel's cartilage. These observations suggest that BMPs do not play essential roles in <em>growth</em>-promoting interactions in the medial region of the developing mandible. However, BMP-mediated signaling is a part of the signaling pathways regulating chondrogenesis of the mandibular mesenchyme.

Serum and <em>growth</em> <em>factors</em> activate both the canonical extracellular signal-regulated kinase (ERK) 1/2 pathway and the ERK5/big mitogen-activated protein kinase 1 (BMK) 1 pathway. Pharmacological inhibition of the ERK1/2 pathway using PD98059 and U0126 prevents cyclin D1 expression and inhibits cell proliferation, arguing that the ERK1/2 pathway is rate limiting for cell-cycle re-entry. However, both PD98059 and U0126 also inhibit the ERK5/BMK1 pathway, raising the possibility that the anti-proliferative effect of such drugs may be due to inhibition of ERK5 or both pathways. Here we characterize the effect of the novel mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD184352, on the ERK1/2 and ERK5 pathways in the Chinese hamster <em>fibroblast</em> cell line CCl39. In quiescent cells, serum-stimulated ERK1 activity was completely inhibited by PD184352 with an IC50 below 1 microM, whereas ERK5 activation was unaffected even at <em>20</em> microM. Serum-stimulated DNA synthesis and cyclin D1 expression was inhibited by low doses of PD184352, which abolished ERK1 activity but had no effect on ERK5. Similarly, in cycling cells PD184352 caused a dose-dependent G1 arrest and inhibition of cyclin D1 expression at low doses, which inhibited ERK1 but were without effect on ERK5. These results indicate that the anti-proliferative effect of PD184352 is due to inhibition of the classical ERK1/2 pathway and does not require inhibition of the ERK5 pathway.

The expression in human <em>fibroblasts</em> of the beta 2-interferon (IFN-beta 2) gene, which is now recognized to be identical to the gene encoding B-cell differentiation <em>factor</em> BSF-2, is enhanced by several cytokines that affect cell <em>growth</em> (tumor necrosis <em>factor</em>, interleukin 1, platelet-derived <em>growth</em> <em>factor</em>, and beta 1-interferon). We have examined the possibility that IFN-beta 2 gene expression is regulated through activation, by diacylglycerol, of the protein kinase C pathway. The synthetic diacylglycerols 1,2-dioctanoylglycerol (diC8) and 1-oleoyl-2-acetylglycerol strongly enhanced IFN-beta 2, but not IFN-beta 1, gene expression in human <em>fibroblasts</em> (FS-4 strain). An increase in IFN-beta 2 mRNA level was detected within 15 min after addition of diC8 (290 microM) to FS-4 cells and was maximal approximately <em>20</em> hr later. An increase in IFN-beta 2 gene transcription was detected within 5 min of addition of diC8, and the rate of transcription was near-maximal by 15-30 min. The enhancement of IFN-beta 2 gene expression by diC8, interleukin 1, or tumor necrosis <em>factor</em> was not prevented by H8, a preferential inhibitor of cAMP- and cGMP-dependent protein kinases, but was blocked by H7, an inhibitor of protein kinase C as well as of cyclic nucleotide-dependent protein kinases. diC8 was found to protect FS-4 cells from the cytopathic effect of vesicular stomatitis virus; this protection was blocked by polyclonal or monoclonal antibodies that neutralize IFN-beta, suggesting that the antiviral effect was due to the secretion of IFN-beta 2 by the diC8-treated <em>fibroblasts</em>. The calcium ionophore A23187 (1-10 microM) also elicited an increase in the level of IFN-beta 2 mRNA in FS-4 <em>fibroblasts</em>; appropriate combinations of A23187 and diC8 had at least an additive effect in enhancing IFN-beta 2 mRNA levels. These results show that protein kinase C-activating or [Ca2+]-elevating agents rapidly increase the expression of the IFN-beta 2 gene in human <em>fibroblasts</em>.

BACKGROUND

Thalidomide is a putative antiangiogenesis agent with activity in several hematologic malignancies.

METHODS

Forty-four patients who had myelofibrosis with myeloid metaplasia received treatment with thalidomide in a Phase II clinical trial at a dose of <em>20</em>0 mg daily with escalation by <em>20</em>0 mg weekly until the best tolerated dose (maximum, 800 mg) was reached.

RESULTS

Seventeen of 41 evaluable patients (41%) who received treatment for at least 15 days had a response. A complete response (without reversal of bone marrow fibrosis) was achieved in 4 patients (10%), a partial response was achieved in 4 patients (10%), and hematologic improvements in anemia, thrombopenia, and/or splenomegaly were observed in 9 patients (21%). Improvements in anemia occurred in 7 of 35 patients (<em>20</em>%) with hemoglobin levels <10.0 g/dL, and improvements in thrombopenia occurred in 5 of 24 patients (21%) with platelet counts <100 x 10(9)/L. Five of 24 patients (21%) became transfusion-independent. Major or minor regression of splenomegaly was noted in 9 of 29 evaluable patients (31%), and complete regression was noted in 5 patients. Responders had a lower baseline median vascular endothelial <em>growth</em> <em>factor</em> levels (77.9 pg/mL vs. 97.7 pg/mL; P <.01) and higher median basis <em>fibroblast</em> <em>growth</em> <em>factor</em> levels (60.8 pg/mL vs. 37.4 pg/mL; P <.01) compared with nonresponders. Nine patients (22%) had deterioration that was attributed to thalidomide (resolved after withdrawal) with either progressive cytopenias or excessive proliferation. Two patients developed Grade 3 neutropenia with recovery and resumed therapy with dose reductions, and both later achieved a complete response. Dose-related toxicities included fatigue (50%), constipation (48%), rash or pruritus (37%), sedation (35%), peripheral edema (29%), tremors (23%), peripheral neuropathy (22%), and orthostasis (16%).

CONCLUSIONS

Thalidomide warrants further evaluation in patients with MMM, particularly in combination regimens, along with the investigation of newer analogs.

Rat mucosal keratinocytes serially propagated under permanently serum-free conditions responded to interleukin (IL)-1 beta/IL-alpha and to transforming <em>growth</em> <em>factor</em> (TGF)-alpha/epidermal <em>growth</em> <em>factor</em> (EGF) (as well as to 12-O-tetradecanoylphorbol-13-acetate (TPA)) by upregulation of M(r) 95,000 gelatinase (MMP-9) (M(r) 95K GL) and <em>fibroblast</em>-type collagenase (MMP-1) (FIB-CL), whereas control cells expressed barely detectable levels of either of these enzymes. The cells secreted 8-10 micrograms/10(6) cells/day (M(r) 95K GL) and 2-3 micrograms/10(6) cells/day (FIB-CL) of enzyme protein for at least 24 h when maximally induced. This level was attained only after a 24-h lag period, and the earliest emergence of enzyme protein in the culture medium required 10-14 h. IL-1 beta was by far the most potent cytokine with maximal effect already at 10(-10) M, whereas IL-1 alpha, TGF-alpha, and EGF required <em>20</em>-100-fold higher concentrations. Pretreatment of the cells with TPA (10(-7) M) abolished the subsequent response to IL-1 beta, TGF-alpha, and EGF and at the same time resulted in>> 90% reduction of cytosolic protein kinase C activity. Surprisingly, staurosporine, a potent kinase inhibitor, not only failed to block <em>growth</em> <em>factor</em>/cytokine responses but itself stimulated expression of the enzymes at a magnitude comparable to TPA. The inducing effect of TGF-alpha/EGF was down-regulated by 70-85% by 10(-7) M dexamethasone. Dexamethasone was less effective in ablating the IL-1 beta response yielding 60% reduction M(r) 95K GL and little or no reduction of FIB-CL. Dexamethasone also failed to block the TPA response.

In an attempt to isolate novel regulatory and/or tumor suppressor genes, we identified cDNAs whose abundance is low in NIH 3T3 cells and further decreased following the expression of the activated oncogene, v-src. The transcription of one such gene, 322, is suppressed at least 15-fold in src-, ras-, and fos-transformed cells and 3-fold in myc-transformed cells but is unaffected in raf-, mos-, or neu-transformed cells. Activation of a ts-v-src allele in confluent 3Y1 <em>fibroblasts</em> resulted in an initial increase in 322 mRNA levels after 1 to 2 h followed by a rapid decrease to suppressed levels after 4 to 8 h. Morphological transformation was not detected until 12 h later, indicating that the accumulation of 322 transcripts is regulated by v-src and not as a consequence of transformation. Addition of fetal calf serum to starved subconfluent NIH 3T3 or 3Y1 <em>fibroblasts</em> resulted in a similar biphasic regulation of 322, indicating that 322 transcription is responsive to mitogenic <em>factors</em>. Sequence analysis of a putative full-length 322 cDNA clone (5.4 kb) identified a large open reading frame (ORF) encoding a 148.1-kDa product. In vitro transcription and translation of the 322 cDNA from a T7 promoter resulted in a <em>20</em>7-kDa product whose electrophoretic mobility on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was unaffected by digestion with endoglycosidase F. The discrepancy in predicted versus measured molecular weights may result from the high percentage of acidic residues (roughly <em>20</em>% Glu or Asp) in the 322 ORF product. Comparison of the 322 cDNA ORF with sequences in data banks indicates that this gene is novel. The 322 ORF product contains a potential Cys-1-His-3 Zn finger, at least five nuclear localization signals of the adenovirus E1a motif K(R/K)X(R/K), and alternating acidic and basic domains. Overexpression of the 322 cells resulted in the selection of rapidly <em>growing</em> cells which had lost the transduced 322 cDNA. Thus, 322 represent a novel src- and ras-regulated gene which encodes a potential regulator of mitogenesis and/or tumor suppressor.

We demonstrated previously tyrosine phosphorylation-dependent modulation of phospholipase C-gamma 1 (PLC-gamma 1) catalytic activity (Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 250, 1253-1256). The increase in PLC-gamma 1 catalytic activity in A-431 cells occurs rapidly, with maximal activation 5 min after epidermal <em>growth</em> <em>factor</em> (EGF) stimulation. Certain other <em>growth</em> <em>factors</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>) also stimulate PLC-gamma 1 catalytic activity, whereas insulin does not. A similar increase in PLC-gamma 1 specific activity (2-3-fold) was observed in both soluble (cytosol) and particulate (membrane) preparations from EGF-treated cells. Tyrosine-phosphorylated PLC-gamma 1 was detected in both cytosol and membrane fractions in lysates from EGF-treated A-431 cells, but the proportion of tyrosine-phosphorylated PLC-gamma 1 was higher in the cytosol (approximately 50%) than in the membrane (approximately <em>20</em>%). Because a micellar concentration of the non-ionic detergent Triton X-100 allows detection of the tyrosine phosphorylation-dependent increase in PLC-gamma 1 catalytic activity in this assay, we evaluated the kinetic properties of PLC-gamma 1, immunoprecipitated from cytosol of control or EGF-treated cells, using substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), solubilized in Triton X-100 at various molar ratios. The behavior of the control enzyme differed from the EGF-activated enzyme with respect to both Ks and Km. The control enzyme has a 7.5-fold higher Ks value than the activated enzyme (1.5 mM as compared with 0.22 mM). Activation by EGF is also a positive allosteric modifier of PLC-gamma 1-catalyzed PtdIns 4,5-P2 hydrolysis, i.e. the activated enzyme displayed apparent Michalis-Menton kinetics, with a Km of 0.6 mol fraction PtdIns 4,5-P2, whereas the control enzyme displayed sigmoidal kinetics with respect to PtdIns 4,5-P2 hydrolysis. At low substrate mol fractions (e.g. 0.07), the reaction velocity of the control enzyme was 4-fold lower than the activated enzyme. However, at a high substrate mol fraction (e.g. 0.33), the estimated maximal reaction velocities (Vmax) for both forms of PLC-gamma 1 were equivalent. PLC-gamma 1 activity from both control and EGF-treated cells was stimulated by increasing nanomolar Ca2+ concentrations. Although the catalytic activity of PLC-gamma 1 from EGF-treated cells was greater than control PLC-gamma 1 at every Ca2+ concentration tested, the relative stimulation of activity was markedly greater at Ca2+ concentrations above approximately 300 nM.