Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified <em>15</em> genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue <em>growth</em> <em>factor</em> (CTGF), a modulator of <em>fibroblast</em> matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming <em>growth</em> <em>factor</em> beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.

A completely serum-free assay method has been used to compare the mitogenic activities of polypeptide <em>growth</em> <em>factors</em> and estrogens with MCF-7 and T47D human breast cancer cells in culture. The lines were maintained in a viable, slowly dividing condition in Ham's F12 and Dulbecco's modified Eagle's medium (1:1) supplemented with sodium bicarbonate (2.2 g/liter), <em>15</em> mM 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid, human transferrin (10 micrograms/ml), and bovine serum albumin (200 micrograms/ml) (designated Tf/BSA). This medium allowed the assay of mitogenic activities as measured by multiple rounds of cell division and permitted comparisons of the biological potencies of <em>growth</em> <em>factors</em> within functional families as well as of dissimilar mitogens. Insulin-like <em>growth</em> <em>factor</em> I (IGF-I) was the most potent mitogen studied, showing ED50 values of 160 pg/ml and 1.7 ng/ml with the MCF-7 and T47D cells, respectively. Insulin-like <em>growth</em> <em>factor</em> II and insulin were less active, with ED50 values of 0.55 and 1.2 ng/ml with MCF-7 cells and 4.3 and 10 ng/ml with the T47D cell line, respectively. Mitogens sharing epidermal <em>growth</em> <em>factor</em>-like functional properties had ED50 values from 35 pg/ml to 2.5 ng/ml, while transforming <em>growth</em> <em>factor</em> type beta and platelet-derived <em>growth</em> <em>factor</em> had no detectable stimulatory effects. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> had ED50 values of 0.42 ng/ml and 3.7 ng/ml for the MCF-7 and T47D cells, respectively, while acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> was nearly inactive. In phenol red-free Tf/BSA, 17 beta-estradiol caused a 60% increase in MCF-7 cell numbers over controls in 8 days while having no effect on <em>growth</em> of the T47D cell line. From MCF-7 conditioned Tf/BSA medium, IGF-I was identified by biological activity, by radioimmunoassay (approximately equal to 2 pg/ml) and by estimation of molecular weight (8,000) under dissociating conditions. The concentration of IGF-I was not affected by 17 beta-estradiol treatment. The data indicate that induction of acid stable, low molecular weight autocrine <em>growth</em> <em>factors</em> involved more regulation than defined by estrogens alone. The minimal effects of 17 beta-estradiol in Tf/BSA opened several possibilities including the putative roles of other serum-borne hormones, <em>growth</em> <em>factors</em> and regulators in autocrine <em>growth</em> <em>factor</em> induction.

The gut microbiota is found to be strongly associated with atherosclerosis (AS). Resveratrol (RSV) is a natural phytoalexin with anti-AS effects; however, its mechanisms of action remain unclear. Therefore, we sought to determine whether the anti-AS effects of RSV were related to changes in the gut microbiota. We found that RSV attenuated trimethylamine-N-oxide (TMAO)-induced AS in ApoE(-/-) mice. Meanwhile, RSV decreased TMAO levels by inhibiting commensal microbial trimethylamine (TMA) production via gut microbiota remodeling in mice. Moreover, RSV increased levels of the genera Lactobacillus and Bifidobacterium, which increased the bile salt hydrolase activity, thereby enhancing bile acid (BA) deconjugation and fecal excretion in C57BL/6J and ApoE(-/-) mice. This was associated with a decrease in ileal BA content, repression of the enterohepatic farnesoid X receptor (FXR)-<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FGF<em>15</em>) axis, and increased cholesterol 7a-hydroxylase (CYP7A1) expression and hepatic BA neosynthesis. An FXR antagonist had the same effect on FGF<em>15</em> and CYP7A1 expression as RSV, while an FXR agonist abolished RSV-induced alterations in FGF<em>15</em> and CYP7A1 expression. In mice treated with antibiotics, RSV neither decreased TMAO levels nor increased hepatic BA synthesis. Additionally, RSV-induced inhibition of TMAO-caused AS was also markedly abolished by antibiotics. In conclusion, RSV attenuated TMAO-induced AS by decreasing TMAO levels and increasing hepatic BA neosynthesis via gut microbiota remodeling, and the BA neosynthesis was partially mediated through the enterohepatic FXR-FGF<em>15</em> axis.

OBJECTIVE

Recently, trimethylamine-N-oxide (TMAO) has been identified as a novel and independent risk <em>factor</em> for promoting atherosclerosis (AS) partially through inhibiting hepatic bile acid (BA) synthesis. The gut microbiota plays a key role in the pathophysiology of TMAO-induced AS. Resveratrol (RSV) is a natural phytoalexin with prebiotic benefits. A growing body of evidence supports the hypothesis that phenolic phytochemicals with poor bioavailability are possibly acting primarily through remodeling of the gut microbiota. The current study showed that RSV attenuated TMAO-induced AS by decreasing TMAO levels and increasing hepatic BA neosynthesis via gut microbiota remodeling. And RSV-induced hepatic BA neosynthesis was partially mediated through downregulating the enterohepatic farnesoid X receptor-<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> axis. These results offer new insights into the mechanisms responsible for RSV's anti-AS effects and indicate that the gut microbiota may become an interesting target for pharmacological or dietary interventions to decrease the risk of developing cardiovascular diseases.

p38 is a member of the mitogen-activated protein (MAP) kinase superfamily activated by stress signals and implicated in cellular processes involving inflammation and apoptosis. Unlike the extracellular signal-regulated kinases (p42 and p44 MAP kinases), which are stimulated by insulin in many cell types, p38 activity is inhibited by insulin in postmitotic fetal neurons for which insulin is a potent survival <em>factor</em> (Heidenreich, K. A., and Kummer, J. L. (1996) J. Biol. Chem. 271, 9891-9894). These data suggested that insulin's effects on neuronal survival are mediated by inhibition of a p38-mediated apoptotic pathway. To better understand the relationship between p38 activity and cell survival, we induced apoptosis in two cell lines and examined the ability of insulin or a specific p38 inhibitor (a pyridinyl imidazole compound PD169316) to block p38 activity and cell death. In Rat-1 <em>fibroblasts</em> grown in the presence of serum, p38 activity was undetectable by immune complex assays, and the number of apoptotic cells was very low (<0.5%). After the removal of serum for 16 h, p38 activity was markedly elevated, and apoptosis increased by 14-<em>15</em>-fold. Insulin (50 ng/ml) inhibited p38 activity by approximately 70% and blocked apoptosis by at least 80%. PD169316 also blocked p38 enzyme activity and apoptosis by approximately 80%. Similar results were obtained in differentiated PC12 cells that were deprived of nerve <em>growth</em> <em>factor</em> (NGF) for 16 h. In the presence of NGF, p38 activity and the number of apoptotic cells was very low (approximately 1.0%). After NGF withdrawal, p38 activity was selectively elevated and apoptosis increased to <em>15</em>%. Both insulin and PD169316 markedly blocked the increase in p38 activity and apoptosis. The MAP kinase kinase inhibitor, PD98059, had no effect on apoptosis in Rat-1 <em>fibroblasts</em> and only partially blocked apoptosis in PC12 cells. PD98059 did not influence insulin's ability to block apoptosis, indicating that the extracellular signal-regulated kinase pathway does not mediate insulin's survival effects. These data further support the role of p38 in cellular apoptosis and support the hypothesis that insulin promotes cell survival, at least in part, by inhibiting the p38 pathway.

The cycle of gallbladder filling and emptying controls the flow of bile into the intestine for digestion. Here we show that <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>15</em>, a hormone made by the distal small intestine in response to bile acids, is required for gallbladder filling. These studies demonstrate that gallbladder filling is actively regulated by an endocrine pathway and suggest a postprandial timing mechanism that controls gallbladder motility.

OBJECTIVE

To evaluate levels of biomarkers in preclinical rheumatoid arthritis (RA) and to use elevated biomarkers to develop a model for the prediction of time to future diagnosis of seropositive RA.

METHODS

Stored samples obtained from 73 military cases with seropositive RA prior to RA diagnosis and from controls (mean 2.9 samples per case; samples collected a mean of 6.6 years prior to diagnosis) were tested for rheumatoid factor (RF) isotypes, anti-cyclic citrullinated peptide (anti-CCP) antibodies, 14 cytokines and chemokines (by bead-based assay), and C-reactive protein (CRP).

RESULTS

Preclinical positivity for anti-CCP and/or ≥2 RF isotypes was >96% specific for future RA. In preclinical RA, levels of the following were positive in a significantly greater proportion of RA cases versus controls: interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, IL-15, fibroblast growth factor 2, flt-3 ligand, tumor necrosis factor α, interferon-γ-inducible 10-kd protein, granulocyte-macrophage colony-stimulating factor, and CRP. Also, increasing numbers of elevated cytokines/chemokines were present in cases nearer to the time of diagnosis. RA patients who were ≥40 years old at diagnosis had a higher proportion of samples positive for cytokines/chemokines 5-10 years prior to diagnosis than did patients who were <40 years old at diagnosis (P < 0.01). In regression modeling using only case samples positive for autoantibodies highly specific for future RA, increasing numbers of cytokines/chemokines were predictive of decreased time to diagnosis, and the predicted time to diagnosis based on cytokines/chemokines was longer in older compared with younger cases.

CONCLUSIONS

Levels of autoantibodies, cytokines/chemokines, and CRP are elevated in the preclinical period of RA development. In preclinical autoantibody-positive cases, the number of elevated cytokines/chemokines is predictive of the time of diagnosis of future RA in an age-dependent manner.

The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostalpha-Ostbeta exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostalpha-Ostbeta have not been investigated. To determine the role of Ostalpha-Ostbeta in intestinal bile acid absorption, the Ostalpha gene was disrupted by homologous recombination in mice. Ostalpha(-/-) mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostalpha(-/-) vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostalpha(-/-)Mrp3(-/-) mice. The bile acid pool size was significantly reduced (>65%) in Ostalpha(-/-) mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostalpha(-/-) mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FGF<em>15</em>). These data indicate that Ostalpha-Ostbeta is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.

The t(4;14) translocation that occurs uniquely in a subset (<em>15</em>%) of patients with multiple myeloma (MM) results in the ectopic expression of the receptor tyrosine kinase (RTK), <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3). Inhibition of activated FGFR3 in MM cells induces apoptosis, validating FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these patients, who have a poor prognosis. We describe here the characterization of a novel, small-molecule inhibitor of class III, IV, and V RTKs, CHIR-258, as an inhibitor of FGFR3. CHIR-258 potently inhibits FGFR3 with an inhibitory concentration of 50% (IC50) of 5 nM in in vitro kinase assays and selectively inhibited the <em>growth</em> of B9 cells and human myeloma cell lines expressing wild-type (WT) or activated mutant FGFR3. In responsive cell lines, CHIR-258 induced cytostatic and cytotoxic effects. Importantly, addition of interleukin 6 (IL-6) or insulin <em>growth</em> <em>factor</em> 1 (IGF-1) or coculture on stroma did not confer resistance to CHIR-258. In primary myeloma cells from t(4;14) patients, CHIR-258 inhibited downstream extracellular signal-regulated kinase (ERK) 1/2 phosphorylation with an associated cytotoxic response. Finally, therapeutic efficacy of CHIR-258 was demonstrated in a xenograft mouse model of FGFR3 MM. These studies support the clinical evaluation of CHIR-258 in MM.

A large number of extracellular polypeptides bound to their cognate receptors activate the transcription <em>factor</em> STAT3 by phosphorylation of tyrosine 705. Supplemental activation occurs when serine 727 is also phosphorylated. STAT3 deletion in mice leads to embryonic lethality. We have produced mice with alanine substituted for serine 727 in STAT3 (the SA allele) to examine the function of serine 727 phosphorylation in vivo. Embryonic <em>fibroblasts</em> from SA/SA mice had approximately 50% of the transcriptional response of wild-type cells. However, SA/SA mice were viable and grossly normal. STAT3 wild-type/null (+/-) animals were also normal and were interbred with SA/SA mice to study SA/- mice. The SA/- mice progressed through gestation, showing 10 to <em>15</em>% reduced birth weight, three-fourths died soon after birth, and the SA/- survivors reached only 50 to 60% of normal size at 1 week of age. The lethality and decreased <em>growth</em> were accompanied by altered insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1) levels in serum, establishing a role for the STAT3 serine phosphorylation acting through IGF-1 in embryonic and perinatal <em>growth</em>. The SA/- survivors have decreased thymocyte number associated with increased apoptosis, but unexpectedly normal STAT3-dependent liver acute phase response. These animals offer the opportunity to study defined reductions in the transcriptional capacity of a widely used signaling pathway.

Pirfenidone, a broad-spectrum antifibrotic agent, is known to have efficacy in certain fibrotic disease models, and is under clinical trials in patients with idiopathic pulmonary fibrosis. We investigated the antifibrotic effect of pirfenidone, and its regulatory effect on various pulmonary cytokines, in bleomycin-induced lung fibrosis in mice at the protein level, using prednisolone as a reference agent. Pirfenidone attenuated the bleomycin-induced pulmonary fibrosis at a minimum effective dose of 30 mg/kg/day t.i.d. from the analysis of lung hydroxyproline content. Both pirfenidone (30, 100 mg/kg/day t.i.d) and prednisolone (3, <em>15</em> mg/kg/day q.d.) suppressed lung inflammatory edema; however, prednisolone failed to suppress pulmonary fibrosis, which was significantly suppressed only by pirfenidone. Both pirfenidone and prednisolone suppressed the increase in lung interleukin (IL)-1beta, IL-6, IL-12p40 and monocyte chemoattractant protein (MCP)-1 levels induced by bleomycin. On the other hand, pirfenidone prevented the bleomycin-induced decrease in lung interferon (IFN)-gamma levels, while prednisolone had no such effect. Furthermore, pirfenidone suppressed elevation of lung basic-<em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and transforming <em>growth</em> <em>factor</em> (TGF)-beta1 levels, but prednisolone had no such effect. The increases in lung stroma cell derived <em>factor</em> (SDF)-1alpha and IL-18 were also suppressed. These findings suggest that pirfenidone exerts its antifibrotic effect through regulation of lung IFN-gamma, bFGF and TGF-beta1 levels during the development of bleomycin-induced pulmonary fibrosis in mice. The effect on SDF-1alpha and IL-18 levels may also be related to the antifibrotic effects of pirfenidone.

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain <em>fibroblasts</em> and another marrow-derived clone, BMC10, were isolated from a transgenic mouse (H-2Kb-tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming <em>growth</em> <em>factor</em> beta and 10-7 M dexamethasone in <em>15</em>-ml polypropylene tubes ("aggregate cultures"). Adipogenic potential was quantitated by flow cytometry of Nile Red-stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate-resistant acid phosphatase-positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10-7 M vitamin D3 and <em>15</em>0 mM ascorbate-2-phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow-derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain <em>fibroblasts</em>, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast-supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult-derived multipotential progenitor cell that is situated early in the mesenchymal lineage.

Most endometrial carcinomas are diagnosed at an early stage. Still, <em>15</em>-20% of these carcinomas recur with limited effect of systemic therapies in metastatic disease. Improved ability to target surgical and systemic therapies to well selected patient populations will increase the likelihood of benefits. Retrospective studies have identified several markers for lymph-node metastasis and poor prognosis. No new targeted treatments are available in the clinic, but recent comprehensive molecular characterisations of tumours have identified drugs targeting the PI3K/PTEN/AKT/mTOR pathway and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) 2 as promising for further studies, also reflected in current clinical trials investigating endometrial carcinoma. A more systematic approach to integration of biomarkers in surgical trials and clinical trials of therapeutics, earlier characterisation and standardisation of diagnostic imaging and biomarker assessment, and prospective implementation studies are needed for clinical implementation. We summarise the present knowledge regarding biomarkers in endometrial carcinoma, assessing how such markers could be applied to address key clinical challenges for the treatment of this disease.

Controlled release of human vascular endothelial <em>growth</em> <em>factor</em> (VEGF) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from hydrogels composed of chemically modified hyaluronan (HA) and gelatin (Gtn) was evaluated both in vitro and in vivo. We hypothesized that inclusion of small quantities of heparin (Hp) in these gels would regulate <em>growth</em> <em>factor</em> (GF) release over an extended period, while still maintaining the in vivo bioactivity of released GFs. To test this hypothesis, HA, Gtn, and Hp (<em>15</em> kDa) were modified with thiol groups, then co-crosslinked with poly (ethylene glycol) diacrylate (PEGDA). Either VEGF or bFGF was incorporated into the gels before crosslinking with PEGDA. Release of these GFs in vitro could be sustained over 42 days by less than 1% Hp content, and was found to decrease monotonically with increasing Hp concentration. As little as 0.03% Hp in the gels reduced the released VEGF fraction from 30% to 21%, while 3% Hp reduced it to 19%. Since the minimum Hp concentration capable of effective controlled GF release in vitro was found to be 0.3% (w/w), this concentration was selected for subsequent in vivo experiments. To evaluate the bioactivity of released GFs in vivo, gel samples were implanted into the ear pinnas of Balb/c mice and the resulting neovascularization response measured. In the presence of Hp, vascularization was sustained over 28 days. GF release was more rapid in vitro from gels containing Gtn than from gels lacking Gtn, though unexpectedly, the in vivo neovascularization response to Gtn-containing gels was decreased. Nevertheless significant numbers of neovessels were generated. The ability to stimulate localized microvessel <em>growth</em> at controlled rates for extended times through the release of GFs from covalently linked, Hp-supplemented hydrogels will ultimately provide a powerful therapeutic tool.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) has been shown to be a potent mediator of angiogenesis that functions as a survival <em>factor</em> for endothelial cells by up-regulating Bcl-2 expression. We have recently reported that human dermal microvascular endothelial cells (HDMECs) seeded in biodegradable sponges and implanted into severe combined immunodeficient (SCID) mice organize into functional human microvessels that transport mouse blood cells. In this study, we implanted sponges seeded with OSCC-3 (oral squamous cell carcinoma) or SLK (Kaposi's sarcoma) together with endothelial cells into SCID mice to generate human tumors vascularized with human microvessels. This model system was used to examine the role of both endothelial cell Bcl-2 and the proangiogenic chemokine interleukin-8 (IL-8) on tumor <em>growth</em> and intratumoral microvascular density. Coimplantation of HDMECs overexpressing Bcl-2 (HDMEC-Bcl-2) and tumor cells resulted in a 3-fold enhancement of tumor <em>growth</em> when compared with the coimplantation of control HDMECs and tumor cells. This was associated with increased intratumoral microvascular density and enhanced endothelial cell survival. To determine whether the enhanced neovascularization mediated by Bcl-2 overexpression in endothelial cells was influenced by the synthesis of endogenous mediators of angiogenesis, we screened these cells for expression of VEGF, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and IL-8 by ELISA. HDMEC-Bcl-2 cells and VEGF-treated HDMECs exhibited a <em>15</em>-fold and 4-fold increase, respectively, in the expression of the proangiogenic chemokine IL-8 in vitro, whereas the expression of VEGF and bFGF remained unchanged. Transfection of antisense Bcl-2 into HDMECs blocked VEGF-mediated induction of IL-8. Conditioned media from HDMEC-Bcl-2 induced proliferation and sprouting of endothelial cells in vitro and neovascularization in rat corneas. Anti-IL-8 antibody added to HDMEC-Bcl-2 conditioned media markedly reduced the potency of these responses. SCID mice bearing VEGF-producing tumor implants that were treated with anti-lL-8 antibody exhibited a 43% reduction in microvessel density and a 50% reduction in tumor weight compared with treatment with a nonspecific antibody. These results demonstrate that the up-regulation of Bcl-2 expression in endothelial cells that constitute tumor microvessels enhances intratumoral microvascular survival and density and accelerates tumor <em>growth</em>. Furthermore, endothelial cells that overexpress Bcl-2 have more angiogenic potential than control cells, and IL-8-neutralizing antibodies attenuate their angiogenic activity in vitro and in vivo.

BACKGROUND

Patients with critical limb ischaemia have a high rate of amputation and mortality. We tested the hypothesis that non-viral 1 fibroblast growth factor (NV1FGF) would improve amputation-free survival.

METHODS

In this phase 3 trial (EFC6145/TAMARIS), 525 patients with critical limb ischaemia unsuitable for revascularisation were enrolled from 171 sites in 30 countries. All had ischaemic ulcer in legs or minor skin gangrene and met haemodynamic criteria (ankle pressure <70 mm Hg or a toe pressure <50 mm Hg, or both, or a transcutaneous oxygen pressure <30 mm Hg on the treated leg). Patients were randomly assigned to either NV1FGF at 0·2 mg/mL or matching placebo (visually identical) in a 1:1 ratio. Randomisation was done with a central interactive voice response system by block size 4 and was stratified by diabetes status and country. Investigators, patients, and study teams were masked to treatment. Patients received eight intramuscular injections of their assigned treatment in the index leg on days 1, 15, 29, and 43. The primary endpoint was time to major amputation or death at 1 year analysed by intention to treat with a log-rank test using a multivariate Cox proportional hazard model. This trial is registered with ClinicalTrials.gov, number NCT00566657.

RESULTS

259 patients were assigned to NV1FGF and 266 to placebo. All 525 patients were analysed. The mean age was 70 years (range 50-92), 365 (70%) were men, 280 (53%) had diabetes, and 248 (47%) had a history of coronary artery disease. The primary endpoint or components of the primary did not differ between treatment groups, with major amputation or death in 86 patients (33%) in the placebo group, and 96 (36%) in the active group (hazard ratio 1·11, 95% CI 0·83-1·49; p=0·48). No significant safety issues were recorded.

CONCLUSIONS

TAMARIS provided no evidence that NV1FGF is effective in reduction of amputation or death in patients with critical limb ischaemia. Thus, this group of patients remains a major therapeutic challenge for the clinician.

BACKGROUND

Sanofi-Aventis, Paris, France.

In an in vitro model of granulation tissue, early passage human diploid <em>fibroblasts</em> under mechanical tension showed little or no apoptosis. Release of mechanical tension triggered an apoptotic response that occurred within 3-6 h and reached a plateau by 24 h. The percentage of apoptotic cells (approximately <em>15</em>%) remained constant up to 7 days, and after 3 days, total cell number declined. Identification of mechanical unloading as a stimulus for apoptosis, without application of pharmacologic or genetic intervention, is a novel observation that permits us to model similar events that occur during wound healing. Studies on the mechanism regulating apoptosis under these conditions established that the apoptotic response does not require differentiation of cells into myo<em>fibroblasts</em> but is governed by a combination of mechanical tension and <em>growth</em> <em>factors</em> in the collagen matrix.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) has been found to increase neuronal survival and neurite extension in a highly purified population of fetal rat hippocampal neurons under well-defined serum-free cell culture conditions. In the presence of FGF, neuronal survival after 7 days in culture on a simple plastic substrate is increased 4-fold, to 54% of the initial population. Survival is increased 2-fold to 40% on polyornithine-laminin. When FGF was bound to plastic or heparin substrates, neurite out<em>growth</em> was significantly increased to lengths comparable to those seen with laminin; however, FGF produced no further increase in neurite out<em>growth</em> on laminin. Half-maximal survival was observed at FGF concentrations of about <em>15</em> pg/ml (1 pM); half-maximal process out<em>growth</em> occurred at about 375 pg/ml (20 pM). The responsive cells were identified as neurons by their labeling with tetanus toxin and by antibodies to neurofilaments and to the neuron-specific enolase. Astrocytes, identified by the presence of glial fibrillary acidic protein, constituted about 10% of cells present at 1 week both in the presence and in the absence of FGF. These results strongly suggest that, in addition to its known mitogenic effects on nonneuronal cells, FGF possesses neurotrophic activity for hippocampal neurons.

Insulin and such insulinlike <em>growth</em> <em>factors</em> as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as <em>growth</em> effects (stimulation of [(3)H]thymidine incorporation into DNA in cultured <em>fibroblasts</em>). In addition, most cells have separate receptors for insulin and insulinlike <em>growth</em> <em>factors</em>, and both peptides have weaker affinity for each other's specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike <em>growth</em> <em>factors</em>, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor. In rat adipocytes, 10 mug/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding <5%. The anti-insulin receptor Fab is without intrinsic biological activity, but acts as a competitive inhibitor of insulin receptors. Blockade of insulin receptors with Fab fragments produced a 30-fold rightward shift in the dose response for stimulation of glucose oxidation by both insulin and MSA. The dose-response curves for stimulation of oxidation by vitamin K(5) and spermine, agents that stimulate glucose oxidation through noninsulin receptor pathways, were not affected by the blockade of insulin receptors with Fab antibody fragments. These data suggest that this acute metabolic effect of both insulin and MSA is mediated via the insulin receptor. In cultured human <em>fibroblasts</em>, 10 mug/ml of Fab inhibited insulin binding by 90% and MSA binding by <em>15</em>%. In <em>fibroblasts</em>, however, blockade of the insulin receptor did not alter the dose response for stimulation of thymidine incorporation into DNA by either insulin or MSA. Furthermore, intact antireceptor antibody immunoglobulin (Ig)G, which produces multiple other insulinlike effects, and Fab fragments of antireceptor antibody did not stimulate thymidine incorporation. These data demonstrate directly that the insulin receptor mediates the metabolic effects of insulin and MSA, whereas the <em>growth</em>-promoting action of both peptides is mediated by the MSA receptor or other <em>growth</em> <em>factors</em>.

Mammalian oocytes are deficient in their ability to carry out glycolysis. Therefore, the products of glycolysis that are necessary for oocyte development are provided to oocytes by companion cumulus cells. Mouse oocytes secrete paracrine <em>factors</em> that promote glycolysis in cumulus cells. The objective of this study was to identify paracrine <em>factors</em> secreted by oocytes that promote glycolysis and expression of mRNA encoding the glycolytic enzymes PFKP and LDHA. Candidates included <em>growth</em> differentiation <em>factor</em> 9 (GDF9), bone morphogenetic protein <em>15</em> (BMP<em>15</em>) and <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs). Bmp<em>15</em>-/- and Gdf9+/- Bmp<em>15</em>-/- (double mutant, DM) cumulus cells exhibited reduced levels of both glycolysis and Pfkp and Ldha mRNA, and mutant oocytes were deficient in promoting glycolysis and expression of Pfkp and Ldha mRNA in cumulus cells of wild-type (WT) mice. Alone, neither recombinant BMP<em>15</em>, GDF9 nor FGF8 promoted glycolysis and expression of Pfkp and Ldha mRNA in WT cumulus cells. Co-treatment with BMP<em>15</em> and FGF8 promoted glycolysis and increased expression of Pfkp and Ldha mRNA in WT cumulus cells to the same levels as WT oocytes; however, the combinations of BMP<em>15</em>/GDF9 or GDF9/FGF8 did not. Furthermore, SU5402, an FGF receptor-dependent protein kinase inhibitor, inhibited Pfkp and Ldha expression in cumulus cells promoted by paracrine oocyte <em>factors</em>. Therefore, oocyte-derived BMP<em>15</em> and FGFs cooperate to promote glycolysis in cumulus cells.

The catalytic subunit of protein phosphatase 2A (PP2A) is inactivated by in vitro phosphorylation of Tyr307 by receptor and nonreceptor protein tyrosine kinases (Chen, J., Martin, B. L., and Brautigan, D. L. (1992) Science 257, 1261-1264). Here we show the phosphorylation of PP2A in cells under different <em>growth</em> conditions. In lysates of nontransformed murine 10T1/2 <em>fibroblasts</em>, there were two forms of PP2A at 36 kDa detected after two-dimensional gel electrophoresis and immunoblotting with anti-PP2A peptide antibody. These two forms exactly comigrated with unphosphorylated purified PP2A and the PP2A 32P-labeled by in vitro phosphorylation with p60v-src kinase. The phosphorylated form of PP2A recovered from red blood cells or produced by in vitro phosphorylation was eliminated by incubation with tyrosine-specific phosphatase (PTP1B). Transformation of 10T1/2 cells by expression of p60v-src resulted in most of the PP2A in the cells being converted to a phosphorylated form that was reactive with anti-phosphotyrosine antibody. Serum starvation of cells reduced the amount of phosphorylated PP2A, whereas serum stimulation of quiescent cells caused an increase to the same relative amount of phosphorylated PP2A as in src-transformed cells. Addition of epidermal <em>growth</em> <em>factor</em> to quiescent NeoR cells (10T1/2 <em>fibroblasts</em> overexpressing epidermal <em>growth</em> <em>factor</em> receptors) temporarily increased the level of phosphorylation of PP2A, with a peak at 5-<em>15</em> min and a return to basal level within 60 min. The results show that PP2A is phosphorylated in intact cells, and the extent of this modification is increased by <em>growth</em> <em>factors</em> or cell transformation, providing evidence for a physiological mechanism of PP2A regulation.

mRNA expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), and hypoxia-inducible <em>factor</em> (HIF) subunits HIF-1alpha and HIF-1beta in human skeletal muscle was studied during endurance exercise at different degrees of oxygen delivery. Muscle biopsies were taken before and after 45 min of one-legged knee-extension exercise performed under conditions of nonrestricted or restricted blood flow (approximately <em>15</em>-20% lower) at the same absolute workload. Exercise increased VEGF mRNA expression by 178% and HIF-1beta by 340%, but not HIF-1alpha and FGF-2. No significant differences between the restricted and nonrestricted groups were observed. The exercise-induced increase in VEGF mRNA was correlated to the exercise changes in HIF-1alpha and HIF-1beta mRNA. The changes in VEGF, HIF-1alpha, and HIF-1beta mRNAs were correlated to the exercise-induced increase in femoral venous plasma lactate concentration. It is concluded that 1) VEGF but not FGF-2 gene expression is upregulated in human skeletal muscle by a single bout of dynamic exercise and that there is a graded response in VEGF mRNA expression related to the metabolic stress and 2) the increase in VEGF mRNA expression correlates to the changes in both HIF-1alpha and HIF-1beta mRNA.

HER2 amplification is found in more than <em>15</em>% of gastric cancers and is associated with poor clinical outcome. Lapatinib, a dual HER2 and epidermal <em>growth</em> <em>factor</em> receptor (EGFR) tyrosine kinase inhibitor, has shown promising in vitro results in treating HER2(+) cancer cells. However, several studies have shown that activation of alternative receptor tyrosine kinases can mediate resistance to HER-targeted therapy. Here, we investigated whether activated MET can confer resistance to lapatinib inhibition of gastric cancer cells. A panel of gastric cancer cell lines was treated with lapatinib, and we observed that cell proliferation was reduced by 70% and that the degree of HER2 amplification corresponds to sensitivity to lapatinib. Immunoblotting analysis indicated that phosphorylation of HER2, EGFR, MET, AKT, and extracellular signal-regulated kinase was inhibited by lapatinib and presumably led to cell-cycle arrest as observed with flow cytometry. Hepatocyte <em>growth</em> <em>factor</em> (HGF) activation of MET receptors rescued cells from lapatinib-induced <em>growth</em> inhibition by restimulating the downstream pathways and restoring normal cell-cycle progression. This rescue effect could be abrogated by inhibiting MET with PHA-665752 (a highly specific MET inhibitor) or downregulating MET expression with short interfering RNA. No synergy in <em>growth</em> inhibition was observed when cells were treated with a combination of lapatinib and PHA-665752. Repeat studies using insulin-like <em>growth</em> <em>factor</em> 1 and <em>fibroblast</em> <em>growth</em> <em>factor</em> 3 could not uniformly rescue the lapatinib-treated gastric cancer cells. In conclusion, HGF/MET-mediated resistance to lapatinib is a novel mechanism of resistance to HER2-targeted agents in gastric cancer cells. Development of inhibitors targeting multiple receptors or common downstream signaling proteins merits further investigation.

BACKGROUND

An unmet medical need exists for patients with metastatic renal cell carcinoma who have progressed on VEGF-targeted and mTOR-inhibitor therapies. Fibroblast growth factor (FGF) pathway activation has been proposed as a mechanism of escape from VEGF-targeted therapies. Dovitinib is an oral tyrosine-kinase inhibitor that inhibits VEGF and FGF receptors. We therefore compared dovitinib with sorafenib as third-line targeted therapies in patients with metastatic renal cell carcinoma.

METHODS

In this multicentre phase 3 study, patients with clear cell metastatic renal cell carcinoma who received one previous VEGF-targeted therapy and one previous mTOR inhibitor were randomly assigned through an interactive voice and web response system to receive open-label dovitinib (500 mg orally according to a 5-days-on and 2-days-off schedule) or sorafenib (400 mg orally twice daily) in a 1:1 ratio. Randomisation was stratified by risk group and region. The primary endpoint was progression-free survival (PFS) assessed by masked central review. Efficacy was assessed in all patients who were randomly assigned and safety was assessed in patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01223027.

RESULTS

284 patients were randomly assigned to the dovitinib group and 286 to the sorafenib group. Median follow-up was 11·3 months (IQR 7·9-14·6). Median PFS was 3·7 months (95% CI 3·5-3·9) in the dovitinib group and 3·6 months (3·5-3·7) in the sorafenib group (hazard ratio 0·86, 95% CI 0·72-1·04; one-sided p=0·063). 280 patients in the dovitinib group and 284 in the sorafenib group received at least one dose of study drug. Common grade 3 or 4 adverse events included hypertriglyceridaemia (38 [14%]), fatigue (28 [10%]), hypertension (22 [8%]), and diarrhoea (20 [7%]) in the dovitinib group, and hypertension (47 [17%]), fatigue (24 [8%]), dyspnoea (21 [7%]), and palmar-plantar erythrodysaesthesia (18 [6%]) in the sorafenib group. The most common serious adverse event was dyspnoea (16 [6%] and 15 [5%] in the dovitinib and sorafenib groups, respectively).

CONCLUSIONS

Dovitinib showed activity, but this was no better than that of sorafenib in patients with renal cell carcinoma who had progressed on previous VEGF-targeted therapies and mTOR inhibitors. This trial provides reference outcome data for future studies of targeted inhibitors in the third-line setting.

BACKGROUND

Novartis Pharmaceuticals Corporation.

Squamous cell carcinomas (SCCs) of the head and neck are malignant tumors with high capacity to invade and metastasize. We have examined expression of the new collagenase, collagenase-3 (MMP-13), in SCCs of the head and neck. MMP-13 mRNAs were detected in 22 of 29 SCC cell lines: in 14 of <em>15</em> primary SCC cell lines and in 8 of 14 SCC cell lines from recurrent tumors or metastases. MMP-13 mRNAs were expressed by all 6 cell lines from highly invasive primary tumors and in all 4 cell lines from small aggressive tumors. Using in situ hybridization, MMP-13 mRNAs were detected in <em>15</em> of 17 SCC tumor samples. In most tumors, MMP-13 was expressed by tumor cells at the invading front of the tumors, but in a subset of SCCs, MMP-13 mRNA was also expressed by stromal <em>fibroblasts</em>. No MMP-13 expression was detected in intact skin or oral mucosa. MMP-13 mRNA levels in SCC cells were enhanced by transforming <em>growth</em> <em>factor</em>-beta, tumor necrosis <em>factor</em>-alpha, transforming <em>growth</em> <em>factor</em>-alpha, and keratinocyte <em>growth</em> <em>factor</em>. Specific expression of MMP-13 by SCC cells in vitro and in vivo strongly suggests a role for MMP-13 in the high invasion capacity of SCC cells.