BACKGROUND

Equol is a polyphenolic/isoflavonoid molecule that can be expressed as isomers. However, the characteristics of the equol isomers on dermal gene/protein expression and human skin percutaneous absorption remain unknown.

OBJECTIVE

Perform a comprehensive investigation on equol as: R-equol, racemic equol or S-equol to determine their differential expression of skin-related genes, quantify collagen expression and determine percutaneous absorption in human skin.

METHODS

Quantified: (i) gene expression/mRNA levels via gene array technology using human skin equivalents with equol exposure at 1.2% in qPCR experiments, (ii) in vitro collagen expression in human fibroblasts, and (iii) percutaneous absorption by Franz cell techniques.

RESULTS

In the qPCR studies, only three genes displayed the greatest significant expression by S-equol, whereas 16 genes displayed the greatest significant levels (either stimulation or inhibition) by R-equol and/or racemic equol, such as extracellular matrix proteins (i.e., collagen and elastin), nerve growth factor, aging genes [FOS, 100 A8 and A9 calcium-binding proteins, 5α-reductase type 1, and matrix metalloproteinases (1, 3, and 9)], and inflammatory genes (e.g., interleukin-1 alpha, interleukin-6, and cyclooxygenase-1). Collagen type I expression in fibroblasts was greater with racemic versus S-equol treatment at 1 and 10 nM. Percutaneous absorption demonstrated high sequestering in keratinocytes with subsequent accumulation/release over time.

CONCLUSIONS

Overall, these results illustrate the significant differences in mirror-image molecules or isomers of equol where R-equol and/or racemic equol are better molecules for skin gene expression compared to S-equol and the percutaneous absorption of equol represents a unique epidermal reservoir delivery mechanism.

Plasma calcium concentration is maintained within a narrow range (8.5-10.5 mg/dL) by the coordinated action of parathyroid hormone (PTH), 1,25(OH)2D3, calcitonin, and ionized calcium (iCa(2+)) itself. The kidney plays a key role in this process by the fine regulation of calcium excretion. More than 95% of filtered calcium is reabsorbed along the renal tubules. In the proximal tubules, 60% of filtered calcium is reabsorbed by passive mechanisms. In the thick ascending limb, 15% of calcium is reabsorbed by paracellular diffusion through paracellin-1 (claudin-<em>16</em>). The calcium sensing receptor (CaSR) in the basolateral membrane of the thick ascending limb senses the change in iCa(2+) and inhibits calcium reabsorption independent to PTH and 1,25(OH)2D3. The fine regulation of calcium excretion occurs in the distal convoluted tubules and connecting tubules despite the fact that only 10-15% of filtered calcium is reabsorbed there. Transient receptor potential vanilloid 5 (TRPV5) and 6 (TRPV6) in the apical membrane act as the main portal of entry, calbindin-D28K delivers Ca(2+) in the cytoplasm, and then Na(2+)/Ca(2+) exchanger (NCX1) and plasma membrane Ca(2+)-ATPase in the basolateral membrane serve as an exit. In the cortical collecting duct, TRPV6 is expressed, but the role might be negligible. In addition to PTH and 1,25(OH)2D3, acid-base disturbance, diuretics, and estrogen affect on these calcium channels. Recently, klotho and <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) are suggested as new players in the calcium metabolism. Klotho is exclusively expressed in the kidney and co-localized with TRPV5, NCX1, and calbindin-D28K. Klotho increases calcium reabsorption through trafficking of TRPV5 to the plasma membrane, and also converts FGF receptor to the specific FGF23 receptor. FGF23:klotho complex bound to FGF receptor inhibits 1α-hydroxylase of vitamin D, and contributes to calcium reabsorption and phosphate excretion in the kidney.

BACKGROUND

Biochemical alterations associated with mechanical stress have been explored as an initiating step in the pathological progression of ligamentum flavum hypertrophy (LFH); however, this mechanism remains poorly understood. Recently, the inflammation induced after mechanical stress and the subsequent response of ligamentum flavum (LF) cells have been implicated in LFH pathology.

OBJECTIVE

To investigate the hypothesis that angiogenesis may be a critical link between hypertrophy and a series of stimulating events, including mechanical stress.

METHODS

LF from 20 lumbar spinal canal stenosis (LSCS) patients and <em>16</em> non-LSCS patients (control group) were collected during surgery. Patient demographic and radiographic data were obtained. The levels of angiogenic <em>factors</em> (vascular endothelial <em>growth</em> <em>factor</em> [VEGF], angiopoietin-1, vascular cell adhesion molecule, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>) in the LF were investigated by using an enzyme-linked immunosorbent assay. Angiogenesis was also quantified by immunohistochemical detection of CD34-positive capillaries. The correlations among clinical <em>factors</em>, including radiographic <em>factors</em>, angiogenic <em>factors</em>, and angiogenesis, were statistically analyzed.

RESULTS

The LSCS group was older and exhibited a longer symptom duration, wider segmental motion, and thicker LF than the control group. The LSCS group showed significantly higher tissue concentrations of VEGF (P < .001) that positively correlated with LF thickness (r = 0.557, P < .001) and segmental motion (r = 0.586, P < .001). The LSCS group showed significantly more CD34-positive capillaries than the control group (P = .004).

CONCLUSIONS

The LSCS group showed greater segmental motion, higher VEGF concentrations, and more CD34-positive capillaries than the control group. These data indicate that VEGF-mediated angiogenesis following mechanical stress may be a critical step within the series of pathological events in LFH.

Glioblastoma multiforme (GBM) is the most common brain malignancies in adults. Most GBM patients succumb to the disease less than 1 year after diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radioresistant and chemoresistant properties to the tumor cells; therefore, there is a critical need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat signaling pathway. Here, it is reported that EGFRvIII activates Stat5 and GBM invasion by inducing the expression of a previously established mediator of glioma cell invasion and survival: <em>fibroblast</em> <em>growth</em> <em>factor</em>-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is Stat5 dependent and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon Src-MEK/ERK-Stat3 activation. Notably, treatment of EGFRvIII-expressing GBM cells with the FDA-approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration and survival. Because EGFR inhibitors display limited therapeutic efficacy in GBM patients, the EGFRvIII-Stat5-Fn14 signaling pathway represents a node of vulnerability in the invasive GBM cell populations.Implications: Targeting critical effectors in the EGFRvIII-Stat5-Fn14 pathway may limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival. Mol Cancer Res; <em>16</em>(7); 1185-95. ©2018 AACR.

Basic phospholipase A2 was identified from the venom of the eastern diamondback rattlesnake. The Crotalus adamanteus toxin-II (CaTx-II) induced bactericidal effects (7.8 µg/ml) on Staphylococcus aureus, while on Burkholderia pseudomallei (KHW), and Enterobacter aerogenes were killed at 15.6 µg/ml. CaTx-II caused pore formation and membrane damaging effects on the bacterial cell wall. CaTx-II was not cytotoxic on lung (MRC-5), skin <em>fibroblast</em> (HEPK) cells and in mice. CaTx-II-treated mice showed significant wound closure and complete healing by <em>16</em> days as compared to untreated controls (**P<0.01). Histological examination revealed enhanced collagen synthesis and neovascularization after treatment with CaTx-II versus 2% Fusidic Acid ointment (FAO) treated controls. Measurement of tissue cytokines revealed that interleukin-1 beta (IL-1β) expression in CaTx-II treated mice was significantly suppressed versus untreated controls. In contrast, cytokines involved in wound healing and cell migration i.e., monocyte chemotactic protein-1 (MCP-1), <em>fibroblast</em> <em>growth</em> <em>factor</em>-basic (FGF-b), chemokine (KC), granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF) were significantly enhanced in CaTx-II treated mice, but not in the controls. CaTx-II also modulated nuclear <em>factor</em>-kappa B (NF-κB) activation during skin wound healing. The CaTx-II protein highlights distinct snake proteins as a potential source of novel antimicrobial agents with significant therapeutic application for bacterial skin infections.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) genetic alterations are frequently observed in cancer, suggesting that FGFR inhibition may be a promising therapy in patients harboring these lesions. Identification of predictive and pharmacodynamic biomarkers to select and monitor patients most likely to respond to FGFR inhibition will be the key to clinical development of this class of agents. Sensitivity to FGFR inhibition and correlation with FGFR pathway activation status were determined in molecularly annotated panels of cancer cell lines and xenograft models. Pathway inhibition in response to FGFR inhibitor treatment was assessed in cell lines (both in vitro and in vivo) and in samples from patients treated with the FGFR inhibitor JNJ-42756493 (erdafitinib). Frequency of FGFR aberrations was assessed in a panel of NSCLC, breast, prostate, ovarian, colorectal, and melanoma human tumor tissue samples. FGFR translocations and gene amplifications present in clinical specimens were shown to display potent transforming activity associated with constitutive pathway activation. Tumor cells expressing these FGFR activating mutants displayed sensitivity to the selective FGFR inhibitor erdafitinib and resulted in suppression of FGFR phosphorylation and downstream signal transduction. Clinically, patients receiving erdafitinib showed decreased Erk phosphorylation in tumor biopsies and elevation of serum phosphate. In a phase I study, a heavily pretreated bladder cancer patient with an FGFR3-TACC3 translocation experienced a partial response when treated with erdafitinib. This preclinical study confirmed pharmacodynamics and identified new predictive biomarkers to FGFR inhibition with erdafitinib and supports further clinical evaluation of this compound in patients with FGFR genetic alterations. Mol Cancer Ther; <em>16</em>(8); 1717-26. ©2017 AACR.

BACKGROUND

Increased blood levels of fibroblast growth factor-23 (FGF-23) are associated with increased mortality. We evaluated the effect of combined therapy with lanthanum carbonate (LaC), a new phosphate binder and calcium carbonate (CaC) on serum levels of phosphate and FGF-23.

METHODS

This was a single-arm, open-label, multicenter study. Hemodialysis patients with a serum phosphate level >6.0 mg/dL despite CaC therapy were also given LaC for 16 weeks at a dose up to 2250 mg/day. CaC was given at a fixed dose throughout the 16-week period.

RESULTS

Of 42 patients enrolled, 36 completed the 16-week study. The serum phosphate level showed a significant decrease from 6.9 ± 1.4 mg/dL at week 0 to 5.5 + 1.2 mg/dL at week 16 (-20.0%, P < 0.05). The median FGF-23 level showed a significant decrease from 8250 ng/L at week 0 to 5000 ng/L at week 16 (-39.2%, P < 0.05). In contrast, corrected serum calcium and the serum parathyroid hormone level showed no significant changes. A significant positive correlation (r = 0.442, P = 0.007) was demonstrated between the percent reduction of serum FGF-23 and that of serum phosphate.

CONCLUSIONS

Both serum phosphate and FGF-23 levels were significantly decreased by treatment with LaC plus CaC.

Angiogenesis is involved in the <em>growth</em> of new blood vessels from the existing one. Consequently, angiogenesis plays an indispensable role in tissue <em>growth</em> and repair including early placentation processes. Besides angiogenic <em>growth</em> <em>factors</em> (vascular endothelial <em>growth</em> <em>factor</em> (VEGF), angiopoietin-like 4 (ANGPTL4), placental <em>growth</em> <em>factor</em> (PlGF), platelet derived <em>growth</em> <em>factor</em> (PDGF), <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF)), dietary fatty acids (c><em>16</em>) also directly or indirectly modulate angiogenic processes in tumors and other cell systems. Usually n-3 fatty acids inhibit whereas n-6 fatty acids stimulate angiogenesis in tumors and other cells. Contrary to this, docosahexaenoic acid, 22:6n-3 (DHA) and other fatty acids including conjugated linoleic acid stimulate angiogenesis in placental first trimester cells. In addition to the stimulation of expression of major angiogenic <em>factors</em> such as VEGF and ANGPTL4, fatty acids also stimulate expression of intracellular fatty acid-binding proteins (FABPs) FABP-4 and FABP-3 those are known to directly modulate angiogenesis. Emerging data indicate that FABPs may be involved in the angiogenesis process. This paper reviews the fatty acid mediated angiogenesis process and the involvement of their binding proteins in these processes.

<strong class="sub-title">Purpose:</strong> NCI-MATCH is a nationwide, histology-agnostic, signal-finding, molecular profile-driven trial for patients with refractory cancers, lymphomas, or myelomas. Patients with tumors harboring actionable aberration(s) in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (<i>FGFR</i>) <i>1-3</i> were treated with AZD4547, an oral FGFR1-3 inhibitor.

Methods: Patients' tumors were screened by next-generation sequencing for predefined FGFR amplification, activating mutations, or fusions. Patients were treated with AZD4547, 80 mg orally twice daily until progression of disease or drug intolerance. A response rate of 16% was considered promising.

Results: Between July 2016 and June 2017, 70 patients were assigned and 48 received protocol therapy and are eligible for analysis. Patients' tumors harbored FGFR1 or FGFR2 amplification (n = 20), FGFR2 or FGFR3 single-nucleotide variants (n = 19), or FGFR1 or FGFR3 fusions (n = 9). The most common primary tumors were breast (33.3%), urothelial (12.5%), and cervical cancer (10.4%).Grade 3 adverse events were consistent with those described in previous clinical trials. Confirmed partial responses were seen in 8% (90% CI, 3% to 18%) and were observed only in patients whose tumors harbored FGFR1-3 point mutations or fusions. Stable disease was observed in 37.5% (90% CI, 25.8% to 50.4%). The median progression-free survival (PFS) was 3.4 months, and the 6-month PFS rate was 15% (90% CI, 8% to 31%). For patients with tumors harboring FGFR fusions, the response rate was 22% (90% CI, 4.1% to 55%), and 6-month PFS rate was 56% (90% CI, 31% to 100%).

Conclusion: Preliminary signals of activity appeared to be limited to cancers harboring FGFR activating mutations and fusions, although AZD4547 did not meet the primary end point. Different FGFR somatic alterations may confer different levels of signaling potency and/or oncogene dependence.

Background Preclinical studies suggest that imatinib resistance in gastrointestinal stromal tumor (GIST) can be mediated by MAP-kinase activation via <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling. In FGF stimulated GIST cell lines, BGJ398, a pan-FGFR kinase inhibitor in combination with imatinib, was cytotoxic and superior to imatinib therapy alone. In FGF-dependent GIST, the combination of BGJ398 and imatinib may provide a mechanism to overcome imatinib resistance. Methods This phase Ib study of BGJ398 and imatinib was performed in patients with imatinib refractory advanced GIST. A standard 3 + 3 dosing schema was utilized to determine the recommended phase II dose (RP2D). Two treatment schedules were evaluated incorporating imatinib 400 mg daily in combination with (A) BGJ398 daily 3 weeks on, 1 week off or (B) BGJ398 daily 1 week on, 3 weeks off. Results <em>16</em> patients enrolled. The median age was 54 years (range: 44-77), 81% were male, and the median number of lines of prior therapy was 4 [range: 2-6, 13 patients had ≥3 prior therapies]. 12 patients received treatment on schedule A [BGJ398 dose range: 25 - 75 mg]: 2 patients experienced dose limiting toxicities (DLT) (n = 1, myocardial infarction & grade (G)4 CPK elevation; n = 1, G3 ALT elevation) on schedule A (BGJ398 75 mg), significant hyperphosphatemia, an on-target effect, was not observed, implying the maximum tolerated dose was below the therapeutic dose. Following protocol amendment, 4 patients enrolled on schedule B [BGJ398 dose range: 75 - 100 mg]: no DLTs were observed. The most common treatment related adverse events occurring in >15% of patients included CPK elevation (50%), lipase elevation (44%), hyperphosphatemia (24%), anemia (19%), and peripheral edema (19%). Among the 12 evaluable patients, stable disease (SD) was the best response observed in 7 patients by RECIST v1.1 and 9 patients by CHOI. Stable disease ≥ 32 weeks was observed in 3 patients (25%). Median progression free survival was 12.1 weeks (95% CI 4.7-19.5 weeks). Conclusions Toxicity was encountered with the combination therapy of BGJ398 and imatinib. Due to withdrawal of sponsor support the study closed before the RP2D or dosing schedule of the combination therapy was identified. In heavily pre-treated patients, stable disease ≥ 32 weeks was observed in 3 of 12 evaluable patients.

BACKGROUND

NCT02257541 .

The prevalence of non-alcoholic fatty liver disease (NAFLD) has increased dramatically worldwide, and subsequently also the risk of developing nonalcoholic steatohepatitis (NASH), hepatic fibrosis, cirrhosis and cancer. Today, weight loss is the only available treatment, but administration of <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) analogues has, in addition to weight loss, shown improvements on liver metabolic health but the mechanisms behind are not entirely clear. The aim of this study was to investigate the hepatic metabolic profile in response to FGF21 treatment. Diet-induced obese (DIO) mice were treated with subcutaneous administration of FGF21 or subjected to caloric restriction by switching from high fat diet (HFD) to chow to induce 20% weight loss, and changes were compared to vehicle dosed DIO mice. Cumulative caloric intake was reduced by chow while no differences was observed between FGF21 and vehicle dosed mice. The body weight loss in both treatment groups was associated with reduced body fat mass and hepatic triglycerides (TG), while hepatic cholesterol was slightly decreased by chow. Liver glycogen was decreased by FGF21 and increased by chow. The hepatic gene expression profiles suggest that FGF21 increased uptake of fatty acids and lipoproteins, channeled TGs towards production of cholesterol and bile acid, reduced lipogenesis and increased hepatic glucose output. Furthermore, FGF21 appeared to reduce inflammation and regulate hepatic leptin receptor-a expression. In conclusion, FGF21 affected several metabolic pathways to reduce hepatic steatosis and improve hepatic health, and markedly more genes than diet restriction (61 vs <em>16</em> out of 89 investigated genes).

<AbstractText>Lenvatinib (an inhibitor of vascular endothelial <em>growth</em> <em>factor</em> (GF) receptors 1-3, <em>fibroblast</em> GF receptors 1-4, platelet-derived GF receptor α, rearranged during transfection, and stem cell <em>factor</em> receptor) was non-inferior to sorafenib in a phase 3 (REFLECT) trial of advanced hepatocellular carcinoma. This study examined the efficacy and safety of lenvatinib in a real-world setting.</AbstractText><AbstractText>This was a retrospective, multicenter, observational study. Inclusion and exclusion criteria were based on the phase 3 trial, and participants were observed for at least 12 weeks. Therapeutic effect was determined using the modified Response Evaluation Criteria In Solid Tumors (m-RECIST) at the 8th week. Patients received oral lenvatinib 12 mg/day (body weight > 60 kg) or 8 mg/day (body weight < 60 kg). Dose interruptions followed by reductions for lenvatinib-related toxicities were permitted. Grades of adverse events (AEs) complied with the Common Terminology Criteria for Adverse Events version 4.0.</AbstractText><AbstractText>All <em>16</em> patients included in this study had prior treatment history, and a median 3.9 years had passed since the first treatment. Fatigue, hypertension, and proteinuria were the most frequent AEs, and were higher than Grade 2. AEs could be controlled by appropriate dose reduction, interruption, and symptomatic treatment according to the protocol. In the m-RECIST evaluation at the 8th week, 0, 6, 8, and 1 patients had achieved complete response, partial response, stable disease, and progressive disease, respectively. The objective response rate was 40%.</AbstractText><AbstractText>Lenvatinib treatment could be accomplished with safety and good response in a real-world setting.</AbstractText>

The tumour microenvironment has an important role in cancer progression and recent reports have proposed that stromal AKT is activated and regulates tumourigenesis and invasion. We have shown, by immuno-fluorescent analysis of oro-pharyngeal cancer biopsies, an increase in AKT activity in tumour associated stromal <em>fibroblasts</em> compared to normal stromal <em>fibroblasts</em>. Using organotypic raft co-cultures, we show that activation of stromal AKT can induce the invasion of keratinocytes expressing the HPV type <em>16</em> E6 and E7 proteins, in a Keratinocyte <em>Growth</em> <em>Factor</em> (KGF) dependent manner. By depleting stromal <em>fibroblasts</em> of each of the three AKT isoforms independently, or through using isoform specific inhibitors, we determined that stromal AKT2 is an essential regulator of invasion and show in oro-pharyngeal cancers that AKT2 specific phosphorylation events are also identified in stromal <em>fibroblasts</em>. Depletion of stromal AKT2 inhibits epithelial invasion through activating a protective pathway counteracting KGF mediated invasions. AKT2 depletion in <em>fibroblasts</em> stimulates the cleavage and release of IL1B from stromal <em>fibroblasts</em> resulting in down-regulation of the KGF receptor (<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2B (FGFR2B)) expression in the epithelium. We also show that high IL1B is associated with increased overall survival in a cohort of patients with oro-pharyngeal cancers. Our findings demonstrate the importance of stromal derived <em>growth</em> <em>factors</em> and cytokines in regulating the process of tumour cell invasion.

BACKGROUND

The mitotic activity of the epithelial cells of odontogenic keratocysts (OKCs) is greater than that of other odontogenic jaw cysts, and the mitotic activity of the epithelial cells decreases after marsupialization. Keratinocyte growth factor (KGF) interacts with its specific receptor (KGFR), and elicits the proliferation and/or differentiation of the various types of epithelial cells. The aim of this study was to investigate the expression of KGF/KGFR in OKCs before and after marsupialization.

METHODS

The expression of KGF was immunohistochemically detected in the specimens of 16 OKCs and 11 dentigerous cysts before and after marsupialization. The expression of KGF mRNA was measured in the fibroblasts isolated from OKCs by real-time PCR.

RESULTS

KGF was expressed in the epithelial cells and fibroblasts of 12 and seven of 16 OKC specimens, respectively. The intensity of the KGF expression in both the epithelial cells and the fibroblasts significantly decreased after marsupialization. KGFR was expressed throughout the epithelium in 15 of 16 OKC specimens, but the intensity of the KGFR expression did not change after marsupialization. The expression of KGF was detected in the epithelium of two of 11 dentigerous cyst specimens, but not in the fibroblasts before marsupialization. Real-time PCR revealed that recombinant human interleukin (IL)-1alpha increased the expression of KGF mRNA in the fibroblasts isolated from OKCs.

CONCLUSIONS

KGF/KGFR signaling may play a crucial role in the epithelial cells of OKCs. Furthermore, the expression of KGF in the fibroblasts of OKCs is regulated by IL-1alpha.

Recent findings have provided much insight into the mechanisms underlying long-term memory formation, and it is now known that long-term memory depends on the activation of a molecular cascade that culminates with structural changes in the brain. However, little is known about the signals that give rise to or regulate these structural changes. In this article we propose that <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2), a mitogen for several cell types, may be one of the molecular signals critically involved in the structural changes underlying long-term memory. If FGF2 is part of the signalling cascade involved in long-term memory, then increasing the activation of FGF2 should facilitate memory. In Experiments 1 and 2, we demonstrated that systemic injection of FGF2 (20ng/g of body weight) facilitated memory for contextual fear in <em>16</em>, 19, and 22 day old male Sprague Dawley rats. Experiment 3 demonstrated that the observed facilitation of memory was not due to FGF2 increasing rats' sensitivity to footshock. These results implicate FGF2 as a possible molecular signal in long-term memory, and further, illustrate a novel means of enhancing memory.

OBJECTIVE

To determine if low oxygen affects growth factor responsiveness in retinal pigment epithelial (RPE) cells and if such effects are mediated through changes in cell surface receptors.

METHODS

Proliferating human RPE cells were exposed to varying concentrations of exogenous basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) at different media oxygen tensions (16 to 147 mm Hg) and cell counts determined after 4 days. Receptor expression was determined by affinity cross-linking and saturation binding studies on confluent RPE cultures exposed to varying media oxygen tensions for 2 days.

RESULTS

Retinal pigment epithelial cells exhibited a greater proliferative response to exogenous growth factors at hypoxia than at higher media oxygen tensions, and they expressed bFGF and demonstrated that hypoxia caused both an increase in the number of EGF receptors per cell and a shift from low to high affinity receptors.

CONCLUSIONS

These results suggest that hypoxia not only can stimulate RPE cell proliferation per se, it also can "prime" cells t respond more markedly to exogenous growth factors. These observations may be important in elucidating the cause of proliferative vitreoretinopathy.

The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription <em>factors</em> POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) for 14-<em>16</em> days, columnar rosette cells formed in the EB out<em>growth</em>s. Sox2, microtubule-associated protein (MAP2), beta-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and beta-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, beta-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen beta-tublinIII (73+/-4.7%) and some expressed intermediate neuron antigen MAP2 (18+/-7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%+/-1.2%), GFAP (4.93+/-1.9%), S100 (7+/-3.5%) and O4 (0.27+/-0.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be efficiently induced to differentiate along neural lineages.

OBJECTIVE

In synovial tissues of patients with rheumatoid arthritis (RA), strong expression of laminins and integrins co-localises with increased expression of inflammatory cytokines. Synovial <em>fibroblasts</em> (SF) contribute to the pathogenesis of RA through increased expression of cytokines and chemoattractant <em>factors</em>, one of which is interleukin-<em>16</em> (IL<em>16</em>). A study was undertaken to investigate the regulatory pathways of IL<em>16</em> in SF from patients with RA (RA-SF) and osteoarthritis (OA-SF).

METHODS

SF were seeded in laminin-coated flasks and activated by the addition of cytokines. The expression of IL<em>16</em> was investigated by quantitative RT-PCR, immunoblotting and ELISA; its biological activity was determined by a cell migration assay. Cell-matrix interactions were investigated by cell binding and attachment assays. Relevant intracellular signalling pathways were studied by immunoblotting and with pharmacological blocking reagents.

RESULTS

Stimulation of SF with transforming growth factor beta(1)(TGF-beta(1)) and growth on laminin-111 (LM-111) significantly increased the expression of IL<em>16</em>. Binding to LM-111 induced significantly more IL<em>16</em> mRNA in RA-SF than in OA-SF (p<0.05). The IL<em>16</em> cytokine was detected in supernatants of TGF-beta(1)-activated and in LM-111+TGF-beta(1)-activated RA-SF (38 to 62 pg/ml), but not in supernatants of OA-SF. This IL<em>16</em> regulation involved p38MAPK, ERK1/2 and SMAD2 signalling, but not NFkappaB.

CONCLUSIONS

Binding of RA-SF to LM-111 in the presence of TGF-beta(1) triggers a significant IL<em>16</em> response and thus may contribute to the infiltration of CD4+ lymphocytes into synovial tissues. This mode of IL<em>16</em> induction represents a novel pathway leading to IL<em>16</em> production in RA-SF but not in OA-SF, which operates independently of NFkappaB signalling.

OBJECTIVE

To determine the localisation and distribution of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta type 1 (TGF-beta1) in uterine tissues from cycling and early pregnant pigs.

METHODS

In situ hybridisation and immunohistochemistry were used to localise CCN2 (CTGF) or TGF-beta1 in uteri obtained from gilts on days 0, 5, 10, 12, 15, and 18 of the oestrous cycle or days 10, 12, 14, 16, 17, and 21 of gestation.

RESULTS

In cycling animals, CCN2 (CTGF) mRNA and protein were abundant in luminal epithelial cells (LECs) and glandular epithelial cells (GECs), with lesser amounts in stromal fibroblasts and little or none in endothelial cells. A similar pattern of staining was seen up to day 10 of pregnancy, except that overall staining intensities for CCN2 (CTGF) mRNA or protein were higher and that stromal and endothelial cells were CCN2 (CTGF) positive. However, on days 12-17 there was a striking decrease in the amount of CCN2 (CTGF) in LECs at the utero-conceptus interface, which was associated with maternal stromal matrix reorganisation and the onset of subepithelial neovascularisation. This differential distribution of CCN2 (CTGF) was localised to those LECs that were in close proximity to or in apposition with trophoblast cells. This decrease in CCN2 (CTGF) staining was transient in nature and high amounts of CCN2 (CTGF) were again apparent in LECs on days 17-21, when endometrial neovascularisation and matrix remodelling were complete. The expression of uterine TGF-beta1 was comparable to that of CCN2 (CTGF) at most stages of the oestrous cycle or early pregnancy. Pre-elongation blastocysts recovered on day 10 were positive for both CCN2 (CTGF) and TGF-beta1 in the extra-embryonic trophectoderm, endoderm, and inner cell mass. On day 12, trophectoderm expressed low amounts of TGF-beta1 mRNA and non-detectable amounts of TGF-beta1 protein or CCN2 (CTGF) mRNA or protein. By days 17-21, the expression of both growth factors in the extra-embyronic/placental membranes increased and frequently exceeded that seen in LECs.

CONCLUSIONS

The pattern of CCN2 (CTGF) production during the initial attachment phase supports a role for this factor in stromal remodelling and neovascularisation, although alternative functions at later stages such as epithelial-epithelial interactions are also possible. In most major cell types in the uterus or utero-placental unit, CCN2 (CTGF) expression was highly correlated with that of TGF-beta(1), indicating that CCN2 (CTGF) may mediate some of the functions of TGF-beta in the reproductive tract during the oestrous cycle and pregnancy. The data further highlight epithelium as an important source of CCN2 (CTGF) in the regulation of uterine function.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors(1-2), much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension(3-4). Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture(3). While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis. The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized(4). Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells(5). Although some <em>factors</em> participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals(6). <em>Fibroblasts</em> are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine <em>growth</em> <em>factors</em>(7). Many groups have used three dimensional culture systems to study the role of <em>fibroblasts</em> on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion(8-15). We have optimized a simple method for assessing the effects of mammary <em>fibroblasts</em> on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)(<em>16</em>-22). With continued co-culture the cells form spheroids with the <em>fibroblasts</em> clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the <em>fibroblasts</em> that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections(23). Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of <em>fibroblast</em> exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

Intermittent claudication is the most common symptom of peripheral arterial disease (PAD), in part due to an inadequate rise in limb blood flow with exercise. Claudication causes a severe impairment in functional capacity and quality of life in over 3 million Americans. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) stimulates angiogenesis in vivo and improves limb blood flow in several animal models of hindlimb ischemia. However, the relative safety and efficacy of angiogenic molecules in the treatment of claudication has not been fully evaluated in prospective, blinded clinical trials. In this study, a randomized, double-blind, placebo-controlled, phase II trial of recombinant human bFGF for the treatment of intermittent claudication was performed. bFGF was administered weekly by intravenous infusions of 2 microg/kg for 6 sequential weeks (total dose 12 microg/kg). The primary efficacy endpoint was change in peak walking time (PWT) on a graded exercise treadmill protocol. Secondary efficacy endpoints included changes in functional status as measured by validated questionnaires. The study was stopped prematurely after treatment of the first 24 subjects due to proteinuria in five of the <em>16</em> subjects who received systemic bFGF, which exceeded 1000 mg/24 h in four of these five subjects. The small sample size limited evaluation of the predefined efficacy endpoints; however, there was no significant difference between the treatment and control groups for any of the measures of efficacy. In conclusion, intravenous administration of bFGF delivered at low doses weekly for 6 weeks was associated with a high rate of severe proteinuria. It is speculated that bFGF-related proteinuria in this study was primarily related to the systemic route of administration and the frequent dosing schedule. Future clinical trials of bFGF protein should carefully monitor renal function and consider alternative dosing schedules and drug administration routes.

The myeloproliferative leukemia virus (MPLV), a novel murine retroviral complex that does not transform <em>fibroblasts</em>, has been shown to cause an acute leukemia in adult mice accompanied by a progressive polycythemia. The present study demonstrates that, on in vivo inoculation, MPLV induces a rapid suppression of <em>growth</em> <em>factor</em> requirement for in vitro colony formation by both the late and the primitive erythroid progenitor cells. CFU-e-derived erythrocytic colonies developed and differentiated in semi-solid medium without the addition of erythropoietin (Epo). In addition, the formation of CFU-e colonies was not altered by the presence of specific neutralizing Epo antibodies. In the spleen, the CFU-e pool size increased rapidly up to 30-fold. By day 6 postinfection, 100% of these progenitor cells were Epo-independent. The in vivo effects of MPLV-infection on early erythroid progenitor cell compartments were examined in cultures grown for seven days. The concentration of erythroid progenitor cells was twofold elevated in spleen from MPLV-infected mice. As early as day 4 postinfection, 50% of these progenitors produced fully hemoglobinized colonies in serum-free cultures without the addition of interleukin-3 (IL-3) and Epo. Most spontaneous colonies were large and contained up to 10(5) cells per colony. They were composed of either erythroblasts only (<em>16</em>%) or erythroblasts and megakaryocytes (70%); few of them were multipotential (14%). In the marrow, the total number of BFU-e was reduced and only few <em>factor</em>-independent bursts were observed, suggesting a rapid migration of infected progenitors from marrow to spleen. Furthermore, the data show that abnormal erythropoiesis was due to the replication defective MPLV information and was not influenced by the Fv-2 locus.

The <em>growth</em> and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment (1). Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions (1-2). However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor <em>growth</em> that lead to changes in gene expression, cell division, differentiation and invasion(3). Matrix density (4), stiffness (5-6), and structure (6-7), interstitial fluid pressure (8), and interstitial fluid flow (8) are all altered during cancer progression. Interstitial fluid flow in particular is higher in tumors compared to normal tissues (8-10). The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 μm s(-1), depending on tumor size and differentiation (9, 11). This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability (12). Interstitial fluid flow has been shown to increase invasion of cancer cells (13-14), vascular <em>fibroblasts</em> and smooth muscle cells (15). This invasion may be due to autologous chemotactic gradients created around cells in 3-D (<em>16</em>) or increased matrix metalloproteinase (MMP) expression (15), chemokine secretion and cell adhesion molecule expression (17). However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of <em>fibroblasts</em> into tumor-promoting myo<em>fibroblasts</em> (18), (b) transporting of antigens and other soluble <em>factors</em> to lymph nodes (19), and (c) modulating lymphatic endothelial cell morphogenesis (20). The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion (13-15, 17). By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

The possibility that the stimulation of hexose transport in human <em>fibroblasts</em> by phorbol myristate acetate (PMA), insulin, platelet-derived <em>growth</em> <em>factor</em> (PDGF) or epidermal <em>growth</em> <em>factor</em> (EGF) is associated with phosphorylation of the glucose transporter has been investigated. The time and concentration dependencies of the stimulation of transport by these agents under conditions identical to those used for phosphorylation were determined. Each agent, when used at the concentration that resulted in the maximal increase in transport rate, elicited this effect within 30 min of exposure. The extent of stimulation ranged from 15 to 70%. For determination of phosphorylation of the glucose transporter, <em>fibroblasts</em> were incubated for <em>16</em> h with [32P]Pi and exposed to the agonist for 30 min; the transporter was then isolated from a detergent lysate of the cells by immunoprecipitation with a monoclonal antibody. Under these conditions, there was no phosphorylation of transporter in basal cells and only PMA caused detectable incorporation of phosphate into the transporter. Thus, it is unlikely that the stimulation of glucose transport by insulin, PDGF and EGF involve transporter phosphorylation.