Among IL-<em>17</em> families, IL-<em>17</em>A and IL-<em>17</em>F share amino acid sequence similarity and bind to IL-<em>17</em>R type A. IL-<em>17</em> signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) both remain to be elucidated. This study revealed that IL-<em>17</em>A expression was significantly increased in the involved skin and sera of SSc patients, whereas the IL-<em>17</em>F levels did not increase. In contrast, the expression of IL-<em>17</em>R type A in SSc <em>fibroblasts</em> significantly decreased in comparison with that in normal <em>fibroblasts</em>, due to the intrinsic TGF-β1 activation in these cell types. Moreover, IL-<em>17</em>A, not IL-<em>17</em>F, reduced the protein expression of α1(I) collagen and connective tissue <em>growth</em> <em>factor</em>. miR-129-5p, one of the downregulated microRNAs in SSc <em>fibroblasts</em>, increased due to IL-<em>17</em>A and mediated the α1(I) collagen reduction. These results suggest that IL-<em>17</em>A signaling, not IL-<em>17</em>F, has an antifibrogenic effect via the upregulation of miR-129-5p and the downregulation of connective tissue <em>growth</em> <em>factor</em> and α1(I) collagen. IL-<em>17</em>A signaling is suppressed due to the downregulation of the receptor by the intrinsic activation of TGF-β1 in SSc <em>fibroblasts</em>, which may amplify the increased collagen accumulation and fibrosis characteristic of SSc. Increased IL-<em>17</em>A levels in the sera and involved skin of SSc may be due to negative feedback. Clarifying the novel regulatory mechanisms of fibrosis by the cytokine network consisting of TGF-β and IL-<em>17</em>A may lead to a new therapeutic approach for this disease.

Meltrin alpha (ADAM12) is a metalloprotease-disintegrin whose specific expression patterns during development suggest that it is involved in myogenesis and the development of other organs. To determine the roles Meltrin alpha plays in vivo, we generated Meltrin alpha-deficient mice by gene targeting. Although the number of homozygous embryos are close to the expected Mendelian ratio at embryonic days <em>17</em> to 18, ca. 30% of the null pups born die before weaning, mostly within 1 week of birth. The viable homozygous mutants appear normal and are fertile. Most of the muscles in the homozygous mutants appear normal, and regeneration in experimentally damaged skeletal muscle is unimpeded. In some Meltrin alpha-deficient pups, the interscapular brown adipose tissue is reduced, although the penetrance of this phenotype is low. Impaired formation of the neck and interscapular muscles is also seen in some homozygotes. These observations suggest Meltrin alpha may be involved in regulating adipogenesis and myogenesis through a linked developmental pathway. Heparin-binding epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em> (HB-EGF) is a candidate substrate of Meltrin alpha, and we found that TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ectodomain shedding of HB-EGF is markedly reduced in embryonic <em>fibroblasts</em> prepared from Meltrin alpha-deficient mice. We also report here the chromosomal locations of Meltrin alpha in the mouse and rat.

Several recent in vitro investigations and experimental studies performed in animal models of osteoarthritis (OA) sustained the previously held view that interleukin (IL)-1 or tumour necrosis <em>factor</em>-alpha (TNFalpha) disrupt the metabolism of synovial joint tissues. The evidence to date indicates that, in addition to IL-1 and TNFalpha, other pro-inflammatory cytokines, including IL-6, members of the IL-6 protein superfamily, IL-7, IL-<em>17</em> and IL-18, can also promote articular cartilage extracellular matrix protein degradation or synergize with other cytokines to amplify and accelerate cartilage destruction. Most importantly, many of these cytokines have been implicated in causing synovial tissue activation and damage to subchondral bone as well as altering cartilage homeostasis in spontaneously occurring or surgically induced animal models of OA and in transgenic mice genetically primed to develop OA. In this regard, these pro-inflammatory cytokines may also play a significant role in the pathogenesis of human OA. However, attempts to modify the progression of human OA in well designed, controlled clinical trials with an IL-1 receptor antagonist protein (IRAP) have not been successful. Several anabolic cytokines (also termed <em>growth</em> <em>factors</em>), including transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), platelet-derived <em>growth</em> <em>factor</em> (PDGF) and connective tissue <em>growth</em> <em>factor</em> (CTGF), have also been proposed as regulators of skeletal long bone <em>growth</em> and development as well as cartilage and bone homeostasis. TGF-beta, IGF-1 and FGF-2, in particular, have been characterized as potential chondroprotective agents. Thus, enzymatic disruption and removal of these <em>growth</em> <em>factors</em> from cartilage extracellular matrix proteins, as in the case of TGF-beta and FGF-2, or disruption of their function, as in the case of the enhanced binding of free IGF-1 with IGF binding proteins in OA joint synovial fluid, may compromise and ultimately be responsible for the inadequate repair of articular cartilage in OA. An improved understanding of the cellular and molecular mechanisms by which pro-inflammatory and/or anabolic cytokines alter both the structure and function of synovial joints may eventually result in the commercial development of disease-modifying OA drugs (DMOADs). Since the prevalence of OA is high in the elderly population, future development of DMOADs must also take into account potential differences in the way DMOADs would be metabolized in the older individual compared with younger people.

The regulation of Schwann cell (SC) responses to injury stimuli by microRNAs (miRNAs) remains to be explored. Here, we identified <em>17</em> miRNAs that showed dynamic expression alterations at five early time points following rat sciatic nerve resection. Then we analyzed the expression pattern of <em>17</em> miRNAs, and integrated their putative targets with differentially expressed mRNAs. The resulting 222 potential targets were mainly involved in cell phenotype modulation, including immune response, cell death and cell locomotion. Among <em>17</em> miRNAs, miR-182 expression was up-regulated. The enhanced expression of miR-182 was correlated with nerve injury-induced phenotype modulation of SCs. Further investigation revealed that <em>fibroblast</em> <em>growth</em> <em>factor</em> 9 (FGF9) and neurotrimin (NTM) were two direct targets of miR-182 in SCs, with miR-182 binding to the 3'-untranslated region of FGF9 and NTM. Silencing of FGF9 and NTM recapitulated the inhibiting effect of miR-182 mimics on SC proliferation and migration, respectively, whereas enforced knockdown of FGF9 and NTM reversed the promoting effect of miR-182 inhibitor on SC proliferation and migration, respectively. Our data indicate that nerve injury inhibits SC proliferation and migration through rapid regulation of miR-182 by targeting FGF9 and NTM, providing novel insights into the roles of miRNAs in nerve injury and repair.

We have examined the effects of the natriuretic peptides on DNA synthesis in primary cultures of neonatal rat cardiac <em>fibroblasts</em>. Binding analysis using 125I-labeled atrial natriuretic peptide identified a single class of high-affinity binding sites (Kd = 0.03 +/- 0.01 nmol/L) in these cells. Of these sites, 80% appear to be of the natriuretic peptide C receptor subtype, with the remainder being A and B receptor subtypes. Northern blot analysis confirmed the presence of all three natriuretic peptide receptors in these cells. Atrial natriuretic peptide (10(-7) mol/L) effected a modest but consistent reduction in both agonist- and stretch-stimulated [3H]thymidine incorporation (<em>17</em>% to 41%). Moreover, brain natriuretic peptide (10(-7) mol/L), C-type natriuretic peptide (10(-7) mol/L), and des-[Gln18,Ser19,Gly20,Leu21,Gly22]-ANF 4-23-NH2 (10(-7) to 10(-6) mol/L) all proved capable of antagonizing <em>growth</em> <em>factor</em>-dependent [3H]thymidine incorporation (the inhibition ranged from 14% to 28%) and cell proliferation, suggesting that all three natriuretic peptide receptor subtypes are involved in the regulation of mitogenesis in these cultures. The inhibition by atrial natriuretic peptide was amplified by cotreatment with phosphodiesterase inhibitors. Similar reduction in [3H]thymidine incorporation was seen after treatment with 8-bromo-cGMP (10(-4) to 10(-3) mol/L) or nitroprusside (10(-4) to 10(-3) mol/L). These results suggest an important paracrine role for the natriuretic peptides in regulating <em>fibroblast</em> <em>growth</em> during cardiac hypertrophy.

The NY99 genotype of West Nile virus (WNV) introduced into North America has demonstrated high virulence for American crows (AMCRs), whilst a closely related WNV strain (KEN-3829) from Kenya exhibits substantially reduced virulence in AMCRs [Brault, A. C., Langevin, S. A., Bowen, R. A., Panella, N. A., Biggerstaff, B. J., Miller, B. R. & Nicholas, K. (2004). Emerg Infect Dis 10, 2161-2168]. Viruses rescued from infectious cDNA clones of both the NY99 and KEN-3829 strains demonstrated virulence comparable to that of their parental strains in AMCRs. To begin to define parameters that might explain the different virulence phenotypes between these two viruses, temperature-sensitivity assays were performed for both viruses at the high temperatures experienced in viraemic AMCRs. <em>Growth</em> curves of the two WNV strains were performed in African green monkey kidney (Vero; 37-42 degrees C) and duck embryonic <em>fibroblast</em> (DEF; 37-45 degrees C) cells cultured at temperatures that were tolerated by the cell line. Unlike the NY99 virus, marked decreases in KEN-3829 viral titres were detected between 36 and 120 h post-infection (p.i.) at temperatures above 43 degrees C. Replication of KEN-3829 viral RNA was reduced 6500-fold at 72 h p.i. in DEF cells incubated at 44 degrees C relative to levels of intracellular virus-specific RNA measured at 37 degrees C. In contrast, replication of virus derived from the NY99 infectious cDNA at 44 degrees C demonstrated only a <em>17</em>-fold reduction in RNA level. These results indicated that the ability of WNV NY99 to replicate at the high temperatures measured in infected AMCRs could be an important <em>factor</em> leading to the increased avian virulence and emergence of this strain of WNV.

BACKGROUND

YKL-40 is secreted by macrophages and neutrophils and is a growth factor for vascular endothelial cells and fibroblasts. Elevated serum concentrations of YKL-40 are found in patients with diseases characterized by inflammation or ongoing fibrosis. The aim of this study was to seek association between serum YKL-40 in patients with ulcerative colitis (UC) and Crohn disease (CD) and clinical disease activity.

METHODS

One-hundred-and-sixty-four patients with UC and 173 patients with CD were studied. The Simple Clinical Colitis Activity Index (SCCAI) and the Harvey-Bradshaw (H-B) score were used to assess disease activity. Serum YKL-40 (determined by ELISA) was related to C-reactive protein (CRP) and disease activity.

RESULTS

In patients with UC, the median serum YKL-40 rose with increasing disease activity, and patients with severe active disease had higher serum YKL-40 (median 59 microg/L (95% CI: 26-258 microg/L), P < 0.001) than patients with inactive UC (33 microg/L (19-163)) and age-matched controls (43 microg/L (20-124)). Patients with severe active CD had higher serum YKL-40 (59 microg/L (21-654), P < 0.001) than age-matched controls, but not higher than inactive CD patients (43 microg/L (17-306)). Serum YKL-40 was elevated in 41% of the patients with severe UC, in 10% with inactive UC, in 46% with severe CD and in 30% with inactive CD. Serum YKL-40 correlated with SCCAI in UC patients but not with H-B score in CD patients. In both patient groups, low correlations were found between serum YKL-40 and CRP, albumin and leucocytes.

CONCLUSIONS

Serum YKL-40 is elevated in patients with active IBD and may be complementary to inflammatory markers and clinical characteristics in the assessment of disease activity.

OBJECTIVE

Rapamycin, a novel macrolide immunosuppressive drug, is increasingly used as an agent for posttransplant immunosuppression and treatment of autoimmune disease. The molecular mechanism related to rapamycin-mediated immunosuppression is that rapamycin binds to FK-506 binding protein 12, and the formed complex inhibits the function of the mammalian target of rapamycin (mTOR), which in turn reduces protein phosphorylation, cell cycle progression, and cytokine production. The aim of this study was to examine the effect of rapamycin against the development of fibrosis and autoimmunity in 2 different types of systemic sclerosis (SSc) model mice.

METHODS

Tight skin (TSK/+) mice and bleomycin- induced SSc model mice were used to evaluate the effect of rapamycin on fibrosis and immunologic abnormalities. Furthermore, the antifibrotic effect of rapamycin was assessed using TSK/+ mouse fibroblasts.

RESULTS

Treatment with rapamycin reduced skin fibrosis of TSK/+ mice and skin and lung fibrosis of bleomycin-induced SSc model mice. The production of fibrogenic cytokines, such as interleukin-4 (IL-4), IL-6, IL-17, and transforming growth factor beta1, was attenuated by rapamycin. Hypergammaglobulinemia and anti-topoisomerase I antibody production were also reduced by rapamycin treatment in TSK/+ mice. In addition, mTOR expression levels were increased in TSK/+ mouse fibroblasts compared with those in wild-type mouse fibroblasts. Rapamycin treatment inhibited proliferation and collagen production of TSK/+ mouse fibroblasts in a dose-dependent manner.

CONCLUSIONS

This study is the first to show that rapamycin has a significant inhibitory effect on fibrosis in both TSK/+ and bleomycin-induced SSc model mice. These results suggest that rapamycin might be an attractive candidate for clinical trials in SSc patients.

Pulmonary fibrosis is a potentially life-threatening disease that may be caused by overt or asymptomatic inflammatory responses. However, the precise mechanisms by which tissue injury is translated into inflammation and consequent fibrosis remain to be established. Here, we show that in a lung injury model, bleomycin induced the secretion of IL-6 by epithelial cells in a transglutaminase 2 (TG2)-dependent manner. This response represents a key step in the differentiation of IL-<em>17</em>-producing T cells and subsequent inflammatory amplification in the lung. The essential role of epithelial cells, but not inflammatory cells, TG2 was confirmed in bone marrow chimeras; chimeras made in TG2-deficient recipients showed reduced inflammation and fibrosis, compared with those in wild-type mice, regardless of the bone marrow cell phenotype. Epithelial TG2 thus appears to be a critical inducer of inflammation after noninfectious pulmonary injury. We further demonstrated that <em>fibroblast</em>-derived TG2, acting downstream of transforming <em>growth</em> <em>factor</em>-β, is also important in the effector phase of fibrogenesis. Therefore, TG2 represents an interesting potential target for therapeutic intervention.

Normal rat kidney (NRK) cells transformed by simian sarcoma virus (SSV) release into the culture medium a biologically active mitogen with properties identical to those of human platelet-derived <em>growth</em> <em>factor</em> (PDGF). Like PDGF, the <em>growth</em> <em>factor</em> derived from SSV-NRK cells was shown to be stable to heat and sensitive to reducing agents. It was capable of inhibiting binding of labeled PDGF to the receptor on human <em>fibroblasts</em>. It also stimulated the phosphorylation of the same membrane protein (185 kilodaltons) in isolated plasma membranes from human <em>fibroblasts</em>. Immunoprecipitation of metabolically labeled proteins released by SSV-NRK cells showed that a 34-kilodalton protein was specifically precipitated by antiserum to PDGF. Upon reduction, this protein had a molecular size of <em>17</em> kilodaltons. PDGF has been shown to consist of two 14- to 18-kilodalton proteins linked by disulfide bonds.

Better prognostic markers are needed for hormone-refractory prostate cancer (HRPC) patients. No single biochemical or clinical parameter can reliably predict patient response to therapy or rapidity of disease progression. Peptide <em>factors</em> involved in major cancer <em>growth</em> pathways, such as tumor angiogenesis, are attractive candidates as markers of low- and high-risk HRPC patients. We analyzed prospectively collected urine specimens from 100 of 390 HRPC patients undergoing therapy with the <em>growth</em> <em>factor</em> antagonist suramin as part of CALGB 9480. Levels of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) were assessed from day 1 of therapy (D1) and day 29 (D29) urine samples from this subset of 100 randomly selected patients. <em>Growth</em> <em>factor</em> levels were determined by standardized ELISA microtiter plate assays from a commercial (bFGF) or proprietary (VEGF) source. Pretreatment urine VEGF levels were predictive of survival. In univariate analysis, patients whose baseline urine VEGF level was < or =28 pg/ml (the median level) had an average survival of <em>17</em> months; those with baseline VEGF >28 pg/ml had a significantly shorter survival of 10 months (P = 0.024). This difference corresponded to a 60% increased risk of dying for the higher urine VEGF patients (hazard ratio, 1.62; P = 0.03) and remained significant in multivariate analysis (hazard ratio, 1.72, P = 0.02). No significant correlations between urine bFGF level or change in bFGF levels and survival were found. These results support the notion that certain peptide <em>growth</em> <em>factor</em>-mediated, mitogenic pathways are important in HRPC and that their levels can predict outcome.

OBJECTIVE

Fibroblast growth factor (FGF) 21, a hormone primarily secreted by liver, has recently been shown to have beneficial effects on glucose and lipid metabolism and insulin sensitivity in animal models. This study investigated the association of serum FGF21 levels with insulin secretion and sensitivity, as well as circulating parameters of lipid metabolism and hepatic enzymes in Chinese subjects.

METHODS

Serum FGF21 levels were determined by ELISA in 134 normal glucose tolerance (NGT), 101 isolated-impaired fasting glucose, and 118 isolated-impaired glucose tolerance (I-IGT) Chinese subjects, and their association with parameters of adiposity, glucose, and lipid profiles, and levels of liver injury markers was studied. In a subgroup of this study, the hyperglycemic clamp technique was performed in 31 NGT, 17 isolated-impaired fasting glucose, and 15 I-IGT subjects to measure insulin secretion and sensitivity to test the associations with serum FGF21.

RESULTS

The serum FGF21 levels in I-IGT were significantly higher than NGT subjects [164.6 pg/ml (89.7, 261.0) vs. 111.8 pg/ml (58.0, 198.9); P < 0.05], and correlated positively with several parameters of adiposity. Multiple stepwise regression analysis showed an independent association of serum FGF21 with serum triglycerides, total cholesterol, and gamma-glutamyltransferase (all P < 0.05). However, FGF21 did not correlate with insulin secretion and sensitivity, as measured by hyperglycemic clamp and a 75-g oral glucose tolerance test.

CONCLUSIONS

Serum levels of FGF21 are closely related to adiposity, lipid metabolism, and biomarkers of liver injury but not insulin secretion and sensitivity in humans.

We evaluated <em>17</em> clinically unaffected members of three families with an autosomal dominant form of idiopathic pulmonary fibrosis for evidence of alveolar inflammation. Each person in the study was examined by gallium-67 scanning for a general estimate of pulmonary inflammation, and by bronchoalveolar lavage for characterization of the types of recovered cells and their state of activation. Eight of the <em>17</em> subjects had evidence of alveolar inflammation on the lavage studies. Supporting data included increased numbers of neutrophils and activated macrophages that released one or more neutrophil chemoattractants, and <em>growth</em> <em>factors</em> for lung <em>fibroblasts</em>--findings similar to those observed in patients with overt idiopathic pulmonary fibrosis. Four of these eight also had a positive gallium scan; in all the other clinically unaffected subjects the scan was normal. During a follow-up of two to four years in seven of the eight subjects who had evidence of inflammation, no clinical evidence of pulmonary fibrosis has appeared. These results indicate that alveolar inflammation occurs in approximately half the clinically unaffected family members at risk of inheriting autosomal dominant idiopathic pulmonary fibrosis. Whether these persons with evidence of pulmonary inflammation but no fibrosis will proceed to have clinically evident pulmonary fibrosis is not yet known.

Interleukin-<em>17</em> (IL-<em>17</em>) is a CD4 T cell cytokine. In this report, we investigated the effects of this cytokine on the elaboration of proangiogenic <em>factors</em> by lung <em>fibroblasts</em>. After stimulation with a wide range of doses of IL-<em>17</em>, <em>fibroblasts</em> produced more amount of various kinds of angiogenic <em>factors</em> including NO, HGF, MCP-1, KC, MIP-2, PGE1, PGE2 and VEGF in a dose-dependent manner. Treatment with a COX-1 and COX-2 inhibitor indomethacin did not impair IL-<em>17</em>-induced HGF and VEGF secretion in <em>fibroblasts</em>. In addition, TNF-alpha alone stimulated the elaboration of KC, MIP-2, PGE2 and VEGF in <em>fibroblasts</em>. IL-<em>17</em> and TNF-alpha in combination up-regulated elaboration of these proangiogenic <em>factors</em> additively or synergistically. Moreover, conditioned media (CM) from IL-<em>17</em>-stimulated <em>fibroblasts</em> showed significantly higher activity on endothelial cell <em>growth</em> than those from non-treated control cells. These results indicate that IL-<em>17</em> up-regulates elaboration of various proangiogenic <em>factors</em>, and modulates macrophage-derived TNF-alpha-induced production of KC, MIP-2, PGE2 and VEGF by <em>fibroblasts</em>. Our findings also demonstrate that IL-<em>17</em> might be a potential contributor to the inflammatory angiogenesis via induction of proangiogenic <em>factors</em> by stromal <em>fibroblasts</em>.

BACKGROUND

Fibroblast growth factor 19 (FGF19) and FGF21 are novel metabolic regulators that improve insulin sensitivity and decrease adiposity in mice. However, little is known about the nutritional regulation of these factors in humans.

OBJECTIVE

The objective of this study was to measure plasma FGF19 and FGF21 levels in patients with anorexia nervosa (AN) and to explore its relationship with anthropometric and endocrine parameters.

METHODS

This was a single-center cross-sectional study.

METHODS

The study was performed in a university hospital.

METHODS

Seventeen untreated women with a restrictive type of AN and 17 healthy women (control group) were included.

METHODS

Fasting plasma FGF19 and FGF21, serum insulin, leptin, soluble leptin receptor, adiponectin, resistin, and C-reactive protein were the main outcome measures.

RESULTS

Plasma FGF19 levels did not significantly differ between the groups studied, whereas plasma FGF21 levels were significantly reduced in AN relative to the control group. Plasma FGF21 positively correlated with body mass index and serum leptin and insulin and was inversely related to serum adiponectin in both groups. In contrast, plasma FGF19 was not related to any of parameters studied. Partial realimentation significantly reduced plasma FGF21 levels in AN.

CONCLUSIONS

Circulating levels of FGF21 but not FGF19 are strongly related to body weight and serum levels of leptin, adiponectin, and insulin in both anorectic and normal-weight women. We suggest that reduced plasma FGF21 levels could be involved in the pathophysiology of AN or in a complex adaptive response to this disease.

Transformation of cloned rat embryo <em>fibroblasts</em> (CREF) with the T24-ras oncogene results in loss of contact inhibition, <em>growth</em> in soft agar and tumor formation in nude mice. Previously we showed that in such cells (CREF T24), the phosphorylation rate of protein synthesis initiation <em>factor</em> 4E (eIF-4E) is increased, correlating with an increase in the general rate of protein synthesis. In the present study, we have expressed antisense RNA complementary to eIF-4E mRNA in CREF T24 cells using a stably integrated vector. Cells expressing antisense RNA (CREF T24/AS) contained 30-50% of the normal level of eIF-4E and exhibited many of the properties of untransformed cells. CREF T24 had a spindle-shaped, refractile appearance, whereas CREF T24/AS grew in ordered, parallel patterns and exhibited contact inhibition similar to untransformed CREF. The rates of <em>growth</em> and protein synthesis in CREF T24/AS were decreased compared to CREF T24 but were not as low as in CREF. The efficiency of <em>growth</em> in soft agar was 11-fold lower for CREF T24/AS compared with CREF T24. The latency period for tumor formation in nude mice was increased from 8 days for CREF T24 to <em>17</em>-27 days for CREF T24/AS and various clonal lines derived from them. Cell lines established from these CREF T24/AS-derived tumors were shown to have partially regained the eIF-4E levels characteristic of CREF T24. These results demonstrate that many of the phenotypic alterations associated with ras-induced malignant transformation can be reversed by a moderate reduction of the translational initiation capacity and therefore may be mediated through a translational mechanism.

Platelets sequester angiogenesis regulatory proteins early in tumor <em>growth</em>, which suggests a new avenue for monitoring disease. To date, there are no clinically relevant reference ranges for markers of early angiogenesis. We introduce a new ELISA-based method for accurate and reproducible measurement of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), platelet <em>factor</em> 4 (PF4), thrombospondin-1 (TSP-1), <em>fibroblast</em> <em>growth</em> <em>factor</em>, basic (bFGF), and endostatin in platelets. To facilitate clinical applicability, the platelet levels in isolated samples were determined utilizing a new actin ELISA method. Platelets from healthy donors at single and repetitive time points were used for the assessment of normal ranges of these proteins. The physiological levels in platelets were: VEGF (0.74 +/- 0.37 pg/10(6) platelets); PDGF (23 +/- 6 pg/10(6)); PF4 (12 +/- 5 ng/10(6)); TSP-1 (31 +/- 12 ng/10(6)); bFGF (0.44 +/- 0.15 pg/10(6)); and endostatin (5.6 +/- 3.0 pg/10(6)). There was an excellent correlation (R(2) = 0.7) between the platelet levels calculated with the actin ELISA and complete blood count. The levels of the platelets were higher than those in platelet-poor plasma by <em>factors</em> of: VEGF (215-fold); PDGF (914-fold); PF-4 (516-fold); TSP-1 (813-fold); and bFGF (<em>17</em>-fold). The endostatin levels were nearly equivalent. The biovariability of the platelet proteins in eight healthy subjects over a 5-week period was found to be minimal. We describe accurate and direct measurements of the concentrations of VEGF, bFGF, PDGF, TSP-1, endostatin, and PF4 in platelets of healthy human subjects. In contrast to the highly variable levels in plasma and serum, the platelet-derived measurements were accurate and reproducible with minimal biovariability.

Platelet derived <em>growth</em> <em>factors</em> have been shown to stimulate cell proliferation efficiently in vivo(1,2) and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), <em>fibroblasts</em> and endothelial colony-forming cells with platelets activated by thrombin(3-5) or lysed by freeze/thaw cycles(6-14) before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived <em>growth</em> <em>factors</em> has already been tested in several trials for tissue engineering and regenerative therapy.(1,15-<em>17</em>) Varying efficiency is considered to be at least in part due to individually divergent concentrations of <em>growth</em> <em>factors</em>(18,19) and a current lack of standardized protocols for platelet preparation.(15,16) This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and <em>growth</em> <em>factors</em> are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

We have characterized a cell, the Schwann cell precursor, that represents a distinct intermediate differentiation stage in the process by which Schwann cells are generated from neural crest cells. The Schwann cell precursor shows radical differences from Schwann cells which include death regulation, antigenic phenotype, pattern of cell-cell interaction, migratory behavior, and morphology. In the nerves of the rat hind limb, Schwann cells are irreversibly generated from these during a brief period, essentially embryonic days 15-<em>17</em>. We also provide evidence that the survival of Schwann cell precursors is regulated by neurons and identify basic <em>fibroblast</em> <em>growth</em> <em>factor</em> as a potential key regulator of apoptosis in Schwann cell precursors and of precursor to Schwann cell conversion. These findings have implications for our understanding of gliogenesis in the peripheral nervous system.

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated <em>fibroblasts</em> (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting <em>growth</em> <em>factors</em>, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell <em>growth</em>, but also by promoting <em>fibroblast</em> activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor <em>growth</em> in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor <em>growth</em> and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers.Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated <em>fibroblasts</em> through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(<em>17</em>); 4957-70. ©2018 AACR.

Overexpression of genes, through genomic amplification and other mechanisms, can critically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that are primarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was defined as containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-<em>17</em>-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in all cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-<em>17</em>-92, in primary samples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the <em>growth</em> <em>factors</em> <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2), hepatocyte <em>growth</em> <em>factor</em> (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and altered the cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4 in Xenopus blastocysts was also enhanced. Our data are consistent with a role of GPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads to mesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeutic intervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene.

Prostate cancer selectively metastasizes to the axial skeleton to produce osteoblastic lesions, which suggests that bidirectional paracrine interactions exist between prostate cancer and bone cells. To evaluate the role of tumor-stromal cell interaction and stromal-specific <em>growth</em> <em>factors</em> in prostate cancer <em>growth</em> and dissemination, we coinoculated nontumorigenic human prostate cancer cells (LNCaP) and various tissue-specific <em>fibroblasts</em> subcutaneously in athymic mice. LNCaP tumors were induced most consistently by human bone <em>fibroblasts</em> (62%), followed by two prostate <em>fibroblast</em> cell lines (31% and <em>17</em>%), but not by lung, kidney, or embryonic 3T3 <em>fibroblasts</em>. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. Immunohistochemical and biochemical techniques confirmed the human prostate component of these tumors and were paralleled by elevations in serum prostate specific antigen. In vitro mitogenic assays revealed a two-to three-fold bidirectional stimulation between LNCaP and bone or prostate <em>fibroblast</em> conditioned media, but not lung, kidney, or 3T3 <em>fibroblast</em> conditioned media. A novel method developed to deliver concentrated bone or prostate <em>fibroblast</em> conditioned media in vivo using a slowly absorbed matrix (gelfoam) also induced tumor formation, emphasizing the importance of <em>fibroblast</em> <em>growth</em> <em>factors</em> in LNCaP tumor formation. Northern analysis identified the stromal compartment as the primary source of extracellular matrix (collagen, fibronectin), while only LNCaP cells expressed transforming <em>growth</em> <em>factor</em> alpha. Although LNCaP and stromal cells express basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), the bidirectional paracrine-mediated mitogenic activity between these cells is not inhibited by anti-bFGF antibodies, suggesting that other undefined <em>growth</em> <em>factors</em> may be involved in stimulating LNCaP <em>growth</em>. These observations illustrate the importance of stromal-epithelial interaction in prostate tumor <em>growth</em> and suggest that extracellular matrix and paracrine-mediated <em>growth</em> <em>factors</em> play a role in prostate cancer <em>growth</em> and metastasis.

The effect of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) administration on regional myocardial function and blood flow in chronically ischemic hearts was studied in 26 pigs instrumented with proximal circumflex coronary artery (LCX) ameroid constrictors. In 13 animals bFGF was administered extraluminally to the proximal left anterior descending (LAD) and LCX arteries with heparin-alginate beads and 13 other animal served as controls. bFGF-treated pigs showed a fourfold reduction in left ventricular infarct size compared to untreated controls (infarct size: 1.2 +/- 0.4% vs. 5.1 +/- 1.3% of LV mass, mean +/- SEM, P < 0.05). Percent fractional shortening (% FS) in the LCX area at rest was reduced compared with the LAD region in both bFGF and control pigs. However, there was better recovery in the LCX area after rapid pacing in bFGF-treated pigs (% FSLCX/% FSLAD, 22.9 +/- 7.3%-->30.5 +/- 8.5%, P < 0.05 vs. prepacing) than in controls (16.0 +/- 7.8%-->14.3 +/- 7.0%, P = NS). Furthermore, LV end-diastolic pressure rise with rapid pacing was less in bFGF-treated than control pigs (pre-pacing; pacing; post-pacing, 10 +/- 1; <em>17</em> +/- 3; 11 +/- 1* mmHg vs 10 +/- 1; 24 +/- 4; 15 +/- 1 mmHg, *P < 0.05 vs. control). Coronary blood flow in the LCX territory (normalized for LAD flow) was also better during pacing in bFGF-treated pigs than in controls. Thus, periadventitial administration of bFGF in a gradual coronary occlusion model in pigs results in improvement of coronary flow and reduction in infarct size in the compromised territory as well as in prevention of pacing-induced hemodynamic deterioration.

OBJECTIVE

Fibroblast-like synoviocytes (FLS) are a major component of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Cyr61 (CCN1) is a product of a growth factor-inducible immediate early gene and is involved in cell adhesion, proliferation, and differentiation. However, the role that Cyr61 plays in FLS proliferation has remained undetermined. The aim of this study was to identify the role of Cyr61 in regulating the proliferation of FLS derived from patients with RA.

METHODS

Expression of Cyr61 in synovial tissue (ST) and in FLS was determined simultaneously using immunohistochemistry, real-time polymerase chain reaction, and Western blotting. Cyr61 levels in synovial fluid (SF) were determined by enzyme-linked immunosorbent assay. FLS proliferation stimulated by SF, Cyr61, and interleukin-17 (IL-17) was measured by thymidine incorporation. Activation of signal transduction pathways was determined by Western blotting and confocal microscopy.

RESULTS

Cyr61 was overexpressed in ST, FLS, and SF samples from RA patients as compared with samples from normal controls. Elevated levels of Cyr61 in RA SF promoted the proliferation of FLS, an effect that was abrogated by a neutralizing monoclonal antibody against human Cyr61. Furthermore, in samples from RA patients, Cyr61 was found to protect FLS from apoptosis and to sustain the expression of Bcl-2 in FLS. Most importantly, the expression of Cyr61 in FLS was regulated by IL-17 mainly via the p38 MAPK and NF-kappaB signaling pathways. Knockdown of expression of the Cyr61 gene inhibited IL-17-stimulated FLS proliferation.

CONCLUSIONS

Our findings indicate that Cyr61 plays a critical role in IL-17-mediated proliferation of FLS in RA and likely contributes to hyperplasia of synovial lining cells and eventually to joint destruction in patients with RA.