Patients with primary biliary cholangitis (PBC) who had an inadequate response to ursodiol have few treatment options. Alkaline phosphatase (ALP) and bilirubin levels correlate with the risk of liver transplant or death in PBC patients. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 19 is a hormone that acts directly in the liver to regulate bile acid synthesis. We evaluated NGM282, an engineered analogue of FGF19, for the treatment of PBC. In this 28-day, double-blind, placebo-controlled phase 2 trial, 45 PBC patients who had an inadequate response to ursodiol were randomly assigned 1:1:1 to receive subcutaneous daily doses of either NGM282 at 0.3 mg (n = 14), 3 mg (n = <em>16</em>), or placebo (n = 15). The primary endpoint was a change in ALP from baseline after 28 days of treatment. At day 28, ALP was significantly reduced with NGM282 treatment at both 0.3 mg (least-squares mean -51.0 IU/L [standard error (SE) 15.4]) and 3 mg (-66.0 IU/L [SE <em>16</em>.0]) versus placebo (3.3 IU/L [SE 14.8]), with least-squares mean differences of -54.3 IU/L (95% confidence interval -104.2 to -4.5; P = 0.0149) and -69.3 IU/L (95% confidence interval -120.5 to -18.3; P = 0.0030), respectively. Fifty percent (7 of 14) of patients receiving NGM282 0.3 mg and 46% (6 of 13) of those receiving NGM282 3mg achieved 15% or greater reduction in ALP levels from baseline, compared with 7% (1 of 15) of patients receiving placebo. NGM282 also significantly reduced serum concentrations of transaminases and immunoglobulins. Most adverse events were grade 1 (mild) to grade 2 (moderate) in severity, with gastrointestinal disorders more frequent in the NGM282 treatment groups. No worsening of pruritus was observed with NGM282 treatment. Conclusion: NGM282 administered for 28 days resulted in significant improvements in ALP and transaminase levels compared with placebo, with an acceptable safety profile in patients with PBC. (Hepatology Communications 2018; 00:000-000).

Prolidase [E.C.3.4.13.9] is a cytosolic exopeptidase that catalyses the hydrolysis of C-terminal proline containing dipeptides or tripeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis. Increase in enzyme activity is correlated with increased rates of collagen turnover but the mechanism and endpoints by which this enzyme is regulated remain largely unknown. We have found that insulin-like <em>growth</em> <em>factor</em>-I (IGF-I), potent stimulator of collagen biosynthesis, induces prolidase activity in cultured human skin <em>fibroblasts</em>. Supporting evidence comes from the following observations: (1) Serum of fasted rats, (IGF-I, 72 +/- <em>16</em> ng/ml) showed about 50% reduced ability to stimulate prolidase activity and collagen biosynthesis in confluent <em>fibroblasts</em> in comparison to the effect of control rat serum (IGF-I, <em>16</em>8 +/- 29). (2) An addition of IGF-I (100 ng/ml) to fasted rat serum restored its ability to stimulate prolidase activity and collagen biosynthesis to control values. (3) In confluent human skin <em>fibroblasts</em>, cultured for 48 h with serum free medium prolidase activity was decreased to 50% of control cells, cultured in the presence of normal rat serum. Supplementation of serum free medium with EGF, PDGF and IGF-I (<em>factors</em> that can replace <em>growth</em> promoting activity of serum) stimulated prolidase activity to control values while the medium deprived IGF-I had no such effect. (4) The relative differences in prolidase activity due to specific treatment of confluent cells with above <em>growth</em> <em>factors</em> were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells as shown by western immunoblot analysis. Thus we conclude that prolidase activity is regulated by IGF-I in confluent <em>fibroblasts</em>.

OBJECTIVE

Non-alcoholic fatty liver (NAFL) associated with obesity is a major cause of liver diseases which can progress to non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) plays an important role in liver metabolism and is also a potential marker for NAFL. Here we aimed to test the effect of FGF21 deficiency on liver pathology in mice consuming a conventional high fat, high sucrose (HFHS) obesogenic diet for up to 52 weeks.

METHODS

C57BL6 WT and FGF21 KO mice were fed a conventional obesogenic diet and were evaluated at 16 and 52 weeks. Evaluation included metabolic assessment, liver pathology, and transcriptomic analysis.

RESULTS

With consumption of HFHS diet, FGF21 deficient mice (FGF21 KO) develop excess fatty liver within 16 weeks. Hepatic pathology progresses and at 52 weeks FGF21 KO mice show significantly worse fibrosis and 78% of mice develop HCC; in contrast only 6% of WT mice develop HCC. Well differentiated hepatocellular carcinomas in FGF21 KO mice were characterized by expanded hepatic plates, loss of reticulin network, cytologic atypia, and positive immunostaining for glutamine synthetase. Microarray analysis reveals enrichment of several fibroblast growth factor signaling pathways in the tumors.

CONCLUSIONS

In addition to attenuating inflammation and fibrosis in mice under a number of dietary challenges, we show here that FGF21 is required to limit the progression from NAFL to HCC in response to prolonged exposure to an obesogenic diet. The induction of hepatic FGF21 in response to the high fat, high sucrose obesogenic diet may play an important role in limiting progression of liver pathology from NAFL to HCC.

OBJECTIVE

Dysregulation of extracellular matrix (ECM) following myocardial infarction is a key contributor to myocardial fibrosis, chamber dilation, and progression to heart failure. Basic fibroblast growth factor is a potent inhibitor of fibrosis. We propose a novel surgical procedure leveraging a commercially available ECM biomaterial for the treatment of ischemic heart failure.

METHODS

Epicardial infarct repair using CorMatrix-ECM biomaterial patch (CorMatrix Cardiovascular Inc, Roswell, Ga) was compared with sham in a rat myocardial infarction model. Key indices of ischemic remodeling, including inflammation, fibrosis, and myocardial performance were evaluated 16 weeks post-treatment.

RESULTS

Histology and immunohistochemistry demonstrated comprehensive integration of CorMatrix-ECM biomaterial patch without evidence of immune reaction and an increase in basic fibroblast growth factor expression in treated animals. Functional analysis by serial echocardiography of normal (n = 13), sham (n = 15), nonenhanced CorMatrix-ECM patch (n = 18), and basic fibroblast growth factor-enhanced CorMatrix-ECM patch (n = 10) animals revealed an improvement in ejection fraction in basic fibroblast growth factor-enhanced CorMatrix-ECM patch animals compared with shams (55.3% ± 8.0% vs 35.1% ± 7.6%; P < .001). Prevention of left ventricle remodeling was also confirmed by pressure volume loop analysis, which demonstrated reduced left ventricular end diastolic volumes in basic fibroblast growth factor-enhanced CorMatrix-ECM patch animals (n = 5) compared with shams (n = 6) (208.0 ± 59.3 μL vs 363. 1 ± 108.7 μL; P < .01) and improved left ventricle contractility in nonenhanced CorMatrix-ECM patch (n = 7) and basic fibroblast growth factor-enhanced CorMatrix-ECM patch animals compared with shams (0.709 ± 0.306 and 0.609 ± 0.160 vs 0.437 ± 0.218; P < .05).

CONCLUSIONS

Epicardial infarct repair with basic growth factor-enhanced CorMatrix-ECM biomaterial patch attenuates myocardial remodeling and improves cardiac performance after subacute myocardial infarction in a rat coronary ligation model. These observations establish proof-of-concept for this novel surgical approach.

OBJECTIVE

To examine the up-regulation of vascular endothelial growth factor (VEGF) expression by hypoxia, a crucial event leading to neovascularization, as the reduction in VEGF expression may facilitate minimization of adhesion development.

METHODS

Prospective experimental study.

METHODS

University medical center.

METHODS

Five patients with adhesions undergoing laparotomy with excision of adhesions and normal peritoneum.

METHODS

Adhesion and normal peritoneal fibroblasts were treated with dichloroacetic acid (DCA) or NS-398 (a cyclooxygenase-2 [COX-2] inhibitor) for 24 to 48 hours.

METHODS

A real-time reverse transcriptase polymerase chain reaction (RT-PCR) to quantify relative changes in mRNA levels of VEGF from each treatment.

RESULTS

In both normal peritoneal and adhesion fibroblasts, VEGF mRNA was present with statistically significantly higher levels in adhesion fibroblasts (32%). The DCA treatment resulted in a statistically significant decrease in VEGF mRNA levels in adhesion (20%) and normal peritoneal (18%) fibroblasts. The NS-398 treatment resulted in a statistically significant decrease in VEGF mRNA levels in adhesion (25%) and normal peritoneal (16%) fibroblasts.

CONCLUSIONS

Stimulation of aerobic metabolism by DCA or inhibition of COX-2 by NS-398 reduces VEGF expression. Angiogenesis, which is an integral component in the development of dense vascular adhesions, may be reduced by either COX-2 inhibitors or stimulation of aerobic metabolism by DCA.

We have produced human papillomavirus type <em>16</em> E7 protein in a bacterial expression system and examined the mitogenic activity of this protein in Swiss 3T3 cells after scrape loading. The ability of E7 to induce cellular DNA synthesis in quiescent mouse <em>fibroblasts</em> is strongly enhanced by the presence of a single <em>growth</em> <em>factor</em> such as insulin. Although only weakly mitogenic, introduction of E7 alone resulted in the rapid induction of the transcriptionally active form of E2F, which was not enhanced further by the addition of insulin. Mutant E7 proteins defective for RB binding failed to induce the active form of E2F or act synergistically with insulin to stimulate DNA synthesis. The ability of E7 to regulate E2F may therefore be necessary, but is not sufficient, for full induction of DNA synthesis.

Microsatellite instability (MSI) is a molecular phenotype present in approximately 25% of endometrial cancers. We examined the global gene expression profiles of early-stage endometrioid endometrial cancers with and without the MSI phenotype to test the hypothesis that MSI phenotype may determine a unique molecular signature among otherwise similar cancers. Unsupervised principal component analysis of the expression data from these cases indicated two distinct groupings of cancers based on MSI phenotype. A relatively small number of array features (392) at high statistical value (P < 0.001) were identified that drive the instability signature in these cancers; 109 of these transcripts differed by at least 2-fold. These data identify distinct gene expression profiles for MSI and microsatellite stable (MSS) cancers, which suggest that cancers with MSI develop in part by different mechanisms from their similar stable counterparts. In particular, we found evidence that two members of the secreted frizzled related protein family (SFRP1 and SFRP4) were more frequently down-regulated in MSI cancers as compared with MSS cancers. Down-regulation was accompanied by promoter hypermethylation for SFRP1. SFRP1 was hypermethylated in 8 of 12 MSI cancers whereas only 3 of <em>16</em> MSS cancers were methylated. The WNT target <em>fibroblast</em> <em>growth</em> <em>factor</em> 18 was found to be up-regulated in MSI cancers. These data classify histologically similar endometrioid endometrial cancers into two distinct groupings with implications affecting therapy and prevention.

In animals, high fibroblast growth factor 21 (FGF21) states improve insulin resistance but induce bone loss. Whether FGF21 relates to bone mineral density (BMD) is unknown in humans. Contrary to prediction from animal findings, we found higher FGF21 levels associating with greater BMD in women, independent of age and body composition.

BACKGROUND

Recent laboratory studies suggest that FGF21 is involved in reciprocal regulation of bone and energy homeostasis. Systemic administration of FGF21 protects animals from obesity and diabetes but causes severe bone loss, smothering the enthusiasm over FGF21 as a potential antiobesity therapeutic. To date, there is no information on whether FGF21 relates to BMD in humans. We thus studied the relationship between plasma FGF21 levels and BMD in healthy adults.

METHODS

Fasting plasma FGF21 levels were measured by enzyme-linked immunosorbent assay and body composition by dual-energy X-ray absorptiometry.

RESULTS

Among 40 healthy volunteers (age 32 ± 10 year, 16 women), men had significantly higher lean body mass (p < 0.01) and total BMD (p < 0.05), and lower percent body fat than women (p < 0.01). Median plasma FGF21 levels were not different between the sexes. While there was no association between FGF21 concentrations and body composition in men, FGF21 levels correlated positively with fat mass (p < 0.01) in women. In men, no significant correlation between FGF21 with BMD was observed. However, in women, FGF21 correlated positively with total BMD (R (2) = 0.69, p = 0.003) and spine BMD (R (2) = 0.76, p = 0.001); the correlation remained significant after adjusting for age, ethnicity, and body composition.

CONCLUSIONS

This study reveals for the first time a strong positive association between plasma FGF21 levels and BMD in healthy women, suggesting the association between bone loss and high FGF21 states in animals may not be directly translated to humans in physiologic states. We hypothesize that FGF21 may increase bone mass particularly in women through paracrine mechanisms in the bone-adipose interface.

OBJECTIVE

To analyze intraocular growth factor and cytokine concentrations in eyes with different stages of age-related macular degeneration (AMD) compared with controls.

METHODS

The Clinical Age-Related Maculopathy Staging (CARMS) system was used for assignment of patients into the respective categories. Aqueous humor specimens were taken before cataract surgery in 21 controls (CARMS 1) and in 17 early (CARMS 2) and 16 intermediate (CARMS 3) AMD patients. In 18 neovascular (CARMS 5) AMD patients, specimens were taken immediately before anti-vascular endothelial growth factor intravitreal therapy. Luminex multiplex bead assays were conducted for endostatin, angiogenin, vascular endothelial growth factor, platelet-derived growth factor AA, placental growth factor, thrombospondin 2, and fibroblast growth factor a.

RESULTS

Vascular endothelial growth factor concentrations were elevated in CARMS 3 (P = 0.037) and tended to be elevated in CARMS 5 (P = 0.093), whereas levels in CARMS 2 (P = 0.425) were similar to CARMS 1. Platelet-derived growth factor levels were diminished in CARMS 2 (P = 0.020), with a trend to lower levels for CARMS 3 (P = 0.099) and CARMS 5 (P = 0.082) compared with CARMS 1. For CARMS 5, antiangiogenic endostatin was elevated (P < 0.002), while antiangiogenic thrombospondin 2 was reduced (P = 0.029).

CONCLUSIONS

Clinical Age-Related Maculopathy Staging 3 dry AMD was associated with higher vascular endothelial growth factor levels than CARMS 5 neovascular AMD. Therefore, intraocular vascular endothelial growth factor concentrations do not seem to reflect choroidal neovascularization activity in neovascular AMD directly. Platelet-derived growth factor was decreased in most forms of AMD. The antiangiogenic endostatin was exclusively elevated in neovascular AMD, while thrombospondin 2 was reduced. Age-related macular degeneration disease seems to be associated with a generally altered cytokine system.

BACKGROUND

Various compounds have been detected in gingival crevicular fluid (GCF) as indicators of periodontal disease activity. Therefore, the analysis of GCF may be especially beneficial for diagnosing current periodontal status and addressing the effects of treatment. Moreover, the identification of new markers in GCF may also contribute to elucidating novel mechanisms involved in periodontal disease. This study sought novel marker proteins specific to chronic periodontitis by profiling cytokines in GCF using a cytokine antibody array system.

METHODS

Human cytokine array V, which detects 79 cytokines on one membrane, was used to determine the profile of cytokines in GCF from seven subjects with chronic periodontitis and seven subjects with healthy periodontia. The profile was exposed to x-ray film and quantified using image analysis software. Healthy and diseased sites were compared statistically.

RESULTS

We detected 10 cytokines in periodontally healthy sites and 36 cytokines in periodontally diseased sites. Interleukin-8 (IL-8) and transforming <em>growth</em> <em>factor</em>-beta 2 (TGF-beta2) were detected at high levels in healthy and diseased subjects. There were significant differences between healthy and diseased subjects in the levels of tissue inhibitor of metalloproteinases-2 (TIMP-2), tumor necrosis <em>factor</em>-beta (TNF-beta), <em>growth</em>-related oncogene (GRO), interferon-inducible protein-10 (IP-10), angiogenin (Ang), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), insulin-like <em>growth</em> <em>factor</em> binding protein-3 (IGFBP-3), osteoprotegerin (OPG), epidermal <em>growth</em> <em>factor</em> (EGF), glial-derived neurotrophic <em>factor</em> (GDNF), pulmonary and activation-regulated chemokine (PARC), oncostatin M (OSM), <em>fibroblast</em> <em>growth</em> <em>factor</em>-4 (FGF-4), IL-<em>16</em>, homologous to lymphotoxins (LIGHT), and placenta <em>growth</em> <em>factor</em> (PlGF). Of these, the newly detected cytokines were GRO, Ang, IGFBP-3, GDNF, PARC, OSM, FGF-4, IL-<em>16</em>, LIGHT, and PlGF.

CONCLUSIONS

In this study, we detected several cytokines in GCF using a cytokine antibody array system, including both inflammatory cytokines and various growth factors. Therefore, periodontal disease may participate in the wound healing process and in tissue destruction via the inflammatory process. Our results suggest that the quantification of these cytokines in GCF provides useful information for the diagnosis of periodontal disease status.

Although best understood as a degradative pathway, recent evidence demonstrates pronounced involvement of the macroautophagic/autophagic molecular machinery in cellular secretion. With either overexpression or inhibition of autophagy mediators, dramatic alterations in the cellular secretory profile occur. This affects secretion of a plethora of <em>factors</em> ranging from cytokines, to granule contents, and even viral particles. Encompassing a wide range of secreted <em>factors</em>, autophagy-dependent secretion is implicated in diseases ranging from cancer to neurodegeneration. With a <em>growing</em> body of evidence shedding light onto the molecular mediators, this review delineates the molecular machinery involved in selective targeting of the autophagosome for either degradation or secretion. In addition, we summarize the current understanding of <em>factors</em> and cargo secreted through this unconventional route, and describe the implications of this pathway in both health and disease. <b>Abbreviations</b>: BECN1, beclin 1; CAF, cancer associated <em>fibroblast</em>; CUPS, compartment for unconventional protein secretion; CXCL, C-X-C motif chemokine ligand; ER, endoplasmic reticulum; FGF2, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2; HMGB1, high mobility group box 1; IDE, insulin degrading enzyme; IL, Interleukin; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAPS, misfolding associated protein secretion; MEF, mouse embryonic <em>fibroblast</em>; MTORC1, MTOR complex I; PtdIns, phosphatidyl inositol; SEC22B, SEC22 homolog B, vesicle trafficking protein (gene/pseudogene); SFV, Semliki forest virus; SNCA, synuclein alpha; SQSTM1, sequestosome 1; STX, Syntaxin; TASCC, TOR-associated spatial coupling compartment; TGFB, transforming <em>growth</em> <em>factor</em> beta; TRIM<em>16</em>, tripartite motif containing <em>16</em>; UPS, unconventional protein secretion; VWF, von Willebrand <em>factor</em>.

Injury to the adult brain results in abortive axon regeneration and the deposition of a dense fibrous glial scar. Therapeutic strategies to promote postinjury axon regeneration are likely to require antiscarring strategies. In neonatal brain wounds, scar material is not laid down and axons grow across the lesion site, either by de novo <em>growth</em> or regeneration. To achieve the therapeutic goal of recapitulating the nonscarring neonatal response in the injured adult, an understanding of how ontogenic differences in scarring reflect developmental diversities in the trophic response to injury is required. Fibrobast <em>growth</em> <em>factor</em>-2 (FGF-2) expression is developmentally regulated and has been implicated as a regulator of the wounding response of the adult rat central nervous system. We have investigated the expression of FGF-2 and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) after penetrating lesions to the cerebral cortex of 5 days post partum (dpp) (nonscarring) and <em>16</em> dpp and adult (scarring) rats. In situ hybridization, immunohistochemistry and Western blotting showed robust and sustained increases in FGF-2 and FGFR1 mRNA and protein in reactive astrocytes around the lesion in scarring rats, a response that was attenuated substantially in the nonscarring neonate. These results demonstrate that changes in astrocyte FGF-2 and FGFR1 expression are coincident with the establishment of a mature pattern of glial scarring after injury in the maturing central nervous system, but it is premature to infer a causal relationship without further experiments.

Ascidian larvae develop mesenchyme cells in their trunk. A <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF9/<em>16</em>/20) is essential and sufficient for induction of the mesenchyme in Ciona savignyi. We have identified two basic helix-loop-helix (bHLH) genes named Twist-like1 and Twist-like2 as downstream <em>factors</em> of this FGF. These two genes are phylogenetically closely related to each other, and were expressed specifically in the mesenchymal cells after the 110-cell stage. Gene-knockdown experiments using a specific morpholino oligonucleotide demonstrated that Twist-like1 plays an essential role in determination of the mesenchyme and that Twist-like2 is a downstream <em>factor</em> of Twist-like1. In addition, both overexpression and misexpression of Twist-like1 converts non-mesenchymal cells to mesenchymal cells. We also demonstrate that the upstream regulatory mechanisms of Twist-like1 are different between B-line mesenchymal cells and the A-line mesenchymal cells called 'trunk lateral cells'. FGF9/<em>16</em>/20 is required for the expression of Twist-like1 in B-line mesenchymal precursor cells, whereas FGF, FoxD and another novel bHLH <em>factor</em> called NoTrlc are required for Twist-like1 to be expressed in the A-line mesenchymal precursor cells. Therefore, two different but partially overlapping mechanisms are required for the expression of Twist-like1 in the mesenchymal precursors, which triggers the differentiation of the mesenchyme in Ciona embryos.

Prior studies have demonstrated that <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-3 (FGFR-3) regulates proliferation of undifferentiated intestinal epithelial cells in vitro. However, the function(s) of FGFR-3-mediated signaling during intestinal development and epithelial differentiation in vivo remain unknown. The goal of this study was to define the temporal, regional, and cell-specific patterns of FGFR-3 expression and its ligands during normal intestinal ontogeny and epithelial regeneration. Both the IIIb and IIIc isoforms of FGFR-3 mRNA, which result from differential splicing of the FGFR-3 primary transcript, were detected in mouse small intestine as early as embryonic day <em>16</em>. FGFR-3 levels peaked in the small intestine from 7 to 21 days after birth and decreased thereafter to reach the low levels observed in adult mice. FGFR-3 IIIb and IIIc mRNA levels were highest in the duodenum and proximal jejunum with lower levels of both seen in the distal jejunum, ileum, and colon. FGFR-3 was expressed in a subset of proliferating undifferentiated crypt epithelial cells located in the intervillous epithelium and in the lower half of nascently forming crypts but not in differentiated epithelial cell types. FGFR-3 IIIb was the dominant isoform expressed in both small intestinal and colonic crypts. Expression of FGF1, FGF2, and FGF9, known ligands of FGFR-3, paralleled patterns of FGFR-3 expression during gut development. These data suggest that signaling through FGFR-3 plays a role in regulating morphogenic events involved in formation of intestinal crypts and/or the fate of epithelial stem cells.

Angiotensin II (ANG II), a potent vasoconstricting peptide, may act as a <em>growth</em> <em>factor</em> for cardiac muscle cells and induce hypertrophy. We examined the molecular phenotype of neonatal rat cardiac <em>fibroblasts</em> in relation to ANG II by studying the expression pattern of three transcription <em>factors</em> (Egr-1, c-fos and c-jun) and the transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1). ANG II did not affect cell proliferation and <em>growth</em> of serum deprived neonatal cardiac <em>fibroblasts</em> as predicted from their DNA and protein contents. The expression of Egr-1 and c-fos was induced as early as 15 min that reached maximal levels at 45 min and declined thereafter, whereas c-jun was induced at 45 min and remained elevated up to 2 hrs of ANG II addition. ANG II up-regulated the expression of TGF-beta 1, which became apparent after 1 hr of incubation and reached a plateau between <em>16</em>-48 hrs. Our results indicate that ANG II transiently stimulates the expression of transcription <em>factors</em>, which may up-regulate TGF-beta 1, that in turn could contribute to the process of myocardial extra-cellular matrix remodeling in hypertrophy.

<em>Growth</em> <em>factors</em> are important substances in the central control of wound healing during the exudative phase. Although these peptides have been applied frequently to chronic wounds in clinical studies, little is known about the naturally occurring levels at the wound site in correlation to healing in superficial wounds. We have therefore investigated the presence of these cytokines in partial thickness wounds. In <em>16</em> patients undergoing reconstructive surgery, split-thickness skin wounds were enclosed in cutaneous vinyl chambers filled with 2.5 ml of saline. Chambers placed over unwounded skin served as controls. After 24 hours, the accumulated wound fluid was harvested and replaced by 2.5 ml of saline until the wounds were healed. Wound fluid was centrifuged, aliquoted, and frozen at -70 degrees C. Samples were analyzed for protein and <em>growth</em> <em>factors</em> (insulin-like <em>growth</em> <em>factor</em>-1, epidermal <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>-AB, interleukin-1alpha, and transforming <em>growth</em> <em>factor</em>-beta1 and -beta2) and insulin-like <em>growth</em> <em>factor</em>-binding proteins 1 and 3 using a monoclonal Sandwich enzyme-linked immunosorbent assay and radioimmunoassay. All wounds healed in the liquid environment within 7 days (macroscopically) and 11 days (barrier function), respectively. In wound fluid, protein concentrations dropped from 5 mg/ml on day 1 to a baseline of 0.1 mg (unwounded skin), indicating a return of the barrier function. All <em>growth</em> <em>factors</em> could be measured already after 24 hours postwounding. However, the concentrations measured varied from 10 to more than 10,000 pg/ml between the different <em>factors</em>. The highest range was found for insulin-like <em>growth</em> <em>factor</em>-1 (21,000 to 41,000 pg/ml), the lowest for epidermal <em>growth</em> <em>factor</em> (3 to 63 and 3 to 88 pg/ml, respectively). Two different patterns of kinetics were distinguished: (1) a high initial peak decreasing to baseline values or below serum levels by the time of healing (insulin-like <em>growth</em> <em>factor</em>-1, insulin-like <em>growth</em> <em>factor</em> binding protein-1, -3, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>-AB, transforming <em>growth</em> <em>factor</em>-beta1) and (2) a low initial concentration followed by an increase to a maximum at the time of epithelialization (interleukin-1alpha, transforming <em>growth</em> <em>factor</em>-beta2). Comparing the <em>growth</em> <em>factor</em> levels measured to serum baseline values, it was found that four of the <em>growth</em> <em>factors</em> appeared in wound fluid at above serum concentrations (interleukin-1alpha, transforming <em>growth</em> <em>factor</em>-beta2, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>); the other <em>factors</em> never reached serum values in wound fluid (insulin-like <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta1, platelet-derived <em>growth</em> <em>factor</em>-AB). It is concluded that the different profiles of secretion might reflect different functions of polypeptide <em>growth</em> <em>factors</em> such as stimulation of epithelialization (epidermal <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-1), matrix synthesis (transforming <em>growth</em> <em>factor</em>-beta), and inflammatory stimulation (interleukin-1alpha). The concentrations determined could serve as guidelines for adapted administration of <em>growth</em> <em>factors</em> once correlations to healing disorders such as overhealing and ulceration are established.

We have developed a silicone nerve regeneration chamber that is partitioned into two compartments by a strip of nitrocellulose paper. The modified two-compartment chamber allows the investigation of the effects on rat sciatic nerve regeneration of trophic or <em>growth</em> <em>factors</em> that are initially bound to the nitrocellulose partition. In this study we compared the effects of untreated nitrocellulose, a siliconized nitrocellulose strip, and a strip that had been soaked in a basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) solution. FGF is a known angiogenic <em>factor</em> and a mitogen for endothelial cells, <em>fibroblasts</em>, and Schwann cells. All of these cell types are present in the peripheral nerve. In vitro analyses, using 3T3 cells as test cells, showed that some of the bound FGF remained active on the nitrocellulose paper for at least 8-10 days. In vivo experiments, examined at <em>16</em> days post-implantation, revealed that spatial migration of all cellular elements (perineurial-like cells, vasculature, and Schwann cells) across the chamber gap was slower with untreated nitrocellulose strips than with siliconized strips but was most advanced with FGF-treated ones. Most striking was the well-developed vascular arborization of the regenerate within the FGF chambers. Histologic sections from the proximal one-half of the chamber revealed that the regenerate in untreated strip chambers consisted of fibrin matrix and erythrocytes, whereas a well-developed structure with all the cellular elements of a regenerating nerve was seen in several of the FGF strip chambers. We conclude that FGF stimulates peripheral nerve regeneration in this model.

<em>Growth</em> <em>factors</em> may play a significant role in regulating the orderly progression of organ <em>growth</em> and differentiation during fetal development. We hypothesized that epidermal <em>growth</em> <em>factor</em> (EGF) would help regulate the development of surfactant synthesis in the fetal lung by influencing <em>fibroblast</em>-epithelial cell interactions. The effect of EGF (10 ng per ml) on the ability of the fetal lung <em>fibroblast</em> to produce <em>fibroblast</em> pneumonocyte <em>factor</em> (FPF) was studied in sex-specific <em>fibroblasts</em> cultured from day <em>16</em>, day 17 or day 18 fetal mouse lungs. FPF which is normally not produced by day <em>16</em> <em>fibroblasts</em>, is found only in female <em>fibroblasts</em> on day 17, and then in both males and females on day 18. EGF advanced this pattern such that female <em>fibroblasts</em> produced activity on day <em>16</em> and <em>fibroblasts</em> from both sexes produced FPF activity on day 17 and day 18. <em>Fibroblasts</em> from an androgen receptor-deficient mouse model confirmed that the effect of EGF was sex-specific and related to the state of development of the fetal lung. We conclude that EGF advances the fetal lung <em>fibroblast</em> through specific stages of development. It appears, therefore, to help control the timing of the clock regulating fetal lung maturation.

Mutated epidermal <em>growth</em> <em>factor</em> receptor (EGF-RvIII, DeltaEGF-R, and de2-7 EGF-R) is the result of an 801-bp deletion within the extracellular domain of wild-type EGF-R and is expressed by breast carcinomas, but not by normal breast tissues. EGF-RvIII is expressed both on the surface and in the cytoplasm of tumor cells. Thus, EGF-RvIII is a potential tumor-specific target for both Abs and T cells. However, it is not known whether breast cancer patients can raise immune responses to EGF-RvIII expressed by their tumors. The demonstration of EGF-RvIII-specific immune responses in patients would suggest that immunization of patients with EGF-RvIII vaccines is feasible, because these vaccines may boost a pre-existing immune response. We have evaluated humoral and cellular immune responses to EGF-RvIII in <em>16</em> breast cancer patients and three healthy donors. Seven of <em>16</em> patients developed EGF-RvIII-specific Abs that bound to isolated EGF-RvIII protein or the protein expressed by EGF-RvIII-transfected mouse <em>fibroblasts</em>. The Abs that bound to EGF-RvIII did not bind to wild-type EGF-R, and anti-EGF-RvIII Abs were not found in the sera of healthy donors. Three patients had EGF-RvIII peptide-specific lymphoproliferative responses, and two of these patients also had humoral immune responses. Humoral and cellular immune responses correlated with EGF-RvIII expression by patients' tumors in most cases. These studies demonstrate that breast cancer patients specifically recognize EGF-RvIII with an overall immune response rate of 50%, suggesting that patients may benefit from vaccination against EGF-RvIII, boosting pre-existing immune responses.

The possible contributions of the angiotensin receptor subtypes 1 (AT1) and 2 (AT2) to angiotensin II-induced changes in collagen secretion and production were studied using the specific angiotensin receptor AT1 and AT2 antagonists telmisartan and P-186. The role of the renin-angiotensin system and its interaction with transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) in collagen deposition in cardiac <em>fibroblasts</em> in relation to the development of myocardial fibrosis is also discussed. Cardiac <em>fibroblasts</em> (from normal male adult rats) from passage 2 were cultured to confluency and incubated in the presence of angiotensin II (ANG II) in a concentration range of 10(-10)-10(-6) M in serum-free Dulbecco's MEM medium for 24 h. Collagen production and secretion were assayed by [3H]-proline incorporation and noncollagen production and secretion were also analyzed. ANG II dose-dependently increased collagen secretion and production in rat adult cardiac <em>fibroblasts</em> in culture. Noncollagen secretion and production were also concentration-dependently increased by ANG II. Addition of 100 nmol/l ANG II increased (p < 0.01) collagen secretion and production by 75 +/- 6 (SEM) and 113 +/- 23%, respectively, and noncollagen secretion and production by 65 +/- 6 and 57 +/- <em>16</em>%, respectively. Pretreatment of cardiac <em>fibroblasts</em> with telmisartan completely blocked the ANG II-induced increase in collagen secretion (p < 0.001) and production (p < 0.05) and in noncollagen secretion (p < 0.01) and production (p < 0.01). P-186 had no effect on the ANG II-induced increase in collagen secretion and production. Addition of telmisartan and P-186 did not affect collagen secretion and production in basal cardiac <em>fibroblasts</em>. TGF-beta 1 also concentration- and time-dependently increased the secretion and production of collagen in cardiac <em>fibroblasts</em>. Our data demonstrate that the effects of ANG II on collagen secretion and production in adult rat cardiac <em>fibroblasts</em> in culture are AT1-receptor mediated since they were abolished by the specific AT1-receptor antagonist telmisartan but not by the specific AT2-receptor antagonist P-186. The ability of ANG II to induce collagen synthesis in cardiac <em>fibroblasts</em> may be mediated by increased TGF-beta 1 production.

The tumor microenvironment is now recognized as a major <em>factor</em> in determining the survival and <em>growth</em> of disseminated tumor cells at potential metastatic sites. Tumor cells send signals to stroma cells and stimulate them to produce <em>factors</em> that in turn create favorable conditions for tumor cell metastasis. Activated <em>fibroblasts</em> constitute an important component of the tumor-associated stroma. We have previously shown that S100A4 protein produced by stromal <em>fibroblasts</em> in the primary tumor stimulates metastasis formation. Here we show that activated <em>fibroblasts</em> also stimulate the formation of metastases independently of S100A4 expression during organ colonization. To identify genes that could potentially interfere with <em>fibroblast</em>-driven metastasis, we used gene expression profiling of S100A4-deficient <em>fibroblasts</em> treated with and without tumor cell-conditioned media. Five differentially expressed genes encoding cell surface and secreted proteins with potential metastasis-modulating activity were selected. Expression of lymphocyte antigen 6 complex (Ly6c) and matrix metalloproteinase 3 (Mmp3) was upregulated in <em>fibroblasts</em> in response to tumor-conditioned medium, whereas expression of cadherin-<em>16</em> (Cdh<em>16</em>), Ccn2, and fibulin-5 (Fbln5) was downregulated. Further analysis showed that Fibulin-5 is able to suppress the metastatic colonization of lungs and liver. Additional studies suggest a mechanism in which Fibulin-5 suppresses metastasis formation by inhibiting production of matrix metalloproteinase 9 (MMP9) and reducing the invasive behavior of <em>fibroblasts</em>. Together our data are consistent with the notion that tumors secrete <em>factors</em> that downregulate expression of Fbln5 in <em>fibroblasts</em> at sites of metastatic colonization, in turn upregulating Mmp9 expression and stimulating metastatic organ colonization.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44-amino-acid, hydrophobic polypeptide which localizes predominantly in Golgi membranes and appears to transform cells through the activation of tyrosine kinase <em>growth</em> <em>factor</em> receptors. In <em>fibroblasts</em>, E5 interacts with both the <em>16</em>-kilodalton vacuolar ATPase subunit and the platelet-derived <em>growth</em> <em>factor</em> receptor (PDGF-R) via its hydrophobic transmembrane domain and induces autophosphorylation of the receptor. To further analyze the correlation between E5 biological activity and its ability to bind these cellular proteins, a series of nine E5 transmembrane mutants was evaluated. In 32D mouse hematopoietic cells, there was an incomplete correlation between the abilities of the E5 mutant proteins to associate the PDGF-R and to transform cells. However, all transforming E5 mutant proteins induced PDGF-R tyrosine phosphorylation. In NIH 3T3 and C127 mouse <em>fibroblasts</em>, both transforming and nontransforming E5 mutant proteins were defective for PDGF-R binding. In addition, while most of the transforming E5 proteins induced PDGF-R phosphorylation, one hypertransforming mutant (serine 17) neither bound nor induced receptor autophosphorylation. These findings support the hypothesis that the transformation of <em>fibroblasts</em> by E5 transmembrane mutants can involve alternative cellular targets or potentially independent activities of the E5 protein. In addition, these results underscore the critical role of the transmembrane domain in mediating E5 biological activities.

A sensitive sandwich enzyme immunoassay for human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (HbFGF) was developed employing three monoclonal antibodies (MAb3H3, MAb98 and MAb52). The Fab' fragment of MAb3H3 which inhibits HbFGF biological activity was conjugated to horseradish peroxidase. A mixture of MAb52 and MAb98 was used in the solid phase. Neither human acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>, hst-1/KS3 product nor acid denatured HbFGF was cross-reactive in this assay system. The detection limit of this assay system was 1 pg/well. Using this assay, some tumor cell lines were revealed to produce a higher level of bFGF than a normal one. Serum samples from normal volunteers were also assayed, and immuno-reactive HbFGF could be detected in <em>16</em> out of 57 samples at range 30 approximately 206 pg/ml.

OBJECTIVE

Tubulointerstitial pathology with the accumulation of extracellular matrix are pathological hallmarks of diabetic nephropathy that are directly related to declining renal function. Tranilast (N-[3,4-dimethoxycinnamoyl]anthranilic acid), an inhibitor of transforming growth factor-beta (TGF-beta), used to treat hypertrophic scars has recently been shown in pilot studies to exert a beneficial effect in advanced diabetic nephropathy in humans. However, its effects on diabetic renal pathology are unknown.

METHODS

Studies were conducted using a transgenic model, the diabetic (mRen-2)27 rat, which develops many of the structural and functional characteristics of human diabetic nephropathy when diabetes is induced with streptozotocin (STZ). An experimental design was chosen to mimic, in part, the clinical context with drug therapy (tranilast 400 mg/kg/ day) initiated in established disease (8 weeks after STZ) and in the presence of persistent hyperglycaemia and hypertension.

RESULTS

At 16 weeks, diabetes was associated with progressive albuminuria, tubulointerstitial fibrosis and tubular atrophy. Without affecting blood pressure or blood glucose, tranilast treatment was associated with a 83% reduction in tubulointerstitial fibrosis (p < 0.001), a 58% reduction in tubular atrophy (p < 0.01) and near normalization of albuminuria (p < 0.05) in diabetic Ren-2 rats. In vitro studies in primary cultures of human renal cortical fibroblasts demonstrated a reduction in TGF-beta-induced hydroxyproline incorporation and fibronectin synthesis with tranilast 100 microM.

CONCLUSIONS

Tranilast, when administered during the course of experimental diabetic nephropathy, attenuates tubulointerstitial pathology and albuminuria. These findings are consistent with the antagonist effects of tranilast on TGF-beta actions in the diabetic kidney.