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). <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) plays an important role in liver metabolism and is also a potential marker for NAFL. Here we aimed to test the effect of FGF<em>21</em> 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 FGF<em>21</em> 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, FGF<em>21</em> deficient mice (FGF<em>21</em> KO) develop excess fatty liver within 16 weeks. Hepatic pathology progresses and at 52 weeks FGF<em>21</em> 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 FGF<em>21</em> 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 <em>growth</em> <em>factor</em> 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 FGF<em>21</em> is required to limit the progression from NAFL to HCC in response to prolonged exposure to an obesogenic diet. The induction of hepatic FGF<em>21</em> 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.

Pharmacological administration of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) improves metabolic profile in preclinical species and humans. FGF<em>21</em> exerts its metabolic effects through formation of beta-klotho (KLB)/FGF receptor 1c FGFR1c complex and subsequent signaling. Data from various in vitro systems demonstrate the intact C- and N-terminus of FGF<em>21</em> is required for binding with KLB, and interaction with FGFR1c, respectively. However the relative roles of the termini for in vivo pharmacological effects are unclear. Here we report PF-05231023, a long-acting FGF<em>21</em> analogue which is unique in that the half-life and subcutaneous (s.c.) bioavailability of the intact C-terminus are significantly different from those of the intact N-terminus (2 vs. 22 hr for half-life and 4~7 vs. ~50% SC bioavailability). Therefore, this molecule serves as a valuable tool to evaluate the relative roles of intact C-terminus vs. N-terminus in in vivo pharmacology studies in preclinical species. We determined the effects of PF-05231023 administration on body weight (BW) loss and glucose reduction during an oral glucose tolerance test (OGTT) following SC and intravenous (i.v.) administration in diet-induced obese (DIO) and leptin-deficient obese (ob/ob) mice, respectively. Our data show that the intact N-terminus of FGF<em>21</em> in PF-05231023 appears to be sufficient to drive glucose lowering during OGTT and sustain BW loss in DIOs. Further, PK/PD modeling suggests that while the intact FGF<em>21</em> C-terminus is not strictly required for glucose lowering during OGTT in ob/ob mice or for BW reduction in DIO mice, the higher potency conferred by intact C-terminus contributes to a rapid initiation of pharmacodynamic effects immediately following dosing. These results provide additional insight into the strategy of developing stabilized versions of FGF<em>21</em> analogs to harness the full spectrum of its metabolic benefits.

In mice of normal weight and with diet-induced obesity, a high-fat, low-carbohydrate ketogenic diet (KD) causes weight loss, reduced circulating glucose and lipids, and dramatic changes in hepatic gene expression. Many of the effects of KD are mediated by <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>). We tested the effects of KD feeding on ob/ob mice to determine if metabolic effects would occur in obesity secondarily to leptin deficiency. We evaluated the effect of prolonged KD feeding on weight, energy homeostasis, circulating metabolites, glucose homeostasis, and gene expression. Subsequently, we evaluated the effects of leptin and fasting on FGF<em>21</em> expression in ob/ob mice. KD feeding of ob/ob mice normalized fasting glycemia and substantially reduced insulin and lipid levels in the absence of weight loss. KD feeding was associated with significant increases in lipid oxidative genes and reduced expression of lipid synthetic genes, including stearoyl-coenzyme A desaturase 1, but no change in expression of inflammatory markers. In chow-fed ob/ob mice, FGF<em>21</em> mRNA was elevated 10-fold compared with wild-type animals, and no increase from this elevated baseline was seen with KD feeding. Administration of leptin to chow-fed ob/ob mice led to a 24-fold induction of FGF<em>21</em>. Fasting also induced hepatic FGF<em>21</em> in ob/ob mice. Thus, KD feeding improved ob/ob mouse glucose homeostasis without weight loss or altered caloric intake. These data demonstrate that manipulation of dietary macronutrient composition can lead to marked improvements in metabolic profile of leptin-deficient obese mice in the absence of weight loss.

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

BACKGROUND

Recent laboratory studies suggest that FGF<em>21</em> is involved in reciprocal regulation of bone and energy homeostasis. Systemic administration of FGF<em>21</em> protects animals from obesity and diabetes but causes severe bone loss, smothering the enthusiasm over FGF<em>21</em> as a potential antiobesity therapeutic. To date, there is no information on whether FGF<em>21</em> relates to BMD in humans. We thus studied the relationship between plasma FGF<em>21</em> levels and BMD in healthy adults.

METHODS

Fasting plasma FGF<em>21</em> 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 FGF<em>21</em> levels were not different between the sexes. While there was no association between FGF<em>21</em> concentrations and body composition in men, FGF<em>21</em> levels correlated positively with fat mass (p < 0.01) in women. In men, no significant correlation between FGF<em>21</em> with BMD was observed. However, in women, FGF<em>21</em> 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 FGF<em>21</em> levels and BMD in healthy women, suggesting the association between bone loss and high FGF<em>21</em> states in animals may not be directly translated to humans in physiologic states. We hypothesize that FGF<em>21</em> 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.

Fish oil has been used effectively in the treatment of cardiovascular disease via triglyceride reduction and inflammation modulation. This study aimed to assess the effects of fish oil on patients with nonalcoholic fatty liver disease (NAFLD) associated with hyperlipidemia. Eighty participants with NAFLD associated with hyperlipidemia were randomly assigned to consume fish oil (n=40, 4 g/d) or corn oil capsules (n=40, 4 g/d) for 3 months in a double-blind, randomized clinical trial. Blood levels of lipids, glucose and insulin, liver enzymes, kidney parameters and cytokines at baseline and the end of the study were measured. Seventy people finished the trial. Plasma concentrations of eicosapentaenoic acid and docosahexaenoic acid significantly increased in the fish oil group after intervention. After adjustment for age, gender and BMI, fish oil significantly decreased fasting serum concentrations of total cholesterol, triglyceride, apolipoprotein B and glucose (by (mean±SD) 0.49±0.43 mmol/L, 0.58±0.89 mmol/L, 0.28±0.33 g/L and 0.76±0.56 mmol/L, respectively, P<0.05), as well as alanine aminotransferase and γ-glutamyl transpeptidase levels (by (median (interquartile)) 9.0(0.5, <em>21</em>.5) and 7.0(2.2, 20.0) IU/L, respectively, P<0.05), significantly increased serum adiponectin levels (by 1.29±0.62 μg/mL, P<0.001), and reduced serum levels of tumor necrosis <em>factor</em> α, leukotrienes B4, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), cytokeratin 18 fragment M30 and prostaglandin E2 (by 1.70±1.18 pg/mL, 0.59±0.28 ng/mL, 1<em>21</em>±31 pg/mL, 83±60 IU/L and 10.9±2.3 pg/mL, respectively, P<0.001). Corn oil had no effect except for increasing serum creatinine concentrations by 7.7±8.9 μmol/L (P=0.008). The effects of fish oil on lipids, glucose and γ-glutamyl transpeptidase were positively correlated with the reductions of serum FGF<em>21</em> and prostaglandin E2 concentrations after adjustment for age, gender and BMI (r = 0.275 to 0.360 and 0.261 to 0.375, respectively, P<0.05). In conclusion, our findings suggest that fish oil can benefit metabolic abnormalities associated with NAFLD treatment.

BACKGROUND

ChiCTR-TRC-12002380.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. However, the role of FGF<em>21</em> in hypertension remains elusive. Here we show that FGF<em>21</em> deficiency significantly exacerbates angiotensin II-induced hypertension and vascular dysfunction, whereas such negative effects are reversed by replenishment of FGF<em>21</em>. Mechanistically, FGF<em>21</em> acts on adipocytes and renal cells to promote induction of angiotensin-converting enzyme 2 (ACE2), which in turn converts angiotensin II to angiotensin-(1-7), then inhibits hypertension and reverses vascular damage. In addition, ACE2 deficiency strikingly abrogates these beneficial effects of FGF<em>21</em> in mice, including alleviation of angiotensin II-associated hypertension and vascular damage. Otherwise, pharmaceutical inhibition of angiotensin-(1-7) attenuates the protective effect of FGF<em>21</em> on angiotensin II-induced vascular dysfunction, but not on hypertension. Thus, FGF<em>21</em> protects against angiotensin II-induced hypertension and vascular impairment by activation of the ACE2/angiotensin-(1-7) axis via fine-tuning the multi-organ crosstalk between liver, adipose tissue, kidney, and blood vessels.

Elevated plasma fibroblast growth factor 23 (FGF23) is a prognostic marker in chronic kidney disease. Recently, FGF23 was reported to also be a predictive factor in chronic congestive heart failure (HF). To date however, plasma levels in acute decompensated HF (ADHF) have not been reported and myocardial production and distribution of FGF23 in HF is poorly defined. We aimed to determine plasma levels and myocardial production of FGF23 in ADHF.

Plasma FGF23, N-terminal pro B-type natriuretic peptide (NT-proBNP) and estimated glomerular filtration rate (eGFR) were assessed in 21 ADHF patients and 19 controls. Myocardial gene expression and distribution of FGF23 was determined on left ventricular samples from HF patients and normal controls.

Plasma FGF23 was markedly higher in ADHF patients compared with controls (1498 ± 1238 versus 66 ± 27 RU/mL, P < 0.0001). There were no correlations between FGF23 and eGFR, NT-proBNP, ejection fraction or age. ADHF subjects with eGFR >60 mL/min/1.73 m(2) had FGF23 levels of 1526 ± 1601 RU/mL versus 55 ± 20 RU/mL in controls (P = 0.007). Quantified myocardial FGF23 gene expression was similar between HF patients and controls. Myocardial FGF23 immunostaining was similar between HF patients and controls, with equal distribution throughout cardiomyocytes.

Patients with ADHF had markedly elevated plasma FGF23 levels. Myocardial FGF23 gene expression was present in HF at a similar level as normal controls, and immunohistochemistry showed similar cellular distribution of FGF23 in HF and controls, suggesting that the myocardium does not contribute to the elevated circulating FGF23 in HF.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a recently identified member of the FGF superfamily, is mainly secreted from the liver and adipose tissues and plays an important role in improving metabolic syndrome and homeostasis. The aim of this study is to evaluate the role of FGF<em>21</em> in alcoholic fatty liver disease (AFLD) and to determine if it has a therapeutic effect on AFLD. In this paper, we tested the effect of FGF<em>21</em> on alcohol-induced liver injury in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. Male KM mice received single dose of 5 g/kg ethanol gavage every day for 6 weeks, which induced significant fatty liver and liver injury. The alcohol-induced fatty liver cell model was achieved by adding ethanol into the medium of HepG2 cell cultures at a final concentration of 75 mM for 9 days. Results showed that treatment with recombinant FGF<em>21</em> ameliorated alcoholic fatty liver and liver injury both in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. In addition, FGF<em>21</em> treatment down-regulated the hepatic expression of fatty acid synthetic key enzyme, activated hepatic AMPK-SIRT1 pathway and significantly down-regulated hepatic oxidative stress protein. Taken together, FGF<em>21</em> corrects multiple metabolic parameters of AFLD in vitro and in vivo by activation of the AMPK-SIRT1 pathway.

Platelet-derived endothelial cell <em>growth</em> <em>factor</em> (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin <em>fibroblasts</em>, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas <em>21</em> other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis.

Cartilage structures from the head and neck possess a certain but limited capacity to heal after injury. This capacity is accredited to the perichondrium. In this study, the role of the inner (cambium) and the outer (fibrous) layers of the perichondrium in cartilage wound healing in vitro is investigated. For the first time, the possibility of selectively removing the outer perichondrium layer is presented. Using rabbit ears, three different conditions were created: cartilage explants with both perichondrium layers intact, cartilage explants with only the outer perichondrium layer dissected, and cartilage explants with both perichondrium layers removed. The explants were studied after 0, 3, 7, 14, and <em>21</em> days of in vitro culturing using histochemistry and immunohistochemistry for Ki-67, collagen type II, transforming <em>growth</em> <em>factor</em> beta 1 (TGFbeta1), and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2). When both perichondrium layers were not disturbed, fibrous cells grew over the cut edges of the explants from day 3 of culture on. New cartilage formation was never observed in this condition. When only the outer perichondrium layer was dissected from the cartilage explants, new cartilage formation was observed around the whole explant at day <em>21</em>. When both perichondrium layers were removed, no alterations were observed at the wound surfaces. The <em>growth</em> <em>factors</em> TGFbeta1 and FGF2 were expressed in the entire perichondrium immediately after explantation. The expression gradually decreased with time in culture. However, the expression of TGFbeta1 remained high in the outer perichondrium layer and the layer of cells <em>growing</em> over the explant. This indicates a role for TGFbeta1 in the enhancement of fibrous over<em>growth</em> during the cartilage wound-healing process. The results of this experimental in vitro study demonstrate the dual role of perichondrium in cartilage wound healing. On the one hand, the inner layer of the perichondrium, adjacent to the cartilage, provides (in time) cells for new cartilage formation. On the other hand, the outer layer rapidly produces fibrous over<em>growth</em>, preventing the good cartilage-to-cartilage connection necessary to restore the mechanical function of the structure.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a key metabolic regulator that is induced by peroxisome proliferator-activated receptor alpha (PPARalpha) activation in response to fasting. We recently reported that bezafibrate, a pan-agonist of PPARs, decreases body temperature late at night through hypothalamic neuropeptide Y (NPY) activation and others have shown that mice overexpressing FGF<em>21</em> are prone to torpor.

OBJECTIVE

We examined whether FGF<em>21</em> is essential for fasting-induced hypothermia using FGF<em>21</em> knockout (KO) mice.

RESULTS

Acute fasting decreased body temperature late at night accompanied by the induction of hepatic FGF<em>21</em> and hypothalamic NPY expression in wild-type mice. A deficiency of FGF<em>21</em> affected neither fasting-induced hypothermia nor hypothalamic NPY induction. Fasting enhanced locomotor activity in both genotypes. On the other hand, a deficiency of FGF<em>21</em> significantly attenuated chronic hypothermia and hypoactivity induced by a ketogenic diet (KD).

CONCLUSIONS

Our findings suggest that FGF<em>21</em> is not essential for the hypothermia that is associated with the early stages of fasting, although it might be involved in the adaptive response of body temperature to chronic starvation.

Brown adipose tissue (BAT) is considered an interesting target organ for the treatment of metabolic disease due to its high metabolic capacity. Non-shivering thermogenesis, once activated, can lead to enhanced partitioning and oxidation of fuels in adipose tissues, and reduce the burden of glucose and lipids on other metabolic organs such as liver, pancreas, and skeletal muscle. Sustained long-term activation of BAT may also lead to meaningful bodyweight loss. In this review, we discuss three different drug classes [the thiazolidinedione (TZD) class of PPARγ agonists, β3-adrenergic receptor agonists, and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) analogs] that have been proposed to regulate BAT and beige recruitment or activation, or both, and which have been tested in both rodent and human. The learnings from these classes suggest that restoration of functional BAT and beige mass as well as improved activation might be required to fully realize the metabolic potential of these tissues. Whether this can be achieved without the undesired cardiovascular side effects exhibited by the TZD PPARγ agonists and β3-adrenergic receptor agonists remains to be resolved.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric, and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody-drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which binds to the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a noncleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nanomolar to subnanomolar range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared with healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor <em>growth</em> inhibition or tumor regression of cell line-based or patient-derived xenograft models of human gastric or breast cancer. Furthermore, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients and a phase I study (NCT02368951) has been initiated. Cancer Res; 76(<em>21</em>); 6331-9. ©2016 AACR.

OBJECTIVE

We tested the hypothesis that intravenous administration of basic fibroblast growth factor (bFGF) during 4 hours of permanent focal ischemia would affect acute brain injury.

METHODS

Halothane-anesthetized cats underwent left middle cerebral artery (MCA) occlusion for 4 hours. Control cats received diluent (n = 14). Experimental cats were treated with bFGF at a rate of 5 (n = 13), 50 (n = 13), or 250 microg/kg per hour (n = 9) intravenously beginning 60 minutes after initiation of ischemia and continuing until the end of the protocol.

RESULTS

As measured by the microsphere method, blood flow to ipsilateral caudate nucleus and ipsilateral inferior temporal cortex was decreased similarly during ischemia, before drug administration, in all groups. Likewise, there was no difference in blood flow to ipsilateral caudate nucleus or inferior temporal cortex as a result of bFGF administration during MCA occlusion. Triphenyltetrazolium-determined injury volume of the ipsilateral cerebral cortex (control, 40+/-7%; bFGF 5 microg/kg per hour, 22+/-5%; bFGF 50 microg/kg per hour, 26+/-7%; bFGF 255 microg/kg per hour, 23+/-6% of ipsilateral cerebral cortex; mean+/-SEM) was less in cats treated with bFGF. There was no difference among groups in injury volume to caudate nucleus (control, 29+/-8%; bFGF 5 microg/kg per hour, 29+/-8%; bFGF 50 microg/kg per hour, 21+/-7%; bFGF 250 microg/kg per hour, 32+/-7% of ipsilateral caudate nucleus). Somatosensory evoked potential amplitude decreased similarly (to <20% of baseline amplitude in all groups) during MCA occlusion and was not altered by bFGF administration. CONCLUSIONS; These data indicate that systemic administration of bFGF ameliorates acute injury in the cerebral cortex without increasing blood flow during focal ischemia in cats. Because bFGF afforded protection when administered after the onset of ischemia, bFGF may provide its beneficial effect by limiting progression of injury in ischemic border regions.

Transforming <em>growth</em> <em>factor</em> (TGF)-beta isoforms (TGF-beta 1, -beta 2, and -beta 3) regulate cell <em>growth</em> and differentiation and have critical regulatory roles in the process of tissue repair and remodeling. Signal transduction for TGF-beta function is transmitted by a heteromeric complex of receptors consisting of two serine/threonine kinase transmembrane proteins (RI and RII). We have previously shown that each TGF-beta isoform is widely expressed in a distinct spatial and temporal pattern throughout the processes of excisional and incisional wound repair. As the presence of TGF-beta receptors determines cellular responsiveness, we have currently examined, by immunohistochemistry, the localization of RI (ALK-1, ALK-5) and RII throughout repair of full-thickness excisional wounds up to <em>21</em> days after wounding. The expression of RI (ALK-5) and RII co-localized in both the unwounded and wounded skin and was present in the same cell types as TGF-beta ligands. However, immunoreactivity for TGF-beta receptors, throughout repair, occurred 1 to 5 days later than TGF-beta isoform immunostaining. This implies that the presence of TGF-beta ligands may up-regulate TGF-beta receptors for function and/or may reflect a lag due to local processing of latent TGF-beta. As observed for the immunohistochemical localization of TGF-beta isoforms in unwounded skin, RI and RII were expressed throughout the four layers of the epidermis, showing a wavy pattern of slight to moderate immunostaining, and hair follicles, sweat glands, and sebaceous glands were moderately immunoreactive. The extracellular matrix, <em>fibroblasts</em>, and blood vessels in the dermis were not immunoreactive. After injury, as observed for TGF-beta ligands, RI and RII expression was increased in the epidermis adjacent to the wound and the epithelium migrating over the wound was completely devoid of TGF-beta receptor immunoreactivity until re-epithelialization was completed by day 7 after wounding. The dermis was only slightly immunoreactive for RI and RII until day 5 when, immediately under the wound, immunostaining for <em>fibroblasts</em>, connective tissue cells, and newly forming vasculature began to increase and remained intense until day 14. Consistent with the role for TGF-beta in scarring, numerous <em>fibroblasts</em>, ostensibly active in the production of extracellular matrix components, continued to be slightly immunoreactive for RI and RII at <em>21</em> days. The ALK-1 (TSR-1) type I receptor, which binds both activin and TGF-beta, showed slight immunostaining early in repair (days 1 to 7) that progressively became more intense later in repair after day 10 and through day <em>21</em>. This suggests that there may be a switch to a different type I receptor, implying different functions for the ALK-1 and ALK-5 receptors. The concomitant expression of TGF-beta isoforms and their signal-transducing receptors denote potential spatial and temporal activity of TGF-beta. Thus, although TGF-beta ligand is present, TGF-beta would not function in wound repair until a later time when RI and RII appear. This information should aid in the development of receptor antagonists as a therapeutic approach to scarring and fibrosis. In addition, these studies underscore the importance of defining the expression of proteins in vivo to establish a basis for the analysis of mechanisms in vitro.

An extract of <em>21</em>-day rainbow trout embryos stimulated <em>growth</em> of several piscine cell lines in the absence of added serum. Established lines from trout (RTG-2 and STE-137), salmon (CHSE-<em>21</em>4), carp (EPC), and goldfish (CAR) and early-passage cells initiated from trout embryos grew in serum-free medium containing the embryo extract. In addition the extract was sufficient for maintaining long-term cultures of CHSE-<em>21</em>4 cells for several months through a minimum of 20 passages (approximately 50 population doublings) in the absence of serum. Optimal response was achieved with 100 micrograms of extract protein per ml, but a significant <em>growth</em>-promoting effect was observed with as little as 2.5 micrograms/ml. The activity was nondialyzable, protease-sensitive, and stable in 200 mM acetic acid. The level of mitogenic response induced by the extract could not be duplicated with purified mammalian <em>growth</em> <em>factors</em> added individually or in combination, and the extract did not stimulate DNA synthesis in quiescent mouse <em>fibroblasts</em>. These results suggest that trout embryo extract may contain a novel <em>growth</em>-promoting activity for fish cells.

Epidermal <em>growth</em> <em>factor</em> and cartilage-derived basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (EGF and CD-bFGF) are mitogens shown to increase the rate of wound repair in animal models. In addition to being a mitogen for granulation tissue, CD-bFGF stimulates the recruitment of cells to the wound site. CD-bFGF and a closely-related chondrosarcoma-derived <em>fibroblast</em> <em>growth</em> <em>factor</em> stimulated chemotaxis of granulation tissue cells in vitro, each <em>factor</em> having a maximum activity at a concentration of 55 pM. Epidermal <em>growth</em> <em>factor</em> was also a potent chemoattractant for rat granulation tissue <em>fibroblasts</em>; however, maximum activity was obtained at 1.7 nM. Cells from all stages of wound repair were chemotactically responsive to these <em>factors</em>, but there was some attenuation of the response to bFGF in cells derived from fully-organized day 28 granulation tissue. Collagenase-catalyzed restructuring of collagen, an additional significant feature of wound repair, is probably critical to cell movement in an extracellular matrix. Cells derived from organizing (6-day old) sponge granulation tissue secreted latent collagenase constitutively in vitro. In the presence of serum, the production of collagenase was stimulated three-four fold by 1.8 nM bFGF derived either from cartilage or chondrosarcoma. When serum was present, as at a wound site, collagenase production was not enhanced by the addition of EGF. Cells from fully organized, day <em>21</em> sponge granulation tissue did not secrete latent collagenase constitutively and could not be stimulated to do so by the addition of EGF, bFGF, or phorbol ester. Human skin <em>fibroblast</em> collagenase production was also stimulated by bFGF and was refractory to EGF. While both classes of <em>growth</em> <em>factor</em> have the ability to promote wound healing, the varying responses they elicit in cell populations from the wound site emphasize the different pathways of cellular activation.

Increasing evidence has shown the potential of neuronal plasticity in adult brain after injury. Neural proliferation can be triggered by a focal sublethal ischemic preconditioning event; whether mild global ischemia could cause neurogenesis has been not clear. The present study investigated stimulating effects of sublethal transient global ischemia (TGI) on endogenous neurogenesis and neuroblast migration in the subventricular zone (SVZ), dentate gyrus, and peri-infarct areas of the adult cortex. Adult mice of 129S2/Sv strain were subjected to 8-min bilateral common carotid artery ligation followed by 5-bromo-2'-deoxyuridine (BrdU; 50 mg/kg, intraperitoneal) administration every day until being sacrificed at 1-<em>21</em> days after reperfusion. The mild TGI did not induce neuronal cell death for up to 7 days after TGI, as evidenced by negative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining among NeuN-positive cells in the hippocampus and neocortex. In TGI animals, BrdU staining revealed enhanced proliferation of neuroblasts and their migration track from the SVZ into the striatum and neocortex. In the corpus callosum, there were more BrdU-positive cells in the TGI group in the first 2 days. Increasing numbers of BrdU-positive cells were seen 7-<em>21</em> days later in the striatum and cortex of TGI mice. The cortex of TGI animals showed increased expression of erythropoietin, erythropoietin receptor, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2, vascular endothelial <em>growth</em> <em>factor</em>, and phosphorylated Jun N-terminal kinase; the expression was peaked 2 to 3 days after reperfusion. BrdU and NeuN double staining in the dentate gyrus, striatum, and cortex implied increased neurogenesis induced by the TGI preconditioning. Doublecortin (DCX)-positive cells increased in the cortex of TGI mice, localized to cortical layers II, III, and V, and many stained positive for the mature neuronal markers NeuN, neurofilament, N-methyl-d-aspartic acid receptor subunit gene NR1, or the gamma-aminobutyric-acid-synthesizing enzyme glutamic acid decarboxylase (GAD67). The atypical localization of DCX-positive cells and the colabeling with mature neuronal markers suggested that, in addition to indentifying migrating neuroblasts, DCX might also be a stress marker in the cortex. It is suggested that the sublethal TGI-induced regenerative responses may contribute to the beneficial effects of ischemic preconditioning.

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 16. FGFR-3 levels peaked in the small intestine from 7 to <em>21</em> 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.

Tumor necrosis <em>factor</em> (TNF)-like weak inducer of apoptosis (TWEAK) is an inflammatory cytokine that modulates several biological responses by inducing chemokines and proinflammatory cytokines. We hypothesized that TWEAK could promote secretion of IL-17, an amplifier of inflammatory arthritis. To test this, we investigated the capacity of TWEAK to induce IL-17 production in T cells via the <em>fibroblast</em> <em>growth</em> <em>factor</em>-inducible gene 14 (Fn14, also known as TWEAK receptor) signal pathway in rheumatoid arthritis (RA). Fn14 and IL-17 were highly expressed in arthritic tissues of collagen-induced arthritis (CIA) mice. TWEAK induced production of IL-17 alone and synergistically with lipopolysaccharide. In naïve murine T cells, TWEAK promoted Th17 differentiation. The expression of Fn14 was predominant in Th17 cells. TWEAK and IL-17 concentrations were significantly higher in synovial fluid and serum in RA patients than OA patients. In addition, we identified CD4(+)IL-17(+)Fn14(+) cells in synovium from RA patients. TWEAK promoted IL-17 production synergistically with IL-23 or IL-<em>21</em> and blockade of Fn14 with Fn14-Fc suppressed Th17 differentiation. Conversely, this treatment enhanced Treg differentiation. These results suggest that TWEAK induces IL-17 production and may be a therapeutic target in the treatment of RA.

Science is marked by the death of dogmas; the discovery that adipocytes are more than just lipid-storing cells but rather produce potent hormones is one such example that caught physiologists by surprise and reshaped our views of metabolism. While we once considered the adipocyte as a passive storage organ for efficient storage of long-term energy reserves in the form of triglyceride, we now appreciate the general idea (once a radical one) that adipocytes are sophisticated enough to have potent endocrine functions. Over the past two decades, the discoveries of these adipose-derived <em>factors</em> ("adipokines") and their mechanistic actions have left us marveling at and struggling to understand the role these <em>factors</em> serve in physiology and the pathophysiology of obesity and diabetes. These hormones may serve an integral role in protecting nonadipose tissues from lipid-induced damage during nutrient-deprived or replete states. As such, adipocytes deliver not only potentially cytotoxic free fatty acids but, along with these lipids, antilipotoxic adipokines such as leptin, adiponectin, and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> that potently eliminate excessive local accumulation of these lipids or their conversion to unfavorable sphingolipid intermediates.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1) is a potent mitogen for mesoderm- and neuroectoderm-derived cell types in vitro. However, a mutant FGF-1 with deletion in its nuclear localization sequence (NLS, residues <em>21</em>-27) is not mitogenic in vitro. We demonstrated that synthetic peptides containing this NLS were able to stimulate DNA synthesis in a FGF receptor-independent manner after they were delivered into living NIH 3T3 cells by a cell-permeable peptide import technique. The stimulation of maximal DNA synthesis by these peptides required the presence of peptides during the entire G1 phase of the cell cycle. The mitogenic effect was specific for the NLS of FGF-1 because a peptide with double point mutations at lysine residues was inactive in stimulating DNA synthesis. Our results suggest that the NLS plays an important role in the mitogenic pathway initiated by exogenous FGF-1 by its direct involvement in the nuclear transport and signaling of internalized FGF-1.

OBJECTIVE

This review focuses on studies from the past year that highlight molecular and cellular mechanisms of pancreatic injury arising from acute and chronic pancreatitis.

RESULTS

<em>Factors</em> that induce or ameliorate injury as well as cellular pathways involved have been examined. Causative or sensitizing <em>factors</em> include refluxed bile acids, hypercalcemia, ethanol, hypertriglyceridemia, and acidosis. In addition, the diabetes drug exendin-4 has been associated with pancreatitis, whereas other drugs may reduce pancreatic injury. The intracellular events that influence disease severity are better understood. Cathepsin-L promotes injury through an antiapoptotic effect, rather than by trypsinogen activation. In addition, specific trypsinogen mutations lead to trypsinogen misfolding, endoplasmic reticulum stress, and injury. Endogenous trypsin inhibitors and upregulation of proteins including Bcl-2, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, and activated protein C can reduce injury. Immune cells, however, have been shown to increase injury via an antiapoptotic effect.

CONCLUSIONS

The current findings are critical to understanding how causative factors initiate downstream cellular events resulting in pancreatic injury. Such knowledge will aid in the development of targeted treatments for pancreatitis. This review will first discuss factors influencing pancreatic injury, and then conclude with studies detailing the cellular mechanisms involved.