The time course and potency of the de novo angiogenic response to vascular endothelial <em>growth</em> <em>factor</em> isoform 165 (VEGF165) at approximate physiologic doses was assessed using the nonsurgical mesenteric-window angiogenesis assay in adult rats. Daily i.p. injections of VEGF165 at 4.8, 48, and 480 pM were given for Days 0-4, controls receiving the vehicle. Groups of 10 animals per treatment group were sacrificed on Days 7, 14, and <em>21</em>. Using microscopic morphometry and image analysis, the vascularized area, a measure of microvascular spatial extension, and the microvascular length, a measure of microvascular density, were measured. At 4.8 and 480 pM, VEGF165 induced significant angiogenesis as early as on Day 7, suggesting a direct angiogenic effect. This very rapid angiogenic response to VEGF165 is distinct from other angiogenesis reactions, including angiogenesis induced by basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, studied using the same methodology. During the early angiogenic phase, the increase in microvascular spatial expansion dominated over the increase in microvascular density. The specific response to VEGF165 peaked on Day 7 for the 4.8 pM dose and on Day 14 for the 480 pM dose, which was the more potent. It is, moreover, noteworthy that no statistically significant response was induced by the intermediate dose of VEGF165 (48 pM) at any observation time. The data thus suggest that, although VEGF165 at near physiologic doses apparently acts as a direct angiogen, its dose-related effect in terms of angiogenesis is nonlinear.

Therapeutic angiogenesis with angiogenic <em>growth</em> <em>factors</em> has been described as a promising approach for tissue engineering, wound healing, and for treating ischemic tissues. Here, we assessed the merit of heparin-entrapped hyaluronic acid-gelatin (HA-G) microspheres for the sustained release of recombinant basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (rbFGF) to promote localized neovascularization. HA-G microspheres were prepared by a water-in-oil emulsion method, and the in vitro release kinetics were first examined using three model proteins. Then, bFGF was incorporated into microspheres, and the bioactivity of the in vitro-released rbFGF was tested on human umbilical vein endothelial cell cultures. The ability to promote microvessel <em>growth</em> was assessed in vivo, at the subcutaneous groin fascia of Wistar rats after 3, 7, 14, and <em>21</em> days. Histological and morphometrical analysis indicated that heparin-entrapped HA-G microspheres have the capacity to release bioactive rbFGF, leading to localized neovascularization in the rat subcutaneous tissue.

Interleukin 2 (IL-2) is a potent <em>growth</em> <em>factor</em> for T lymphocytes, playing a crucial role in the immune response. In view of the considerable evidence that the immunoregulatory cytokines (or lymphokines) also play a role in the <em>growth</em> and differentiation of cells in the central nervous system (CNS), we examined the operation of the IL-2 system in a cell line of CNS origin by expressing a cDNA encoding the beta chain of the human IL-2 receptor (IL-2R beta, a 75-kDa protein). When the cDNA was expressed in a human oligodendroglioma cell line, ONS-<em>21</em>, the IL-2R beta bound IL-2 with an affinity similar to that in lymphoid cells (Kd, approximately 2 nM). Furthermore, cell proliferation ([3H]thymidine incorporation) was stimulated by IL-2. These results demonstrate that the same cytokine receptor is functional in cells of the immune system and CNS and point to a molecular mechanism that is similar for <em>growth</em>-signal transduction between lymphoid and neural cells but that may be different in other cells, such as <em>fibroblasts</em>.

Acute pancreatitis leads to pancreatic damage followed by subsequent regeneration. The aim of our study was to evaluate the presence of growth factors in the course of spontaneous pancreatic regeneration after ischemia/reperfusion (I/R)-induced pancreatitis.

METHODS

In rats, I/R was evoked by clamping of splenic artery for 30 min followed by reperfusion. Rats were sacrificed 1, 5, 12 h or 1, 2, 3, 5, 7, 9 or 21 days after removal of vascular clips. Pancreatic blood flow (PBF), plasma lipase, interleukin-1beta (IL-1beta), interleukin-10, pancreatic cells proliferation and morphological signs of pancreatitis were determined. Pancreatic presence of fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta type II receptor (TGFbeta RII) was detected by immunohistochemisty.

RESULTS

Exposure to I/R led to the development of acute necrotizing pancreatitis followed by regeneration. Morphological features showed maximal pancreatic damage between the 1(st) and 2(nd) day of reperfusion. It was correlated with a maximal increase in plasma lipase, and pro-inflammatory IL-1beta concentration, as well as, a reduction in PBF and pancreatic DNA synthesis. I/R increased FGF-2 content in pancreatic acinar cells between the 12(th) and 24(th) h, and between 5(th) and 9(th) day of reperfusion. At the 2(nd) day the presence of FGF-2 in pancreatic acinar cells was reduced. After I/R PDGF-A appeared in pancreatic vessels from the 12(th) h to 5 (th) day of reperfusion. PDGF-A was not observed in pancreatic acinar cells in the control or in I/R group. In pancreatic ducts, the presence of PDGF-A was reduced between the 1(st) and 3(rd), and between 7(th) and 9(th) day of reperfusion. In acinar cells, VEGF content was increased after I/R at the time between the 1(st) and 24(th) h, and between 3(rd) and 7(th) day of reperfusion. At the 2(nd) day of reperfusion, VEGF was not detected in the pancreatic acinar cells. Moreover, VEGF was found in the inflammatory infiltration, in the tubular complexes between the 2(nd) and 5(th) day, and in granulation tissue at the 9(th) day of reperfusion. In pancreatic acinar cells, I/R caused an increase in TGFbeta RII presence between the 5(th) and 24(th) h, and between 7(th) and 9(th) day of reperfusion. Between the 2(nd) and 5(th) day of reperfusion the acinar presence of TGFbeta RII was reduced. In the pancreatic ducts, the presence of TGFbeta RII was increased after I/R from the 1(st) h to 9(th) day of observation. Four weeks after induction of acute pancreatitis, the pancreatic regeneration was completed and the presence of growth factors tested returned to control value.

CONCLUSIONS

The presence of FGF, VEGF, PDGF-A and TGFbeta RII is modified in the course of I/R-induced acute pancreatitis. Maximal content of FGF, VEGF and TGFbeta RII has been observed in early stage of pancreatic regeneration suggesting the involvement these factors in pancreatic recovery.

The liver plays a pivotal role in the physiological adaptation to fasting and a better understanding of the metabolic adaptive responses may give hints on new therapeutic strategies to control the metabolic diseases. The liver X receptors (LXRs) are well-established regulators of lipid and glucose metabolism. More recently <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has emerged as an important regulator of energy homeostasis. We hypothesized that the LXR transcription <em>factors</em> could influence Fgf<em>21</em> expression, which is induced in response to fasting. Wild-type, LXRα(-/-), and LXRβ(-/-) mice were treated for 3 d with vehicle or the LXR agonist GW3965 and fasted for 12 h prior to the killing of the animals. Interestingly, serum FGF<em>21</em> levels were induced after fasting, but this increase was blunted when the mice were treated with GW3965 independently of genotypes. Compared with wild-type mice, GW3965-treated LXRα(-/-) and LXRβ(-/-) mice showed improved insulin sensitivity and enhanced ketogenic response at fasting. Of note is that during fasting, GW3965 treatment tended to reduce liver triglycerides as opposed to the effect of the agonist in the fed state. The LXR-dependent repression of Fgf<em>21</em> seems to be mainly mediated by the recruitment of LXRβ onto the Fgf<em>21</em> promoter upon GW3965 treatment. This repression by LXRβ occurs through the recruitment and stabilization of the repressor complex composed of retinoid-related orphan receptor-α/Rev-Erbα/histone deacetylase 3 onto the Fgf<em>21</em> promoter. Our data clearly demonstrate that there is a cross talk between the LXR and FGF<em>21</em> signaling pathways in the adaptive response to fasting.

Sprouty1 (Spry1) is a negative regulator of <em>fibroblast</em> <em>growth</em> <em>factor</em> signaling with a potential tumor suppressor function in prostate cancer (PCa). Spry1 is downregulated in human PCa, and Spry1 expression can markedly inhibit PCa proliferation in vitro. We have reported DNA methylation as a mechanism for controlling Spry1 expression. However, promoter methylation does not seem to explain gene silencing in all PCa cases studied to suggest other mechanisms of gene inactivation, such as alterations in trans-acting <em>factors</em> and/or post-transcriptional activity may be responsible for the decreased expression in those cases. Binding sites for Wilm's tumor (WT1) transcription <em>factors</em> EGR1, EGR3 and WTE are highly conserved between the mouse and human Spry1 promoter regions, suggesting an evolutionary conserved mechanism(s) involving WT1 and EGR in Spry1 regulation. Spry1 mRNA contains multiple microRNA (miRNA) binding sites in its 3'UTR region suggesting post-transcriptional control. We demonstrate that Spry1 is a target for miR-<em>21</em>-mediated gene silencing. miRNA-based therapeutic approaches to treat cancer are emerging. Spry1 is highly regulated by miRNAs and could potentially be an excellent candidate for such approaches.

Bone morphogenetic protein family members (BMPs) are essential signaling molecules during limb development and, in this process, <em>fibroblast</em> <em>growth</em> <em>factor</em> family members (FGFs) cooperate with BMPs. FGFs also exert anabolic effects in bone when systemically or locally applied. Thus, it is likely that the cooperation with FGFs also occurs in BMP-induced ectopic bone formation and that the exogenous FGF application would promote this bone formation. In the present study, after subcutaneously implanting recombinant human BMP-2 (rhBMP-2) in rats, we examined the expression of FGF-4 and FGF receptors (FGFRs) mRNAs and the effect of exogenous recombinant human FGF-4 (rhFGF-4) on bone formation. Three days after implantation, the pellets containing rhBMP-2 were surrounded by <em>fibroblast</em>ic mesenchymal cells; on day 7, cartilage tissue appeared; on day 10, hypertrophic chondrocytes and a small amount of mineralized tissue were observed; and, on day 14, the amount of mineralized tissue increased. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that FGF-4 expression appeared at early stages (days 3 and 7) and its expression decreased at later stages (days 10, 14, and <em>21</em>), whereas FGFRs were expressed continuously. In situ hybridization revealed that, on days 3 and 7, FGF-4, and FGFR subtypes 1 and 2 (FGFR-1 and FGFR-2) were expressed in mesenchymal cells and chondrocytes, and in the area of alkaline phosphatase (ALP) expression. On day 10, FGF-4 was not detected, whereas the expression of FGFR-1 and FGFR-2 was detectable in the area of alkaline phosphatase (ALP) expression. Injection of rhFGF-4 on days 2, 3, and 4 enhanced the mineralized tissue formation induced by rhBMP-2; however, neither rhFGF-4 treatment on days 6, 7, and 8 nor rhFGF-4 treatment on days 9, 10, and 11 influenced the amount of rhBMP-2-induced mineralization. Our results indicate that FGF-4 and FGFR signals play important roles during rhBMP-2-induced bone formation. We further suggest that the combination of rhBMP-2 and rhFGF-4 would be useful for bone augmentation.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an effective metabolic regulator of glucose and lipid homeostasis in the context of insulin resistance, glucose intolerance and dyslipidemia in diabetic rodents and monkeys, and peroxisome proliferator-activated receptor α (PPARα) directly induces FGF<em>21</em> expression in the rodent liver. Recent findings suggest that the effects and regulation of FGF<em>21</em> qualitatively differ between rodents and humans. Here, we examined the effects of PPARα and PPARγ agonists on FGF<em>21</em> mRNA expression in the mouse liver and in cultured hepatocytes. Intraperitoneal injection of both bezafibrate and pioglitazone induced FGF<em>21</em> mRNA expression in the mouse liver. Rosiglitazone and pioglitazone as well as bezafibrate significantly induced FGF<em>21</em> mRNA expression in cultured mouse hepatocytes. On the other hand, both rosiglitazone and pioglitazone significantly induced, whereas bezafibrate did not affect FGF<em>21</em> mRNA expression in the human liver carcinoma cell line HepG2. Bezafibrate significantly induced pyruvate dehydrogenase kinase 4 mRNA expression, suggesting that HepG2 cells are sensitive to bezafibrate like the mouse liver. Our findings suggest that PPARγ-activating antidiabetic drugs such as rosiglitazone and pioglitazone induce FGF<em>21</em> expression in mouse and human hepatocytes, and that PPARγ rather than PPARα might play an important role in human FGF<em>21</em> production.

Chronic treatment with <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>) favorably improves obesity and nonalcoholic fatty liver disease (NAFLD) outcomes; however, FGF-<em>21</em> expression is paradoxically elevated in obese conditions. Here, we sought to determine the effects of obesity prevention by daily exercise (EX) vs. caloric restriction (CR) on hepatic FGF-<em>21</em> in the hyperphagic, Otsuka Long-Evans Tokushima Fatty (OLETF) rat. Four-week-old male OLETF rats were randomized into groups (n = 7-8 per group) of ad libitum fed, sedentary (OLETF-SED), voluntary wheel running exercise (OLETF-EX), or CR (OLETF-CR; 70% of SED) until 40 weeks of age. Nonhyperphagic, Long-Evans Tokushima Otsuka (LETO-SED) rats served as controls. Both daily EX and CR prevented obesity and NAFLD development observed in the OLETF-SED animals. This was associated with significantly (p < 0.01) lower serum FGF-<em>21</em> (~80% lower) and hepatic FGF-<em>21</em> mRNA expression (~65% lower) in the OLETF-EX and OLETF-CR rats compared with the OLETF-SED rats. However, hepatic FGF-<em>21</em> protein content was reduced to the greatest extent in the OLETF-EX animals (50% of OLETF-SED) and did not differ between the OLETF-SED and OLETF-CR rats. Hepatic FGF-<em>21</em> signaling mediators - hepatic FGF-<em>21</em> receptor 2 (FGFR2, mRNA expression), hepatic FGF-<em>21</em> receptor substrate 2 (FRS2, protein content), and co-receptor β-Klotho (protein content) - were all elevated (60%-100%, ~40%, and +30%-50%, respectively) in the OLETF-EX and OLETF-CR animals compared with the OLETF-SED animals. Daily exercise and caloric restriction modulate hepatic FGF-<em>21</em> and its primary signaling mediators in the hyperphagic OLETF rat. Enhanced metabolic action of FGF-<em>21</em> may partially explain the benefits of exercise and caloric restriction on NAFLD outcomes.

In <em>21</em> human small cell lung cancer (SCLC) cell lines, we determined the expression of mRNA and secreted protein levels of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The VEGF expression was highly variable between cell lines, with a>> 100-fold variation, under identical in vitro conditions. The bFGF expression in cell lines was generally very low. Nine of the cell lines were further analyzed during <em>growth</em> as solid tumor xenografts in nude mice (in vivo). A more uniform VEGF protein expression was present in vivo. Compared with the variable in vitro expression, VEGF was relatively up-regulated in the tumor lines CPH 54A and CPH 54B and down-regulated in GLC 3. One line, DMS 79, had a high VEGF expression in vivo as well as in vitro. The vessel density was determined by Chalkley point counting on CD31 immunostained cryosections of tumors of each of the nine SCLC lines. We found a strong positive correlation between vessel density and tissue VEGF protein expression (r(s) = 0.75; P = 0.02) and a comparatively strong negative correlation (r(s) = -0.80; P = 0.01) between vessel density and tissue bFGF expression. No significant correlation was present between vessel density and in vitro VEGF expression. We conclude that VEGF and bFGF expression is dependent on microenvironmental conditions, as well as cell line-specific <em>factors</em>, and that a strong positive correlation exists between in vivo VEGF expression and vessel density, whereas high tissue levels of bFGF are not correlated with higher vessel densities in SCLC xenografts.

We raised antiserum to human recombinant basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (rbFGF) in rabbits. With this affinity-purified antiserum, other antisera to rbFGF, and commercial antiserum to bovine pituitary bFGF, we undertook immunocytochemical detection of bFGF in histological sections of rat mammary glands at different developmental stages. In non-<em>growing</em> ducts, anti-bFGF serum stains the basement membrane/myoepithelial cells, whereas in serial sections most of this stain is observed to be associated with anti-laminin-staining basement membranes rather than with anti-callus-keratin-staining myoepithelial cells. The weak staining of the myoepithelial cells is enhanced when NiCl2 is included in the detection system, but little staining for bFGF is observed in the epithelial cells. In <em>growing</em> neonatal ducts from 1-day-old rats, in <em>growing</em> terminal end buds (TEBs) and, to a lesser extent, in <em>growing</em> alveolar buds (ABs) in prepubescent (<em>21</em>-day) and pubescent (50-day) rats, both their inner and outer cells are stained moderately by anti-bFGF sera. In non-<em>growing</em> ducts from rats aged 6 days, in non-<em>growing</em> ABs of rats aged 60 days and more, and in alveoli from pregnant and lactating rats, only the basement membrane/myoepithelial cell area is stained by anti-bFGF sera; the epithelial cells are unstained. Staining of the myoepithelial cells is enhanced by mixtures of rbFGF and anti-bFGF sera in non-<em>growing</em> ducts, but there is little change in the staining of <em>growing</em> TEBs. All staining by anti-bFGF sera is abolished with heparin in the reactions. We suggest that the immunoreactive bFGF is present mainly bound to heparan sulfate glycosaminoglycans in the basement membrane of resting structures, but that immunoreactive bFGF becomes associated with proliferating cells, particularly those intermediate in characteristics between epithelial and myoepithelial cells in <em>growing</em> structures such as TEBs.

The effects of acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (aFGF and bFGF) on the morphology of cultured rat astroblasts and on the expression of glial fibrillary acidic protein (GFAP) were compared. The addition of either aFGF or bFGF affected the morphology of the flat, irregular, polygonal-shaped astroblasts, which formed processes and acquire a fibrous appearance. Appreciable different morphological aspects were observed between aFGF- and bFGF-induced cells, essentially between 11 and 14 days in culture. In the presence of bFGF the astroglial cells were more fibrous with a more compact perikaryon as compared to aFGF treated cells. At the ultrastructural level abundant intermediate filaments were observed in astroglial cells as an effect of aFGF and rare filaments but numerous microtubules were seen in bFGF-treated cells. The immunoreactivity for GFAP increased with time in culture and was much stronger in aFGF-treated cells compared to bFGF-treated cells at day 14. An intense positive staining was observed in the somata of the astroglial cells and their processes in the presence of aFGF, while essentially the processes were stained in the presence of bFGF. After <em>21</em> days in culture GFAP immunoreaction was also found in the perikarya of cells treated with bFGF. These results show that rat astroglial cells respond somewhat differently to aFGF and bFGF.

Nonalcoholic fatty liver disease is a major public health concern in the obese and type 2 diabetic populations. The high-fat lard diet induces obesity and fatty liver in C57BL/6J mice and suppresses expression of the PPAR-target gene, FA elongase 5 (Elovl5). Elovl5 plays a key role in MUFA and PUFA synthesis. Increasing hepatic Elovl5 activity in obese mice lowered hepatic TGs and endoplasmic reticulum stress markers (X-box binding protein 1 and cAMP-dependent transcription <em>factor</em> 6α) and increased TG catabolism and fatty acyl carnitines. Increased hepatic Elovl5 activity did not increase hepatic capacity for β-oxidation. Elovl5 effects on hepatic TG catabolism were linked to increased protein levels of adipocyte TG lipase (ATGL) and comparative gene identification 58 (CGI58). Elevated hepatic Elovl5 activity also induced the expression of some (pyruvate dehydrogenase kinase 4 and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>), but not other cytochrome P450 4A10 (CYP4A10), PPAR-target genes. FA products of Elovl5 activity increased ATGL, but not CGI58, mRNA through PPARβ-dependent mechanisms in human HepG2 cells. Treatment of mouse AML12 hepatocytes with the PPARβ agonist (GW0742) decreased (14)C-18:2,n-6 in TGs but did not affect β-oxidation. These studies establish that Elovl5 activity regulates hepatic levels of FAs controlling PPARβ activity, ATGL expression, and TG catabolism, but not FA oxidation.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) analogs and FGF<em>21</em> receptor agonists (FGF<em>21</em>RAs) that mimic FGF<em>21</em> ligand activity constitute the new "FGF<em>21</em>-class" of anti-obesity and anti-diabetic molecules that improve insulin sensitivity, ameliorate hepatosteatosis and promote weight loss. The metabolic actions of FGF<em>21</em>-class proteins in obese mice are attributed to stimulation of brown fat thermogenesis and increased secretion of adiponectin. The therapeutic utility of this class of molecules is being actively investigated in clinical trials for the treatment of type 2 diabetes and non-alcoholic steatohepatitis (NASH). This review is focused on various FGF<em>21</em>-class molecules, their molecular designs and the preclinical and clinical activities. These molecules include modified FGF<em>21</em> as well as agonistic antibodies against the receptor for FGF<em>21</em>, namely the complex of FGF receptor 1 (FGFR1) and the obligatory coreceptor βKlotho (KLB). In addition, a novel approach to increase endogenous FGF<em>21</em> activity by inhibiting the FGF<em>21</em>-degrading protease <em>fibroblast</em> activation protein (FAP) is discussed.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is positively associated with body mass index, potentially as a compensatory mechanism to mediate obesity related metabolic and inflammatory insult due to chronic low-grade elevations of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis <em>factor</em> alpha (TNF-α). Therefore, FGF<em>21</em> response in obese subjects and the associations with increased pro-inflammatory cytokines, insulin resistance, and energy utilization warrants investigation.

RESULTS

Twenty four untrained subjects (12 obese and 12 normal-weight) performed 30 min of continuous submaximal aerobic exercise. Following exercise, obese subjects exhibited a blunted FGF<em>21</em> response to exercise compared to normal-weight subjects as indicated by area-under-the-curves "with respect to increase" (AUCi) analyses (p = 0.005). Furthermore, while exercise-induced plasma FGF<em>21</em> was not associated with any inflammatory cytokine (IL-6 and TNF-α) response, FGF<em>21</em> AUCi was positively correlated with glucose AUCi (r = 0.495, p = 0.014), total relative energy expenditure (r = 0.562, p = 0.004), and relative maximal oxygen consumption (VO2max; r = 0.646, p = 0.001) in all subjects.

CONCLUSIONS

Impaired cardiorespiratory fitness may influence the sensitivity of FGF<em>21</em> response to acute exercise in obese individuals, potentially contributing to the attenuated metabolic response (e.g., glucose) and total exercise energy expenditure. Therefore, exercise training aimed at improving cardiorespiratory fitness and/or body composition may augment cardioprotective properties against obesity-associated CVD through enhanced FGF<em>21</em> flux.

We have studied insulin-like <em>growth</em> <em>factors</em> (IGFs) and IGF-binding proteins released by human <em>fibroblasts</em>. Conditioned medium was obtained after incubation of 2 X 10(6) cells in 2 ml serum-free medium for 72 h. IGF binding protein was identified in aliquots of conditioned medium at 4 C for 16 h with [125]IGF II after charcoal separation. After gel filtration in neutral phosphate buffer through Sephadex G-150, the binding activity eluted with an apparent size greater than 100,000 daltons. After gel filtration through Bio-Rad P-100 in 1 M acetic acid, binding activity had a molecular size of about 50,000 daltons. When [125I]IGF-II bound to conditioned medium binding protein was cross-linked with disuccimidyl suberate and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex had an estimated molecular size of 67,000 daltons. Competitive binding studies with labeled and unlabeled IGF-I and IGF-II showed that IGF-II was preferentially bound by <em>fibroblast</em> binding protein. The above findings are characteristic of serum binding protein but not shed IGF surface receptors. To eliminate possible interference from binding proteins in the IGF-I RIA and the IGF-II radioreceptor assay, conditioned medium was subjected to acid gel filtration, and the peptide fractions were pooled. We found that conditioned medium of seven <em>fibroblast</em> lines contained 0.20 +/- 0.06 ng/ml IGF-I. After the addition of 20 ng/ml human GH (hGH), the conditioned medium contained 0.48 +/- 0.09 ng/ml. These results are lower than those previously reported. One of the two lines of <em>fibroblasts</em> from patients apparently resistant to GH had a minimal increase in IGF-I in conditioned medium after hGH addition. We were able to detect IGF-II in <em>fibroblast</em> conditioned medium in concentrations of 4.4 to <em>21</em> ng/ml but there was no consistent response to GH either in the normal <em>fibroblast</em> lines or in <em>fibroblasts</em> obtained from children with short stature.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has potent effects on normalizing glucose, lipid, and energy homeostasis, and represents an attractive novel therapy for type 2 diabetes mellitus and obesity. Approaches to improve the pharmacokinetic properties of FGF<em>21</em>, such as conjugation with polyethylene glycol, have been explored for therapeutic development. However, not only is there room for further pharmacokinetic improvements, additional re-engineering approaches to improve the potency and stability of FGF<em>21</em> have not been reported. Here, we describe a novel approach to modify and improve the function of FGF<em>21</em> by altering its C-terminal βKlotho interaction domain.

METHODS

We first identified Avimer proteins that are capable of binding βKlotho. Then we explored replacing the C-terminal βKlotho interaction domain of FGF<em>21</em> with a βKlotho-binding Avimer protein.

RESULTS

Such a βKlotho-binding Avimer protein was able to fully complement the C-terminal domain function of FGF<em>21</em>. The resulting FGF<em>21</em>-Avimer fusion is functionally indistinguishable from wild type FGF<em>21</em>, and more tolerant of C-terminal modification.

CONCLUSIONS

These results demonstrate a viable strategy to modulate the affinity, potency, and engineering of FGF<em>21</em>, paving the way for further improvements of FGF<em>21</em> as a therapeutic.

Antagonizing glucagon action represents an attractive therapeutic option for reducing hepatic glucose production in settings of hyperglycemia where glucagon excess plays a key pathophysiological role. We therefore generated REGN1193, a fully human monoclonal antibody that binds and inhibits glucagon receptor (GCGR) signaling in vitro. REGN1193 administration to diabetic ob/ob and diet-induced obese mice lowered blood glucose to levels observed in GCGR-deficient mice. In diet-induced obese mice, REGN1193 reduced food intake, adipose tissue mass, and body weight. REGN1193 increased circulating levels of glucagon and glucagon-like peptide 1 and was associated with reversible expansion of pancreatic α-cell area. Hyperglucagonemia and α-cell hyperplasia was observed in <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>-deficient mice treated with REGN1193. Single administration of REGN1193 to diabetic cynomolgus monkeys normalized fasting blood glucose and glucose tolerance and increased circulating levels of glucagon and amino acids. Finally, administration of REGN1193 for 8 weeks to normoglycemic cynomolgus monkeys did not cause hypoglycemia or increase pancreatic α-cell area. In summary, the GCGR-blocking antibody REGN1193 normalizes blood glucose in diabetic mice and monkeys but does not produce hypoglycemia in normoglycemic monkeys. Thus, REGN1193 provides a potential therapeutic modality for diabetes mellitus and acute hyperglycemic conditions.

Cellular oncogenes appear to be involved in the control of normal cell <em>growth</em> and differentiation. The abnormal activation of these genes in naturally occurring and experimentally induced cancers may have an important role in the expression of the malignant phenotype in cancer cells. Mechanisms for the activation of these genes include chromosomal translocation, point mutation, and DNA amplification. The amplification of specific oncogenes correlates with clinical prognosis in several human malignancies, including breast cancer and neuroblastoma. We examined <em>21</em> fresh-frozen human squamous cell carcinomas of the aerodigestive tract for amplification of 10 known cellular oncogenes (c-myc, N-myc, L-myc, N-ras, H-ras, K-ras, erb-B, erb-B2, raf, and int-2), using Southern blotting techniques. Eleven of <em>21</em> tumors demonstrated a two-fold to 11-fold amplification of the int-2 oncogene, one member of a family of genes related to basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Amplification of c-myc, a gene that codes for a DNA-binding protein involved in the regulation of cell <em>growth</em>, was seen in two tumors. None of the other eight genes studied were amplified in any of the tumor specimens.

We used fetal bovine serum (FBS) and different <em>growth</em> <em>factors</em> to investigate their potential for inducing chondrogenic differentiation of synovium-derived stromal cells. Human synovium was harvested from patients suffering from osteoarthritis and expanded in monolayer. To evaluate the effect of serum and <em>growth</em> <em>factors</em> on chondrogenic differentiation, 10 ng/mL of transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1), 100 ng/mL of bone morphogenic protein-2 (BMP-2), 100 ng/mL of insulin-like <em>growth</em> <em>factor</em>-1, 20 ng/mL of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and 10% FBS were added to the chemically defined chondrogenic medium singly or in combination during pellet culture for <em>21</em> days. The cell size and weight, glycosaminoglycan content, histology, and cartilage matrix-associated genes expression were analyzed. TGF-beta1 alone and TGF-beta1 + BMP-2 induced chondrogenic differentiation of synovium-derived stromal cells and synthesized cartilage-like matrix confirmed by histological analysis and immunohistochemistry. FBS, BMP-2, insulin-like <em>growth</em> <em>factor</em>-1, and bFGF as a single <em>factor</em> or other combinations except for TGF-beta1 + BMP-2 hardly induced chondrogenesis. Chondrogenic differentiation appeared to be inhibited when bFGF or the serum was added to the chondrogenic medium during pellet culture. The results of this study demonstrate the negative or positive role of serum and <em>growth</em> <em>factors</em> on chondrogenic differentiation of synovium-derived stromal cells.

Intracerebral neural xenografts elicit a host immune response that results in their rapid rejection. This forms a key barrier to the therapeutic use of xenogeneic tissue transplantation for conditions such as Parkinson's disease. The current study sought to provide insight into the cellular components of donor cell suspensions that are important in stimulating the host rejection response and thereby to suggest rational manipulations of xenogeneic donor tissue that might ultimately enhance its clinical utility. The neural stem cell mitogens, epidermal <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, have been used to isolate and expand populations of primordial neural precursor cells from the embryonic pig brain. The immune response elicited by these cells on transplantation into the non-immunosuppressed rat has been fully characterised. In the first experiments, expanded neural precursors were grafted into the hemi-parkinsonian, non-immunosuppressed Sprague-Dawley rat and graft status and host response examined 10, <em>21</em>, 35 and 60 days post-transplantation. While equivalent primary tissue grafts were completely eliminated at 35 days, grafts of expanded neural precursors with healthy neurofilament-positive projections were present at all time-points, and two large grafts remained even at 60 days. Some grafts appeared to elicit minimal host immune responses at the time-points they were examined, although most did appear to be undergoing a rejection process since a co-ordinated response involving host cytotoxic T-lymphocytes, microglia/macrophages, immunoglobulin M and complement could be demonstrated to varying degrees. Subsequent experiments went on to demonstrate further that expanded precursor populations and primary tissue suspensions differed in their immunogenic profile. Firstly, when primary tissue was injected intraperitoneally into immunocompetent rats a vigorous primary humoral response was generated. No such response was detected following injection of expanded neural precursors. Secondly, flow cytometric analysis revealed small but significant levels of class II porcine major histocompatibility complex expression in primary cell suspensions but no such expression in expanded precursor populations.The results of this study therefore demonstrate that the immunogenicity of porcine neural cell suspensions used for intracerebral grafting is reduced when neural stem cell mitogens are used to expand precursor cells. The implications of these findings in the development of novel xenogeneic cellular therapies for neurodegenerative conditions such as Parkinson's disease are discussed.

BACKGROUND

In this Phase 4 international study, efficacy and safety of paricalcitol-centred therapy were compared with that of cinacalcet-centred therapy for the treatment of chronic kidney disease (CKD)-associated secondary hyperparathyroidism (SHPT) in patients undergoing haemodialysis (ClinicalTrials.gov identifier NCT00977080).

METHODS

Patients ≥ 18 years of age with Stage 5 CKD and SHPT [intact parathyroid hormone (iPTH) level of 300-800 pg/mL, calcium level of 8.4-10.0 mg/dL and phosphate concentration of ≤ 6.5 mg/dL] who were undergoing haemodialysis were included. Patients were randomized by mode of paricalcitol administration [i.e. intravenous (IV) or oral strata] to receive paricalcitol- or cinacalcet-centred therapy for ≤ 28 weeks. Changes in metabolic markers [total alkaline phosphatase (AP), bone-specific AP and <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23)] and the proportion of patients in each treatment group who achieved an iPTH level of 150-300 pg/mL during Weeks 8, 16 and <em>21</em>-28 as a composite value were evaluated.

RESULTS

Compared with cinacalcet-centred therapy, levels of both bone turnover markers were significantly reduced from baseline with IV and oral paricalcitol-centred treatment (P < 0.05 for both dosing strata) at Weeks 8, 16 and 28. Levels of FGF-23 were increased with paricalcitol versus cinacalcet-centred treatment. A greater proportion of patients receiving paricalcitol-centred therapy achieved target iPTH levels (i.e. 150-300 pg/mL) throughout the study in the IV and oral dosing strata compared with patients receiving cinacalcet-centred treatment.

CONCLUSIONS

In patients with CKD and SHPT undergoing haemodialysis, paricalcitol-centred therapy reduced circulating bone turnover markers and iPTH levels and increased FGF-23 levels compared with cinacalcet-centred treatment.

BACKGROUND

ClinicalTrials.gov identifier NCT00977080.

BACKGROUND

Thalidomide has been shown to have antiangiogenic effects in preclinical models as well as a significant antitumor effect in hematologic tumors such as multiple myeloma. The authors performed this Phase II study to determine the activity, toxicity profile, and antiangiogenic effect of thalidomide in patients with locoregionally recurrent or metastatic squamous cell carcinoma of the head and neck.

METHODS

Twenty-one patients with recurrent or metastatic squamous cell carcinoma of the head and neck were treated with single-agent thalidomide. All patients had received radiation therapy, and most had undergone surgery (95%) and/or chemotherapy (90%). Thalidomide was initiated at 200 mg;3>daily and increased to a target dose of 1000 mg daily. Patients continued treatment until disease progression, unacceptable toxicity, or death occurred.

RESULTS

All <em>21</em> patients eventually developed progressive disease. Median time to progression was 50 days (95% confidence interval, 28-70), with median overall survival time of 194 days (95% lower confidence boundary, 151), similar to the progression and survival times reported for this patient group with other agents. Thalidomide was generally well tolerated, with few patients experiencing Grades 3 to 4 toxicities. Serum vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> levels increased in six of seven patients, for whom paired serum samples were available and all of whom had progressive disease.

CONCLUSIONS

In this heavily pretreated population of patients with advanced squamous cell carcinoma of the head and neck, thalidomide does not appear to have single-agent antitumor activity. Further evaluation of the mechanism of action of thalidomide is indicated. Potentially, future evaluations of thalidomide may be performed in combination with other antiangiogenic or cytotoxic agents in patients with earlier stage disease or in patients with minimal residual disease.

OBJECTIVE

This study examined the bioactivity of porcine small intestinal submucosa (SIS Wound Matrix [SISWM], USP) and oxidized regenerated cellulose/collagen (ORC).

METHODS

Bioactivity was assessed in vitro as the ability to stimulate neurite out<em>growth</em> in rat pheochromocytoma (PC12) cells, proliferation of human <em>fibroblasts</em>, secretion of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) from human <em>fibroblasts</em>, and in an in vivo angiogenesis model. In the angiogenesis model, SISWM and ORC were implanted subcutaneously into the mice, and vessel in<em>growth</em> was assessed at day <em>21</em> after implantation using fluorescence microangiography and histology.

METHODS

The change in cellular differentiation, proliferation, growth factor secretion, and angiogenesis over the negative control was measured after exposure to SISWM or ORC.

RESULTS

SISWM increased neurite out<em>growth</em> in PC12 cells by approximately 22% over negative controls and induced proliferation in 50.8% of human <em>fibroblasts</em>. These increases were comparable to positive controls. ORC was not active in either of these assays. SISWM also stimulated fibroblast VEGF secretion to a greater extent (422.4 pg/mL) than ORC (4.2 pg/mL) (P < .001). At <em>21</em> days, fluorescence microangiography showed dense infiltration of blood vessels in the SISWM that extended approximately 3 mm from the edge of the disc. In contrast, the ORC implant showed blood vessel incursion less than 1 mm from the edge of the disc, and it dissolved in the site.

CONCLUSIONS

SISWM shows much greater bioactivity than ORC. This is likely related to its close structural and biochemical approximation to natural dermal extracellular matrix and may help explain the strong clinical successes of SISWM.