BACKGROUND

Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling.

METHODS

Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters.

RESULTS

A median 15% (0-48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-beta1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-beta1 stimulation.

CONCLUSIONS

This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.

OBJECTIVE

To determine whether fibroblast growth factor-2 (FGF-2) treatment of equine mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chondrogenesis in a 3-dimensional culture system.

METHODS

6 healthy horses, 6 months to 5 years of age.

METHODS

Bone marrow-derived MSCs were obtained from 6 horses. First-passage MSCs were seeded as monolayers at 10,000 cells/cm(2) and in medium containing 0, 1, 10, or 100 ng of FGF-2/mL. After 6 days, MSCs were transferred to pellet cultures (200,000 cells/pellet) and maintained in chondrogenic medium. Pellets were collected after 15 days. Pellets were analyzed for collagen type II content by use of an ELISA, total glycosaminoglycan content by use of the dimethylmethylene blue dye-binding assay, and DNA content by use of fluorometric quantification. Semiquantitative PCR assay was performed to assess relative concentrations of collagen type II and aggrecan mRNAs.

RESULTS

Use of 100 ng of FGF-2/mL significantly increased pellet DNA and glycosaminoglycan content. Collagen type II content of the pellet was also increased by use of 10 and 100 ng of FGF-2/mL. Collagen type II and aggrecan mRNA transcripts were increased by treatment with FGF-2. Some control samples had minimal evidence of collagen type II and aggrecan transcripts after 35 cycles of amplification.

CONCLUSIONS

FGF-2 treatment of bone marrow-derived MSC monolayers enhanced subsequent chondrogenic differentiation in a 3-dimensional culture. This result is important for tissue engineering strategies dependent on MSC expansion for cartilage repair.

Evidence in animal models indicates that signaling networks functioning in the developing pharyngeal arches regulate stereotyped processes critical for proper development of the aortic arch and cardiac outflow tract. Here, we describe the phenotype of mice lacking <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (Fgf<em>15</em>), which encodes a secreted signaling molecule expressed within the developing pharyngeal arches. Homozygous Fgf<em>15</em> mutants present heart defects consistent with malalignment of the aorta and pulmonary trunk. These defects correlate with early morphological defects of the outflow tract due to aberrant behavior of the cardiac neural crest. We demonstrate that Fgf<em>15</em> expression within the pharyngeal arches is unaltered by a loss of Tbx1, a key regulator of pharyngeal arch development implicated in DiGeorge syndrome. In addition, Fgf<em>15</em> and Tbx1 do not interact genetically, suggesting that Fgf<em>15</em> operates through a pathway independent of Tbx1. These studies reveal a novel role of Fgf<em>15</em> during development of the cardiac outflow tract.

In the present study, the ability of tumor necrosis <em>factor</em>-alpha (TNF) to stimulate hexose transport in quiescent 3T3-L1 <em>fibroblasts</em> has been examined. Activation of transport occurred in a dose- and time-dependent manner, with maximal stimulation (6-8-fold) observed 16 h after exposure to 2.5 nM TNF. Early activation of hexose transport by TNF (2-fold within 30 min) was associated with increased plasma membrane immunoreactive glucose transporters. Prolonged exposure to TNF (16 h) resulted in a 2-fold increase in glucose transporter content of both plasma and inner membrane compartments. The magnitude of increased glucose transport (6-8-fold) was greater than the increased content of plasma membrane glucose transporters (2-fold), suggesting that the TNF-treatment altered the intrinsic activity of the glucose transporters. Increased transcription of the glucose transporter (GLUT-1) gene, as well as several immediate-early genes (c-fos, c-jun, jun-B, and beta-actin) was observed within <em>15</em> min of exposure to TNF. Transcriptional activation of immediate-early genes was tightly coupled to subsequent accumulation of their respective mRNAs. However, increased GLUT-1 mRNA (8 h after TNF treatment) was due to an apparent 3-fold increase in the stability of this message and not to increased transcription. The time course of TNF-induced hexose transport occurred concomitant with a 6-fold increase in total RNA synthesis which preceded a 3-fold increase in protein synthesis. Moreover, TNF induced cell-cycle progression through S-phase, as measured by aphidicolin-sensitive thymidine uptake. Phorbol myristate acetate also stimulated hexose transport as well as expression of the GLUT-1 gene and several immediate-early genes in quiescent 3T3-L1 cells. TNF-induced immediate-early gene expression was intact in PMA-pretreated cells (with the exception of GLUT-1 and beta-actin genes where the response was muted), suggesting the involvement of multiple pathways in TNF signal transduction. Our results indicate that TNF initiates mitogenic events in quiescent 3T3-L1 <em>fibroblasts</em> reminiscent of serum-derived <em>growth</em> <em>factors</em>.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) interacts with a dual receptor system consisting of tyrosine kinase receptors and heparan sulfate proteoglycans (HSPGs). In rat mammary <em>fibroblasts</em>, FGF-2 stimulated DNA synthesis and induced a sustained phosphorylation of p42/44(MAPK) and of its downstream target, p90(RSK). Moreover, FGF-2 also stimulated the transient degradation of IkappaBalpha and IkappaBbeta. PD098059, a specific inhibitor of p42/44(MAPK) phosphorylation, inhibited FGF-2-stimulated DNA synthesis, phosphorylation of p42/44(MAPK) and p90(RSK), and degradation of IkappaBbeta. In contrast, in chlorate-treated and hence sulfated glycosaminoglycan-deficient cells, FGF-2 was unable to stimulate DNA synthesis. However, FGF-2 was able to trigger a transient phosphorylation of both p42/44(MAPK) and p90(RSK), which peaked at <em>15</em> min and returned to control levels at 30 min. In these sulfated glycosaminoglycan-deficient cells, no degradation of IkappaBalpha and IkappaBbeta was observed after FGF-2 addition. However, in chlorate-treated cells, the addition of heparin or purified HSPGs simultaneously with FGF-2 restored DNA synthesis, the sustained phosphorylation of p42/44(MAPK) and p90(RSK), and the degradation of IkappaBalpha and IkappaBbeta. These results suggest that the HSPG receptor for FGF-2 not only influences the outcome of FGF-2 signaling, e.g. cell proliferation, but importantly regulates the immediate-early signals generated by this <em>growth</em> <em>factor</em>.

BACKGROUND

Tumor angiogenesis, or new blood vessel formation, is regulated by a balance between pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and anti-angiogenic factors such as endostatin.

METHODS

To investigate this angiogenic balance in soft tissue sarcomas (STS), blood samples were collected from 76 STS patients and 15 healthy controls, and analyzed for VEGF, bFGF and endostatin using quantitative enzyme-linked immunosorbent assays (ELISA).

RESULTS

Forty-one patients (54%) had primary tumors, 20 (26%) had local recurrences and 15 (20%) had metastatic disease with or without local disease. Levels of all three angiogenic factors were highly variable in STS patients. Mean levels of VEGF and bFGF were 12 and 14 times higher, respectively, in patients compared with controls (P<0.0001). VEGF levels correlated with size of tumor, with the highest levels found in tumors >10 cm in size. Patients with metastases had endostatin levels 45% lower than patients without metastases (P=0.047). In 54 patients who underwent resection of primary disease or local recurrence, low pre-operative bFGF level was associated with a higher risk of subsequent recurrence (P=0.044).

CONCLUSIONS

STS secrete widely variable levels of angiogenic factors, and levels of specific factors may correlate with extent of disease, predict risk of recurrence and possibly guide the use of anti-angiogenic agents.

The proposal that epidermal <em>growth</em> <em>factor</em> (EGF) activates phospholipase D (PLD) by a mechanism(s) not involving phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis was examined in Swiss 3T3 <em>fibroblasts</em>. EGF, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), bombesin, and platelet-derived <em>growth</em> <em>factor</em> (PDGF) activated PLD as measured by transphosphatidylation of butanol to phosphatidylbutanol. The increase in inositol phosphates induced by bFGF, EGF, or bombesin was significantly enhanced by Ro-31-8220, an inhibitor of protein kinase C (PKC), suggesting that PtdIns(4,5)P2-hydrolyzing phospholipase is coupled to the receptors for these agonists but that the response is down-regulated by PKC. Activation of PLD by EGF was inhibited dose dependently by the PKC inhibitors bis-indolylmaleimide and Ro-31-8220, which also inhibited the effects of bFGF, bombesin, and PDGF. Down-regulation of PKC by prolonged treatment with 4 beta-phorbol 12-myristate 13-acetate also abolished EGF- and PDGF-stimulated phosphatidylbutanol formation. EGF and bombesin induced biphasic translocations of PKC delta and epsilon to the membrane that were detectable at <em>15</em> s. In the presence of Ro-31-8220, translocation of PKC alpha became evident, and membrane association of the delta- and epsilon-isozymes was enhanced and/or sustained in response to the two agonists. The inhibitor also enhanced EGF-stimulated [3H]diacylglycerol formation in cells preincubated with [3H]arachidonic acid, which labeled predominantly phosphatidylinositol, but inhibited [3H]diacylglycerol production in cells preincubated with [3H]myristic acid, which labeled mainly phosphatidylcholine. These data support the conclusion that EGF can stimulate diacylglycerol formation from PtdIns(4,5)P2 and that PKC performs the dual role of down-regulating this response as well as mediating phosphatidylcholine hydrolysis. In summary, all of the results of the study indicate that PLD activation by EGF is downstream of PtdIns(4,5)P2-hydrolyzing phospholipase and is dependent upon subsequent PKC activation.

Liver fibrogenesis is sustained by pro-fibrogenic myofibroblast-like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)<em>fibroblasts</em>, and is favoured by hypoxia-dependent angiogenesis. Human HSC/MFs were reported to express vascular-endothelial <em>growth</em> <em>factor</em> (VEGF) and VEGF-receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia-induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non-oriented and oriented migration were evaluated by using wound healing assay and the modified Boyden's chamber assay, respectively. The data indicate that hypoxia-induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (<em>15</em> min) and mitochondria-related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF-1α-dependent increase in VEGF expression (facilitated by ROS) and its progressive, time-dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV-related fibrotic/cirrhotic livers revealed HIF-2α and haem-oxygenase-1 positivity in hepatocytes and α-SMA-positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia-induced migration of HSC/MFs involves an early, mitochondrial-dependent ROS-mediated activation of ERK and JNK, followed by a delayed- and HIF-1α-dependent up-regulation and release of VEGF.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding <em>growth</em> <em>factor</em> 1 (HBGF-1) was studied by using the murine <em>fibroblast</em> cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr <em>15</em>0,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-<em>15</em>0,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-<em>15</em>0,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after <em>15</em> to 30 min. Phosphoamino acid analysis of the Mr-<em>15</em>0,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-<em>15</em>0,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-<em>15</em>0,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and <em>15</em>0,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.

The aim of this paper was to determine if <em>growth</em> <em>factors</em>, known to be upregulated in proliferative diabetic retinopathy, exerted combined effects on retinal endothelial cells. The authors explored the individual and collective actions of insulin-like <em>growth</em> <em>factor</em> I (IGF-I), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), platelet-derived <em>growth</em> <em>factor</em>-BB (PDGF-BB), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) and placenta <em>growth</em> <em>factor</em> (PlGF) on several parameters that reflect the angiogenic potential of endothelial cells. The effect of <em>growth</em> <em>factors</em> on cell migration and survival/proliferation was examined using primary cultures of bovine retinal endothelial cells (BREC). The authors also determined the <em>growth</em> <em>factor</em> action on capillary-like tube formation on a reconstituted basement membrane matrix and on the newly described phenomenon of secondary sprouting, in which endothelial cell colonies spontaneously survive, proliferate, migrate and invade the matrix after the original capillary-like tubes have collapsed. Sprouting cells were positive for von Willebrand <em>factor</em> and could aggregate into larger tubes with lumens. Incubation with VEGF+IGF-I or PlGF+FGF-2 enhanced tube stability by 40-50%, more than each <em>growth</em> <em>factor</em> alone or other combinations (5-20%). The concurrent addition of four <em>growth</em> <em>factors</em> did not improve the response seen with <em>growth</em> <em>factor</em> pairs. Surprisingly, PDGF-BB induced tube collapse. IGF-I and FGF-2 mildly enhanced BREC proliferation/survival (5-<em>15</em>%). However, VEGF+IGF-I or PlGF+FGF-2 increased BREC proliferation/survival by 25% under low serum conditions, whereas combinations of all four <em>growth</em> <em>factors</em> exerted a clearly synergistic effect (250% increase). PDGF-BB or FGF-2 stimulated secondary sprouting and were the only <em>factors</em> capable of exerting this effect alone. Even though VEGF, IGF-I or PlGF were not effective, if administered in pairs, they demonstrated increased responses. PDGF-BB was also able to enhance the effect of FGF-2+IGF-I+VEGF on BREC secondary sprouting, but not of any of them individually. No other <em>growth</em> <em>factor</em> tested was able to significantly improve the action of combinations of three other <em>growth</em> <em>factors</em>. VEGF increased cell migration in a wounded monolayer assay two-fold and PDGF-BB, 2.5 times, but other individual <em>growth</em> <em>factors</em> were ineffective. PlGF+FGF-2 enhanced cell migration more than each <em>factor</em> alone. VEGF+IGF-I+PlGF+FGF-2, however, increased cell migration four-fold. In summary, this study indicates that <em>growth</em> <em>factors</em>, overexpressed in diabetic retinopathy eyes, enhance the angiogenic characteristics of cultured cells (tube formation, proliferation, secondary sprouting and migration). Their effects, however, can be greatly augmented by other <em>growth</em> <em>factors</em> that alone exert little or no action. Therefore, diabetic retinal neovascularization may result from the additive or synergistic action of several <em>growth</em> <em>factors</em>.

BACKGROUND

Low vitamin D concentrations are prevalent in patients with chronic kidney disease (CKD). We investigated the relationship between plasma 25-hydroxyvitamin D (25[OH]D) or 1,25-dihydroxyvitamin D (1,25[OH](2)D) concentrations with death, cardiovascular events, and dialysis therapy initiation in patients with advanced CKD.

METHODS

The HOST (Homocysteinemia in Kidney and End Stage Renal Disease) Study was a randomized double-blind trial evaluating the effects of high doses of folic acid on death and long-term dialysis therapy initiation in patients with advanced CKD (stages 4 and 5 not yet on dialysis therapy). 25(OH)D and 1,25(OH)(2)D were measured in stored plasma samples obtained 3 months after trial initiation and evaluated at clinically defined cutoffs (<10, 10-30, and >30 ng/mL) and tertiles ((<em>15</em>, <em>15</em>-22, and >22 pg/mL), respectively. Cox proportional hazard models were used to examine the association between vitamin D concentrations and clinical outcomes.

METHODS

1,099 patients with advanced CKD from 36 Veteran Affairs Medical Centers.

METHODS

25(OH)D and 1,25(OH)(2)D concentrations.

RESULTS

Death, cardiovascular events, and time to initiation of long-term dialysis therapy.

RESULTS

After a median follow-up of 2.9 years, 41% (n = 453) died, whereas 56% (n = 6<em>15</em>) initiated dialysis therapy. Mean 25(OH)D and 1,25(OH)(2)D concentrations were 21 ± 10 ng/mL and 20 ± 11 pg/mL, respectively. After adjustment for potential confounders, the lowest tertile of 1,25(OH)(2)D was associated with death (HR, 1.33; 95% CI, 1.01-1.74) and initiation of long-term dialysis therapy (HR, 1.78; 95% CI, 1.40-2.26) compared with the highest tertile. The association with death and initiation of dialysis therapy was moderately attenuated after adjustment for plasma fibroblast growth factor 23 (FGF-23) concentrations (HRs of lower tertiles of 1.20 [95% CI, 0.91-1.58] and 1.56 [95% CI, 1.23-1.99], respectively, compared with highest tertile). There was no association between 25(OH)D concentrations and outcomes.

CONCLUSIONS

Participants were mostly men.

CONCLUSIONS

Low plasma 1,25(OH)(2)D concentrations are associated with death and initiation of long-term dialysis therapy in patients with advanced CKD. FGF-23 level may attentuate this relationship.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FGF<em>15</em>), FGF19 in humans, is a gut-derived hormone and a key regulator of bile acids and carbohydrate metabolism. FGF<em>15</em> also participates in liver regeneration after partial hepatectomy inducing hepatocellular proliferation. FGF19 is overexpressed in a significant proportion of human hepatocellular carcinomas (HCC), and activation of its receptor FGFR4 promotes HCC cell <em>growth</em>. Here we addressed for the first time the role of endogenous Fgf<em>15</em> in hepatocarcinogenesis. Fgf<em>15</em>(+/+) and Fgf<em>15</em>(-/-) mice were subjected to a clinically relevant model of liver inflammation and fibrosis-associated carcinogenesis. Fgf<em>15</em>(-/-) mice showed less and smaller tumors, and histological neoplastic lesions were also smaller than in Fgf<em>15</em>(+/+) animals. Importantly, ileal Fgf<em>15</em> mRNA expression was enhanced in mice undergoing carcinogenesis, but at variance with human HCC it was not detected in liver or HCC tissues, while circulating FGF<em>15</em> protein was clearly upregulated. Hepatocellular proliferation was also reduced in Fgf<em>15</em>(-/-) mice, which also expressed lower levels of the HCC marker alpha-fetoprotein (AFP). Interestingly, lack of FGF<em>15</em> resulted in attenuated fibrogenesis. However, in vitro experiments showed that liver fibrogenic stellate cells were not direct targets for FGF<em>15</em>/FGF19. Conversely we demonstrate that FGF<em>15</em>/FGF19 induces the expression of the pro-fibrogenic and pro-tumorigenic connective tissue <em>growth</em> <em>factor</em> (CTGF) in hepatocytes. These findings suggest the existence of an FGF<em>15</em>-triggered CTGF-mediated paracrine action on stellate cells, and an amplification mechanism for the hepatocarcinogenic effects of FGF<em>15</em> via CTGF production. In summary, our observations indicate that ileal FGF<em>15</em> may contribute to HCC development in a context of chronic liver injury and fibrosis.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about <em>15</em>-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.

OBJECTIVE

This study investigated fibroblast growth factor 21 (FGF21)-mediated cardiac protection against apoptosis caused by diabetic lipotoxicity and explored the protective mechanisms involved.

METHODS

Cardiac Fgf21 mRNA expression was examined in a diabetic mouse model using real-time PCR. After pre-incubation of palmitate-treated cardiac H9c2 cells and primary cardiomyocytes with FGF21 for 15 h, apoptosis and Fgf21-induced cell-survival signalling were investigated using small interfering (si)RNA and/or pharmacological inhibitors. We also examined the cardiac apoptotic signalling and structural and functional indices in wild-type and Fgf21-knockout (Fgf21-KO) diabetic mice.

RESULTS

In a mouse model of type 1 diabetes, cardiac Fgf21 expression was upregulated about 40-fold at 2 months and 3-1.5-fold at 4 and 6 months after diabetes. FGF21 significantly reduced palmitate-induced cardiac apoptosis. Mechanistically, palmitate downregulated, but FGF21 upregulated, phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2, mitogen-activated protein kinase 14 (p38 MAPK) and AMP-activated protein kinase (AMPK). Inhibition of each kinase with its inhibitor and/or siRNA revealed that FGF21 prevents palmitate-induced cardiac apoptosis via upregulating the ERK1/2-dependent p38 MAPK-AMPK signalling pathway. In vivo administration of FGF21, but not FGF21 plus ERK1/2 inhibitor, to diabetic or fatty-acid-infused mice significantly prevented cardiac apoptosis and reduced inactivation of ERK1/2, p38 MAPK and AMPK and prevented cardiac remodelling and dysfunction. The Fgf21-KO mice were more susceptible to diabetes-induced cardiac apoptosis, and this could be prevented by administration of FGF21. Deletion of Fgf21 did not further exacerbate cardiac dysfunction.

CONCLUSIONS

These results demonstrate that FGF21 prevents lipid- or diabetes-induced cardiac apoptosis by activating the ERK1/2-p38 MAPK-AMPK pathway. FGF21 may be a therapeutic target for the treatment of diabetes-related cardiac damage.

Pancreatic cancer is often associated with an intense production of interstitial collagens, known as the desmoplastic reaction. To understand more about desmoplasia in pancreatic cancer, the expression of mRNA for type I and III collagens and potent desmoplastic inducing <em>growth</em> <em>factors</em> transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), connective tissue <em>growth</em> <em>factor</em> (CTGF), acidic and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF) A and C and epidermal <em>growth</em> <em>factor</em> (EGF) was analysed by quantitative RT-PCR. Expression of both collagens in 23 frozen primary pancreatic cancer nodules was significantly higher than that in <em>15</em> non-neoplastic pancreatic tissues. The expressions of mRNAs for TGF-beta, acidic FGF, basic FGF and PDGF C were likewise higher in surgical cancer nodules, while that of CTGF, PDGF A and EGF were not. Among these <em>growth</em> <em>factors</em>, the expression of TGF-beta mRNA showed the most significant correlation with that of collagens (P<0.0001). By immunohistochemistry, TGF-beta showed faint cytoplasmic staining in cancer cells. In contrast, isolated cells, mainly located on the invasive front surrounding cancerous nests, were prominently and strongly stained. These TGF-beta-positive cells contained a segmented nucleus, were negative for anti-macrophage (CD68) and positive for anti-granulocyte antibodies, indicating their granulocytic nature. In conclusion, TGF-beta seemed to play a major role among the various <em>growth</em> <em>factors</em> in characteristic overproduction of collagens in pancreatic cancer. Moreover, the predominant cells that express TGF-beta were likely to be infiltrated granulocytes (mostly are neutrophils) and not pancreatic cancer cells.

Mesenchymal stem cells (MSCs) that overexpress secreted frizzled-related protein 2 (sFRP2) exhibit an enhanced reparative phenotype. The secretomes of sFRP2-overexpressing MSCs and vector control-MSCs were compared through liquid chromatography tandem mass spectrometry. Proteomic profiling revealed that connective tissue <em>growth</em> <em>factor</em> (CTGF; CCN2) was overrepresented in the conditioned media of sFRP2-overexpressing MSCs and MSC-derived CTGF could thus be an important paracrine effector. Subcutaneously implanted, MSC-loaded polyvinyl alcohol (PVA) sponges and stented excisional wounds were used as wound models to study the dynamics of CTGF expression. Granulation tissue generated within the sponges and full-thickness skin wounds showed transient upregulation of CTGF expression by MSCs and <em>fibroblasts</em>, implying a role for this molecule in early tissue repair. Although collagen and COL1A2 mRNA were not increased when recombinant CTGF was administered to sponges during the early phase (day 1-6) of tissue repair, prolonged administration >><em>15</em> days) of exogenous CTGF into PVA sponges resulted in <em>fibroblast</em> proliferation and increased deposition of collagen within the experimental granulation tissue. In support of its physiological role, CTGF immunoinhibition during early repair (days 0-7) reduced the quantity, organizational quality and vascularity of experimental granulation tissue in the sponge model. However, CTGF haploinsufficiency was not enough to reduce collagen deposition in excisional wounds. Similar to acute murine wound models, CTGF was transiently present in the early phase of human acute burn wound healing. Together, these results further support a physiological role for CTGF in wound repair and demonstrate that when CTGF expression is confined to early tissue repair, it serves a pro-reparative role. These data also further illustrate the potential of MSC-derived paracrine modulators to enhance tissue repair.

BACKGROUND

Hypertrophic scars and keloids respond to dermal disruption with excessive collagen deposition and increased transforming growth factor (TFG)-beta expression. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta activity that is associated with scar and fibrosis. The authors hypothesize that there is increased expression of CTGF by hypertrophic scar and keloid fibroblasts in response to TGF-beta stimulation.

METHODS

Primary fibroblasts were isolated in culture from human hypertrophic scar (n = 2), keloid (n = 2), and normal skin (n = 2). After 18 hours of serum starvation, the cells were stimulated with 10 ng/ml of TGF-beta1, TGF-beta2, and TGF-beta3 for 24 hours. Quantitative real-time polymerase chain reaction was performed on extracted RNA samples to assay for CTGF mRNA expression.

RESULTS

Baseline CTGF expression was increased 20-fold in unstimulated hypertrophic scar fibroblasts and 15-fold in keloid fibroblasts compared with normal fibroblasts. CTGF expression increased greater than 150-fold when stimulated with TGF-beta1 (p < 0.002) and greater than 100-fold when stimulated by TGF-beta2 or TGF-beta3 compared with normal fibroblasts (p < 0.02 and p < 0.002, respectively). CTGF expression was greatest after TGF-beta1 stimulation in hypertrophic scar fibroblasts compared with TGF-beta2 (p < 0.04) and TGF-beta3 (p < 0.02). Keloid fibroblast CTGF expression also increased greater than 100-fold after stimulation with TGF-beta1 (p = 0.16) and greater than 75-fold after addition of TGF-beta2 and TGF-beta3 (p = 0.06 and p = 0.22, respectively).

CONCLUSIONS

Hypertrophic scar fibroblasts have both intrinsic up-regulation of CTGF transcription and an exaggerated capacity for CTGF transcription in response to TGF-beta stimulation. These data suggest that blockage of CTGF activity may reduce pathologic scar formation.

Motoneuron death can occur over several spinal levels with disease or trauma, resulting in muscle denervation. We tested whether cotransplantation of embryonic neurons with 1 or more neurotrophic <em>factors</em> into peripheral nerve improved axon regeneration, muscle fiber area, reinnervation, and function to a greater degree than cell transplantation alone. Sciatic nerves of adult Fischer rats were cut to denervate muscles; 1 week later, embryonic ventral spinal cord cells (days 14-<em>15</em>) were transplanted into the tibial nerve stump as the only source of neurons for muscle reinnervation. <em>Factors</em> that promote motoneuron survival (cardiotrophin 1; <em>fibroblast</em> <em>growth</em> <em>factor</em> 2; glial cell line-derived neurotrophic <em>factor</em>; insulin-like <em>growth</em> <em>factor</em> 1; leukemia inhibitory <em>factor</em>; and hepatocyte <em>growth</em> <em>factor</em>) were added to the transplant individually or in combinations. Inclusion of a single <em>factor</em> with the cells resulted in comparable myelinated axon counts, muscle fiber areas, and evoked electromyographic activity to cells alone 10 weeks after transplantation. Only cell transplantation with glial cell line-derived neurotrophic <em>factor</em>, hepatocyte <em>growth</em> <em>factor</em>, and insulin-like <em>growth</em> <em>factor</em> 1 significantly increased motoneuron survival, myelinated axon counts, muscle reinnervation, and evoked electromyographic activity compared with cells alone. Thus, immediate application of a specific combination of <em>factors</em> to dissociated embryonic neurons improves survival of motoneurons and the long-term function of reinnervated muscle.

BACKGROUND

Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been used in preclinical studies to induce new blood vessel growth in ischemic cardiac muscle with promising results. However, clinical trials have been much less convincing and further work is needed. This study expands on prior work by comparing the long-term proangiogenic effects of direct intramyocardial (IM) injection of bFGF, as well as IM and intravenous (IV) VEGF in a porcine model of chronic hibernating myocardium.

METHODS

Mini-swine with proximal 90% left circumflex (LCx) coronary stenosis subtending chronically ischemic, viable (hibernating) myocardium by positron emission tomography (PET) and dobutamine stress echocardiography (DSE) were randomized to IM bFGF (n = 5), IM VEGF(165) (n = 5), IV VEGF(165) (n = 5), IM vehicle (n = 5), or sham redo-thoracotomy (n = 4). The bFGF protein was administered in a total dose of 1.35 microg divided into 30 IM injections. IM VEGF(165) protein was administered in a total dose of 15 microg/kg divided into 30 injections; IV VEGF(165) was given at a dose of 50 ng. kg(-1). min(-1) for 200 minutes at three 72-hour intervals (30 microg/kg total dose). After 3 and 6 months the PET and DSE studies were repeated, and the animals were sacrificed for tissue vascular density and angiogenic protein analysis.

RESULTS

Myocardial blood flow (MBF) by PET was significantly improved 3 months posttreatment in the IM bFGF and IM VEGF(165) groups, differences that were sustained at 6 months. There was no significant increase in MBF 3-months posttreatment in the IV VEGF(165) group; however, at 6 months MBF was significantly improved. No change in MBF was seen in the IM vehicle or sham groups. Regional wall motion at rest and peak stress in the LCx region demonstrated small but statistically significant improvements by 6 months in the IM bFGF and IV VEGF(165) groups only; no improvement was seen in the IM VEGF(165), IM vehicle, or sham groups. Quantitative vascular density was significantly increased in the LCx regions of all treatment groups (IM bFGF, IM VEGF(165), IV VEGF(165)) 6-months postoperatively. No significant increase in LCx region myocardial bFGF or VEGF protein levels was seen in the treated animals at 6 months.

CONCLUSIONS

The IM bFGF, IM VEGF(165), and IV VEGF(165) all improve regional perfusion and vascular density 6-months posttherapy in the animal model utilized. Functional improvements were less consistent. Both bFGF and VEGF(165) may be useful therapies for improving regional perfusion in chronically ischemic myocardium, although combination therapy with additional growth factors or cellular therapies may be necessary if concomitant improvements in function are to be seen.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) are angiogenic molecules whose combined mitogenic activity is potently synergistic. However, the molecular mechanism underlying this synergy is incompletely understood. We examined whether VEGF and bFGF affect expression of each other or alter expression of the VEGF receptor KDR in retinal capillary endothelial cells. In addition, we investigated the intracellular signaling mechanisms involved in this response. VEGF-induced [3H]thymidine uptake was tightly correlated with KDR mRNA and protein concentrations, suggesting that increased KDR expression might account for VEGF's synergistic activity in the presence of bFGF. bFGF (10 ng/ml) induced KDR mRNA expression within 4 h and attained a 4.0-fold increase after 24 h. KDR protein expression was increased 7.5-fold after 48 h. VEGF (= 50 ng/ml) did not alter bFGF, VEGF, or KDR mRNA expression under serum-deprived conditions. In contrast, VEGF increased KDR mRNA expression 87% under <em>growth</em> conditions and 2.9-fold under serum-deprived conditions in the presence of bFGF. The protein kinase C (PKC) agonist phorbol myristate acetate (PMA) induced KDR mRNA expression 5.1-fold at 100 nmol/l. bFGF increased p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation within 5 min, reaching a maximum within <em>15</em> min and remaining significantly elevated for >6 h. bFGF-induced MAPK phosphorylation and KDR mRNA expression were almost completely inhibited by 5 micromol/l GFX, a non-isoform-selective PKC inhibitor. MAPK inhibitor PD98059 reduced KDR mRNA expression 72% at concentrations that inhibited bFGF-induced MAPK phosphorylation 100%, suggesting that pathways in addition to MAPK might also be involved. Inhibitors of the beta isoform of PKC (LY333531), protein kinase A (PKA) (H89), and phosphotidylinositol (PI) 3 kinase (wortmannin) had no significant effect. These data suggest that bFGF stimulates KDR expression through a PKC and p44/p42 MAPK-dependent pathway not primarily involving the beta isoform of PKC, PKA, or PI-3 kinase. Since bFGF induces VEGF expression and since increased KDR expression potentiates VEGF action, resulting in additional KDR expression and marked mitogenic activity, these data provide a novel mechanistic explanation for the angiogenic synergy between VEGF and bFGF.

Chronic lymphocytic leukemia (CLL) is characterized by delayed senescence and slow accumulation of monoclonal, small lymphocytes. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a pleiotropic cytokine that plays a role in hematopoiesis and apoptosis. Elevated bFGF levels have been detected in urine from patients with a variety of neoplastic diseases including various leukemias; however, the cellular source of the bFGF has not been determined. In this study, the intracellular bFGF level in lymphocytes of 36 patients with B-CLL and <em>15</em> normal donors was determined using an enzyme-linked immunoassay. In cells derived from patients with high-risk disease, the median level of intracellular bFGF was 381.5 pg/2 x 10(5) cells, compared with a median of 90.5 pg/2 x 10(5) cells in patients with intermediate disease. In patients with low-risk disease, the median bFGF level was 4.9 pg/2 x 10(5) cells, and in normal controls, it was 6.0 pg/2 x 10(5) cells. The difference in the bFGF levels was significant for the comparison between low- and intermediate-risk (P = .00119), low- and high-risk (P < .0001), and intermediate- and high-risk disease (P = .0001). Immunofluorescent stains of peripheral blood mononuclear cells confirmed CLL lymphocytes as a cellular source of bFGF. To evaluate the potential contribution of elevated intracellular bFGF levels to the phenotype of CLL cells, leukemic cells were cultured in vitro with an apoptotic stimulus (fludarabine). CLL cells with high intracellular levels of bFGF appeared to be more resistant to fludarabine treatment. The addition of bFGF to fludarabine-treated CLL cells resulted in a delay of apoptosis and prolonged survival. These data suggest that bFGF may contribute to the resistance of CLL cells to an apoptotic stimulus.

This chapter will review the various biological actions of the mouse <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (Fgf<em>15</em>) and human <em>fibroblast</em> <em>growth</em> <em>factor</em> 19 (FGF19). Unlike other members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family, the Fgf<em>15</em> and FGF19 orthologs do not share a high degree of sequence identity. Fgf<em>15</em> and FGF19 are members of an atypical subfamily of FGFs that function as hormones. Due to subtle changes in tertiary structure, these FGFs have low heparin binding affinity enabling them to diffuse away from their site of secretion and signal to distant cells. FGF signaling through the FGF receptors is also different for this sub-family, requiring klotho protein co<em>factor</em>s rather than heparin sulfate proteoglycan. Mouse Fgf<em>15</em> and human FGF19 play key roles in enterohepatic signaling, regulation of liver bile acid biosynthesis, gallbladder motility and metabolic homeostasis.

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-<em>15</em>) tumor <em>growth</em> in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor <em>growth</em>. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.

Sex myoblast migration in the Caenorhabditis elegans hermaphrodite represents a simple, genetically amenable model system for studying how cell migration is regulated during development. Two separable components of sex myoblast guidance have been described: a gonad-independent mechanism sufficient for the initial anterior migration to the mid-body region, and a gonad-dependent mechanism required for precise final positioning (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041-1052). Here, we demonstrate a role for a Ras-mediated signal transduction pathway in controlling sex myoblast migration. Loss-of-function mutations in let-60 ras, ksr-1, lin-45 raf, let-537/mek-2 or sur-1/mpk-1 cause defects in sex myoblast final positions that resemble those seen in gonad-ablated animals, while constitutively active let-60 ras(G13E) trans-genes allow fairly precise positioning to occur in the absence of the gonad. A mosaic analysis demonstrated that let-60 ras is required within the sex myoblasts to control proper positioning. Our results suggest that gonadal signals normally stimulate let-60 ras activity in the sex myoblasts, thereby making them competent to sense or respond to positional cues that determine the precise endpoint of migration. let-60 ras may have additional roles in sex myoblast guidance as well. Finally, we have also investigated genetic interactions between let-60 ras and other genes important for sex myoblast migration, including egl-<em>15</em>, which encodes a <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor tyrosine kinase (D. L. DeVore, H. R. Horvitz and M. J. Stern (1995) Cell 83, 611-623). Since mutations reducing Ras pathway activity cause a different phenotype than those reducing egl-<em>15</em> activity and since constitutive Ras activity only partially suppresses the migration defects of egl-<em>15</em> mutants, we argue that let-60 ras and egl-<em>15</em> do not act together in a single linear pathway.