Cell transplantation using bone marrow stromal cells (BMSCs) to alleviate neurological deficits has recently become the focus of research in regenerative medicine. Evidence suggests that secretion of various <em>growth</em>-promoting substances likely plays an important role in functional recovery against neurological diseases. In an attempt to identify a possible mechanism underlying the regenerative potential of BMSCs, this study investigated the production and possible contribution of neurotrophic <em>factors</em> by transected sciatic nerve defect in a rat model with a <em>15</em> mm gap. Cultured BMSCs became morphologically homogeneous with <em>fibroblast</em>-like shape after ex vivo expansion. We provided several pieces of evidence for the beneficial effects of implanted <em>fibroblast</em>-like BMSCs on sciatic nerve regeneration. When compared to silicone tube control animals, this treatment led to (i) improved walking behavior as measured by footprint analysis, (ii) reduced loss of gastrocnemius muscle weight and EMG magnitude, and (iii) greater number of regenerating axons within the tube. Cultured <em>fibroblast</em>-like BMSCs constitutively expressed trophic <em>factors</em> and supporting substances, including nerve <em>growth</em> <em>factor</em> (NGF), brain-derived neurotrophic <em>factor</em> (BDNF), glial cell line-derived neurotrophic <em>factor</em> (GDNF), ciliary neurotrophic <em>factor</em> (CNTF), collagen, fibronectin, and laminin. The progression of the regenerative process after BMSC implantation was accompanied by elevated expression of neurotrophic <em>factors</em> at both early and later phases. These results taken together, in addition to documented Schwann cell-like differentiation, provide evidence indicating the strong association of neurotrophic <em>factor</em> production and the regenerative potential of implanted BMSCs.

Regulatory T cells (Tregs) are crucial for the induction and maintenance of self-tolerance and are present in peripheral tissues such as skin and gut under normal, noninflamed conditions. We report isolation and expansion of the Treg population resident in normal human skin. Cutaneous Tregs expressed high levels of CD25, L-selectin, GITR, FOXP3, and intracellular CTLA-4, low levels of CD69, and high levels of the skin-homing addressins CLA, CCR4, and CCR6. Skin Tregs suppressed the proliferation of CD25(lo) T cells from the same skin sample in response to CD3 and CD28 antibodies. Suppression was dependent on cell contact and not affected by neutralizing antibodies to interleukin-10 (IL-10) and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). Surprisingly, cutaneous Tregs proliferated in an antigen-independent manner when cultured in contact with dermal <em>fibroblasts</em> and IL-<em>15</em>, conditions similar to those found in chronically inflamed skin. We hypothesize that local proliferation of Tregs may occur within inflamed skin and could serve as a brake for cutaneous inflammation as well as a mechanism for the homeostatic proliferation of natural Tregs that has been observed within intact organisms.

The effects of thyroid hormone analogues on modulation of angiogenesis have been studied in the chick chorioallantoic membrane model. Generation of new blood vessels from existing vessels was increased 3-fold by either l-thyroxine (T4; 10(-7) mol/L) or 3,5,3'-triiodo-l-thyronine (10(-9) mol/L). T4-agarose reproduced the effects of T4, and tetraiodothyroacetic acid (tetrac) inhibited the effects of both T4 and T4-agarose. Tetrac itself was inactive and is known to block actions of T4 on signal transduction that are initiated at the plasma membrane. T4 and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) were comparably effective as inducers of angiogenesis. Low concentrations of FGF2 combined with submaximal concentrations of T4 produced an additive angiogenic response. Anti-FGF2 inhibited the angiogenic effect of T4. The proangiogenic effects of T4 and FGF2 were blocked by PD 98059, a mitogen-activated protein kinase (MAPK) pathway inhibitor. Endothelial cells (ECV304) treated with T4 or FGF2 for <em>15</em> minutes demonstrated activation of MAPK, an effect inhibited by PD 98059 and the protein kinase C inhibitor CGP41251. Reverse transcription-polymerase chain reaction of RNA extracted from endothelial cells treated with T4 revealed increased abundance of FGF2 transcript at 6 to 48 hours, and after 72 hours, the medium of treated cells showed increased FGF2 content, an effect inhibited by PD 98059. Thus, thyroid hormone is shown to be a proangiogenic <em>factor</em>. This action, initiated at the plasma membrane, is MAPK dependent and mediated by FGF2.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF) stimulates the incorporation of 32P from [gamma-32P]ATP into a Mr approximately 170,000 protein by an endogenous tyrosine-specific protein kinase in membrane preparations of Swiss mouse 3T3 cells. Epidermal <em>growth</em> <em>factor</em> (EGF), but not <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) or insulin, stimulates limited incorporation of 32P into a protein of similar molecular weight. The ligand concentration required for half-maximal activity (S0.5) for PDGF stimulation of phosphorylation is 50 ng/ml; saturation is achieved at 300 ng/ml. The S0.5 for ATP is <em>15</em> microM. Mg2+ or Mn2+ is required for protein kinase activity. Stimulation of PDGF results in the preferential phosphorylation of tyrosine residues in this Mr approximately 170,000 membrane protein. The Mr approximately 170,000 protein can be resolved into Mr approximately 180,000 and 160,000 components in 4% NaDodSO4 gels. PDGF stimulates 32P incorporation preferentially into the Mr approximately 180,000 and less extensively into the Mr approximately 160,000 protein. EGF stimulates 32P incorporation predominantly into a protein of Mr approximately 160,000. The similarity of PDGF and EGF in stimulating phosphotyrosine-specific protein kinase activity and the stimulation of a similar activity by viral transformation (src) genes suggest that a common mechanism may exist for the phenotypic expression of increased DNA synthesis and cell <em>growth</em> stimulated by these separate <em>factors</em>.

There is much evidence that rheumatoid arthritis is closely linked to angiogenesis. Important angiogenic mediators have been demonstrated in synovium and tenosynovium of rheumatoid joints. VEGF (Vascular Endothelial <em>Growth</em> <em>Factor</em>), expressed in response to soluble mediators such as cytokines and <em>growth</em> <em>factors</em> and its receptors are the best characterized system in the angiogenesis regulation of rheumatoid joints. Moreover, other angiogenic mediators such as platelet-derived <em>growth</em> <em>factor</em> (PDGF), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), epidermal <em>growth</em> <em>factor</em> (EGF), insulin-like <em>growth</em> <em>factor</em> (IGF), hepatocyte <em>growth</em> <em>factor</em> (HGF), transforming <em>growth</em> <em>factor</em> beta (TGF-beta), tumor necrosis <em>factor</em> alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, IL-8, IL-13, IL-<em>15</em>, IL-18, angiogenin, platelet activating <em>factor</em> (PAF), angiopoietin, soluble adhesion molecules, endothelial mediator (endoglin) play an important role in angiogenesis in rheumatoid arthritis. On the other hand, endostatin, thrombospondin-1 and -2 are angiogenic inhibitors in rheumatoid arthritis. The persistence of inflammation in rheumatoid joints is a consequence of an imbalance between these inducers and inhibitors of angiogenesis.

Gut microbiota influences host health status by providing trophic, protective, and metabolic functions, including bile acid (BA) biotransformation. Microbial imprinting on BA signature modifies pool size and hydrophobicity, thus contributing to BA enterohepatic circulation. Microbiota-targeted therapies are now emerging as effective strategies for preventing and/or treating gut-related diseases. Here, we show that gut microbiota modulation induced by VSL#3 probiotics enhances BA deconjugation and fecal excretion in mice. These events are associated with changes in ileal BA absorption, repression of the enterohepatic farnesoid X receptor-<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FXR-FGF<em>15</em>) axis, and increased hepatic BA neosynthesis. Treatment with a FXR agonist normalized fecal BA levels in probiotic-administered mice, whereas probiotic-induced alterations in BA metabolism are abolished upon FXR and FGF<em>15</em> deficiency. Our data provide clear in vivo evidence that VSL#3 probiotics promote ileal BA deconjugation with subsequent fecal BA excretion and induce hepatic BA neosynthesis via downregulation of the gut-liver FXR-FGF<em>15</em> axis.

In vivo tumor cells interact with a variety of host cells such as endothelial cells and platelets, and these interactions are mediated by integrins GPIIb/IIIa and alphavbeta3. We used chimeric (c) 7E3 Fab (ReoPro) and murine (m) 7E3 F(ab')(2) to elucidate the role of these integrins in angiogenesis, tumor <em>growth</em>, and metastasis. These antibodies are potent inhibitors of GPIIb/IIIa and alphavbeta3. c7E3 Fab inhibited alphavbeta3-mediated human umbilical vein endothelial (HUVEC) and melanoma cell adhesion, migration, invasion, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> stimulated proliferation of HUVECs (IC(50) values range from 0.<em>15</em> to 5 microg/ml for different assays). In an in vitro angiogenesis assay, c7E3 Fab inhibited basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and platelet-stimulated capillary formation of HUVECs (IC(50) = 10 microg/ml and <em>15</em> microg/ml, respectively), demonstrating that endothelial alphavbeta3 is important for sprouting, and platelet-stimulated sprouting is mediated by GPIIb/IIIa. In an experimental metastasis assay, a single pretreatment of human melanoma cells with c7E3 Fab (2.5 microg/ml) inhibited lung colonization of the tumor cells in severe combined immunodeficient mice. In vivo, m7E3 F(ab')(2) partially inhibited <em>growth</em> of human melanoma tumors in nude mice compared with control-treated animals. These data suggest that tumor cell-expressed integrins are important but not the only component involved in tumor <em>growth</em>. Because c7E3 Fab and m7E3 F(ab')(2) do not cross-react with murine integrins, this inhibition of metastasis and tumor <em>growth</em> is attributable to direct blockade of human tumor alphavbeta3 integrins. m7E3 F(ab')(2) completely blocked tumor formation and <em>growth</em> of human melanoma tumors <em>growing</em> in nude rats. In this xenograft model, m7E3 F(ab')(2) simultaneously binds to both human tumor and host platelet GPIIb/IIIa and endothelial alphavbeta3 integrins, thus participating as an antiangiogenic and an antitumor agent. Collectively, these results indicate that combined blockade of GPIIb/IIIa and alphavbeta3 affords significant antiangiogenic and antitumor benefit.

We report on the clinical and molecular findings in 25 males and three females with Kallmann syndrome (KS) aged 10-53 yr. Ten males were from five families, and the remaining <em>15</em> males and three females were apparently sporadic cases. Molecular studies were performed for Kallmann syndrome 1 (KAL1) and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1, also known as KAL2) by sequence analysis for all the coding exons, by PCR-based deletion analysis, and by fluorescence in situ hybridization (FISH) analysis, showing six novel and two recurrent intragenic KAL1 mutations in seven familial and four sporadic male cases and two novel intragenic FGFR1 mutations in two sporadic male cases. In addition, submicroscopic deletions at Xp22.3 involving VCX-A, STS, KAL1, and OA1 were identified in three familial cases and one sporadic male case affected by a contiguous gene syndrome. Clinical assessment in the <em>15</em> males with KAL1 mutations showed normal and borderline ol<em>factor</em>y function in two males and right-side dominant renal lesion in seven males, in addition to variable degrees of hypogonadotropic hypogonadism (HH) in all the <em>15</em> males and ol<em>factor</em>y dysfunction in 13 males. The two males with FGFR1 mutations had HH and anosmia and lacked other features. Clinical features in the remaining 11 cases with no demonstrable KAL1 or FGFR1 mutations included right renal aplasia in one female, cleft palate in one male, cleft palate and perceptive deafness in one male, and dental agenesis and perceptive deafness in one male, in addition to a variable extent of HH and ol<em>factor</em>y dysfunction. The results suggest the following: 1) KAL1 mutations might be more prevalent in the Japanese patients than previously estimated in the Caucasian patients and can be associated with apparently normal ol<em>factor</em>y function; 2) FGFR1 mutations account for approximately 10% of KS patients, as previously reported in the Caucasian patients, and can result in HH and ol<em>factor</em>y dysfunction-only phenotype; and 3) renal aplasia, which is characteristic of KAL1 mutations, and cleft palate and dental agenesis, which are characteristic of FGFR1 mutations, can occur in patients without KAL1 and FGFR1 mutations.

The sex myoblasts (SMs) in C. elegans hermaphrodites undergo anteriorly directed cell migrations that allow for the proper localization of the egg-laying muscles. These migrations are controlled in part by a signal emanating from gonadal cells that allows the SMs to be attracted to their precise final positions flanking the center of the gonad. Mutations in egl-<em>15</em> alter the nature of the interaction between the gonad and the SMs, resulting in the posterior displacement of the SMs. Here we show that egl-<em>15</em> encodes a receptor tyrosine kinase of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) subfamily with multiple roles in development. Three genes were identified that behave genetically as activators or mediators of egl-<em>15</em> activity. One of these genes, sem-5, encodes an adaptor molecule that transduces signals from a variety of receptor tyrosine kinases. Like egl-<em>15</em> and sem-5, the other two genes may similarly act in FGFR signaling pathways in C. elegans.

Autosomal dominant polycystic liver disease (ADPLD) is a distinct clinical and genetic entity that can occur independently from autosomal dominant polycystic kidney disease (ADPKD). We previously studied two large kindreds and reported localization of a gene for ADPLD to an approximately 8-Mb region, flanked by markers D19S586/D19S583 and D19S593/D19S579, on chromosome 19p13.2-13.1. Expansion of these kindreds and identification of an additional family allowed us to define flanking markers CA267 and CA048 in an approximately 3-Mb region containing >70 candidate genes. We used a combination of denaturing high-performance liquid chromatography (DHPLC) heteroduplex analysis and direct sequencing to screen a panel of <em>15</em> unrelated affected individuals for mutations in genes from this interval. We found sequence variations in a known gene, PRKCSH, that were not observed in control individuals, that segregated with the disease haplotype, and that were predicted to be chain-terminating mutations. In contrast to PKD1, PKD2, and PKHD1, PRKCSH encodes a previously described human protein termed "protein kinase C substrate 80K-H" or "noncatalytic beta-subunit of glucosidase II." This protein is highly conserved, is expressed in all tissues tested, and contains a leader sequence, an LDLa domain, two EF-hand domains, and a conserved C-terminal HDEL sequence. Its function may be dependent on calcium binding, and its putative actions include the regulation of N-glycosylation of proteins and signal transduction via <em>fibroblast</em> <em>growth</em>-<em>factor</em> receptor. In light of the focal nature of liver cysts in ADPLD, the apparent loss-of-function mutations in PRKCSH, and the two-hit mechanism operational in dominant polycystic kidney disease, ADPLD may also occur by a two-hit mechanism.

Proteoglycans have been shown in vitro to bind multiple components of the cellular microenvironment that function during wound healing. To study the composition and function of these molecules when derived from an in vivo source, soluble proteoglycans released into human wound fluid were characterized and evaluated for influence on <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 activity. Immunoblot analysis of wound fluid revealed the presence of syndecan-1, syndecan-4, glypican, decorin, perlecan, and versican. Sulfated glycosaminoglycan concentrations ranged from <em>15</em> to 65 microgram/ml, and treatment with chondroitinase B showed that a large proportion of the glycosaminoglycan was dermatan sulfate. The total glycosaminoglycan mixture present in wound fluid supported the ability of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 to signal cell proliferation. Dermatan sulfate, and not heparan sulfate, was the major contributor to this activity, and dermatan sulfate bound FGF-2 with Kd = 2.48 microM. These data demonstrate that proteoglycans released during wound repair are functionally active and provide the first evidence that dermatan sulfate is a potent mediator of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 responsiveness.

OBJECTIVE

Cholestasis is a liver disorder characterized by impaired bile flow, reduction of bile acids (BAs) in the intestine, and retention of BAs in the liver. The farnesoid X receptor (FXR) is the transcriptional regulator of BA homeostasis. Activation of FXR by BAs reduces circulating BA levels in a feedback mechanism, repressing hepatic cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme for the conversion of cholesterol to BAs. This mechanism involves the hepatic nuclear receptor small heterodimer partner and the intestinal <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 19 and <em>15</em>. We investigated the role of activation of intestine-specific FXR in reducing hepatic levels of BAs and protecting the liver from cholestasis in mice.

METHODS

We generated transgenic mice that express a constitutively active FXR in the intestine. Using FXR gain- and loss-of-function models, we studied the roles of intestinal FXR in mice with intrahepatic and extrahepatic cholestasis.

RESULTS

Selective activation of intestinal FXR induced FGF<em>15</em> and repressed hepatic Cyp7a1, reducing the pool size of BAs and changing the BA pool composition. Activation of intestinal FXR protected mice from obstructive extrahepatic cholestasis after bile duct ligation or administration of α-naphthylisothiocyanate. In Mdr2(-/-) mice, transgenic expression of activated FXR in the intestine protected against liver damage, whereas absence of FXR promoted progression of liver disease.

CONCLUSIONS

Activation of FXR transcription in the intestine protects the liver from cholestasis in mice by inducing FGF<em>15</em> expression and reducing the hepatic pool of BA; this approach might be developed to reverse cholestasis in patients.

Members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibres; a low dose of FGF can stimulate cell proliferation (but not fibre differentiation), whereas higher doses of FGF are required to induce fibre differentiation. To determine if these cellular events are regulated by the same signalling pathways, we examined the role of mitogen-activated protein kinase (MAPK) signalling in FGF-induced lens cell proliferation and differentiation. We show that FGF induced a dose-dependent activation of extracellular regulated kinase 1/2 (ERK1/2) as early as <em>15</em> minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fibre differentiation. Interestingly, inhibition of ERK signalling can block the morphological changes associated with FGF-induced lens fibre differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, beta-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, our data indicate that different levels of ERK signalling may be important for the regulation of lens cell proliferation and early morphological events associated with fibre differentiation; however, multiple signalling pathways are likely to be required for the process of lens fibre differentiation and maturation.

BACKGROUND

The efficacy of adult stem cells is known to be compromised as a function of age. This therefore raises questions about the effectiveness of autologous cell therapy in elderly patients.

RESULTS

We demonstrated that the expression profile of stemness markers was altered in BM-MSCs derived from old rats. BM-MSCs from young rats (4 months) expressed Oct-4, Sox-2 and NANOG, but we failed to detect Sox-2 and NANOG in BM-MSCs from older animals (<em>15</em> months). Chondrogenic, osteogenic and adipogenic potential is compromised in old BM-MSCs. Stimulation with a cocktail mixture of bone morphogenetic protein (BMP-2), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) and insulin-like <em>growth</em> <em>factor</em> (IGF-1) induced cardiomyogenesis in young BM-MSCs but not old BM-MSCs. Significant differences in the expression of gap junction protein connexin-43 were observed between young and old BM-MSCs. Young and old BM-MSCs fused with neonatal ventricular cardiomyocytes in co-culture and expressed key cardiac transcription <em>factors</em> and structural proteins. Cells from old animals expressed significantly lower levels of VEGF, IGF, EGF, and G-CSF. Significantly higher levels of DNA double strand break marker γ-H2AX and diminished levels of telomerase activity were observed in old BM-MSCs.

CONCLUSIONS

The results suggest age related differences in the differentiation capacity of BM-MSCs. These changes may affect the efficacy of BM-MSCs for use in stem cell therapy.

Cardiac <em>fibroblasts</em> are responsible for synthesis and deposition of fibrillar collagen types I and III. Transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) has been proved to increase collagen biosynthesis in various systems, both in vivo and in vitro. We have investigated the effect of TGF-beta 1 on collagen gene expression in cultured cardiac <em>fibroblasts</em> and have compared this effect with that of a mitogenic agent, phorbol myristate acetate (PMA). The regulation of collagen types I and III gene expression was examined by using cDNA probes to rat alpha 2 (I) and mouse alpha 1 (III) procollagens. Quiescent cultured cardiac <em>fibroblasts</em> from rabbit heart were treated with TGF-beta 1 (10-<em>15</em> ng/ml) and PMA (200 ng/ml). After 24 hours of treatment with TGF-beta 1, the abundance of mRNA for pro-alpha 2 (I) and pro-alpha 1 (III) collagens was increased by 112% (p less than 0.001) and 97% (p = 0.05), respectively, in treated <em>fibroblasts</em> compared with untreated cells. However, PMA-treated cells showed an opposite response: a 42% (p = 0.01) decrease in mRNA levels for pro-alpha 2 (I) collagen was observed. Immunofluorescent staining of cardiac <em>fibroblasts</em> in culture with anti-type I collagen antibody showed that alterations in mRNA levels led to altered collagen synthesis: cellular collagen was relatively increased in TGF-beta 1-treated cells and significantly diminished in PMA-treated cells. The abundance of mRNA for pro-alpha 1 (III) collagen was not affected by PMA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

METHODS

We collected the specimens of lumbar intervertebral disc (i.e., the symptomatic degenerative disc) from patients with discogenic low back pain to study the histopathologic features and growth factor expressions.

OBJECTIVE

To study the pathogenesis of disc degeneration, meanwhile discriminating between common disc degeneration (aging disc) (i.e., black asymptomatic disc, not clinically relevant) and painful disc degeneration (i.e., symptomatic disc, clinically relevant).

BACKGROUND

The pathogenesis of intervertebral disc degeneration is poorly understood, mainly because of the difficulty to establish the experimental model with good reproducibility. Recently, the popularity of spinal fusion leads to more opportunities to obtain disc specimens, which could be applied to explore the pathogenesis of disc degeneration with modern biologic techniques.

METHODS

There were 21 specimens of lumbar intervertebral discs from 15 patients with discogenic low back pain during posterior lumbar interbody fusion, 16 aging discs from patients without low back pain, and 10 normal discs as control collected for the study of their histopathologic features, as well as the expressions of basic fibroblast growth factor (bFGF) and its receptor (Flg), transforming growth factor-beta1 (TGF-beta1) and its receptor (TGF-betaRI) by immunohistochemistry. The distribution of macrophages and mast cells was also noted. Proliferating cell nuclear antigen was assessed to evaluate proliferating activities of disc cells.

RESULTS

The distinct histologic characteristic of the disc from the patient with discogenic low back pain was the ingrowth of vascularized granulation tissue along torn fissures, extending from the external layer of the anulus fibrosus into the nucleus pulposus. The immunohistochemical staining showed that there were strong expressions of bFGF and TGF-beta1 and their receptors, as well as a strong expression of proliferating cell nuclear antigen in the zones of granulation tissue in the painful discs. However, there were only weak expressions in the nongranulation tissue zones in the painful discs and aging discs, and no expression in the control discs. In addition, abundant macrophages and mast cells were found in the granulation tissue zones of painful discs but absent in the nongranulation tissue zones of painful discs or aging discs and the normal control discs.

CONCLUSIONS

The findings indicated that degeneration of the painful disc might originate from the injury and subsequent repair of anulus fibrosus. Growth factors, such as bFGF and TGF-beta1, macrophages and mast cells might play a key role in the repair of the injured anulus fibrosus and subsequent disc degeneration.

We have examined the kinetics of 1,2-diacylglycerol production in quiescent IIC9 <em>fibroblasts</em>. alpha-Thrombin and epidermal <em>growth</em> <em>factor</em> (EGF) both stimulate an increase in the mass of cellular 1,2-diacylglycerol. The generation of 1,2-diacylglycerol is biphasic when stimulated by a high concentration of alpha-thrombin (500 ng/ml), with an early phase peaking at <em>15</em> s and a late phase peaking at 5 min. Production of 1,2-diacylglycerol is monophasic when stimulated by: (a) a low concentration of alpha-thrombin (100 pg/ml); (b) a high concentration of alpha-thrombin added to cultures which had been pretreated with chymotrypsin; or (c) EGF. In all cases the stimulation of 1,2-diacylglycerol was sustained for at least 30 min. In a previous report (Raben, D. M., Yasuda, K., and Cunningham, D. D. (1987) Biochemistry 26, 2759-2765), it was demonstrated that alpha-thrombin stimulates lipid metabolism in <em>fibroblasts</em> via two coupling mechanisms designated R1 and R2. We now present evidence that the early phase of alpha-thrombin-stimulated 1,2-diacylglycerol production is related to R1, which is characterized by: 1) increased release of arachidonic acid, 2) hydrolysis of polyphosphoinositides, and 3) inhibition by pretreating cultures with chymotrypsin. The late phase is related to R2 which is characterized by 1,2-diacylglycerol production in the absence of stimulated phosphoinositide hydrolysis and arachidonic acid release. In addition, EGF activates an R2-like mechanism in that it does not stimulate the release of arachidonic acid or hydrolysis of polyphosphoinositides but does stimulate a 2-fold increase in 1,2-diacylglycerol mass.

OBJECTIVE

Cholestasis is associated with increased liver injury and morbidity after partial hepatectomy (PH), yet bile acids (BAs) are emerging as important mediators of liver regeneration. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (Fgf<em>15</em>, human FGF19) is a BA-induced ileum-derived enterokine that governs BA metabolism. We evaluated the relevance of Fgf<em>15</em> in the preservation of BA homeostasis after PH and its potential role in the regenerative process.

METHODS

Liver regeneration after PH was studied in Fgf<em>15</em> (-/-) and Fgf<em>15</em> (+/+) mice. The effects of the BA sequestrant cholestyramine and adenovirally delivered Fgf<em>15</em> were examined in this model. The role of Fgf<em>15</em> in BA-induced liver <em>growth</em> was tested in Fgf<em>15</em> (-/-) mice upon cholic acid (CA) feeding. The direct mitogenic effect of Fgf<em>15</em> was evaluated in cultured mouse hepatocytes and cholangiocytes.

RESULTS

Fgf<em>15</em> (-/-) mice showed marked liver injury and mortality after PH accompanied by persistently elevated intrahepatic BA levels. Cholestyramine feeding and adenovirally delivered Fgf<em>15</em> reduced BA levels and significantly prevented this lethal outcome. Fgf<em>15</em> also reduced mortality after extensive hepatectomy in Fgf<em>15</em>(+/+) animals. Liver <em>growth</em> elicited by CA feeding was significantly diminished in Fgf<em>15</em> (-/-) mice. Proliferation of hepatocytes and cholangiocytes was also noticeably reduced in CA-fed Fgf<em>15</em> (-/-) mice. Fgf<em>15</em> induced intracellular signalling and proliferation of cultured hepatocytes and cholangiocytes.

CONCLUSIONS

Fgf<em>15</em> is necessary to maintain BA homeostasis and prevent liver injury during liver regeneration. Moreover, Fgf<em>15</em> is an essential mediator of the liver <em>growth</em>-promoting effects of BA. Preoperative administration of this enterokine to patients undergoing liver resection might be useful to reduce damage and foster regeneration.

Purpose This two-part, first-in-human study was initiated in patients with advanced solid tumors harboring genetic alterations in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) to determine the maximum tolerated dose (MTD), the recommended phase II dose (RP2D), and the schedule, safety, pharmacokinetics, pharmacodynamics, and antitumor activity of oral BGJ398, a selective FGFR1-3 tyrosine kinase inhibitor. Patients and Methods Adult patients were treated with escalating dosages of BGJ398 5 to <em>15</em>0 mg once daily or 50 mg twice daily continuously in 28-day cycles. During expansion at the MTD, patients with FGFR1-amplified squamous cell non-small-cell lung cancer (sqNSCLC; arm 1) or other solid tumors with FGFR genetic alterations (mutations/amplifications/fusions) received BGJ398 daily on a continuous schedule (arm 2), or on a 3-weeks-on/1-week-off schedule (arm 3). Results Data in 132 patients from the escalation and expansion arms are reported (May <em>15</em>, 20<em>15</em>, cutoff). The MTD, 125 mg daily, was determined on the basis of dose-limiting toxicities in four patients (100 mg, grade 3 aminotransferase elevations [n = 1]; 125 mg, hyperphosphatemia [n = 1]; <em>15</em>0 mg, grade 1 corneal toxicity [n = 1] and grade 3 aminotransferase elevations [n = 1]). Common adverse events in patients treated at the MTD (n = 57) included hyperphosphatemia (82.5%), constipation (50.9%), decreased appetite (45.6%), and stomatitis (45.6%). A similar safety profile was observed using the 3-weeks-on/1-week-off schedule (RP2D). However, adverse event-related dose adjustments/interruptions were less frequent with the 3-weeks-on/1-week-off (50.0%) versus the continuous (73.7%) schedule. Antitumor activity (seven partial responses [six confirmed]) was demonstrated with BGJ398 doses ≥ 100 mg in patients with FGFR1-amplified sqNSCLC and FGFR3-mutant bladder/urothelial cancer. Conclusion BGJ398 at the MTD/RP2D had a tolerable and manageable safety profile and showed antitumor activity in several tumor types, including FGFR1-amplified sqNSCLC and FGFR3-mutant bladder/urothelial cancers.

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand <em>factor</em>, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and <em>fibroblasts</em>. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial <em>growth</em> <em>factors</em> (VEGF121, VEGF165) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) induced proliferation of all endothelial cell types. Placental <em>growth</em> <em>factors</em> PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and <em>15</em> +/- 20%) and HPEC-A2 (<em>15</em> +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

The kinetics and second messenger regulation of three cloned mammalian intestinal Na+/H+ exchangers were studied using fluorometric techniques. These exchangers, NHE1, NHE2, and NHE3, were stably expressed in PS120 <em>fibroblasts</em>, which lack an endogenous Na+/H+ exchanger. H+ kinetic data indicated cooperativity by internal protons, with Hill coefficients of approximately 2 for all three isoforms. In contrast, Na+ kinetic data fit Michaelis-Menten kinetics, with Km (Na+) <em>15</em>-18 mM and a Hill coefficient of approximately 1. The exchangers were all activated by <em>growth</em> <em>factors</em> and thrombin; in NHE1 these agonists increased the apparent affinity for intracellular H+, but did not change Vmax, while for NHE2 and NHE3 the effect was on Vmax alone. Phorbol ester stimulated NHE1 and NHE2, but inhibited NHE3 with a decrease in Vmax. ATP-depletion decreased Vmax and the apparent affinity for H+ for all three isoforms, and reduced the Hill coefficient to approximately 1, suggesting that a basal level of phosphorylation was required for the cooperativity. The differences in kinetics and second messenger regulation suggest that the NHE isoforms may serve different cellular functions. The up- and down-regulation of NHE3 by kinases indicates that this isoform may be involved in a specialized function such as Na+ absorption.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs), such as FGF-1, have been shown to induce differentiation of lens epithelial cells both in tissue culture and in transgenic mice. In the present study, using the alpha A-crystallin promoter, we generated transgenic mice that express different FGFs (FGF-4, FGF-7, FGF-8, FGF-9) specifically in the lens. All four FGFs induced changes in ocular development. Microphthalmic eyes were evident in transgenic mice expressing FGF-8, FGF-9 and some lines expressing FGF-4. A developmental study of the microphthalmic eyes revealed that, by embryonic day <em>15</em>, expression of these FGFs induced lens epithelial cells to undergo premature fiber differentiation. In less severely affected lines expressing FGF-4 or FGF-7, the lens epithelial cells exhibited a premature exit from the cell cycle and underwent a fiber differentiation response later in development, leading to cataract formation. The responsiveness of lens cells to different FGFs indicates that these proteins stimulate the same or overlapping downstream signalling pathway(s). These overlapping effects of different FGFs on a common cell type indicate that the normal developmental roles for these genes are determined by the temporal and spatial regulation of their expression patterns. The fact that any of these FGFs can induce ocular defects and loss of lens transparency implies that it is essential for the normal eye to maintain very specific spatial control over FGF expression in order to prevent cataract induction.

Transforming <em>growth</em> <em>factor</em>-betas (TGF-betas) constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation, and tissue remodeling. In the developing nervous system, TGF-beta2 and -beta3 occur in radial and astroglial cells as well as in many populations of postmitotic, differentiating neurons. TGF-beta1 is restricted to the choroid plexus and meninges. In addition to functions related to glial cell maturation and performances, TGF-beta2 and -beta3 are important regulators of neuron survival. In contrast to neurotrophic <em>factors</em>, as for example, neurotrophins, TGF-betas are most likely not neurotrophic by themselves. However, they can dramatically increase the potency of select neurotrophins, <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, ciliary neurotrophic <em>factor</em>, and glial cell line-derived neurotrophic <em>factor</em> (GDNF). In the case of GDNF, we have shown that GDNF fails to promote the survival of highly purified neuron populations in vitro unless it is supplemented with TGF-beta. This also applies to the in vivo situation, where antibodies to all three TGF-beta isoforms fully prevent the trophic effect of GDNF on axotomized, target-deprived neurons. In addition to the TGF-beta isoforms -beta2 and -beta3, other members of the TGF-beta superfamily are expressed in the nervous system having important roles in embryonic patterning, cell migration, and neuronal transmitter determination. We have cloned and expressed a novel TGF-beta, named <em>growth</em>/differentiation <em>factor</em>-<em>15</em> (GDF-<em>15</em>). GDF-<em>15</em> is synthesized in the choroid plexus and released into the CSF, but also occurs in all regions investigated of the developing and adult brain. GDF-<em>15</em> is a potent trophic <em>factor</em> for developing and 6-OHDA-lesioned midbrain dopaminergic neurons in vitro and in vivo, matching the potency of GDNF.

The role of epidermal <em>growth</em> <em>factor</em> (EGF) and its receptors in human cancers was studied using 24 human cell cultures including <em>15</em> of squamous cell carcinoma (SCC) of the skin, oral cavity, and esophagus. EGF was found to inhibit the <em>growth</em> and colony formation of all the SCC cells at doses that are mitogenic in many other cells, including epidermal keratinocytes and dermal <em>fibroblasts</em>. This inhibitory effect of EGF on SCCs was specific, because EGF did not inhibit and in some cases slightly stimulated the <em>growth</em> of other tumor cells, such as adenocarcinomas of the stomach, cervix, and breast and sarcomas. The amounts of EGF receptors on these SCC cells were measured by immunoprecipitation of labeled proteins with anti-EGF receptor polyclonal antibody and binding assay of membrane preparations using 125I-EGF. Of 13 SCC cell cultures tested, all except 3 of esophageal SCC showed higher levels of EGF receptor than normal epidermal keratinocytes, which contain 1.5 X 10(5) binding sites/cell. In general, SCCs of the skin and oral cavity had large amounts of EGF receptor on the order of 10(6)/cell, whereas the receptor of esophageal SCCs was on the order of 10(5)/cell. Some SCC cells had about twice as many EGF receptors as A431 cells. The values for the equilibrium dissociation constant (Kd) of these cells were on the order of nM. The sensitivity to the inhibitory effect of EGF correlated well with the elevated level of EGF receptors in 12 SCC cell lines, and higher significance was obtained when data on esophageal SCCs were excluded. The present observations suggest that EGF and EGF receptors play a role in the development of SCCs.