The control of neuronal number is critical for coordinating innervation and target organ requirements. Although basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is known to regulate neuron number in the developing embryonic cortex, its potential role during postnatal brain development remains undefined. To address this issue, the cerebellum, a site of postnatal neurogenesis, was used. Previously, we found that a single peripheral injection of bFGF in newborn rats elicited mitosis of neuronal precursors in the external germinal layer (EGL) 8 h after administration. We now define the sustained effects of bFGF treatment on postnatal granule cell production and cerebellar <em>growth</em>. Seventy-two h after a single injection of bFGF (20 ng/g) in newborn rats, the fraction of BrdU-labeled cells in the EGL increased by 46% without altering apoptotic cell number, consistent with enhanced precursor proliferation. Moreover, bFGF increased mitotically labeled cells by 100% and total cell density by 33% in the internal granular layer (IGL), the final destination of the EGL precursors. Because cerebellar volume also increased by 22%, bFGF-induced proliferation enhanced generation of total IGL neurons and increased cerebellar <em>growth</em>. These morphometric measures were corroborated independently by using DNA quantitation: cerebellar DNA content increased <em>16</em>% after bFGF injection, consistent with increased neuron number. Furthermore, using DNA quantitation as an index, increased total cerebellar cell number elicited by bFGF injection persisted beyond the neurogenetic period, until P35. We conclude that a single postnatal injection of bFGF increases granule neuron number and enhances cerebellar <em>growth</em> following mitotic stimulation.

Keloid scars after skin trauma are a significant clinical problem, especially in black populations, in which the incidence of keloids has been estimated at 4-<em>16</em>%. Keloids are abnormal dermal proliferative scars secondary to dysregulated wound healing. Despite several biochemical studies on the role of extracellular matrix proteins and <em>growth</em> <em>factors</em> during keloid formation, we still do not know what molecules and signals induce this change. <em>Fibroblasts</em> are thought to be the major inductive cell for keloid scar formation. The aim of this study was to identify gene expression patterns that characterize keloid <em>fibroblasts</em>; identifying such genetic disequilibrium may shed light on the molecular signaling events responsible for keloid formation. In this study, we performed gene expression analysis of <em>fibroblasts</em> isolated from keloid lesions from three individuals in comparison with the <em>fibroblasts</em> isolated from normal skin using the Affymetrix U133a chip (22,284 genes and expression sequence tags). We found through J5 test score expression analysis that among 22,284 genes, there were 43 genes that were overexpressed and five genes were underexpressed in keloid <em>fibroblasts</em> when compared with dermal <em>fibroblasts</em> from persons without keloids. The overexpression of three genes not previously reported as being up-regulated in keloids (annexin A2, Transgelin, and RPS18) was confirmed by real-time polymerase chain reaction. Certain overexpressed genes were similar to previous biochemical observations on the protein levels of these overexpressed genes during keloid formation. We also report for the first time that a few tumor-related genes are overexpressed in keloid <em>fibroblasts</em>.

Stromal myo<em>fibroblasts</em> (SMF) associated with various types of carcinomas are believed to emerge under the influence of the tumor cells. Recent studies have shown that SMF may originate from <em>fibroblasts</em> within the tumor stroma or even from carcinoma cells by the process of epithelial-mesenchymal transition. The aim of this study was to investigate the concomitant expression of epithelial membrane antigen and alpha-smooth muscle actin in cells at the tumor-connective tissue interface in human tongue carcinoma, as a possible reflection of epithelial-mesenchymal transition. Given its key role in this process, expression of transforming <em>growth</em> <em>factor</em>-beta in the malignant cells was assessed as well. Immunostaining with alpha-smooth muscle actin was performed on cases of hyperplasia (n = <em>16</em>), mild dysplasia (n = 12), moderate-to-severe dysplasia (n = 11) and carcinoma (n = 22). Transforming <em>growth</em> <em>factor</em>-beta assessment and double immunostaining with epithelial membrane antigen and alpha-smooth muscle actin were performed only in cases of carcinoma. SMF were significantly associated with carcinomas, while their number in pre-malignant lesions (hyperplasia and dysplasia) was significantly lower (P < 0.001). Although SMF were found in all carcinomas, they were heterogeneous in their frequency and patterns of distribution. In addition, 95% of the carcinomas expressed transforming <em>growth</em> <em>factor</em>-beta and 41% exhibited cells positive for both epithelial membrane antigen and alpha-smooth muscle actin. SMF were almost exclusively associated with established carcinomas and not with pre-malignant lesions. Cells that co-expressed epithelial membrane antigen and alpha-smooth muscle actin can be a manifestation of epithelial-mesenchymal transition and, as such, may serve as a source for SMF in these tumors. These findings appear to be linked to the frequent expression of transforming <em>growth</em> <em>factor</em>-beta by the malignant cells.

While stimulating the <em>growth</em> of <em>fibroblasts</em>, transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta 1) inhibits the <em>growth</em> of various normal and malignant cell lines in vitro. We studied the effects of TGF-beta 1 in vivo. The level of TGF-beta 1 in serum was maximally elevated 2 h after injecting 1 muCi of 125I-TGF-beta 1 into the peritoneal cavity of nude mice. Five h after the i.p. administration of 10 micrograms of unlabeled TGF-beta 1, 20 ng/ml of TGF-beta-like material in serum were detected by a radioreceptor assay on A549 lung carcinoma cells. Trichloracetic acid-precipitable 125I-TGF-beta 1 was taken up by liver, spleen, lungs, kidneys, and tumor tissue but not by the brain. At doses exceeding 2 micrograms/day, TGF-beta 1 induced a generalized interstitial fibrosis and a cachexia, which was not mediated by elevated serum levels of tumor necrosis <em>factor</em> alpha as determined by Western blot analysis and enzyme-linked immunosorbent assay. A total of 200,000 cells of the estrogen receptor-negative human breast cancer line MDA-MB-231, which had been shown to be maximally <em>growth</em> inhibited in vitro by 40 pM TGF-beta 1 and to have high-affinity receptors (9, 11, 12), were injected into the mammary fat pad of each nude mouse. The duration of treatment was <em>16</em> days with ten animals in the control group and five animals in the treated groups. The dose ranged from 1 to 4 micrograms per animal daily. The treatment was started 24 h after the injection of the tumor cells. Tumor <em>growth</em> was not significantly affected at either nontoxic or toxic doses of TGF-beta 1. Thus, we have demonstrated that TGF-beta 1, apart from being a local <em>growth</em> <em>factor</em>, has systemic effects, such as cachexia and multiple fibrosis. Its role as an antitumor agent may be limited.

Insulin-like <em>growth</em> <em>factor</em> I (IGF-I) is known to exert an anabolic effect on tendon <em>fibroblast</em> production of collagen. IGF-I's regulation is complex and involves six different IGF binding proteins (IGFBPs). Of these, IGFBP-4 and -5 could potentially influence the effect of IGF-I in the tendon because they both are produced in <em>fibroblast</em>; however, the response of IGFBP-4 and -5 to mechanical loading and their role in IGF-I regulation in tendinous tissue are unknown. A splice variant of IGF-I, mechano-<em>growth</em> <em>factor</em> (MGF) is upregulated and known to be important for adaptation in loaded muscle. However, it is not known whether MGF is expressed and upregulated in mechanically loaded tendon. This study examined the effect of mechanical load on tendon collagen mRNA in relation to changes in the IGF-I systems mRNA expression. Data were collected at 2, 4, 8 and <em>16</em> days after surgical removal of synergistic muscle to the plantaris muscle of the rat, thus increasing the load to plantaris muscle and tendon. Nearly a doubling of the tendon mass was observed after <em>16</em> days of loading. A rapid rise in tendon procollagen III mRNA was seen after 2 days whereas the increase in procollagen I mRNA was significant from day 8. MGF was expressed and upregulated in loaded tendon tissue with a faster response than IGF-I, which was increased from day 8. Finally, IGFBP-4 mRNA was increased with a time pattern similar to procollagen III, whereas IGFBP-5 decreased at day 8. In conclusion, loading of tendon tissue results in an upregulation of IGF-I, IGFBP-4, and procollagen and is associated with an increase in tendon mass. Also, MGF is expressed with an early upregulation in loaded tendon tissue. We suggest that the IGF-I system could be involved in collagen synthesis in tendon in response to mechanical loading.

OBJECTIVE

Preclinical studies suggest antiangiogenesis strategies may be effective in the treatment of prostate cancer. In tumor models, the copper-chelating agent tetrathiomolybdate (TM) has been shown to be antiangiogenic. We evaluated the antitumor activity of TM in patients with hormone-refractory prostate cancer (HRPC).

METHODS

Nineteen patients with asymptomatic HRPC enrolled. Copper depletion was monitored using serum ceruloplasmin levels. Once the target ceruloplasmin level of 5-15 mg/dl was attained, patients underwent staging evaluation. Patients were reassessed every 12 weeks, and TM was continued until they developed evidence of disease progression or intolerable toxicity. Prostate-specific antigen and levels of vascular endothelial growth factor, basic fibroblast growth factor, interleukin (IL)-6 and IL-8 were measured at study entry, at the time of copper depletion, and monthly while on therapy.

RESULTS

Seventeen of 19 patients achieved copper deficiency on TM therapy. Of the 16 evaluable patients, 14 developed progressive disease, 1 discontinued therapy because of toxicity and 1 patient opted to discontinue therapy because of rising prostate-specific antigen level without objective evidence of progressive disease. Levels of vascular endothelial growth factor, IL-6 and IL-8, but not basic fibroblast growth factor, were elevated when compared to normal controls prior to TM therapy, but there was no significant change during therapy. There was no correlation between prostate-specific antigen and levels of angiogenesis factors.

CONCLUSIONS

Copper depletion with TM did not delay disease progression in patients with asymptomatic metastatic HRPC.

OBJECTIVE

Numerous studies have evaluated the prevalence and importance of vitamin D deficiency among patients with chronic kidney disease and end-stage renal disease; however, little is known about vitamin D levels in acute kidney injury (AKI). We evaluated the association between vitamin D metabolites and clinical outcomes among patients with AKI.

METHODS

Prospective cohort study.

METHODS

A total of 30 participants with AKI and 30 controls from general hospital wards and intensive care units at a tertiary care hospital were recruited for the study.

METHODS

Plasma levels of 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2 D], 24R,25-dihydroxyvitamin D3 , vitamin D binding protein (VDBP) and fibroblast growth factor 23 (FGF23) were measured within 24 hours of AKI onset and 5 days later. Bioavailable 25(OH)D and 1,25(OH)2 D levels, defined as the sum of free- and albumin-bound 25(OH)D and 1,25(OH)2 D, were estimated using equations.

RESULTS

Compared to controls, participants with AKI had lower levels of 1,25(OH)2 D [17 (10-22) vs 25 (15-35) pg/ml, P = 0·01], lower levels of VDBP [23 (15-31) vs 29 (25-36) mg/dl, P = 0·003] and similar levels of bioavailable 25(OH)D and 1,25(OH)2 D at enrolment. Levels of bioavailable 25(OH)D were inversely associated with severity of sepsis in the overall sample (P < 0·001). Among participants with AKI, bioavailable 25(OH)D, but not other vitamin D metabolites, was significantly associated with mortality after adjusting for age and serum creatinine (adjusted odds ratio per 1 SD ln [bioavailable 25(OH)D] = 0·16, 95% confidence interval = 0·03-0·85).

CONCLUSIONS

Bioavailable 25(OH)D could have a role as a biomarker or mediator of adverse outcomes among patients with established AKI.

Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.

OBJECTIVE

This study was undertaken to investigate the role of the angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor in the development and progression of Barrett esophagus and adenocarcinomas of the esophagus and gastroesophageal junction.

METHODS

Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels, relative to the control gene encoding beta-actin, were measured by using a quantitative reverse transcription-polymerase chain reaction method (ABI 7700 Sequence Detector system) in specimens of Barrett intestinal metaplasia (n = 16), dysplasia (n = 11), adenocarcinoma (n = l 5), and matching normal squamous esophageal tissues (n = 35). Vascular endothelial growth factor and basic fibroblast growth factor protein expression and CD31(+) microvessel density were assessed by means of immunohistochemistry in 25 tissue sections that included representative areas for each of these Barrett stages.

RESULTS

Expression levels were significantly increased in adenocarcinoma compared with in either normal squamous mucosa (P <.0001 for both genes) or intestinal metaplasia (vascular endothelial growth factor, P =.002; basic fibroblast growth factor, P <.0001). Vascular endothelial growth factor levels were also significantly higher in cancer tissues compared with dysplasia tissues (P =.024, Mann-Whitney U test). Basic fibroblast growth factor expression was also significantly increased in Barrett dysplastic mucosa compared with in intestinal metaplasia or normal esophageal mucosa. Microvessel density was generally higher in adenocarcinoma compared with in preneoplastic Barrett tissues. The pattern of vascular endothelial growth factor and basic fibroblast growth factor protein expression was similar to the messenger RNA expression pattern, with the exception that mucin-containing goblet cells stained intensely for vascular endothelial growth factor and only weak vascular endothelial growth factor staining was present in some adenocarcinomas.

CONCLUSIONS

Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels are significantly upregulated in esophageal and gastroesophageal junction adenocarcinomas, suggesting a role for these angiogenic factors in the development of these cancers. Vascular endothelial growth factor and basic fibroblast growth factor messenger RNA expression levels are also increased in some Barrett esophagus tissues, with this increase occurring at an earlier stage for basic fibroblast growth factor than for vascular endothelial growth factor. Basic fibroblast growth factor protein expression pattern is similar to the messenger RNA expression pattern, but unlike the messenger RNA findings, vascular endothelial growth factor protein expression is strongest in goblet cells.

Epithelial ovarian cancers (EOCs) arise in the Ovarian Surface Epithelium (OSE). This tissue is a simple, poorly committed mesothelium which exhibits characteristics of epithelial and mesenchymal cells when grown in culture. In contrast, EOCs frequently exhibit properties of complex epithelial tissues of the female reproductive tract, such as oviductal, endometrial and cervical epithelia, and show induction of expression of epithelial markers such as E-cadherin. <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> Receptor 2 isoform IIIb (FGF receptor 2-IIIb) is a spliced variant of FGF receptor 2 that binds the ligands FGF-1 and FGF-7 with high affinity, and is expressed exclusively by epithelial cells. We have studied the expression of FGF receptor 2-IIIb and its ligands in primary cultures of normal human OSE, EOC cell lines and snap frozen tissue from EOCs. Expression of FGF receptor 2-IIIb mRNA is undetectable in normal OSE, but is expressed in <em>16</em>/20 (80%) of EOCs. FGFs 1 and 7 mRNAs are expressed in normal OSE, whilst only 4/20 (20%) and 12/20 (60%) of EOCs demonstrated expression for these ligands respectively. However, FGF-7 protein was detected in 70% (mean level=0.7 ng/ml) of ascitic fluids obtained from patients with EOC. FGFs 1 and 7 stimulate DNA synthesis in EOC cell lines that express FGF receptor 2-IIIb. Moreover, DNA synthesis in these cell lines can be partially blocked by blocking antisera to FGFs 1 and 7. It is suggested that induction of expression of FGF receptor 2-IIIb may play a role in the development of EOCs by rendering the OSE susceptible to paracrine and/or autocrine stimulation by its requisite FGF ligands.

<AbstractText>X-linked hypophosphataemia in children is characterised by elevated serum concentrations of <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), hypophosphataemia, rickets, lower extremity bowing, and <em>growth</em> impairment. We compared the efficacy and safety of continuing conventional therapy, consisting of oral phosphate and active vitamin D, versus switching to burosumab, a fully human monoclonal antibody against FGF23, in paediatric X-linked hypophosphataemia.</AbstractText><AbstractText>In this randomised, active-controlled, open-label, phase 3 trial at <em>16</em> clinical sites, we enrolled children with X-linked hypophosphataemia aged 1-12 years. Key eligibility criteria were a total Thacher rickets severity score of at least 2·0, fasting serum phosphorus lower than 0·97 mmol/L (3·0 mg/dL), confirmed PHEX (phosphate-regulating endopeptidase homolog, X-linked) mutation or variant of unknown significance in the patient or a family member with appropriate X-linked dominant inheritance, and receipt of conventional therapy for at least 6 consecutive months for children younger than 3 years or at least 12 consecutive months for children older than 3 years. Eligible patients were randomly assigned (1:1) to receive either subcutaneous burosumab starting at 0·8 mg/kg every 2 weeks (burosumab group) or conventional therapy prescribed by investigators (conventional therapy group). Both interventions lasted 64 weeks. The primary endpoint was change in rickets severity at week 40, assessed by the Radiographic Global Impression of Change global score. All patients who received at least one dose of treatment were included in the primary and safety analyses. The trial is registered with ClinicalTrials.gov, number NCT02915705.</AbstractText><AbstractText>Recruitment took place between Aug 3, 20<em>16</em>, and May 8, 2017. Of 122 patients assessed, 61 were enrolled. Of these, 32 (18 girls, 14 boys) were randomly assigned to continue receiving conventional therapy and 29 (<em>16</em> girls, 13 boys) to receive burosumab. For the primary endpoint at week 40, patients in the burosumab group had significantly greater improvement in Radiographic Global Impression of Change global score than did patients in the conventional therapy group (least squares mean +1·9 [SE 0·1] with burosumab vs +0·8 [0·1] with conventional therapy; difference 1·1, 95% CI 0·8-1·5; p<0·0001). Treatment-emergent adverse events considered possibly, probably, or definitely related to treatment by the investigator occurred more frequently with burosumab (17 [59%] of 29 patients in the burosumab group vs seven [22%] of 32 patients in the conventional therapy group). Three serious adverse events occurred in each group, all considered unrelated to treatment and resolved.</AbstractText><AbstractText>Significantly greater clinical improvements were shown in rickets severity, <em>growth</em>, and biochemistries among children with X-linked hypophosphataemia treated with burosumab compared with those continuing conventional therapy.</AbstractText><AbstractText>Ultragenyx Pharmaceutical and Kyowa Kirin International.</AbstractText>

OBJECTIVE

Gangliogliomas (GGs) are neuronal-glial tumors highly associated with epilepsy. We hypothesized that the expression of select gene families including neurotransmitter receptor subunits and growth factors would be distinct in neurons and astrocytes within GG compared with adjacent cortex and that these changes would yield insights into seizure onset and lesion formation.

METHODS

Candidate gene expression was defined in single immunohistochemically labeled neurons and astrocytes microdissected from GG specimens compared with neurons and astrocytes microdissected from morphologically intact cortex adjacent to the GG or normal control cortex.

RESULTS

Differential expression of 16 genes including glutamate transporter (EAAC1) and receptor (NMDA2C, mGluR5), growth factor (hepatocyte growth factor), and receptor (platelet derived growth factor receptor beta, fibroblast growth factor receptor 3) mRNAs was detected in GG neurons compared with control neurons. In astrocytes, altered expression of p75NGF, mGluR3, TGFbeta3 and Glt-1 mRNAs was detected. Nestin mRNA, a gene that exhibits enhanced expression in balloon cell cortical dysplasia, was increased in GG neurons. Because of the morphological similarities between GG and cortical dysplasia, we show that there is activation of the mTOR cascade in GG as evidenced by enhanced expression of phospho-p70S6kinase and phosphoribosomal S6 proteins.

CONCLUSIONS

We find differential candidate gene expression in neurons and astrocytes in GG compared with adjacent cortex and show that there is activation of the mTOR pathway. These changes highlight pathways that may be pivotal for epileptogenesis and lesion growth.

<AbstractText>X-linked hypophosphatemia (XLH) is characterized by excess <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), hypophosphatemia, skeletal abnormalities, and <em>growth</em> impairment. We aimed to understand the burden of disease of XLH across the lifespan.</AbstractText><AbstractText>Responses were collected from adults with XLH and parents/caregivers of a child with XLH in an online survey, including multiple-choice and open-ended questions on demographics, disease manifestations, treatment history, assistive device use, and age-specific patient-reported outcomes (PROs).</AbstractText><AbstractText>Data were collected from 232 adults with XLH (mean age, 45.6 years; 76% female) and 90 parents/caregivers of a child with XLH (mean age, 9.1 years; 56% female). Mean age recalled for symptom onset was 3.2 years for adults and 1.3 years for children. When surveyed, nearly all children (99%) and 64% of adults were receiving oral phosphate, active vitamin D, or both. Prior participation in a trial investigating burosumab, a fully human monoclonal antibody against FGF23, was reported in 3% of children and 10% of adults; of these respondents, only one child reported current treatment with burosumab at the time of the survey. Both children and adults reported typical features of XLH, including abnormal gait (84% and 86%, respectively), bowing of the tibia/fibula (72% and 77%), and short stature (80% and 86%). Nearly all adults (97%) and children (80%) reported bone or joint pain/stiffness. Adults reported a history of fractures (n/N = 102/232; 44%), with a mean (SD) age at first fracture of 26 (<em>16</em>) years. Adults reported osteophytes (46%), enthesopathy (27%), and spinal stenosis (19%). Mean scores for PROs evaluating pain, stiffness, and physical function were worse than population norms. Analgesics were taken at least once a week by 67% of adults.</AbstractText><AbstractText>Despite the common use of oral phosphate and active vitamin D established in the 1980s, children with XLH demonstrate a substantial disease burden, including pain and impaired physical functioning that persists, as demonstrated by similar responses reported in adults with XLH.</AbstractText>

Aloe vera has long been used as a traditional medicine for inducing wound healing. Gingival <em>fibroblasts</em> (GFs) play an important role in oral wound healing. In this study, we investigated the effects of acemannan, a polysaccharide extracted from Aloe vera gel, on GF proliferation; keratinocyte <em>growth</em> <em>factor</em>-1 (KGF-1), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), and type I collagen production; and oral wound healing in rats. [(3)H]-Thymidine incorporation assay and ELISA were used. Punch biopsy wounds were created at the hard palate of male Sprague Dawley rats. All treatments (normal saline; 0.1% triamcinolone acetonide; plain 1% Carbopol; and Carbopol containing 0.5%, 1%, and 2% acemannan (w/w)) were applied daily. Wounded areas and histological features were observed at day 7 after treatment. From our studies, acemannan at concentrations of 2, 4, 8, and <em>16</em> mg/ml significantly induced cell proliferation (P<0.05). Acemannan concentrations between 2 - <em>16</em> mg/ml significantly stimulated KGF-1, VEGF, and type I collagen expressions (P<0.05). Wound healing of animals receiving Carbopol containing 0.5% acemannan (w/w) was significantly better than that of the other groups (P<0.05). These findings suggest that acemannan plays a significant role in the oral wound healing process via the induction of <em>fibroblast</em> proliferation and stimulation of KGF-1, VEGF, and type I collagen expressions.

Angiogenic therapy with individual <em>growth</em> <em>factors</em> or "master switch" genes is being evaluated for treatment of advanced coronary artery disease. In this study, we investigated the efficacy and mechanism of PR39, a gene capable of activating VEGF and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2-dependent pathways. PR39 enhances hypoxia-inducible <em>factor</em>-1alpha (HIF-1alpha)-dependent gene expression by selectively inhibiting proteasome degradation of this transcription <em>factor</em>. In addition, PR39 also stimulates expression of the FGF receptors (FGFR)-1 and syndecan-4. In a pig model of chronic myocardial ischemia, we used angiography, MRI, and microsphere regional blood flow to evaluate the efficacy of intramyocardial adenoviral protein arginine-rich peptide (Ad-PR39) injections. Ad-PR39 improved collateral scores, regional perfusion, and regional function in a dose-dependent manner. Local VEGF, VEGFR-1, VEGFR-2, syndecan, and FGFR-1 levels were <em>16</em>-75% upregulated after Ad-PR39 injections as assessed by real-time PCR, suggesting upregulation of VEGF and FGF pathways. PR39 is an angiogenic peptide that improves perfusion and function of ischemic myocardium, at least in part, through collateral formation. The dual mechanism, i.e., stimulation of HIF-1alpha and FGF receptor expression, likely accounts for the functional benefits of PR39.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF)-like molecules secreted by alveolar macrophages have been postulated to be mediators of lung fibrogenesis since these cytokines stimulate the proliferation and chemotaxis of lung <em>fibroblasts</em>. We are studying the biology and biochemistry of rat macrophage-derived PDGF and have identified for the first time the specific isoforms of PDGF (-AA, -AB, and -BB) that these macrophages secreted in vitro following activation with either chrysotile asbestos or carbonyl iron spheres. Subsequently, the proliferative response of rat lung <em>fibroblasts</em> (RLF) to the different PDGF isoforms was established. Using several antibodies raised against the distinct isoforms, we established that two different PDGF-like <em>factors</em> with molecular masses of 30 to 34 kD and <em>16</em> to 18 kD were contained in alveolar macrophage-conditioned medium. Within each of these molecular mass regions was a mixture of all three PDGF isoforms. We estimated that the 30- to 34-kD PDGF was mainly PDGF-BB (approximately 50%), while the remaining consisted of PDGF-AA (approximately 13%) and PDGF-AB (approximately 37%). Purified recombinant PDGF isoforms were tested for their ability to stimulate the <em>growth</em> of early-passage RLF and Swiss 3T3 cells in a 3-day cell proliferation assay. PDGF-BB and PDGF-AB were the most potent inducers of RLF proliferation and stimulated <em>growth</em> half-maximally at approximately 1 ng/ml and approximately 7 ng/ml, respectively. While these two B-chain-containing dimers stimulated lung <em>fibroblast</em> <em>growth</em> by as much as 150% above control medium, the PDGF-AA homodimer stimulated lung <em>fibroblast</em> proliferation less than 25% above control medium at the highest concentrations tested (20 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of the transforming <em>growth</em> <em>factor</em> beta (TGF-beta) on the <em>growth</em> and glycosaminoglycan synthesis of rabbit <em>growth</em> plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of <em>16</em>-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified <em>growth</em> <em>factors</em> such as platelet-derived <em>growth</em> <em>factor</em> (PDGF), epidermal <em>growth</em> <em>factor</em> (EGF) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the <em>growth</em> <em>factors</em> for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of <em>growth</em>-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like <em>growth</em> <em>factor</em> I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal <em>growth</em> cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating <em>growth</em> and phenotypic expression of chondrocytes in the <em>growth</em> plate, possibly via a paracrine mechanism.

OBJECTIVE

Ovarian cancer has very heterogeneous histological classification, and response to therapy of the same grade and type varies. We studied genes in the Wnt and hedgehog (Hh) pathways, which are essential for embryonic development and which play critical roles in proliferation in a variety of human cancers. Variations in these pathway genes causing proliferation could play a role in the variation in tumor progression and response to therapy.

METHODS

Using real-time polymerase chain reaction, we studied <em>16</em> primary grade 3 International Federation of Gynecology and Obstetrics stage III serous ovarian cancer samples for expression of the Wnt pathway gene AXIN2, <em>fibroblast</em> <em>growth</em> <em>factor</em> 9, and Hh pathway gene expressions of glioma-associated oncogene 1, glioma-associated oncogene 2, patched homolog 1, patched homolog 2, Indian Hedgehog (HH), sonic HH, and Smoothened, a G protein-coupled receptor protein. Normal ovary epithelial cell line was used as control.

RESULTS

We found wide variation of up-regulation of pathway component and target genes in the primary tumor samples and apparent cross talk between the pathways. AXIN2, a Wnt target gene, showed increased expression in all serous ovarian cancer samples. Fibroblast growth factor 9 was also overexpressed in all tumors with greater than 1000-fold increase in gene expression in 4 tumors. Expression of Hh pathway genes varied greatly. More than half of the tumor samples showed involvement of Hh signaling or pathway activation either by expression of transcription factors and Hh ligands or by overexpression of Indian HH/sonic HH and the receptor-encoding patched homolog 1/patched homolog 2.

CONCLUSIONS

We found a wide variation in fold expression of genes involved in the Wnt and Hh pathway between patient samples.

Addition of exogenous acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) to NBT-II epithelial carcinoma cells results in <em>fibroblast</em>ic transformation and cell motility. We have generated aFGF-producing NBT-II cells by transfection with recombinant expression vectors containing human aFGF cDNA, or the human aFGF cDNA coupled to a signal peptide (SP) sequence. The effects of the nonsecreted and the secreted <em>16</em>-kDa <em>growth</em> <em>factor</em> on the morphology, motility, and cell invasive potential (gelatinase activity) were compared. aFGF coupled to a SP was actively secreted out of the producing cells. The secretion of aFGF was not necessary for induction of gelatinase activity, as this was observed in NBT-II cells producing aFGF with or without SP. Production of aFGF, whether secreted or not secreted, resulted in increased in vitro motility of most isolated clones; however, there was no correlation between aFGF level and motility rate. The data suggest that expression of aFGF in NBT-II cells induces metastatic potential through an autocrine or intracrine mechanism.

The E5 polypeptide of bovine papillomavirus type 1 is a small membrane-bound protein which induces the transformation of immortalized <em>fibroblasts</em>, apparently via the formation of a ternary complex with the platelet-derived <em>growth</em> <em>factor</em> receptor (PDGFR) and the <em>16</em>-kDa V-ATPase protein. This interaction seems to be mediated, at least in part, by their respective transmembrane domains. E5 also cooperates with transfected beta PDGFR to induce interleukin-3 (IL-3)-independent <em>growth</em> of a mouse myeloid precursor cell line (32D) which normally lacks expression of most known tyrosine kinase <em>growth</em> <em>factor</em> receptors. Cell proliferation induced by beta PDGFR and E5 is also highly specific, since the highly conserved alpha PDGFR and other related receptors did not physically or functionally interact with E5 in these cells. In the current study, analysis of chimeric alpha and beta PDGFRs confirmed that a short region encompassing the beta PDGFR transmembrane domain was sufficient for complex formation with E5, receptor autophosphorylation, and sustained proliferation of 32D cells in the absence of IL-3. Furthermore, a deletion mutant lacking the entire extracellular domain efficiently bound E5 and induced IL-3-independent <em>growth</em>. These data provide direct evidence that the interaction between E5 and the beta PDGFR involves amino acids 531 to 556 of the receptor transmembrane region and that this specific interaction is critical for activation of the PDGFR signaling complex.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) regulate bone <em>growth</em>, but their expression in human cartilage is unclear. Here, we determined the expression of entire FGF family in human fetal <em>growth</em> plate cartilage. Using reverse transcriptase PCR, the transcripts for FGF1, 2, 5, 8-14, <em>16</em>-19, and 21 were found. However, only FGF1, 2, 17, and 19 were detectable at the protein level. By immunohistochemistry, FGF17 and 19 were uniformly expressed within the <em>growth</em> plate. In contrast, FGF1 was found only in proliferating and hypertrophic chondrocytes whereas FGF2 localized predominantly to the resting and proliferating cartilage. In addition, only the 18 kD isoform of FGF2 was found in resting chondrocytes while proliferating chondrocytes also synthesized 22 kD and 24 kD FGF2, similar to in vitro cultivated chondrocytes. In cell <em>growth</em> experiments, FGF1, 2, and 17 but not FGF19 inhibited the proliferation of FGFR3-expressing rat chondrosarcoma chondrocytes (RCS) with relative potency FGF2>>) FGF1 = FGF17. We conclude that FGF1, 2, 17, and 19 are the predominant FGF ligands present in developing human cartilage that are, with the exception of FGF19, experimentally capable of inhibiting chondrocyte proliferation.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) may be critical in the development of diabetic nephropathy (DN), and genetic predisposition is an important determinant of DN risk. We evaluated mRNA expression levels of TGF-beta system components in cultured skin <em>fibroblasts</em> (SFs) from type 1 diabetic patients with fast versus slow development of DN. A total of 125 long-standing type 1 diabetic patients were ranked by renal mesangial expansion score (MES) based on renal biopsy findings and diabetes duration. Patients in the highest quintile of MES who were also microalbuminuric or proteinuric (n = <em>16</em>) were classified as "fast-track" for DN, while those in the lowest quintile who were also normoalbuminuric (n = 23) were classsified as "slow-track" for DN. Twenty-five normal subjects served as control subjects. SFs were cultured in medium with 25 mmol/l glucose for 36 h. SF mRNA expression levels for TGF-beta1, TGF-beta type II receptor (TGF-beta RII), thrombospondin-1, and latent TGF-beta binding protein-1 (LTBP-1) were measured by real-time RT-PCR. LTBP-1 mRNA expression was reduced in slow-track (0.99 +/- 0.38) versus fast-track patients (1.65 +/- 0.52, P = 0.001) and control subjects (1.41 +/- 0.7, P = 0.025). mRNA levels for TGF-beta1, TGF-beta RII, and thrombospondin-1 were similar in the three groups. Reduced LTBP-1 mRNA expression in SFs from slow-track patients may reflect genetically determined DN protection and suggests that LTBP-1 may be involved in the pathogenesis of DN through the regulation of TGF-beta activity.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF), a key regulatory <em>factor</em> of cell <em>growth</em> and differentiation, is involved in embryonic development, angiogenesis, and tumorigenesis. To date, four different FGF receptors (FGFRs) have been cloned and characterized. We examined the expression of four FGFRs in human gastric cancer tissues and cell lines using Northern analysis, ribonuclease protection assay, and immunohistochemistry. The mRNAs of FGFR-1 (10/14), FGFR-2 (9/14), and FGFR-4 (9/14) were up-regulated in cancer compared with normal tissues. FGFR-3 mRNAs were barely detectable in both normal and cancer tissues. These FGFR mRNAs were co-expressed in various combinations of two or three in the same tissue. Immunohistochemistry confirmed specific staining of multiple FGFRs, except FGFR-3, in the cancer specimens. To investigate the functional significance of FGFR co-expression we examined the invasive property of SNU-<em>16</em> cells, which exhibited gene amplification of FGFR-2, -3, and -4 as well as over-expression of keratinocyte <em>growth</em> <em>factor</em> receptor (KGFR), a splice variant of FGFR-2, and FGFR-4 mRNA. KGF plus acidic FGF (aFGF), KGF, and aFGF treatment enhanced the invasive potential of SNU-<em>16</em> cells over the control by 100%, 107%, and 47%, respectively, indicating that neither additive nor synergistic effect was induced by stimulation with aFGF plus KGF. These results suggest that co-expression of FGFRs in various combinations may cause subtle changes in the progression of gastric cancer.

The idea that the gene(s) that cause diabetes mellitus can be expressed in extrapancreatic cells has been examined by tissue culture techniques. Skin biopsies were obtained from 25 normal subjects (N), 26 overt diabetics (D), <em>16</em> of juvenile onset (JOD) and 9 of maturity onset (MOD), and 21 subjects genetically predisposed to diabetes (P) on the basis of maturity-onset diabetes in both parents. Each biopsy was subdivided, multiple skin fragments were explanted in vitro, and several parameters of cellular out<em>growth</em> were monitored in primary and secondary cultures until cell division ceased because of senescence. In general, the rank order of <em>growth</em> vigor was N greater than P greater than D although differences were often marginal and statistically significant between N and JOD and(or) MOD. Out<em>growth</em> of epithelial cells was more vigorous in N explants in early stages, but later, JOD and MOD cells grew better than those of N. Out<em>growth</em> of <em>fibroblast</em> cells from N explants was more vigorous both at early and later stages and required less time to achieve maximum percent out<em>growth</em>. In secondary cultures, N cells grew faster than the other three groups so that fewer days elapsed between subcultures but significant differences were only seen between N and one or two of the other groups over some of the first seven subcultures. The onset of cellular senescence occurred earlier in P and JOD cultures both in mean population doublings and calendar time. N cultures had a higher percent surviving clones after picking than MOD, and a shorter recloning time than clones of JOD. The replicative life-spans of cultures (mean population doublings +/- SE) were N = 52.54 +/- 2.24, P = 47.84 +/- 2.43, JOD = 47.12 +/- 2.99, and MOD = 46.40 +/- 4.04, but differences did not reach significance for N vs the other three groups. The data demonstrate that cellular <em>growth</em> is impaired in both JOD and MOD types of cultures and to a generally lesser extent in P cultures. This is consistent with intrinsic genetic defects but the possibility that persistent deleterious effects of in vivo pathophysiology contribute alone or in combination cannot be ruled out. Therefore, the diabetic defect(s) can be expressed in extrapancreatic cells of mesenchymal origin. This system should prove useful in exploring the interplay between genetic and environmental <em>factors</em> in diabetes, the mechanisms(s) of hyperglycemia and other metabolic derangements, and the propensity that affected individuals have to develop degenerative diseases.