<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) have been proved to participate in a wide variety of processes, including <em>growth</em>, differentiation, cell proliferation, migration, sex determination and sex differentiation. The roles of FGF9/<em>16</em>/20 subfamily members in the gonadal development of teleost fish have not yet been reported. Three FGFs (<em>16</em>, 20a and 20b) of the FGF9/<em>16</em>/20 subfamily were cloned from the Nile tilapia by RT-PCR and RACE. Phylogenetic, bioinformatic and syntenic analyses demonstrated that these cloned FGFs are genuine FGF<em>16</em>, 20a and 20b. Our analyses further supported the non-existence of FGF9 ortholog and the existence of two FGF20 paralogs in teleost genomes. Tissue distribution analysis by RT-PCR demonstrated that FGF<em>16</em> was expressed in a wide range of tissues including the testis and ovary, FGF20b in the brain, pituitary, intestine and ovary, but not in the testis, while FGF20a in the brain, pituitary and spleen, but not in the gonad. These results were consistent with the Northern blot analysis. The expression profiles of FGF<em>16</em> and FGF20b during normal and sex reversed gonadal development were investigated by real-time PCR. Both showed much higher expression in the XX ovary and 17 beta-estradiol induced XY ovary compared with the XY testis and fadrozole and tamoxifen induced XX testis, with the highest in both sexes at 120 dah. Strong signals of FGF<em>16</em> and FGF20b were detected in phase II oocytes by in situ hybridization. These data suggest that FGF9/<em>16</em>/20 subfamily is involved in the early oocyte development of the female.

This study aimed to systematically assess the parameter-specific effects of the diode low-level laser on human gingival <em>fibroblasts</em> (HGFs) and human periodontal ligament <em>fibroblasts</em> (HPDLFs). An extensive search was performed in major electronic databases including PubMed (1997), EMBASE (1947) and Web of Science (1956) and supplemented by hand search of reference lists and relevant laser journals for cell culture studies investigating the effect of diode low-level lasers on HGFs and HPDLFs published from January 1995 to December 2015. A total of 21 studies were included after screening 324 independent records, amongst which eight targeted HPDLFs and 13 focussed on HGFs. The diode low-level laser showed positive effects on promoting <em>fibroblast</em> proliferation and osteogenic differentiation and modulating cellular inflammation via changes in gene expression and the release of <em>growth</em> <em>factors</em>, bone-remodelling markers or inflammatory mediators in a parameter-dependent manner. Repeated irradiations with wavelengths in the red and near-infrared range and at an energy density below <em>16</em> J/cm(2) elicited favourable responses. However, considerable variations and weaknesses in the study designs and laser protocols limited the interstudy comparison and clinical transition. Current evidence showed that diode low-level lasers with adequate parameters stimulated the proliferation and modulated the inflammation of <em>fibroblasts</em> derived from human periodontal tissue. However, further in vitro studies with better designs and more appropriate study models and laser parameters are anticipated to provide sound evidence for clinical studies and practice.

OBJECTIVE

We present the main morphological modifications in the human gubernaculum during testicular migration in humans.

METHODS

We obtained 12 gubernacula from fresh, macroscopically normal human fetuses at 15 to 29 weeks of gestation. Collagen was evidenced using trichrome and Sirius red staining procedures, while Weigert's resorcinol-fuchsin and anti-human elastin antibody were used to reveal elastic system fibers. Smooth muscle cells were detected by anti-human smooth muscle alpha-actin antibody.

RESULTS

When the testes were still located in the abdomen at 15 to <em>16</em> weeks of gestation, collagen fibers were sparse and embedded in a loose extracellular matrix. The amount of fibers then gradually increased with age and at 28 weeks of gestation the gubernaculum was mostly collagenous in composition. Elastic fibers had a similar <em>growth</em> pattern, although they were located mainly at the distal end of the gubernaculum. <em>Fibroblasts</em> largely predominated over other cell types and decreased in number with gestational age, whereas smooth muscle cells were restricted to the walls of blood vessels. Striated muscle cells were detected at the scrotal end of the gubernaculum, where they were disposed as isolated and scattered bundles running in various directions. Like <em>fibroblasts</em>, their number also decreased with age.

CONCLUSIONS

During testicular migration gubernacular connective tissue undergoes extensive remodeling and ultimately becomes an essentially fibrous structure rich in collagen and elastic fibers. Such changes should decrease the size of the gubernaculum and, thus, contribute to other forces that cause the testes to move toward the scrotum. In fact, because of the lack of smooth muscle cells, and the amount and organization of striated muscle cells, active contraction of the gubernaculum is less likely to be an important factor in testicular descent.

OBJECTIVE

Interruption of the enterohepatic circulation of bile acids induces hepatic bile acid synthesis, increases hepatic cholesterol demand, and increases clearance of apoB-containing lipoproteins in plasma. Based on these effects, bile acid sequestrants have been used for many years to treat hypercholesterolemia and the associated atherosclerosis. The objective of this study was to determine the effect of blocking ileal apical versus basolateral membrane bile acid transport on the development of hypercholesterolemia and atherosclerosis in mouse models.

RESULTS

ApoE(-/-) and Ldlr(-/-) mice deficient in the apical sodium-dependent bile acid transporter (Asbt) or apoE(-/-) mice deficient in the basolateral bile acid transporter (Ostα) were fed an atherogenic diet for <em>16</em> weeks. Bile acid metabolism, cholesterol metabolism, gene expression, and development of atherosclerosis were examined. Mice deficient in Asbt exhibited the classic response to interruption of the enterohepatic circulation of bile acids, including significant reductions in hepatic and plasma cholesterol levels, and reduced aortic cholesteryl ester content. Ileal <em>Fibroblast</em> <em>Growth</em> <em>Factor</em>-15 (FGF15) expression was significantly reduced in Asbt(-/-)apoE(-/-) mice and was inversely correlated with expression of hepatic cholesterol 7-hydroxylase (Cyp7a1). Ileal FGF15 expression was directly correlated with plasma cholesterol levels and aortic cholesterol content. In contrast, plasma and hepatic cholesterol levels and atherosclerosis development were not reduced in apoE(-/-) mice deficient in Ostα.

CONCLUSIONS

Decreases in ileal FGF15, with subsequent increases in hepatic Cyp7a1 expression and bile acid synthesis appear to be necessary for the plasma cholesterol-lowering and atheroprotective effects associated with blocking intestinal bile acid absorption.

BACKGROUND

Coronary calcifications (CC) portend increased mortality in adults receiving hemodialysis (HD), however the risk factors associated with CC progression are not well known in pediatric patients. Our previous cross-sectional studies demonstrated high CC prevalence (31 %) in pediatric patients, which were significantly associated with high serum phosphorus (P), fibroblast growth factor 23 (FGF) levels, dialysis vintage, and low cholesterol. The current study was undertaken to determine and elucidate CC progression in pediatric HD patients.

METHODS

A 1-year prospective longitudinal study of 16 pediatric patients (ten male; mean age, 16.9 ± 3 years; range, 10.1-20.4 years) receiving chronic HD was conducted.

RESULTS

CC were observed in five of 16 (31.3 %) patients on baseline computed tomogram (CT) scan; 14/16 patients underwent 1-year CT. All patients with initial CC who completed CT at 1 year (3/5) progressed; one patient had new CC and none of the patients had resolved CC. Mean Agatston score increased from 23.4 ± 18.06 HU (baseline) to 169 ± 298.9 HU. Patients with CC progression had higher mean serum P (8.6 ± 1.8 mg/dl vs. 6.3 ± 1.1 mg/dl, p = 0.015) and FGF 23 levels (3,994 ± 860.5 pg/ml vs. 2,327 ± 1,206.4 pg/ml, p = 0.028). Serum P and FGF 23 levels were positively correlated with final Agatston scores (R = 0.65, p = 0.01 for serum P and R = 0.54, p = 0.045 for FGF 23) and change in Agatston scores (R = 0.65, p = 0.01 for serum P and R = 0.52, p = 0.048 for FGF 23).

CONCLUSIONS

Our study shows that CC is progressive in pediatric patients receiving HD and that increased serum P and FGF 23 levels are associated with this progression.

The vertebrate brain is regionalized during development into forebrain, midbrain and hindbrain. <em>Fibroblast</em> <em>growth</em> <em>factor</em> 8 (FGF8) is expressed in the midbrain/hindbrain boundary (MHB) and functions as an organizer molecule. Previous studies demonstrated that the brain of basal chordates or ascidians is also regionalized at least into fore/midbrain and hindbrain. To better understand the ascidian brain regionalization, the expression of the Ciona Fgf8/17/18 gene was compared with the expression of Otx, En and Pax2/5/8 genes. The expression pattern of these genes resembled that of the genes in the vertebrate forebrain, midbrain, MHB and hindbrain, each of those domains being characterized by sole or combined expression of Otx, Pax2/5/8, En and Fgf8/17/18. In addition, the putative forebrain and midbrain expressed Ci-FgfL and Ci-Fgf9/<em>16</em>/20, respectively. Therefore, the regionalization of the ascidian larval central nervous system was also marked by the expression of Fgf genes.

The type-1 inhibitor of plasminogen activator (PAI-1) is a major physiologic regulator of pericellular proteolytic activity and, as such, influences matrix integrity, cell-to-substrate adhesion, and cellular proliferation. Excessive accumulation of both PAI-1 mRNA and protein correlates with the progressive acquisition of morphological and <em>growth</em> traits characteristic of the senescent phenotype (Mu and Higgins, 1995, J. Cell. Physiol., <em>16</em>5:647-657). Compared to early-passage IMR-90 human diploid <em>fibroblasts</em>, a late-passage senescence-associated 11-fold elevation in steady-state PAI-1 mRNA content reflected a 15-fold increase in constitutive PAI-1 gene transcription. Differential mRNA stability was not a <em>factor</em> in age-associated PAI-1 overexpression in IMR-90 cells. Upon removal of serum, early-passage human <em>fibroblasts</em> enter into a state of <em>growth</em> arrest with marked down-regulation of PAI-1 synthesis. Rapid induction of both the 3.0- and 2.2-kb PAI-1 mRNA species was evident upon serum-induced "activation" of quiescent early-passage <em>fibroblasts</em>; induced PAI-1 transcripts were maximal at 2 hr post-serum stimulation and declined in late G1 prior to entry into S phase. In contrast, late-passage (p32) <em>fibroblasts</em> maintained a significant level of PAI-1 expression under serum-free culture conditions. Although the PAI-1 gene was further responsive to serum in senescent cells, transcript abundance remained elevated and actually increased over the 12 to <em>16</em> hr post-serum addition period (a time when early-passage <em>fibroblasts</em> down-regulate PAI-1 mRNA content). Development of the senescent phenotype in human <em>fibroblasts</em> is associated, therefore, with significant changes in PAI-1 gene regulation. Such reprogramming involves predominantly transcriptional events and results in a marked increase in steady-state PAI-1 transcript abundance involving both the 3.0- and 2.2-kb mRNA species.

BACKGROUND

It is now clear that the progression of renal disease is closely correlated to the degree of renal interstitial fibrosis. We have previously demonstrated that the renal proximal tubular epithelial cell may contribute to the fibrotic response by the generation of profibrotic cytokines. Transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) are two of a group of profibrotic cytokines that have been associated with the development of renal interstitial fibrosis. In this study, we have examined the influence of TGF-beta1 on the generation of bFGF by renal tubular epithelial cells.

METHODS

HK2 cells were grown to confluence and were serum deprived and stimulated with recombinant TGF-beta1 under serum-free conditions. Subsequently, supernatant, cell-associated, intracellular, and matrix-associated bFGF concentrations were determined by enzyme-linked immunosorbent assay (ELISA). bFGF mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS

The exposure of confluent serum-deprived HK2 cells to TGF-beta1 led to a significant increase in bFGF concentration in the cell culture supernatant. Twenty-four hours following the addition of 10 ng/ml TGF-beta1, this represented a twofold increase in bFGF concentration (control, 102 pg/ml, N = 24, vs. 202 pg/ml, N = 19, P = 0.0001). Despite the increase in bFGF concentration in the supernatant, there was no change in the expression of bFGF mRNA following the addition of TGF-beta1. The addition of 10 ng/ml of TGF-beta1 led to a 30% decrease in the total cell-associated bFGF concentration (control, 8.51 ng/ml, N = 16, TGF-beta1, 6.01 ng/ml, N = 13, P = 0.0042). This decrease in intracellular bFGF was associated with a 15% reduction in anti-bFGF antibody binding to fixed permeabilized cells, following the addition of 10 ng/ml of recombinant TGF-beta1 (N = 9, P = 0.0007), suggesting that the mechanism of stimulation of bFGF by TGF-beta1 involved the release of preformed bFGF from within the cells. In addition, following the addition of TGF-beta1, there was a significant dose-dependent decrease in the amount of bFGF sequestered in the extracellular matrix. At a dose of 10 ng/ml TGF-beta, this represented a greater than sevenfold decrease (N = 9, P = 0.0007) in matrix-bound bFGF, although this represented less than 3% of the total bFGF released into the supernatant.

CONCLUSIONS

The data presented suggest that the main mechanism by which TGF-beta1 stimulates bFGF generation by proximal tubular epithelial cells is by stimulation of the secretion of preformed cytokine from within the cells.

Bovine brain-derived <em>growth</em> <em>factor</em> (BDGF), a <em>16</em>-17 kDa protein with biochemical properties resembling brain-derived acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (acidic FGF) and endothelial cell <em>growth</em> <em>factor</em>, was found to have potent chemotactic activity for bovine ligament <em>fibroblasts</em>, human skin <em>fibroblasts</em> and rat astroglial cells, maximal at 100-200 pg/ml. The chemotactic activity was completely blocked by protamine sulfate (5 ug/ml), an inhibitor of receptor-binding and mitogenic activity of BDGF. BDGF did not stimulate migration of human monocytes. These results indicate that the effects of BDGF 'in vivo' might extend to mesenchymal cell recruitment.

Human papillomaviruses (HPV) have been etiologically linked to human cervical cancer. Transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) is a cytokine which is a potent <em>growth</em> inhibitor of most epithelial, endothelial, lymphoid, and myeloid cells, but is mitogenic for mesenchymal cells and bone cells. In this study, we analyzed the effects of HPV <em>16</em> oncoproteins E6 and E7 on the TGF-beta 1 promoter. The results showed that the HPV <em>16</em> E6 significantly induced (sixfold) the TGF-beta 1 promoter activity while HPV <em>16</em> E7 showed no significant effect. The E6 effect was cell type-specific and was observed only in the <em>fibroblast</em> cell lines, not in epithelial cells. Promoter analysis revealed that a 9-bp sequence, GGGGCGGGG, representing the consensus Sp1-binding site between -109 and -100 of the TGF-beta 1 promoter, was the major target for E6-mediated transactivation. Mutation analysis of the E6 polypeptide showed that the retention of amino acids between 123 and 136 of the HPV <em>16</em> E6 protein was critical for the transactivation of the TGF-beta 1 promoter. Previous studies have shown that the adenovirus 12S E1A oncoprotein represses the TGF-beta 1 promoter by targeting an adjacent (-90 to -81) but different GC-rich sequence (TGGGTGGGG). These studies provide evidence that variant GC-rich promoter elements are not functionally identical and are differentially regulated by the DNA virus oncoproteins.

BACKGROUND

Airway angiogenesis may be an important part of structural remodelling in the pathogenesis of asthma. The development of asthma is frequently preceded by rhinitis.

OBJECTIVE

We sought to determine whether the levels of angiogenesis-related factors are elevated in airways of patients with rhinitis or controlled asthma.

METHODS

We analysed the induced sputum of 18 rhinitis patients, 16 asthmatic patients, and 15 healthy controls. The concentrations of angiogenin, vascular endothelial growth factor (VEGF), IL-8, fibroblast growth factor (bFGF), and TNF-alpha were measured by cytometric bead arrays.

RESULTS

We found significantly increased angiogenin and VEGF concentrations in the induced sputum supernatant of both rhinitis and asthma patients compared with that of the healthy control group (P< or =0.0005). With the exception of TNF-alpha, there was no difference in the other angiogenic factors; TNF-alpha levels were higher in the rhinitis group than in the control group (P=0.02).

CONCLUSIONS

These in vivo results suggest increased airway angiogenesis in patients with rhinitis without asthma as well as in corticosteroid-treated and well-controlled asthma patients.

Vascular participation manifested by a runny nose (rhinorrhea) is a prominent feature of the acute consequences of rhinovirus infection. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is an angiogenic <em>factor</em> that also induces potent increases in vascular permeability; it is a candidate mediator of rhinorrhea in response to rhinovirus infection as well as contributing to enhanced vascular leakage in rhinovirus-linked asthma exacerbations. It has been shown that rhinovirus induces significant increases in both VEGF protein and mRNA in primary airway <em>fibroblasts</em> [Ghildyal et al. (2005): J Med Virol 75:608-615]. The current studies assessed VEGF responses to rhinovirus in primary culture airway epithelium, in epithelial and <em>fibroblast</em> cell lines and in rhinovirus-infected nasal secretions. Epithelial and <em>fibroblast</em> cells were infected with rhinovirus serotype <em>16</em> and VEGF protein and isoforms assessed by ELISA and RT-PCR, respectively. VEGF protein was released by both epithelial and <em>fibroblast</em> cell lines and primary airway epithelial cells in culture but was not increased following rhinovirus infection. PCR products coding for four or five of the six known VEGF isoforms were produced (121, 145, <em>16</em>5 and 183, and/or 189 amino acids) in cell lines and primary culture cells, but no specific isoform was linked to rhinovirus infection. Nasal VEGF was also measured in a cohort of asthmatics with verified rhinovirus and respiratory syncytial virus (RSV) infection. VEGF was not raised following rhinovirus infection alone, but was increased significantly if concomitant RSV infection was present. The data suggest that <em>fibroblasts</em> rather than the epithelium may play a key role in VEGF mediated vascular responses after rhinovirus infection. This may aid recruitment of inflammatory cells and contribute to airway inflammation and bronchial obstruction.

Ischemia-induced angiogenic response is reduced in spontaneously hypertensive rats (SHR). To study whether exogenous basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) infusion is effective in expanding collateral circulation in frankly hypertensive SHR, femoral arteries of male SHR (weighing approximately 250 g) were kept intact (nonoccluded control; n = 9) or occluded for 4h(n = 12) or for <em>16</em> days with vehicle (n = 14) or bFGF [0.5 (n = 17), 5.0 (n = 13), and 50.0 (n = 14) microg. kg-1. day-1 for 14 days] intraarterially. Maximal collateral-dependent blood flows (BF) to the hindlimbs were determined with 85Sr- and 141Ce-labeled microspheres during running at 20 and 25 m/min (15% grade). Preexercise heart rates (approximately 530 beats/min) and blood pressures (BP; approximately 200 mmHg) were similar across groups except in the high-dose bFGF group, where BP was reduced by approximately 12% (P < 0.05). Femoral artery occlusion for 4 h resulted in approximately 95% reduction of BF in calf muscles [199 +/- 18.7 (nonoccluded group) to 10 +/- 1.0 ml. min-1. 100 g-1; P < 0.001]. BF to calf muscles of the vehicle and low-dose bFGF (0.5 microg. kg-1. day-1) groups increased to 36 +/- 3.2 and 45 +/- 2.0 ml. min-1. 100 g-1, respectively (P < 0.001). bFGF infusion at 5.0 and 50.0 microg. kg-1. day-1 further increased (P < 0.001) BF to calf muscles (62 +/- 4.6 and 62 +/- 2.2 ml. min-1. 100 g-1, respectively). Our results show that bFGF can effectively increase BF in hypertensive rats. The reduced hypertension with high-dose bFGF suggests that a critical signal in arteriogenesis (nitric oxide bioavailability) may be restored. These findings suggest that the dulled endothelial nitric oxide synthase of SHR does not preempt collateral vessel remodeling.

BACKGROUND

Poor healing of the sternum often limits the use of bilateral internal thoracic arteries (BITAs) after coronary bypass surgery in diabetic patients. We have reported that a gelatin sheet that incorporates basic fibroblast growth factor (bFGF) accelerates sternal healing after BITA removal in normal rats. This study evaluated the effects of the above method for sternal healing in diabetic animals.

RESULTS

Diabetic Wistar rats with blood glucose levels >400 mg/dL and body-weight loss >20 g were established by a single intravenous injection of streptozotocin (55 mg/kg). After median sternotomy and BITA removal, 16 diabetic rats received either a gelatin sheet that incorporated bFGF (100 microg/sheet) on the posterior table of the sternum (FGF group, n=9) or no gelatin sheet (control, n=7). Peristernal blood flow, as measured by a noncontact laser Doppler 4 weeks after surgery in the FGF group, recovered to the preoperative level (106+/-10% versus 82+/-9%, P<0.01), and marked angiogenesis was also observed around the sternum in the FGF group (30.5+/-3.2 versus 15.8+/-2.7 vessels/unit area, P<0.01). Deep sternal wound complications developed in 5 control rats but only in 1 rat in the FGF group (P<0.05). In the FGF group, histological examination showed improved sternal healing (excellent in 6 rats and slow/poor healing in 3). Bone mineral content as assessed by dual-energy x-ray absorptometry was greater in the FGF group (75.9+/-18.1 versus 48.9+/-10.7 mg, P<0.05). Bone mineral density of the sternum was similar between the 2 groups.

CONCLUSIONS

A gelatin sheet that incorporates bFGF may offset sternal ischemia and accelerate sternal bone regeneration and healing, even in diabetic patients.

The survival of cultured mouse hippocampal neurons was found to be greatly enhanced by micromolar concentrations of the excitatory neurotransmitter glutamate. Blockade of kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptors increased the rate of neuron death, suggesting that endogenous glutamate in the cultures promotes survival. Addition of glutamate (0.5-1 microM) further increased neuron survival, whereas glutamate in excess of 20 microM resulted in increased death. Thus, the survival vs. glutamate dose-response relation is bell-shaped with an optimal glutamate concentration near 1 microM. We found that hippocampal neurons from mice with the genetic defect trisomy <em>16</em> (Ts<em>16</em>) died 2-3 times faster than normal (euploid) neurons. Moreover, glutamate, at all concentrations tested, failed to increase survival of Ts<em>16</em> neurons. In contrast, the neurotrophic polypeptide basic <em>fibroblast</em> <em>growth</em> <em>factor</em> did increase the survival of Ts<em>16</em> and euploid neurons. Ts<em>16</em> is a naturally occurring mouse genetic abnormality, the human analog of which (Down syndrome) leads to altered brain development and Alzheimer disease. These results demonstrate that the Ts<em>16</em> genotype confers a defect in the glutamate-mediated survival response of hippocampal neurons and that this defect can contribute to their accelerated death.

BACKGROUND

Hair greying is an obvious sign of ageing in humans. White (nonpigmented) hair is thicker than black (pigmented) hair. The growth rate of white hair is also significantly higher than that of black hair. However, the mechanism underlying this is largely unknown.

OBJECTIVE

To examine the association between hair greying and hair growth patterns by evaluating expression of the genes or proteins related to hair growth in white and black hairs.

METHODS

Morphological characteristics were observed in eyebrow and scalp hairs. The differential expression of genes was analysed in black and white hairs from human scalp by a microarray analysis. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry for genes and proteins related to hair growth were performed in black and white hairs.

RESULTS

Keratin and keratin-associated protein (KRTAP) genes in white hair were upregulated at least two-fold in comparison with black hair in a microarray analysis. Upregulation of selected keratin genes and KRTAP4 isoform genes in white hair was validated by RT-PCR. Immunoreactivity for KRT6, KRT14/16 and KRT25 was increased in the hair follicle of white hair compared with black hair. Gene expression of fibroblast growth factor 5 (FGF5) was downregulated in white hair compared with black hair. However, gene expression of FGF7 was upregulated in white hair compared with black hair.

CONCLUSIONS

Expression of genes and proteins associated with active hair growth is upregulated in white (nonpigmented) hair compared with black (pigmented) hair. These results suggest that hair greying is associated with active hair growth.

Prenatal exposure to ethanol is the cause of fetal alcohol syndrome, which is characterized by brain abnormalities and decreased mental capacity. In the current study, cultured neurons from embryonic rat cortices were used to study the reversal of ethanol toxicity on neuronal survival and neurite out<em>growth</em>. Ethanol treatment followed by treatment with estrogen and certain <em>growth</em> <em>factors</em> were used to assess the potential of these <em>growth</em> <em>factors</em> and estrogen to reverse the effects of ethanol damage. Cortical neurons from embryonic day (E) <em>16</em> rats were grown in defined medium with a glial plane at a distance of 1mm from the neurons. Ethanol (45 mM) was administered on day in vitro 1 (DIV 1) and DIV 4. Insulin-like <em>growth</em> <em>factor</em>-I (IGF-I, 10 ng/ml), insulin-like <em>growth</em> <em>factor</em>-II (IGF-II, 10 ng/ml), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 5 ng/ml), nerve <em>growth</em> <em>factor</em> (NGF, 100 ng/ml), and estrogen (Es, 10 ng/ml) were administered on DIV 4 and DIV 5. Cell viability was determined on DIV 6 using the intravital dyes fluorescein diacetate and propidium iodide. IGF-I and bFGF reduced ethanol's toxic effect on neuronal survival. Estrogen, bFGF, and NGF increased total neurite length after ethanol treatment. Although none of the treatments had a statistically significant effect on the mean number of primary neurites, all caused a statistically significant increase in the mean number of secondary neurites per cell (a measure of neuritic branching) relative to the ethanol treatment alone.

Neurons from various brain regions of postnatal (15 days after birth) and fetal (<em>16</em> days gestation) rats were cultured in the presence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). bFGF increased the survival of neurons from postnatal septum, striatum, midbrain, and hippocampus. Fetal neurons derived from cerebral cortex, septum, striatum, midbrain, thalamus, and colliculus were far more dependent on bFGF for survival in comparison with postnatal neurons. In contrast, cerebellum neurons of postnatal and fetal rat brain did not respond to bFGF. The increase of postnatal and fetal neuronal survival with bFGF treatment (0.01-10 ng/ml) was dose-dependent and reached 2-4-fold and 5-10-fold more than the control, respectively. Fetal cortical neurons showed almost complete dependence on bFGF since almost all neurons died in control cultures. Nerve <em>growth</em> <em>factor</em> was slightly effective only on postnatal septal and striatal neurons, being ineffective on the other neurons tested. These results indicate that bFGF can function as a neurotrophic <em>factor</em> not only on fetal but also on postnatal neurons of the central nervous system, and that bFGF has great potential for application in vivo.

By using a recently modified method of isolating and culturing rabbit corneal cells, this study investigated the presence of a diffusible substance(s) in stromal <em>fibroblast</em> conditioned medium that stimulated the <em>growth</em> of cultured corneal epithelial cells. The <em>growth</em> stimulation involved initiation of DNA synthesis (assayed by [3H]-thymidine incorporation) and enhanced cell proliferation (quantified by cell counting). Among the three corneal cell types, only <em>fibroblasts</em> (rabbit and human) released the stimulatory substance, which acted only on epithelial cells. The effect of this stromal <em>fibroblast</em> <em>factor</em> (SFF) was observed after an exposure period of less than <em>16</em> hr and persisted as long as it was present. Its action was concentration-dependent and was not a result of improvement in the survival of epithelial cells during culture. Both sparse and confluent epithelial cultures were susceptible to SFF. The release of SFF was correlated with the number of <em>fibroblasts</em> in the culture and appeared to be sensitive to the <em>growth</em> condition of the cells. Both the release and action of SFF did not depend on the presence of serum in the culture medium. The <em>factor</em> was heat resistant and insensitive to proteolytic enzymes. From ultrafiltration studies, the size of SFF was estimated to be in the approximate range of 50-1000 daltons. By direct comparison of the stimulatory effect with other previously studied <em>growth</em> promoting agents, it was concluded that SFF was not epidermal <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, putrescine, cyclic AMP, hydrocortisone or acetate. The implication of SFF in the regulation of epithelial <em>growth</em> by endogenous, intercellular mechanisms is discussed.

BACKGROUND

Despite numerous articles on the use of artificial nerve conduits, autologous nerve transplants remain the most effective for nerve repair. To improve this technique, the authors examined conduits containing gelatin hydrogel as a carrier enabling the sustained release of basic fibroblast growth factor (bFGF).

METHODS

To confirm sustained bFGF release in vivo, nerve-guide tubes containing iodine-125-labeled bFGF with or without gelatin hydrogel were implanted under the skin of mice, and the remaining radioactivity was measured. Next, a 15-mm segment of the sciatic nerve was resected and repaired with autologous nerve (group 1), a tube with gelatin hydrogel and bFGF (group 2), a tube with bFGF alone (group 3), or a tube only (group 4). Histologic and functional analyses were performed for 16 weeks after surgery.

RESULTS

The radioactivity from iodine-125-labeled bFGF incorporated into gelatin hydrogel decreased more slowly than iodine-125-labeled bFGF alone. Four weeks after surgery, significantly more regenerating axons were detected in group 2 than in groups 3 and 4, but the axonal density in group 2 was lower than in group 1. Similarly, the animals in group 2 showed significantly better motor performance than those in groups 3 and 4, but worse than those in group 1. The animals in groups 1 and 2 showed significantly better sensory recovery than those in groups 3 and 4.

CONCLUSIONS

The nerve-guide tube containing gelatin hydrogel and bFGF promoted axonal regeneration after peripheral nerve injury, but not as well as autologous transplants. Understanding the limitations of this technique will facilitate its improvement for clinical applications.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone-like matrix. During the initial <em>growth</em> period (days 1-10), addition of acidic FGF (100 micrograms/ml) to actively proliferating cells increased (P < 0.05) 3H-thymidine uptake (2,515 +/- 137, mean +/- SEM vs. 5,884 +/- 818 cpm/10(4) cells). During the second stage of maturation (days 10-15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days <em>16</em>-29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14-29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF-induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell <em>growth</em> and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7-8) enhanced histone H4, osteopontin, type I collagen, and TGF-beta mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14-15) reactivated H4, osteopontin, type I collagen, and TGF-beta gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 micrograms/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose-related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF-beta and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) is a multifunctional protein involved in the control of proliferation, differentiation and other functions in many cell types. The anchorage-independent <em>growth</em> of some established lines of untransformed <em>fibroblasts</em> in soft agar is induced by TGF-beta and requires in addition exogenous EGF for certain target cells, notably rat NRK-49 cells. The formation of colonies of NRK-49F cells is completely inhibited by the synthetic 11-beta substituted nor-steroid RU38486 added at a final concentration of 1.3 X 10(-5) M. We also explored the effect of TGF-beta on Daudi and Raji lymphoma cells by measuring the production of Epstein-Barr Virus (EBV) early antigens (EA). In Daudi cells an induction capacity giving rise to 10-<em>16</em>% positive EA-cells was observed; in Raji cells the induction only reached between 6 and 8%. The induction was partially inhibited by the anti-steroid RU38486 in both systems. Thus, RU38486 not only antagonizes the glucocorticoid hormone action but also interferes with the effects of TGF-beta in <em>fibroblasts</em> and in lymphoma cells. The molecular basis of the interactions observed was investigated by considering (1) the binding to specific receptors, (2) transfection experiments, in order to examine if the interference of the anti-steroid with TGF-beta activities occurs at the transcriptional level as in the case of glucocorticoid induction. The results suggest that the blocking by antiglucocorticoids of the effects of TGF-beta and glucocorticoids, in <em>fibroblasts</em> and lymphoma cells, occurs by different mechanisms.

The wound healing process concludes with downregulation of <em>fibroblast</em> activity. Clinical observations suggest that the regenerating epidermis suppresses this activity. An important regulator of <em>fibroblast</em> activity is the fibrogenic cytokine connective tissue <em>growth</em> <em>factor</em>. We hypothesized that epidermal keratinocytes may affect <em>fibroblast</em> activity via this cytokine. We demonstrate keratinocyte-mediated suppression of connective tissue <em>growth</em> <em>factor</em> at both the mRNA and protein levels by around 50% or more when <em>fibroblasts</em> were cultured in multiwell plates with keratinocyte cultures in accompanying semipermeable cell culture inserts, or stimulated by keratinocyte-conditioned media. Both basal and transforming-<em>growth</em>-<em>factor</em>-beta1-stimulated levels of connective tissue <em>growth</em> <em>factor</em> were inhibited. A 3 h coculture period with keratinocytes was sufficient to suppress connective tissue <em>growth</em> <em>factor</em> expression by <em>fibroblasts</em>, but the inhibition developed over a time period of around <em>16</em> h. The putative keratinocyte-derived <em>factor</em>(s) responsible for these effects was found to be soluble and stable. By analyzing cytokines secreted by keratinocytes we identified interleukin-1alpha as a potent inhibitor of connective tissue <em>growth</em> <em>factor</em> mRNA expression in <em>fibroblasts</em>. Involvement of this cytokine in keratinocyte-mediated connective tissue <em>growth</em> <em>factor</em> suppression was confirmed by using anti-interleukin-1alpha antibodies. Tumor necrosis <em>factor</em> alpha or prostaglandins did not appear to be involved. In conclusion, our results indicate that interleukin-1alpha secretion by keratinocytes provides a mechanism for the downregulation of connective tissue activity during the end-stage of wound healing, when epithelia coverage has developed over the wound area.

SPARC (Secreted Protein, Acidic, Rich in Cysteine/osteonectin/BM-40) is a highly conserved metal-binding extracellular matrix (ECM) glycoprotein which is first expressed by Xenopus embryos during late gastrulation/early neurulation (stage 12/13), by presumptive notochord and somitic cells. When animal cap explants of stage 9 embryos were cultured in vitro, SPARC expression was not detected until sibling embryos reached late neurula stage (stage 19). Addition of activin, a potent dorsal mesoderm inducer, to animal caps resulted in SPARC being expressed by the time sibling embryos reached stage <em>16</em>. While basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a ventral mesoderm inducer, had modest effects on SPARC mRNA expression, the combination of both activin and bFGF was synergistic. The appearance, however, of SPARC transcripts 11 h after the addition of activin and bFGF, indicates that unknown intermediates were likely to be involved in activating SPARC expression. In order to identify the potential intermediate regulatory <em>factors</em> which may activate and control SPARC expression, we examined the genomic organization of the 5' end of the Xenopus SPARC gene. No significant homology to the equivalent region that is highly conserved in the mouse, bovine and human SPARC genes was observed. Thus, while mammalian SPARC promoters lack TATA or CAAT boxes, the Xenopus gene contains a consensus TATA box. Moreover, promoter-proximal GGA-box repeats necessary for high level expression of mammalian SPARC are absent in Xenopus. When reporter constructs containing the 5' flanking region of the Xenopus gene were microinjected into two-cell embryos, 868 bp of 5' flanking DNA was sufficient to mimic the temporal and tissue-specific pattern of SPARC expression observed in whole embryos. While a bovine SPARC promoter reporter construct containing 740 bp of the 5' flanking DNA was expressed at a significant level in Xenopus embryos, significant differences in the cell-type expression of the reporter genes were obtained between the bovine and Xenopus constructs. The data indicate that zygotic activation of SPARC mRNA is mediated by regulatory <em>factors</em> acting downstream of major mesoderm induction events. The high DNA sequence conservation at the 5' end of mammalian SPARC genes is not conserved in Xenopus. These differences led to differences in their ability to direct tissue-specific gene expression in early Xenopus embryos.