FGF-2 mRNA Levels Research Articles

Roles of exogenous and endogenous FGF-2 in animal models of depression

Several members of the fibroblast growth factor (FGF) family have been shown to be dysregulated in individuals with major depression, and treatment with antidepressants has been reported to increase FGF-2 mRNA levels in the forebrain. We have used the tail suspension test (TST), and olfactory bulbectomy (OBX), and FGF-2 deficient mice to investigate putative roles of FGF-2 as an antidepressant and mediator of antidepressive drug actions. FGF-2 applied intraventricularly generated antidepressant-like effects in the TST. FGF-2, similar to the antidepressant amitriptyline, attenuated neuron demise in the piriform cortex and posterolateral cortical nucleus of the amygdala following OBX. Moreover, OBX induced reduction in hippocampal neurogenesis could be ameliorated by subsequent treatment with either amitriptyline or FGF-2. Furthermore, FGF-2 was effective in reversing depressive-like behavior induced by OBX, monitored in the locomotor activity and the passive avoidance test. In bulbectomized FGF-2 deficient mice, treatment with amitriptyline protected neurons, but failed to reverse behavioral alterations. Together, these results suggest that FGF-2 constitutes both a potential target for antidepressive treatments and an important growth factor in the cytokine network underlying the actions of antidepressive drugs. The results further suggest a requirement of endogenous FGF-2 for mediating behavioral, but not neuroprotective actions of amitriptyline.

Elevated extracellular calcium increases fibroblast growth factor-2 gene and protein expression levels via a cAMP/PKA dependent pathway in cementoblasts

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Elevated levels of extracellular Ca 2+ have been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of extracellular Ca 2+ signaling in cementogenesis has not been examined. Using RT-PCR, we found that elevated levels of extracellular Ca 2+ increase fibroblast growth factor (FGF)-2 gene expression with a peak at 6 h. Pretreatment with a protein kinase A (PKA) inhibitor, H89, or an adenylate cyclase inhibitor, MDL-12,330A, inhibited Ca 2+-stimulated Fgf-2 expression. In contrast, pretreatment with the protein kinase C (PKC) inhibitor GF-109203X or the phospholipase C (PLC) inhibitor U73122 did not affect the expression of Fgf-2 transcripts, suggesting that the increase in Fgf-2 expression was dependent on the PKA but not the PLC/PKC signaling pathway. Treatment with an activator of adenylate cyclase, forskolin, or a cell-permeable analog of cAMP, 8-Br-cAMP, enhanced Ca 2+-stimulated Fgf-2 expression, but a single treatment with forskolin or 8-Br-cAMP did not, suggesting that cAMP generation is indispensable but not sufficient for Ca 2+-stimulated FGF2 expression. Next, we examined the cation specificity of the putative receptor and showed that treatment with trivalent/divalent inorganic ions, Ca 2+, Gd 3+, Sr 2+, or Al 3+, caused a dose-dependent increase in Fgf-2 mRNA levels in a cAMP-dependent fashion, whereas Mg 2+ and the organic ions neomycin and spermine had no effect on Fgf-2 gene expression levels. These findings suggest that an extracellular Ca 2+-sensing mechanism is present in cementoblasts and its activation leads to FGF-2 stimulation in a cAMP/PKA dependent fashion. Understanding the pathway regulating key genes involved in modulating the regeneration of oral tissues will assist in designing regenerative therapies based on reliable biological principles.

Dysregulation of Leptin and Testosterone Production and Their Receptor Expression in the Human Placenta with Gestational Diabetes Mellitus

Whether the placenta contributes to some of the abnormal hormonal profiles in gestational diabetes mellitus (GDM) pregnancies and whether GDM affects placental endocrine signaling pathways are yet to be established. The objective of this study was to investigate differences in the expression of the placental steroid and peptide hormone synthesis-related factors, enzymes and their receptors between normal and GDM pregnancies. Nine term placentae from GDM pregnancies and twelve from healthy pregnancies were collected. The results of immunohistochemistry, Western blotting and semiquantitative RT-PCR indicated that mRNA and protein levels of leptin, leptin receptors, androgen receptor and FGF2 were significantly higher in the GDM placentae than non-GDM placentae; while NRIH3, NRIH2, StARD3, CYP11A1, HSD3B, HSD11B, HSD17B, ERα, ERβ, progesterone receptor, FGF receptor-2, insulin receptor-α and -β showed no differences. Interestingly, Western blotting and immunohistochemistry revealed that aromatase protein concentrations in the GDM placentae were significantly reduced without a change in mRNA levels. Moreover, androgen upregulated FGF2 expression in the placental villous explants. These findings suggest that the placentae of GDM pregnancies contribute to elevated testosterone and leptin levels due to a decrease in the conversion of testosterone to estrogens and to an increase in leptin production. The androgen and leptin signaling pathways may be over-activated by the presence of excessive ligands and overexpressed receptors in GDM placentae. Dysregulation of these two endocrine networks may contribute to placental abnormalities eventually increasing the frequency of maternal and fetal complications associated with GDM.

Autocrine/Paracrine Action of Vitamin D on FGF23 Expression in Cultured Rat Osteoblasts

To explore the local mechanisms of fibroblast growth factor (FGF) 23 regulations, we examined the FGF23 expression patterns in an osteoblast culture model. The characteristics of cultured rat calvaria osteoblasts in half-confluence, confluence, osteoid deposition, and osteoid mineralization stages might reflect the proliferation, differentiation, maturation, and matrix mineralization stages, respectively. Compared with proliferating cells in half-confluence, FGF23 expression was upregulated by 7.5-fold at the mRNA level and 126% at the protein level in confluent differentiated cells as determined by real-time RT-PCR and Western blot analysis. Interestingly, mRNA levels of CYP27B1 (the gene coding for 1alpha-hydroxylase enzyme which catalyses the conversion of 1alpha,25-dihydroxyvitamin D, 1alpha,25[OH]2D, from its inactive form, 25-hydroxycholecalciferol, 25[OH]D) and CYP24A (the gene coding for 24-hydroxylase, a target gene of 1alpha,25[OH]2D) were significantly increased by twofold and 34-fold, respectively, in differentiated osteoblasts compared with proliferating cells. We next examined if the local production of 1alpha,25(OH)2D might contribute to the FGF23 upregulation. We cultured osteoblasts in serum-free medium with or without 25-(OH)D (the substrate of 1alpha-hydroxylase). FGF23 mRNA levels were increased in proliferating cells (16-fold) and in differentiated cells (28-fold) by the physiological dose of 25-(OH)D3 treatment. CYP27B1 was slightly but significantly upregulated and CYP24A was increased by 1,700-fold and 800-fold, respectively, in transcriptional levels. Because FGF23 was upregulated in confluent osteoblasts regardless of the presence or absence of 25-(OH)D in serum-free medium, we further examined the possible impact of cell communication on FGF23 expression. We treated osteoblasts with carbenoxolone, a gap junction Cx43 blocker in serum-free medium. The FGF23 mRNA level was reduced by 50% in confluent differentiated cells and slightly but not significantly reduced in half-confluent cells by carbenoxolone treatments. The results suggested that upregulation of FGF23 in differentiated osteoblast appeared to be due to increased autocrine/paracrine action of osteoblast-derived 1alpha,25(OH)2D and increased cell communication, which were identified in cultured rat calvaria osteoblasts. These results indicate that FGF23 expression was stimulated not only by circulating calcitriol but also by locally produced 1alpha,25(OH)2D. The local mechanisms of FGF23 expression remain to be characterized.

Growth factor expression in ophthalmic pterygia and normal conjunctiva

The expression of growth factors in ophthalmic pterygium and phenotypically normal conjunctiva was examined and correlated with the clinical findings. Fifteen specimens of ophthalmic pterygia and 8 specimens of phenotypically normal conjunctiva were examined. Total RNA was extracted from all specimens and mRNA levels for transforming growth factor (TGFB1), vascular endothelial growth factor (VEGFA), basic fibroblast growth factor (FGF2), epidermal growth factor (EGF) and insulin-like growth factor (IGF1) were measured using real-time reverse-transcription PCR (qRT-PCR). Differences in the expression of these factors between pterygium and conjunctival specimens were examined, as well as correlations between RNA levels and clinical parameters. mRNA levels for VEGFA and FGF2 were significantly higher in pterygium, compared with conjunctival specimens, whereas the respective differences for other factors examined were statistically not significant. The mRNA levels for VEGFA and FGF2 were also significantly higher in recurrent compared with primary pterygium. Correlations between the mRNA levels and other clinical parameters were statistically not significant for the growth factors examined. The higher levels of FGF2 or VEGFA mRNA in pterygium imply that these factors may be involved in the pathogenesis or clinical behavior of the pterygium, including postoperative recurrence. VEGFA expression may have potential for use in the selective treatment of pterygium with anti-VEGFA agents.

Autocrine/paracrine action of Vitamin D on FGF23 expression in cultured rat osteoblasts

To explore the local mechanisms of fibroblast growth factor (FGF) 23 regulations, we examined the FGF23 expression patterns in an osteoblast culture model. The characteristics of cultured rat calvaria osteoblasts in half-confluence, confluence, osteoid deposition, and osteoid mineralization stages might reflect the proliferation, differentiation, maturation, and matrix mineralization stages, respectively. Compared with proliferating cells in half-confluence, FGF23 expression was upregulated by 7.5-fold at the mRNA level and 126% at the protein level in confluent differentiated cells as determined by real-time RT-PCR and Western blot analysis. Interestingly, mRNA levels of CYP27B1 (the gene coding for 1α-hydroxylase enzyme which catalyses the conversion of 1α,25-dihydroxyvitamin D, 1α,25[OH]2D, from its inactive form, 25-hydroxycholecalciferol, 25[OH]D) and CYP24A (the gene coding for 24-hydroxylase, a target gene of 1α,25[OH]2D) were significantly increased by twofold and 34-fold, respectively, in differentiated osteoblasts compared with proliferating cells. We next examined if the local production of 1α,25(OH)2D might contribute to the FGF23 upregulation. We cultured osteoblasts in serum-free medium with or without 25-(OH)D (the substrate of 1α-hydroxylase). FGF23 mRNA levels were increased in proliferating cells (16-fold) and in differentiated cells (28-fold) by the physiological dose of 25-(OH)D3 treatment. CYP27B1 was slightly but significantly upregulated and CYP24A was increased by 1,700-fold and 800-fold, respectively, in transcriptional levels. Because FGF23 was upregulated in confluent osteoblasts regardless of the presence or absence of 25-(OH)D in serum-free medium, we further examined the possible impact of cell communication on FGF23 expression. We treated osteoblasts with carbenoxolone, a gap junction Cx43 blocker in serum-free medium. The FGF23 mRNA level was reduced by 50% in confluent differentiated cells and slightly but not significantly reduced in half-confluent cells by carbenoxolone treatments. The results suggested that upregulation of FGF23 in differentiated osteoblast appeared to be due to increased autocrine/paracrine action of osteoblast-derived 1α,25(OH)2D and increased cell communication, which were identified in cultured rat calvaria osteoblasts. These results indicate that FGF23 expression was stimulated not only by circulating calcitriol but also by locally produced 1α,25(OH)2D. The local mechanisms of FGF23 expression remain to be characterized.

TGF-β1 RNA Interference in Mouse Primary Dura Cell Culture: Downstream Effects on TGF Receptors, FGF-2, and FGF-R1 mRNA Levels

Transforming growth factor (TGF)-beta1 and fibroblast growth factor (FGF)-2 have both been shown to have significant roles in the regulation of murine calvarial suture fusion. Methods to decrease gene expression of these cytokines and their respective receptors have been established, but because of side effects, clinical applications are limited. In this study, the authors examined the effect of TGF-beta1-specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-beta1, its TGF-betaR1 and TGF-betaR2 receptors, and FGF-2 and its R1 receptor in murine dura cells. A primary dura cell line was established from CD-1 mice. Transfection efficiency using Lipofectamine was determined using BLOCKiT. Dura cells were transfected with serial concentrations of TGF-beta1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-beta1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction. Optimal inhibition of TGF-beta1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-beta1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-betaR1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-betaR2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4. These results indicate that TGF-beta1 siRNA has the potential to alter the murine dura cytokines responsible for suture fusion in vitro. Manipulating underlying cranial suture biology with siRNA technology may ultimately allow control over suture fusion. This intervention may ultimately function as an effective adjunct to surgical intervention for craniosynostosis.

Stimulatory Effects of Thyroid Hormone on Brain Angiogenesis in vivo and in vitro

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day 21 (P21) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an altered expression of vascular endothelial growth factor A (Vegfa) and basic fibroblast growth factor (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain-derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfa upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing factor to the neurologic deficits induced by thyroid hormone deficiency.

The Periphery of the Human Fetal Adrenal Gland Is a Site of Angiogenesis: Zonal Differential Expression and Regulation of Angiogenic Factors

Although the inner fetal zone (FZ) of the mid-gestation human fetal adrenal (HFA) produces dehydroepiandrosterone sulfate, the function of the outer definitive zone (DZ) remains less clear. We have proposed that the DZ phenotype is that of a pool of progenitor cells, many of which are mitotically active. Recently, we studied HFA expression of a family of vascular endothelial cell-specific angiogenic factors, the angiopoietins (Angs), and demonstrated that Ang2 was localized predominantly in the periphery of the gland. Ang1 stabilizes, whereas Ang2 destabilizes, vessels, increasing responsiveness to angiogenic stimuli such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF)-2. Our objective was to test the hypothesis that the periphery of the HFA is a site of angiogenesis. Studies were conducted involving RNA, frozen sections, and primary cell cultures from midgestation HFAs. Immunofluorescence, laser capture microdissection, and real-time quantitative RT-PCR were used. Double immunostaining demonstrated that proliferating endothelial cells were limited to the DZ and DZ/FZ border. Ang2 mRNA was primarily expressed in the DZ, whereas Ang1 mRNA was primarily in the FZ. VEGF-A and FGF-2 mRNA levels were higher in the DZ. FGF-2 (10 ng/ml) induced Ang2 mRNA by 4-fold in both zones of cells (P < 0.01, at 24 h), but not Ang1 or VEGF-A mRNA. Data suggest that angiogenesis occurs at the periphery of the HFA. The DZ-predominant expression of Ang2 may be explained, in part, by the parallel pattern of FGF-2 expression.

Homocysteine Inhibits Arterial Endothelial Cell Growth Through Transcriptional Downregulation of Fibroblast Growth Factor-2 Involving G Protein and DNA Methylation

Homocysteine (Hcy) contributes to atherogenesis and angiostasis by altering the phenotype of arterial endothelial cells (ECs). The present study was aimed at elucidating potential mechanisms by which Hcy can slow EC proliferation and induce EC apoptosis, thereby disrupting endothelial integrity. Given the strong mitogenic and antiapoptotic properties of fibroblast growth factor (FGF)2, we examined whether Hcy can modulate its expression. In cultured human coronary and bovine aortic ECs, Hcy exerted time- and concentration-dependent (0 to 500 micromol/L) reduction of the mRNA and protein levels of FGF2, whereas vascular endothelial growth factor expression was not affected until Hcy reached a proapoptotic 500 micromol/L. By testing a panel of signal transduction inhibitors, we found that the Hcy-induced downregulation of FGF2 was specifically attenuated by pertussis toxin, an inhibitor of Gi protein signaling. Hcy induced cell cycle arrest at the G(1)/S transition and increased TUNEL-positive apoptotic cells in a graded manner. These effects were effectively counteracted by exogenous FGF2. Reporter gene assays showed that Hcy downregulated FGF2 by transcriptional repression of the gene promoter encompassed in a CpG dinucleotide-rich island. This region was heavily methylated at the cytosine residues by Hcy despite decreased methylation potential (S-adenosylmethionine to S-adenosylhomocysteine ratio). Normal levels of FGF2 transcription were restored to ECs simultaneously exposed to Hcy and 5-aza-deoxycytidine. We conclude that homocysteine disrupts the growth and survival of ECs through a G protein-mediated pathway associated with altered promoter DNA methylation and the transcriptional repression of FGF2.

Adrenalectomy counteracts the local modulation of astroglial fibroblast growth factor system without interfering with the pattern of 6-OHDA-induced dopamine degeneration in regions of the ventral midbrain

The present study investigated the effects of bilateral adrenalectomy (ADX) on the synthesis of basic fibroblast growth factor (bFGF, FGF-2) mRNA and on the expression of its FGF receptor subtype-2 (FGFR2) mRNA after a 6-hydroxydopamine (6-OHDA)-induced lesion of nigrostriatal dopamine system. In previous papers we have demonstrated that corticosterone increases FGF-2 immunoreactivity mainly in the astrocytes of the substantia nigra [Chadi, G., Rosen, L., Cintra, A., Tinner, B., Zoli, M., Pettersson, R.F., Fuxe, K., 1993b. Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. Neuroreport 4, 783–786.] and that 6-OHDA injected in the ventral midbrain upregulates FGF-2 synthesis in reactive astrocytes in the ascending dopamine pathways [Chadi, G., Cao, Y., Pettersson, R.F., Fuxe, K., 1994. Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience 61, 891–910.]. Rats were adrenalectomized and received a 6-OHDA stereotaxical injection in the ventral midbrain 2 days later. Seven days after the dopamine lesion, Western blot analysis showed a decreased level of tyrosine hydroxylase in the lesioned side of the midbrain, an event that was not altered by ADX or corticosterone replacement. Moreover, the degeneration of nigral dopamine neurons, which was confirmed by the disappearance of acidic FGF (FGF-1) mRNA and the decrement of tyrosine hydroxylase mRNA labeled nigral neurons, was not altered by ADX. The FGF-2 protein (23 kDa isoform but not 21 kDa fraction) levels increased in the lesioned side of the ventral midbrain. This elevation was counteracted by ADX, an effect that was fully reversed by corticosterone replacement. In situ hybridization revealed that ADX counteracted the elevated FGF-2 mRNA levels in putative glial cells of the ipsilateral pars compacta of the substantia nigra and in the ventral tegmental area. The ADX also counteracted the increased density and intensity of the astroglial FGF-2 immunoreactive profiles within the lesioned pars compacta of the substantia nigra and the ventral tegmental area as determined by stereology. The stereotaxical mechanical needle insertion triggered the expression of FGFR2 mRNA in putative glial cells, spreading to the entire ipsilateral ventral midbrain from the region of needle track, an occurrence that was partially reversed by ADX. In conclusion, bilateral ADX counteracted the increased astroglial FGF-2 synthesis in the dopamine regions of the ventral midbrain following a 6-OHDA-induced local lesion and interfered with FGF receptor regulation around injury. These findings give further evidence that adrenocortical hormones may regulate the astroglial FGF-2-mediated trophic mechanisms and wound repair events in the lesioned central nervous system.

Effects of Direct Current Electrical Stimulation on Gene Expression of Osteopromotive Factors in a Posterolateral Spinal Fusion Model

An in vivo model was used to determine levels of mRNA expression in response to direct current (DC) electrical stimulation in a rabbit posterolateral fusion model. This study tested the possibility that DC stimulation at the surgery site would increase expression of genes related to bone formation relative to expression in autograft alone. DC electrical stimulation as an adjunct treatment in spinal surgery has shown increased fusion rates when compared with autograft alone, yet the biology of such treatment is not fully understood. Thirty New Zealand White rabbits were entered into the study. A posterolateral, intertransverse process fusion was performed bilaterally at L4-L5, with autogenous bone graft. An implantable DC stimulator was placed across the decorticated transverse processes before placement of autograft. Animals were killed at 3, 7, 14, 21, and 28 days. mRNA levels of BMP-2, 4, 6, 7, VEGF, FGF-2, TGF-beta, ALK-2, and ALK-3 were evaluated with real-time RT-PCR. mRNA expression was significantly higher in the DC stimulated animals versus the control animals for several of the genes studied. In particular, levels of mRNA were elevated for BMP-2, BMP-6, and BMP-7. This study shows for the first time that DC stimulation results in a sustained increase of multiple osteogenic genes, suggesting that the biologic mechanism for the DC-induced increase in the rate and extent of bone formation observed clinically may be mediated by the up-regulation of these osteoinductive factors.

Sulfisoxazole, an endothelin receptor antagonist, protects retinal neurones from insults of ischemia/reperfusion or lipopolysaccharide

Endothelins exert pathological effects in the eye and much interest centres on their role in causing retinal neuronal death in ischemic diseases like glaucoma. In the present study the influence of the non-selective endothelin antagonist, sulfisoxazole on raised intraocular pressure-induced ischemia to the rat retina was investigated. Moreover, in vitro studies on primary rat retinal cultures were undertaken to see whether sulfisoxazole is able to blunt the toxic effect of lipopolysaccharide (LPS) to retinal neurones. In order to determine whether sulfisoxazole provides protection to the retina the a- and b-wave amplitudes of the electroretinogram (ERG), the localisation of retinal choline acetyltransferase (ChAT), nitric oxide synthase (nNOS) and Thy-1 and the retinal mRNA levels of Thy-1 and FGF-2 were deduced in retinas subjected to ischemia in the absence or presence of sulfisoxazole. The results showed that the ischemia-induced changes to the a- and b-wave amplitudes of the ERG and changes associated with the localisation of ChAT, nNOS and Thy-1 to be significantly blunted by sulfisoxazole. However, while the ischemia-induced changes to Thy-1 and FGF-2 mRNAs were reduced by sulfisoxazole, the reduction was non-significant. The in vitro studies provided support for the protective effect of sulfisoxazole. Here, it was clearly shown that sulfisoxazole attenuated the elevation of nitric oxide (deduced by measuring nitrite) and the reduction in numbers of GABA-containing neurones caused by LPS. The present study provides evidence for the first time that endothelin antagonist can protect the retina from ischemic-like insults as occurs in glaucoma.

Prenatal stress elicits regionally selective changes in basal FGF-2 gene expression in adulthood and alters the adult response to acute or chronic stress

Exposure to stress during pregnancy influences the trajectory of brain development resulting in permanent alterations that may contribute to increased susceptibility to subsequent cognitive or neuropsychiatric disorders. In this manuscript, we examined the effects of prenatal stress on the expression of basic fibroblast growth factor (FGF-2), an important molecular regulator of development and plasticity, in adult male rats under basal conditions as well as in response to acute or chronic stress. Baseline FGF-2 mRNA levels were differentially influenced by gestational stress in a variety of brain regions, with significant decreases in prefrontal cortex and increases in entorhinal cortex and striatum. By itself, postnatal stress similarly decreased trophic factor expression in prefrontal cortex while evoking stimulation elsewhere. Gestational stress altered the pattern of FGF-2 expression in response to adult stress, completely reversing the pattern in the prefrontal cortex (stimulatory instead of inhibitory), blunting the response in the entorhinal cortex and desensitizing the response in the striatum. These effects point to a unique interference of chronic prenatal stress with both ongoing FGF-2 expression and its responses to subsequent stressors, lasting into adulthood. Given the multifaceted role of FGF-2 in synaptic development, maintenance and plasticity, these data provide detailed mechanistic evidence as to how prenatal stress elicits lifelong effects on synaptic function. The abnormal modulation of FGF-2 gene expression in specific brain regions in response to subsequent stress in adulthood may impair the normal adaptive responses of the cell to challenging situations.

Renin angiotensin system inhibition prevents naturally advanced left ventricular hypertrophy via oxidized stress suppression

We designed in vivo study using high aged spontaneously hypertensive rats (SHR) treated with angiotensin converting enzyme inhibitor (ACEI), angiotensin receptor blocker (ARB) or their combined therapy to clarify the role of reactive oxygen species (ROS) for preventing left ventricular hypertrophy (LVH) and heart failure. We used 30 male SHR aged 50 weeks (6 rats in each group). Each group was treated for 4 weeks using osmotic mini-pump with ACEI (temocaprilat 1mg/kg/day), ARB (olmesartan 0.2mg/kg/day), combined therapy (CT; temocaprilat 0.5mg/kg/day and olmesartan 0.1mg/kg/day) or hydralazine (H; 15mg/kg/day) to reduce equivalently blood pressure (BP) and with saline as control (C). We measured Heart/body weight (Heart Weight Index), BP by tail cuff method and plasma BNP. We measured histologically myocytes size by hematoxylin eosin stain and myocardial fibrotic area by Azan stain using computerized morphometry system (MacSCOPE ver2.2). We also measured p22phox, p47phox, gp91phox, TGFβ1 and FGF2 mRNA levels by real-time PCR and serum NOx by ELISA. Heart/body weight (Heart Weight Index) treated with ARB (p<0.05) or CT (p<0.1) was reduced. Histological myocyte size in ACEI (p<0.05), ARB (p<0.01) or CT (p<0.001) and myocardial fibrosis in ACEI (p<0.05) or CT (p<0.01) was significantly reduced. Plasma BNP and NOx level in ACEI, ARB or CT was reduced (P<0.05). By real-time PCR, TGFβ1 mRNA in ACEI (p<0.05), ARB (p<0.05) or CT (p<0.01) or FGF2 mRNA in ACEI, ARB or CT (p<0.01) were significantly suppressed. The p22phox in ACEI (p<0.05), ARB (p<0.05) or CT (p<0.01), p47phox in ACEI (p<0.01 vs. C, p<0.05 vs. H), ARB (p<0.05 vs. C) or CT (p<0.01 vs. C, p<0.05 vs. H) or gp91phox in ACEI (p<0.01), ARB (p<0.01) or CT (p<0.005) were also significantly suppressed compared those in H or C. We conclude that the dual inhibition of RAS showed additional beneficial effects on improvement of cardiac hypertrophy via ROS suppression even in naturally advanced LVH.

Time Course of Neurotrophic Factor Upregulation and Retinal Protection against Light-Induced Damage after Optic Nerve Section

To assess neurotrophic factor upregulation in the retina after damage to the optic nerve and relate that regulation to changes in photoreceptor stability and function. Retinas of adult pigmented (Long-Evans) rats were examined at successive times (1-60 days) after unilateral optic nerve section. The distribution and expression of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (FGF-2) and their receptor elements FGFR1 and CNTFRalpha were studied with immunohistochemistry and Western blot analysis. FGF-2 and CNTF mRNA levels were also assessed, with semiquantitative reverse transcription-PCR. Levels and localization of the intracellular signaling molecule ERK and its activated, phosphorylated form pERK, were examined by immunohistochemistry. To assess the correlation between neurotrophic factor levels and their protective effect against light damage, albino (Sprague-Dawley) rats were exposed to bright continuous light (1000 lux) for 24 or 48 hours at successive times after nerve section. The TUNEL technique was used to visualize neuronal cell death in the retina. CNTF upregulation was detected 1 week after optic nerve section, peaked at 2 weeks, and fell to control levels at 4 weeks. CNTF appeared first in the inner retina in the ganglion cells, then in the Muller cells in which it became prominent at the outer limiting membrane (OLM) and in the outer segment (OS) region of photoreceptors. FGF-2 upregulation became prominent, particularly in photoreceptors, 21 to 28 days after surgery, continued to 2 months, and slowly declined thereafter. Double labeling with antibodies to ligand and the receptor showed colocalization of CNTF to its receptor at the OS region, whereas FGF-2-to-FGFR1 binding was found in the outer nuclear (ONL) and outer plexiform (OPL) layers. Optic nerve section provided a significant protective effect against light-induced damage in the first 2 weeks. There was no protection when animals were exposed to damaging light 1 month after nerve section. The upregulation of CNTF 7 to 14 days after nerve section correlates with a reduction in the a-wave described previously. Colocalization of CNTF and CNTFRalpha on the outer segments suggests that CNTF acts at the photoreceptor membrane. The slower upregulation of FGF-2 correlates with a reduction of the b-wave. FGF-2/FGFR1 colocalization in the OPL suggests that this factor acts at the synaptic terminals of photoreceptors, modulating the release of neurotransmitters. The time course of pERK upregulation suggests that the successive upregulation of CNTF and FGF-2 activates the ERK pathway. Based on the time course of protection against bright continuous light, it seems that CNTF plays a major role in this effect, and FGF-2 has a less important role in the protection against light-induced damage.

Irradiation of mechanically-injured human arterial endothelial cells leads to increased gene expression and secretion of inflammatory and growth promoting cytokines

Radiation therapy is applied to inhibit neointima formation after percutaneous transluminal coronary angioplasty (PTCA). In this study, we evaluated the effect of irradiation on re-endothelialisation of circular denuded tracks made in post-confluent cultures of arterial endothelial cells (ECs) and on cellular factors involved in this process. Image analysis and time-lapse microcinematography revealed cell migration into denuded areas starting 4 h after injury. Fifty percent coverage was achieved at 14.8±2.0 h. Using competitive PCR and flow cytometry techniques, no significant changes in mRNA expression of interleukin-1β (IL-1β), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B) and tissue factor (TF), and surface molecule expression of anti-intercellular adhesion molecule-1 (ICAM-1), anti-vascular cell adhesion molecule-1 (VCAM-1), anti-platelet/endothelial cell adhesion molecule-1 (PECAM-1), MHC-1, TF and Fas were observed. However, injury did significantly ( P<0.05) elevate the release of IL-8 and FGF-2 protein in the cell culture supernatant, as assessed by ELISA. Radiation (15 Gy) given immediately after injury did not affect the kinetics of re-endothelialisation up to 48 h, in spite of the fact that no cell divisions were observed. Thereafter cell density decreased and cultures deteriorated. Compared to cultures exposed to injury alone, radiation induced significant ( P<0.05) increases in mRNA levels of IL-8 (1.35±0.10-fold increase at 4 h), FGF-2 (1.62±0.10-fold at 4 h; 1.76±0.33-fold at 24 h) and IL-1β (2.76±0.40-fold at 24 h), whereas mRNA levels of TGF-β1, PDGF-A and PDGF-B increased about 1.2-fold. IL-8 and FGF-2 protein concentrations in the media were higher than those observed in non-irradiated injured cell cultures; however, this difference was not significant. Radiation induced a 2.3±0.3-fold increase ( P<0.05) in Fas surface expression only. In conclusion, irradiation of mechanically-injured human EC leads to increased gene expression and protein secretion of inflammatory and growth promoting cytokines.

Irradiation of mechanically-injured human arterial endothelial cells leads to increased gene expression and secretion of inflammatory and growth promoting cytokines

<h2>Abstract</h2> Radiation therapy is applied to inhibit neointima formation after percutaneous transluminal coronary angioplasty (PTCA). In this study, we evaluated the effect of irradiation on re-endothelialisation of circular denuded tracks made in post-confluent cultures of arterial endothelial cells (ECs) and on cellular factors involved in this process. Image analysis and time-lapse microcinematography revealed cell migration into denuded areas starting 4h after injury. Fifty percent coverage was achieved at 14.8±2.0h. Using competitive PCR and flow cytometry techniques, no significant changes in mRNA expression of interleukin-1β (IL-1β), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor-β1 (TGF-β1), platelet-derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B) and tissue factor (TF), and surface molecule expression of anti-intercellular adhesion molecule-1 (ICAM-1), anti-vascular cell adhesion molecule-1 (VCAM-1), anti-platelet/endothelial cell adhesion molecule-1 (PECAM-1), MHC-1, TF and Fas were observed. However, injury did significantly (<i>P</i><0.05) elevate the release of IL-8 and FGF-2 protein in the cell culture supernatant, as assessed by ELISA. Radiation (15Gy) given immediately after injury did not affect the kinetics of re-endothelialisation up to 48h, in spite of the fact that no cell divisions were observed. Thereafter cell density decreased and cultures deteriorated. Compared to cultures exposed to injury alone, radiation induced significant (<i>P</i><0.05) increases in mRNA levels of IL-8 (1.35±0.10-fold increase at 4h), FGF-2 (1.62±0.10-fold at 4h; 1.76±0.33-fold at 24h) and IL-1β (2.76±0.40-fold at 24h), whereas mRNA levels of TGF-β1, PDGF-A and PDGF-B increased about 1.2-fold. IL-8 and FGF-2 protein concentrations in the media were higher than those observed in non-irradiated injured cell cultures; however, this difference was not significant. Radiation induced a 2.3±0.3-fold increase (<i>P</i><0.05) in Fas surface expression only. In conclusion, irradiation of mechanically-injured human EC leads to increased gene expression and protein secretion of inflammatory and growth promoting cytokines.

MRNA expression of fibroblast growth factors and hepatocyte growth factor in rat plantaris muscle following denervation and compensatory overload.

We addressed the question of whether hypertrophy induced by compensatory overload differs according to innervation status, and how fibroblast growth factors (FGF) and hepatocyte growth factor (HGF) mRNAs are expressed in the rat plantaris muscle during overload (OL) and/or denervation. Male Wistar rats were divided into four groups (Normal-Cont, Normal-OL, Denervated-Cont, and Denervated-OL). according to the plantaris denervation and/or overload. Three weeks later, plantaris weight in Denervated-Cont and Denervated-OL was significantly lower than in the Normal-Cont. The muscle weights in the Normal-OL were higher than in the Normal-Cont. The muscle weights in the Denervated-OL were higher than in the Denervated-Cont. Three days after the treatment, FGF-2, FGF-6, FGF-7 and HGF mRNAs in the Normal-OL were significantly higher than those in the Normal-Cont. FGF-2, FGF-6, FGF-7 and HGF mRNAs in the Denervated-OL were also significantly higher after 3 days than those in the Denervated-Cont. After 7 days, FGF-2, FGF-5, FGF-6, FGF-7 and HGF mRNAs were significantly higher in the Normal-OL than those in the Normal-Cont. At 21 days, FGF-1, FGF-6 and HGF mRNA levels were significantly increased. In the Denervated-OL, FGF-2, FGF-7 and HGF mRNAs at 7 days, and FGF-2 mRNA at 21 days were significantly higher than those in the Denervated-Cont. FGF-2 and FGF-6 mRNA levels decreased significantly following denervation; however, FGF-1, FGF-5, FGF-7 and HGF mRNA levels increased and maintained this increase for the 21-days treatment period. Muscle hypertrophy was thus induced by compensatory overload irrespective of innervation status, possibly in association with certain FGFs and HGF. The differential mRNA expression patterns of FGFs and HGF observed following compensatory overload and/or denervation suggest distinct roles for individual FGFs and HGF in muscle hypertrophy and/or atrophy.

THROMBIN- AND FACTOR-XA-INDUCED PROLIFERATION AND MIGRATION IN HUMAN VASCULAR SMOOTH MUSCLE CELLS ARE MEDIATED BY BASIC FIBROBLAST GROWTH FACTOR AND SYNDECAN-4

TGF-β1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2).The prognosis of primary renal disease is often dependent on the degree of tubulointerstitial scarring. Scarring is caused by proliferation and excessive matrix production of renal fibroblasts and possibly other cellular elements. Transforming growth factor-β (TGF-β) is the most important cytokine for the induction of matrix synthesis in the kidney. However, its effects on renal fibroblast proliferation have not been determined. We have recently demonstrated that the expression of basic fibroblast growth factor (FGF-2) is robustly up-regulated in human kidneys with tubulointerstitial fibrosis and that FGF-2 is a potent inducer of fibroblast proliferation. The present study examined the interaction between TGF-β1 and FGF-2 in human renal fibroblasts.Experiments were performed on a transformed medullary fibroblast line and on primary cortical kidney and skin fibroblasts isolated from human biopsies. mRNA levels of FGF-2 and TGF-β1 were analyzed by Northern blot analyses. Changes in protein expression were examined by immunoblots and enzyme-linked immunosorbent assay (ELISA). Bromodeoxyuridine incorporation assays and cell counts were used to analyze cell proliferation. The expression of cell cycle-regulatory proteins cyclin-dependent kinase (cdk) 2 and the cdk inhibitor p27kip1 were determined by immunoblots.Stimulation of renal fibroblasts with FGF-2 resulted in no change of TGF-β1 mRNA expression, whereas incubation of the cells with TGF-β1 induced FGF-2 mRNA up to 3.51 ± 0.21-fold after six hours. This increase could be blocked almost completely by the addition of cyclohexamide, indicating that the process is in large part dependent on protein synthesis. The up-regulation in FGF-2 mRNA expression was paralleled by de novo detection of FGF-2 protein in the supernatant, peaking after 12 to 24 hours, as determined by Western blot and ELISA, whereas cellular protein was only increased up to 2.1-fold. Interestingly, both methods detected release of FGF-2 protein to the supernatant already at three hours, indicating a role for TGF-β1 in directly releasing preformed FGF-2. Since TGF-β1 induced FGF-2, which results in fibroblast proliferation, we hypothesized that TGF-β1 may cause fibroblast proliferation mediated by FGF-2. This hypothesis was verified by cell proliferation assays demonstrating that stimulation of renal fibroblasts with TGF-β1 resulted in an up to 3.21 ± 0.28-fold increase in bromodeoxyuridine incorporation and a 1.95 ± 0.16-fold increase in cell number after 72 hours. This mitogenic effect of TGF-β1 could be blocked completely by the addition of a neutralizing antibody to FGF-2 or the tyrosine kinase inhibitor tyrphostin AG1296, which blocks FGF receptor (FGFR) tyrosine kinase activity. Conversely, a neutralizing antibody to epidermal growth factor (EGF) or the tyrphostin B42, which inhibits EGF receptor signal transduction, had no effect. Interestingly, a neutralizing antibody to PDGF had only minor effects in primary kidney fibroblasts but reduced TGF-β1–induced proliferation considerably in primary skin fibroblasts. Finally, TGF-β1–induced proliferation in kidney fibroblasts was paralleled by a robust increase in cdk 2 protein expression up to 72 hours, whereas p27kip1, whose activity is maintained by TGF-β in epithelial cells, was down-regulated up to 48 hours.Our studies demonstrate, to our knowlege for the first time, that TGF-β1 induces proliferation in human renal fibroblasts and that this process is mediated largely by FGF-2. The induction of proliferation by TGF-β1 via induction of FGF-2 may play an important role in the autonomy of renal fibroblast growth and thus in the pathogenesis of human fibrogenesis.