OBJECTIVE

To develop a rabbit model of reproducible corneal haze after excimer laser keratectomy and to characterize expression of transforming growth factor beta (TGFbeta) and basic fibroblast growth factor (bFGF) in rabbit corneas during haze formation.

METHODS

Seven rabbits underwent a 100 microm deep phototherapeutic keratectomy (PTK) in one eye and a 15-microm shallow PTK in the contralateral eye. Corneal haze was compared at 1-20 weeks after surgery. Subsequently, 16 rabbits underwent 100-microm PTK in one eye and 15-microm PTK in the contralateral eye. Four rabbits were killed at 1, 2, 3, and 4 weeks, respectively, after surgery. Immunohistochemistry was performed on the corneas to localize the expression of TGFbeta and bFGF. Control subjects were rabbits that underwent either epithelial debridement alone or no surgery.

RESULTS

A 100-microm PTK resulted in significantly more corneal haze than a 15-microm PTK at every postoperative examination (p < 0.05). Both TGFbeta and bFGF were expressed in the scars at 1-4 weeks after deep and shallow excimer ablations. bFGF was expressed in the keratocytes of both treated and control corneas. Minimal TGFbeta was detected in the keratocytes of the control corneas, whereas prominent TGFbeta expression was noted in the keratocyte-like cells adjacent to the postkeratectomy scars.

CONCLUSIONS

The 100-microm PTK ablation resulted in significantly more corneal scarring than the 15-microm PTK ablation. Even though there was no immunohistochemical difference in the pattern of TGFbeta and bFGF expression after deep and shallow ablations, there was an association between the expression of the growth factors and corneal scarring after excimer laser keratectomy.

Local angiogenic therapy with recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (rhbFGF) has been used to promote wound healing. To obtain useful information for the development of optimal angiogenic therapy, we chronologically evaluated the effects of a single local application of rhbFGF on angiogenesis in a rabbit ear chamber model of wound healing by observing the subcutaneous vessel bed intravitally. New vessel formation during wound healing was macroscopically and microscopically evaluated for 5 wk. Each rabbit ear chamber received a single dose of 6 microg rhbFGF (treatment B1: n = 13), 18 microg rhbFGF (treatment B2: n = <em>16</em>), or physiological saline as control (n = 13). At 1 wk the newly vascularized area was significantly larger in groups B1 and B2 than in control. At 2 wk, the vascularized areas in groups B1, B2, and control were similar. At 5 wk, the percentage of rabbits with complete vascularization was significantly larger in group B1 than in control. Capillary density at 5 wk was similar among the three groups. These results suggest that locally applied rhbFGF accelerated angiogenesis during early wound healing in rabbits; however, this effect was transient and no increase in capillary density occurred at the completion of vascularization.

Recent studies have suggested that hypercholesterolemia may aggravate glomerulosclerosis. Mesangial cells actively participate in this process. To elucidate mechanisms by which lipids act on human mesangial cells (HMC), we measured the receptor-specific uptake of apolipoprotein (Apo) B- and Apo B- and E-containing lipoproteins in the presence and absence of <em>growth</em> <em>factors</em> and studied the <em>growth</em>-related mechanisms in HMC after exposure to low-density lipoprotein (LDL). Human LDL and very low density beta-lipoprotein (beta-VLDL) isolated from cholesterol-fed rabbits were bound, internalized, and degraded by a receptor-specific mechanism (dissociation constants for degradation LDL 30.0 and for beta-VLDL 4.1 micrograms protein/ml medium). Maximal capacities were 30-50% of those of human <em>fibroblasts</em>. Acetylated and copper-oxidized LDL were not taken up specifically, suggesting no active scavenger-receptor activity. Preexposure to endothelin-1 (5 x 10(-7) M) and platelet-derived <em>growth</em> <em>factor</em> (PDGF A, B, 83 x 10(-12) M) for <em>16</em> or 15 h, respectively, doubled the uptake of LDL by HMC. In addition, PDGF synergized with LDL in stimulating DNA synthesis. Exposure of HMC to LDL resulted in a transient elevation of mRNA that encodes c-fos and c-jun, with a maximal effect seen after 30-60 min. In addition, PDGF A- and B-chain mRNAs were transiently elevated, peaking at 3 h in response to LDL (25 micrograms protein/ml medium) and continued to increase in a concentration-dependent manner (25-75 micrograms protein/ml medium). These data demonstrate that HMC take up lipoproteins via a receptor-specific mechanism with a high affinity for Apo E-containing lipoproteins which are often found in plasma of patients with renal disease. Vasoconstrictor and mitogenic peptides enhance lipoprotein receptor activity and have a synergistic effect on the mitogenic effect of LDL. LDL stimulates a number of <em>growth</em>-related genes. These data suggest that lipoproteins may play a critical role in mediating mesangial cell hypertrophy or proliferation, events intimately involved in the development of glomerulosclerosis.

The IR-IGF1 production by rabbit epiphyseal chondrocytes cultured in serum-free medium was analyzed. Cell proliferation was induced by the addition of 10 ng/ml basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) without or with 100 ng/ml recombinant human <em>growth</em> hormone (hGH). GH alone induced no cell multiplication. Chondrocytes treated with bFGF alone secreted an IR-IGF1 activity proportional to the mitotic activity of the cells. A specific positive IGF1 immunostaining was localized in the Golgi of control and hGH-treated cells. The IR-IGF1 activity recovered into culture medium was mainly composed of three fractions of apparent MW 6-8 kDa, 9-14 kDa, and <em>16</em>-18 kDa. [35S]Methionine pulse-chase experiments indicated that the radiolabeled <em>16</em>-18 kDa IR-IGF1 fraction was partly converted into the 9-14 kDa and 6-8 kDa fractions. At equilibrium, 70% of the chondrocyte IR-IGF1 activity was recovered as 9- to 18-kDa forms which contained high IR-proIGF1A activity. The 6-8 kDa fraction had biochemical characteristics similar to those of the mature IGF1 peptide. Similar results were observed when 4% fetal calf serum was added to the culture. The addition of 100 ng/ml of hGH significantly and specifically increased IGF1 precursor material, which thus represented 90% of total IR-IGF1 activity. On Day <em>16</em> of the culture, when cells stopped dividing, the amount of chondrocyte IR-IGF1 was significantly lower than during cell proliferation, and hGH had no effect on this production. These data indicate that cultured chondrocytes produce more IGF1 precursors than mature IGF1 and that GH specifically stimulates biosynthesis of IGF1 precursors but not IGF1 per se. A GH-dependent biological function of IGF1 proforms in chondrocytes remains to be demonstrated.

Satellite cells are multipotential stem cells responsible for muscle <em>growth</em> and regeneration. Satellite cell proliferation, differentiation, and responsiveness to <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) is, in part, regulated by the heparan sulfate proteoglycans syndecan-4 and glypican-1. Syndecan-4 and glypican-1 expression declines with satellite cell age and may be associated with decreased satellite cell activity. The objective of the current study was to determine if overexpression of syndecan-4 and glypican-1 would increase proliferation, differentiation and FGF2 responsiveness in satellite cells isolated from pectoralis major muscle from <em>16</em>-wk-old turkeys. Overexpression of syndecan-4 and glypican-1 did not have a significant effect on proliferation and differentiation in 1d, 7 wk, and <em>16</em> wk satellite cells, and did not affect FGF2 responsiveness during proliferation. Expression of syndecan-4 and glypican-1 increased differentiation at 48 h in 1d, 7 wk, and <em>16</em> wk cells treated with FGF2. Expression of myogenic regulatory <em>factors</em> MyoD, myogenin, and MRF4 was affected by the overexpression of syndecan-4 and glypican-1. However, changes in myogenic regulatory <em>factor</em> expression did not have a significant effect on proliferation or differentiation. These data demonstrate that syndecan-4 and glypican-1 are likely not directly associated with the age related decrease in satellite cell activity.

BACKGROUND

Despite refinements in radiotherapy, radiation-impaired wound healing continues to be a major source of postoperative morbidity with few treatment options. The application of polypeptide growth factors has been investigated in both the clinical and laboratory settings. The authors used a novel sustained-release delivery system to examine the effect of transforming growth factor (TGF)-beta and fibroblast growth factor (FGF) on radiation-impaired wound healing in a rodent model.

METHODS

Eighty Sprague-Dawley rats underwent dorsal skin surface irradiation of 2500 cGy using a medical linear accelerator producing energy of 6 MeV followed by creation of a full-thickness skin incision. Six groups of 16 animals underwent either sham irradiation (irradiation control); irradiation only; irradiation and unimpregnated delivery system only; or irradiation and either TGF-beta, FGF, or TGF-beta plus FGF combined. Four animals from each group were euthanized at 4, 7, 14, and 28 days, and the harvested specimens underwent ultimate tensile strength testing and histologic evaluation.

RESULTS

All five irradiated groups had significantly lower ultimate tensile strength than the sham-irradiated control group at all time points (p < 0.05), thus validating the authors' model of radiation-impaired wound healing. Functional analysis demonstrated that all three growth factor-treated groups had significantly higher tensile strengths than either of the untreated irradiated groups at 14 days after wounding (p < 0.05). Histologic evaluation of the irradiated groups revealed increased cellularity and more organized collagen architecture of all treated groups when compared with the untreated groups, with the most pronounced differences seen at 7 days and 14 days after wounding.

CONCLUSIONS

This study effectively demonstrates that TGF-beta and FGF act individually and synergistically when delivered locally by means of a sustained release system to improve ultimate tensile strength in an acute postirradiation impaired wound-healing model.

The role of epithelial-stromal interactions in the progression of human papillomavirus-associated squamous intraepithelial lesions to invasive cervical cancer is poorly understood. Using the Matrigel artificial basement membrane assay as a model of keratinocyte invasion, the effects of selected <em>growth</em> <em>factors</em> on penetration of human papillomavirus <em>16</em>-immortalized keratinocytes through Matrigel were studied. Also studied in this model were the effects of conditioned media from <em>fibroblast</em> lines derived from normal cervical tissues (normal <em>fibroblasts</em>) and adjacent cervical cancer biopsies (tumor-associated <em>fibroblasts</em>) and from primary keratinocytes. Addition of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-alpha, and hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> or conditioned media from tumor-associated <em>fibroblasts</em> to the Matrigel resulted in near-doubling of penetration of human papillomavirus <em>16</em>-immortalized keratinocytes, whereas transforming <em>growth</em> <em>factor</em>-beta, platelet derived <em>growth</em> <em>factor</em>-B, or conditioned media from primary keratinocytes decreased penetration 10-fold. Antibodies to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> abrogated the stimulatory effects of conditioned media from tumor-associated <em>fibroblasts</em> on keratinocyte penetration, whereas antibodies to transforming <em>growth</em> <em>factor</em>-beta abrogated the inhibitory effects of conditioned media from normal <em>fibroblasts</em> on keratinocyte penetration. S1 nuclease protection and enzyme-linked immunosorbent assay showed increased expression of transforming <em>growth</em> <em>factor</em>-beta and decreased expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in normal compared with tumor-associated <em>fibroblasts</em>. Messenger RNA in situ hybridization of five cervical cancer biopsies demonstrated basic <em>fibroblast</em> <em>growth</em> <em>factor</em> expression in stromal cells surrounding nests of invading keratinocytes. Epithelial-stromal interactions mediated by <em>growth</em> <em>factors</em> such as transforming <em>growth</em> <em>factor</em>-beta and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> modulate penetration of human papillomavirus <em>16</em>-immortalized keratinocytes through Matrigel in vitro and these interactions may also be operative in vivo.

BACKGROUND

Basic fibroblast growth factor (bFGF) was administered intramyocardially together with CABG to induce myocardial neovascularizaton and collateral growth in patients with ungraftable coronary arteries. Coronary angiographic and myocardial scintigraphic findings revealed that the effects of CABG were potentially confounding.

RESULTS

Patients in the bFGF group (n = 16) underwent angiogenic therapy using bFGF for ungraftable territory, and incomplete revascularization (IR) patients (n = 22) underwent only CABG. The magnitude of collateral development was assessed by the Rentrop score and collateral connection (CC) grade. Rentrop scores tended to increase among patients in the bFGF group (before vs. after surgery, 1.9 ± 1.2 vs. 2.3 ± 1.2, p = 0.05), but not in the IR group. The CC grade significantly increased among patients in the bFGF group (before vs. after surgery, 1.0 ± 0.9 vs. 1.4 ± 0.5, p <0.05), but not in the IR group. Myocardial perfusion in territories injected with bFGF improved in 13 patients (81%) of the bFGF group, and also in the nonbypassed territory in 4 IR patients (25%) (p <0.05).

CONCLUSIONS

Angiogenic therapy with bFGF induced collateral development and improved myocardial perfusion in territories injected with bFGF.

Insulin-like <em>growth</em> <em>factors</em> (IGFs) contribute to the maintenance of the cartilage matrix by stimulating proteoglycan synthesis. In contrast, interleukin-1 (IL-1), an inflammatory cytokine, suppresses the synthesis of proteoglycans. In pathological conditions the chondrocytes' responsiveness to IGF-I is decreased, and elevated levels of IGF-binding proteins (IGFBPs) have been implicated as a possible cause. The aim of this study was to investigate the effects of IGF-I and IL-1 on IGFBP production by ovine articular chondrocytes (OAC) and the roles of these IGFBPs in the regulation of proteoglycan synthesis. As revealed by Western ligand and immunoblotting, OACs secreted IGFBP-2 and a 24-kDa IGFBP in culture medium under basal conditions. Exposure of the cells to IGF-I for 48 h resulted in the appearance of IGFBP-5 in the medium. Des(1-3)IGF-I, an IGF-I analog with reduced affinity for IGFBPs, also increased the level of IGFBP-5, but to a lesser extent than IGF-I, whereas LR3IGF-I, which has virtually no affinity for IGFBPs, had no effect on IGFBP-5. Furthermore, IGFBP-5 underwent a time-dependent limited proteolysis when incubated with OAC-conditioned medium, degrading into 22- and <em>16</em>-kDa fragments. The degradation of IGFBP-5 was significantly inhibited by IGF-I, but not by des(1-3)IGF-I or LR3IGF-I. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta, and platelet-derived <em>growth</em> <em>factor</em> had no effect on OAC IGFBPs. However, IL-1alpha increased the IGFBP-5 level in a dose-dependent manner, showing maximum activity at 200 U/ml. Furthermore, IL-1alpha, but not IGF-I, induced IGFBP-5 messenger RNA expression, as assessed by Northern blot analysis. Coincubation of IGF-I with IL-1alpha resulted in a substantially increased IGFBP-5 protein level, suggesting a synergism between the mechanisms of action of these two <em>factors</em>. Des(1-3)IGF-I and LR3IGF-I were 10 times more potent than IGF-I in stimulating proteoglycan synthesis, indicating inhibition of IGF-I activity by endogenous IGFBPs. IL-1alpha reduced the IGF-I bioactivity, but had no effect on the activities of the IGF-I analogs, thus implying that locally produced IGFBPs, particularly IGFBP-5, which was substantially increased when IGF-I and IL-1alpha were coincubated, mediated the reduction of the IGF-I activity. Our results demonstrate that IGF-I and IL-1alpha synergistically increase the level of IGFBP-5 in OAC by inhibiting the proteolysis and stimulating the expression of IGFBP-5, respectively. Furthermore, the attenuation of IGF-I-stimulated proteoglycan synthesis by IL-1alpha in OAC appears to be mediated by chondrocyte IGFBPs. We conclude that locally produced IGFBPs, in particular IGFBP-5, may play a critical role in the regulation of cartilage matrix degradation in inflammatory and degenerative arthritides.

Normal adult human dermal <em>fibroblasts</em> were cultured in the presence of sera from 142 subjects. Results of a <em>fibroblast</em> proliferation assay revealed that <em>16</em> of 42 patients with scleroderma, 1 of 25 with rheumatoid arthritis, 3 of 10 with systemic lupus erythematosus, 2 of 3 with mixed connective tissue disease and 0 of 42 normal controls had values outside the normal range. The activity of <em>fibroblast</em> <em>growth</em> promoting <em>factor</em> (FGPF) in the scleroderma group correlated with the skin involvement but not with involvement of any other organ system. The levels of FGPF were higher in the first 2 years of disease duration than at any other time. Our data suggest that <em>fibroblast</em> activation may be a key process in the pathogenesis of scleroderma.

BACKGROUND

Immunological mechanisms are suspected in sensory neuropathy (SN) occurring with systemic autoimmune diseases and in some idiopathic cases, but so far there are no antibodies (Abs) identifying these neuropathies.

METHODS

In the search for such specific antibodies, serum samples were collected from 106 patients with SN of these 72 fulfilled the diagnosis criteria of sensory neuronopathy (SNN) and 211 control subjects including patients with sensorimotor neuropathies, other neurological diseases (ONDs), systemic autoimmune diseases and healthy blood donors.

RESULTS

In the first step, a protein array with 8000 human proteins allowed identification of the intracellular domain of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) as a target of Abs in 7/<em>16</em> SNN and 0/30 controls. In the second step, an ELISA method was used to test the 317 patients and controls for anti-FGFR3 Abs. Abs were detected in <em>16</em>/106 patients with SN and 1/211 controls (p<0.001). Among the 106 patients with SN, anti-FGFR3 Abs were found in 11/38 patients with autoimmune context, 5/46 with idiopathic neuropathy and 0/22 with neuropathy of other aetiology (p=0.006). The only control patient with anti-FGFR3 Abs had lupus and no recorded neuropathy. Sensitivity, specificity, and positive and negative predictive values of anti-FGFR3 Abs for a diagnosis of idiopathic or dysimmune SN were 19%, 99.6%, 94.1% and 77.3%, respectively. A cell-based assay confirmed serum reactivity against the intracellular domain of FGFR3. The neuropathy in patients with anti-FGF3 Abs was non-length dependent in 87% of patients and fulfilled the criteria of probable SNN in 82%. Trigeminal nerve involvement and pain were frequent features.

CONCLUSIONS

A anti-FGFR3 Abs identify a subgroup of patients with SN in whom an underlying autoimmune disorder affecting sensory neurons in the dorsal root and trigeminal nerve ganglia is suspected.

The primary patterning event in early vertebrate development is the formation of mesoderm and subsequent induction of the neural tube by the mesoderm. Some of the transforming <em>growth</em> <em>factor</em> (TGF)-beta family (Activin, Vg1) and the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family molecules have been implicated for their roles in mesoderm induction. Here we show first the evidence that neuregulin, an epidermal <em>growth</em> <em>factor</em> (EGF)-like <em>growth</em> <em>factor</em> known for its role in neural and muscle differentiation, participates in mesoderm induction. Neuregulin could induce the ectopic expression of mesoderm specific gene Xbra in animal cap explants reared to the midgastrula stage, when animal caps dissected from late blastula were cultured with Neuregulin at a low concentration (10 ng/ml). In situ hybridization study showed that alpha-cardiac actin was expressed in animal caps that were treated with Neuregulin overnight. Skeletal and cardiac muscle specific genes such as MyoD family genes (myoD, MRF4, myf5) and SL1 as well as NCAM, a pan neural marker, were also ectopically expressed by treatment with Neuregulin. However, the expression of NCAM is presumed to be a secondary result of the initial mesoderm induction by Neuregulin. The temporal expression pattern of neuregulin during the early developmental stages was analyzed by RT-PCR in order to determine if neuregulin is expressed at the time of mesoderm induction. It has been found that the neuregulin transcript was already detected from the <em>16</em>-cell stage (stage 5) and continued to be expressed till the tailbud stage (stage 25), the latest embryonic stage analyzed in this study. Considering that the mesoderm is induced at early blastula before the start of zygotic transcription, maternal neuregulin is expressed at the right time to participate in mesoderm induction. These data strongly suggest that neuregulin plays an important role in mesoderm induction.

We examined the expression of FGF-2 mRNA in <em>16</em> early and 14 advanced gastric cancer by in situ hybridisation to elucidate its role in cancer progression. Anti-sense RNA probes were synthesized by transcribing the subcloned vector with T7 RNA polymerase in the presence of digoxigenin-labeled UTP. FGF-2 mRNA was located mainly in the cytoplasm around the nuclei of endothelial cells, <em>fibroblasts</em> and carcinoma cells. The expression was more frequently in the diffuse type carcinomas (4/7, 57%) than in the intestinal type tumours (5/23, 22%). The survival rates of advanced gastric cancers with FGF-2 mRNA expression were significantly lower than those without FGF-2 mRNA expression (p < 0.01). No significant correlation was seen with other clinicopathological <em>factors</em>. These results suggest that FGF-2 may play an important role for the <em>growth</em> of diffuse type gastric cancers, particularly at their advanced stage.

Previous studies have localized basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and its mRNA in normal and neoplastic endometrium but the expression of bGFG mRNA in endometriosis cells is virtually unknown. Our purpose was to investigate the presence of bFGF and its receptor mRNAs in highly purified primary cultures of stromal cells isolated from six eutopic endometrial samples obtained from patients without evidence of endometriosis and five endometriosis cyst linings. Using reverse transcriptase-polymerase chain reaction (RT-PCR), single major DNA bands of the expected sizes for bFGF and its receptor (354 and 661 bp, respectively) were detected in both endometrial and endometriosis samples. A competitive RT-PCR, that uses coprimer extension and amplification of a bovine RNA as an internal standard, was developed for semiquantitative estimation of bFGF gene expression. The target to standard mean ratios +/- SEM in six normal endometrial samples and in five endometriosis cultures were 26.7 +/- 10.7 and 9.2 +/- 3.0, respectively. Furthermore, when data were analyzed according to the cyst diameter, levels of bFGF mRNA resulted statistically higher (P < 0.05) in bigger cysts when compared to those detected in smaller ones (<em>16</em> +/- 2.7 and 4.7 +/- 1.8, respectively). These results demonstrate that the genes coding for bFGF and its receptor are expressed in endometriosis cells, but levels of bFGF mRNA are generally lower than those detected in their eutopic counterpart. Moreover, they indicate that endometriosis cells derived from large cysts have increased bFGF mRNA levels. Thus, bFGF could be one of the <em>factors</em> responsible for a more or less active behavior of the endometnotic lesion.

<AbstractText>We aim to investigate the effects of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>16</em> (FGF<em>16</em>) on Leydig cell regeneration in ethane dimethane sulphonate (EDS)-treated rat testis.</AbstractText><AbstractText>We intraperitoneally inject 75 mg/kg EDS to adult male Sprague Dawley rats and then intratesticularly inject FGF<em>16</em> (0, 10 and 100 ng/testis/day) from post-EDS day 14 for 14 days. We investigate serum hormone levels, Leydig cell number, gene and protein expression in vivo. We also explore the effects of FGF<em>16</em> treatment on stem Leydig cell proliferation in vitro.</AbstractText><AbstractText>FGF<em>16</em> lowers serum testosterone levels (21.6% of the control at a dose of 100 ng/testis) without affecting the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) on post-EDS day 28 in vivo. FGF<em>16</em> increases Leydig cell number at doses of 10 and 100 ng/mg without affecting Sertoli cell number, increases the percentage of PCNA-positive Leydig cells, and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) and Sertoli cell genes (Fshr, Dhh and Sox9) and their proteins in vivo. FGF<em>16</em> increases phosphorylation of AKT1 and AKT2 as well as EKR1/2 in vivo, indicating that it possibly acts via AKT1/ATK2 and ERK1/2 pathways. FGF<em>16</em> also lowers medium testosterone levels and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) but increases EdU incorporation into stem Leydig cells in vitro.</AbstractText><AbstractText>These data suggest that FGF<em>16</em> stimulates stem and progenitor Leydig cell proliferation but blocks their differentiation, thus lowering testosterone biosynthesis.</AbstractText>

OBJECTIVE

We determined the effects of pregnancy on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in ovine uterine and omental (systemic control) artery endothelial cells (UAEC, OAEC). We also determined in primary cultures of UAEC the effects of exposure to either exogenous nitric oxide (NO) or angiogenic growth factors (basic fibroblast growth factor [bFGF], vascular endothelial growth factor [VEGF], and epidermal growth factor [EGF]) on UAEC GAPDH expression.

METHODS

We isolated UAEC to high purity from both nonpregnant (NP; n = 4) and pregnant ewes (P; 110-120 days' gestation, n = 4) by limited collagenase dispersion and immediately extracted total RNA. In additional experiments performed in vitro, ovine UAEC isolated from NP ewes and maintained in culture (n = 3) were exposed to 1) 100 mumol/L sodium nitro-prusside for 0, 6, 12, or 24 hours or 2) 10 ng/mL bFGF, VEGF, EGF, or vehicle for 24 hours. Total RNA was then immediately extracted. A partial ovine GAPDH (oGAPDH) cDNA was isolated by reverse transcriptase polymerase chain reaction (RT/PCR) and sequenced. A one-tube semiquantitative RT/PCR amplification assay was established, and GAPDH mRNA was subsequently quantified in all samples of total RNA. The PCR products were separated by size, quantified by Southern hybridization analysis, and normalized to 28S rRNA content. Expression of GAPDH protein was also measured by Western analysis of endothelium-derived protein from omental (n = 14) and uterine (n = 16) arteries from NP and P ewes.

RESULTS

Pregnancy was associated with a 4.5-fold increase in GAPDH mRNA levels in UAEC, although in vitro exposure of primary cultures of UAEC from NP ewes to NO or angiogenic growth factors did not significantly change GAPDH mRNA expression. A 1.6-fold increase in GAPDH protein was detected in the uterine artery endothelium of P versus NP ewes, but no corresponding increase was found in omental artery endothelium.

CONCLUSIONS

Pregnancy increases the expression of both GAPDH mRNA and protein in UAEC. Furthermore, the pregnancy-induced increase in GAPDH protein in the endothelium of the uterine artery appears specific, as it is not observed in the omental (systemic) artery. This induction is not, however, reproduced in vitro with exogenous NO or angiogenic growth factor treatment (up to 24 hours).

Type II cells were isolated from rats with a purity of 80-95% with less than 4% macrophages. These cells, after plating for approximately <em>16</em> h, were cultured with 50% RPMI <em>16</em>40 and 50% (vol/vol) conditioned medium obtained from confluent hamster lung <em>fibroblasts</em>, together with 0.1% fetal calf serum (FCS). Conditioned media were obtained from either <em>fibroblasts</em> derived from normal hamsters breathing room air [normoxic-conditioned medium (NCM)] or from hamsters exposed for 4 days to 100% O2 [hyperoxic-conditioned medium (HCM)]. Controls consisted of 100% minimal essential medium (MEM) containing 0.1% FCS. Over a 96-h culture period, NCM stabilized cell populations but was unable to induce proliferation. In contrast, at low cell densities, HCM could cause a two- to threefold increase in type II cell number within 24-48 h after introduction. This effect could not be demonstrated at high cell densities. When tested with FCS concentrations ranging from 0 to 10%, maximum effects were obtained using 0.1-0.2% FCS. We conclude that lung <em>fibroblasts</em> from oxidant-injured hamsters produce <em>growth</em> <em>factors</em> that can stimulate at least one mitotic division in cultured type II cells, which are plated at low density. These <em>factors</em> are absent, or present in much lower concentration, in lung <em>fibroblasts</em> from normal animals.

In the present study, we attempted to clarify the controversial question whether basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, FGF-2) mRNA is present or absent in the embryonic central nervous system (CNS). For this purpose we analyzed the expression of the FGF-2 mRNA in the embryonic and adult forebrain, brainstem, and spinal cord using the highly specific ribonuclease protection assay. Using this method we were able to detect FGF-2 mRNA in the rat CNS of embryonic day (E) <em>16</em> and 17, however, at lower levels compared to adult FGF-2 mRNA levels. In addition, we show that a FGF-2 antisense transcript is expressed in embryonic CNS tissue. Furthermore, using this method, we demonstrate FGF receptor 1 mRNA in the rat embryonic and adult CNS. The presence of FGF-2 and FGF receptor 1 suggests a physiological role for this <em>growth</em> <em>factor</em> during the development of the embryonic CNS.

BACKGROUND

Wound contraction and scar formation after cleft palate repair impair the growth of the maxilla. The implantation of a growth factor-loaded scaffold might solve these problems.

METHODS

The tissue response to fibroblast growth factor (FGF)-2 loaded collagen scaffolds was evaluated after implantation in the palate of rats. Scaffolds, with and without FGF-2, were implanted submucoperiosteally in the palate of 25 rats and evaluated after up to 16 weeks. On hematoxylin and eosin (H&E)-stained sections, the cell density and the number of giant cells within the scaffolds were quantified. Infiltration of inflammatory cells, myofibroblasts, and the number of blood vessels were quantified after immunohistochemistry.

RESULTS

The cell density was significantly higher in the FGF-2 group up to 4 weeks after implantation (102% at 2 weeks, P < 0.001). The number of blood vessels was also significantly higher in the FGF-2 group at 1 and 2 weeks (316% at 1 week, P = 0.003), but the myofibroblast score was lower (100% at 2 weeks, P = 0.008). A comparable mild and rapidly subsiding inflammatory response and foreign body reaction were found in both groups.

CONCLUSIONS

FGF-2-loaded scaffolds displayed a faster influx of host cells, an increased rate of vascularization, and a reduced differentiation of myofibroblasts. These scaffolds might therefore be highly suitable for intra-oral reconstructions, such as cleft palate repair.

To provide data about the frequency of prenatal misdiagnosis in achondroplasia (Ach), we retrospectively abstracted data from 37 consecutive referrals of infants with Ach where ultrasound was performed prenatally. Nine of 37 (24 per cent) had a positive family history of Ach; all nine were correctly diagnosed prenatally. Of the 28 with no family history of Ach, <em>16</em> (57 per cent) were recognized to have abnormalities on ultrasound but none was given a definite diagnosis of Ach. Five families received an appropriate diagnosis of "most likely' Ach and four others were given a non-specific (but appropriate) diagnosis of some dwarfing disorder, not otherwise specified. In seven instances (25 per cent), an incorrect diagnosis of a lethal or very severe disorder was provided. These results illustrate the difficulty of making a specific prenatal diagnosis of Ach. In the face of the resulting uncertainty, physicians appear to elect to emphasize the most severe of alternative diagnoses. Given the homogeneity of mutations within the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) gene in the vast majority of patients with Ach, FGFR3 mutational analysis can be offered in every instance where a short-limb disorder is ultrasonographically detected in the latter stages of pregnancy. This would reduce the amount of incorrect and potentially harmful information provided to families.

To clarify the effects of recombinant human <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (rhFGF-2) on the osteoconduction ability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in vivo, rhBMP-2 (2 microg) was mixed with different doses of rhFGF-2 (0, <em>16</em>, 80, 400, or 2,000 ng), and implanted into the lower leg muscle of rats using type I collagen as a carrier. Twenty-one days later, ectopic neoplastic bones had bone mineral content, bone area, and bone mineral density measured by means of dual energy X-ray absorptiometry and imaged by soft X-ray. The values for alkaline phosphatase activity and calcium content were determined, and histology obtained. In the group treated with rhFGF-2 at <em>16</em> ng, alkaline phosphatase activity, calcium content, bone mineral content, bone area, and bone mineral density were the greatest of all treatment groups, and the richest trabeculae were histologically observed in this group. In the groups treated with rhFGF-2 at 80, 400, or 2,000 ng, bone formation was suppressed in a dose-dependent manner. These results indicate that rhFGF-2 promotes ectopic rhBMP-2-related osteoinduction at a low concentration (<em>16</em> ng) in vivo, and that it suppresses osteoinduction at a higher amount (>80 ng).

OBJECTIVE

Hyperthyroidism is a well-described cause of hyperphosphatemia. We aimed to clarify the physiological role of fibroblast growth factor (FGF)-23 in serum phosphate homeostasis in patients with Graves' disease during the course of treatment for hyperthyroidism.

BACKGROUND

The study group comprised 56 patients (45 for a cross-sectional study and 11 for a longitudinal study) with Graves' disease. For the cross-sectional study, patients were assigned, on the basis of their serum phosphate level, to a hypophosphatemia group (n = 14), a normophosphatemia group (n = 16), or a hyperphosphatemia group (n = 15). Serum FGF-23, calcium, phosphate, PTH, and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels were compared between the three groups. For the longitudinal study, we assessed changes in these biochemical indices before and after antithyroid treatment.

RESULTS

In the cross-sectional study, the serum FGF-23 level was significantly higher (P < 0.05) in the hyperphosphatemia group than in the other groups (61 +/- 36 ng/liter vs. 31 +/- 22 ng/liter and 30 +/- 9 ng/liter). In the longitudinal study, serum levels of FGF-23 decreased significantly (P < 0.05) from a high of 54 +/- 12 ng/liter before treatment to 29 +/- 14 ng/liter after treatment. In contrast, the serum 1,25(OH)(2)D level increased significantly (P < 0.005) from 55 +/- 22 pmol/liter before treatment to 185 +/- 76 pmol/liter 3 months after treatment. Serum FGF-23 levels were positively correlated with serum phosphate levels (P < 0.0001) and negatively correlated with serum 1,25(OH)(2)D levels (P < 0.0001).

CONCLUSIONS

The significant positive correlation between serum levels of phosphate and FGF-23 indicates that FGF-23 may play an important role in serum phosphate homeostasis by its up-regulation in the hyperphosphatemic condition.

Dominant mutations in three <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor genes (FGFRs1-3) cause Crouzon, Jackson-Weiss, Pfeiffer, and Apert syndromes. In the present study, 50 Brazilian patients with these four syndromes (27 Apert, 17 Crouzon, 5 Pfeiffer, and 1 Jackson-Weiss patients) were screened for mutations in the FGFR1-3 genes. Except for one, all the Apert patients had either S252W (n = <em>16</em>) or P253R (n = 10) mutations. The remaining Apert case is atypical with a mutation altering the splice site of FGFR2 exon IIIc. The Pfeiffer patients had mutations in one of the FGFR genes: three in FGFR2, one in FGFR1, and one in FGFR3. In contrast, only 8 of the 17 Crouzon patients studied had a mutation in either FGFR2 (n = 7) or FGFR3 locus (n = 1). Mutations in the FGFR2 locus account for most (93%) of our syndromic craniosynostotic cases, whereas 5% had mutations in the FGFR3 locus and only 2% had mutations in the FGFR1 gene. Except for one, all the other mutations were reported previously in craniosynostotic patients from other populations. Interestingly, the mutation C278F, previously described in Crouzon and Pfeiffer cases, was here identified in a familial case with Jackson-Weiss. Also, unexpectedly, a common mutation altering the splice site of the FGFR2 exon IIIc was found in one Apert and two Pfeiffer patients. In addition, we identified a new mutation (A337P) in the FGFR2 exon IIIc associated with Crouzon phenotype.

BACKGROUND

Vitamin D deficiency or insufficiency is prevalent in kidney transplant recipients. Little is known about post-transplantation changes in vitamin D forms, which are essential for bone health and other health outcomes. The aim was to measure the levels of calcidiol and calcitriol during the first 6 months after kidney transplantation and examine their relation with other bone mineral metabolic parameters.

METHODS

A prospective study was performed on 98 patients recruited between April 2010 and June 2011. Calcidiol and calcitriol levels were measured at baseline and at days 15, 30, 90, and 180 after kidney transplantation.

RESULTS

Serum calcidiol levels remained persistently low: 14.3 (9-22) ng/ml at baseline and <em>16</em>.3 (10.1-20.6) ng/ml at 6 months (P=0.641). At 6 months, calcidiol levels showed an inverse correlation with simultaneously measured parathyroid hormone levels. Calcidiol showed a trend to be higher in patients transplanted in spring but with no statistically significant difference. Calcitriol levels increased from 17 (13-23.7) pg/ml at baseline to 24 (<em>16</em>-32) pg/ml (P=0.002) in the first 2 weeks after transplantation and reached 37 (25-50) pg/ml (P=0.000) after 6 months. During the follow-up, calcitriol levels showed a significant inverse correlation with baseline <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 levels. At month 6, calcitriol levels were inversely correlated with baseline <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 levels and directly correlated with calcidiol levels.

CONCLUSIONS

In most patients, calcidiol levels remain low 6 months after kidney transplantation, whereas calcitriol levels rapidly return to normal. Lower calcidiol blood levels promoted lower calcitriol blood levels and higher parathyroid hormone concentrations.