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Objectives: To assess the effect of transdermal electromotive drug therapy (EMDT) on transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) expression and their receptors in plaques in patients with Peyronie's disease., Methods: Tissue was obtained from 13 patients with stable Peyronie's disease who had undergone plaque excision because of penile curvature. Of the 13 patients, 7 underwent EMDT with dexamethasone, verapamil, and lidocaine as first-line therapy before plaque excision and 6 were therapy naive. TGF-beta and bFGF mRNA and protein expression and that of their receptors were measured using real-time polymerase chain reaction and Western blotting., Results: The mean patient age was 52.83 years. The mean interval from the end of EMDT to plaque excision was 7.6 months, with stable disease for >or=5 months. The comparison of TGF-beta mRNA expression in the plaques showed no difference between the EMDT and therapy-naive patients (P = .17). Also, TGF-beta protein expression in the plaques was not significantly different between the EMDT and therapy-naive patients (P = .443). TGF-beta receptor 1 mRNA expression in the plaques was significantly different between the EMDT and therapy-naive patients (P = .023), but no difference was found for TGF-beta receptor 2 mRNA (P = .292). The expression of bFGF mRNA (P = .0005) and bFGF protein expression (P = .034) in the plaques was significantly lower after EMDT. bFGF receptor mRNA expression (P = .619) showed no significant differences., Conclusions: Patients with Peyronie's had significantly lower bFGF mRNA and bFGF protein expression in the plaques after EMDT. Also, overexpression of TGF-beta protein and the TGF-beta receptor was identified in the EMDT plaques compared with the therapy-naive plaques.

Osteofibrous dysplasia (OFD) and fibrous dysplasia (FD) are both benign bone lesions which comprise the proliferation of fibroblast-like cells with bone formation, and these fibroblast-like cells have the phenotype of osteoprogenitor cells. The roentgenograph of OFD shows a heterogeneous osteolytic lesion with surrounding osteosclerosis, whereas FD is typically characterized by a rather homogeneous osteolytic lesion, or 'ground-glass appearance', with a smaller amount of surrounding osteosclerosis. Growth factors of transforming growth factor-beta1 (TGF-beta1), fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) modulate bone differentiation. Expression of these growth factors was examined in the fibroblast-like cells of 16 cases of OFD and 16 cases of FD, immunohistochemically. TGF-beta1 in fibroblast-like cells was frequently expressed both in the OFD (16/16) and the FD (15/16) cases. The frequency of FGF-2 (16/16) expression and BMP-2 (13/16) expression in the fibroblast-like cells of OFD was higher than that of those [FGF-2 (8/16) and BMP-2 (6/16)] in the fibroblast-like cells of FD, with a statistical significance. These results seem to suggest that fibroblast-like cells of OFD have greater bone-forming ability than those of FD, and may explain the roentgenographic difference between OFD and FD and a difference in the nature of fibroblast-like cells between these two types of lesions.

Background: The success of anterior cruciate ligament (ACL) reconstruction using tendon grafts depends on biological integration between the tendon and bone. Growth factors play a significant role in this integration process, but few studies have defined the regulating mechanisms of these growth factors during tendon-bone healing. The aim of the present study was to clarify the relationship between the histological changes and the expression of endogenous growth factors at the tendon-bone interface., Methods: Using intra-articular tendon transfer in rabbits to stimulate ACL reconstruction, the presence of fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), and BMP-7 at the interface between the tendon and bone was evaluated immunohistochemically. Histological and immunohistochemical investigations were performed at 1, 3, 6, and 12 weeks after surgery., Results: Fibrous integration of the tendon graft to the bone was observed immediately after tendon transfer and followed remodeling of the bone tunnel. Fibroblast and vascular growth factors were found in abundance at the tendon-bone interface in the first 3 weeks of graft incorporation, but were absent in the 12-week specimens. BMPs were found throughout the 12-week study period and were observed at high concentrations near the bone., Conclusions: These results indicate that FGF-2 and VEGF contribute to fibrous integration between the tendon and bone during the early postoperative stage, and that BMP-2 and BMP-7 are specifically involved in bone remodeling leading to osseous integration. The early stages of tendon-bone healing might be important in controlling the integration process of the interface in ACL reconstruction surgery as seen in this rabbit model.

During the development and regeneration of skeletal muscle, many growth factors, such as basic fibroblast growth factor (bFGF, FGF-2) and myostatin, have been shown to play regulating roles. bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle, whereas myostatin plays a series of contrasting roles. In order to elucidate whether the expression of bFGF has any relationship with the expression of myostatin in skeletal muscle cells, we constructed a eukaryotic expression vector for the expression of exogenous bFGF in murine C2C12 myoblasts. Quantitative RT-PCR assays indicated that with the increase of the expression of exogenous bFGF gene, the expression of endogenous myostatin gene was suppressed at mRNA level and protein level.

Objective: To investigate the characteristics of the expression of transforming growth factor-beta(1) (TGF-beta(1)) and basic fibroblast growth factor (bFGF) in the apical articular process cartilages of adolescent idiopathic scoliosis (AIS) patients., Methods: The specimens of articular processes of 22 AIS patients and 18 congenital scoliosis (CS) patients were collected during operation. The pathology of the processes was observed with H.E staining. Immunohistochemistry and in situ hybridization were adopted to detect the expression of TGF-beta(1) and bFGF. The differences in the pathological changes, and expression of TGF-beta(1) and bFGF between the apical processes and the end processes, the convex processes and the concave processes, and the AIS processes and CS processes. The images of immunohistochemistry and in situ hybridization were input into the image analysis system and were analyzed semi-quantitatively. The SAS (8.01) software was adopted and (128.1 +/- 50.6 vs 165.4 +/- 59.2, 126.5 +/- 47.0 vs 168.3 +/- 46.8; 76.6 +/- 29.9 vs 96.4 +/- 28.4, 73.7 +/- 31.6 vs 101.8 +/- 39.4; 77.1 +/- 52.2 vs 114.4 +/- 59.4, and 69.5 +/- 40.1 vs 109.8 +/- 51.0 (P < 0.05) was defined as the significant levels., Results: The expression of TGF-beta(1) and the expression of bFGF were not significantly different between the AIS patients and the CS patients. The TGF-beta(1) expression in the concave side of the apical vertebrae of the AIS group was 165.4 +/- 59.2, significantly higher than that in the convex sides (128.1 +/- 50.6, P = 0.03), and the TGF-beta(1) expression in the concave side of the apical vertebrae of the CS group was 168.3 +/- 46.8, significantly higher than that of the convex side (126.5 +/- 47.0, P = 0.02). However, there was no statistically significant differences in the TGF-beta(1) expression between the concave and convex sides of the upper and lower end vertebrae and between the upper and lower end vertebrae. The bFGF expression in the concave side of the apical vertebrae of the AIS group, and the bFGF expression in the concave side of the apical vertebrae of the CS group was 101.8 +/- 39.4, significantly higher than that of the convex side (73.7 +/- 31, P = 0.02). However, there were no statistically significant differences in the bFGF expression between the concave and convex sides of the upper and lower end vertebrae and between the upper and lower end vertebrae., Conclusion: The cartilages of the apical processes show some signs of regression and hypoplasia in AIS patients, especially at the concave side in comparison with the convex side. Increase of TGF-beta(1) and bFGF in the concave sides of apical processes in AIS may be the results of reconstruction of extracellular matrix and the compensation reactions which are caused by abnormal biomechanical forces, especially compressive stresses.

Background and Objective: The study was designed to determine the temporal expression of insulin-like growth factor (IGF)-I, IGF-II, basic fibroblast factor 2 (bFGF-2), and transforming growth factor beta 1 (TGF-beta1) in recessed extraocular muscles., Materials and Methods: Sixteen eyes of eight rabbits were subjected to conventional 4-mm recession of superior rectus muscles. Two rabbits were untreated as control. The rabbits were killed and their eyes were enucleated at 3 (group 3), 6 (group 6), 24 (group 24), and 72 (group 72) hours after the operation (two rabbits per group), and the expression of IGF-I, IGF-II, bFGF-2, and TGF-beta1 was immunohistochemically examined., Results: The peak levels of IGF-I, IGF-II, and TGF-beta1 expression were observed in groups 24, 6, and 3, respectively. However, bFGF-2 was less expressed than the other growth factors in all groups., Conclusions: IGF-I, IGF-II, bFGF-2, and TGF-beta1 in regenerating muscle cells were expressed by different kinetics, suggesting a distinct role of each growth factor during wound healing after recession of extraocular muscles.

In the present study, we determined the interactive effects of ethanol and transforming growth factor beta-3 (TGF-beta3) on basic fibroblast growth factor (bFGF) release from folliculostellate (FS) cells and the role of the mitogen-activated protein kinase (MAPK) pathway in this interaction. We found that TGF-beta3 and ethanol alone increased release of bFGF from FS cells, but together they showed markedly increased levels of bFGF compared with the individual effect. Ethanol and TGF-beta3 alone moderately increased activation of MAPK p44/42, but together they produced marked activation of MAPK p44/42. TGF-beta3 alone increased the activation of smad2. Ethanol did not activate smad2 or alter TGF-beta3 activation of smad2. Pretreatment of FS cells with a mitogen-activated protein kinase kinase 1/2 inhibitor or with a protein kinase C (PKC) inhibitor suppressed the TGF-beta3 and ethanol actions on MAPK p44/42 activation and bFGF release. Ethanol and TGF-beta3, either alone or in combination, increased the levels of active Ras. Furthermore, the MAPK p44/42 activation by TGF-beta3 and ethanol was blocked by overexpression of Ras N17, a dominant negative mutant of Ras p21. These data suggest that the PKC-activated Ras-dependent MAPK p44/42 pathway is involved in the cross talk between TGF-beta3 and ethanol to increase bFGF release from FS cells.

Collagen overproduction characteristic for dilated cardiomyopathy (DCM) is coregulated by endothelin (ET)-1, transforming growth factor (TGF)-beta1, basic fibroblast growth factor (bFGF) and matrix metalloproteases (MMPs). Whether these molecules affect grafts transplanted to heart failure patients is unknown. In 67 idiopathic DCM patients, 31 patients with ischemic cardiomyopathy (ICM) and 16 controls, the myocardial bFGF, TGF-beta1, pro-collagen (PrCol) type 1 (PrCol1-alpha1, -alpha2) and MMP expressions were examined using real-time RT-PCR or Western blotting. mRNA expression was measured in grafts for 1 year. TGF-beta1/bFGF stimulation or gene silencing was used to examine their effect on collagen synthesis in cardiac tissue cultures. TGF-beta1 and PrCol1 were upregulated in DCM only, while bFGF was upregulated in both groups versus controls. TGF-beta1 downregulated MMP-1 and upregulated collagen 1, whereas bFGF upregulated MMP-13 in DCM tissue. Post-transplant PrCol1-alpha1, -alpha2 and ET-1 mRNA increased over time in grafts of DCM patients only, while other factors returned to control baseline levels in DCM and ICM. These data indicate that cardiac transplantation corrects the dysregulated TGF/bFGF/MMP-1/MMP-13, but not the excess collagen and ET-1 synthesis in cardiac grafts transplanted to DCM patients. ET-1 might be a major pathologic trigger for graft fibrosis in DCM.

Objective: To explore the effects of exogenous transforming growth factor-beta 1 (TGFbeta1) on peripheral nerve regeneration after the peripheral nerve injury and if TGFbeta1 regulates the expression of basic fibroblast growth factor (bFGF) in the anterior horn motoneurons of spinal cord during regeneration., Methods: Forty-eight rats were crushed on the right sciatic nerve and then randomly divided into 2 groups: TGFbeta1 group and NS group. In TGFbeta1 group, TGFbeta1 50 microL (0.1 microg/mL) was injected into the proximal nerve near to the crushed nerve and after the operation the injured leg was injected with equal TGFbeta1 whereas the NS was replaced in the NS group. The rats of each group survived for 3, 7, 14 and 21 days after the lesion. The bFGF expression in the anterior horn motoneurons of spinal cord was detected by immunohistochemistry (IHC). Semi-thin section and Fast Blue retrograde tracing were also performed with the rats surviving for 21 days to observe the regeneration of distal end in the injured right sciatic nerve., Results: The number of bFGF immunoreactive positive motoneurons in TGFbeta1 group was obviously higher than that of the NS group (P < 0.05). In the distal sciatic nerve of the rats treated with TGFbeta1, the number and diameter of regenerating myelinated axons and the thickness of myelinated sheath were more than those of the NS group (P < 0.05). The number of motoneurons in spinal cord and neurons in dorsol root ganglia (DRG) labelled with Fast Blue in the NS group was obviously lower than in the TGFbeta1 group (P < 0.01)., Conclusion: Exogenous TGFbeta1 plays an important role in promoting the peripheral nerve regeneration; TGFbeta1 up-regulates the bFGF expression in the anterior horn motoneurons of spinal cord during the peripheral nerve regeneration.

Objective: To evaluate the effects of copper tripeptide (GHK-Cu) on the growth and autocrine production of basic fibroblast growth factor, transforming growth factor beta1, and vascular endothelial growth factor by normal and irradiated fibroblasts in a serum-free in vitro environment., Methods: Primary human dermal fibroblast cell lines were established after explantation from intraoperative specimens obtained from patients who had undergone radiation therapy for head and neck cancer. Normal and irradiated fibroblasts were propagated in serum- and growth factor-free media. Treatment groups were exposed to GHK-Cu (1 x 10(-9) mol/L). We measured cell counts and production of basic fibroblast growth factor, transforming growth factor beta1, and vascular endothelial growth factor., Results: Irradiated fibroblasts survived and replicated in serum-free media. The population-doubling times of normal and irradiated fibroblasts exposed to GHK-Cu were faster than those of nontreated controls. Irradiated fibroblasts treated with GHK-Cu doubled at a rate that approximated that of untreated controls, and produced significantly more basic fibroblast growth factor and vascular endothelial growth factor than untreated controls early after GHK-Cu exposure., Conclusions: Irradiated fibroblasts survive and replicate in serum-free media, establishing this model as ideal for evaluating growth factor production in vitro. Copper tripeptide accelerates the growth of normal and irradiated fibroblasts to the point where treated irradiated fibroblasts approximate the population-doubling time of normal controls. An early increase in basic fibroblast growth factor and vascular endothelial growth factor production by GHK-Cu-treated irradiated fibroblasts may improve wound healing.

Muscle contusions represent a major part of sports injuries. The suggested treatments are generally sufficient to support muscle healing, but require a relatively long period of time. Given that autologous blood products are safe treatments, we have used a technique which stimulates the release of certain growth factors in the autologous conditioned serum (ACS). Those growth factors are known to improve the proliferative activity of myogenic precursor cells. Mice were subjected to an experimental contusion injury to their gastrocnemius muscle; one group received local injections of ACS at 2 hrs, 24 hrs, and 48 hrs after injury, a control group received saline injections. The histology results showed that satellite cell activation at 30/48 hrs post injury was accelerated and the diameter of the regenerating myofibers was increased compared to the controls within the first week after injury. ELISA results on the ACS have shown that the elevations in FGF-2 (460 %) and TGF-beta1 (82 %) could be partly responsible for the accelerating effects on regeneration due to proliferative and chemotactic properties. We conclude that ACS injection is a promising approach to reduce the time of recovery from muscle injury. In terms of clinical targets, this new approach could be used in the treatment of sports injuries and may also be interesting in postoperative situations.

This study examined temporal changes in growth plate apoptosis molecules and growth factors in an animal model of radiation injury with and without a radioprotectant. Thirty weanling 5-week Sprague-Dawley rats underwent right knee irradiation with single-fraction 17.5 Gy while the left served as internal control. Six animals each were sacrificed at 0.5, 1, 2, 3, or 4 weeks after irradiation. Half of the animals received pretreatment with amifostine (WR-2721) radioprotectant. Immunohistochemical staining for PTHrP, Bcl-2, Bax, caspase-3, FGF-2, and TGF-beta was performed. PTHrP decreased to a nadir at 1 week after irradiation but rebounded to above control levels at 2 weeks in the reserve and transitional zones. The radioprotectant amifostine blunted the decrease in PTHrP but kept PTHrP expression lower than controls during the rebound phase in untreated irradiated animals. Hypertrophic zone Bax expression was decreased by amifostine in both irradiated and non-irradiated limbs at 1 and 2 weeks. FGF, TGF-beta, Bcl-2, and caspase levels generally decreased at 1 week and returned thereafter toward control levels. These findings underscore the importance of PTHrP in response to growth plate irradiation and show the novel finding of a decrease in Bax expression with amifostine pretreatment.

Radiation therapy for cancer permanently damages tissue in the line of treatment. This study sought to establish a serum-free protocol to evaluate the growth of irradiated fibroblasts and to analyze the levels of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) compared with normal fibroblasts. One irradiated cell line of human dermal fibroblasts was established from an intraoperative specimen obtained from a patient who had undergone radiation therapy for head and neck cancer. Irradiated and normal fibroblasts were then plated in UltraCULTURE (serum and growth factor free), modified Webber's medium (bFGF 50 ng/ml, insulin-like growth factor 100 ng/ml), and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (serum with undefined basal growth factors). Irradiated cells were also seeded in UltraCULTURE with 50 and 100 ng/ml of bFGF. Cell counts were performed at 0, 1, 3, 5, and 7 days, and cell supernatants were assayed for bFGF and TGF-beta. Irradiated and normal fibroblasts exhibited stronger growth in modified Webber's medium than in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum. Growth of irradiated fibroblasts under bFGF modulation was similar to their growth in Webber's medium. Furthermore, irradiated fibroblasts remained viable in a serum-free and growth factor-free environment for at least 7 days; however, their growth and autocrine growth factor production was less than that of normal cells. This confirms the results of previous studies suggesting that cells from irradiated tissue undergo cellular changes. This study provides an effective model for the first-line evaluation of agents to improve wound healing, and it helps to establish standard levels of bFGF and TGF-beta production for irradiated fibroblasts.

Background: Heparin decreases dermal fibroblast proliferation and collagen production according to several studies. Heparin may mediate these effects by altering the levels of growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor-beta 1 (TGF-b1). This study sought to delineate the effect of heparin on proliferation and bFGF and TGF-b1 production by human normal, keloid, and fetal dermal fibroblasts., Material/methods: Human normal, keloid, and fetal dermal fibroblasts were propagated in a serum-free in vitro model, with exposure to 0 microg/ml, 50 microg/ml, 300 microg/ml, or 600 microg/ml heparin for 0, 24, 72, or 96 hours. Cell counts were determined by phase contrast microscopy. Levels of bFGF and TGF-b1 in the supernatants were determined by enzyme-linked immunosorbant assay (ELISA)., Results: Heparin inhibited keloid and fetal fibroblast proliferation. All doses of heparin significantly stimulated production of bFGF by normal (341% to 1137% increase), keloid (237% to 1955% increase), and fetal fibroblasts (292% to 1866% increase) at all time points (p<0.05). Heparin (300 microg/ml and 600 microg/ml) also stimulated production of TGF-b1 by normal (56% to 75%), keloid (105% to 269%), and fetal fibroblasts (25% to 57%), with statistical significance (p<0.05) at various time points. 600 microg/ml heparin generally caused the greatest increase in growth factor levels., Conclusions: Heparin inhibits proliferation by keloid and fetal fibroblasts and significantly stimulates production of bFGF and TGF-b1 by normal, keloid, and fetal dermal fibroblasts. These effects of heparin on dermal fibroblasts may have implications for wound healing in vivo.

Objective: To evaluate differences in fibroblast autocrine growth factor production by human fetal, keloid, and normal adult dermal fibroblasts., Design: Serum-free cell lines of fetal, keloid, and normal adult dermal fibroblasts were established. Cell counts were performed and supernatants collected at 4, 24, and 72 hours. Cell-free supernatants were quantitatively assayed for transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF)., Results: Population doubling times for fetal, keloid, and normal adult fibroblasts were 120.0, 88.1, and 128.4 hours, respectively. Differences in population doubling times did not reach statistical significance. Statistically significant differences between TGF-beta1 levels from fetal and normal adult fibroblasts were seen at 24 and 72 hours. Significant differences between TGF-beta1 levels from keloid and normal adult fibroblasts were also seen at 24 and 72 hours. Fetal fibroblasts demonstrated higher levels of bFGF than normal adult fibroblasts at each time point, but these differences were not statistically significant. No significant differences were observed between keloid and normal adult bFGF levels., Conclusions: Both fetal and keloid fibroblasts produce significantly more TGF-beta1 than normal adult fibroblasts. Our data and the data of others suggest that fetal fibroblasts produce more bFGF than adult fibroblasts. The serum-free model we describe can be used to quantitatively measure autocrine growth factor production by cells that underlie clinically different types of wound healing. This model provides information that may allow us to better treat and prevent undesirable scarring.

To investigate the changes in the expression of basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in glomus cell grafts of carotid body in the rat model of 6-hydroxydopamine-induced Parkinson disease, immunohistochemical staining of bFGF and TGFbeta2 in the sections of striate body was done on the 2nd, 4th and 12th week after transplantation. The results showed that on the 2nd week after transplantation, bFGF and TGFbeta2 were not detectable in the glumous cell grafts. On the 4th week after graft, bFGF and TGFbeta2 immunoreactivity was increased within the grafts and at the graft-host interface but was restricted only to astrocytes. In the striatum surrounding the graft, bFGF was expressed persistently, while TGFbeta2 showed transient expression. It was suggested that the transient expression of TGFbeta2 was likely due more to the trauma imposed by the graft procedure than to an intrinsic. The deficiency in astrocytic bFGF early after graft may be responsible for the poor survival of grafted glomus cells of carotid body.

Background: Triamcinolone acetonide has been shown to decrease both cellular proliferation and collagen production by dermal fibroblasts. An alteration of cytokine levels may mediate these effects., Objective: To delineate the effect of triamcinolone acetonide on both cellular proliferation and the production of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1) by human fibroblasts grown in a serum-free in vitro model., Methods: Human normal and keloid dermal fibroblasts were propagated in a serum-free in vitro model with exposure to 0, 5, 10, or 20 microm triamcinolone acetonide for 0, 24, 72, or 96 hours. Cell counts were determined by phase contrast microscopy. Levels of bFGF and TGF-beta1 in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA)., Results: In our study, 20 microm triamcinolone acetonide caused statistically significant increases in the peak levels of bFGF for normal and keloid fibroblast cell lines (P < 0.05). It also caused statistically significant decreases in the level of TGF-beta1 for normal and keloid fibroblast cell lines. For the keloid fibroblasts, 10 microm triamcinolone acetonide also caused a statistically significant decrease in the level of TGF-beta1., Conclusion: We conclude from these results that triamcinolone acetonide increases the production of bFGF and decreases production of TGF-beta1 by human dermal fibroblasts.

Arteritis induced in rats by vasodilators, fenoldopam and theophylline, was examined immunohistochemically for expressions of inducible type of nitric oxide synthase (iNOS), basic fibroblast growth factor (bFGF) and tumor growth factor-beta1 (TGF-beta1). Rats were administered fenoldopam for 24 hours by intravenous infusion with or without following repeated daily oral administrations of theophylline. Irrespective of theophylline administration, iNOS antigens were remarkably abundant in ED-1-positive cells on day 5 and 8 post-fenoldopam-infusion (DPI); bFGF antigens were remarkably abundant in ED-1-positive cells on 1 and 3 DPI; TGF-beta1 antigens were observed in ED-1-positive cells on and after 5 DPI. These results suggest that the peak expression of iNOS antigen was followed by that of bFGF antigen, and bFGF may have a suppressive effect on iNOS expression in these rat arteritis models. On the other hand, TGF-beta1 was not considered to have a suppressive effect on iNOS expression in these models.

Aims: To evaluate the expression of transforming growth factor beta1 (TGF-beta1) and fibroblast growth factor 2 (FGF-2) mRNA in stromal cells in response to injury in the presence of either TGF-beta1 or FGF-2. It has been shown previously that heparan sulfate proteoglycans and FGF-2 are present transiently during wound repair in vivo and that an increase in TGF-beta1 mRNA is detected rapidly after injury., Methods: Primary corneal fibroblasts were cultured to confluency, serum starved, and linear wound(s) were made in medium containing TGF-beta1 or FGF-2. TGF-beta1 and FGF-2 mRNA expression were evaluated using both northern blot analysis and in situ hybridisation. Both dose dependent and time course experiments were performed. Whole eye organ culture experiments were also carried out and growth factor expression was assessed., Results: Injury and exogenous TGF-beta1 increased TGF-beta1 mRNA values. The increase in expression of FGF-2 mRNA was not detected until wound closure. In contrast, FGF-2 inhibited the expression of TGF-beta1. TGF-beta1 increased TGF-beta1 mRNA stability but did not alter that of FGF-2. Migration assay data demonstrated that unstimulated stromal cells could be activated to migrate to specific growth factors., Conclusions: TGF-beta1 specifically enhances cellular responsiveness, as shown by increased stability after injury and the acquisition of a migratory phenotype. These data suggest that there is an integral relation during wound repair between TGF-beta1 and FGF-2.

Background: A serum-free in vitro model was used to determine the effect of combined carbon dioxide (CO2) and erbium (Er):YAG laser (Derma K; ESC/Sharplan Medical Systems, Yokneam, Israel) irradiation on keloid-producing fibroblasts (KFs) from 2 distinct facial sites. Transforming growth factor beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) play an integral part in wound healing and were assayed using this model. It has always been a clinical impression that fibroblasts from different regions of the face behave differently. This is exemplified by patients prone to lobule keloid formation after ear piercing, who heal normally after a facial incision., Design: Laboratory-based wound healing., Methods: Human KF cell lines were established from operative specimens using standard explant techniques. At 48 hours after seeding, 20% of each well was exposed to 1.7 J/pulse of Er:YAG laser energy and CO2 delivered at 3 or 5 W and at a duty cycle of 25%, 50%, or 100%. Using a quantitative enzyme-linked immunosorbent assay, TGF-beta1 and bFGF were assayed from collected supernatants., Results: Laser-treated ear lobule KFs demonstrated decreased TGF-beta1 production when compared with preauricular KFs. Statistical significance (P<.005) was seen in the 3-W CO2 25% duty cycle; a trend was seen in the 3-W CO2 50% duty cycle (P<.08). Preauricular KFs secreted increased bFGF when compared with lobule KFs. Significance was seen in the 3-W CO2 25% and 50% duty cycles (P<.05). Laser-treated preauricular KFs had increased bFGF secretion when compared with non-laser-treated preauricular KFs in the 3-W CO2 25%, 50%, and 100% duty cycles., Conclusions: Combined CO2 and Er:YAG laser treatment decreases the production of TGF-beta1 in preauricular and ear lobule KFs. This laser may have clinical promise in the treatment of keloids. Finally, the different growth factor profiles obtained suggest that KFs from the ear lobule and preauricular regions are different.

The primary heart tube is an endocardial tube, ensheathed by myocardial cells, that develops from bilateral primary heart fields located in the lateral plate mesoderm. Earlier mapping studies of the heart fields performed in whole embryo cultures indicate that all of the myocardium of the developed heart originates from the primary heart fields. In contrast, marking experiments in ovo suggest that the atrioventricular canal, atria and conotruncus are added secondarily to the straight heart tube during looping. The results we present resolve this issue by showing that the heart tube elongates during looping, concomitant with accretion of new myocardium. The atria are added progressively from the caudal primary heart fields bilaterally, while the myocardium of the conotruncus is elongated from a midline secondary heart field of splanchnic mesoderm beneath the floor of the foregut. Cells in the secondary heart field express Nkx2.5 and Gata-4, as do the cells of the primary heart fields. Induction of myocardium appears to be unnecessary at the inflow pole, while it occurs at the outflow pole of the heart. Accretion of myocardium at the junction of the inflow myocardium with dorsal mesocardium is completed at stage 12 and later (stage 18) from the secondary heart field just caudal to the outflow tract. Induction of myocardium appears to move in a caudal direction as the outflow tract translocates caudally relative to the pharyngeal arches. As the cells in the secondary heart field begin to move into the outflow or inflow myocardium, they express HNK-1 initially and then MF-20, a marker for myosin heavy chain. FGF-8 and BMP-2 are present in the ventral pharynx and secondary heart field/outflow myocardium, respectively, and appear to effect induction of the cells in a manner that mimics induction of the primary myocardium from the primary heart fields. Neither FGF-8 nor BMP-2 is present as inflow myocardium is added from the primary heart fields. The addition of a secondary myocardium to the primary heart tube provides a new framework for understanding several null mutations in mice that cause defective heart development.

Transforming growth factor (TGF)-beta(1) is an inflammatory cytokine that plays multiple roles in pulmonary fibrosis. In vascular epithelium, it has been shown to regulate production and activity of fibroblast growth factor (FGF)-2, a potent type II cell mitogen in the lung. Such a relationship could have important consequences in prefibrotic change in the lung alveolus, where reepithelialization of alveolar surfaces is crucial. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-beta(1) or FGF-1, another type II cell mitogen. Isolated rat type II cells were exposed to 0 to 40 ng/mL of TGF-beta(1) or 0 to 500 ng/mL of FGF-1 in serum-free medium for 1 to 3 days. Using a specific immunoassay, significant increases in FGF-2 protein in type II cell lysates were achieved after 1 day of exposure to 100 ng/mL of FGF-1 and after 3 days of treatment with 8 ng/mL of TGF-beta(1). Similarly, transcripts for FGF-2 were dramatically increased with TGF-beta(1) or FGF-1, as were those for FGF receptor (FGFR)-1. These interactions were dramatically effected by the addition of heparin, a model sulfated extracellular matrix (ECM). Heparin as low as 0.01 mg/mL significantly downregulated expression of TGF-beta(1) and FGF-1-stimulated FGF-2 and FGFR-1. These results demonstrate important regulatory links between FGF-2, sulfated ECMs, and both TGF-beta(1) and FGF-1, which could contribute to the modulation of normal cell turnover, development, and repair processes attendant to fibrosis in the lung.

Objective: To study the expression of beta-Fibroblastic Growth Factor (bFGF) and Transforming Growth Factor beta 1 (TGF beta 1) in filtering area following sclerectomy and the interaction between growth factors and interferon alpha-2b (IFN alpha-2b)., Methods: Immunohistochemical technique were applied to test the expression bFGF and TGF beta 1 in frozen sections of filtration area following sclerectomy on postoperative day three, five, seven and 14 in white rabbits, respectively. 16 eyes of eight rabbits in all were randomly and equally divided into two groups. The experimental eyes had subconjunctivally been administered IFN alpha-2b 5 x 10(5) IU 0.2 ml each time at filtering bleb following operation whereas the control eyes hadn't taken., Results: In control group, positive cell and substance expressing bFGF and TGF beta 1 on postoperative day three were revealed in the filtration area, on postoperative day five and seven, they were gradually increased. On the postoperative day 14, all significant cells and substance markly decreased. In experimental group only small amount of positive cell and bFGF or TGF beta 1-expressing substance were sparely distributed in the filtration area., Conclusion: The filtration area after glaucoma filtering surgery was "bathed" in growth factors such as bFGF and TGF beta 1 which well-known play an important role in stimulating wound healing response, IFN alpha-2b had an inhibitory effect on bFGF or TGF beta 1 expression in wound environment.

Objective: To map the expression of transforming growth factor (TGF)-beta(1), TGF-beta(3), and basic fibroblast growth factor (bFGF) in full-thickness skin wounds of the horse. To determine whether their expression differs between limbs and thorax, to understand the pathogenesis of exuberant granulation tissue., Study Design: Six wounds were created on one lateral metacarpal area and one midthoracic area of each horse. Sequential wound biopsies allowed comparison of the temporal expression of growth factors between limb and thoracic wounds., Animals: Four 2- to 4-year-old horses., Methods: Wounds were assessed grossly and histologically at 12 and 24 hours, and 2, 5, 10, and 14 days postoperatively. ELISAs were used to measure the growth factor concentrations of homogenates of wound biopsies taken at the same timepoints., Results: TGF-beta(1) peaked at 24 hours in both locations and returned to baseline in thoracic wounds by 14 days but remained elevated in limb wounds for the duration of the study. Expression kinetics of TGF-beta(3) differed from those of TGF-beta(1). TGF-beta(3) concentrations gradually increased over time, showing a trend toward an earlier and higher peak in thoracic compared with limb wounds. bFGF expression kinetics resembled those of TGF-beta(1), but no statistically significant differences existed between limb and thoracic wounds., Conclusions: Growth factor expression is up-regulated during normal equine wound repair. TGF-beta(1) and TGF-beta(3) show a reciprocal temporal regulation. Statistically significant differences exist between limb and thoracic wounds with respect to TGF-beta(1) expression., Clinical Relevance: The persistence of TGF-beta(1) expression in leg wounds may be related to the development of exuberant granulation tissue in this location, because TGF-beta(1) is profibrotic., (Copyright 2001 by The American College of Veterinary Surgeons)

The activation of hepatic stellate cells (HSC) during liver fibrogenesis has been shown to be mediated by paracrine and autocrine loops involving transforming growth factor-beta1 (TGF-beta1) as the main fibrogenic mediator secreted by activated macrophages, endothelial cells and liberated by disintegrated platelets. The cell-associated plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We have studied whether TGF-beta1 could modulate the plasminogen activation system in human HSC and the role of such protease system in the activity of TGF-beta1 on HSC. Urokinase plasminogen activator receptors (u-PAR), u-PA and plasminogen activator inhibitor type 1 (PAI-1) were determined by immunoassay and RNase protection assay. Cell migration, evaluated either as chemotaxis or as chemoinvasion, was studied in Boyden chambers after addition of TGF-beta1, and inhibition with anti-u-PA and anti-u-PAR antagonists [antibodies against u-PA and u-PAR and antisense oligonucleotides (aODN) against u-PAR mRNA]. We have shown that TGF-beta1 is not mitogenic for HSC, while it is a powerful motogen either in chemotaxis or chemoinvasion assays. TGF-beta1 up-regulates the synthesis and expression of PAI-1, as well as u-PAR expression and exposure at the cell membrane, while it does not affect u-PA levels. TGF-beta1-dependent chemoinvasion of reconstituted basement membrane exploits the cell-associated plasminogen activation system, since it is blocked by monoclonal antibodies against u-PA and against various u-PAR domains, as well as by anti-u-PAR aODN. We have also observed a cumulative effect of TGF-beta1, b-FGF and PDGF in the invasion assay of HSC: in the presence of low amounts of TGF-beta1 the chemoinvasive activity of PDGF and bFGF is dramatically increased. Also this cooperation requires u-PAR and is inhibited by monoclonal antibodies against u-PAR domains I, II and III.

Objective: To evaluate the expression and distribution of transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) in the lung tissue of chronic obstructive pulmonary (COPD) rat models and the relationship between these growth factors and the airway wall remodeling. The effects of drugs on them were also investigated., Methods: The COPD rat model (model group) was established by intratracheal instillation of lipopolysaccharide twice and daily exposure to cigarette smoking. Drug intervention groups received daily inhalation of heparin since the second week and TGF-beta1 monoclonal antibody (TB21) 0.5 mg twice through the tail veins. At the end of four weeks, the thickness of the smooth muscle and collagen in bronchi and pulmonary arterioles were measured by computer image analyzer, also the protein and gene relative content of these growth factors as well as the effects of drugs on them were observed., Results: There was a significant increase in the smooth muscle and collagen thickness in the bronchi and pulmonary arterioles of the model group in comparison with that of the control group (P < 0.01), the relative contents for TGF-beta1, EGF and bFGF in the epithelial cells of the bronchi, endothelial cells of the pulmonary arterioles and alveolar macrophages of the model group were significantly higher than those of control group (P < 0.001 approximately 0.05). The relative content for TGF-beta1 in TB21 group was significantly lower than that of model group (P < 0.01). These were statistical positive relationships between the smooth muscle e thickness of bronchi and the relative contents for TGF-beta1, EGF and bFGF in the epithelial cells, between the collagen thickness of the bronchi and the relative content for TGF-beta1, between the smooth muscle thickness of the pulmonary arterioles and the relative content for TGF-beta1 and EGF in the endothelial cells (P < 0.05 approximately 0.01)., Conclusion: TGF-beta1, EGF and bFGF may play an important role in the airway wall and pulmonary arteriole structure remodeling in COPD, the intervention against TGF-beta1 and long term inhalation of heparin mat be helpful for the inhibition of airway wall remodeling in human COPD and worth of further observation.

Transforming growth factor-beta (TGF-beta) is involved in multiple processes including cell growth and differentiation. In particular, TGF-beta has been implicated in the pathogenesis of fibrotic lung diseases. In this study, we examined regulation of the mitogen-activated protein kinase pathway by TGF-beta1 in primary human lung fibroblasts. TGF-beta1 treatment resulted in extracellular signal-regulated kinase (ERK) pathway activation in a delayed manner, with maximal activity at 16 h. ERK activation occurred concomitantly with the induction of activator protein-1 (AP-1) binding, a nuclear factor required for activation of multiple genes involved in fibrosis. AP-1 binding was dependent on ERK activation, since the MEK-1 (mitogen-activated protein kinase kinase) inhibitor PD98059 inhibited TGF-beta1-induced binding. Induction of the receptor tyrosine kinase-linked growth factor, basic fibroblast growth factor (bFGF) protein expression temporally paralleled the activation of ERK/AP-1. Induction of AP-1 by TGF-beta1-conditioned medium was observed at 2 h, similar to AP-1 induction in response to exogenous bFGF. Dependence of ERK/AP-1 activation on bFGF induction was demonstrated by inhibition of TGF-beta1-induced ERK/AP-1 activation when conditioned medium from TGF-beta1-treated cells was incubated with bFGF-neutralizing antibody. Together, these results demonstrate that TGF-beta1 regulates the autocrine induction of bFGF, resulting in activation of the ERK mitogen-activated protein kinase pathway and induction of AP-1 binding.

Objective: The review the effect of cytokines on repair of tendon injury and the relevant mechanism., Methods: By broadly consulting recent issues about cytokines involved in tendon repair, a variety of cytokines with effects in repairing injured tendon was made and the possible mechanisms were summarized, with unsolved problems discussed., Results: There were many cytokines participated in the procedure of tendon repair, among which insulin-like growth factor (IGF-1), transforming growth-beta 1 (TGF-beta 1) played significant roles. Most of the relevant researches were limited in experimental study in vitro., Conclusion: Cytokines possibly can accelerate tendon repair and show great potentials in future clinical application.

Objective: To localize the distribution of basic fibroblast growth factor (bFGF) and transforming growth factor-beta(TGF-beta) in tissues from dermal chronic ulcer and hypertrophic scar and to explore their effects on tissue repair., Methods: Twenty-one cases were detected to localize the distribution of bFGF and TGF-beta, among them, there were 8 cases with dermal chronic ulcers, 8 cases with hypertrophic scars, and 5 cases of normal skin., Results: Positive signal of bFGF and TGF-beta could be found in normal skin, mainly in the keratinocytes. In dermal chronic ulcers, positive signal of bFGF and TGF-beta could be found in granulation tissues. bFGF was localized mainly in fibroblasts cells and endothelial cells and TGF-beta mainly in inflammatory cells. In hypertrophic scar, the localization and signal density of bFGF was similar with those in granulation tissues, but the staining of TGF-beta was negative., Conclusion: The different distribution of bFGF and TGF-beta in dermal chronic ulcer and hypertrophic scar may be the reason of different results of tissue repair. The pathogenesis of wound healing delay in a condition of high concentration of growth factors may come from the binding disorder of growth factors and their receptors. bFGF may be involved in all process of formation of hypertrophic scar, but TGF-beta may only play roles in the early stage.

Objective: To study the mechanism of haze formation and investigate the expression changes of transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1(TGF-beta 1) and basic fibroblast growth factor (bFGF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK)., Methods: Sixteen white rabbits were randomly divided into 4 groups, and PRK was performed on each eye of 12 rabbits. The haze formation was examined under a slit-lamp microscope at the 1st, 2nd and 3rd month after PRK, and the expressions of TGF-alpha, TGF-beta 1 and bFGF mRNA were detected with in situ hybridization., Results: The corneal haze formed at the 1st month after PRK. The most prominent haze formation was observed at the 2nd month, and declined gradually at the 3rd month after ablation. TGF-alpha mRNA expression was presented on the normal corneal epithelium and not on the corneal stroma. TGF-beta 1 and bGFG mRNA were expressed by both corneal epithelium and stroma. The capacities for cornea tissue expression of three growth factors mRNA increased after PRK, and the peaks appeared on the 1st, 2nd month. The extent for expressions of three growth factors related proportionally to the haze formation., Conclusion: Three growth factors took part in promoting corneal wound healing after PRK, and might contribute to corneal haze formation and development.

Osteoarthritis is characterized by marked osteophyte formation consisting of new cartilage and bone. Because several growth factors are known to be involved in chondrogenesis and osteogenesis, the expression of transforming growth factor-beta 1 and basic fibroblast growth factor in the osteophytes of the human femoral heads in osteoarthritis were examined. Transforming growth factor-beta 1 messenger ribonucleic acid was detected in the osteophytes by reverse transcription-polymerase chain reaction. All of the nine examined osteophytes expressed transforming growth factor-beta 1 messenger ribonucleic acid, whereas one of four osteoarthritic femoral heads and none of four osteonecrotic femoral heads expressed transforming growth factor-beta 1 messenger ribonucleic acid. The extent of transforming growth factor-beta 1 messenger ribonucleic acid expression varied among the osteophytes. Transforming growth factor-beta 1 or basic fibroblast growth factor was analyzed in osteophytes immunohistochemically. Transforming growth factor-beta 1 was localized in the superficial cells in the osteophyte cartilage, but it was scarcely detected in the superficial cells in the degenerative articular cartilage. Basic fibroblast growth factor was detected in the cells in the whole layer of osteophyte cartilage and in the articular cartilage. There was a difference in the localization, which suggests the different roles of transforming growth factor-beta 1 and basic fibroblast growth factor in bone and cartilage metabolism in osteophyte formation.

Numerous studies have found dura mater-calvarial mesenchyme interactions during calvarial bone induction; however, the exact molecular mechanisms governing these inductive events remain unknown. Recent studies have implicated basic fibroblast growth factor (FGF-2) and transforming growth factor-beta1 (TGF-beta1) in regulating bone formation. The purpose of this study was, therefore, to investigate the expression of FGF-2 and TGF-beta1 during calvarial bone formation in rats. Eight rats were killed on embryonic days 14, 18, and 20 and neonatal day 1 (n = 32). Four animals at each time point were analyzed by in situ hybridization, and the remainder were analyzed by immunohistochemistry. The results indicated that the dura mater underlying the developing calvarial bone strongly expressed FGF-2 and TGF-beta1 mRNA at all time points examined. In contrast, minimal growth factor expression was noted in the overlying calvarial mesenchyme until embryonic day 18, but it increased significantly with increasing age. Importantly, FGF-2 and TGF-beta1 mRNA expression in the dura mater underlying the developing calvarium preceded and was significantly greater than expression in the calvarium mesenchyme (p < 0.05). Interestingly, minimal expression of FGF-2 and TGF-beta1 mRNA was noted for all time points in the dura mater underlying the posterior frontal suture and within the posterior frontal suture connective tissue (p < 0.01 when compared with the dura mater underlying the developing calvarium). Immunohistochemical findings closely paralleled mRNA expression, with intense staining for FGF-2 and TGF-beta1 in the dura mater underlying the developing calvarial mesenchyme. Increasing FGF-2 and TGF-beta1 staining was noted within calvarial osteoblasts with increasing age, particularly in cells located near the endocranial surface (i.e., in contact with the developing dura mater). These findings, together with the known biologic functions of FGF-2 and TGF-beta1, implicate these growth factors in the regulation of calvarial bone growth by the developing dura mater. The possible mechanisms of this interaction are discussed.

Objective: To study the mechanism of guided bone regeneration combined with osteoinduction by observation of expression of BMP-2, TGF-beta, bFGF., Methods: 42 adult, male, New Zealand rabbits were randomly divided into 6 groups, each group being 7 rabbits. 10mm standard bone defect model was produced bilaterally in the middle radial shaft of each rabbit. Randomly, one defect enveloped with silicon membrane was tested, and the other served as control, sacrificed at 3 days, 1, 2, 3, 4, 5 weeks after surgery for histological observation, immunohistochemical staining of BMP-2, TGF-beta, bFGF and in situ hybridization of their cDNA probes., Results: Histologically bone defects was sealed by silicon membrane, in which inflammation tissue and callus were formed from cells in bone ends. Similar expression of BMP-2, TGF-beta, bFGF between the test groups and control groups was noted. Inflammatory cells in the hematoma expressed bFGF and proliferating periosteal cells, endosteal cells, medullary mesenchymal cells expressed TGF-beta at 3 days postoperatively. Osteocytes in the bone ends and osteoblasts lining callus surface started to express BMP-2, TGF-beta at first week after surgery. Following the formation of inflammation tissue, in which giant cells, macrophages, mesenchymal cells expressed TGF-beta, bFGF respectively. Although the expression of three osteoinductors was the same between the test and control sides, their content was very different in both sides. The whole content in all test sides was much more than all control sides (P < 0.01), the content of test side was also much more than control side in each group., Conclusions: It is a key reason for successful guided bone regeneration that membrane tube formed a relatively independent bone regenerative situation to prevent around tissue from interruption, and to prevent osteoinductors from diffusion, so that enhanced osteoinductors induced bone regeneration to repair bone defects.

Distraction osteogenesis is a viable method for regenerating large amounts of bone. In contrast to fracture healing, the mode of bone formation in distraction osteogenesis is primarily intramembranous ossification. The basic biology of the process is still not well understood. The growth factor cascade is likely to play an important role in distraction. This study examines the growth factor cascade in a lengthened ovine mandible model. Twenty-four animals were divided into four groups with varying rates of distraction (1, 2, 3, and 4 mm/day). A unilateral distractor at the angle of the mandible was used. The mandibles were lengthened to 24 mm and fixed for a period of 5 weeks, after which the animals were killed. The sections were probed for transforming growth factor-beta, basic fibroblast growth factor, and insulin-like growth factor I. The growth factors studied were present in all four groups. Transforming growth factor-beta, basic fibroblast growth factor, and insulin-like growth factor I were present in both the bony matrix of the sections and the cytoplasm of the cells, osteoblasts, and a small number of mesenchymal cells. The sections obtained from groups distracted at faster rates showed stronger presence of the growth factors examined by more intense staining. In fracture healing, the localization of transforming growth factor-beta in stage I of healing corresponded with the precise region of intramembranous ossification in stage II. Diffuse presence of transforming growth factor-beta throughout the lengthened region corresponded with the process of intramembranous ossification observed in distraction. In fracture healing, insulin-like growth factor I and basic fibroblast growth factor have been shown to promote proliferation and differentiation of osteoblasts from precursor cells. The intense presence of insulin-like growth factor I and basic fibroblast growth factor in the distracted region may account for osteoblast proliferation and formation from precursor mesenchymal cells. Mechanical strain has been shown to increase the expression of transforming growth factor-beta and insulin-like growth factor I. Distraction may serve as a source of mechanical strain, which may explain, in part, the expression of these growth factors, particularly in the faster groups.

It has been proposed that, in skeletal muscle, the angiogenic response to exercise may be signaled by the increase in muscle blood flow, via biomechanical changes in the microcirculation (increased shear stress and/or wall tension). To examine this hypothesis, we compared the change in abundance of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta1 (TGF-beta1) mRNA in skeletal muscles of the canine leg after 1 h of pump-controlled high blood flow alone (passive hyperperfusion; protocol A) and electrical stimulation of the femoral and sciatic nerves producing muscle contraction (protocol B). The increase in leg blood flow (5.4- and 5. 9-fold change from resting values, respectively) was similar in both groups. Passive hyperperfusion alone did not increase message abundance for VEGF (ratio of mRNA to 18S signals after vs. before hyperperfusion, 0.94 +/- 0.08) or bFGF (1.08 +/- 0.05) but slightly increased that of TGF-beta1 (1.14 +/- 0.07; P < 0.03). In contrast, as previously found in the rat, electrical stimulation provoked more than a threefold increase in VEGF mRNA abundance (3.40 +/- 1.45; P < 0.02). However, electrical stimulation produced no significant changes in either bFGF (1.16 +/- 0.13) or TGF-beta1 (1.31 +/- 0.27). These results suggest that the increased muscle blood flow of exercise does not account for the increased abundance of these angiogenic growth factor mRNA levels in response to acute exercise. We speculate that other factors, such as local hypoxia, metabolite concentration changes, or mechanical effects of contraction per se, may be responsible for the effects of exercise.

The mechanisms involved in normal cranial suture development and fusion as well as in the pathophysiology of craniosyostosis are not well understood. The purpose of this study was to investigate the expression of several cytokines--transforming growth factor-beta-1 (TGF-beta1), basic fibroblast growth factor (bFGF), and interleukin-6 (IL-6)--during cranial suture fusion. TGF-beta exists in three mammalian isoforms that are abundant in bone and stimulate calvarial bone formation when delivered locally. Other bone growth factors including basic fibroblast growth factor and the interleukins regulate bone growth and are mitogenic for bone marrow cells and osteoblasts. The involvement of growth factors in the pathophysiology of craniosynostosis is supported by recent genetics data linking fibroblast growth factor receptor mutations to syndromal craniosynostoses. In this experimental study, in situ hybridization was used to localize and quantify the gene expression of TGF-beta1, bFGF, and IL-6 during cranial suture fusion. In the Sprague-Dawley rat, the posterior frontal cranial suture normally undergoes fusion between 12 and 22 days of age, whereas all other cranial sutures remain patent. All in situ analyses of fusing posterior frontal sutures were compared with the patent, control, sagittal sutures. Posterior frontal and sagittal sutures, together with underlying dura, were harvested from rats at 8, 12, 16, and 35 days of postnatal life to analyze posterior frontal suture activity before, during, and after fusion. In situ hybridization was performed on frozen sections of these specimens using DNA probes specific for TGF-beta1, bFGF, and IL-6 mRNA. A negative control probe to IL-6 in the sense orientation was also used to validate the procedure. Cells expressing cytokine-specific mRNA were quantified (in cells positive per 10(-1) mm2) and analyzed using the unpaired Student's t test. Areas encompassing the fibrous suture and the surrounding bone plates were analyzed for cellular mRNA activity. IL-6 mRNA expression showed a minimal rise in the posterior frontal suture at days 12 and 16, with an average count of 10 and 6 cells per 10(-1) mm2, respectively. The sagittal suture remained negative for IL-6 mRNA at all time points. TGF-beta1 and bFGF analyses were most interesting, showing marked increases specifically in the posterior frontal suture during the time of active suture fusion. On postnatal day 8, a 1.5-fold increase in posterior frontal suture TGF-beta1 mRNA was found compared with sagittal sutures (p = 0.1890, unpaired Student's t test). This difference was increased 26-fold on day 12 in posterior frontal suture TGF-beta1 expression (p = 0.0005). By day 35, posterior frontal suture TGF-beta1 mRNA had nearly returned to prefusion levels, whereas TGF-beta1 mRNA levels in the sagittal suture remained low. A similar upregulation of bFGF mRNA, peaking at day 12, was observed in posterior frontal but not sagittal sutures (p = 0.0003). Furthermore, both TGF-beta1 and bFGF mRNA samples with intact dura showed an intense dural mRNA expression in the time preceding and during active posterior frontal suture fusion but not in sagittal tissues. Our data demonstrate that TGF-beta1 and bFGF mRNA are up-regulated in cranial suture fusion, possibly signaling in a paracrine fashion from dura to suture. TGF-beta1 and bFGF gene expression were dramatically increased both in and surrounding the actively fusing suture and followed the direction of fusion from endocranial to epicranial. These experimental data on bone growth factors support the recent human genetics data linking growth factor/fibroblast growth factor receptor deletions to syndromal craniosynostoses. The ultimate aim of these studies is to understand the underlying mechanisms regulating suture growth, development, and fusion so surgeons may one day manipulate the biology of premature cranial suture fusion.

Purpose: To understand the mechanism of fibrosis after filtering surgery for glaucoma, the effect of transforming growth factor-beta (TGF-beta) was studied in subconjunctival fibroblasts (SCFs). TGF-beta, universal inhibitor of cell proliferation, stimulates the cell proliferation of fibroblasts. SCFs were evaluated for their production of TGF-beta and fibroblast growth factor 2 (FGF-2) to determine whether TGF-beta may be an indirect mitogen acting through the induction of an endogenous growth factor, or factors, that then acts as the direct mitogen in an autocrine manner., Methods: Cell proliferation was determined either by counting cell numbers or by analyzing the incorporation of [3H]thymidine into DNA. The synthesis of TGF-beta and FGF-2 was analyzed by immunoprecipitation and immunoblotting., Results: TGF-beta 1, TGF-beta 2, and TGF-beta 3 stimulated the cell proliferation of SCFs in a dose-dependent manner. The media conditioned by SCFs, which were subsequently activated by acid, stimulated cell proliferation of corneal stromal fibroblasts. When the acid-activated media conditioned by SCFs were immunoprecipitated, respectively, either with anti-TGF-beta 1 and TGF-beta 2 antibodies or with anti-TGF-beta 3 antibody, TGF-beta s, with an apparent molecular size of 25 kDa, were detected, whereas SCFs produced an 80-kDa latent form of TGF-beta 1. Interestingly, SCFs produced and secreted an 18-kDa extracellular isoform of FGF-2, the synthesis of which is further stimulated by TGF-beta 1 and TGF-beta 3, respectively, whereas the neutralizing antibody to FGF-2 and the FGF-2-specific antisense oligonucleotide primers inhibited the stimulatory activities of TGF-beta 1 in SCFs., Conclusions: These findings indicate that SCFs produce TGF-beta and FGF-2 and that FGF-2 seems to be the direct stimulator of TGF-beta-mediated cell proliferation in SCFs.

Purpose: Although transforming growth factor-betas (TGF-beta s) inhibit epithelial cell proliferation, these same substances stimulate cell proliferation of fibroblasts. In order to elucidate the mechanism of stimulatory activity of TGF-beta on fibroblast, the present study was performed to determine whether TGF-beta might be an indirect mitogen acting through induction of an endogenous growth factor(s) that then acts as the direct mitogen in an autocrine manner in corneal stromal fibroblasts (CSFs)., Methods: Cell proliferation was determined either by counting cell numbers or by analyzing the incorporation of [3H]-thymidine into DNA. The synthesis of TGF-beta, TGF-beta receptors, FGF-2 and p27 was analyzed by immunoprecipitation and immunoblotting., Results: TGF-beta 1, TGF-beta 2, and TGF-beta 3 significantly stimulated cell proliferation of CSFs in a dose-dependent manner. The medium conditioned by CSFs and subsequently activated by acid-inhibited cell proliferation of corneal endothelial cells by 40%. When the acid-activated media conditioned by CSFs were immunoprecipitated with either combined anti-TGF-beta 1 and TGF-beta 2 antibodies or anti-TGF-beta 3 antibody, all three TGF-beta s, with an apparent molecular size of 25 kDa, were detected, whereas CSFs produced an 80-kDa latent form of TGF-beta 1. These cells can also express TGF-beta type II receptor and betaglycan. Interestingly, CSFs produced and secreted 18-kDa FGF-2, the synthesis of which is further stimulated by either TGF-beta 1 or TGF-beta 3, while both the neutralizing antibody to FGF-2 and the FGF-2 specific antisense oligonucleotide primers significantly inhibited the stimulatory activities of TGF-beta 1 in CSFs. The expression of p27, a negative regulator in cell cycle, was not altered by TGF-beta., Conclusions: These findings indicate that CSFs produce both TGF-beta s and FGF-2 and that FGF-2 appears to be a direct stimulator for TGF-beta-mediated cell proliferation in CSFs.

Chronic hepatic regeneration constitutes an important part of the cirrhotic process. The factors regulating chronic hepatic regeneration, however, remain unclear. We therefore analyzed the intrahepatic messenger RNA (mRNA) expression of growth factors (epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], hepatocyte growth factor [HGF], transforming growth factor [TGF]-alpha, and TGF-beta) at progressive time points (postoperative days 2, 7, 14, and 21) in a rat bile duct-ligated (BDL) model of cirrhosis versus sham controls. Intrahepatic growth factor mRNA expression was quantitatively assessed by polymerase chain reaction (PCR) using a dot-blot hybridization technique. Cirrhosis was associated with statistically significant (P < .05) progressive increases in the intrahepatic mRNA expression of bFGF (80-fold), EGF (25-fold), and TGF-beta (fourfold) in BDL animals versus controls. Furthermore, immunohistochemistry of hepatic sections showed a progressive up-regulation of bFGF protein in areas of bile duct proliferation. These areas also showed a dramatic increase in the number of hepatic stellate cells (HSC). In contrast, the intrahepatic expression of hepatocyte growth factor (HGF) mRNA was only significantly increased at postoperative days 7 and 14 in BDL animals before returning to control levels as cirrhosis developed. There were no significant differences found at any timepoint in the expression of TGF-alpha in BDL animals versus controls. In conclusion, the development of cirrhosis in this BDL rat model was associated with a progressive increase in the intrahepatic expression of EGF, bFGF, and TGF-beta. Early increased expression of HGF was not maintained in established cirrhosis. The findings suggest that these growth factors may play important roles in the pathogenesis of chronic hepatic regeneration in cirrhosis.

Myelofibrosis with myeloid metaplasia (MMM) is a myeloproliferative disorder characterized by clonal expansion of hematopoiesis and marrow fibrosis. Previous results from our group have shown an increased production of two potent fibrogenic factors also involved in the regulation of primitive hematopoietic cells, namely transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF), in patients with MMM. It is likely to assume that the myeloproliferation characteristic of this disease may result from an abnormal proliferation of CD34+ hematopoietic progenitors. Thus, we were particularly concerned in studying the gene and protein expression of these cytokines and their receptors in CD34+ progenitors purified from the peripheral blood of MMM patients by using semiquantitative reverse transcriptase-polymerase chain reaction and immunolabeling methods. Our data showed that the expression of TGF-beta1 is not altered in patients CD34+ cells; in contrast, the expression of TGF-beta type II receptor is significantly decreased in such cells, as compared with CD34+ cells from healthy subjects. Regarding bFGF, the very low expression of the cytokine and its type I and II receptors detected in normal CD34+ cells contrasts with that observed in patients' CD34+ cells, which is significantly higher. Our results might be a clue for a better understanding of the mechanism(s) involved in the dysregulation of hematopoiesis in MMM. Actually, the increased expression of bFGF and its receptors associated with the reduction of the TGF-beta binding receptor in CD34+ progenitors from MMM patients might facilitate the expansion of hematopoietic progenitors, not only by stimulating their growth and/or survival, but also by overcoming negative regulatory signals.

Although drug-induced gingival hyperplasia has been extensively studied, the pathogenesis of this disorder has not been clarified to date. Transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) have been shown to be implicated in diverse fibrotic and hyperplastic diseases. Heparan sulphate proteoglycan (HSPG), which is composed of heparan sulphate glycosaminoglycan (HSGAG), has also been shown to play an important role in the pathogenesis of tissue overgrowth by enhancing the functions of bFGF. However, the possible implication of these growth factors in gingival hyperplasia has not been studied. Immunohistochemical localization of TGF beta, bFGF, their receptors and HSGAG was studied in 4 nifedipine-induced and 5 phenytoin-induced hyperplastic gingival tissues, and 5 non-hyperplastic control gingival tissues to elucidate the pathogenesis of this disease. Significant immunostaining against TGF beta, bFGF, the receptors of these two growth factors and HSGAG was observed in the lamina propria of hyperplastic gingival tissues while less immunostaining was observed in the controls. The mean numbers of immunostained cells against TGF beta, bFGF, their receptors in a square unit (0.1 x 0.1 mm) of the lamina propria, which were counted to 10 units of each hyperplastic gingival tissue, were significantly higher than those of the controls. The results suggest that the increased synthesis of TGF beta, bFGF, their receptors and HSGAG may be related to the pathogenesis of drug-induced gingival hyperplasia.

When ES-5 cells were transfected with an exogenous porcine TGF-beta 1 gene, one can obtain clones of genetically modified ES cells with over-expression of the transfected gene. We called the genetically modified ES-5 cells as ES-T cells. When ES-T cells were used to study their differentiation in vitro by all trans-retinoic acid (RA), it was soon noticed that embryoid bodies of ES-T cells can exclusively differentiate into endothelial cells and vessel-like structures, but not in their parent ES-5 cells. The above result is the first indication that the differentiation of tubular structures in embryoid bodies of ES-T cells may somehow be related to TGF-beta 1. To demonstrate further the role of TGF-beta 1 in the formation of vessel-like structures, the cultured ES-5 cells in the presence of added rhTGF-beta 1 were closely followed in the course of their differentiation. We have, thus, demonstrated the promoting effects of exogenous rhTGF-beta 1 in the formation of vessel-like structures, morphologically similar to those structures derived from ES-T6 cells, during the differentiation of ES-5 cells, both in monolayer culture, in three dimensional collagen gel and in embryoid bodies cultured on gelatin-coated tissue culture wells. Addition of suitable amount of anti-TGF-beta 1 monoclonal antibody IgG (TB21) to the culture medium of embryoid bodies of ES-T6 cells could effectively abolish the formation of vessel-like structures induced by retinoic acid. The percentage of the inhibition was very high, giving a figure comparable to that of atypical vessel-like structures formed in the control embryoid bodies from their parent ES-5 cells. The flat epithelial-like cells and round cells differentiated from embryoid bodies of ES-T6 cells were stained rather strongly for laminin and type IV collagen by immunofluorescent procedure. The above results indicate clearly that TGF-beta 1 is a crucial factor in organizing the differentiated derivatives (endothelial-like cells and their immediate progenitor cells) from ES-T6 cells to form vessel-like structures, and that the role of TGF-beta 1 in vasculogenesis might be performed, in part, through the modulation of the composition and organization of the extracellular matrix. In addition, the enhanced expression of bFGF mRNA in derivatives differentiated from both ES-5 cells treated with rhTGF-beta 1 and ES-T6 cells were detected by Northern blot analysis. Thus, aside from its effects on extracellular matrix, TGF-beta 1 might also modulate the bioactivity of bFGF in relation to the growth of vascular endothelial cells in the present system.

A major adaptation to exercise is new capillary formation in skeletal muscle. On the basis of angiogenesis in tumors and during development, several angiogenic growth factors may be involved, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta 1 (TGF-beta 1). In 9-wk-old female Wistar rats, mRNA expression for these three growth factors in gastrocnemius muscle was examined by quantitative Northern analysis after a single 1-h run at 15 or 20 m/min at 10 degrees incline in room air. A third group ran at 15 m/min in 12% O2, and resting control groups were included at inspired O2 fractions of 0.21 and 0.12. Exercise significantly increased mRNA levels two- to fourfold, which was evident over the first 4 h postexercise; by 8 and 24 h, mRNA levels returned to baseline. For all three factors, mRNA levels were significantly higher after exercise at 20 than at 15 m/min. Hypoxia at rest doubled VEGF and TGF-beta 1 message but had no effect on bFGF. Hypoxic exercise further raised VEGF mRNA levels but had no effect on the other factors. We suggest that VEGF, bFGF, and TGF-beta 1 may be involved in the angiogenic response to exercise and that reduced intracellular PO2 (as occurs during normoxic exercise) may be part of the stimulus to such growth factor production.

Basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGFbeta1) are potential autocrine growth regulators of the prostatic stroma, and therefore may play a role in the development of benign prostatic hyperplasia (BPH). We reported that TGFbeta1 increased bFGF and bFGF mRNA expression in cultured human prostate stromal cells (PS). The current study extends those studies and investigates the mechanism by which TGFbeta1 upregulates the level of bFGF mRNA. A solution hybridization assay was used to quantitate bFGF mRNA. Treatment of PS for 6 hr with TGFbeta1 (1 ng/ml) maximally stimulated bFGF mRNA expression. TGFbeta2 and TGFbeta3 were similarly active in upregulating bFGF mRNA. TGFbeta1 or cycloheximide each increased bFGF mRNA about 3-fold. The effect of these agents was not additive. This suggested that a labile protein was involved in processing bFGF mRNA. Determination of message stability indicated that the half-life of bFGF mRNA in TGFbeta1-treated PS was 6.8 hr, as compared to 4.3 hr in untreated cells. The data indicated that posttranscriptional mechanisms that increased message stability were, at least in part, responsible for upregulation of bFGF mRNA by TGFbeta1 in PS. Our studies suggest that growth of the prostatic stroma is regulated by the interaction of members of two families of growth modulators, bFGFand TGFbeta. It remains to be determined if an imbalance in this system in favor of stroma hyperplasia plays a role in the development of BPH.

Kaposi's sarcoma is a highly vascularized multifocal tumor which frequently appears as a complication of HIV infection. It has been suggested that a disorder in the cytokine network may contribute to the development of the disease. We examined the expression of several cytokines in human sporadic Kaposi's-sarcoma specimens, as well as in spindle cells cultured from human lesions, and consistently found high levels of expression of hepatocyte growth factor (HGF). In addition, human lesion-derived spindle cells synthesize and secrete biologically active hepatocyte growth factor and express the hepatocyte-growth-factor receptor (c-MET). Moreover, elevated levels of transforming growth factor beta 1 (TGF beta 1) mRNA were found in lesions of human sporadic Kaposi's sarcoma and in lesion-derived spindle cells which also over-express urokinase. Since HGF, TGF beta 1 and urokinase are all involved in capillary-vessel organization, dysregulation of these interacting agents may play a role in the initiation and/or progression of Kaposi's sarcoma, stimulating the growth of spindle cells and recruiting endothelial cells into the lesion.

Transforming growth factors beta (TGF-beta), a family of pleiotropic cytokines, are widely distributed in the developing and adult nervous system. In order to further determine the neural functions of TGF-beta, we have localized the TGF-beta isoforms 1, 2 and 3 in the adult rat adrenal medulla and studied the neuroprotective capacity of one representative family member, TGF-beta 2, for those spinal cord neurons which innervate adrenal chromaffin cells and which die after destruction of the adrenal medulla. Unilateral electrothermal destruction of the adrenal medulla led to the disappearance of 25% of sympathetic preganglionic neurons, which are located in the intermediolateral (IML) column of thoracic spinal cord segments 7-10 and can be selectively marked by NADPH-diaphorase. The neurons which disappeared following adrenomedullectomy constitute the full set of neurons that innervate the adrenal medulla. Implantation of gelfoam soaked with 0.5 micrograms TGF-beta 2 into the adrenal wound cavity rescued all spinal cord neurons in the IML ipsilaterally to the lesioned side. Cytochrome c was not effective. Injections of [125I]TGF-beta 2 into the adrenal medulla did not result in retrograde transport and subsequent labelling of spinal cord neurons, suggesting that TGF-beta may exert its neuroprotective actions by indirect mechanisms. TGF-beta applied to cultured adrenocortical cells did not overtly increase the amount of mRNA for fibroblast growth factor-2, an established trophic molecule for sympathetic preganglionic spinal cord neurons. The mechanisms by which TGF-beta exerts its neurotrophic effect are therefore unclear. Even so, our data provide the first evidence that TGF-beta may play an important role in vivo in the control of maintenance of a population of spinal cord neurons.

Nodular palmar fibromatosis is a self-limited proliferation of fibro-/myofibroblasts associated with growth factor synthesis and abundant fibronectin extracellular matrix deposition. bFGF and TGF beta are potent modulators of fibro-/myofibroblast proliferation and differentiation. Moreover, in vitro investigations evidenced a TGF beta 1-dependent regulation of alternative splicing of fibronectin mRNA. To investigate a possible implication of these growth factors in the tissue formation process of palmar fibromatosis, TGF beta 1/2 and bFGF synthesis, as well as TGF beta 1/3 and bFGF tissue distribution, is demonstrated by RNA in situ hybridization and/or immunohistochemistry in relation to myofibroblast phenotype development (alpha-smooth muscle actin, desmin immunohistochemistry), expression of different fibronectin isoforms (ED-A+, ED-B+ and oncofetal glycosylated fibronectin immunohistochemistry, fibronectin RNA in situ hybridization) and cellular activity (cyclin RNA in situ hybridization, Ki-67 immunolabelling). The myofibroblast phenotype (alpha-smooth muscle actin, desmin), the growth factor synthesis (TGF beta 1 and 2, bFGF), fibronectin matrix synthesis (RNA in situ hybridization with cDNA) and ED-A+, ED-B+ and oncofetal glycosylated fibronectin immunostaining are exclusively localized in the active proliferative nodules (Ki-67 immunolabelling and cyclin mRNA demonstration). Whereas the growth factor synthesis is restricted to the proliferative areas of the fibromatosis only, TGF beta 1, TGF beta 3 and bFGF proteins can also be detected immunohistochemically with a lower intensity in the surrounding aponeurotic tissue. The spatial correlation of myofibroblast phenotype, TGF beta and bFGF synthesis and the occurrence of the oncofetal molecular fibronectin variants (ED-B+ and oncofetal glycosylated fibronectin) in the active proliferative fibromatosis nodules suggests a pathogentic role of these growth factors and matrix components in the tumorous tissue formation process. The presence of the bFGF and TGF beta 1/3 proteins in fibroblasts neighbouring the proliferative nodules may point to a recruitment of quiescent aponeurotic fibroblasts in the fibromatous tissue formation process.

Purpose: To determine factors that control ocular enlargement in experimental form-deprivation myopia and to clarify the mechanism of form-deprivation myopia., Methods: After the left eyes of 20 chicks were monocularly occluded for 2 weeks, protein, basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 2 contents in samples of constant area (circular button, diameter = 8.5 mm) in the retina-retinal pigment epithelium (RPE)-choroid and the sclera in the posterior region of control and myopic eyes were determined by enzyme-linked immunosorbent assay., Results: The bFGF content (per circular button) and bFGF concentration (per mg protein) were significantly lower in the sclera in the posterior region of the myopic eyes than in control eyes. The bFGF content and concentration were similar in the retina-RPE-choroid in myopic and control eyes. The TGF-beta 2 content and concentration were significantly higher in myopic eyes in both the retina-RPE-choroid and the sclera (P < 0.05)., Conclusions: These results are consistent with the possibility that bFGF and TGF-beta 2 regulate ocular enlargement or respond to myopiagenic mechanisms in form-deprivation myopia.

Vasoproliferative retinopathies result from retinal capillary non-perfusion and consequent inner retinal hypoxia. However, it is not known whether oxygen mediates vasoproliferation directly (at the nuclear level) or indirectly by regulating the production of growth factors. We have investigated the effect of oxygen on the production of basic fibroblast growth factor and transforming-growth-factor-beta by a variety of retinal cell types in culture. Confluent cultures were maintained for 48 hr under varying oxygen tensions ranging from 135 to 18 mmHg. A reduction in basic fibroblast growth factor levels was observed in the cell lysates and extracellular matrix from retinal microvascular endothelial cell, retinal microvascular pericyte and retinal pigment epithelial cell cultures when the oxygen tension of the medium was reduced from 135 to 18 mmHg. Levels of basic fibroblast growth factor in conditioned media from microvascular endothelial and retinal pigment epithelial cell cultures also decreased when the oxygen tension of the medium was reduced from 135 to 18 mmHg. Total transforming-growth-factor-beta (and specifically isoforms 1 and 2) in the conditioned media from all three cell types was similarly modulated by oxygen i.e. it decreased as the oxygen tension of the medium was reduced from 135 to 18 mmHg. In contrast, the steady state messenger RNA levels for both basic fibroblast growth factor and transforming-growth-factor-beta 1 genes in RPE cells increased significantly when the oxygen tension of the medium was reduced from 135 to 18 mmHg. These results support the putative role of oxygen in influencing the balance of growth factors during the development of preretinal new vessels.