Anti-basic Fibroblast Growth Factor Research Articles

Endothelial Cell Growth Promoting Activity in Graves’ Disease Sera is Neutralized by Anti-Basic Fibroblast Growth Factor Antibodies in Patients with Fat Expansive but Not Infiltrative Orbitopathy

To report a case of orbital fat expansion leading to globe prolapse in a Graves' disease patient undergoing high-dose glucocorticoid therapy. To evaluate the growth factor receptor specificities of plasma autoantibodies in Graves' disease patients who exhibited contrasting subtypes of thyroid-associated ophthalmopathy, i.e. orbital fat expansion-type vs. infiltrative. Sera from Graves' orbitopathy and control patients with or without Graves' disease were subjected to protein-A affinity chromatography to obtain immunoglobulin G. A (1/50th to 1/1600th) range in dilutions of the protein-A eluate fraction was incubated for four days at 37 degrees C with bovine pulmonary artery endothelial cells to test for endothelial cell inhibition or stimulation. Growth stimulatory autoantibodies were co-incubated with specific neutralizing anti-insulin like growth factor 1 receptor antibodies or anti-basic fibroblast growth factor antibodies to assess autoantibody specificity in contrasting Graves' orbitopathy subtypes. We observed increased mean endothelial cell growth promoting activity in the protein-A eluates of serum from eighteen patients with active Graves' disease (117 ± 28%, n = 18) compared to mean endothelial cell activity (89 ± 10%, n = 13, P = 0.003) in thirteen adults without Graves' disease. The protein-A eluate fraction in acute infiltrative-type Graves' orbitopathy contained a high titer (> 1:1000) of endothelial cell stimulatory activity which was significantly neutralized by specific monoclonal anti-human insulin-like growth factor 1 receptor antibodies. The protein-A eluate fraction in fat expansion-type Graves' orbitopathy contained endothelial cell inhibitory activity (at low titers) and stimulatory activity (at high titers), and the latter stimulatory activity was completely neutralized by specific anti-basic fibroblast growth factor antibodies. Graves' disease suffering globe prolapse secondary to marked orbital fat-expansion had coexisting plasma fibroblast growth factor-inhibitory and -stimulatory autoantibodies. The latter was completely neutralized by anti-basic fibroblast growth factor antibodies.

(125)I-labeled anti-bFGF monoclonal antibody inhibits growth of hepatocellular carcinoma.

To investigate the inhibitory efficacy of (125)I-labeled anti-basic fibroblast growth factor (bFGF) monoclonal antibody (mAb) in hepatocellular carcinoma (HCC). bFGF mAb was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with (125)I through the chloramine-T method, bFGF mAb was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of (125)I-bFGF mAb. The murine H22 HCC xenograft model was established and randomized to interventions with control (phosphate-buffered saline), (125)I-bFGF mAb, (125)I plus bFGF mAb, bFGF mAb, or (125)I. The ratios of tumor inhibition were then calculated. Expression of bFGF, fibroblast growth factor receptor (FGFR), platelet-derived growth factor, and vascular endothelial growth factor (VEGF) mRNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction. The purified bFGF mAb solution was 8.145 mg/mL with a titer of 1:2560000 and was stored at -20 °C. After coupling, (125)I-bFGF mAb was used at a 1: 1280000 dilution, stored at 4 °C, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the (125)I-bFGF mAb group than in the other groups (P < 0.05). Expression of bFGF and FGFR mRNA in the (125)I-bFGF mAb group was significantly decreased in comparison with other groups (P < 0.05). Groups under interventions revealed increased expression of VEGF mRNA (except for (125)I group) compared with the control group. (125)I-bFGF mAb inhibits growth of HCC xenografts. The coupling effect of (125)I-bFGF mAb is more effective than the concomitant use of (125)I and bFGF mAb.

Synergistic tumor growth-inhibitory effect of the prostate-specific antigen-activated fusion peptide BSD352 for prostate cancer therapy

Prostate-specific antigen (PSA), a serine protease, is a promising target for the development of prodrugs in prostate cancer treatment. In this study, we designed a novel fusion peptide, BSD352, containing three functional domains: a protein transduction domain from HIV transactivating regulatory protein (TAT) followed by the BH3 domain of the p53 upregulated modulator of apoptosis (TAT-BH3), an anti-vascular endothelial growth factor peptide (SP5.2), and an anti-basic fibroblast growth factor peptide (DG2). These different domains in BSD352 were linked together by a linker sequence corresponding to a PSA hydrolytic substrate peptide. The BSD352 fusion peptide could be selectively cleaved by PSA in PSA-producing LNCaP prostate cancer cells. Furthermore, the BSD352 fusion peptide was efficiently transduced into tumor cells both in vitro and in vivo, and the BH3 domain was found to induce tumor cell apoptosis by elevating the expression of Bax, cytochrome C release, and caspase-9 cleavage. Moreover, the SP5.2 and DG2 domains in the BSD352 fusion peptide also exhibited in-vitro endothelial cell growth inhibition and in-vivo antiangiogenic activities. Direct injection of BSD352 into an established LNCaP xenograft tumor in mice inhibited tumor growth, whereas a synergistic effect was observed with the combined use of wild-type BH3, SP5.2, and DG2 functional domains. These results suggest that BSD352 could be beneficial for the treatment of accessible prostate tumors and may provide a complementary strategy for prostate cancer therapy.

Effects of inhibitory substance P, capsaicin and anti-basic fibroblast growth factor antibody on collagen deposition of excision scar formation in mice

ObJective To observe the effects of inhibitory substance P (capsaicin) and anti-bas-ic fibroblast growth factor antibody on collagen deposition during excision scar formation and explore their effects on scar formation.Methods The excision injury model on the back of mice skin was developed,and the different final concentrations of capsaicin (20 and 80 μg/0.1 ml), anti-basic fibroblast growth fac-tor antibody (20 and 80 μg/0.1 ml),or capsaicin (20 μg/0.1 ml) in combination with anti-basic fibro-blast growth factor antibody (20 μg/0.1) were used in the excision injury.Results The high dose of capsaicin (80 μg/0.1 ml) or high anti-basic fibroblast growth factor antibody (80 μg/0.1 ml) markedly inhibited collagen deposition in the excision site, but low dose of capsaicin (20 μg/0.1) or anti-basic fi-broblast growth factor antibody (20 μg/0.1) alone did not influence collagen deposition significantly.However,the combined use of low dose of capsaicin (20 μg/0.1 ml) and low dose of anti-basic fibroblast growth factor antibody (20 μg/0.1) decreased collagen deposition significantly.Conclusion The colla-gen deposition of the excision site in mice could be inhibited by capsaicin or bFGF antibodies in a dose-de-pendent manner.Combined use of capsaicin and anti-bFGF antibody could jointly inhibit scars formation during wound healing. Key words: Hypertrophic scar; Capsaicin; bFGF; Antibody; Collagen

Effect of γδ T cell supernatant on human skin fibroblast proliferation and collagen production – Possible role of transforming growth factor‐β and basic fibroblast growth factor

Perivascular mononuclear cell infiltration, vascular injury and diffuse interstitial fibrosis has been shown in systemic sclerosis (SSc). Objective To study a role of cellular immune mechanism inducing fibroblast proliferation and collagen synthesis. The interaction of gammadelta T-cell receptor bearing lymphocyte supernatant to human fibroblast proliferation and collagen synthesis was studied. The fibroblasts added with gammadelta T cell supernatant showed increased proliferation compared with those without it and that added with alphabeta supernatant. The endothelial cells added with gammadelta T cell supernatants, however, showed significantly decreased proliferation compared with those without it and that added with alphabeta T cell supernatant. The fibroblasts added with endothelial cell supernatant showed increased proliferation compared with those without it. A doubling of collagen synthesis was observed when human skin fibroblasts were maintained in all of the supernatant. The collagen synthesis was inhibited by antitransforming growth factor-beta antibody and antibasic fibroblast growth factor antibody. gammadelta T-cell-receptor bearing lymphocyte has proliferative and collagen synthesis activity to human skin fibroblasts, but suppressive to endothelial cells.

Structural and Sequence Motifs in Dermatan Sulfate for Promoting Fibroblast Growth Factor-2 (FGF-2) and FGF-7 Activity

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.

Hypoxia-induced angiogenesis of cultured human salivary gland carcinoma cells enhances vascular endothelial growth factor production and basic fibroblast growth factor release.

The angiogenic activity of two human salivary gland tumor cell lines, ACCS from adenoid cystic carcinoma and IT-2 from mucoepidermoid carcinoma, was examined by stimulating tube formation by bovine capillary endothelial cells (BCE). ACCS and IT-2 were cultured in 20 or 3% oxygen, representing normoxic and hypoxic conditions, respectively, and conditioned medium (CM) was obtained from each culture. The BCE tubes stimulated by hypoxic CM were 1.59 (ACCS) and 1.42 (IT-2) times longer than those stimulated by normoxic CM. The tube-forming activity of CM was inhibited by preincubation with either anti-vascular endothelial growth factor (VEGF) IgG or anti-basic fibroblast growth factor (bFGF) IgG, suggesting that both VEGF and bFGF with angiogenic activity were present in the CM. This was confirmed by ELISA, which also demonstrated increased concentrations of both proteins in the hypoxic CM. Northern blot analysis showed an increased VEGF mRNA level in both carcinoma cells with hypoxia, while hypoxia did not affect the bFGF mRNA level in either cell line. The results suggest that both VEGF and bFGF are major angiogenesis factors in salivary gland tumors, and hypoxia-induced angiogenesis results from upregulation of VEGF and increased release of bFGF.

Increased Density of Interstitial Mast Cells in Amyloid A Renal Amyloidosis

Renal interstitial fibrosis is the final common pathway leading to end-stage renal disease in various nephropathies including renal amyloidosis. However, the role of mast cells (MCs) in the fibrotic process of renal amyloidosis is not fully understood. We compared the distribution of MCs in renal biopsies from 30 patients with AA type renal amyloidosis and 20 control cases. Immunoreactivity of renal MCs to anti-tryptase and anti-chymase was studied. Interstitial myofibroblasts were stained with anti-α-smooth muscle actin (α-SMA) antibody, and inflammatory cells were identified by anti-CD45, -CD20, and -CD68 mAbs. Positively stained cells were counted, and the relative interstitial and fractional areas of anti-α-SMA stained cells were measured. Anti-CD29 mAb was used to detect β1 integrin and anti-basic fibroblast growth factor (bFGF) mAb for the growth factor on MCs. MCs were rarely found in control samples. In contrast, samples showing amyloid deposition contained numerous tryptase-positive (MCT) (940.17 ± 5.4 versus 6.74 ± 1.1/mm2) but fewer chymase-positive (MCTC) cells (20.7 ± 2.86 versus 1.7 ± 0.76/mm2) in the renal interstitium. There was a significant relationship between interstitial MCT and creatinine clearance (r = −0.72), and between interstitial MCT and glomerular amyloid-index (GAI) (r = 0.723) and interstitial amyloid area (r = 0.824). Accumulation of MCs correlated significantly with the number of T lymphocytes (MCT: r = 0.694). There was also a significant relationship between mast cell (MC) number and the fractional area of α-SMA positive interstitium (r = 0.733) and interstitial fibrotic area (r = 0.6). Double immunostaining demonstrated intracytoplasmic presence of β1 integrin on 87% of MCT and correlated significantly with the interstitial amyloid area (r = 0.818, P = .001) and T-cell number (r = 0.639, P = .002). bFGF was also detected on 85.5% of MCTC correlating well with the interstitial α-SMA-area (r = 0.789). Our results indicate that MCs constitute an integral part of the overall inflammatory process and play a crucial role in interstitial fibrosis in renal amyloidosis.

Lactate elicits vascular endothelial growth factor from macrophages: a possible alternative to hypoxia.

Macrophages respond to various stimuli to produce angiogenic factors but few mechanistic details are known. We examined the effects of hypoxia, lactate and nicotinamide on the expression of vascular endothelial growth factor by cultured macrophages. These agents were chosen because they down-regulate polyadenosine diphosphoribose levels. Following exposure, conditioned media were analyzed for vascular endothelial growth factor protein. Nicotinamide adenine dinucleotide, polyadenosine diphosphoribose, and vascular endothelial growth factor mRNA were measured in the cellular fraction. Angiogenic capacity of the conditioned media was tested in rabbit corneas and Matrigel implants. All three agents, hypoxia, lactate and nicotinamide, elicited significantly increased levels of vascular endothelial growth factor mRNA and vascular endothelial growth factor in the conditioned media, and these levels were paralleled by their angiogenic activity. Polyadenosine diphosphoribose in the cellular fraction was correspondingly depressed. Anti-vascular endothelial growth factor antibody inhibited most of the angiogenic response whereas anti-basic fibroblast growth factor antibody had little effect. We propose that redox changes associated with the alteration of cellular nicotinamide adenine dinucleotide and polyadenosine diphosphoribose are involved in lactate-mediated VEGF expression.

Lactate elicits vascular endothelial growth factor from macrophages: a possible alternative to hypoxia

Macrophages respond to various stimuli to produce angiogenic factors but few mechanistic details are known. We examined the effects of hypoxia, lactate and nicotinamide on the expression of vascular endothelial growth factor by cultured macrophages. These agents were chosen because they down-regulate polyadenosine diphosphoribose levels. Following exposure, conditioned media were analyzed for vascular endothelial growth factor protein. Nicotinamide adenine dinucleotide, polyadenosine diphosphoribose, and vascular endothelial growth factor mRNA were measured in the cellular fraction. Angiogenic capacity of the conditioned media was tested in rabbit corneas and Matrigel implants. All three agents, hypoxia, lactate and nicotinamide, elicited significantly increased levels of vascular endothelial growth factor mRNA and vascular endothelial growth factor in the conditioned media, and these levels were paralleled by their angiogenic activity. Polyadenosine diphosphoribose in the cellular fraction was correspondingly depressed. Anti-vascular endothelial growth factor antibody inhibited most of the angiogenic response whereas anti-basic fibroblast growth factor antibody had little effect. We propose that redox changes associated with the alteration of cellular nicotinamide adenine dinucleotide and polyadenosine diphosphoribose are involved in lactate-mediated VEGF expression.

Human oral squamous cell carcinoma cell lines promote angiogenesis via expression of vascular endothelial growth factor and upregulation of KDR/flk-1 expression in endothelial cells.

Angiogenesis is an important phenomenon for the growth and metastasis of solid tumors. The present study examined the characterization of angiogenic factors produced by human oral squamous cell carcinoma (oral SCC) cell lines established from lymph node metastatic tumors and primary tumor in different patients. The conditioned medium of HSC3 with the strongest metastatic ability among the examined lines enhanced a tube-forming activity of bovine carotid artery endothelial (BAE) cells in collagen gel cultures. The treatment of HSC3 with anti-vascular endothelial growth factor (VEGF) antibody or anti-basic fibroblast growth factor (bFGF) antibody, either alone or in combination, attenuated the activity of urokinase-type plasminogen activator (uPA) in the endothelial cells stimulated by the conditioned medium of HSC3. In contrast, neither anti-interleukin-8 (IL-8) antibody nor anti-hepatocyte growth factor (HGF beta) antibody affected uPA activity in the endothelial cells. Among these HSC cell lines, HSC3 secreted VEGF with the highest (1.92 +/- 0.24 ng/10(6) cells/24 h) level and bFGF. The level of bFGF secreted by HSC3 was lower than that secreted by BAE cells. Other oral SCC cell lines secreted lower levels of VEGF and undetectable levels of bFGF. By reverse transcriptase-polymerase chain reaction analysis of mRNA the production of VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206 in these cell lines was able to be detected. Moreover, the conditioned medium of HSC3 enhanced the tyrosine phosphorylation and expression of kinase insert domain-containing receptor (KDR/flk-1) in the endothelial cells. These results suggest that oral SCC promotes angiogenesis via expression of VEGF and upregulation of their receptor KDR/flk-1 expression in endothelial cells.

Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism.

In this study, we demonstrate that human umbilical cord vein-derived endothelial cells (HUVECs) expressed c-Mpl, the thrombopoietin (TPO) receptor, and that TPO activates HUVECs in vitro, as indicated by directional migration, synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF) and interleukin-8 (IL-8), and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT5B. The observation that WEB 2170 and CV3988, 2 structurally unrelated PAF receptor antagonists, prevented the motility of HUVECs induced by TPO suggests a role of PAF as secondary mediator. Moreover, kinetic analysis of TPO-induced tyrosine phosphorylation of STAT demonstrated that STAT5B activation temporally correlated with the synthesis of PAF. PAF, in turn, induced a rapid tyrosine phosphorylation of STAT5B and PAF receptor blockade, by WEB 2170, preventing both TPO- and PAF-mediated STAT5B activation. The in vivo angiogenic effect of TPO, studied in a mouse model of Matrigel implantation, demonstrated that TPO induced a dose-dependent angiogenic response that required the presence of heparin. Moreover, the in vivo angiogenic effect of TPO was inhibited by the PAF receptor antagonist WEB 2170 but not by the anti-basic fibroblast growth factor neutralizing antibody. These results indicate that the effects of TPO are not restricted to cells of hematopoietic lineages, because TPO is able to activate endothelial cells and to induce an angiogenic response in which the recruitment of endothelial cells is mediated by the synthesis of PAF. Moreover, biochemical analysis supports the hypothesis that STAT5B may be involved in the signaling pathway leading to PAF-dependent angiogenesis.

Suppression of smooth muscle cell proliferation after experimental PTFE arterial grafting: a role for polyclonal anti-basic fibroblast growth factor (bFGF) antibody

To determine the role of polyclonal anti-basic Fibroblast Growth Factor (bFGF) antibody in inhibiting the proliferation of smooth muscle cells after experimental polytetrafluorethilene (PTFE) arterial grafting. In 14 male inbred Lewis rats (weight 250 mg) a 1 cm long segment of PTFE was interposed at the level of abdominal aorta. Animals were randomised to receive polyclonal anti-bFGF antibody (group A: n = seven animals) or aspecific immunoglobulin (group B: n = seven animals). Anti-bFGF antibody or aspecific immunoglublin were given intraperitoneally at the end of operation, and for the first 2 postoperative days. Animals were sacrificed 7 days after surgery, 24 h after intraperitoneal injection of BromodeoxyUridin (BrdU) to label proliferating smooth muscle cells. One animal in each group died in the immediate postoperative period due to anaesthetic problems. All grafts were patent at the time of sacrifice. BrdU labelling index was statistically higher in the control group B animals at the level of the anastomotic regions (proximal anastomosis: group B 7.9% vs. group A 4.1%. Distal anastomosis: group B 5.1% vs. group A 2.6% p = 0.009) and at the level of PTFE graft (group B 3.8% vs. group A 2.6% p = 0.002), while there was no statistical difference between the control thoracic aorta of the two groups. bFGF plays a major role in the proliferation of smooth muscle cells at the level of the anastomoses after arterial PTFE grafting. Agents able to block the action of bFGF may be useful in inhibiting the formation of myointimal hyperplasia.

Mitogenic effect of angiotensin II on rat carotid arteries and type II or III mesenteric microvessels but not type I mesenteric microvessels is mediated by endogenous basic fibroblast growth factor.

In this study, anti-basic fibroblast growth factor (anti-bFGF) antibody was used to determine whether the mitogenic effect of angiotensin II in vivo could be blocked by neutralizing bFGF in the vessel wall. Animals, divided into six experimental groups, were given (1) angiotensin II, (2) angiotensin II + anti-bFGF antibody, (3) angiotensin II + normal goat IgG (ngIgG), (4) anti-bFGF antibody, (5) ngIgG, and (6) Ringer's solution. Angiotensin II at 435 ng x kg(-1) x min(-1) was infused into rats continuously for 1 week to induce smooth muscle cell replication, and anti-bFGF antibody or ngIgG was injected intravenously 4 times over the 1-week period at a dose of 60 mg/injection. Bromodeoxyuridine (30 mg/mL) was also continuously infused during the 1-week period. The left carotid artery of all animals was balloon-injured on day 4 of the treatment, and all groups were killed for study on day 7. The results showed that angiotensin II significantly stimulated smooth muscle replication in the balloon-injured carotid artery, intact carotid artery, and three branch levels of the mesenteric vascular tree. Anti-bFGF was able to block the mitogenic effect of angiotensin II in larger vessels but not the smallest (type I) microvessels of the mesenteric arterial tree. This differential response may be attributable to the nature of the lesions in type I vessels versus larger vessels: the type I vascular lesion has a large component of proliferating macrophages, whereas the larger vessels show less injury, few macrophages, and varying levels of smooth muscle replication. Our data suggest that the vessel wall remodeling in the angiotensin II-treated larger vessels involves DNA replication that is dependent on the presence of bFGF.

Identification and Partial Purification of a Basic Fibroblast Growth Factor-Like Growth Factor Derived from Bovine Colostrum

Bovine colostrum that had been collected up to 6h postpartum was fractionated by ammonium sulfate precipitation, and various fractions were examined for basic fibroblast growth factor activity. Activity that stimulated cell growth was detected in the cream fraction, which was purified by isoelectric focusing and heparin affinity chromatography. Three peaks were eluted from the heparin affinity column at approximately 0.5, 1, and 1.75 M NaCl. Although activity that stimulated cell growth was detected in the second and third peaks, a reaction with antibasic fibroblast growth factor antibody was observed only in the third peak. Fractions in the second and third peaks were examined by SDS-PAGE and Western blot analysis. Activity that stimulated cell growth was detected in the second and third peaks; however, after Western blot analysis using antibasic fibroblast growth factor, only the third peak yielded positive bands at 15 and 28 kDa. These fractions were further subjected to a neutralization test using antibasic fibroblast growth factor antibody. The activity that stimulated cell growth in the second peak was virtually unchanged; however, the activity in the third peak was diminished, showing a relative activity of less than 10% at 1.25μg/ml. Therefore, neutralization of the activity that stimulates cell growth by antibasic fibroblast growth factor antibody suggests that the third peak, which was eluted at approximately 1.5 to 2 M NaCl in heparin affinity chromatography, might be a basic fibroblast growth factor-like growth factor.

The 27-kDa Heat Shock Protein Facilitates Basic Fibroblast Growth Factor Release from Endothelial Cells

Basic fibroblast growth factor is an important mitogenic and angiogenic factor that stimulates endothelial cell growth and migration. This hormone is not secreted via the classical vesicular pathway, and the identification of intracellular proteins that facilitate its release remains lacking. Transfection and expression of the 27-kDa human heat shock protein in bovine arterial endothelial cells doubles the rate of estrogen-induced basic fibroblast growth factor secretion, preferentially inducing the release of high molecular weight forms of the hormone. The secreted basic fibroblast growth factor is mitogenic to breast adenocarcinoma cells cultured in the conditioned medium obtained from the transfected endothelial cells. In contrast, decreasing the level of the endogenous heat shock protein homolog with an antisense vector markedly decreases basic fibroblast growth factor release. Anti-heat shock protein or anti-basic fibroblast growth factor antibodies co-precipitate both proteins from endothelial cell extracts, demonstrating a direct association between the two proteins. This interaction is likely to be an important step in the mechanism of basic fibroblast growth factor secretion.

Diabetic state-modified macrophages in GK rat release platelet-derived growth factor-BB for tube formation of endothelial cells in rat aorta

Cultured peritoneal macrophages (Mø) of GK rats, a non insulin-dependent diabetes mellitus model established from normal Wistar rats, increased the tube formation of aortic endothelial cells (EC). A polyclonal anti-platelet-derived growth factor (PDGF)-BB (0.21–3.3 μg/ml) inhibited (by 66.5 ± 6.6%) the tube-forming activity of conditioned medium (CM) from Mø in diabetic GK rats, but not that in age-matched normal Wistar rats. A polyclonal anti-interleukin (IL)-α (0.16–0.33%) also inhibited (by 37.7 ± 5.8%) the activity of diabetic Mø-CM, and its inhibitory effect was smaller than that of anti-PDGF-BB. A monoclonal anti-basic fibroblast growth factor (bFGF) (0.6–60 ng/ml) inhibited the activities of CM from both Mø, respectively. The tube-forming activity of the normal MO-CM was increased by the serum isolated from the diabetic GK rat more than by that from the age-matched normal Wistar rat. The activity of normal MO-CM was also increased by 16.7-50 mM glucose-exposed serum. These tube-forming activities of the CM of Mø stimulated by diabetic serum and by the high concentration of glucose-treated serum were completely inhibited by anti-PDGF-BB. In conclusion, PDGF-BB was selectively released from the diabetic state-modified Mø in the GK rat to increase tube formation, suggesting a key role in the cause of angiogenesis. The diabetic state-induced activation may depend on advanced glycosylation endproducts produced in the serum in the diabetic rat.

Effects of fibrin on the angiogenesis in vitro of bovine endothelial cells in collagen gel.

The effect of fibrin on angiogenesis in vitro was investigated using an experimental model of tube formation by bovine capillary endothelial cells (BCEs) in type I collagen gel. One milligram per milliliter of fibrin added into type I collagen gel significantly increased the length of the tubular structures formed by BCEs in the gel by about 180% compared with type I collagen only. The facilitating effect of fibrin on tube formation by BCEs was inhibited by either anti-basic fibroblast growth factor (bFGF) IgG (25 micrograms/ml) or anti-urokinase type plasminogen activator (uPA) IgG (10 micrograms/ml) added to the gel and culture medium, but not by anti-tissue type plasminogen activator (uPA) IgG (10 micrograms/ml) added to the gel and culture medium, but not by anti-tissue type plasminogen activator (10 micrograms/ml) or non-immune IgG. The Arg-Gly-Asp (RGD) containing peptides (100 micrograms/ml) added to the culture medium also suppressed tube formation by BCEs in fibrin-containing type I collagen gel, but not in type I collagen gel. These results suggest that the increased release of bFGF and uPA by BCEs therefore plays a role in the angiogenic effect of fibrin in vitro, and the angiogenic effect of fibrin is mediated by the RGD sequence in fibrin, probably via the function of integrin receptor of the BCEs.

Antiproliferative effect of multiple autocrine loop blockade in human malignant glioma cell lines.

The effects of specific antibodies against growth factors and receptors on deoxyribonucleic acid (DNA) synthesis in two established human glioma cell lines, A172 and TM-1, were examined. Anti-platelet-derived growth factor (PDGF), anti-basic fibroblast growth factor (bFGF), and anti-epidermal growth factor receptor (EGF-R) antibodies inhibited thymidine incorporation by both cell lines in serum-free medium. Antibody specific to transforming growth factor-alpha only slightly suppressed DNA synthesis by both cell lines. Although the antiproliferative effects of anti-PDGF and anti-bFGF antibodies decreased in serum-supplemented medium, the effect of anti-EGF-R antibody was little changed. The combination of anti-PDGF, anti-bFGF, and anti-EGF-R antibodies significantly inhibited thymidine incorporation by the two cell lines even in serum-supplemented medium. This preliminary study suggests that simultaneous blockade of multiple autocrine loops may provide a new approach to the treatment of human malignant gliomas.

Conditioned media of carcinoma cells cultured in hypoxic microenvironment stimulate angiogenesis in vitro; relationship to basic fibroblast growth factor

Conditioned media (CM) harvested from human pulmonary squamous cell carcinoma (QG56), pulmonary small cell carcinoma (QG90) and gastric adenocarcinoma (MKN28) cultivated under hypoxic conditions (3% oxygen), enhanced the angiogenic activity in vitro more than those obtained under normoxic cultivation (20% oxygen). The total length of the tube structures formed by bovine capillary endothelial cells (BCEs) in the CM cultured at 3% oxygen was about 1.5 (QG56 and MKN28) or 1.9 (QG90) times longer than that at 20% oxygen. Tube formation was diminished by the preincubation of CM with anti-basic fibroblast growth factor (bFGF) IgG. After performing the fractionations of the CM and the crude extracts of cell lysates cultured using a heparin-Sepharose column, the mitogenic activity in the CM from all cancer cells at 3% oxygen was about twice that of CM at 20% oxygen, while it decreased in the cell lysates at 3% oxygen to about 40% of those at 20% oxygen. This mitogenic activity of BCEs in the CM from all cancer cells was almost totally suppressed by anti-bFGF IgG, but not with anti-vascular endothelial growth factor IgG. Hypoxia is an important factor in tumor angiogenesis by bFGF or bFGF-like molecule(s) derived from tumour cells.