Distinct regulation of genes by bFGF and VEGF-A in endothelial cells

A finely tuned balance of angiogenic inhibitors and inducers controls the activity of angiogenesis characterized by proliferation, migration and differentiation of endothelial cells. Among many angiogenic factors, basic fibroblast growth factor (bFGF) was first identified to be angiogenic whereas vascular endothelial growth factor A (VEGF-A) is an endothelial cell specific mitogen. In addition to being a specific mitogen, VEGF-A is also known as a vascular permeability factor. The majority of growth factors transduce their mitogenic signals from cell surface to nucleus where gene expression occurs. Whether these ligands utilize a distinct or a common molecular pathway to exert their biological effects on human endothelial cells remains elusive. We thus studied the expression profile of 884 human genes under the influence of either bFGF or VEGF-A alone in the context of human endothelial cells. A total of ninety-four genes were differentially regulated by more than two folds. The expression patterns of 32 genes are similar between the treatment of either factor alone whereas those of the remaining 62 genes are only regulated by one but not the other factor. Their function in the control of angiogenesis will be discussed and apoptotic signaling in the regulation of angiogenesis is also implicated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Concentrations of hepatocyte growth factor, basic fibroblast growth factor, and vascular endothelial growth factor in pericardial fluid and plasma

Some angiogenic factors, including hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), have been reported to promote angiogenesis and improve myocardial perfusion in experimental models of ischemic heart disease. These factors are produced in various tissues, including myocardium. We measured the concentrations of HGF, bFGF, and VEGF by enzyme-linked immunosorbent assay in plasma and in pericardial fluid sampled during open heart surgery (12 patients with ischemic heart disease and 17 with nonischemic heart disease). HGF levels were significantly higher in plasma than in pericardial fluid (12.0 +/- 1.8 versus 0.26 +/- 0.04 ng/mL, P < 0.0001). On the other hand, bFGF levels were significantly higher in pericardial fluid than in plasma (243.5 +/- 50.9 versus 49.6 +/- 7.8 pg/mL, P = 0.009). VEGF levels were not significantly different between pericardial fluid and plasma (47.2 +/- 17.6 versus 24.5 +/- 3.6 pg/mL, P = 0.23). Concentrations of angiogenic factors in pericardial fluid and in plasma were not significantly different between patients with ischemic and nonischemic heart disease. These results suggest that the production, secretion, and kinetics of HGF, bFGF, and VEGF are different. These angiogenic factors may have different pathophysiologic roles.     

Angiogenic factors in multiple myeloma: higher levels in bone marrow than in peripheral blood

BACKGROUND AND OBJECTIVES: To study the role of some soluble factors in the process of angiogenesis that accompanies multiple myeloma (MM). DESIGN AND METHODS: The concentrations of three well-known angiogenic peptides, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) were evaluated by an ELISA method. All of these factors were measured in the plasma obtained from peripheral blood (PB) and bone marrow (BM) aspirates of 34 patients affected by plasma cell disorders. This series included one patient with a solitary extramedullary plasmacytoma, 17 patients with MM at diagnosis, and 16 with previously treated MM. RESULTS: In all the patients, the concentration of each angiogenic factor was higher in bone marrow than in peripheral blood. Mean values of the three angiogenic factors in BM or in PB were lower in stage I than stage II-III. One patient with extramedullary solitary myeloma had high levels of VEGF and bFGF but this increase was not found in the other 6 patients with extramedullary disease when compared with patients without extramedullary disease. VEGF and bFGF did not correlate with each other while HGF showed a weak correlation with VEGF and a stronger one with bFGF. Moreover, VEGF correlated with features of disease activity, such as C-reactive protein, and 2-microglobulin, while both bFGF and HGF showed an inverse correlation with albumin level. No correlation was found between VEGF, bFGF and HGF levels and age, M protein level, osteolytic lesions, or percentage of BM plasma cells. Since angiogenic factors may be released by normal cells in response to hypoxia, we also evaluated erythropoietin (EPO) levels (which correlate with the hypoxic stimulus) both in PB and BM plasma of these patients but none of the measured angiogenic factors correlated with EPO levels. Interpretation and Conclusions. Several soluble factors may play a role in the angiogenic activity described in MM but their contribution to the progression of disease may be different. The finding of higher levels of these factors in BM than in PB might indicate that the bone marrow environment is their major source. Concentrations of angiogenic factors parallel the activity of disease and are independent of the hypoxic stimulus.     

Oncostatin M induces angiogenesis in vitro and in vivo.

Neovascularization of the atherosclerotic plaque is responsible for its weakening and consequently for the complications of vascular disease. Macrophages are a source of growth factors that can modulate angiogenesis. In this study, we analyzed the effect of oncostatin M (OSM) on angiogenesis, as it could be involved in the development of atherosclerosis. The effect of OSM was compared with those of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). On human dermal microvasculature endothelial cells (HMEC-1s), OSM (22.5 to 112.5 pmol/L) induced a dose-dependent increase in cell proliferation greater than that induced by the classic angiogenic factors vascular endothelial growth factor (VEGF; 543 pmol/L) and basic fibroblast growth factor (bFGF; 1.1 nmol/L). LIF (19 to 475 pmol/L) induced only a 30% increase in cell proliferation, and IL-6 had no effect. Furthermore, in a modified Boyden-chamber model, OSM, LIF, and IL-6 were chemoattractant for HMEC-1s. In a tridimensional gel of fibrin, OSM increased tube formation and tube length, which were already noticeable by day 3. LIF and IL-6 induced a weaker effect that was only obvious by day 10. The angiogenic effect of OSM was also demonstrated in vivo in a rabbit corneal model: OSM was more potent than LIF, the length of the neovessels being longer with OSM than with LIF, whereas IL-6 was without effect. We tested factors that could be involved in the proliferative effect of OSM on HMEC-1s. OSM induced only a slight increase in the urokinase receptor and a 60% increase in VEGF secretion, whereas it does not modify IL-8 secretion or bFGF levels. The effect of OSM seems to depend on endothelial cell origin and cell species: OSM (up to 112.5 pmol/L) did not induce human umbilical vein endothelial cell proliferation and even had a small inhibitory effect (17%) on calf pulmonary artery endothelial cells. In conclusion, OSM induces an angiogenic effect on capillary endothelial cells, which could be, at least in part, implicated in pathological processes such as atherosclerosis or tumor growth.     

Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes

Angiogenesis has been associated with the growth, dissemination, and metastasis of solid tumors. The aims of this study were to evaluate the vascularity and the levels of angiogenic factors in patients with acute and chronic leukemias and myelodysplastic syndromes (MDS). The numbers of blood vessels were measured in 145 bone marrow biopsies and the levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis growth factor-alpha (TNF-alpha), tumor growth factor-alpha (TGF-alpha), and hepatocyte growth factor (HGF) were determined in 417 plasma samples. Except for chronic lymphocytic leukemia (CLL), vascularity was significantly higher in all leukemias and MDS compared with control bone marrows. The highest number of blood vessels and largest vascular area were found in chronic myeloid leukemia (CML). VEGF, bFGF, and HGF plasma levels were significantly increased in acute myeloid leukemia (AML), CML, CLL, chronic myelomonocytic leukemia (CMML), and MDS. HGF, TNF-alpha, and bFGF but not VEGF were significantly increased in acute lymphoblastic leukemia (ALL). TNF-alpha levels were significantly increased in all diseases except for AML and MDS. No significant increase was found in TGF-alpha in any leukemia or MDS. The highest plasma levels of VEGF were in CML, and the highest plasma levels of bFGF were in CLL. The levels of HGF were highest in CMML. These data suggest that vascularity and angiogenic factors are increased in leukemias and MDS and may play a role in the leukemogenic process.     

Elevation of serum hepatocyte growth factor during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization

We examined serum levels of the angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), in normal donors for allogeneic peripheral blood stem cell (PBSC) transplantation. Granulocyte colony-stimulating factor (G-CSF) (filgrastim 400 microg/m2/d) was administered to 23 donors for 5 d and aphereses were performed on days 4 and 5. Although bFGF remained at similar levels after G-CSF treatment, serum VEGF and HGF levels increased 1.5-fold (n = 13; P = 0.02) and 6.8-fold (n = 23; P < 0.0001) respectively. The serum HGF level before G-CSF administration on day 1 correlated inversely with mobilized CD34+ cell numbers. Time course kinetics of HGF showed that on the day after G-CSF administration (day 2), serum HGF levels increased to 3678 pg/ml. For auto PBSC mobilization with chemotherapy and G-CSF 200 microg/m2/d (n = 8), we observed similar HGF elevation, which appeared to be dose-dependent on the G-CSF administered.     

Rhu-Epo down-regulates pro-tumorigenic activity of cancer-associated fibroblasts in multiple myeloma

We have previously demonstrated that recombinant human erythropoietin (rHuEpo) is involved in the regulation of the angiogenic response in multiple myeloma (MM) through a direct effect on macrophages and endothelial cells isolated from the bone marrow of patients with MM. The aim of the present study was designed to determine the effects of rHuEpo on cancer-associated fibroblasts (CAFs) from monoclonal gammopathy of undetermined significance (MGUS) and MM patients by means of in vitro and in vivo assays. rHuEpo treatment reduces the expression of mRNA levels of fibroblast activation markers, namely alpha smooth actin (αSMA) and fibroblast activation protein (FAP) in MGUS and MM CAFs, and of pro-inflammatory and pro-angiogenic cytokines, including interleukin (IL)-6 and IL-8, vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor (HGF) in MM CAFs. Moreover, rHuEpo inhibits the proliferative activity of MM CAFs and increased the apoptosis of MGUS and MM CAFs. Overall, these data suggest that rHu-Epo down-regulates CAFs pro-tumorigenic activity. Moreover, these results are not suggestive for a pro-angiogenic activity of rHuEpo on CAFs. In fact, rHuEpo pre-treatment induces a low angiogenic response in vivo in the chorioallantoic membrane (CAM) assay of MGUS and MM CAFs conditioned medium, not comparable to that of a well-known angiogenic cytokine, VEGF-A, tested in the same assay.     

Acidosis inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.     

Adipokine resistin promotes in vitro angiogenesis of human endothelial cells

OBJECTIVE: Resistin may be associated with obesity and cardiovascular diseases. However, it is unknown whether resistin directly contributes to angiogenesis. In the present study, we evaluated the effects of resistin on angiogenic potential, including endothelial cell proliferation, migration, and capillary-like tube formation. METHODS: Human coronary artery endothelial cells (HCAECs) were treated with resistin. Cell proliferation was evaluated by [3H]thymidine incorporation and MTS assays. Cell migration was assessed by a modified Boyden chamber assay. Capillary-like tube formation was studied with a Matrigel model. Several gene expression levels were determined by real-time PCR. Activation of mitogen-activated protein kinases (MAPKs) was determined by Bio-Plex luminex analyzer. Basic fibroblast growth factor (bFGF) was used as a control. Human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVEC-L) were also included. RESULTS: Resistin induced both endothelial proliferation and migration in a dose- and time-dependent manner with the maximal effect at 40 ng/ml. Both resistin-induced cell proliferation and migration could be effectively blocked by a resistin-neutralization antibody. In addition, resistin promoted capillary-like tube formation of HCAECs on Matrigel. Resistin also significantly upregulated the mRNA expression of vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2) and matrix metalloproteinases (MMP-1 and MMP-2) at both mRNA and protein levels. Furthermore, transient phosphorylation of ERK1/2 and p38 was observed after the addition of resistin to HCAECs. The resistin-induced cell proliferation and migration were both completely blocked by specific ERK1/2 and p38 inhibitors. CONCLUSIONS: Resistin induces human endothelial cell proliferation and migration, promotes capillary-like tube formation, upregulates the expression of VEGFRs and MMPs, and activates ERK1/2 and p38 pathways. Thus, resistin may play an important role in angiogenesis-associated vascular disorders.     

Platelets release CXCL4L1, a nonallelic variant of the chemokine platelet factor-4/CXCL4 and potent inhibitor of angiogenesis

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.     

Autocrine Role of Interleukin-8 in Induction of Endothelial Cell Proliferation,Survival, Migration and MMP-2 Production and Angiogenesis

Interleukin-8 (IL-8/CXCL8), a paracrine angiogenic factor, modulates multiple biologic functions in CXCR1 and CXCR2 expressing endothelial cells. Several reports suggest that inflammation, infection, cellular stress and tumor presence regulate IL-8 production in endothelial cells. In the present study, we test the hypothesis that IL-8 regulates multiple biological effects in endothelial cells in an autocrine manner. We examined the autocrine role of IL-8 in regulating angiogenesis by using a neutralizing antibody to IL-8, CXCR1 or CXCR2 in human vein umbilical endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMEC). Neutralizing antibody to IL-8, CXCR1 or CXCR2 inhibited endothelial cell proliferation, and MMP-2 production as compared to cells cultured with medium alone or control antibody. In addition, we observed that the number of apoptotic cells was significantly higher in anti-IL-8, anti-CXCR1 and anti-CXCR2 treated endothelial cells, which coincided with decreased survival-associated gene expression. We observed reduced migration of endothelial cells treated with anti-IL-8 and anti-CXCR2 antibody, but not anti-CXCR1 antibody as compared to controls. Further, we observed an inhibition of capillary tube formation and neovascularization following treatment with anti-IL-8, anti-CXCR1 and anti-CXCR2 antibodies. Together these data suggest that IL-8 functions as an important autocrine growth and angiogenic factor in regulating multiple biological activities in endothelial cells.     

Fetuin-A influences vascular cell growth and production of proinflammatory and angiogenic proteins by human perivascular fat cells

Aims/hypothesis

Fetuin-A (alpha2-Heremans-Schmid glycoprotein), a liver-derived circulating glycoprotein, contributes to lipid disorders, diabetes and cardiovascular diseases. In a previous study we found that perivascular fat cells (PVFCs) have a higher angiogenic potential than other fat cell types. The aim was to examine whether fetuin-A influences PVFC and vascular cell growth and the expression and secretion of proinflammatory and angiogenic proteins, and whether TLR4-independent pathways are involved.

Methods

Mono- and co-cultures of human PVFCs and endothelial cells were treated with fetuin-A and/or palmitate for 6–72 h. Proteins were quantified by ELISA and Luminex, mRNA expression by real-time PCR, and cell growth by BrDU-ELISA. Some PVFCs were preincubated with a nuclear factor κB NFκBp65 inhibitor, or the toll-like receptor 4 (TLR4) inhibitor CLI-095, or phosphoinositide 3-kinase (PI3K)/Akt inhibitors and/or stimulated with insulin. Intracellular forkhead box protein O1 (FoxO1), NFκBp65 and inhibitor of κB kinase β (IKKβ) localisation was visualised by immunostaining.

Results

PVFCs expressed and secreted IL-6, IL-8, plasminogen activator inhibitor 1 (PAI-1), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-BB, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), placental growth factor (PLGF) and hepatocyte growth factor (HGF). Fetuin-A upregulated IL-6 and IL-8, and this was potentiated by palmitate and blocked by CLI-095. Immunostaining and electrophoretic mobility shift assay (EMSA) showed partial NFκBp65 activation. MCP-1 was upregulated and blocked by CLI-095, but not by palmitate. However, HGF was downregulated, which was slightly potentiated by palmitate. This effect persisted after TLR4 pathway blockade. Stimulation of insulin–PI3K–Akt signalling by insulin resulted in nuclear FoxO1 extrusion and HGF upregulation. Fetuin-A counteracted these insulin effects.

Conclusions/interpretation

Fetuin-A and/or palmitate influence the expression of proinflammatory and angiogenic proteins only partially via TLR4 signalling. HGF downregulation seems to be mediated by interference with the insulin-dependent receptor tyrosine kinase pathway. Fetuin-A may also influence angiogenic and proinflammatory proteins involved in atherosclerosis.     

Syndecan-1 and angiogenic cytokines in multiple myeloma: correlation with bone marrow angiogenesis and survival

Angiogenesis is a complex process involved in the proliferation and metastasis of malignant tumours, and partly triggered by the secretion of various angiogenic factors by tumour cells or cells in the stromal environment. We investigated the correlation between bone marrow angiogenesis, estimated as microvessel density (MVD), and interleukin-6 (IL-6), basic fibroblastic growth factor (bFGF), hepatocyte growth factor (HGF) and syndecan-1 in 67 patients with newly diagnosed multiple myeloma, and evaluated the prognostic value of these parameters. Circulating levels of IL-6, bFGF, HGF and syndecan-1 were significantly higher in patients than in controls. Moreover, in patients, bone marrow levels of bFGF, HGF and syndecan-1 were higher than peripheral blood levels. Positive correlations were found between MVD and syndecan-1 blood levels (r = 0.33, P = 0.017), syndecan-1 bone marrow levels (r = 0.49, P = 0.046) and HGF blood levels (r = 0.36, P = 0.008) respectively. High MVD and high blood levels of IL-6, HGF and syndecan-1 were predictive of a shorter survival. In a multivariate survival analysis MVD and blood levels of IL-6 retained independent prognostic significance, while in a survival analysis without MVD the peripheral blood levels of HGF and syndecan-1 were strong independent prognostic factors.     

Marked decrease of plasma VEGF after implantation of autologous bone marrow mononuclear cells in a patient with critical limb ischemia--a case report

The authors performed autologous bone marrow mononuclear cells implantation (BMI) in a 79-year-old man with critical limb ischemia. After BMI, the resting pain of the ischemic leg improved gradually. They measured the plasma concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and serum hepatocyte growth factor (HGF) in the blood from bilateral femoral veins. Before BMI, the plasma VEGF and bFGF concentrations were much greater in the ischemic leg than in the other lower limb, but decreased to the same concentrations as those in the contralateral lower extremity after BMI. The large concentrations of the angiogenic factors VEGF and bFGF in plasma indicate the severity and extent of the leg ischemia. BMI resulted in lower levels of VEGF and bFGF, and this fall is the hallmark of the effectiveness of BMI in the treatment of peripheral artery disease.     

Resveratrol and its synthetic derivatives exert opposite effects on mesothelial cell-dependent angiogenesis via modulating secretion of VEGF and IL-8/CXCL8

We examined the effect of resveratrol (RVT) and its two derivatives (3,3',4,4'-tetrahydroxy-trans-stilbene and 3,3',4,4',5,5'-hexahydroxy-trans-stilbene) on human peritoneal mesothelial cell (HPMC)-dependent angiogenesis in vitro. To this end, angiogenic activity of endothelial cells (HUVEC, HMVEC, and HMEC-1) was monitored upon their exposure to conditioned medium (CM) from young and senescent HPMCs treated with stilbenes or to stilbenes themselves. Results showed that proliferation and migration of endothelial cells were inhibited in response to indirect (HPMC-dependent) or direct RVT activity. This effect was associated with decreased secretion of VEGF and IL-8/CXCL8 by HPMCs treated with RVT, which confirmed the experiments with recombinant forms of these angiogenic agents. Angiogenic activity of endothelial cells treated with CM from HPMCs exposed to RVT analogues was more effective. Improved migration was particularly evident in cells exposed to CM from senescent HPMCs. Upon direct treatment, RVT derivatives stimulated proliferation (but not migration) of HUVECs, and failed to affect the behaviour of HMVEC and HMEC-1 cells. These compounds stimulated production of VEGF and IL-8/CXCL8 by HPMCs. Studies with neutralizing antibodies against angiogenic factors revealed that augmented angiogenic reactions of endothelial cells exposed to CM from HPMC treated with RVT analogues were related to enhanced production of VEGF and IL-8/CXCL8. Collectively, these findings indicate that RVT and its synthetic analogues divergently alter the secretion of the angiogenic factors by HPMCs, and thus modulate HPMC-dependent angiogenic responses in the opposite directions. This may have implications for the attempts of practical employment of the stilbenes for treatment of pathologies proceeding with abnormal vascularisation of the peritoneal tissue.     

CD14+ CD163+ IL-10+ monocytes/macrophages: Pro-angiogenic and non pro-inflammatory isolation, enrichment and long-term secretion profile

The establishment of a stable endothelial layer on a biomaterial suture is a well known strategy to achieve hemocompatibility. The endothelialisation is supported by factors as e.g. vascular endothelial growth factor (VEGF), which can be secreted by monocytes/macrophages (mo/mΦ) in an angiogenic milieu. In order to avoid detrimental inflammation triggered by these mo/mΦ, we established a protocol for the generation of alternatively activated macrophages and studied their response towards VEGF-A165. We could generate sufficient amounts of these CD14+ CD163+ IL-10+ mo/mΦ from buffy coats(8.6 ± 4.7 × 105 cells/mlbuffy coat). Furthermore, we achieved a VEGF-A165 secretion in the nanogram range. The VEGF-A165 secretion increased 2.1-fold within 14 days from 7.6 ± 2.2 to 16.1 ± 2.5ng/ml when the cells were grown with a VEGF-A165 supplemented (10ng/ml) cell culture medium. Within this time period the secretion levels of other pro-angiogenic growth factors (bFGF, PDGF-BB) and immunomodulatory cytokines (IL-10, IL-12, IL-1ra, TNFα, IFNγ) reached only the picogram range. These results suggest that angiogenically stimulated CD14+ CD163+ IL-10+ mo/mΦ might be useful as a cellular cytokine delivery system supporting endothelialisation of biomaterials without inducing pro-inflammatory effects.     

Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.     

Angiogenic factors stimulate mast-cell migration

Mast cells accumulate at sites of angiogenesis. The factor(s) that control mast-cell recruitment at these sites have yet to be defined. We sought to determine if angiogenic factors result in mast-cell chemotaxis. In this study, we observed that platelet-derived growth factor-AB (PDGF-AB), vascular endothelial cell growth factor (VEGF), and basic fibroblast growth factor (bFGF) each cause directed migration of murine mast cells at picomolar concentrations, with a typical bell- shaped dose-response curve. Another potent angiogenic factor, platelet- derived endothelial cell growth factor (PD-ECGF), appears to promote chemokinesis of mast cells, whereas tumor necrosis factor-alpha, a weak angiogenic factor, is less robust but still functions as a mast cell chemotactic factor. Epidermal growth factor (EGF), a growth factor with minimal angiogenic properties, was ineffective as a mast cell chemotactic factor. A checkerboard analysis confirmed the directional chemotactic response of PDGF-AB, VEGF, and bFGF, while indicating the chemokinetic response induced by PD-ECGF. Cross-desensitization of growth-factor-induced directed migration was observed between PDGF-AB and bFGF, and also between PDGF-AB and PD-ECGF. Tyrosine kinase- inhibitor genistein effectively dampened the chemotactic responses, whereas pertussis toxin had no effect. In summary, our findings suggest that factors known to act on endothelial cells and stimulate neovascularization may simultaneously serve to recruit mast cells to these sites. The local accumulation of mast cells is believed to facilitate new vessel formation through complex cell:cell interactions.