Showing papers in "Angiogenesis in 1999"
TL;DR: This in vitro process is validated as a model of in vivo angiogenesis that can be quantified to assay stimulatory and inhibitory agents, signals and drugs.
Abstract: This report describes a model of angiogenesis which develops in admixtures (co-cultures) of human umbilical vein endothelial cells (HUVEC) and human diploid fibroblasts of dermal origin from adult patients. The system does not require the addition of further growth factors other than those normally present in endothelial growth medium (EGM), nor matrix proteins, and cell growth and proliferation are allowed to occur in a standard low (2%) concentration of fetal calf serum. Angiogenesis was specifically stimulated in response to vascular endothelial growth factor (VEGF), resulting in an increased development of structures resembling a microvasculature bed. Alternatively, angiogenesis was inhibited by addition of an excess of neutralising anti-VEGF antibodies, and the anti-angiogenic drugs such as suramin. We briefly show that stimulatory and inhibitory activities can be easily and quickly quantified by image analysis. Tubule formation was confirmed by confocal and electron microscopy, and the development and disposition of these structures within the co-cultures has been analysed immunochemically to show expression of specific endothelial cell determinants, such as PECAM-1. On this and a number of other criteria, the findings validate this in vitro process as a model of in vivo angiogenesis that can be quantified to assay stimulatory and inhibitory agents, signals and drugs.
306 citations
TL;DR: It is demonstrated that the zebrafish is a viable whole animal model for screening small molecules that affect blood vessel formation as a primary screen for compounds that affect angiogenesis.
Abstract: Angiogenesis is necessary for tumor growth, making inhibition of vessel formation an excellent target for cancer therapy. Current assays for angiogenesis, however, are too complex to be practical for drug screening. Here, we demonstrate that the zebrafish is a viable whole animal model for screening small molecules that affect blood vessel formation. Blood vessel patterning is highly characteristic in the developing zebrafish embryo and the subintestinal vessels (SIVs) can be stained and visualized microscopically as a primary screen for compounds that affect angiogenesis. Small molecules added directly to the fish culture media diffuse into the embryo and induce observable, dose-dependent effects. To evaluate the zebrafish as a model, we used two angiogenesis inhibitors, SU5416 and TNP470, both of which have been tested in mammalian systems. Both compounds caused a reduction in vessel formation when introduced to zebrafish embryos prior to the onset of angiogenesis. Short duration (1 h) exposure of SU5416 was sufficient to block new angiogenic and vasculogenic vessel formation. In contrast, TNP470 required continuous exposure to block SIV formation and had no apparent effect on vasculogenic vessel formation. To ascertain whether blood vessels in the zebrafish embryo respond to angiogenic compounds, we introduced human VEGF into embryos. Injection of VEGF caused an observable increase in SIV formation.
281 citations
TL;DR: Recent experimental evidence obtained using inhibitors of uPA and uPAR has validated this system as a therapeutic target for the development of anti-angiogenic and anti-metastatic therapeutic agents.
Abstract: Substantial evidence exists which implicates the urokinase plasminogen activator system [urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1)] in the neo-vascularization, invasion and metastasis of many solid tumors Clinical studies have demonstrated an association between high levels of expression of the components of this system in tumors and poor patient prognosis and outcome Components of the uPA/uPAR system are differentially expressed or activated on motile cells including invading tumor cells and leukocytes, and migrating endothelial cells In contrast, there is little or no expression on most normal, quiescent cells Studies performed in vitro have demonstrated the regulation of the expression of uPA and uPAR by growth and differentiation factors as well as by oncogenes In this review, we summarize recent findings on the role of the components of the uPA/uPAR system in angiogenesis, invasiveness and tumor metastasis The activities of this system in endothelial and leukocyte cell biology and the relevance of these activities to angiogenesis and tumor metastasis will be considered Recent experimental evidence obtained using inhibitors of uPA and uPAR has validated this system as a therapeutic target for the development of anti-angiogenic and anti-metastatic therapeutic agents These studies, as well as additional therapeutic and diagnostic implications for uPAR targeting, will be discussed
178 citations
TL;DR: The anti-angiogenic activity of TSP is localized in its heparin binding domain and type 1 and type 3 repeats and TSP inhibits angiogenesis by at least two separate mechanisms, displacement of VEGf165 from endothelial cell HS and direct binding to VEGF165.
Abstract: Vascular endothelial growth factor (VEGF) is a specific angiogenic factor, and thrombospondin (TSP), is a potent inhibitor of angiogenesis. To better understand the role of TSP as an anti-angiogenic agent, we have identified its specific domains that participate in its anti-angiogenic activity and examined the mechanism of its inhibitory effect on VEGF(165) induced angiogenesis. Exogenously added TSP inhibited VEGF(165) induced angiogenesis (proliferation and tube formation of human dermal microvascular endothelial cells [HDMEC] and neovascular outgrowth from human arterial rings). Although both VEGF(165) and TSP are heparin binding proteins, TSP had a higher affinity for (125)I-heparin than VEGF(165) (K(d1) 4 nM and K(d2) 14 nM for TSP; K(d) 91 nM for VEGF(165)). TSP displaced 36% of (125)I-VEGF(165) from HDMEC and this was comparable to the 27% reduction in (125)I-VEGF(165) binding to HDMEC upon cleavage of cell surface heparan sulfate (HS). About 35% of the mitogenic activity of VEGF(165) was attributable to its heparin binding region. These results indicate that a proportion of the mitogenic activity of VEGF(165) is inhibited by TSP via competition for cell surface HS. Further, (125)I-VEGF(165) bound directly to TSP in a saturable, concentration dependent manner, and heparin modulated this binding. The mAbs to the heparin binding domain to the type 1 and type 3 repeats of TSP inhibited the binding of VEGF(165) to TSP, and also blocked the inhibitory effect of TSP on VEGF(165) induced HDMEC proliferation. We conclude that (i) the anti-angiogenic activity of TSP is localized in its heparin binding domain and type 1 and type 3 repeats (ii) TSP inhibits angiogenesis by at least two separate mechanisms, (a) displacement of VEGF(165) from endothelial cell HS and (b) direct binding to VEGF(165).
167 citations
TL;DR: It is demonstrated that Aloe vera gel and its extracts are angiogenic on the chorioallantoic membrane (CAM) of chick embryo and β-sitosterol is a novel plant-derivedAngiogenic factor which may have potential pharmaceutical applications for the management of chronic wounds.
Abstract: Aloe vera gel has a beneficial effect on wound healing. Because angiogenesis is an essential process in wound healing, we hypothesized that Aloe vera gel might contain potent angiogenic compounds. Here we demonstrate that Aloe vera gel and its extracts are angiogenic on the chorioallantoic membrane (CAM) of chick embryo. Out of the three compounds purified from the final fraction of Aloe vera gel, beta-sitosterol showed a potent angiogenic activity in the CAM assay. In the presence of heparin, beta-sitosterol stimulated neovascularization in the mouse Matrigel plug assay and the motility of human umbilical vein endothelial cells in an in vitro wound migration assay. Thus beta-sitosterol is a novel plant-derived angiogenic factor which may have potential pharmaceutical applications for the management of chronic wounds.
147 citations
TL;DR: It is shown that exogenous addition of thymosin β4 (in the ng–μg range) to HUVEC in culture can induce several biological responses, which include increased tube formation in vitro and rearrangement of the actin cytoskeleton in vitro.
Abstract: When human umbilical vein endothelial cells (HUVEC) differentiate into capillary-like tubes, there is a five-fold upregulation of the mRNA for thymosin β4 (Tβ4) (Grant et al. J Cell Sci 1995; 108: 3685–94 [1]) and this endogenous expression plays an important role in endothelial cell attachment to and spreading on matrix components. We now show that exogenous addition of thymosin β4 (in the ng–μg range) to HUVEC in culture can induce several biological responses. These responses include increased tube formation in vitro. Additionally, exogenous thymosin β4 enhances vascular sprouting in the coronary artery ring angiogenesis assay. Measurements of these vascular sprouts show a doubling of the vessel area (via increased branching) with as little as 100 ng of synthetic thymosin β4. These processes appear to involve the binding of thymosin β4 to an unknown cell surface receptor and internalization of the protein. This cell surface-binding appears not to be mediated through the thymosin β4-actin binding domain LKTET. An increase in thymosin β4 cytoplasmic staining in HUVEC exposed 10 μg of the peptide appears to occur without increased mRNA translation. In summary Tβ4 induces an increase in cell-matrix attachment, proliferation, tube formation, internalization of the peptide and rearrangement of the actin cytoskeleton. The data now defines both an autocrine and paracrine role for thymosin β4 in vessel formation.
144 citations
TL;DR: The term ‘neovascularization’, which is more prevalent in the ophthalmic literature, will be used to describe the development of pathological new vessels and should be considered synonymous with ‘angiogenesis’.
Abstract: Ocular angiogenesis is responsible for the majority of irreversible blindness in the developed world [1]. This debilitating complication affects all age groups and characterizes such diverse and widespread diseases as trachoma, retinopathy of prematurity, diabetic retinopathy, neovascular glaucoma and age-related macular degeneration. Although numerous relatively rare conditions also exhibit ocular angiogenesis, the aim of this review will be to briefly summarize our current knowledge regarding the clinical and laboratory findings of the most epidemiologically significant diseases. We will also describe current concepts regarding the pathogenesis of ocular angiogenesis. In this review the term ‘neovascularization’, which is more prevalent in the ophthalmic literature, will be used to describe the development of pathological new vessels and should be considered synonymous with ‘angiogenesis’.
108 citations
TL;DR: In this paper, the authors examined the expression of galectin-1 in capillaries associated to the carcinoma cells or present in the remote non-tumoral stroma of 100 human prostate carcinoma samples by immunoperoxidase staining.
Abstract: Besides providing tumors with nutrients, newly formed capillaries constitute a potential escape route for tumor cells favoring metastatic dissemination, and constitute an access for the anti-tumoral host immune cells. Galectin-1, a soluble human lectin, is involved in numerous biological functions including cell–cell and cell–substrate interactions. In addition, galectin-1 is able to induce apoptosis of activated T-lymphocytes. In this study, we have examined galectin-1 expression in capillaries associated to the carcinoma cells or present in the remote non-tumoral stroma of 100 human prostate carcinoma samples by immunoperoxidase staining. Galectin-1 was expressed by endothelial cells from capillaries infiltrating the tumor tissue in 64% (64/100) of the cases. On the contrary, endothelial cells in the adjacent non-tumoral stroma expressed galectin-1 in very few cases (7/100). Increased frequency of galectin-1-positive capillaries in the tumor-associated compared to the tumor-free areas was observed in 63% of the cases. This striking contrast led us to set up an in vitro model to test whether tumor cells could induce galectin-1 expression by endothelial cells. Incubation of human umbilical vein endothelial cells with conditioned media from PC-3 or DU 145 prostate carcinoma cells led to a significant increase of galectin-1 protein expression (+32.97% and 37.91% P < 0.01 and P < 0.05, respectively). PC-3 conditioned medium also induced increased adhesion values of PC-3 cells to the endothelial cells (53.4 ± 4.7 vs. 38.5 ± 3.5 after 30 min; 66.6 ± 7.8 vs. 46.2 ± 6.4 after 60 min). An anti-galectin-1 antiserum abolished this modulation, and recombinant galectin-1 also induced increased adhesion values in a dose-dependent fashion. This effect was specific as no such modulations were observed using normal lymphocytes instead of PC-3 cells. Preferential galectin-1 expression in the endothelial cells close to the cancer cells could provide these latter with increased abilities to interact with the endothelial cells as well as a defense against the host immune system.
95 citations
TL;DR: A possible role for PECAM-1 in the complex process of tumor angiogenesis is suggested and additional evidence of the importance of endothelial cell adhesion molecules to the formation of new vessels is provided.
Abstract: Platelet endothelial cell adhesion molecule (PECAM-1/CD31), a member of the immunoglobulin superfamily expressed at high levels on endothelial cells, has been recently implicated in angiogenesis. Although antagonism of PECAM-1 inhibited neovascularization in two different animal models of growth factor/chemokine-induced angiogenesis, its participation in tumor angiogenesis has not been established. We therefore investigated its involvement in models of tumor angiogenesis in mice. An antibody against murine PECAM-1 that was shown to block in vitro murine endothelial tube formation inhibited the subcutaneous growth and tumor vascularity of three tumors in mice: A549 human non-small cell lung cancer in SCID mice, B16 murine melanoma in C57BL/6 mice and AB12 murine mesothelioma in Balb/c mice. These studies suggest a possible role for PECAM-1 in the complex process of tumor angiogenesis and provide additional evidence of the importance of endothelial cell adhesion molecules to the formation of new vessels.
87 citations
TL;DR: How dysregulation of apoptosis can lead to aberrant angiogenesis as demonstrated in the pathogenesis of retinopathy of prematurity and cancer is discussed.
Abstract: Angiogenesis, the process of new microvessel development, is encountered in a select number of physiological processes and is central to the pathogenesis of a wide variety of diseases. There is now convincing evidence that regulated patterns of endothelial cell survival and death, a process known as apoptosis, play a central role in the periodic remodeling of the vasculature, and in the timely evolution and regression of angiogenic responses. In this review we discuss the current evidence suggesting a role for inducers and inhibitors of angiogenesis as well as other mediators that modify endothelial cells functions in the survival and death of endothelial cells. We also discuss how dysregulation of apoptosis can lead to aberrant angiogenesis as demonstrated in the pathogenesis of retinopathy of prematurity and cancer.
69 citations
TL;DR: The production system yields a well characterized, soluble and potent rhEndostatin at quantities sufficient for human use, and the preclinical studies described herein are an important first step toward the assessment of Endostatin in the clinic.
Abstract: Endostatin, a potent endogenous inhibitor of angiogenesis, inhibits the growth of primary tumors without induction of acquired drug resistance in mice. We report that a soluble recombinant human (rh) Endostatin produced with characteristics of the native Endostatin, effectively inhibited the growth of primary tumors and pulmonary metastases in a dose-dependent manner. We also show that deletion of two of the four zinc ligands of rhEndostatin did not affect this potent tumor inhibiton. The growth of established Lewis lung primary tumors implanted into mice was inhibited (80–90%) upon systemic treatment with 50 mg/kg/12 h of rhEndostatin. Using the B16-BL6 murine experimental pulmonary metastases model, rhEndostatin administered at 1.5 mg/kg/day or 4.5 mg/kg/day beginning 3- or 11-days post tumor cell injection, respectively, resulted in an approximate 80% inhibition of tumor growth. At effective anti-tumor doses of 1.5 and 50 mg/kg, pharmacokinetic modeling in mice showed (a) the protein was 100% bioavailable, (b) the AUC ranged from 16 to 700 ngml/h and (c) the Cmax ranged from 161 to 4582 ng/ml. At the highest dose tested (300 mg/kg), delivered as a single bolus, no drug-related toxicity was observed in a Cynomolgus monkey infused with rhEndostatin. No toxicity was observed even at AUC and Cmax values that were 1.3- to 56-fold higher than those observed in mice with tumors that were potently inhibited. Our production system yields a well characterized, soluble and potent rhEndostatin at quantities sufficient for human use. The preclinical studies described herein are an important first step toward the assessment of Endostatin in the clinic.
TL;DR: It is concluded that contortrostatin binds to αvβ3, and interferes with the anchorage-dependent survival mechanism of the vascular endothelial cells, and the mobility of the cells.
Abstract: Contortrostatin, a 135 kDa disulfide-linked homodimeric polypeptide possessing an Arg–Gly–Asp sequence, was isolated from venom of the southern copperhead snake Daily injection of contortrostatin into the primary tumor of human breast cancer MDA-MB-435 carried in nude mice significantly inhibited tumor growth and neovascularization of the tumor tissue On the chick embryo chorioallantoic membrane, contortrostatin inhibited angiogenesis induced by MDA-MB-435 cells, basic fibroblast growth factor, and vascular endothelial growth factor In addition, contortrostatin effectively blocked adhesion of human umbilical vein endothelial cells (HUVEC) to immobilized vitronectin and significantly inhibited invasion of HUVEC through a Matrigel barrier Competitive binding assays and adhesion assays with different integrin antibodies suggested that integrin αvβ3 is a binding site for contortrostatin on vascular endothelial cells Detachment of HUVEC from vitronectin by contortrostatin induced apoptosis HUVEC adhered and spread well on immobilized contortrostatin without undergoing apoptosis, suggesting that it is the inhibition of adhesion and spreading of HUVEC on extracellular matrix proteins, rather than binding of contortrostatin to integrins per se, that triggers apoptosis We conclude that contortrostatin binds to αvβ3, and interferes with the anchorage-dependent survival mechanism of the vascular endothelial cells, and the mobility of the cells The consequent suppression of angiogenesis is an important component of the antineoplastic activity of contortrostatin
TL;DR: A naturally occurring soluble VEGFR-1 has been discovered in the supernatant from endothelial cells and at present appears to be the key regulator for the availability of VEGF secreted from different cells and tissues.
Abstract: The three human VEGF receptors 1–3 mediate biological signals important for new blood vessel formation and lymphangiogenesis. Soluble VEGF receptors contain all the information necessary for high affinity ligand binding and have been used as experimental tools and regulators in several angiogenic in vitro and in vivo models. Recombinant receptor molecules can be used for specific inhibition of VEGF mediated signal transduction and for blocking tumor angiogenesis by limiting the amount of VEGF secreted from tumor cells or stroma cells. A naturally occurring soluble VEGFR-1 has been discovered in the supernatant from endothelial cells and at present appears to be the key regulator for the availability of VEGF secreted from different cells and tissues. The exact physiological role has not yet been demonstrated.
TL;DR: The role of the angiogenin family in both normal and abnormal physiology and in development will only fully be realised by genetic approaches involving gene deletion.
Abstract: Angiogenin is a member of the ribonuclease superfamily, which shows an ever expanding collection of molecules being identified and cloned. It was initially isolated from the conditioned medium of cultured tumour cells. Its angiogenic activity appears to be critical for the maintenance and support of tumour growth. Angiogenin also plays a role in a number of non-malignant vasculoproliferative pathological conditions. Along with other related molecules, it has been identified in a wide variety of somatic tissues in adult and embryonic stages of vertebrate development. This suggests that angiogenin and related molecules are likely to play a vital role in normal physiology. Angiogenin is detectable in serum and to date has been implicated as a mitogen for vascular endothelial cells, an immune modulator with suppressive effects on polymorphonuclear leukocytes, an activator of certain protease cascades such as matrix metalloproteases and plasminogen-activated plasmin pathways, as well as an adhesion molecule. However, the role of the angiogenin family in both normal and abnormal physiology and in development will only fully be realised by genetic approaches involving gene deletion.
TL;DR: Data show that matrix-embedded fibroblasts regulate the motility of vascular endothelial cells and enhanceAngiogenesis, an effect partly attributed to production of angiogenesis-promoting cytokines HGF/SF and/or VEGf.
Abstract: This study sought to examine the effect of matrix-bound fibroblasts on angiogenesis and endothelial cell motility. Promotion of angiogenesis by matrix-bound fibroblasts was assessed using rat aortic ring and endothelial tube formation assays. Enhancement of human endothelial cell motility by matrix-bound fibroblasts was assessed using cytodex-2 bead and colloidal gold phagokinetic motility assays. Antibody to hepatocyte growth factor/scatter factor (HGF/SF) but not vascular endothelial growth factor (VEGf) decreased fibroblast-enhanced motility of endothelial cells. The promotion of tube formation by matrix-bound fibroblasts was neutralised with antibodies to HGF/SF and VEGf, both known promoters of angiogenesis. HGF/SF presence was detected by ELISA; whilst the presence of VEGf was detected by Western blotting. These data show that matrix-embedded fibroblasts regulate the motility of vascular endothelial cells and enhance angiogenesis, an effect partly attributed to production of angiogenesis-promoting cytokines HGF/SF and/or VEGf.
TL;DR: The low environmental pH encountered by endothelial cells in solid tumours would not necessarily be detrimental to neovascularisation: a prominent in vitro angiogenic response is still observed at low pHe when stimulated by exogenous growth factors, high concentrations of which would be present in vivo.
Abstract: The microenvironment of the majority of solid tumours in which new vessels must grow and survive is acidic. Whilst recent reports suggest a role of the low tumour pH in the invasive and metastatic potential of tumour cells, little is known as to its impact on angiogenesis. The three-dimensional in vitro rat aortic ring model was used to study the effects of low extracellular pH (pHe) on microvascular growth. The spontaneous angiogenic response in collagen gels was seen to be highly dependent on the pH of the culture medium, with optimal outgrowth at pH 7.4, and a marked delay in microvascular growth at pH 6.9. This inhibition of vascular development was reversible. The absence of similar effects of medium pH on monolayer outgrowths of endothelial cells from rat aortic rings suggested an effect of pHe on aspects specific to three-dimensional growth. Vascular endothelial growth factor and basic fibroblast growth factor, whilst having limited effects at pH 7.4, were seen to reduce the time to onset of vessel outgrowth at pH 7.1, and lead to an initial growth rate similar to that observed at pH 7.4 in the absence of growth factors. Thus, the low environmental pH encountered by endothelial cells in solid tumours would not necessarily be detrimental to neovascularisation: a prominent in vitro angiogenic response is still observed at low pHe when stimulated by exogenous growth factors, high concentrations of which would be present in vivo.
TL;DR: The results support the previous findings that blockade of integrin αvβ3 inhibits angiogenesis and tumor growth and indicates that dual inhibitors of αv β3 and αIIbβ3 are effective in blocking tumor Growth and Angiogenesis.
Abstract: Angiogenesis plays an essential role in the growth and dissemination of solid tumor cancers. The expression of endothelial cell integrin αvβ3 has been shown to increase during vascular proliferation associated with human tumors. Selective antagonists of αvβ3 can block angiogenesis and tumor growth by inducing programmed cell death in proliferating endothelial cells. Monoclonal antibody 7E3, an antagonist of the human, but not murine, integrins αvβ3 and αIIbβ3 (GPIIb/IIIa), inhibits platelet aggregation. It is the parent antibody of a mouse/human chimeric antibody fragment approved for adjunctive therapy of patients undergoing percutaneous coronary interventions to prevent ischemic complications (c7E3Fab; abciximab; ReoPro). To evaluate the potential of 7E3 to inhibit human angiogenesis and tumor growth independent of its antiplatelet effects, we established integrin αvβ3-negative human melanoma tumors in full-thickness human skin grafted onto SCID mice. The resulting tumors induce a human angiogenic response as assessed by the immunoreactivity of vascular cells with monoclonal antibodies specific for human CD31. Administration of 7E3 prevented or significantly inhibited the growth of tumors, and this effect correlated with a significant reduction in the number of blood vessels supplying the tumors. These results support the previous findings that blockade of integrin αvβ3 inhibits angiogenesis and tumor growth and indicates that dual inhibitors of αvβ3 and αIIbβ3 are effective in blocking tumor growth and angiogenesis.
TL;DR: It is indicated that PI3-kinase activity is required for migration, mitogenicity and survival but not for differentiation of endothelial cells during angiogenesis.
Abstract: Angiogenesis involves endothelial cell invasion and migration into the surrounding tissue where cells differentiate, to form new lumen-containing vessels. We have investigated the role of phosphoinositide 3-kinase (PI3-kinase) in vascular endothelial growth factor (VEGF)- and fibroblast growth factor (FGF)-induced angiogenesis. Angiogenesis in vivo in chick embryos was inhibited by treatment with the PI3-kinase inhibitors wortmannin and LY294002. Stimulation of primary bovine capillary endothelial (BCE) cells with FGF-2, VEGF-A165, or a combination of the two induced PI3-kinase activity in vitro and subsequent activation of the serine/threonine kinase Akt. The combination of FGF-2 and VEGF-A165 led to an additive response. Activation of PI3-kinase was strictly required for FGF-2- and VEGF-A165-induced migration and DNA synthesis of BCE cells. Tubular morphogenesis was unaffected by treatment with wortmannin or LY294002, but survival of the tubular structures was dependent on PI3-kinase activity. VEGF-A165 and FGF-2 induced increased stability of the tubular structures in a synergistic manner. These data indicate that PI3-kinase activity is required for migration, mitogenicity and survival but not for differentiation of endothelial cells during angiogenesis.
TL;DR: It is shown that 17β-estradiol increases human umbilical vein endothelial cell attachment to the extracellular matrix proteins laminin-1, type IV collagen, type I collagen, and fibronectin and integrin-mediated signaling was enhanced by estrogens since an increase in tyrosine-phosphorylation of focal adhesion kinase induced by cell attachment was observed in estrogen-treated endothelial cells.
Abstract: Premenopausal women have a lower cardiovascular risk and a higher incidence of several autoimmune diseases involving blood vessels than men. Although the precise effects of estrogens on the cardiovascular system are largely unknown, recent data suggest that estrogens can exert direct regulatory effects on endothelial cells. In the present study, we show that 17β-estradiol increases human umbilical vein endothelial cell attachment to the extracellular matrix proteins laminin-1, type IV collagen, type I collagen, and fibronectin. Estradiol enhanced adhesion most significantly to laminin-1 and to fibronectin-derived synthetic peptides containing an RGD sequence. Upon exposure to estradiol, an increase in β1, α5 and α6 integrin mRNA was observed in subconfluent cells which was abrogated by treatment with cycloheximide. This increase was followed by a later enhancement in surface expression of the above integrins. In addition, integrin-mediated signaling was also enhanced by estrogens since an increase in tyrosine-phosphorylation of focal adhesion kinase induced by cell attachment was observed in estrogen-treated endothelial cells. Since integrins have an important role in mediating endothelial cell attachment, migration and differentiation, the increase in integrin expression and function induced by estradiol may be an important mechanism through which estrogens can promote neovascularization and vessel repair.
TL;DR: Of particular interest was the increase in expression of the mRNA for the cell surface proteins vascular endothelial (VE-) and neural (N-) cadherin and α5, αv, β1 and β3 integrins, which offers a previously unrecognized opportunity for vascular targeting.
Abstract: Endothelial cells are known to be a rich source of transcriptional gene expression. Recent technological advances now permit the detailed profiling of mRNA expression using arrays of known cDNAs on blots. We have used such arrays to examine expression of mRNA by primary isolates of human foreskin microvascular endothelial cells in the proliferative and quiescent state. Cells were stimulated by a combination of known growth factors for these cells including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and ‘endothelial cell growth supplement (ECGS)’ either alone or in combination. Analysis showed the expression of many mRNAs but of the 588 examined, only one, namely monocyte chemotactic protein-1 (MCP-1), showed a decrease on treatment with EGF. A combination of image grabbing followed by subtractive densitometry enabled identification of the mRNAs upregulated in proliferating endothelium. In consideration of the possibility of selective vascular targeting, of particular interest was the increase in expression of the mRNA for the cell surface proteins vascular endothelial (VE-) and neural (N-) cadherin and α5, αv, β1 and β3 integrins. The α5 integrin offers a previously unrecognized opportunity for vascular targeting.
TL;DR: The role of FGF-2 in wound healing of the chick embryo chorioallantoic membrane is demonstrated and it is suggested that CAM can also be utilized as an in vivo model for the easy, rapid, and economic screening of molecules potentially able to affect the wound healing process.
Abstract: Re-epithelization and the formation of a granulation tissue consisting of inflammatory cells, newly formed blood vessels, and fibroblasts embedded in a loose collagenous extracellular matrix, are critical events occurring during wound healing. In this study, utilizing the chick embryo chorioallantoic membrane (CAM) as an in vivo model of wound healing, we investigated the role of endogenous and exogenous fibroblast growth factor-2 (FGF-2) in the wound healing reparative processes. The results showed that: (1) neutralizing anti-FGF-2 antibodies (400 ng/embryo) decreased significantly the rate of wound healing (occurring only in 25% of specimens) when applied close to the edge of the wound, causing a significant decrease of microvessel and fibroblast density, and of an inflammatory macrophage infiltrate in the wounded area; (2) conversely, the application of exogenous recombinant FGF-2 (1.0 μg/embryo) greatly accelerated the wound repair occurring approximately 24h earlier than in untreated CAMs, stimulating angiogenesis, fibroblast proliferation, and macrophage infiltration. These findings demonstrate the role of FGF-2 in wound healing of the CAM and suggest that CAM, usually employed as an in vivo assay to study angiogenesis, can also be utilized as an in vivo model for the easy, rapid, and economic screening of molecules potentially able to affect the wound healing process.
TL;DR: It is predicted that the appearance of macrophages in inflammatory areas is necessary for angiogenic responses in various inflammatory diseases and MIP-1α and VEGF may have an important role in recruiting macrophage and neovascularization.
Abstract: We predicted that the appearance of macrophages in inflammatory areas is necessary for angiogenic responses in various inflammatory diseases. Using a mouse inflammatory corneal model in which model mouse corneas were cauterized with silver nitrate, we examined the infiltration of macrophages immunohistochemically and the total area of neovascularization quantitively. The expression of macrophage inflammatory protein-1α (MIP-1α) and vascular endothelial growth factor (VEGF) levels were also examined. A day after cauterization, short capillaries began to develop into the corneal stroma, and after 4 or 5 days the neovascularization became maximal and then began to regress. The number of macrophages within the cauterized cornea increased to a maximum at day 3 and began to decrease at day 5. The number of infiltrated macrophages reached maximum at day 3. Both MIP-1α and VEGF protein levels increased markedly immediately after the chemical cauterization, and production of MIP-1α (85.8 pg/4 corneas) and VEGF (206.5 pg/4 corneas) was maximal at 1 day and 0.5 day after cauterization, respectively. MIP-1α and VEGF mRNA levels also increased at 0.5 day after cauterization. In situ hybridization showed that MIP-1α was localized in corneal epithelial cells, and VEGF was localized in corneal epithelial cells and infiltrating inflammatory cells. MIP-1α and VEGF may have an important role in recruiting macrophages and neovascularization.
TL;DR: Although heparin decreases binding of VEGF to V EGF receptor-1, the remaining binding results in more efficient kinase activation, and there is no loss of VegF/VEGF receptor- 1 function in the presence of he parin.
Abstract: Vascular endothelial growth factor (VEGF) regulates blood vessel formation by binding to the receptor tyrosine kinases VEGF receptor-1 (Flt-1) or VEGF receptor-2 (KDR) and to the structurally unrelated neuropilins. As exon 7-containing isoforms of VEGF bind to heparin, angiogenesis may be modulated by heparin/heparan sulfate. We analyzed the effect of heparin on VEGF165-binding and activation of VEGF receptor-1 in porcine aortic endothelial cells, which lack expression of VEGF receptor-2 and neuropilins. Heparin decreased binding of 125I-VEGF to 50% at 5 μg/ml and cross-linking of 125I-VEGF to VEGF receptor-1 on intact cells was similarly decreased. Schatchard analyses showed that the affinity for binding of 125I-VEGF to VEGF receptor-1 was decreased in the presence of heparin. In contrast, VEGF receptor-1 kinase activity was elevated when cells were treated simultaneously with VEGF and heparin. In accordance, VEGF-induced tyrosine phosphorylation of phospholipase Cγ (PLCγ) and DNA synthesis were augmented by heparin. However, basal PLCγ tyrosine phosphorylation and DNA synthesis levels were to some extent increased by incubation of cells with heparin alone. We conclude that although heparin decreases binding of VEGF to VEGF receptor-1, the remaining binding results in more efficient kinase activation. Taken together, there is no loss of VEGF/VEGF receptor-1 function in the presence of heparin.
TL;DR: Observations indicate that membrane-type MMP-1 (MT1-MMP) is involved in the angiogenic process of endothelial cells in vitro.
Abstract: Matrix metalloproteinases (MMPs) have been reported to play critical roles in endothelial cell migration and matrix remodeling during the angiogenic process. Among these MMPs, membrane-type MMP-1 (MT1-MMP) is an important molecule that can trigger the invasion of tumor cells by activating MMP-2 on their plasma membrane. However, the precise involvement of MT1-MMP in the angiogenic process has not been determined. To investigate the roles of the MT1-MMP by the matrix remodeling of endothelial cells, MT1-MMP expression vector was transfected into bovine aortic endothelial cells (BAECs). Increased expression of MT1-MMP in BAECs enhanced the activation of MMP-2, invasion and migration of BAECs. Moreover, the capacity of tube formation was increased in MT1-MMP transfectants. However, cotransfection with antisense MT1-MMP expression vector abolished the effects of MT1-MMP overexpression. These observations indicate that MT1-MMP is involved in the angiogenic process of endothelial cells in vitro.
TL;DR: A 31-year-old female with severe Crohn's disease who had been treated with corticosteroids and 6-mercaptopurine, was treated with thalidomide initially for erythema nodosum and all symptoms of Crohn’s disease disappeared and she was able to discontinue all other drugs.
Abstract: A 31-year-old female with severe Crohn's disease for 15 years who had been treated with corticosteroids and 6-mercaptopurine, was treated with thalidomide initially for erythema nodosum. While on thalidomide all symptoms of Crohn's disease disappeared and she was able to discontinue all other drugs. At this writing she has been on thalidomide as sole therapy for over 4 years with the exception of a 5-week hiatus, during which time her symptoms recurred, but again disappeared after resumption of thalidomide therapy. This case suggests that thalidomide may be a useful therapy for Crohn's disease and provides impetus for a clinical trial of thalidomide for Crohn's disease.
TL;DR: The data presented here suggest FGF-2 is capable of stimulating smooth muscle cell proliferation through an FGFR independent, HLGAG dependent mechanism.
Abstract: On the surface of smooth muscle cells there are two types of receptors for the mitogenic and angiogenic growth factor fibroblast growth factor-2 (FGF-2); a high affinity tyrosine kinase FGF receptor (FGFR1) and low affinity heparin./heparan-like glycosaminoglycan (HLGAG) component of surface expressed proteoglycans. It is believed that all three components; FGFR1, FGF-2, and the HLGAG chains, must form a ternary complex for maximal cellular stimulation. To carefully examine the role surface HLGAGs play in FGF-2-mediated proliferation of SMCs we have utilized HLGAG degrading enzymes heparinase I, II and III. We report that heparinase treatment of bovine smooth muscle cells inhibits the binding of 125I-FGF-2 to FGFR1, but does not inhibit FGF-2 induced cellular proliferation. Through the use of both sodium chlorate and FGF-2 mutants with deficient HLGAG-binding capabilities, we show the FGF-2-HLGAG interaction is important for FGF-2's ability to induce SMC proliferation. Finally, we report conditioned media from heparinase treated SMCs is capable of supporting FGF-2 induced proliferation in an HLGAG-free lymphoid F32 cells, suggesting that the heparinase generated fragments are responsible for the proliferative response. The data presented here suggest FGF-2 is capable of stimulating smooth muscle cell proliferation through an FGFR independent, HLGAG dependent mechanism.
TL;DR: The results demonstrate the possibility of using cationic lipid delivery of an antiangiogenic gene for cancer treatment and increase the anti-tumor activities of endostatin.
Abstract: Inhibition of the neovascularization of tumors has proven efficacious in reducing tumor growth and metastases Attaining antiangiogenesis through cationic lipid-based systemic gene therapy presents an attractive approach to the treatment of disseminated and primary cancers Intravenous administration of an endostatin plasmid, complexed with a cationic lipid system, produced significant levels of endostatin in the lung and the circulation The expressed endostatin blocked systemic angiogenesis and inhibited tumor growth in murine models Cytokine induction by cationic lipid/DNA complex increased the anti-tumor activities of endostatin These results demonstrate the possibility of using cationic lipid delivery of an antiangiogenic gene for cancer treatment
TL;DR: It is suggested that blockade of NO synthesis induces expression of angiogenic growth factors as well as vessel wall remodeling, and TGF-β may counteract angiogens growth factors and limit subsequent alterations in capillary network remodeling.
Abstract: Long-term blockade of nitric oxide synthesis with Nω-nitro-L-arginine methyl ester (L-NAME) induces cardiac perivascular fibrosis in rats. Its relationship to expression of angiogenic growth factors and capillary network remodeling is not understood. This study was designed to determine whether capillary proliferation and angiogenic growth factor regulation occur in response to L-NAME. Three groups of rats were studied: C, control; L1, L-NAME 13 mg/kg/day; L2, 130 mg/kg/day. One and eight weeks later the hearts were removed and subjected to morphometric analysis and analysis of gene expressions of molecules related to angiogenesis. Arterial hypertension was observed within 8 weeks in the L1 and L2 groups compared with control. After 1 week immunohistochemical assays demonstrated basic fibroblast growth factor (bFGF) in the arteriolar media. Northern blot analysis revealed increase in bFGF and transforming growth factor-β (TGF-β) mRNA during this period. At 8 weeks arteriolar medial thickening and perivascular fibrosis were seen microscopically in the L1 and L2 groups, which were accompanied by only a modest remodeling of capillary network due to increase in venular or intermediate capillary portions. Concomitantly immunoreactivity for vascular endothelial growth factor (VEGF) and TGF-β were detected in perivascular area. These results suggest that (1) blockade of NO synthesis induces expression of angiogenic growth factors as well as vessel wall remodeling, and (2) TGF-β may counteract angiogenic growth factors and limit subsequent alterations in capillary network remodeling.
TL;DR: Among PTP inhibitors, bpV(phen) had powerful angiostatic properties at a low concentration, dependent upon both the compound's dose and the nature of the ancillary ligand.
Abstract: Angiogenesis is a complex process that involves the activation of endothelial cells through the triggering of several intracellular signaling pathways including those involving tyrosine phosphorylation. In the present study, we analyzed the angiogenic properties of two phosphotyrosyl phosphatase (PTP) inhibitors that are composed of a peroxovanadium core containing different ancillary ligands. In cell monolayer and 3D culture systems examined in this study, the administration of potassium bisperoxo(1,10-phenanthroline)oxovanadate(V) [bpV(phen)] or potassium bisperoxo(pyridine-2-carboxylato)oxovanadate(V) [bpV(pic)], but not oxovanadiums, interfered markedly with endothelial cell growth, organization, and terminal differentiation. This effect was dependent upon both the compound's dose and the nature of the ancillary ligand. Rat aortic ring assay showed a significant inhibition by low dose of bpV(phen) on cell migration. In addition, a chick embryo angiogenesis assay demonstrated that bpV(phen) is a potent inhibitor of angiogenesis. Among PTP inhibitors, bpV(phen) had powerful angiostatic properties at a low concentration.
TL;DR: There is histological, molecular and clinical evidence supporting VI as a useful `in vivo' indicator of tumor angiogenesis, particularly for predicting lymph node metastases in cervical carcinomas.
Abstract: The role of angiogenesis in tumorigenesis is widely accepted. Therefore, it is mandatory to develop a clinically useful method for assessing tumor angiogenesis. This study was designed to compare the `in vivo' and `in vitro' methods for assessing angiogenesis and to evaluate their clinical application using cervical carcinoma as a model. Ninety women with stages IB-IIA cervical carcinoma exhibiting visible cervical tumors by transvaginal ultrasound were enrolled in this study. All patients underwent radical abdominal hysterectomy and pelvic lymph node dissection. Vascularity index (VI) was assessed by power Doppler ultrasound and a quantitative image processing system. The microvessel density (MVD) of the excised tumors was immunohistochemically assessed. Both the enzyme immunoassay and immunohistochemistry methods were performed for assessing the protein levels of vascular endothelial growth factor (VEGF) in tumor tissues. Significantly higher VI, MVD and cytosol VEGF concentrations were detected in tumors with deep stromal invasion (≥1/2 thickness) (11.43 ± 7.25 vs. 5.87 ± 6.81, P < 0.001; 53.0 vs. 37.0, P = 0.006, 550.0 vs. 86.0 pg/mg, P < 0.001), lymphatic invasion (12.21 ± 7.89 vs. 6.86 ± 6.29, P < 0.001; 53.0 vs. 40.0, P = 0.038; 930.0 vs. 110.0 pg/mg, P = 0.002), and pelvic lymph node metastasis (17.15 ± 8.58 vs. 7.83 ± 6.41, P < 0.001; 54.0 vs. 39.0, P = 0.02; 964.0 vs. 131.0 pg/mg, P = 0.002). VEGF-rich tumors detected by immunohistochemistry also revealed higher VI (12.26 ± 7.96 vs. 8.05 ± 7.62, P = 0.012), MVD (53.0 vs. 37.5, P = 0.01) and cytosol VEGF levels (745.0 vs. 98.0 pg/mg, P = 0.002). The relationships between VI values, MVD values and cytosol VEGF concentrations were linear (VI vs. MVD, r = 0.38, P < 0.001; VI vs. VEGF, r = 0.78, P < 0.001; MVD vs. VEGF, r = 0.29, P = 0.006). As revealed by the receiver operating characteristic (ROC) curve analysis, VI is better than MVD and VEGF in predicting lymph node metastasis. In conclusion, there is histological, molecular and clinical evidence supporting VI as a useful `in vivo' indicator of tumor angiogenesis, particularly for predicting lymph node metastases in cervical carcinomas.