Fibrin fragment E stimulates the proliferation,migration and differentiation of human microvascular endothelial cells in vitro

Various factors involved in haemostasis also regulate the development of new blood vessels by a process called angiogenesis. Enzymatic cleavage of fibrin yields a variety of fibrin degradation products, particularly in areas of intense angiogenesis such as in healing wounds and active atherosclerotic plaques. One of these, fibrin fragment E (FnE), is a potent angiogenic factor in the chick chorioallantoic membrane assay of angiogenesis. Here, we extend these studies to show that FnE stimulates the proliferation, migration and differentiation of human dermal microvascular endothelial cells (HuDMECs) in vitro, both in the absence and presence of such additional endothelial growth factors as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We also show that these stimulatory effects occur at concentrations of the protein known to be present in angiogenic tissues in vivo. FnE enhanced the angiogenic effects of VEGF or bFGF, indicating a possible synergy between the signalling pathways used by these three angiogenic factors. This revised version was published online in June 2006 with corrections to the Cover Date.     

Angiocidal effect of Cyclosporin A: a new therapeutic approach for pathogenic angiogenesis.

AIM: Angiogenesis is essential in the development of several disorders such as cancer, arthritis, and autoimmune diseases. Several agents prevent angiogenesis but only a few destroy established angiogenesis. In this study we tested whether local or systemic administration of Cyclosporin A (CyA) would inhibit as well as destroy established angiogenesis in an in vivo assay of angiogenesis. METHODS: We utilized an in vivo assay of angiogenesis in which an angiogenic mixture of Matrigel, FGF, VEGF, and heparin was injected subcutaneously into mice. Angiogenesis in the subcutaneous plugs was quantified by ANOVA. CyA or the vehicle for CyA was administered to the experimental or the control groups by three routes: by addition to the angiogenic mixture, by local injection into the angiogenic plug at various time points or by systemic administration at high doses. Angiogenesis was quantified by pointing method and expressed as an angiogenic index (AI). RESULTS: In control animals the subcutaneous plug of Matrigel with the angiogenic mixture revealed exuberant angiogenesis at day 4 and day 7. This angiogenesis was completely inhibited when CyA was included in the angiogenic mixture; the vehicle for CyA had no such effect. Angiogenesis that had progressed was found to regress after local subcutaneous injection of CyA at day 4 and 7. Similar regression of angiogenesis was noted when CyA was administered systemically after allowing angiogenesis to proceed for 4 days. CONCLUSIONS: Our experiments strongly suggest that CyA is both angiocidal and angiostatic in vivo. These results provide a basis for future therapy directed against established angiogenesis in malignancies and autoimmune diseases.     

Differential role of platelet granular mediators in angiogenesis

OBJECTIVES: Platelets contain numerous substances regulating angiogenic response. However, the regulatory role of platelets in blood vessel development remains to be elucidated. We investigated the comprehensive effect of platelets as a cellular system on angiogenesis. METHODS: The following approaches were applied: (a) in vitro-aortic ring assay and chemotaxis assay; (b) in vivo-injection of platelet-containing matrigel plug subcutaneously into a mouse followed by immunohistochemical analysis of angiogenic response. RESULTS: Platelets stimulated formation of blood vessels in vitro in the rat aortic ring model via VEGF and bFGF, while blocking of platelet factor-4 promoted this effect. Addition of platelets to the matrigel followed by its subcutaneous injection into a mouse resulted in an intensive migration of fibroblasts into the matrigel as well as formation of blood capillaries de novo. This platelet effect was mediated through bFGF, VEGF, and heparanase. Furthermore, platelet releasate was found to induce endothelial cell chemotaxis. This effect was mediated by a concerted action of intraplatelet bFGF, PDGF, VEGF, and heparanase. CONCLUSION: Platelets affect different stages of the angiogenic response with a trend to a pro-angiogenic net effect despite the presence of angiogenesis inhibitors such as platelet factor 4. While a concomitant effect of bFGF and VEGF seemed to be essential for the entire process of vessel formation (aortic ring and matrigel models), PDGF and heparanase were effective only at the migration stage.     

A ncovel angiogenic factor derived from Aloe vera gel: β-sitosterol, a plant sterol

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, β-sitosterol showed a potent angiogenic activity in the CAM assay. In the presence of heparin, β-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 β-sitosterol is a novel plant-derived angiogenic factor which may have potential pharmaceutical applications for the management of chronic wounds. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Anti-Angiogenic Effect of Sinomenine

Sinomenine is an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, which has been utilized to treat rheumatoid arthritis (RA) in China for over 2000 years. Sinomenine has been shown to mediate a wide range of pharmacological actions which includes anti-inflammatory and anti-rheumatic effects. RA has been classified as a chronic immune-mediated disease that exhibits overlapping manifestation of inflammatory, abnormal cellular and hormonal immune responses with synovial hyperplasia. Since, angiogenesis is recognized to play a critical role in the development of RA and anti-angiogenic therapy has been proposed as a new therapeutic strategy for treatment of RA, we would like to see if sinomenine possesses anti-angiogenic property. In this study, sinomenine inhibited bFGF-induced proliferation of human umbilical vein endothelial cells (HUVEC) and arrested its cell cycle in G1 phase. Sinomenine disrupted tube formation of HUVEC on Matrigel and suppressed the chemotaxis of HUVEC. In addition, sinomenine reduced neovascularization in Matrigel plug assay as well as microvascular outgrowth in rat aorta ring sprouting assay. These results suggest that sinomenine inhibited bFGF-induced angiogenesis in vitro and in vivo. As the leukocytes–endothelial adhesive interactions also play an important role in inflammation, we found that sinomenine reduced the transmigration of granulocytic differentiated HL60 cells across IL-1β activated HUVEC monolayer. Therefore, the inhibition of leukocytes migration across blood vessel walls and the anti-angiogenic effect of sinomenine may contribute towards its therapeutic mechanisms in alleviating the pathogenesis of RA.     

Platelet-derived growth factor inhibits basic fibroblast growth factor angiogenic properties in vitro and in vivo through its alpha receptor

Basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) modulate vascular wall cell function in vitro and angiogenesis in vivo. The aim of the current study was to determine how bovine aorta endothelial cells (BAECs) respond to the simultaneous exposure to PDGF-BB and bFGF. It was found that bFGF-dependent BAEC migration, proliferation, and differentiation into tubelike structures on reconstituted extracellular matrix (Matrigel) were inhibited by PDGF-BB. The role played by PDGF receptor alpha (PDGF-Ralpha) was investigated by selective stimulation with PDGF-AA, by blocking PDGF-BB-binding to PDGF-Ralpha with neomycin, or by transfecting cells with dominant-negative forms of the receptors to selectively impair either PDGF-Ralpha or PDGF-Rbeta function. In all cases, PDGF-Ralpha impairment abolished the inhibitory effect of PDGF-BB on bFGF-directed BAEC migration. In addition, PDGF-Ralpha phosphorylation was increased in the presence of bFGF and PDGF, as compared to PDGF alone, whereas mitogen-activated protein kinase phosphorylation was decreased in the presence of PDGF-BB and bFGF compared with bFGF alone. In vivo experiments showed that PDGF-BB and PDGF-AA inhibited bFGF-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane assay and that PDGF-BB inhibited bFGF-induced angiogenesis in Matrigel plugs injected subcutaneously in CD1 mice. Taken together these results show that PDGF inhibits the angiogenic properties of bFGF in vitro and in vivo, likely through PDGF-Ralpha stimulation.     

Comparison of three in vitro human ‘angiogenesis’ assays with capillaries formed in vivo

Angiogenesis assays are an important tool for studying both the mechanisms of angiogenesis and the potential development of therapeutic strategies to modulate neovascularisation. In vivo angiogenesis assays are considered to be the most informative of these but are often expensive, time-consuming and require specialist training to perform. In vitro assays tend to be more rapid, less expensive and easier to interpret. In vitro angiogenesis assays operate on the principle that endothelial cells form tubule-like structures when cultured on a supportive matrix. Assays involving a matrix derived from murine tumours, Matrigel (or a growth factor reduced form of this), are now the most common in vitro tubule formation assays. However, another tubule formation assay has recently been developed in which endothelial cells are co-cultured with fibroblasts. Here, we have used quantitative image analysis to compare the morphological features of tubules formed in the Matrigel assay and this new ‘Co-culture’ assay, with those of capillaries formed in a microvascular bed in vivo. Tubules formed in standard and growth factor reduced Matrigel assays were short and relatively homogeneous, whereas those formed in the Co-culture assay were significantly more heterogeneous, consisting of both short and long interconnecting tubules that more closely resembled capillaries than Matrigel tubules. Moreover, cells on Matrigel, and to a lesser extent growth factor reduced (GFR) Matrigel, often clumped into large cell aggregates, a feature rarely seen in the Co-culture assay. In addition, we demonstrate that Matrigel stimulates tubule formation by various non-endothelial cell types, suggesting that tubule formation by endothelial cells may not represent true differentiation of this cell type. In summary, the morphology of tubules in the Co-culture assay appears more representative of capillary formation in vivo, than the endothelial cell changes that occur in either form of Matrigel assay. This revised version was published online in June 2006 with corrections to the Cover Date.     

A bone-derived mixture of TGFβ-superfamily members forms a more mature vascular network than bFGF or TGF-β2 in vivo

Clinical trials of therapeutic angiogenesis for the treatment of cardiovascular ischemia have failed to meet the expectations with the use of single growth factors, namely VEGF and bFGF. We show here that a bovine bone-derived growth factor mixture (GFM) of TGFβs, BMPs, and no more than 0.1% aFGF can initiate a dose-dependent angiogenic response in subcutaneously implanted Growth Factor Reduced Matrigel plugs that includes abundant smooth muscle actin positive (SMA+) tubes and functional CD31+, red blood cell filled, capillaries. Tube forming activity of the single factors, recombinant bFGF and bone-derived TGF-β2, were comparable to GFM, but only the bone-derived factors were able to create a larger fraction of SMA+ tubes than Matrigel alone at an equal dose. Basic FGF formed a greater number of RBC-filled capillaries within the plugs than GFM or TGF-β2 at the highest doses, although GFM created RBC-filled capillaries that penetrated deeper into the plugs than bFGF. However, bFGF showed the greatest number of non-cell-lined, RBC-filled pools, suggestive of vessel rupture, and the largest number of plugs showing signs of fluid accumulation in the form of large, cell-lined clefts in the implants. TGF-β2 showed less RBC-filled pools, but a significant number of implants with signs of fluid accumulation. At high doses of GFM penetration by blood vessels and mesenchymal cells was obstructed by cartilage development within the plugs accompanied by a prominent band of SMA+ granulation tissue with abundant RBC-filled capillaries encapsulating the implants. Thus, GFM is also capable of dramatically remodeling the vascular system in the interstitial space surrounding the plug. These results show that GFM is capable of inducing the formation of a more mature vascular system than that formed by the single factors bFGF and TGFβ-2. Natural mixtures of TGFβs, BMPs, and FGFs may have superior clinical utility in therapeutic angiogenesis applications.     

A bone-derived mixture of TGF beta-superfamily members forms a more mature vascular network than bFGF or TGF-beta 2 in vivo

Clinical trials of therapeutic angiogenesis for the treatment of cardiovascular ischemia have failed to meet the expectations with the use of single growth factors, namely VEGF and bFGF. We show here that a bovine bone-derived growth factor mixture (GFM) of TGFbetas, BMPs, and no more than 0.1% aFGF can initiate a dose-dependent angiogenic response in subcutaneously implanted Growth Factor Reduced Matrigel plugs that includes abundant smooth muscle actin positive (SMA+) tubes and functional CD31+, red blood cell filled, capillaries. Tube forming activity of the single factors, recombinant bFGF and bone-derived TGF-beta2, were comparable to GFM, but only the bone-derived factors were able to create a larger fraction of SMA+ tubes than Matrigel alone at an equal dose. Basic FGF formed a greater number of RBC-filled capillaries within the plugs than GFM or TGF-beta2 at the highest doses, although GFM created RBC-filled capillaries that penetrated deeper into the plugs than bFGF. However, bFGF showed the greatest number of non-cell-lined, RBC-filled pools, suggestive of vessel rupture, and the largest number of plugs showing signs of fluid accumulation in the form of large, cell-lined clefts in the implants. TGF-beta2 showed less RBC-filled pools, but a significant number of implants with signs of fluid accumulation. At high doses of GFM penetration by blood vessels and mesenchymal cells was obstructed by cartilage development within the plugs accompanied by a prominent band of SMA+ granulation tissue with abundant RBC-filled capillaries encapsulating the implants. Thus, GFM is also capable of dramatically remodeling the vascular system in the interstitial space surrounding the plug. These results show that GFM is capable of inducing the formation of a more mature vascular system than that formed by the single factors bFGF and TGFbeta-2. Natural mixtures of TGFbetas, BMPs, and FGFs may have superior clinical utility in therapeutic angiogenesis applications.     

A study of metabolites as intermediate effectors in angiogenesis

Metabolites released from hypoxic tissues have been reported as angiogenic factors in circumstances of reduced tissue oxygenation or an increased rate of metabolism. However, in more recent reports their possible role in angiogenesis prior to the induction of hypoxia-inducible genes appears to have been neglected. In a systematic attempt to evaluate their role, metabolites common to the glycolytic and oxidative pathways (nicotinamide adenine dinucleotide, adenosine diphosphate and adenosine triphosphate), exclusively glycolytic metabolites (pyruvate and lactic acid) and exclusively oxidative metabolites (malate, succinate, fumarate and citrate) were tested to assess their effects upon in vivo angiogenesis and in vitro endothelial cell migration and proliferation. In addition, adenosine was tested due to its proposed role in hypoxia-induced angiogenesis. The angiogenic effects in vivo were examined using the chick chorioallantoic membrane assay and in vitro on chick embryonic capillary endothelial cells using a phagokinetic track/migration assay and crystal violet dye binding/proliferation assay. Metabolites common to the glycolytic and oxidative metabolic pathways and exclusively glycolytic metabolites produced an angiogenic response in vivo and in vitro on endothelial cell proliferation and migration, whereas exclusively oxidative metabolites, with the exception of malate, did not. Adenosine caused an increased proliferation of blood vessels in vivo and stimulated endothelial cell migration and proliferation. Overall, these results implicate metabolites as effectors in angiogenesis and it is proposed that they have a role which is possibly independent of the upregulation of hypoxia-inducible genes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Continuous endothelial cell activation increases angiogenesis: evidence for the direct role of endothelium linking angiogenesis and inflammation

There is increasing evidence that chronic inflammation is tightly linked to diseases associated with endothelial dysfunction, including the induction of aberrant angiogenesis. While leukocytes have been described as mediators of inflammation-associated angiogenesis, the effects of direct chronic endothelial activation have not been addressed in this context. Using an uncleavable mutant of the transmembrane form of tumor necrosis factor-alpha (TNF-alpha), we have established models of stable TNF-alpha expression in endothelial cells in vitro and in transgenic mice in vivo. In the in vitro model, continuous endothelial activation leads to increased leukocyte cellular adhesion molecule expression and intracellular reactive oxygen species, hallmarks of a proinflammatory and dysfunctional endothelium. In addition, stable expression of TNF-alpha in endothelial cells increased angiogenic sprout formation in the presence but also in the absence of angiogenic growth factors. The partial neutralization of this effect by TNF-alpha antibodies and the inability of conditioned media from stable TNF-alpha-expressing endothelial cells to induce angiogenic activities in control endothelial cells suggest that this effect does not require expression of additional autocrine factors, but is an autonomous effect of the transmembrane TNF on the endothelial cells. Furthermore, using the Matrigel plug assay in vivo, increased angiogenesis was observed in endothelial TNF-alpha-expressing transgenic versus control mice. In conclusion, chronic inflammatory changes mediated by TNF-alpha can induce angiogenesis in vitro and in vivo, suggesting endothelial cell activation as a direct link between inflammation and angiogenesis.     

The adaptor protein Shc integrates growth factor and ECM signaling during postnatal angiogenesis

Angiogenesis requires integration of cues from growth factors, extracellular matrix (ECM) proteins, and their receptors in endothelial cells. In the present study, we show that the adaptor protein Shc is required for angiogenesis in zebrafish, mice, and cell-culture models. Shc knockdown zebrafish embryos show defects in intersegmental vessel sprouting in the trunk. Shc flox/flox; Tie2-Cre mice display reduced angiogenesis in the retinal neovascularization model and in response to VEGF in the Matrigel plug assay in vivo. Functional studies reveal a model in which Shc is required for integrin-mediated spreading and migration specifically on fibronectin, as well as endothelial cell survival in response to VEGF. Mechanistically, Shc is required for activation of the Akt pathway downstream of both integrin and VEGF signaling, as well as for integration of signals from these 2 receptors when cells are grown on fibronectin. Therefore, we have identified a unique mechanism in which signals from 2 critical angiogenic signaling axes, integrins and VEGFR-2, converge at Shc to regulate postnatal angiogenesis.     

An ex vivo angiogenesis assay utilizing commercial porcine carotidartery: Modification of the rat aortic ring assay

The study of angiogenesis as a therapeutic target requires a reliable, physiologically relevant, and technically straightforward assay. An ex vivo assay bridges the gap between cell-based assays, which may not realistically represent the complex process of vessel sprouting, and in vivo assays, which are time consuming and expensive. Porcine carotid arteries provide an ideal tissue source for angiogenesis inhibitor screens due to their availability, physiological relevance and large size. 1.5 mm² fragments of porcine carotid arteries were incubated in 48-well culture plates and sandwiched between two 100 μl layers of Matrigel. Sprouting was observed from the explants and quantitated, using a digital imaging system, after two weeks of incubation. Histological analysis using Factor VIII-related antigen (von Willebrand Factor) as an endothelial cell-specific marker identified these sprouts, which were consistent with endothelial cell morphology, supporting the system as a model of angiogenesis. Accordingly, the angiogenesis inhibitors suramin, 2-methoxyestradiol, and the matrix metalloprotease inhibitor Batimastat were shown to completely inhibit sprouting at 50, 0.5, and 5.0 μg/ml, respectively and to have ED₅₀ values of 23, 0.15, and 0.14 μg/ml. This assay shows good reproducibility and eliminates animal to animal variation. The system should prove adaptable to other forms of angiogenic stimulation, ultimately making a variety of assays for angiogenesis available to laboratories of limited resources. This revised version was published online in June 2006 with corrections to the Cover Date.     

Histologic evidence that basic fibroblast growth factor enhances the angiogenic effects of transmyocardial laser revascularization

Objectives. To determine whether addition of basic fibroblast growth factor (bFGF), an angiogenic growth factor, enhances the angiogenic effects of transmyocardial laser revascularization (TMR). Background. TMR is an investigational therapy for treating patients with medically refractory angina not amenable to traditional therapies. Histologic and blood flow studies in animals have suggested that TMR enhances angiogenesis above that normally seen in ischemic myocardium. We tested the hypothesis that bFGF administered into TMR channels further enhance the angiogenic effects of TMR. Methods. Chronic ischemia was created in 3 groups of dogs using an ameroid constrictor on the proximal LAD. In the bFGF group (n = 5) non-transmyocardial channels were created in the LAD territory and bFGF, (100 ng/ml) dissolved in pluronic gel was injected into the each channel. In the TMR group (n = 7), transmyocardial channels were created without bFGF. A control group (n = 7) had ischemia without TMR of bFGF. 5-bromo-2'-deoxyuridine (BrdU) was administered to mark proliferating cells. After 8 weeks survival, colored microspheres were injected to assess the regional myocardial blood flow. Results. TMR and TMR+bFGF increased total vascular density by ∼40% over that observed in the control group. However, the number of large vessels (internal diameter ≥ 50 μm) was doubled by the addition of bFGF, and this correlated with a 50% increase in the density of proliferating vascular cells and a tripling of the total estimated vascular cross sectional area. Blood flow to the LAD territory was increased by TMR compared to controls, with no further benefit observed in the bFGF group. Conclusions. On a histologic basis, basic fibroblast growth factor further enhances angiogenesis following TMR in ischemic myocardium mainly by increased the size but not the total number of vessels. Received: 18 June 1998, Returned for revision: 10 September 1998, Revision received: 2 August 1999, Accepted: 11 August 1999     

Utilizing Zebrafish to Identify Anti‐(Lymph)Angiogenic Compounds for Cancer Treatment: Promise and Future Challenges

Cancer metastasis which predominantly occurs through blood and lymphatic vessels, is the leading cause of death in cancer patients. Consequently, several anti‐angiogenic agents have been approved as therapeutic agents for human cancers such as metastatic renal cell carcinoma. Also, anti‐lymphangiogenic drugs such as monoclonal antibodies VGX‐100 and IMC‐3C5 have undergone phase I clinical trials for advanced and metastatic solid tumors. Although anti‐tumor‐associated angiogenesis has proven to be a promising therapeutic strategy for human cancers, this approach is fraught with toxicities and development of drug resistance. This emphasizes the need for alternative anti‐(lymph)angiogenic drugs. The use of zebrafish has become accepted as an established model for high‐throughput screening, vascular biology, and cancer research. Importantly, various zebrafish transgenic lines have now been generated that can readily discriminate different vascular compartments. This now enables detailed in vivo studies that are relevant to both human physiological and tumor (lymph)angiogenesis to be conducted in zebrafish. This review highlights recent advancements in the zebrafish anti‐vascular screening platform and showcases promising new anti‐(lymph)angiogenic compounds that have been derived from this model. In addition, this review discusses the promises and challenges of the zebrafish model in the context of anti‐(lymph)angiogenic compound discovery for cancer treatment.