Vessel Sprouts Research Articles

Eph receptor tyrosine kinases in angiogenesis: from development to disease.

Angiogenesis, the process by which new blood vessels sprout and branch from existing vasculature, is crucial for vascular remodeling during embryogenesis and in normal tissue homeostasis, such as in the female reproductive tract. Angiogenesis can also contribute to the pathogenesis of diseases such as cancer and retinopathy. The Eph family of receptor tyrosine kinases and their ligands, called ephrins, has emerged as critical regulators of vascular remodeling in the embryo. More recently, these molecules have been associated with post-natal angiogenic remodeling and tumor neovascularization. This review provides an overview of recent advances in our understanding of Eph/ephrins in angiogenesis, with an emphasis on development and disease, and the potential for targeting these molecules in anti-angiogenic therapy.

An Introduction to Vascular Biology: From Basic Science to Clinical Practice

The revised and updated second edition of An Introduction to Vascular Biology is broader in scope than its predecessor and more clinically oriented. The extent to which vascular biology and pathophysiology impacts upon clinical medicine is perfectly illustrated by the varied departmental affiliations of the four editors—haematology, rheumatology, obstetrics and vascular surgery. The book includes nineteen short, digestible chapters grouped under basic science, pathophysiology and clinical practice. Contributors include respected names such as Peter Weissberg, Patrick Vallance, Caroline Savage, Allun Hughes and John Tooke. Each chapter has a list of useful up-to-date references, but the illustrations would have benefited from a splash of colour (especially the clinical photographs and histological sections). The basic science section is comprehensive, with useful introductory chapters on topical subjects such as angiogenesis, vascular cell apoptosis, and the biology of wound healing. The whole area of angiogenesis is particularly relevant clinically. Sprouting of new blood vessels from existing vascular structures plays an important part in collateral vessel formation, tumour growth and tissue remodelling and regeneration. This chapter describes the regulation of endothelial cell migration and proliferation, with connective tissue expansion and the gradual creation of new vascular architecture. Numerous growth factors stimulate angiogenesis while other anti-angiogenic molecules inhibit new vessel formation. The basic science is outlined in a clinical context with reference to conditions in which therapeutic manipulation of angiogenic pathways has potential benefits. This might be achieved, for example, by gene therapy whereby DNA for a powerful angiogenic growth factor, VEGF, is administered locally in the leg or the heart to enhance new vessel formation in patients with critical ischaemia. Conversely, there is a rationale for inhibition of angiogenesis as a way to block tumour growth in primary or metastatic disease. The chapter on angiogenesis will appeal to many different clinical and research specialties. The section on pathophysiology also includes topical updates in fields that are rapidly changing—for example, the status of candidate genes for hypertension, the many aspects of endothelial dysfunction, and the growing importance of nitric oxide in health and disease. Imaging of the cardiovascular system has undergone enormous change over the past ten years, particularly with techniques such as spiral CT and MR. Alan Moody (radiologist) has written a chapter illustrating the versatility of these techniques not simply in arterial disease but also in assessing patients with venous disease and venous thrombosis. CT and MR angiography are becoming especially useful in the assessment of patients with carotid and renovascular disease. It is a pity that the images have not reproduced well, but in other respects the chapter is excellent. The third section becomes more focused on therapeutic strategies for improving symptoms and prognosis in patients with assorted vascular conditions, including the cardiovascular complications of diabetes, aortic aneurysms, pulmonary hypertension, and the different forms of vasculitis. Each of these chapters is well illustrated with simple, practical guides to diagnosis and treatment based on a clear understanding of the pathophysiological processes. Beverley Hunt and her co-editors have succeeded in putting together a collection of varied chapters in which the underlying structural and functional abnormalities in blood vessels are described scientifically in the context of clinical disorders. Individual chapters are easy to read and should provide helpful guidance to clinicians and those interested in the pathophysiological basis of vascular medicine.

Inhibition of angiogenic initiation and disruption of newly established human vascular networks by juice from Morinda citrifolia (noni).

noni, the juice of the fruit from the Morinda citrifolia plant, has been used for centuries as a medicinal agent. We tested the effects of noni juice in a three-dimensional fibrin clot matrix model using human placental vein and human breast tumor explants as sources for angiogenic vessel development. Noni in concentrations of 5% (vol/vol) or greater was highly effective in inhibiting the initiation of new vessel sprouts from placental vein explants, compared with initiation in control explants in media supplemented with an equivalent amount of saline. These concentrations of noni were also effective in reducing the growth rate and proliferation of newly developing capillary sprouts. When used at a concentration of 10% in growth media, noni was able to induce vessel degeneration and apoptosis in wells with established capillary networks within a few days of its application. We also found that 10% noni juice in media was an effective inhibitor of capillary initiation in explants from human breast tumors. In tumor explants which did show capillary sprouting, the vessels rapidly degenerated (2-3 days) in those exposed to media supplemented with 10% noni.

Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis.

Neuropilins (NP1 and NP2) are vascular endothelial growth factor (VEGF) receptors that mediate developmental and tumor angiogenesis. Transgenic mice, in which both NP1 and NP2 were targeted (NP1(-/-)NP2(-/-)) died in utero at E8.5. Their yolk sacs were totally avascular. Mice deficient for NP2 but heterozygous for NP1 (NP1(+/-)NP2(-/-)) or deficient for NP1 but heterozygous for NP2 (NP1(-/-)NP2(+/-)) were also embryonic lethal and survived to E10-E10.5. The E10 yolk sacs and embryos were easier to analyze for vascular phenotype than the fragile poorly formed 8.5 embryos. The vascular phenotypes of these E10 mice were very abnormal. The yolk sacs, although of normal size, lacked the larger collecting vessels and had less dense capillary networks. PECAM staining of yolk sac endothelial cells showed the absence of branching arteries and veins, the absence of a capillary bed, and the presence of large avascular spaces between the blood vessels. The embryos displayed blood vessels heterogeneous in size, large avascular regions in the head and trunk, and blood vessel sprouts that were unconnected. The embryos were about 50% the length of wild-type mice and had multiple hemorrhages. These double NP1/NP2 knockout mice had a more severe abnormal vascular phenotype than either NP1 or NP2 single knockouts. Their abnormal vascular phenotype resembled those of VEGF and VEGFR-2 knockouts. These results suggest that NRPs are early genes in embryonic vessel development and that both NP1 and NP2 are required.

Patterning of angiogenesis in the zebrafish embryo.

Little is known about how vascular patterns are generated in the embryo. The vasculature of the zebrafish trunk has an extremely regular pattern. One intersegmental vessel (ISV) sprouts from the aorta, runs between each pair of somites, and connects to the dorsal longitudinal anastomotic vessel (DLAV). We now define the cellular origins, migratory paths and cell fates that generate these metameric vessels of the trunk. Additionally, by a genetic screen we define one gene, out of bounds (obd), that constrains this angiogenic growth to a specific path. We have performed lineage analysis, using laser activation of a caged dye and mosaic construction to determine the origin of cells that constitute the ISV. Individual angioblasts destined for the ISVs arise from the lateral posterior mesoderm (LPM), and migrate to the dorsal aorta, from where they migrate between somites to their final position in the ISVs and dorsal longitudinal anastomotic vessel (DLAV). Cells of each ISV leave the aorta only between the ventral regions of two adjacent somites, and migrate dorsally to assume one of three ISV cell fates. Most dorsal is a T-shaped cell, based in the DLAV and branching ventrally; the second constitutes a connecting cell; and the third an inverted T-shaped cell, based in the aorta and branching dorsally. The ISV remains between somites during its ventral course, but changes to run mid-somite dorsally. This suggests that the pattern of ISV growth ventrally and dorsally is guided by different cues. We have also performed an ENU mutagenesis screen of 750 mutagenized genomes and identified one mutation, obd that disrupts this pattern. In obd mutant embryos, ISVs sprout precociously at abnormal sites and migrate anomalously in the vicinity of ventral somite. The dorsal extent of the ISV is less perturbed. Precocious sprouting can be inhibited in a VEGF morphant, but the anomalous site of origin of obd ISVs remains. In mosaic embryos, obd somite causes adjacent wild-type endothelial cells to assume the anomalous ISV pattern of obd embryos. Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.

VEGFR-3 in adult angiogenesis.

Vascular endothelial growth factor receptor 3 (VEGFR-3, Flt-4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and may play a role in lymphangiogenesis. In embryonic life, VEGFR-3 is essential for blood vessel development. The purpose of this study was to investigate whether VEGFR-3 is also involved in blood vessel angiogenesis in the adult. This was studied in human tissues showing angiogenesis and in a model of VEGF-A-induced iris neovascularization in the monkey eye, by the use of immunohistochemistry at the light and electron microscopic level. VEGFR-3 was expressed on endothelium of proliferating blood vessels in tumours. In granulation tissue, staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis in granulation tissue formation. At the ultrastructural level, VEGFR-3 was localized in the cytoplasm and on the cell membrane of endothelial cells of sprouting blood vessels and sprouting lymphatics. In monkey eyes injected with VEGF-A, blood vessel sprouts on the anterior iris surface and pre-existing blood vessels in the iris expressed VEGFR-3. In conclusion, these results support a role for VEGFR-3 and its ligands VEGF-C and/or VEGF-D in cell-to-cell signalling in adult blood vessel angiogenesis. The expression of VEGFR-3 in VEGF-A-induced iris neovascularization and in pre-existing blood vessels exposed to VEGF-A suggests that this receptor and possibly its ligands are recruited in VEGF-A-driven angiogenesis.

Alteration of the vascular supply in the rabbit ciliary body by transscleral diode laser cyclophotocoagulation.

Besides the direct destruction of the non-pigmented ciliary epithelium by cyclodestructive procedures, further mechanisms are responsible for the decrease of intraocular pressure. This study evaluates the alteration of the ciliary body vascularization by contact transscleral diode laser cyclophotocoagulation in rabbit eyes. Pigmented chinchilla bastard rabbits were used. Preliminary experiments were conducted to determine the parameters for diode laser cyclophotocoagulation of the pars plana or pars plicata. Then, treatment of the pars plicata (three rabbits) or pars plana (three rabbits) was performed in the right eye of six rabbits. After 2, 6 and 12 weeks histologic and transmission electron microscopic studies were performed. Furthermore, three rabbits received pars plicata cyclophotocoagulation of the right and pars plana cyclophotocoagulation of the left eye. After 2, 6 and 12 weeks, vascular casts of the ciliary body were investigated by scanning electron microscopy. Histologic and transmission electron microscopic studies showed a marked coagulation necrosis with subsequent ciliary body atrophy, destruction of the ciliary epithelium, pigment dispersion in the ciliary body stroma and peripheral anterior synechiae. Examination of vascular casts of the ciliary body revealed a marked rarefication of the capillary network within the treated areas of the ciliary body in all eyes and at every time of investigation. Anterior to the laser burns the capillary network was not markedly affected in the eyes with cyclophotocoagulation of the pars plana. After 3 months short vessel sprouts were seen, but regeneration was mostly incomplete. The vascular casting technique is an excellent method for the investigation of changes in ciliary body vascularization after cyclodestruction. This study is the first to demonstrate a marked rarefication of the ciliary body vascularization after diode laser cyclophotocoagulation using vascular casts. The results suggest that alteration of vascularization probably acts as a strong synergistic mechanism in the decrease of intraocular pressure after cyclophotocoagulation of the pars plicata.

Inhibition of tumor growth and microvascular angiogenesis by the potent angiogenesis inhibitor, TNP-470, in rats.

The antiangiogenic effects of TNP-470 on the neovascularization of tumors were studied by examining ultrastructural alterations in the vasculature and interstitial fluid pressure (IFP) of tumors. Wistar rats were first inoculated subcutaneously (s.c.) with the Walker 256 carcinosarcoma cell line, then either vehicle medium or TNP-470, 30 mg/kg, was injected s.c. on day 1. A tumor growth assay, the necrotic area, and the IFP in the tumor were all measured on day 12. The antiangiogenic effects of TNP-470 were studied by scanning electron microscopic images of tumor vascular casts. TNP-470 was observed to inhibit tumor growth and increase the necrotic area significantly. In the TNP-470-treated group, the IFP in the superficial layer, defined as 2-3 mm from the tumor capsule, and in the deep layer, defined as 8-10 mm from the tumor capsule, were significantly higher than the corresponding values in the control. Moreover, vascular casts showed a significant reduction in the budding of sprouts in the superficial layer, and a decrease in the maximum diameter of the tumor vessels in the deep layer. It is possible that the higher IFP in the TNP-470-treated tumors might have prevented tumor vessel dilation. The findings of this study demonstrated that TNP-470 inhibited the budding of tumor vessel sprouts, and increased the IFP. These processes seem to act synergistically to suppress tumor angiogenesis.

Plasma membrane-dependent activation of gelatinase A in human vascular endothelial cells.

The initiation of the angiogenic process requires a locally confined and time-limited proteolysis of the basement membrane (BM) components at the site of new vessel sprout. Gelatinase A, a member of the matrix metalloproteinase family, degrades BM type IV collagen and is involved in the BM breakdown by migrating tumor cells and endothelial cells (EC). Gelatinase A is synthesized as latent proenzyme and must be activated in order to express its proteolytic activity. A plasma membrane-dependent mechanism of activation has been described for several tumor and transformed cells lines. In the present study, we show that latent (72 kD) and mature (62-59 kD) forms of gelatinase A are present in EC membrane fraction from Triton X-114 extract while only latent form is found in the cytosolic fraction. The incubation of EC membrane fraction with exogenous latent gelatinase A resulted in a significant activation giving rise to 62-59 kD mature forms. 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong potentiator of angiogenesis in vitro and in vivo, increases the amount of both latent and activated forms of gelatinase A in EC membrane fraction as well as the ability of this latter fraction to activate exogenous latent gelatinase A. We show that the mRNA transcript coding for the membrane-integrated MMP, the MT-MMP, previously described as a potential gelatinase A activator in invasive tumor cells is also expressed in vascular EC and is regulated through a TPA sensitive process. This enzyme may be responsible for membrane-dependent gelatinase A activation in normal vascular EC and may therefore be a determinant in the control of BM proteolysis during angiogenesis.

Revascularization of skeletal muscle transplanted into the hamster cheek pouch: Intravital and light microscopy

Small fiber bundles from the extensor digitorum longus (EDL) muscle were transplanted into the hamster cheek pouch to observe the process of revascularization. We tested the hypothesis that blood vessels of a graft degenerate and that revascularization results from the ingrowth of blood vessels from the cheek pouch into the graft. Degeneration and regeneration of muscle fibers followed a pattern similar to that reported for autografts of whole muscles in rats. Blood vessels in the graft that were adjacent to vascularized host tissue survived. At 1 day, surviving blood vessels in the graft had normal structure but erythrocytes were packed tightly in the lumen. At 2 days, sprouts from surviving graft vessels had grown into the cheek pouch. Between 2 and 2.5 days, anastomoses of blood vessels from the graft with those of the cheek pouch reestablished circulation. After circulation was established, the blood vessels contained well-spaced erythrocytes. By 3 days, blood vessels which increased in number and diameter throughout the graft occupied 40–60% of the graft. Over the next 24 hr, blood vessels in the graft regressed toward the control value. We conclude that blood vessels in muscle grafts in the cheek pouch survive transplantation, and that circulation in grafts is reestablished by the anastomoses of blood vessel sprouts from the graft with the blood vessels of the host.

Development of the vascular system in the hamster malignant neurilemmoma

The development of the vasculature in the hamster malignant neurilemmoma was studied during growth in the transparent cheek pouch chamber. Prominent features in this process are: (1) Progressive dilatation, tortuosity and some bulbous formations in host venules in the immediate vicinity of the tumor implant. Occasional focal dilatation of some host arterioles. (2) Small focal erythrocytic extravasations. (3) Sprouting of host venous vessels in the perimeter of the implant and subsequent anastomosing of these sprouts and/or their branches to form loops directed toward the center of the implant. Continued sprouting, in effect, produces remodeling of the host venules. (4) Establishment of the tumor capillary net in 4–7 days. The bed assumes a dense net-like arrangement of short, profusely anastomosing, thin-walled endothelial tubes which conduct blood at a brisk rate. (5) Progressive increase in capillary caliber during tumor growth. (6) Prominent focal or general reduction in blood flow after about 10 days of growth. This appears related to the development of tissue growth pressure within the rigid cheek pouch chamber.

KINETICS OF WOUND HEALING IN RABBIT EAR CHAMBERS, A TIME LAPSE CINEMICROSCOPIC STUDY.

Wound healing in rabbit ear chambers was studied by time lapse cinemicroscopy. Endothelial cells for the growth of blood vessel sprouts arose in a zone proximal to the growing fringe both by the mitotic division of existing endothelium and by the conservation for re‐use of the endothelium of resorbing blood vessels. The migration of endothelial cells distally along the luminal surfaces of the walls of circulating blood vessels, into the proximal ends of developing blood vessel sprouts, and along the mid part and finally into the tips of blood vessel sprouts was demonstrated. New lumen of sprouts was produced by the formation, enlargement and coalescence of loculated spaces between apposed layers of endothelium. This process was considered to reflect secretory activity of the endothelial cells. Anastomosis between sprouts involved similar lumen formation and the spatial rearrangement of endothelial cells.Evidence supporting the conversion of fibroblasts into smooth muscle cells was obtained. Growth of lymphatic sprouts was also studied. The virtual absence of locomotion by macrophages during wound healing was confirmed and a possible explanation proposed. Mathematical analysis of the data obtained from film analysis revealed that the curves for blood vessel endothelial migration and sprout growth and lymphatic sprout growth were all derivable from the same general pseudo generating mathematical function.

Observations on healing tissue: A combined light and electron microscopic investigation

1. The process of healing in the rabbit ear chamber has been investigated in detail by correlating light microscopy, mainlyin vivo, and electron microscopy. 2. During healing new vessels are formed from existing vessels by a process of sprouting and anastomosis, with subsequent remodelling of the loops so formed. 3. The fundamental process in the formation of vessels by sprouting is the mitotic division of existing endothelium, during which it retains its characteristic properties. 4. Blood vessel sprouts are composed of strands of tightly apposed cells formed in continuity with the walls of existing vessels. The subsequent canalization of such strands takes place extra-cellularly by a series of events largely as described by Billroth (1856). 5. The endothelium of recently formed vessels has a fine structure which distinguishes it clearly from that of more mature vessels. Certain features of this structure are compatible with a secretory activity by the endothelium during the formation of new vessels. 6. Evidence was obtained that in the course of differentiation of recently formed vessels fibroblast-like cells are incorporated into vessel walls to become adventitial cells, and that adventitial cells may undergo conversion to vascular smooth muscle cells. 7. Lymphatic endothelium exhibits properties during regeneration that confirm the specificity of this form of endothelium. 8. Cells with the characteristic fine structure of fibroblasts were frequently found in mitosis. The fibroblasts in the regions of active fibrogenesis had a highly developed cisternal form of endoplasmic reticulum. Vesicles and corresponding caveolae identifiable in such fibroblasts may provide a communication between the endoplasmic reticulum and the sites of fibrogenesis at the external surfaces of the cells. 9. Cells sharing characteristic features of fine structure formed a series which grouped together the monocyte, macrophage and foreign body giant cell. 10. Highly fibrillary intracytoplasmic tracts were found in both fibroblasts and macrophages. These tracts were equated with the fibroglial fibres of light microscopy. 11. ‘ Clear spaces ’ in advance of the growing fringe of blood vessels were temporary structures lined by a pavement of mesothelium-like cells. 12. No evidence was found of the formation of primitive mesenchymal tissue during healing in the mammal.