Partial degradation products of sodium hyaluronate produced by the action of testicular hyaluronidase induced an angiogenic response (formation of new blood vessels) on the chick chorioallantoic membrane. Neither macromolecular hyaluronate nor exhaustively digested material had any angiogenic potential. Fractionation of the digestion products established that the activity was restricted to hyaluronate fragments between 4 and 25 disaccharides in length.

Background and Purpose Stroke is one of the most common causes of mortality and morbidity in the Western world. It results from the occlusion of a cerebral artery followed by severe disturbances in blood supply through microvessels to brain tissue. Despite an extensive literature its pathophysiology is poorly understood, and this has severely impeded the logical development of therapy.Methods Brains were obtained from 10 patients aged 46 to 85 years with survival times of 5 to 92 days after their stroke. Infarcted areas and representative control tissues from the contralateral uninvolved brain hemisphere were collected. Microvessel density was measured microscopically. A total of 6520 microvessels were scored in 10 801 areas. The level of activation of the endothelial cells was studied by immunohistochemistry using three monoclonal antibodies, viz, E-9, raised against activated endothelial cells; IG11, recognizing vascular cell adhesion molecule-1; and anti-proliferating cell nuclear antigen. Angiogenic activity in tissue extracts was examined using an in vivo chicken chorioallantoic membrane assay.Results There was a statistically significant increase in the number of microvessels (Wilcoxon log-rank test;Ps.01) in 9 of

Angiogenesis is the propelling force for tumor growth and metastasis, and antiangiogenic therapy represents one of the most promising modalities for cancer treatment. CD105 (endoglin) is a proliferation-associated and hypoxia-inducible protein abundantly expressed in angiogenic endothelial cells (EC). It is a receptor for transforming growth factor (TGF) -beta1 and -beta3 and modulates TGF-beta signaling by interacting with TGF-beta receptors I and/or II. Immunohistochemistry studies have revealed that CD105 is strongly expressed in blood vessels of tumor tissues. Intratumoral microvessel density (MVD) determined using antibodies to CD105 has been found to be an independent prognostic indicator, wherein increased MVD correlates with shorter survival. CD105 is able to be shed into the circulation, with elevated levels detected in patients with various types of cancer and positively correlated with tumor metastasis. Tangible evidence of its proangiogenic role comes from knockout studies in which CD105 null mice die in utero as a result of impaired angiogenesis in the yolk sac and heart defects. The potential usefulness of CD105 for tumor imaging has been evaluated in tumor-bearing mice and dogs that have shown the rapid accumulation of radiolabeled anti-CD105 monoclonal antibody in the tumors with a high tumor-to-background ratio. The anti-CD105 antibody conjugated with immunotoxins and immunoradioisotopes efficiently suppressed/abrogated tumor growth in murine models bearing breast and colon carcinoma without any significant systemic side effects. Immunoscintigraphy in patients with renal cell carcinomas has shown specific localization of 99Tcm-labeled CD105 mab in tumor endothelial cells. Thus, CD105 is a promising vascular target that can be used for tumor imaging, prognosis, and bears therapeutic potential in patients with solid tumors and other angiogenic diseases.

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Hyaluronan (HA) is a large nonsulfated glycosaminoglycan and an important regulator of angiogenesis, in particular, the growth and migration of vascular endothelial cells. We have identified some of the key intermediates responsible for induction of mitogenesis and wound recovery. Treatment of bovine aortic endothelial cells with oligosaccharides of hyaluronan (o-HA) resulted in rapid tyrosine phosphorylation and plasma membrane translocation of phospholipase C␥1 (PLC␥1). Cytoplasmic loading with inhibitory antibodies to PLC␥1, G␤, and G␣ i/o/t/z inhibited activation of extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the G␣ i/o inhibitor, pertussis toxin, reduced o-HA-induced PLC␥1 tyrosine phosphorylation, protein kinase C (PKC) ␣ and ␤1/2 membrane translocation, ERK1/2 activation, mitogenesis, and wound recovery, suggesting a mechanism for o-HA-induced angiogenesis through Gproteins, PLC␥1, and PKC. In particular, we demonstrated a possible role for PKC␣ in mitogenesis and PKC␤1/2 in wound recovery. Using antisense oligonucleotides and the Ras farnesylation inhibitor FTI-277, we showed that o-HA-induced bovine aortic endothelial cell proliferation, wound recovery, and ERK1/2 activation were also partially dependent on Ras activation, and that o-HA-stimulated tyrosine phosphorylation of the adapter protein Shc, as well as its association with Sos1. Binding of Src to Shc was required for its activation and for Ras-dependent activation of ERK1/2, cell proliferation, and wound recovery. Neither Src nor Ras activation was inhibited by pertussis toxin, suggesting that their activation was independent of heterotrimeric G-proteins. However, the specific Src kinase inhibitor PP2 inhibited G␤ subunit co-precipitation with PLC␥1, suggesting a possible role for Src in activation of PLC␥1 and interaction between two distinct o-HA-induced signaling pathways.

CD105 (endoglin), a receptor for transforming growth factor beta (TGFbeta), is highly expressed in tissue-cultured, activated endothelial cells in vitro and in tissues undergoing angiogenesis in vivo. The absence of CD105 in knockout mice leads to their death from defective vascular development, but the role of CD105 in the modulation of angiogenesis has not been elucidated. TGFbeta1 is a well-recognized regulator of angiogenesis. Using an antisense approach, we have shown that inhibition of CD105 protein translation in cultured human endothelial cells enhances the ability of TGFbeta1 to suppress growth and migration in these cells. The ability of endothelial cells to form capillary tubes was evaluated by the use of a 3-dimensional collagen matrix system where TGFbeta1 not only reduced the length of capillary-like structures, but also caused massive mortality in CD105-deficient cells compared to control cultures. These results provide direct evidence that CD105 antagonizes the inhibitory effects of TGFbeta1 on human vascular endothelial cells and that normal cellular levels of CD105 are required for the formation of new blood vessels.

Tumour growth and metastasis are totally dependant upon neovascularization. The target cell for tumour neovascularization is the blood-vessel endothelial cell, and specific angiogenic molecules produced or induced by the tumour are believed to initiate the process. In this report, we review one of these angiogenic molecules, the glycosaminoglycan hyaluronan (HA), which appears to have differing roles in neovascularization depending on its molecular mass. High-molecular-mass HA is anti-angiogenic whereas oligosaccharides of HA, of specific size, actively stimulate endothelial-cell proliferation and migration, 2 of the key events associated with neovascularization, and induce angiogenesis in vivo. We provide details of the action of HA oligosaccharides on endothelial cells, from binding to cell-surface receptors, through activation of signal transduction pathways and gene expression to protein synthesis, cell proliferation and cell migration. We also suggest a model to account for HA of differing molecular mass being present, at different locations, within a single tumour and how this HA aids both general tumour growth and tumour metastasis.

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