Shant Kumar

CD105 is important for angiogenesis: evidence and potential applications

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) ‐β1 and ‐β3 and modulates TGF‐β signaling by interacting with TGF‐β 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.—Duff, S. E., Li, C., Garland, J. M., Kumar, S. CD105 is important for angiogenesis: evidence and potential applications. FASEB J. 17, 984–992 (2003)

The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity

Hyaluronidase treatment of hyaluronic acid produced a series of oligosaccharides. Those between 3 and 16 disaccharides in length stimulated angiogenesis in vivo and the proliferation of tissue cultured endothelial cells in vitro. This effect appears to be cell type specific, as no stimulation of fibroblasts or smooth muscle cells was observed. Endothelial cells were found to endocytose both high- and low-molecular-mass hyaluronate, which might be receptor mediated. Fibroblasts and smooth muscle cells, cultured under the same conditions, showed negligible uptake of hyaluronate. Thus, the cell-specific effects may be due to the differences in internalization of hyaluronate. High-molecular-weight hyaluronate both inhibited endothelial cell proliferation and disrupted newly formed monolayers. These data are consistent with the ability of hyaluronate to inhibit new blood vessel formation in vivo and also suggest that hyaluronate metabolism plays a pivotal role in the regulation of angiogenesis.

A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells

A rapid and simple procedure for the direct solubilization of DNA from adherent cultured cells, or sedimented cells, has been developed and employed in the measurement of DNA in small numbers of endothelial cells (1 X 10(3).

Angiogenic Oligosaccharides of Hyaluronan Induce Multiple Signaling Pathways Affecting Vascular Endothelial Cell Mitogenic and Wound Healing Responses

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 Cγ1 (PLCγ1). Cytoplasmic loading with inhibitory antibodies to PLCγ1, Gβ, and Gαi/o/t/z inhibited activation of extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the Gαi/o inhibitor, pertussis toxin, reduced o-HA-induced PLCγ1 tyrosine phosphorylation, protein kinase C (PKC) α and β1/2 membrane translocation, ERK1/2 activation, mitogenesis, and wound recovery, suggesting a mechanism for o-HA-induced angiogenesis through G-proteins, PLCγ1, and PKC. In particular, we demonstrated a possible role for PKCα in mitogenesis and PKCβ1/2 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 Gβ subunit co-precipitation with PLCγ1, suggesting a possible role for Src in activation of PLCγ1 and interaction between two distinct o-HA-induced signaling pathways.

Increased Expression of TGF-β1 in Brain Tissue After Ischemic Stroke in Humans

Background and Purpose Occlusion in cerebral vessels results in ischemic stroke and is followed by proliferation of microvessels, ie, angiogenesis. The process is particularly marked in the border ...

Early‐response gene signalling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Inhibition by non‐angiogenic, high‐molecular‐weight hyaluronan

The degradation products of hyaluronan are known to stimulate endothelial‐cell proliferation and to promote neovascularization associated with angiogenesis, whilst native high‐molecular‐weight hyaluronan is inhibitory to these processes. To investigate the cellular signalling pathways coupled to hyaluronan‐induced responses in angiogenesis, we have analyzed early‐response gene expression in vitro, in cultured bovine aortic endothelial cells. Angiogenic oligosaccharides of hyaluronan induced rapid transient up‐regulation of the immediate early genes c‐fos, c‐jun, jun‐B, Krox‐20 and Krox‐24. In contrast, native hyaluronan when used alone failed to elicit a significant change in expression of any of the genes tested, and when used in combination with angiogenic oligosaccharides of hyaluronan, gave a dose‐dependent inhibition of induced gene expression. However, prior addition of angiogenic hyaluronan, as little as one minute before addition of high‐molecular‐weight hyaluronan, abrogated this inhibition, suggesting that positive or negative responses associated with hyaluronan signalling are integrated at a very early stage following receptor binding. Conversely, prior addition of high‐molecular‐weight hyaluronan led to an irreversible block in gene expression and proliferative response. These data are consistent with native hyaluronan antagonizing the angiogenic response in part by blocking a signalling cascade at or immediately following ligand‐receptor interaction. Finally, we demonstrated that chronic exposure to oligosaccharides of hyaluronan is essential for cell proliferation, indicating that short‐term immediate early‐gene signalling is insufficient to elicit the proliferation of endothelial cells. Int. J. Cancer 71:251–256, 1997.

CD105 antagonizes the inhibitory signaling of transforming growth factor beta1 on human vascular endothelial cells.

CD105 (endoglin), a receptor for transforming growth factor β (TGFβ), 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. TGFβ1isa 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 TGFβ1 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 TGFβ 1 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 TGFβ1 on human vascular endothelial cells and that normal cellular levels of CD105 are required for the formation of new blood vessels.—Li, C., Hampson, I. N., Hampson, L., Kumar, P., Bernabeu, C., Kumar, S. CD105 antagonizes the inhibitory signaling of transforming growth factor b1 on human vascular endothelial cells. FASEB J. 14, 55–64(1999)

Radiation-induced normal tissue injury: role of adhesion molecules in leukocyte-endothelial cell interactions.

The late onset of necrosis and fibrosis in normal tissues can be a serious consequence of radiotherapy in cancer patients. Because radiation‐induced vascular injury precedes the tissue damage, vascular injury is regarded as crucial in the pathogenesis of tissue damage. An understanding of the processes responsible is essential to develop strategies for the amelioration of radiation‐induced normal tissue damage. Leukocyte infiltration is commonly observed at sites of irradiation and is likely to lead to the acceleration and/or induction of parenchymal atrophy, fibrosis and necrosis in normal tissues following radiotherapy. The molecular mechanisms mediating leukocyte infiltration of tissues during inflammation have been studied extensively. It is now well established that cell adhesion molecules (CAMs) expressed on leukocytes and endothelial cells control the trafficking of leukocytes from the blood vessel lumen in these conditions. CAMs including E (endothelial), P (platelet) and L (leukocyte)‐selectins, intercellular adhesion molecule 1 (ICAM‐1), vascular cell adhesion molecule 1 (VCAM‐1), β1 and β2 integrins and CD31 are involved in the cascade of events resulting in rolling, arrest and transmigration of leukocytes through the inflamed endothelium. Whether a similar sequence of molecular events induces leukocyte sequestration in irradiated normal tissues is not known. This review is focussed on the role of CAMs in radiation‐induced leukocyte infiltration of normal tissues and the therapeutic implications of these findings. Int. J. Cancer 82:385–395, 1999.

CD105 prevents apoptosis in hypoxic endothelial cells

CD105, a marker of endothelial cells, is abundantly expressed in tissues undergoing angiogenesis and is a receptor for transforming growth factorβ. The pivotal role of CD105 in the vascular system was demonstrated by the severe vascular defects that occur in CD105-knockout mice, but the exact mechanisms for CD105 regulation of vascular development have not been fully elucidated. In light of the function of CD105 and the importance of hypoxia in neovascularisation, we speculated that CD105 is involved in hypoxia-initiated angiogenesis. Using tissue-cultured human microvascular endothelial cells, we have investigated the effects of hypoxic stress on CD105 gene expression. Hypoxia induced a significant increase in membrane-bound and secreted CD105 protein levels. CD105 mRNA and promoter activity were also markedly elevated, the latter returning to the basal level after 16 hours of hypoxic stress. Hypoxia induced cell cycle arrest at the G0/G1 phases and massive cell apoptosis after 24 hours through a reduction in the Bcl-2 to Bax ratio, downregulation of Bcl-XL and Mcl-1, and upregulation of caspase-3 and caspase-8. The consequence of CD105 upregulation was revealed using an antisense approach and a TUNEL assay. Suppression of CD105 increased cell apoptosis under hypoxic stress in the absence of TGFβ1. Furthermore, hypoxia and TGFβ1 synergistically induced apoptosis in the CD105-deficient cells but not in the control cells. We conclude that hypoxia is a potent stimulus for CD105 gene expression in vascular endothelial cells, which in turn attenuates cell apoptosis and thus contributes to angiogenesis.

Plasma levels of soluble CD105 correlate with metastasis in patients with breast cancer

CD105 (endoglin), a receptor for transforming growth factor (TGF) β1 and β3 in vascular endothelial cells, is highly up‐regulated in blood vessels of tissues where neovascularisation occurs. It modulates endothelial‐mesenchymal signalling and is essential for angiogenesis. Indeed, CD105 knock‐out mice die from malvascularisation by 11.5 day p.c. In the present study CD105, TGFβ1 and CD105/TGFβ1 complexes were quantified in plasma samples from 77 healthy individuals and 92 patients with early stage breast cancer prior to any treatment. When compared with normal controls, both CD105 and CD105/TGFβ1 complex levels were significantly elevated in breast cancer patients, whereas TGFβ1 levels were lower in cancer patients. The most important finding to emerge was that CD105 levels were significantly increased in patients who developed distant metastasis compared with disease‐free patients. While there was no significant difference between CD105 levels in controls compared to disease‐free patients, it was significantly higher in patients with metastatic disease. Thus patients who had died following local relapse or distant metastases possessed the highest levels of CD105. Neither CD105/TGFβ1 complex nor TGFβ1 levels correlated with tumour progression. Our data indicate that CD105 might be a valuable novel angiogenic marker for identifying breast cancer patients who are at high risk of developing metastasis. Int. J. Cancer (Pred. Oncol.) 89:122–126, 2000.