Patricia Kumar

Role of angiogenesis in patients with cerebral ischemic stroke.

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;P≤.01) in 9 of 10 infarcted brain tissues when compared with their contralateral normal hemisphere. In these patients the higher blood vessel counts correlated with longer survival, as ascertained by Spearmans p analysis (P<.02). The number of microvessels filled with blood cells was significantly lower in the infarcted hemispheres (P<.01). In contrast, statistically significant increased numbers of empty microvessels occurred in infarcted tissues compared with the contralateral hemisphere. Monoclonal antibody E-9 reacted weakly with normal-brain vascular endothelial cells; anti-proliferating cell nuclear antigen and IG11 were virtually negative. All three antibodies strongly stained the blood vessels of stroke tissues. The stroke tissues contained angiogenic activity, as shown by the induction of new blood vessels in a chorioallantoic membrane assay. Conclusions We have shown that stroke causes active angiogenesis that is more developed in the penumbra. Further experiments are needed to determine if this angiogenesis has beneficial effect.

Increased intrathecal levels of the angiogenic factors VEGF and TGF-β in Alzheimer’s disease and vascular dementia

The aim of the present study was to investigate, in patients with Alzheimers disease (AD), and vascular dementia (VAD), patterns of local release of vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta), two cytokines having a pivotal role in hypoxia-induced angiogenesis. The intrathecal levels of these molecules were related to the clinical severity of these diseases and to the intrathecal levels of beta-amyloid protein. Significantly increased cerebrospinal fluid (CSF) levels of both VEGF and TGF-beta were observed in 20 patients with AD and in 26 patients with VAD compared to healthy controls. Interestingly, there was significant correlation between the CSF levels of TGF-beta and VEGF in all the individuals studied. Our study demonstrates, both in patients with AD and in patients with VAD, an intrathecal production of VEGF, a cytokine which plays a pivotal role in angiogenesis. These results suggest that vascular factors might not only play a role in the pathogenesis of VAD but also in the pathogenesis of AD. In addition, we show in AD and VAD an intrathecal production of TGF-beta, a cytokine exerting on one hand anti-inflammatory and angiogenic properties, but on the other promoting amyloidogenesis.

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.

A putative role for platelet-derived growth factor in angiogenesis and neuroprotection after ischemic stroke in humans.

BACKGROUND AND PURPOSE Growth factors control two important processes in infarcted tissue, ie, angiogenesis and gliosis. We recently reported that transforming growth factor-beta1 (TGF-beta1) might be involved in angiogenesis after ischemic stroke in humans; here we present data of an extensive study on platelet-derived growth factor (PDGF) and its receptors. METHODS We studied brain samples from patients who suffered from ischemic stroke for the expression of mRNA encoding PDGF-A, PDGF-B, and PDGF receptors (PDGF-R). Proteins were examined by Western blotting and immunohistochemistry using the antibodies to PDGF-AB, PDGF-BB, PDGF-R alpha, and PDGF-R beta. RESULTS At the mRNA level, PDGF-A and PDGF-B were expressed mainly in neurons in penumbra. PDGF-R mRNA was strongly expressed in some astrocytes but mainly in type III/IV neurons in infarct and penumbra. The least expression was seen in the contralateral hemisphere (P<.001). In contrast, both PDGF-AB and PDGF-BB immunoreactive products were present in most cell types: PDGF-R alpha and PDGF-R beta mainly on neurons, and PDGF-R beta on some endothelial cells, with less staining of all the isoforms in the contralateral hemisphere. On Western blots, PDGF-AB and -BB were expressed more within white matter than gray matter of infarct/penumbra, whereas both isoforms of receptor were expressed mainly in gray matter compared with contralateral hemisphere. There was no or very weak expression of the receptor in white matter. CONCLUSIONS PDGF proteins are highly expressed in white matter, suggesting that PDGF may exert its function in white matter participating either in regeneration of damaged axons or in glial scar formation. PDGF-BB and its receptor expressed on microvessel endothelial cells might be involved in angiogenesis after stroke. Thus, PDGF is likely to be angiogenic and neuroprotective in stroke.

Pax genes in embryogenesis and oncogenesis

•  Introduction •  PAX proteins and embryogenesis •  PAX genes and cancer •  PAX3 gene in embryogenesis and cancer ‐  PAX3 in myogenesis and RMS ‐  PAX3 in melanogenesis and melanoma ‐  PAX3 in neurogenesis and neuroblastoma ‐  PAY3splicing and tumours •  PAX2 in tumourigenesis •  PAX5 in tumourigenesis •  PAX8 in tumourigenesis •  PAX and the treatment of cancer •  Summary

Pathophysiology of Acute Ischaemic Stroke: An Analysis of Common Signalling Mechanisms and Identification of New Molecular Targets

Stroke continues to be a major cause of death and disability. The currently available therapies have proven to be highly unsatisfactory (except thrombolysis) and attempts are being made to identify and characterize signalling proteins which could be exploited to design novel therapeutic modalities. The pathophysiology of stroke is a complex process. Delaying interventions from the first hours to days or even weeks following blood vessel occlusion may lead to worsening or impairment of recovery in later stages. The objective of this review is to critically evaluate the major mechanisms underlying stroke pathophysiology, especially the role of cell signalling in excitotoxicity, inflammation, apoptosis, neuroprotection and angiogenesis, and highlight potential novel targets for drug discovery.