Previous studies have shown that changes in expression of the glycosaminoglycan, hyaluronan (HA) were associated with erosion in areas of post-mortem coronary artery liable to rupture. Angiogenesis is an important feature of ulcerating haemorrhagic plaques prone to rupture. HA is a glycosaminoglycan known to possess potent angiogenic properties on metabolism to oligosaccharides of HA (o-HA) in the presence of hyaluronidase (HYAL) enzymes. In this study we have examined HA receptor and HYAL enzyme expression in a series of carotid artery specimens used as vascular transplants and exhibiting various stages of atherosclerotic lesions as determined by anatomo-pathology. Our results demonstrated dramatically increased expression of HYAL-1 in regions of inflammation associated with complicated plaques. Receptor for HA-mediated motility (RHAMM), which is known to be important in transducing angiogenic signals in vascular endothelium, was strongly expressed on intimal blood vessels from complicated lesions but almost absent from other regions including adventitial vessels. Metabolism of HA, together with up-regulation of RHAMM in complicated plaque lesions might be partly responsible for over-production of leaky neovessels and predisposition to plaque rupture.

Cellular Prion Protein (PrPc) is a ubiquitous glycoprotein present on the surface of endothelial cells. Resting vascular endothelial cells show minimum expression of PrPc and can constitutively release PrPc. PrPc participes in cell survival, differentiation and angiogenesis. During development, neonatal brain endothelial cells transiently express PrPc. Our group recently reported upregulation of PrPc in microvessels from ischemic brain regions in stroke patients. Ischemia/hypoxia induces PrPc expression through the activation of extracellular signal-regulated kinase (ERK). All these data suggest that PrPc plays an important role in angiogenic responses. In addition, PrPc participates in cellular function in the central nervous system, since PrPc is also highly expressed in neurons. PrPc binds copper, suggesting a role in copper metabolism. PrPc also protects cells against oxidative stress and it seems to be involved in neuroprotection. Several studies have demonstrated that PrPc prevents cells from apoptosis and subsequent tissue damage. Moreover, PrPc plays an important role in the immune response. Here, we review the multiple functions of PrPc with a special attention to its recently reported role in angiogenesis.

Intimal plaque neovascularization is associated with the development of symptomatic disease and thrombosis, with new 'leaky' fragile microvessels prone to haemorrhage. Perforin or pore forming protein is involved in vascular cell death by forming pores in target cells. Enzymes, in particular, granzyme B are secreted by immune infiltrates present in inflammatory plaque regions and have been shown to induce endothelial cell apoptosis. Similarly, dynamin-2 is a GTPase which mediates oxidised low density lipoprotein-induced apoptosis and is also required for granzyme B-mediated exocytosis and apoptosis. Our pilot studies identified increased expression of these proteins in complicated atherosclerotic plaques. Here we demonstrate by immunohistochemistry that both proteins are over-expressed in angiogenic regions of complicated carotid plaques. Dynamin-2 was extensively localised around microvessels and in immune infiltrating cells whilst perforin was localised in immune infiltrating cells, endothelial cells and smooth muscle cells. Over-expression of these proteins may contribute to plaque destabilisation by increasing cellular apoptosis in vulnerable atherosclerotic plaques.

Ischaemic stroke is a leading cause of death and disability in the Western world and usually occurs as a consequence of progressing atherothrombosis resulting in embolism and associated local tissue damage due to loss of cell membrane integrity and altered signal transduction activity. Survival of neurones, particularly in peri-infarcted regions determines the extent of patient recovery. A significant proportion of neurones in these areas undergo programmed cell death by apoptosis, resulting in a worse prognosis. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion, enhanced neuronal survival and improved recovery. Recent evidence has suggested that both angiogenesis and neuronal survival may be affected following activation of cyclindependent kinase-5 (Cdk5). In this review, the functional roles of Cdk5 in stroke will be described, followed by an analysis and comparison of available pharmacological inhibitors with a view to their potential use in the future treatment of this disease. Keywords: Cdk5, stroke, hypoxia-reperfusion, neurones, angiogenesis, pharmacological inhibition, apoptosis

BACKGROUND:Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain.RESULTS:Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11.CONCLUSION:Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents.

Normal cellular prion protein (PrPc) is expressed by a variety of cells of the immune system and is present in endothelial cells (EC). Our aim was to study local and systemic PrPc expression in advanced human carotid lesions and identify its relationship to unstable angiogenic vascular regions. PrPc expression was examined by Enzyme Immunometric Assay in plasma samples from patients with advanced carotid disease (n=22) and controls (n=21). Carotid specimens obtained during endarterectomy (n=22) and control (n=10) arteries from post-mortem were studied for PrPc and CD105 expression by Western blotting and immunohistochemistry. TaqMan arrays were used to study PrPc and CD105 gene expression. In vitro studies examined expression of PrPc in coronary EC. Patients with carotid disease had significantly higher levels of plasma PrPc than the control group (4,35ng/ml vs. 1,95ng/ml, p<0,001). CD105-positive unstable plaques had higher PrPc protein expression, associated with EC and inflammatory infiltrates. There was a strong correlation between PrPc and CD105 mRNA (p <0,001; r =0,7). Increased gene and protein expression of PrPc was seen in coronary EC exposed to staurosporin and growth factor stimulation. We show that PrPc is expressed in advanced carotid specimens and may be associated with the pro-angiogenic switch and therefore a higher risk of hemorrhagic transformation. Supportorted by HEFCE.

The physiologic properties of the normal cellular prion protein (PrPC) have not been established fully, although recent evidence showed its upregulation in cerebral ischaemia. Using patients, animal models, and in vitro studies we aimed to identify in detail the expression and localization of PrPC in ischemic stroke. Patients in acute phase of ischaemic stroke had increased plasma levels of circulating PrPC as compared to healthy age‐ and gender‐matched controls (3.1 ± 1.4 vs. 1.9 ± 0.7 ng/ml, P = 0.002). Immunohistochemistry showed increased expression of PrPC in the soma of peri‐infarcted neurones as well as in the endothelial cells (EC) of micro‐vessels and inflammatory cells in peri‐infarcted brain tissue from patients who survived for 2–34 days after an initial stroke. The same pattern was repeated 1–48 hr after MCAO. RT‐PCR showed increased gene expression of PrPC by human foetal neurons (HFN) after 12 hr of oxygen glucose deprivation (OGD), which remained increased after 24 hr reperfusion. Western blotting confirmed that protein expression was similarly upregulated, and fluorescent labeling showed a notable increase in peri‐nuclear and axonal PrPC staining intensity. Increased plasma PrPC seems to reflect endogenous expression in acute stroke‐affected brain tissue. Increased cellular expression in peri‐infarcted regions may influence hypoxia‐induced cell damage, although the effects on EC survival and angiogenesis remain to be elucidated. © 2006 Wiley‐Liss, Inc.

In the present study, we perfused the rhesus lung vascular bed in situ with sulfo-NHS-LC-Biotin to biotinylate its luminal surface membrane proteins. After homogenization, dialysis, and affinity chromatography, biotinylated endothelial membrane proteins were successfully isolated and characterized as enriched endothelial membrane proteins with no contamination of intracellular proteins. When they were used as immunogens to develop monoclonal antibodies (MAbs), three MAbs--TX111, TX112, and TX113--were obtained. Among them, TX111 was demonstrated to specifically bind to rhesus lung tissue by Western blotting and enzymelinked immunosorbent assay (ELISA)--that is, positively stained capillary endothelium of rhesus lung. The molecular weight of the corresponding antigen for TX111 was approximately 70 kDa under reducing conditions. TX111 also reacted with human lung homogenate, but not with rat lung homogenate. These results suggest that (1) the biotinylation method is applicable for isolating endothelial proteins in situ from large animals; (2) anti-human protein MAbs are likely to be obtained using monkey proteins; and (3) TX111 is potentially useful for pulmonary vascular targeting.

Objectives: This study aims to identify plaque neovascularisation using antibodies to CD31, CD105 and TGFβ1, and to compare their patterns of expression. Methods: Tissue expression of CD31, CD105 and TGFβ1 was examined immunohistologically in atherosclerotic plaques from 53 patients who had undergone carotid endarterectomy and in 10 controls. Results: CD31 was observed in a proportion of the microvessels within atheroma. The expression of CD105 was barely visible in normal arteries, but was markedly enhanced in atherosclerotic plaques. The vast majority of the microvessels in atheroma were positive for CD105 with pronounced expression around the periphery of the lipid core. In consecutive sections, microvessels showing negative staining for CD31 were positive for CD105. Although TGFβ1 was seen in the thickened intima, it was more strongly expressed in well-formed fibrous plaques. Consecutive sections showed that some microvessels were stained by both CD105 and TGFβ1, but in certain areas microvessels were exclusively CD105 positive. Conclusions: These observations highlight the distinctive expression patterns of CD31, CD105 and TGFβ1, suggesting their specific roles in the development of atherosclerotic plaques. CD105 is almost universally expressed in microvessels within the atheroma and is therefore a better vascular marker than CD31 and TGFβ1 for assessing neovascularisation in atherosclerotic plaques.

In normal colon epithelium, cell proliferation is followed by cell differentiation. The purpose of this work was to investigate, in the HT29-D4 colon adenocarcinoma cell line, the occurrence of a temporal sequence of changes in cell proliferation and differentiation, the role of autocrine EGF family ligands and to determine which transduction pathway(s) are involved in these processes. In a medium lacking both growth factor and serum, HT29-D4 cells secreted amphiregulin (AR), which was shown to be strongly involved in cell adhesion, growth and differentiation. In the main, integrins alpha2beta1 and alphavbeta6 intervened in these processes. Using tyrphostins, it was demonstrated that AR involvement was mediated through the ErbB1/ERK1,2 and ErbB1/FAK pathways. These signalling molecules were directly involved in pRb inhibition and, thus, in cyclin A expression. Concomitantly, colon differentiation markers were also expressed. Furthermore, terminal cell maturation resulted in a colon absorptive cell with strong polarisation, the growth of which was inhibited by tyrphostin and an ERK1,2 inhibitor. It was concluded that in a colon adenocarcinoma, cell proliferation and differentiation can occur concomitantly and that these deregulated processes are controlled by autocrine secretion through the ErbB1/ERK1,2 and FAK pathways.

Recent developments in our understanding of the pathophysiological events that follow acute ischaemic stroke suggest an important role for angiogenesis which, through new blood vessel formation, results in improved collateral circulation and may impact on the medium-to-long term recovery of patients. Future treatment regimens may focus on optimization of this process in the ischaemic boundary zones or 'penumbra' region adjacent to the infarct, where partially affected neurons exposed to intermediate perfusion levels have the capability of survival if perfusion is maintained or normalized. In this review, we present evidence that angiogenesis is a key feature of ischaemic stroke recovery and neuronal post-stroke re-organization, examine the signalling mechanisms through which it occurs, and describe the therapeutic potential of treatments aimed at stimulating revascularization and neuroprotection after stroke.

Alveolar rhabdomyosarcoma (ARMS) cells express high levels of PAX3-FKHR and IGF-II. In this study, we have investigated the effects of PAX3-FKHR and IGF-II on the expression of muscle regulatory factors (myf5, MyoD and myogenin), and platelet derived growth factor-B (PDGF-B) and vascular endothelial growth factor (VEGF) in mouse C2C12 myoblasts in vitro. PAX3-FKHR induced cell cycling of C2C12 cells and promoted proliferation whilst blocking myogenesis. IGF-II inhibited their differentiation without influencing proliferation. Western blotting showed that PAX3-FKHR and IGF-II blocked the expression of myogenin and MyoD respectively. Since MyoD affects early myogenesis and myogenin controls terminal differentiation, a combination of PAX3-FKHR and IGF-II synergistically blocks myogenesis at several different stages in differentiation. We have also shown that the major survival and angiogenic cytokines, PDGF-B and VEGF, were induced by IGF-II and PAX3-FKHR respectively. A combination of PAX3-FKHR and IGF-II could synergistically up regulate the expression of PDGF-B and VEGF and stabilize their high expression levels. Our results suggest that high expression of PAX3-FKHR and IGF-II in ARMS synergistically play a key role in oncogenesis and tumour progression of ARMS.

Our previous work has demonstrated that angiogenesis occurs in the damaged brain tissue of patients surviving acute ischaemic stroke and increased microvessel density in the penumbra is associated with longer patient survival. The brain is one of the richest sources of FGF-2 and several studies have noted its angiogenic and neuroprotective effects in the nervous system. These findings led us to investigate the expression and localisation of both FGF-2 mRNA and protein in brain tissue collected within 12 h of death from 10 patients who survived for between 24 h and 43 days after acute stroke caused by thrombosis or embolus. Western blot analysis demonstrated increased FGF-2 protein expression in both grey and white matter in the infarcted core and the penumbra region compared to the normal contralateral hemisphere of all 10 patients studied. Using indirect immunoperoxidase staining of paraffin embedded sections, we observed the presence of FGF-2 in neurones, astrocytes, macrophages and endothelial cells. In situ hybridisation was used to localise and quantify mRNA expression in ischaemic brain tissue of the same 10 patients. The expression of FGF-2 in the penumbra of all patients was significantly raised compared with infarcted tissue and normal-looking contralateral hemisphere. In addition, serum FGF-2 was significantly increased between 1 and 14 days ( P < 0.001) in many patients with both ischaemic stroke ( n = 28) and intra-cerebral haemorrhage ( n = 16) compared with age-matched control subjects undergoing routine medical examinations ( n = 20). We suggest that up-regulation of FGF-2 is one of the mechanisms that leads to angiogenesis and neuro-protection in the penumbra region after acute stroke in man.

BMRTC, bone-metastasising renal tumour of childhood; CAM, chorioallantoic membrane; EC, endothelial cell; ECM, extracellular matrix; HA, hyaluronan; HAase, hyaluronidase; nHA, native HA; o-HA, oligosaccharides of HA. Dear Sir, We congratulate Dr. Lokeshwar's group for another landmark paper in this journal, wherein they demonstrated that the saliva of patients with high-grade head-and-neck squamous cell carcinoma contained both high and low m.w. HA.1 The latter, i.e., o-HA, is generated when HAase, an endoglycosidase, degrades HA. Although the authors did allude to the issue of HA and its relevance to angiogenesis, we highlight here its pathologic relevance in human malignancy and present our supporting unpublished data. The angiogenic activity of HA is dependent on its molecular mass. High m.w. nHA is antiangiogenic, whereas almost 2 decades ago we reported that HA degradation products of a specific size (3–25 disaccharide units) induce angiogenesis in vivo.2 Subsequently, we have shown that o-HA fragments of 3–10 disaccharides were angiogenic in several in vivo assays and stimulated EC migration, proliferation and sprout formation.3, 4, 5, 6 The biological activities of HA are mediated through interaction with cell surface receptors such as CD44, resulting in activation of intracellular signalling events. In collaboration with Dr. R. Montesano's group, we found that HA degradation products influence EC invasion of a 3-D ECM.7 o-HA acted synergistically with vascular endothelial growth factor but not basic fibroblast growth factor. We reported that o-HA induces phosphorylation and activation of mitogen-activated protein kinase in bovine ECs, as well as upregulating the early response genes c-fos, c-jun and jun-B, which control the expression of other genes, including those of matrix-degrading proteases.8 Because HA acts via its receptor, CD44, it is conceivable that o-HA promotes cell invasion and tube formation by activating intracellular signalling pathways that result in modulation of pericellular proteolysis. Also, o-HA induced multiple signalling pathways involved in EC migration, proliferation and wound healing.9, 10 More relevant to Lokeshwar's publication is our finding that sera of children with renal tumours contain o-HA. Briefly, renal tumours are one of the major groups of childhood solid malignancy,Wilms' tumour (nephroblastoma) being the most frequent in this group. Another kidney tumour BMRTC has been distinguished from Wilms' tumour.11 BMRTC comprises about 4% of all primary renal neoplasms in childhood. In the Manchester region, with a population of 1 million children, we expect to encounter approximately one BMRTC case every 5 years. BMRTC has been associated with poor prognosis, unlike Wilms' tumour.11 Furthermore, in contrast to Wilms' tumour, BMRTC occurs predominantly in boys and, as the name implies, frequently metastasises to bone (in 60% of cases compared to 1% for Wilms' tumour). HA is a normal component of human serum. Its half-life in the circulation is 2.5–5.5 min and its mean serum concentration, 42 ± 25 μg/l in healthy adults.12 In contrast to patients with Wilms' tumour and mesothelioma, patients with many other types of cancer have serum HA levels within the normal range.13, 14 It is possible that the m.w. of HA, rather than its level in serum, may be pathognomonic for certain cancers. Indeed, we found that the HA level in the sera of normal children was barely detectable and had a m.w. of 1 to 5 x 10.5 In both Wilms' and BMRTC patients, very high levels of HA were found in preoperative serum samples; these fell markedly following surgical excision of tumours.15 We also demonstrated the presence of low m.w. HA (similar to the angiogenic fragment of HA) in the sera of BMRTC patients. In contrast, high m.w. HA (which is not angiogenic) was found in the sera of Wilms' patients. Following surgery in BMRTC patients, not only did serum HA levels fall to a value within normal ranges but also the HA which remained was of high m.w. We now have unpublished data to show that tissue culture medium from BMRTC cells, in contrast to Wilms' tumours, contains low m.w. o-HA. Furthermore, FPLC separation of HAase-digested n-HA (kindly provided by Dr. I. Scott, Unilever, London, UK) has enabled us to narrow down the HA size to between 5 and 7 disaccharides, which continue to retain their angiogenic potential. None of these disaccharides individually was able to induce angiogenesis in chicken CAM assay. However, in combination, they had the ability to induce angiogenesis in vivo and EC migration in vitro. Future studies should be directed toward ascertaining whether the size of o-HA is a hallmark of a certain type of malignancy. Yours sincerely, Mark Slevin, David West, Pat Kumar, Paul Rooney, Shant Kumar

CD105 (endoglin) is an important component of the transforming growth factor-beta (TGF-beta) receptor complex and is highly expressed in endothelial cells in tissues undergoing angiogenesis such as healing wounds, infarcts and in a wide range of tumours. In an attempt to understand the molecular mechanism by which CD105 exerts its effects on angiogenesis by modulating TGF-beta1 signalling, in this preliminary communication, CD105 transfected rat myoblasts were utilized as an in vitro model. Overexpression of CD105 in these transfectants antagonised TGF-beta1-mediated inhibition of cell proliferation and reduced TGF-beta1-mediated p3TP-Lux (PAI-1 promoter) luciferase activity. It also reduced (CAGA)12-Luc luciferase activity in response to TGF-beta1. The CAGA sequence is specific for Smad3/4 binding, implying that CD105 is involved in inhibition of TGF-beta1/Smad3 signalling. Furthermore, CD105 overexpression reduced serine phosphorylation of Smad3 and inhibited subsequent nuclear translocation of Smad3. CD105 resulted in high phosphorylation of JNK1, which is able to activate c-Jun. c-Jun is known to inhibit Smad3 transcriptional activity on CAGA sites, suggesting that CD105 may also inhibit Smad3 signalling through JNK1.

Vascular endothelium has attracted extensive attention due to its important role in many physiological and pathological processes. Many methods have been developed to study the components and their functions in vascular endothelium. Here we report a novel approach to investigate vascular endothelium using normal rat lungs as the model. We perfused lung vascular beds with sulfosuccinimidyl-6-(biotinamido) hexanoate, a biotin analog, to label endothelial membrane proteins. The biotinylated proteins were isolated from lung homogenate with immobilized monomeric avidin and confirmed to be highly pure endothelial membrane proteins with little contamination of intracellular proteins. These biotinylated proteins were used as immunogens for development of monoclonal antibodies. Indeed, newly generated monoclonal antibodies have revealed different expression patterns of proteins across tissues. Some proteins were found highly specifically expressed to capillary vessels of pulmonary vasculature. This method has also been proven useful for investigating vasculature of other organs, as this study explored.

CD105 is a receptor for transforming growth factor-beta but it is also considered to be involved in cellular functions such as cell adhesion and migration. Using CD105 transfected rat myoblasts, we have investigated the role of CD105 in cell adhesion, spreading, growth and migration. CD105 transfected myoblasts expressed abundant CD105, which was preferentially located within focal adhesion sites. These cells took on a bipolar morphology whereas mock cells remained polygonal or rounded, and when wounded, CD105 expressing cells realigned their long axis prior to migrating and migrated as a cohort of cells. CD105 expression promoted cellular attachment, spreading, survival and growth in serum-free conditions and each of these parameters could be inhibited by a RGD-containing peptide but not a RAD-containing peptide. Mock-transfected cells could not attach, spread or grow under these conditions. Attachment, spreading and growth in CD105 expressing cells could be promoted by the addition of a monoclonal antibody against CD105. Expression of CD105 resulted in the phosphorylation of JNK1 but had no effect on beta1 integrin expression. From this preliminary study, we conclude that in addition to acting as a transforming growth factor-beta receptor, CD105 has an important role in cell adhesion, migration and survival.

CD105, a marker of endothelial cells, is abundantly expressed in tissues undergoing angiogenesis and is a receptor for transforming growth factorbeta. 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 TGFbeta1. Furthermore, hypoxia and TGFbeta1 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.