Mark Slevin

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.

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

Activation of MAP kinase (ERK-1/ERK-2), tyrosine kinase and VEGF in the human brain following acute ischaemic stroke.

We examined expression of vascular endothelial growth factor (VEGF), phosphorylation of mitogen activated protein kinase (MAP) kinase (ERK1 and ERK2) and tyrosine phosphorylation in 19 patients (aged 58–90 years; mean 75) who died 1–44 days after acute ischaemic stroke. In the grey matter penumbra, 13 of 19 patients showed an increase in MAP kinase tyrosine phosphorylation (ERK1; 2.0- to 8-fold, ERK2; 2.2- to 11-fold) compared with normal contralateral tissue. In almost all cases, ERK-2 phosphorylation was higher than ERK1. Of these 13 patients, 11 also showed a general increase in tyrosine kinase phosphorylation, and eight expressed increased levels of VEGF protein (2.5- to 5-fold). In tissue examined directly from the infarct core, activation of the above proteins was not observed in the, majority of patients. In the white matter, seven of 19 patients (penumbra), and nine of 19 patients (stroke) had an increase in MAP kinase tyrosine phosphorylation (ERK1; 2.0- to 4.6-fold and ERK-2; 2.3- to 5.4-fold respectively) compared with normal contralateral tissue. There was no relationship between activation of MAP kinase and expression of VEGF. Examination of phosphorylated MAP kinase by immunohistochemistry revealed an increase in immunoreactivity in neurones, astroglial cells, reactive microglia and endothelial cells in areas surrounding infarcts, especially in areas with the highest density of microvessels. In conclusion, chronic activation of tyrosine phosphorylated events, in particular redistribution and phosphorylation of MAP kinase (ERK1/ERK2) occurs consistently in the grey matter penumbra of brain tissue following ischaemic stroke, and may be associated with increase in expression of VEGF. These signal transduction events could be important determinants of the extent of neuronal survival and/or angiogenic activity in the recovering brain tissue.

C-Reactive Protein Isoforms Differ in Their Effects on Thrombus Growth

Objective—We studied the impact of native (natCRP) and modified CRP (mCRP) isoforms on platelet adhesion and thrombus growth under arterial flow. Methods and Results—Blood was perfused over type I collagen at a wall shear rate of 1500 s−1, and platelet deposition and thrombus growth were evaluated by confocal microscopy. natCRP and mCRP were either incubated with blood before perfusion experiments or immobilized in the collagen surface and exposed to flowing blood. mCRP significantly increased platelet adhesion and thrombus growth when directly incubated with blood and when immobilized on a collagen surface (P<0.05). In contrast, natCRP did not exert any effect. Confocal immunohistochemistry revealed the presence of CRP on the surface of adhered platelets and within the thrombus and showed an upregulation of P-selectin and CD36 in effluent platelets preincubated with mCRP (P<0.05). Flow cytometry analysis of agonist-induced platelet activation demonstrated that mCRP, but not natCRP, significantly increased platelet surface P-selectin (P<0.05) without modifying CD63 and PAC-1. Conclusions—Our data indicate that whereas serum natCRP may not affect thrombus growth, mCRP displays a prothrombotic phenotype enhancing not only platelet deposition, but also thrombus growth under arterial flow conditions.

Vascular MMP-9/TIMP-2 and neuronal MMP-10 up-regulation in human brain after stroke: a combined laser microdissection and protein array study.

Matrix Metalloproteinases (MMPs) play an important role in brain injury after ischemic stroke. In the present study, we aimed to assess the global expression of MMP-Family proteins in the human brain after stroke by using a combination of Searchlight Protein Array and Laser Microdissection to determine their cellular origin. This study demonstrated that MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, and TIMP-1 were upregulated in the infarcted tissue compared to healthy control areas. Using laser microdissection we obtained specific neuronal and vascular populations from both infarcted and control areas. From these fractions, we showed that MMP-9 and TIMP-2 were highly produced in brain microvessels while MMP-10 was notably increased in neurons of the ischemic brain but not in healthy areas. These findings demonstrate a selective cell-dependent MMP secretion, opening the possibility of selectively targeting specific MMPs for neuroprotection or vasculoprotection following stroke.

Endogenous Expression of C-Reactive Protein Is Increased in Active (Ulcerated Noncomplicated) Human Carotid Artery Plaques

Background and Purpose— There is growing evidence suggesting that C-reactive protein (CRP) is an effecter molecule able to induce and promote atherothrombosis. The presence of CRP in atherosclerotic plaques may reflect local production or infiltration from circulating CRP increased in general inflammatory responses. Our aim was to analyze the presence of CRP in human advanced carotid artery plaques with differential anatomo-pathological characteristics and to assess local expression of CRP and other proinflammatory genes in these lesions. Methods— Human carotid artery specimens from 38 patients undergoing scheduled endarterectomy were classified into 3 groups: ulcerated (noncomplicated) (UNC, n=19), fibrous (F, n=12) and ulcerated (complicated/hemorrhagic) plaques (UC, n=7). The presence of CRP was evaluated by immunohistochemistry, and plasma samples were screened for circulating high-sensitivity C-reactive protein. TaqMan Low-density Arrays were used for study of genes related to inflammation (CRP, interleukin-6, macrophage colony-stimulating factor-1, monocyte chemotactic protein-1, cyclooxygenase-2). Results— CRP mRNA levels were predominantly detected in UNC-high risk plaques but not in UC (P=0.001). UNC also exhibit the highest expression levels of other genes involved in the inflammatory responses: cyclooxygenase-2 (P<0.005 versus F and versus UC), IL-6 (P<0.005 versus F and versus UC) and monocyte chemoattractant protein-1 (P<0.01 versus F and versus UC). Plaque CRP mRNA levels correlated with immunohistochemical findings but were independent of plasma high-sensitivity CRP. In UNC plaques endothelial cells and inflammatory cells were strongly positive for CRP around areas of newly formed microvessels. Conclusions— In human high-risk carotid artery plaques (UNC) CRP expression reflects an active proinflammatory stage. Local synthesis of CRP could be involved in plaque neovascularization and increased risk of hemorrhagic transformation.

Effect of glycation on basic fibroblast growth factor induced angiogenesis and activation of associated signal transduction pathways in vascular endothelial cells: Possible relevance to wound healing in diabetes

Ineffectual wound healing in hyperglycaemic patients suffering from diabetes mellitus is characterised by a reduction in capillary reformation (angiogenesis). Basic fibroblast growth factor (FGF-2) is secreted by fibroblasts, macrophages and in particular endothelial cells (EC) in response to tissue injury and is important in promotion of neovascularisation. Recently, glycation of FGF-2 has been shown to significantly reduce its activity in vitro. We have examined the kinetics of FGF-2 glycation and compared its ability with that of native FGF-2 to activate mitogenesis, capillary formation and associated signal transduction in bovine aortic EC (BAEC). FGF-2 was exposed to 0.25 M glucose-6-phosphate (G-6-P) for 24–72 h and the degree of glycation determined by matrix assisted laser desorption ionisation mass spectrometry. Native FGF-2 was heterogeneous with Mw in the range 15,153.6–17,903 Da. After 24 h incubation with G-6-P there was evidence of glycation, and the mass increase corresponded to addition of 2.7 mol of G-6-P residues; after 48 h, 4 mol sugar was added and this increased to 8.7 after 72 h. Dimerisation of FGF-2 was observed after 72 h of treatment. Induction of mitogenesis in BAEC was significantly reduced by 25%–40% after treatment for 48–96 h with glycated (24 h) FGF-2 (gFGF-2;100 pg/ml–5 ng/ml; P < 0.05), whilst capillary tubule formation was significantly reduced by between 60% and 90% (100 pg/ml–1 ng/ml; P < 0.05) after 5 days compared to native FGF-2. Subsequent investigation of the signal transduction molecules associated with mitogenesis showed a reduction in FGF-2 induced tyrosine phosphorylated proteins of approximate Mw 20–150 kDa between 10 min and 24 h, in particular, mitogen activated protein kinase (MAPK)/early response kinase (ERK-1, ERK-2), after glycation. To determine the reason for reduced angiogenic activity of gFGF-2, we compared its binding characteristics to that of native FGF-2. Total binding of gFGF-2 to the cell surface was significantly reduced in BAEC analysed by FACS compared to native FGF-2 (P < 0.05). Further investigation using 125I-labelled differentially washed samples, demonstrated a significant reduction in gFGF-2 binding to the high affinity tyrosine kinase receptor (46%) compared to native FGF-2. In summary, glycation of FGF-2 in vitro occurs rapidly within 24 h in the presence of elevated levels of G-6-P. Glycation caused a significant reduction in the ability of FGF-2 to bind to the tyrosine kinase receptor and activate signal transduction pathways responsible for both mitogenesis and capillary formation in BAEC. These results could help to explain the mechanism behind impaired wound healing in patients with diabetes mellitus.

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.

C-reactive protein exerts angiogenic effects on vascular endothelial cells and modulates associated signalling pathways and gene expression

BackgroundFormation of haemorrhagic neovessels in the intima of developing atherosclerotic plaques is thought to significantly contribute to plaque instability resulting in thrombosis. C-reactive protein (CRP) is an acute phase reactant whose expression in the vascular wall, in particular, in reactive plaque regions, and circulating levels increase in patients at high risk of cardiovascular events. Although CRP is known to induce a pro-inflammatory phenotype in endothelial cells (EC) a direct role on modulation of angiogenesis has not been established.ResultsHere, we show that CRP is a powerful inducer of angiogenesis in bovine aortic EC (BAEC) and human coronary artery EC (HCAEC). CRP, at concentrations corresponding to moderate/high risk (1–5 μg/ml), induced a significant increase in proliferation, migration and tube-like structure formation in vitro and stimulated blood vessel formation in the chick chorioallantoic membrane assay (CAM). CRP treated with detoxi-gel columns retained such effects. Western blotting showed that CRP increased activation of early response kinase-1/2 (ERK1/2), a key protein involved in EC mitogenesis. Furthermore, using TaqMan Low-density Arrays we identified key pro-angiogenic genes induced by CRP among them were vascular endothelial cell growth factor receptor-2 (VEGFR2/KDR), platelet-derived growth factor (PDGF-BB), notch family transcription factors (Notch1 and Notch3), cysteine-rich angiogenic inducer 61 (CYR61/CCN1) and inhibitor of DNA binding/differentiation-1 (ID1).ConclusionThis data suggests a role for CRP in direct stimulation of angiogenesis and therefore may be a mediator of neovessel formation in the intima of vulnerable plaques.