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ABSTRACT: Ageing poses one of the largest risk factors for the development of cardiovascular disease (CVD). The increased propensity towards vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells (EPCs) to contribute to vascular repair and regeneration. While there is evidence to suggest that colony forming unit-Hill (CFU-Hill) cells and circulating angiogenic cells (CACs) are subject to age-associated changes that impair their function, the impact of ageing on human OEC function has been less studied. We demonstrate that OECs isolated from cord blood or peripheral blood samples from young and old individuals exhibit different characteristics in terms of their migratory capacity. In addition, age-related structural changes were discovered in OEC heparan sulfate (HS), a glycocalyx component that is essential in many signalling pathways. An age-associated decline in the migratory response of OECs towards a gradient of VEGF significantly correlated with a reduction in the relative percentage of the trisulfated disaccharide, 2-O-sulfated-uronic acid, N, 6-O-sulfated-glucosamine (UA[2S]-GlcNS[6S]), within OEC cell surface HS polysaccharide chains. Furthermore, disruption of cell surface HS reduced the migratory response of peripheral blood derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Together these findings suggest that ageing is associated with alterations in the fine structure of HS on the cell surface of OECs. Such changes may modulate the migration, homing and engraftment capacity of these repair cells, thereby contributing to the progression of endothelial dysfunction and age-related vascular pathologies. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
Aging cell 11/2012; · 7.55 Impact Factor
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ABSTRACT: Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs) play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells. In this review, we discuss the current understanding of the cells termed "EPCs," examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.
Frontiers in physiology. 01/2012; 3:30.
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ABSTRACT: Accumulating evidence indicates that vascular repair by endothelial progenitor cells (EPCs) is impaired with age. However, the molecular mechanisms underlying this functional impairment are not understood. Cell-surface heparan sulphate (HS) proteoglycans, by virtue of specific sulphated domains within the glycosaminoglycan chain, are able to bind a variety of ligands essential for EPC mobilisation, homing and differentiation. We hypothesise that structural changes of HS on EPCs contribute to vascular dysfunction in age and disease. Using umbilical cord blood, and adult peripheral blood from young and old subjects, we routinely isolate a rare population of EPCs, termed outgrowth endothelial cells (OECs). These cells are highly proliferative, express a panel of endothelial but not haematopoietic markers, can ingest Ac-LDL and form tubes in Matrigel. Structural analysis of HS by high performance liquid chromatography (HPLC) demonstrates a reduction of 6-O-sulphation of HS on the surface of OECs with vascular age. There is 21.9% 6-O-sulphation of HS chains on cord blood OECs compared to 14.6% and 10.1% on adult peripheral blood OECs from young (20-30 years) and older (>55 years) subjects respectively. Moreover, these HS structural changes correlate with a decrease in the proliferative and migratory capacities of these cells. The effects of these changes on the response of EPCs to signalling molecules implicated in EPC mobilisation and homing (VEGF and SDF-1) are currently being investigated. Whether the impairment in function can be rescued by the addition of soluble heparin is also being assessed. This work could have clinical relevance for therapeutic angiogenesis in patients with limb ischemia or vascular damage.
Heart (British Cardiac Society) 10/2011; 97(20):e7. · 4.22 Impact Factor
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ABSTRACT: Rationale Rheumatoid arthritis (RA) is associated with accelerated atherosclerosis and premature cardiovascular death. Chronic inflammation mediated by tumour necrosis factor (TNF) may contribute to this by promoting endothelial activation, dysfunction and leukocyte recruitment. Anti-TNF therapy has been associated with improved vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. We aim to establish whether the anti-TNF therapy certolizumab pegol (CZP) (Cimzia®; UCB, Belgium) modulates the inflammatory response by activated human endothelial cells. Methods and Results Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to (i) TNF alone, (ii) TNF plus CZP, or (iii) neither agent. Microarray analysis detected at least twofold higher expression of 115 genes after exposure of HAoEC to TNF, compared to control untreated cells. 22.6% of the increased genes were associated with the immune response and 9.6% encoded adhesion/cell surface molecules. In particular, genes for E-selectin, VCAM-1 and ICAM-1 were significantly upregulated by TNF treatment, which was validated by qPCR. Notably, treatment of HAoEC with the TNF/CZP cocktail prevented the up-regulation of these genes, resulting in an expression pattern similar to that detected in control cells. Immunocytochemistry confirmed the NFkB pathway as a downstream target of TNF-induced HAoEC activation, since the TNF-induced nuclear translocation of NFkB was prevented in the presence of CZP. Conclusions The clinically available anti-inflammatory agent CZP eliminates upregulation of adhesion molecules by endothelial cells. Whether this TNF inhibitor contributes to improved endothelial function in patients is currently under investigation.
Heart (British Cardiac Society) 10/2011; 97(20):e7. · 4.22 Impact Factor
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ABSTRACT: Systemic Lupus Erythematosus (SLE) is associated with an increased risk of cardiovascular disease. Endothelial dysfunction in SLE correlates with circulating interferon (IFN) levels. Late-outgrowth endothelial progenitor cells (LO-EPCs) from lupus patients have impaired function which correlates with an increased expression of IFN-response genes (the IFN-signature). We aim to establish an in vitro model of SLE endothelial pathology, using IFN-α treatment of LO-EPC and human aortic endothelial cells (HAoEC), to develop novel preventative or reparative strategies for this disease. Late-outgrowth EPCs were isolated from human umbilical cord blood and characterised by immunocytochemistry and RT-PCR. HAoECs were cultured using standard protocols. Cell proliferation and tubule formation was analysed by MTT and Matrigel assays respectively. Connected tubes and branch points were counted 18 h after treatment with IFN2b or serum from SLE patients. Current studies involve establishing the interferon signature in endothelial cells, using a combination of microarray and Bioplex analysis. IFN-α-2b (10 ng/ml) results in reduced tubule formation by LO-EPCs but not HAoECs. SLE serum inhibits the proliferation of LO-EPCs but not HAoEcs in a dose-dependent manner. However, proliferation was not affected in either cell type by the addition of IFN-α-2b at concentrations up to 100 ng/ml at 24, 48 or 72 h. LO-EPCs are significantly affected in terms of their tube-forming capacity and proliferative response to IFN-α-2b and SLE serum respectively, while mature HAoECs lack this response to IFN2b. We conclude that LO-EPCs could be a target cell in SLE and offer a potential model for development of vasculoprotective therapies in these patients.
Heart (British Cardiac Society) 10/2011; 97(20):e7. · 4.22 Impact Factor
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ABSTRACT: SLE is associated with endothelial dysfunction. Endothelial microparticles (EMPs) are subcellular particles considered to reflect endothelial damage. We aimed to quantify endothelial function and EMPs in a cohort of SLE patients with active disease, and investigate changes in response to treatment in a smaller subset of patients. Methods 12 patients (mean age 41.2 (SD 14.9) years) with active SLE were assessed for disease activity, repeated at 4 months in 5 patients. Four age-matched healthy controls (mean age 40.5 (SD 14.5) years) were assessed once. Endothelial function was assessed using peripheral arterial tonometry (EndoPAT 2000©). EMPs were quantified (number/ml) using flow cytometry after staining platelet-poor-plasma with the cell surface markers CD31, CD42 and Annexin-V. Events positive for annexin-V and CD31, and negative for CD42, were classified as EMPs. Results At baseline, mean EMP count was 21 782/ml (SD 11 946) in SLE cases, and 13 441/ml (SD 2347) in controls (p=0.04). There was a trend towards reduced endothelial function in SLE (mean Reactive Hyperaemic Index (RHI) 2.04 (SD 0.7) vs 2.62 (SD 0.5); p=0.18). In those with follow-up data, the mean EMP count decreased from 32 709/ml (SD 7186) to 15 662/ml (SD 5658; p=0.04) and RHI improved (mean RHI 1.75 (SD 0.2) vs 2.72 (SD 1.2; p=0.16) with better disease control. Conclusions Active SLE is associated with higher levels of EMPs compared to controls, reflecting endothelial damage. Reducing disease activity reduces EMP numbers and may improve endothelial function. EMPs may therefore serve as markers of both disease activity and vascular injury.
Heart (British Cardiac Society) 10/2011; 97(20):e7. · 4.22 Impact Factor
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ABSTRACT: Introduction Stroke is the third most common cause of death world wide. Most cases of stroke are caused by atherosclerotic plaque rupture. A challenge facing clinicians is identifying asymptomatic patients at risk of plaque instability. Locally released angiogenic growth factors may contribute to microvessel instability within plaques. Growth factors and inflammatory cytokines are potential serum biomarkers to identify patients at risk of stroke. Materials and methods Immunohistochemistry and quantitative PCR (Q-PCR) were used to establish localisation and expression of angiogenic growth factors within carotid endarterectomy specimens from symptomatic and asymptomatic patients. Stable or unstable microvessels were distinguished by CD31 and CD105 staining. Systemic levels of circulating angiogenic growth factors and inflammatory cytokines were measured in venous blood using Bio-Plex arrays. Results Hepatocyte growth factor (HGF) and its receptor c-Met were detected in CD31-positive endothelia, and alpha-SMA-positive cells, respectively. Q-PCR demonstrated upregulation of the angiogenic factors CD105, HGF (p<0.001) and c-Met (p=0.011) in symptomatic versus asymptomatic plaques. A significantly greater neovessel density was detected in symptomatic plaques (p=0.042), associated with elevated expression of HGF and c-Met. Suspension arrays demonstrated elevated HGF (p=0.002) and decreased platelet-derived growth factor (PDGF; p=0.036) serum levels in symptomatic versus asymptomatic patients. Twenty-seven cytokines were examined; seven endarterectomy patients demonstrated significantly increased levels in comparison with controls. No differences were observed between preoperative and postoperative serum. Discussion Plaque instability may be mediated by HGF-induced formation of microvessels, and decreased PDGF. We will investigate the effects of inflammatory cytokines with a view to comparing symptomatic versus asymptomatic patients. Targeting surgery to those who will benefit would eliminate unnecessary risk.
Heart (British Cardiac Society) 09/2010; 96(17):e12-3. · 4.22 Impact Factor
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ABSTRACT: The increased risk of cardiovascular disease with age may, in part, be due to a decline in the function of endothelial progenitor cells (EPCs). Cell surface heparan sulphate proteoglycans (HSPGs) can bind a plethora of factors that are essential for EPC function. However, these interactions are dependent upon specific structures of HS. We aim to establish whether structural changes of HS on EPCs underlie the age-associated functional deterioration of these cells. The number and function of EPCs in patients with systemic lupus erythematosus (SLE), a disease associated with accelerated vascular ageing, was compared with age-matched healthy controls (52 patients and 30 controls; mean age 52 and 50 years, respectively). To enumerate EPCs, mononuclear cells were labelled with CD133 and CD34 and analysed by flow cytometry. The formation of colony-forming units (CFUs) after 7 days in culture was a measure of EPC function. Cell surface HS structure was analysed using high performance liquid chromatography. While EPC levels did not significantly differ, impairment in EPC function with vascular ageing was evident from the significantly reduced mean number of CFU (7 (SD=5) vs 17 (SD=18), p=0.01), with fewer large CFU (17% vs 40%; p<0.05) in patients than in controls. Preliminary data suggest decreased 2-O-sulphation of HS in association with vascular age. Ongoing studies are investigating if this affects EPC migration, proliferation and integration into vascular structures. Proving our hypothesis will improve our understanding of age-associated endothelial dysfunction and ascertain whether EPCs and/or HS oligosaccharides have therapeutic potential to attenuate age-associated vascular pathologies.
Heart (British Cardiac Society) 09/2010; 96(17):e27-8. · 4.22 Impact Factor
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ABSTRACT: Vascular calcification is an established pathological process that contributes to several forms of cardiovascular morbidity-notably, atherosclerosis. The molecular mechanisms involved continue to be explored. Hepatocyte growth factor (HGF) signalling, via its receptor c-MET, has been identified in association with atherosclerotic plaque development. We have demonstrated that overexpression of HGF in human smooth muscle cells (hSMC) accelerates their mineralisation. Reports demonstrating upregulation of the notch ligand delta, and the presence of a feedback loop linked to the c-MET pathway, raise the possibility that the effects of HGF on mineralisation may be mediated via notch signalling. We aim to test the hypothesis that notch signalling is involved in Ad-HGF-induced in vitro mineralisation of hSMCs. We demonstrate accelerated mineralisation in response to adenoviral-mediated HGF overexpression, confirmed by alizarin red staining, calcium incorporation and increased alkaline phosphatase activity. In addition, we show upregulation and phosphorylation of c-MET and reduction of the mineralisation inhibitor osteopontin. We identify upregulation of the notch-3 intracellular domain via western blot analysis and, using immunocytochemistry, show an altered distribution of notch-3 in Ad-HGF-infected cells. Finally, we show (i) an attenuation of mineralisation in hSMCs following overexpression of NK4, the c-MET antagonist and (ii) that treatment of hSMCs with DAPT, the notch inhibitor, also decreased the rate of mineralisation compared with Ad-HGF infected cells and controls. These findings suggest a link with the notch pathway as a possible downstream effector of HGF and further elucidate a novel mechanism underpinning vascular calcification.
Heart (British Cardiac Society) 09/2010; 96(17):e27. · 4.22 Impact Factor
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ABSTRACT: Vascular calcification is a progressive pathology that occurs in many diseases, including atherosclerosis, diabetes, end-stage renal disease and valve disease. This study aimed to investigate whether decorin and transforming growth factor (TGF)-beta signal modulation occurs during differentiation of vascular smooth muscle cells (SMCs). Decorin and osteopontin expression was identified in calcified human femoral arteries using immunohistochemistry and biochemical staining. A positive staining pattern of bone-related proteins and mineralisation was also found in aortic root sections from 8-16 week ApoE-/- mice fed a high-fat diet, particularly in the valve leaflets. These data suggest that this model, as well as being an established model of atherosclerosis, is also a suitable model to study vascular calcification. When adenoviral infection of human vascular SMC in vitro was used, decorin overexpression increased SMC osteogenic differentiation fourfold in comparison with controls, as assessed using alizarin red staining and alkaline phosphatase activity. The enhancement of mineralisation was reduced using two approaches to antagonise the TGF-beta pathway-namely, adenoviral-mediated overexpression of the latency-associated particle of TGF-beta- 1 (LAP-beta1) and also a chemical inhibitor of TGF-beta type I receptor, SB431542. In conclusion, these results suggest that vascular calcification involves the modulation of the decorin and TGF-beta pathway in SMCs. The data may have implications for therapeutic targeting of this devastating pathology.
Heart (British Cardiac Society) 09/2010; 96(17):e15. · 4.22 Impact Factor
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ABSTRACT: Charcot neuroarthropathy (CN) is characterised by pathological foot fractures and osteopenia in patients with diabetes, often resulting in debilitating deformity. Paradoxically, these patients show evidence of medial vascular calcification. Recently, accentuated signalling of the receptor activator of nuclear factor kappa-B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG) have been implicated in the development of diabetic CN. This study aims to investigate the role of RANKL and OPG signalling in vascular calcification in patients with diabetes and CN, compared with healthy controls. RANKL and OPG serum levels were measured using ELISA in 12 patients with CN, 10 diabetic patients and five healthy controls. Serum RANKL and OPG levels were elevated in acute CN and in diabetic patients compared with healthy controls (p<0.05). Immunohistochemistry identifies upregulation of RANKL in calcified tibial arterial sections versus non-calcified controls. Human vascular smooth muscle cells (hVSMC) were grown in osteogenic conditions, as our in vitro model of calcification. When hVSMCs were treated with serum from patients with diabetes and CN, we demonstrated (i) accelerated mineralisation of hVSMC, confirmed by Alizarin red staining, and elevated alkaline phosphatase activity compared with control cells and (ii) reduced mineralisation when co-incubated with OPG. These findings demonstrate that RANKL/OPG signalling is modulated in diabetic and CN patients. Furthermore, serum from these patients accelerates vascular calcification in vitro, an effect attenuated by OPG treatment. These are the first human data implicating RANKL/OPG in diabetic vascular calcification and suggest that OPG/anti-RANKL therapy may be a potential target in combating disease progression.
Heart (British Cardiac Society) 09/2010; 96(17):e13. · 4.22 Impact Factor
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ABSTRACT: A challenge facing clinicians is identifying patients with asymptomatic carotid disease at risk of plaque instability. We hypothesise that locally released angiogenic growth factors contribute to plaque instability.
Carotid endarterectomy specimens from eight symptomatic and eight asymptomatic patients were interrogated for microvessel density and angiogenic growth factor expression histologically using immunofluorescence, and biochemically using quantitative real-time polymerase chain reaction (q-RT-PCR). Bio-Plex suspension array was used to assess circulating biomarkers in venous blood from the same patients and six healthy age-matched controls.
Immunofluorescence demonstrated significantly greater neovessel density in symptomatic plaques (P=0.010) with elevated expression of hepatocyte growth factor (HGF) (P=0.001) and its receptor MET (P=0.011) than in asymptomatic plaques. The q-RT-PCR demonstrated up-regulation of Endoglin (CD105), HGF (P=0.001) and MET (P=0.011) in the plaques of symptomatic versus asymptomatic patients. Bio-Plex suspension array demonstrated elevated HGF (P=0.002) serum levels in symptomatic versus asymptomatic patients and healthy controls, and decreased platelet-derived growth factor (PDGF) (P=0.036) serum levels in symptomatic versus asymptomatic patients.
Plaque instability may be mediated by HGF-induced formation of new microvessels, and decreased vessel stability resulting from decreased PDGF. Suspension array technology has the potential to identify circulating biomarkers that correlate with plaque rupture risk.
European journal of vascular and endovascular surgery: the official journal of the European Society for Vascular Surgery 04/2010; 39(4):388-95. · 2.92 Impact Factor
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ABSTRACT: Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c-Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31-positive endothelial cells. c-Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT-PCR, we demonstrated expression of HGF and c-Met in a rat pericyte cell-line, TR-PCT1, and in primary pericytes. HGF treatment of TR-PCT1 cells induced their migration, but not their proliferation, in a dose-dependent manner (10-100 ng/ml, p<0.01), an effect mediated by activation of the serine/threonine kinase Akt, shown by western blot analysis. Treating the cells with the PI3K inhibitors Wortmannin (0.1 microM) or LY294002 (10 microM) abolished these effects. This work demonstrates the expression of c-Met and HGF in human atherosclerotic arteries, in association with SM-actin-positive cells and CD-31-positive cells, respectively. HGF induces pericyte migration via PI3-kinase and Akt activation in vitro. HGF and c-Met may be involved in neovascularization during plaque development, and may recruit pericytes to neovessels. Since pericytes are thought to mechanically stabilize new blood vessels, these factors may function to protect against haemorrhage.
The Journal of Pathology 05/2007; 212(1):12-9. · 6.32 Impact Factor
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ABSTRACT: Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF-positive cells were detected within the intima and adventitia in areas where sporadic 3G5-positive pericytes were localized. Furthermore, VCAF-positive cells were identified in newly formed microvessels in association with CD34-positive/CD146-positive/c-kit-positive cells as well as in intact CD31-positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease.
The Journal of Pathology 03/2007; 211(3):362-9. · 6.32 Impact Factor
