Gareth J. Padfield

Understanding the Role of Endothelial Progenitor Cells in Percutaneous Coronary Intervention

Percutaneous coronary intervention is associated with mechanical endovascular injury and endothelial denudation. Re-endothelialization is essential for restoration of normal vascular homeostasis and regulation of neointimal hyperplasia. The endothelial progenitor cell recently emerged as an important component of the response to vascular injury, having the potential to accelerate vascular repair through rapid re-endothelialization. There remains considerable uncertainty over the precise identity and function of endothelial progenitor cells, and harnessing their therapeutic potential remains a challenge. A better understanding of the role of circulating progenitors in the response to vascular injury is necessary if we are to develop effective strategies to enhance vascular repair after percutaneous coronary intervention. In this review, we examine the preclinical and clinical evidence of a role for bone marrow-derived putative endothelial progenitor cells after iatrogenic vascular injury associated with balloon angioplasty and stent deployment. Therapies designed to mobilize endothelial progenitors or to increase their ability to home to the site of stent implantation may have a role in the future management of patients undergoing percutaneous coronary intervention.

Circulating endothelial progenitor cells are not affected by acute systemic inflammation

Vascular injury causes acute systemic inflammation and mobilizes endothelial progenitor cells (EPCs) and endothelial cell (EC) colony-forming units (EC-CFUs). Whether such mobilization occurs as part of a nonspecific acute phase response or is a phenomenon specific to vascular injury remains unclear. We aimed to determine the effect of acute systemic inflammation on EPCs and EC-CFU mobilization in the absence of vascular injury. Salmonella typhus vaccination was used as a model of acute systemic inflammation. In a double-blind randomized crossover study, 12 healthy volunteers received S. typhus vaccination or placebo. Phenotypic EPC populations enumerated by flow cytometry [CD34(+)VEGF receptor (VEGF)R-2(+)CD133(+), CD14(+)VEGFR-2(+)Tie2(+), CD45(-)CD34(+), as a surrogate for late outgrowth EPCs, and CD34(+)CXCR-4(+)], EC-CFUs, and serum cytokine concentrations (high sensitivity C-reactive protein, IL-6, and stromal-derived factor-1) were quantified during the first 7 days. Vaccination increased circulating leukocyte (9.8 + or - 0.6 vs. 5.1 + or - 0.2 x 10(9) cells/l, P < 0.0001), serum IL-6 [0.95 (0-1.7) vs. 0 (0-0) ng/l, P = 0.016], and VEGF-A [60 (45-94) vs. 43 (21-64) pg/l, P = 0.006] concentrations at 6 h and serum high sensitivity C-reactive protein at 24 h [2.7 (1.4-3.6) vs. 0.4 (0.2-0.8) mg/l, P = 0.037]. Vaccination caused a 56.7 + or - 7.6% increase in CD14(+) cells at 6 h (P < 0.001) and a 22.4 + or - 6.9% increase in CD34(+) cells at 7 days (P = 0.04). EC-CFUs, putative vascular progenitors, and the serum stromal-derived factor-1 concentration were unaffected throughout the study period (P > 0.05 for all). In conclusion, acute systemic inflammation causes nonspecific mobilization of hematopoietic progenitor cells, although it does not selectively mobilize putative vascular progenitors. We suggest that systemic inflammation is not the primary stimulus for EPC mobilization after acute vascular injury.

Cardiovascular effects of tumour necrosis factor α antagonism in patients with acute myocardial infarction: a first in human study

Objective The inflammatory cytokine, tumour necrosis factor α (TNF-α), exerts deleterious cardiovascular effects. We wished to determine the effects of TNF-α antagonism on endothelial function and platelet activation in patients with acute myocardial infarction. Design and setting and patients A double-blind, parallel group, randomised controlled trial performed in a tertiary referral cardiac centre. 26 patients presenting with acute myocardial infarction randomised to receive an intravenous infusion of etanercept (10 mg) or saline placebo. Main outcome measures Leucocyte count, plasma cytokine concentrations, flow cytometric measures of platelet activation and peripheral vasomotor and fibrinolytic function were determined before and 24 h after study intervention. Results Consistent with effective conjugation of circulating TNF-α, plasma TNF-α concentrations increased in all patients following etanercept (254±15 vs 0.12±0.02 pg/ml; p<0.0001), but not saline infusion. Etanercept treatment reduced neutrophil (7.4±0.6 vs 8.8±0.6×109 cells/l; p=0.03) and plasma interleukin-6 concentrations (5.8±2.0 vs 10.6±4.0 pg/ml; p=0.012) at 24 h but increased platelet–monocyte aggregation (30±5 vs 20±3%; p=0.02). Vasodilatation in response to substance P, acetylcholine and sodium nitroprusside, and acute tissue plasminogen activator release were unaffected by either treatment (p>0.1 for all). Conclusions Following acute myocardial infarction, etanercept reduces systemic inflammation but increases platelet activation without affecting peripheral vasomotor or fibrinolytic function. We conclude that TNF-α antagonism is unlikely to be a beneficial therapeutic strategy in patients with acute myocardial infarction.

Cardiovascular effects of tumour necrosis factor α antagonism in patients with acute myocardial infarction

Objective The inflammatory cytokine, tumour necrosis factor α (TNF-α), exerts deleterious cardiovascular effects. We wished to determine the effects of TNF-α antagonism on endothelial function and platelet activation in patients with acute myocardial infarction. Design and setting and patients A double-blind, parallel group, randomised controlled trial performed in a tertiary referral cardiac centre. 26 patients presenting with acute myocardial infarction randomised to receive an intravenous infusion of etanercept (10 mg) or saline placebo. Main outcome measures Leucocyte count, plasma cytokine concentrations, flow cytometric measures of platelet activation and peripheral vasomotor and fibrinolytic function were determined before and 24 h after study intervention. Results Consistent with effective conjugation of circulating TNF-α, plasma TNF-α concentrations increased in all patients following etanercept (254±15 vs 0.12±0.02 pg/ml; p<0.0001), but not saline infusion. Etanercept treatment reduced neutrophil (7.4±0.6 vs 8.8±0.6×109 cells/l; p=0.03) and plasma interleukin-6 concentrations (5.8±2.0 vs 10.6±4.0 pg/ml; p=0.012) at 24 h but increased platelet–monocyte aggregation (30±5 vs 20±3%; p=0.02). Vasodilatation in response to substance P, acetylcholine and sodium nitroprusside, and acute tissue plasminogen activator release were unaffected by either treatment (p>0.1 for all). Conclusions Following acute myocardial infarction, etanercept reduces systemic inflammation but increases platelet activation without affecting peripheral vasomotor or fibrinolytic function. We conclude that TNF-α antagonism is unlikely to be a beneficial therapeutic strategy in patients with acute myocardial infarction.

Endothelial progenitor cells, atheroma burden and clinical outcome in patients with coronary artery disease

Objective We wished to determine the effect of an acute coronary syndrome (ACS) on putative endothelial progenitor cell (EPC) populations, and define their relationship to coronary artery disease (CAD) severity and clinical outcome, in order to clarify their clinical relevance. Design and setting A prospective cohort study conducted in a tertiary referral cardiac centre. Patients Two-hundred-and-one patients undergoing coronary angiography for suspected angina or ACS. Main outcome measures Putative EPC populations were determined by flow cytometry. CAD was quantified using the Gensini scoring system. Survival free from revascularisation, recurrent myocardial infarction and death were determined at 3 years. Results Circulating CD34+VEGFR-2+ and CD34+VEGFR-2+CD133+ cells were rare (<0.007% of mononuclear cells), were not increased in patients with ACS, and were unrelated CAD severity or clinical outcome (p>0.1 for all). By contrast, CD34+CD45− cells were increased in patients with CAD compared with those with normal coronary arteries (p=0.008) and correlated with atheroma burden (r=0.44, p<0.001). Increased concentrations of circulating CD34+CD45− cells were associated with a shorter cumulative event-free survival (p<0.02). Proangiogenic monocytes (CD14+VEGFR-2+Tie-2+) and endothelial cell-colony forming units were increased in patients with ACS (p<0.01 for both), however, concentrations reflected myocardial necrosis, and did not predict the extent of CAD or clinical outcome. Conclusions Traditional EPC populations, CD34+VEGFR-2+ and CD34+VEGFR-2+CD133+ are not related to the extent of CAD or clinical outcome. However, CD34+CD45− cells are increased in patients with CAD and predict future cardiovascular events. It is likely that CD34+CD45− concentrations reflect the extent of vascular injury and atheroma burden.

Dissociation of phenotypic and functional endothelial progenitor cells in patients undergoing percutaneous coronary intervention

Objectives: Endothelial progenitor cells (EPCs) are circulating mononuclear cells with the capacity to mature into endothelial cells and contribute to vascular repair. We assessed the effect of local vascular injury during percutaneous coronary intervention (PCI) on circulating EPCs in patients with coronary artery disease. Design and setting: Prospective case-control study in a university teaching hospital. Patients: 54 patients undergoing elective coronary angiography. Interventions and main outcome measures: EPCs were quantified by flow cytometry (CD34+KDR+ phenotype) complemented by real-time polymerase chain reaction (PCR), and the colony forming unit (CFU-EC) functional assay, before and during the first 24 hours after diagnostic angiography (n = 27) or PCI (n = 27). Results: Coronary intervention, but not diagnostic angiography, resulted in an increase in blood neutrophil count (p<0.001) and C-reactive protein concentrations (p = 0.001) in the absence of significant myocardial necrosis. Twenty-four hours after PCI, CFU-ECs increased threefold (median [IQR], 4.4 [1.3–13.8] vs 16.0 [2.1–35.0], p = 0.01), although circulating CD34+KDR+ cells (0.019% (SEM 0.004%) vs 0.016% (0.003%) of leucocytes, p = 0.62) and leucocyte CD34 mRNA (relative quantity 2.3 (0.5) vs 2.1 (0.4), p = 0.21) did not. There was no correlation between CFU-ECs and CD34+KDR+ cells. Conclusions: Local vascular injury following PCI results in a systemic inflammatory response and increases functional CFU-ECs. This increase was not associated with an early mobilisation of CD34+KDR+ cells, suggesting these cells are not the primary source of EPCs involved in the immediate response to vascular injury.

Novel Biopolymers to Enhance Endothelialisation of Intra-vascular Devices

Rapid endothelisation is of critical importance in the prevention of adverse remodelling after device implantation. Currently, there is a need for alternative strategies to promote re-endothelialisation for intravascular stents and vascular grafts. Using polymer microarray technology 345 polymers are comprehensively assessed and a matrix is identified that specifically supports both progenitor and mature endothelial cell activity in vitro and in vivo while minimising platelet attachment.

Endothelial progenitor cells in patients with chronic obstructive pulmonary disease

The pathogenesis of chronic obstructive pulmonary disease is not fully understood. The objective of this study was to compare circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease to age, sex, and cigarette smoking matched healthy controls. Patients with chronic obstructive pulmonary disease (n = 37) and healthy controls (n = 19) were matched by age, sex, and smoking status. Circulating hematopoietic progenitor cells (CD34(+) or CD133(+) mononuclear cells) and endothelial progenitor cells (CD34(+)KDR(+) or CD34(+)CD133(+)KDR(+) mononuclear cells) were quantified by flow cytometry. Endothelial cell-colony forming units from peripheral blood mononuclear cells were quantified in vitro and phenotypic analysis carried out using immunocytochemistry. Patients with chronic obstructive pulmonary disease had more circulating mononuclear cells compared with controls (8.4 ± 0.6 vs. 5.9 ± 0.4 × 10(9) cells/l; P = 0.02). CD34(+) hematopoietic progenitor cells were reduced as a proportion of mononuclear cells in patients compared with controls (0.99 ± 0.12 vs. 1.9 ± 0.12%; P = 0.02); however, there were no differences in the absolute number of CD34(+), CD34(+)KDR(+), or CD34(+)CD133(+)KDR(+) cells (P > 0.05 for all). Endothelial cell-colony forming units were increased in patients with chronic obstructive pulmonary disease compared with controls (13.7 ± 5.2 vs. 2.7 ± 0.9 colonies; P = 0.048). In contrast to previous studies, the number of circulating progenitor cells was not reduced in patients with chronic obstructive pulmonary disease compared with carefully matched controls. It seems unlikely that circulating endothelial progenitor cells or failure of angiogenesis plays a central role in the development of emphysema.

Platelet receptor polymorphisms do not influence Staphylococcus aureus-platelet interactions or infective endocarditis

Cardiac vegetations result from bacterium–platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen PlA1/A2 and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus–platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa PlA1/A1 genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa PlA1/A2 nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus–platelet interactions in vitro or the clinical course of infective endocarditis.

Percutaneous coronary intervention causes a rapid but transient mobilisation of CD34+CD45− cells

Objective Circulating CD34+CD45− cell concentrations are increased in patients with coronary artery disease, however their pathophysiological significance is unknown. We determined CD34+CD45− cell concentrations following percutaneous coronary intervention (PCI) in order to explore their role in acute vascular injury. Methods In a prospective time-course analysis, we quantified using flow cytometry circulating CD34+CD45− cells, traditional CD34+VEGFR-2+ putative endothelial progenitor cells (EPCs), CD14+ VEGFR− 2+Tie-2+ angiogenic monocytes and intercellular adhesion molecule expression (CXCR-4 and CD18) in patients, before and during the first week following diagnostic angiography (n=13) or PCI (n=23). Vascular endothelial growth factor-A (VEGF-A) and C reactive protein (CRP) were quantified by ELISA. Results Unlike diagnostic angiography, PCI increased circulating neutrophil and CRP concentrations at 24 and 48 h, respectively (p<0.002 for both). CD34+CD45− cell concentrations were unaffected by angiography (p>0.4), but were transiently increased 6 h following PCI (median (IQR) 0.93 (0.43–1.49) vs 1.51 (0.96–2.15)×106 cells/L; p=0.01), returning to normal by 24 h. This occurred in the absence of any change in serum VEFG-A concentration, adhesion molecule expression on CD34+ cells, or mobilisation of traditional EPCs or angiogenic monocytes (p>0.1 for all). Conclusions PCI transiently increases circulating CD34+CD45− cells, without increasing CD34+ adhesion molecule expression or VEGF-A concentrations, suggesting that CD34+CD45− cells may be mobilised from the vessel wall directly as a result of mechanical injury. Traditional putative EPC and angiogenic monocytes are unaffected by PCI, and are unlikely to be important in the acute response to vascular injury.