Differential modulatory effects of Propofol and Sevoflurane anesthesia on blood monocyte HLA-DR and CD163 expression during and after cardiac surgery with cardiopulmonary bypass: a preliminary randomized flow cytometry study.

INTRODUCTION: The increase of the anti-inflammatory CD163highHLA-DRlow blood monocyte subset is one of the mechanisms dampening inflammation during cardiac surgery with cardiopulmonary bypass. We evaluated the effect of two different anesthetic protocols, intravenous Propofol infusion or Sevoflurane-gas administration, on the perioperative frequency of this subset. METHODS: Blood from patients (Propofol = 11, Sevoflurane = 13) undergoing minimally invasive mitral valve surgery was drawn preoperatively (T1), before declamping (T2), at 6 (T3), 24 (T4), 48 (T5), and 72 hours (T6) after declamping. C-reactive protein, haptoglobin, and lactate dehydrogenase were measured. A hemolytic index, as C-reactive protein/haptoglobin ratio, was introduced. Monocyte expression of HLA-DR, CD163, and the CD163highHLA-DRlow subset fraction was quantified by flow cytometry. Baseline-referred variations of plasmatic and cellular data at T2 were normalized for clamping times. Subsequent time-point variations were normalized for the final cardiopulmonary bypass times. RESULTS: Variations of hemolytic index and lactate dehydrogenase were higher with Propofol at T3 (p = 0.004 and p = 0.02, respectively) when compared with Sevoflurane. At T2, the down-modulation of CD163 was higher with Propofol (p = 0.005). Starting from T3, the up-regulatory trend of CD163 was basically higher with Propofol, although not significantly. Propofol induced higher increments of HLA-DR low fractions, at T2 (p = 0.04) and, to a lesser extent, at T4 (p = 0.06). Starting from T3, the CD163highHLA-DRlow subset variations were higher with Propofol, especially at T4 and T6. CONCLUSION: Propofol seems to induce a higher postoperative fraction of the CD163highHLA-DRlow monocyte subset. This could represent either a compensatory mechanism dampening the higher inflammatory condition observed with Propofol at T2 or a consequence of a higher postoperative Propofol-induced hemolysis.

Association of Circulating Neutrophils with Relative Volume of Lipid-Rich Necrotic Core of Coronary Plaques in Stable Patients: A Substudy of SMARTool European Project.

BACKGROUND AND AIMS: Coronary atherosclerosis is a chronic non-resolving inflammatory process wherein the interaction of innate immune cells and platelets plays a major role. Circulating neutrophils, in particular, adhere to the activated endothelium and migrate into the vascular wall, promoting monocyte recruitment and influencing plaque phenotype and stability at all stages of its evolution. We aimed to evaluate, by flow cytometry, if blood neutrophil number and phenotype-including their phenotypic relationships with platelets, monocytes and lymphocytes-have an association with lipid-rich necrotic core volume (LRNCV), a generic index of coronary plaque vulnerability, in a group of stable patients with chronic coronary syndrome (CCS). METHODS: In 55 patients, (68.53 ± 1.07 years of age, mean ± SEM; 71% male), the total LRNCV in each subject was assessed by a quantitative analysis of all coronary plaques detected by computed tomography coronary angiography (CTCA) and was normalized to the total plaque volume. The expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1, CXCR4 and CD41a cell surface markers was quantified by flow cytometry. Adhesion molecules, cytokines and chemokines, as well as MMP9 plasma levels, were measured by ELISA. RESULTS: On a per-patient basis, LRNCV values were positively associated, by a multiple regression analysis, with the neutrophil count (n°/µL) (p = 0.02), neutrophil/lymphocyte ratio (p = 0.007), neutrophil/platelet ratio (p = 0.01), neutrophil RFI CD11b expression (p = 0.02) and neutrophil-platelet adhesion index (p = 0.01). Significantly positive multiple regression associations of LRNCV values with phenotypic ratios between neutrophil RFI CD11b expression and several lymphocyte and monocyte surface markers were also observed. In the bivariate correlation analysis, a significantly positive association was found between RFI values of neutrophil-CD41a+ complexes and neutrophil RFI CD11b expression (p < 0.0001). CONCLUSIONS: These preliminary findings suggest that a sustained increase in circulating neutrophils, together with the up-regulation of the integrin/activation membrane neutrophil marker CD11b may contribute, through the progressive intra-plaque accumulation of necrotic/apoptotic cells exceeding the efferocytosis/anti-inflammatory capacity of infiltrating macrophages and lymphocytes, to the relative enlargement of the lipid-rich necrotic core volume of coronary plaques in stable CAD patients, thus increasing their individual risk of acute complication.

The Positive Impact of Early Frailty Levels on Mortality in Elderly Patients with Severe Aortic Stenosis Undergoing Transcatheter/Surgical Aortic Valve Replacement.

Background: Frailty is highly common in older patients (pts) undergoing transcatheter aortic valve replacement (TAVR), and it is associated with poor outcomes. The selection of patients who can benefit from this procedure is necessary and challenging. The aim of the present study is to evaluate outcomes in older severe aortic valve stenosis (AS) pts, selected by a multidisciplinary approach for surgical, clinical, and geriatric risk and referred to treatment, according to frailty levels. Methods: A total of 109 pts (83 ± 5 years; females, 68%) with AS were classified by Fried's score in pre-frail, early frail, and frail and underwent surgical aortic valve replacement SAVR/TAVR, balloon aortic valvuloplasty, or medical therapy. We evaluated geriatric, clinical, and surgical features and detected periprocedural complications. The outcome was all-cause mortality. Results: Increasing frailty was associated with the worst clinical, surgical, geriatric conditions. By using Kaplan-Meier analysis, the survival rate was higher in pre-frail and TAVR groups (p < 0.001) (median follow-up = 20 months). By using the Cox regression model, frailty (p = 0.004), heart failure (p = 0.007), EF% (p = 0.043), albumin (p = 0.018) were associated with all-cause mortality. Conclusions: According to tailored frailty management, elderly AS pts with early frailty levels seem to be the most suitable candidates for TAVR/SAVR for positive outcomes because advanced frailty would make each treatment futile or palliative.

Blood M2-like Monocyte Polarization Is Associated with Calcific Plaque Phenotype in Stable Coronary Artery Disease: A Sub-Study of SMARTool Clinical Trial.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease. The balance between pro- and anti-inflammatory factors, acting on the arterial wall, promotes less or more coronary plaque macro-calcification, respectively. We investigated the association between monocyte phenotypic polarization and CTCA-assessed plaque dense-calcium volume (DCV) in patients with stable coronary artery disease (CAD). METHODS: In 55 patients, individual DCV component was assessed by quantitative CTCA and normalized to total plaque volume. Flow cytometry expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1 and CXCR4 was quantified. Adhesion molecules and cytokines were measured by ELISA. RESULTS: DCV values were significantly associated, by multiple regression analysis, with the expression (RFI) of CCR5 (p = 0.04), CX3CR1 (p = 0.03), CCR2 (p = 0.02), CD163 (p = 0.005) on all monocytes, and with the phenotypic M2-like polarization ratio, RFI CCR5/CD11b (p = 0.01). A positive correlation with the increased expression of chemokines receptors CCR2, CCR5 and CX3CR1 on subsets Mon1 was also present. Among cytokines, the ratio between IL-10 and IL-6 was found to be strongly associated with DCV (p = 0.009). CONCLUSIONS: The association between DCV and M2-like phenotypic polarization of circulating monocytes indicates that plaque macro-calcification in stable CAD may be partly modulated by an anti-inflammatory monocyte functional state, as evidenced by cell membrane receptor patterns.

Primary and Memory Response of Human Monocytes to Vaccines: Role of Nanoparticulate Antigens in Inducing Innate Memory.

Innate immune cells such as monocytes and macrophages are activated in response to microbial and other challenges and mount an inflammatory defensive response. Exposed cells develop the so-called innate memory, which allows them to react differently to a subsequent challenge, aiming at better protection. In this study, using human primary monocytes in vitro, we have assessed the memory-inducing capacity of two antigenic molecules of Schistosoma mansoni in soluble form compared to the same molecules coupled to outer membrane vesicles of Neisseria lactamica. The results show that particulate challenges are much more efficient than soluble molecules in inducing innate memory, which is measured as the production of inflammatory and anti-inflammatory cytokines (TNFα, IL-6, IL-10). Controls run with LPS from Klebsiella pneumoniae compared to the whole bacteria show that while LPS alone has strong memory-inducing capacity, the entire bacteria are more efficient. These data suggest that microbial antigens that are unable to induce innate immune activation can nevertheless participate in innate activation and memory when in a particulate form, which is a notion that supports the use of nanoparticulate antigens in vaccination strategies for achieving adjuvant-like effects of innate activation as well as priming for improved reactivity to future challenges.

Contact-dependent stimulation and inhibition of dendritic cells by natural killer cells.

Natural killer (NK) cells and dendritic cells (DCs) are two distinct cell types of innate immunity. It is known that the in vitro interaction of human NK cells with autologous DCs results in DC lysis. Here we show that contact-dependent interactions between activated human NK cells and immature DCs (iDCs) provides a "control switch" for the immune system. At low NK/DC ratios, this interaction dramatically amplifies DC responses, whereas at high ratios it completely turns off their responses. Specifically, culture of activated human NK cells with iDCs, at low NK/DC ratios (1:5), led to exponential increases in DC cytokine production, which were completely dependent on cell-to-cell contact. DC maturation was also driven by cognate interactions with NK cells and maturation was dependent on endogenously produced TNF-alpha in the culture. At slightly higher NK/DC ratios (5:1), inhibition of DC functions was the dominant feature due to potent killing by the autologous NK cells. Resting NK cells also stimulated autologous DC maturation in a TNF-alpha/contact-dependent manner, however, increasing the NK/DC ratio only led to an enhancement of this effect.

Vascular and valvular calcification biomarkers.

Vascular and valvular calcification constitutes a major health problem with serious clinical consequences. It is important for medical laboratorians to improve their knowledge on this topic and to know which biological markers may have a potential interest and might be useful for diagnosis and for management of ectopic calcifications. This review focuses on the pathophysiological mechanisms of vascular and valvular calcification, with emphasis on the mechanisms that are different for the two types of events, which underscore the need for differentiated healthcare, and explain different response to therapy. Available imaging and scoring tools used to assess both vascular and valvular calcification, together with the more studied and reliable biological markers emerging in this field (e.g., Fetuin A and matrix Gla protein), are discussed. Recently proposed functional assays, measuring the propensity of human serum to calcify, appear promising for vascular calcification assessment and are described. Further advancement through omic technologies and statistical tools is also reported. Clinical chemistry and laboratory medicine practitioners overlook this new era that will engage them in the near future, where a close cooperation of professionals with different competencies, including laboratorists, is required. This innovative approach may truly revolutionize practice of laboratory and of whole medicine attitude, making progression in knowledge of pathways relevant to health, as the complex calcification-related pathways, and adding value to patient care, through a precision medicine strategy.

Blood Monocyte Phenotype Fingerprint of Stable Coronary Artery Disease: A Cross-Sectional Substudy of SMARTool Clinical Trial.

BACKGROUND AND AIMS: Atherosclerosis is an inflammatory disease with long-lasting activation of innate immunity and monocytes are the main blood cellular effectors. We aimed to investigate monocyte phenotype (subset fraction and marker expression) at different stages of coronary atherosclerosis in stable coronary artery disease (CAD) patients. METHODS: 73 patients with chronic coronary syndrome were evaluated by CT coronary angiography (CTCA) and classified by maximal diameter stenosis of major vessels into three groups of CAD severity: CAD1 (no CAD/minimal CAD, n° = 30), CAD2 (non-obstructive CAD, n° = 21), and CAD3 (obstructive CAD, n° = 22). Flow cytometry for CD14, CD16, and CCR2 was used to quantify Mon1, Mon2, and Mon3 subsets. Expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1, and CXCR4 was also measured. Adhesion molecules and cytokines were quantified by ELISA. RESULTS: Total cell count and fraction of Mon2 were higher in CAD2 and CAD3 compared to CAD1. By multivariate regression analysis, Mon2 cell fraction and Mon2 expression of CX3CR1, CD18, and CD16 showed a statistically significant and independent increase, parallel to stenosis severity, from CAD1 to CAD2 and CAD3 groups. A similar trend was also present for CX3CR1 and HLA-DR expressions on total monocyte population. A less calcified plaque composition was associated to a higher Mon2 expression of CD16 and higher TNF-α levels. IL-10 levels were lower at greater stenosis severity, while the IFN-γ/IL-10 ratio, a marker of a systemic pro-inflammatory imbalance, was directly correlated to stenosis degree and number of noncalcified plaques. CONCLUSIONS: The results of this study suggest that a specific pattern of inflammation-correlated monocyte marker expression is associated to higher stenosis severity and less calcified lesions in stable CAD. The clinical trial Identifier is NCT04448691.

Relationships Between Phenotype and Function of Blood CD4+ T-Cells and Ascending Thoracic Aortic Aneurysm: an Experimental Study.

INTRODUCTION: Non-familial ascending thoracic aorta dilation and aneurysms (TAAs) are silent diseases in elderly patients. Histopathology revealed that functionally polarized infiltrating CD4+ T-cells play a key role in aortic wall weakening. OBJECTIVE: To evaluate the possible associations between phenotype and cytokine production of circulating CD4+ T-lymphocytes and the presence of TAA in patients with aortic valve disease (AVD). METHODS: We studied blood samples from 10 patients with TAA and 10 patients with AVD. Flow cytometry was used to quantify: a) CD4+ T-lymphocytes surface expression of CD25, CD28, and chemokine receptors (CCR5, CXCR3, CX3CR1); b) fractions of in vitro stimulated CD4+ T-cells producing cytokines (interferon gamma [IFN-γ], interleukin [IL]-17A, IL-21, IL-10); c) CD4+CD25highFoxP3+ regulatory T-cells (Treg) fraction. Enzyme-linked immunosorbent assays (ELISA) were performed for cytokines (IFN-γ, IL-6, IL-10, IL-17A, IL-23, transforming growth factor beta [TGF-ß]) and chemokines (RANTES, CX3CL1). RESULTS: The total CD4+CD28±CD4+/CX3CR1+ T-cells fraction was higher (P=0.0323) in AVD (20.452±4.673) than in TAA patients (8.633±2.030). The frequency ratio of CD4+ T-lymphocytes producing IFN-γ vs. IL-17A+IL-21 cytokine-producing CD4+ T-cells was higher (P=0.0239) in AVD (2.102±0.272) than in TAA (1.365±0.123) patients. The sum of CD4+CD28±CD4+/CX3CR1+ T-cells correlated positively with values of the previous cytokine ratio (P=0.0002, R=0.732). The ratio of CD4+CD28±CD4+/CX3CR1+ T-cells vs. Treg was higher (P=0.0008) in AVD (20.859±3.393) than in TAA (6.367±1.277) patients. CONCLUSION: Our results show that the presence of TAA in subjects with AVD is associated with imbalance between phenotypic and cytokine-producing subsets of circulating CD4+ T-lymphocytes, prevalently oriented towards a pro-fibrotic and IFN-γ counteracting effect to functional polarization.

Different inflammatory profile in young and elderly STEMI patients undergoing primary percutaneous coronary intervention (PPCI): Its influence on no-reflow and mortality.

BACKGROUND: Coronary no-reflow phenomenon in ST-segment elevation myocardial infarction (STEMI) is associated with a poor clinical prognosis. Although its pathophysiology is not fully elucidated, a deregulated systemic inflammatory response plays an important role. Specifically, the relationship between age-associated differences in inflammatory markers and either no-reflow or mortality in STEMI patients undergoing primary percutaneous coronary intervention (pPCI) has never been investigated. METHODS AND RESULTS: We retrospectively enrolled 625 consecutive STEMI patients undergoing pPCI for whom a complete laboratory inflammatory pattern was available. Routinely blood measured laboratory parameters were collected at the moment of admission. No reflow was defined as Thrombolysis in Myocardial Infarction (TIMI) flow-grade lower than 3. The population was divided into two groups using a cut-off centered at 65 years. Compared to younger patients, elderly patients had higher mean values of fibrinogen, brain natriuretic peptide (BNP), leukocytes, neutrophil-to-lymphocyte ratio (NLR), C reactive protein/albumin ratio (CAR). Conversely, lymphocyte count and albumin levels were higher in young patients. In elderly patients, the values of NLR, CAR as well as leukocytes, fibrinogen and neutrophils were associated with no-reflow, while in young patients only BNP value was associated. At multivariate Cox regression analysis, only BNP and NLR resulted as independent predictors of all-cause mortality in the whole population and in elderly patients. CONCLUSIONS: Elderly STEMI patients on admission had a higher acute pro-inflammatory profile than young patients, associated to coronary no-reflow and mortality outcome. These results suggest that a different therapeutic approach between elderly and young STEMI patients should be agreed.

Vascular Function Is Improved After an Environmental Enrichment Program: The Train the Brain-Mind the Vessel Study.

Environmental enrichment may slow cognitive decay possibly acting through an improvement in vascular function. Aim of the study was to assess the effects of a 7-month cognitive, social, and physical training program on cognitive and vascular function in patients with mild cognitive impairment. In a single-center, randomized, parallel-group study, 113 patients (age, 65-89 years) were randomized to multidomain training (n=55) or usual care (n=58). All participants underwent neuropsychological tests and vascular evaluation, including brachial artery flow-mediated dilation, carotid-femoral pulse wave velocity, carotid distensibility, and assessment of circulating hematopoietic CD34+ and endothelial progenitor cells. At study entry, an age-matched control group (n=45) was also studied. Compared with controls, patients had at study entry a reduced flow-mediated dilation (2.97±2.14% versus 3.73±2.06%; P=0.03) and hyperemic stimulus (shear rate area under the curve, 19.1±15.7 versus 25.7±15.1×10-3; P=0.009); only the latter remained significant after adjustment for confounders (P=0.03). Training improved Alzheimer disease assessment scale cognitive (training, 14.0±4.8 to 13.1±5.5; nontraining, 12.1±3.9 to 13.2±4.8; P for interaction visit×training=0.02), flow-mediated dilation (2.82±2.19% to 3.40±1.81%, 3.05±2.08% to 2.24±1.59%; P=0.006; P=0.023 after adjustment for diameter and shear rate area under the curve), and circulating hematopoietic CD34+ cells and prevented the decline in carotid distensibility (18.4±5.3 to 20.0±6.6, 23.9±11.0 to 19.5±7.1 Pa-1; P=0.005). The only clinical predictor of improvement of cognitive function after training was established hypertension. There was no correlation between changes in measures of cognitive and vascular function. In conclusion, a multidomain training program slows cognitive decline, especially in hypertensive individuals. This effect is accompanied by improved systemic endothelial function, mobilization of progenitor CD34+ cells, and preserved carotid distensibility. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01725178.

Granulocyte- and monocyte-platelet adhesion index in coronary and peripheral blood after extracorporeal circulation and reperfusion.

BACKGROUND: Neutrophil-granulocyte and mononuclear-cell functional changes occur during cardiopulmonary bypass and cardiovascular surgery. In particular, leukocyte-platelet interaction, leading to generation of heterotypic coaggregates, represents an amplification mechanism of the local inflammatory response and tissue damage. METHODS: Samples of 20 patients were drawn from venous coronary sinus before cardioplegic arrest and immediately after reperfusion, as well as from peripheral blood at 5 and 24 h postoperatively. The granulocyte and monocyte surface expression of CD162, CD15s, CD18, and CD11b were quantified by flow cytometry at the different times. Parallel variations of circulating leukocyte-platelet conjugates (percentages) and a derived (cell number-normalized) leukocyte-platelet adhesion index were measured using a combination of antibodies against CD45, CD14, and CD41a. The evaluation of platelet functional state was carried out using antibodies against CD62P (P-selectin) and PAC-1. RESULTS: Monocyte and granulocyte cell number increased markedly in coronary blood at reperfusion and in peripheral blood postoperatively when compared with measurements done before cardioplegia. A very different course characterized the changes of the leukocyte-platelet adhesion index with respect to the variations of circulating leukocyte-platelet coaggregates (percentages). Leukocyte molecules expression showed no significant variations for CD15s on both the leukocyte subsets, while a significant up-modulation for CD162 was observed on monocytes at 24 h after extracorporeal circulation (P = 0.0002), and for CD11b on granulocytes at 5 h postoperatively (P = 0.033). A loss of CD162 expression was observed in coronary blood at reperfusion (P = 0.0038) on granulocytes, associated to a down-modulation of CD18 (P = 0.0033) and CD11b (P = 0.0184) in peripheral blood at 24 h postoperatively. No significant up-regulation of platelet activatory molecules expression was found at coronary reperfusion, as well as postoperatively in the peripheral blood, when compared with the before-cardioplegia derived data. CONCLUSIONS: The over time variations of a normalized leukocyte-platelet adhesion index seem to reflect the cumulative leukocyte-platelet functional interaction more accurately than the parallel measurements of cellular conjugates. The absence of platelet activation suggests that the leukocyte membrane modifications play a main role in controlling the formation and stability of heterotypic leukocyte-platelet coaggregates after cardiac surgery with extracorporeal circulation.

Platelet activation predicts recurrent ischemic events after percutaneous coronary angioplasty: a 6 months prospective study.

INTRODUCTION: An increasing amount of evidence indicates that platelet reactivity, despite a standard anti-thrombotic therapy, is a potential risk factor for recurrent myocardial ischemia in patients with coronary artery disease. We now hypothesize that this condition, measured by collagen-epinephrine (CEPI) or collagen-ADP (CADP) closure times (CT) by Platelet Function Analyzer (PFA-100), may predict the recurrence of coronary events after percutaneous coronary intervention (PCI). MATERIALS AND METHODS: CEPI and CADP-CT were measured 30+/-8 h after PCI in 175 consecutive patients admitted with a diagnosis of stable angina (n=94) or acute coronary syndromes (n=81) and prospectively followed up for a mean period of 6 months. We stratified the patients in accordance to both the CEPI-CT ( 190 s), reflecting the intensity of cycloxygenase inhibition by aspirin and the distribution into quartiles for CADP-CT. RESULTS: CEPI-CT<190 s as well as CADP-CT<82 s were associated with a higher rate of clinical recurrence (hazard ratio 8.5, p<0.001 and 22.9, p<0.001, respectively). Multivariate analysis after adjustment for other risk factors confirmed that the lowest CADP-CT quartile significantly correlates with the risk of recurrent coronary events (hazard ratio 36.5, p<0.01), as well as CEPI-CT<190 s (hazard ratio 6.7, p=0.01). CONCLUSIONS: An enhanced platelet function after PCI when measured under high shear rates by PFA-100 is an independent predictor of a worst clinical outcome, even during a short term follow-up and may help in patients risk stratification.

Primary and memory response of human monocytes to vaccines: role of nanoparticulate antigens in inducing innate memory

Innate immune cells such as monocytes and macrophages are activated in response to microbial and other challenges and mount an inflammatory defensive response. Exposed cells develop the so-called innate memory, which allows them to react differently to a subsequent challenge, aiming at better protection. In this study, using human primary monocytes in vitro, we have assessed the memory-inducing capacity of two antigenic molecules of Schistosoma mansoni in soluble form compared to the same molecules coupled to outer membrane vesicles of Neisseria lactamica. The results show that particulate challenges are much more efficient than soluble molecules in inducing innate memory, which is measured as the production of inflammatory and anti-inflammatory cytokines (TNFα, IL-6, IL-10). Controls run with LPS from Klebsiella pneumoniae compared to the whole bacteria show that while LPS alone has strong memory-inducing capacity, the entire bacteria are more efficient. These data suggest that microbial antigens that are unable to induce innate immune activation can nevertheless participate in innate activation and memory when in a particulate form, which is a notion that supports the use of nanoparticulate antigens in vaccination strategies for achieving adjuvant-like effects of innate activation as well as priming for improved reactivity to future challenges.

Neoangiogenesis, T-lymphocyte infiltration, and heat shock protein-60 are biological hallmarks of an immunomediated inflammatory process in end-stage calcified aortic valve stenosis.

OBJECTIVES: We investigated the main biomolecular features in the evolution of aortic stenosis, focusing on advanced lesions. BACKGROUND: "Degenerative" aortic valve stenosis shares risk factors and inflammatory similarities with atherosclerosis. METHODS: We compared nonrheumatic stenotic aortic valves from 26 patients undergoing surgical valve replacement (group A) and 14 surgical control patients (group B). We performed semiquantitative histological and immunohistochemical analyses on valve leaflets to measure inflammation, sclerosis, calcium, neoangiogenesis, and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. We assessed heat shock protein 60 (hsp60) gene expression as an index of cellular stress and C-reactive protein, erythrocyte sedimentation rate, and fibrinogen as systemic inflammatory markers. RESULTS: In group A valves, we found a prevalence of calcium nodules surrounded by activated inflammatory infiltrates, neovessels, and abundant ICAM-1, VCAM-1, and hsp60 gene expression. Specimens from group B were negative for all of these markers, except 2 of 14 positivity for hsp60. The presence of active inflammatory infiltrates correlated with an abundance of thin neovessels (p < 0.01) and hsp60 gene expression (p = 0.01), whereas neoangiogenesis correlated with inflammation (p = 0.04), calcium (p = 0.01), and hsp60 gene expression (p = 0.04). CONCLUSIONS: "Degenerative" aortic valve stenosis appears to be a chronic inflammatory process associated with atherosclerotic risk factors. The coexistence of neoangiogenesis, T-lymphocyte infiltration, adhesion molecules, and hsp60 gene expression indicates an active immunomediated process in the final phases of the disease.

Post-reperfusion changes of monocyte function in coronary blood after extracorporeal circulation.

BACKGROUND: Neutrophil and mononuclear cell functional changes represent a hallmark of inflammation during cardiopulmonary bypass and cardiovascular surgery. Knowledge of mechanisms underlying monocyte functional modulation in coronary blood may be useful to develop protective interventions that can limit ischemia/reperfusion injury. METHODS: Samples of 13 patients were drawn from venous coronary sinus before cardioplegic arrest and after reperfusion. The following parameters were studied: surface molecules expression (CD18, CD11b, CD44, CD162, CD15s, CD80, CD86, CD16, CD49d, CD29, CD25, HLA-DR, Toll-like receptor-4 [TLR-4], CXCR1, CCR2, CCR5, CX3CR1), oxidative burst response, monocyte-platelet conjugates (using antibodies against CD45, CD14, CD41a), and platelet activation (CD62P, PAC-1). Enzyme-linked immunosorbent assays were performed to measure levels of interleukin (IL)-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-alpha). RESULTS: Coronary reperfusion down-modulated monocyte molecules expression, especially for CD18 (P = 0.048), CD44 (P = 0.0035), CD49d (P = 0.0029), CD29 (P = 0.032), HLA-DR (P < 0.0001), TLR-4 (P = 0.0109), CCR2 (P = 0.0184), CCR5 (P = 0.0396), and CX3CR1 (P < 0.0001). A marginal increase (P = 0.062) of a normalized adhesion index between monocytes and platelets was observed at reperfusion. No variations were detected for the monocyte oxidative burst and platelet activation. Increased levels of IL-6 (P = 0.013), TNF-alpha (P = 0.0272), and IL-10 (P = 0.0008) were measured after cardioplegia. CONCLUSIONS: The lack of CD11b and CD25 variations and of the oxidative burst exclude monocyte activation at reperfusion. The high after-cardioplegia level of IL-10, the decreased expression of HLA-DR and TLR-4, and the absence of IL-1beta and IL-8 suggest an IL-10-mediated functional depression of monocyte, including their adhesive and migratory capacities. The lack of an after-cardioplegia orientation toward IL-10 producing a "macrophage-like" CD14+/CD16+ phenotype might mean that myocardial infiltrating lymphocytes are the main source of IL-10. Moreover, the increased after-cardioplegia levels of IL-6 and TNF-alpha might be due to myocardial and endothelial activations. The increased adhesion index between monocyte and platelets, without receptor variations, suggests a monocyte membrane modification induced by extracorporeal circulation.

Oligonucleotide biofunctionalization enhances endothelial progenitor cell adhesion on cobalt/chromium stents.

As the endothelium still represents the ideal surface for cardiovascular devices, different endothelialization strategies have been attempted for biocompatibility and nonthrombogenicity enhancement. Since endothelial progenitor cells (EPCs) could accelerate endothelialization, preventing thrombosis and restenosis, the aim of this study was to use oligonucleotides (ONs) to biofunctionalize stents for EPC binding. In order to optimize the functionalization procedure before its application to cobalt-chromium (Co/Cr) stents, discs of the same material were preliminarily used. Surface aminosilanization was assessed by infrared spectroscopy and scanning electron microscopy. A fluorescent endothelial-specific ON was immobilized on aminosilanized surfaces and its presence was visualized by confocal microscopy. Fluorescent ON binding to porcine blood EPCs was assessed by flow cytometry. Viability assay was performed on EPCs cultured on unmodified, nontargeting ON or specific ON-coated discs; fluorescent staining of nuclei and F-actin was then performed on EPCs cultured on unmodified or specific ON-coated discs and stents. Disc biofunctionalization significantly increased EPC viability as compared to both unmodified and nontargeting ON-coated surfaces; cell adhesion was also significantly increased. Stents were successfully functionalized with the specific ON, and EPC binding was confirmed by confocal microscopy. In conclusion, stent biofunctionalization for EPC binding was successfully achieved in vitro, suggesting its use to obtain in vivo endothelialization, exploiting the natural regenerative potential of the human body.

Synthetic analogues of flavonoids with improved activity against platelet activation and aggregation as novel prototypes of food supplements.

We investigated the ability of quercetin and apigenin to modulate platelet activation and aggregation, and compared the observed efficacy with that displayed by their synthetic analogues 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 1-4, and 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 5-7. Platelet aggregation was explored through a spectrophotometric assay on platelet-rich plasma (PRP) treated with the thromboxane A2 mimetic U46619, collagen and thrombin in presence/absence of various bioisosteres of flavonoids (12.5-25-50-100 µM). The platelet density, (mean platelet component, MPC), was measured by the Advia 120 Hematology System as a marker surrogate of platelet activation. The induced P-selectin expression, which reflects platelet degranulation/activation, was quantified by flow cytometry on PRP. Our synthetic compounds modulated significantly both platelet activation and aggregation, thus turning out to be more effective than the analogues quercetin and apigenin when tested at a concentration fully consistent with their use in vivo. Accordingly, they might be used as food supplements to increase the efficacy of natural flavonoids.

Monitoring of monocyte functional state after extracorporeal circulation: a flow cytometry study.

BACKGROUND: Cardiovascular surgery with cardiopulmonary bypass (CPB) induces systemic inflammation and postoperative complications depending on pro- and anti-inflammatory mechanisms. Activated polymorphonuclear cells and monocytes may be responsible for morbidity associated with CPB. Knowledge of the monocyte functional state in particular may help to develop protective interventions. METHODS: Samples were drawn from venous peripheral blood (basal condition, at 4 and 24 h after CPB) and coronary blood (before and after cardioplegic arrest) of 14 patients undergoing cardiac surgery. The following phenotypic and functional parameters of the monocyte population were studied by flow cytometry: surface molecules expression (CD18, CD11a, CD11b, CD14, CD15, CD45, HLA-DR, and Toll-like receptor [TLR]-4), myeloperoxidase (MPO) content, and intracellular cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, and IL-8). RESULTS: Cardiac surgery with CPB induced down-modulation of surface molecules expression on peripheral monocytes, especially at 24 h after CPB, for CD18, CD11a, and CD11b (P < 0.003) and for the CD15 adhesive cluster (P = 0.0028) and HLA-DR (P < 0.001). At 4 h after CPB, downregulation was observed for CD14 (P = 0.004), CD45 (P = 0.014), and CD15 (P = 0.0056). A loss of MPO was detected in venous peripheral (at 24 h after CPB, P = 0.01) or coronary (at reperfusion, P < 0.02) blood. The CD15 cluster complex exhibited a down-modulation in coronary blood (at reperfusion, P = 0.0003). Spontaneous intracellular production of IL-1beta, IL-6, and IL-8 decreased at 24 h after CPB (P < 0.05). CONCLUSIONS: The down-modulation of integrins and adhesive receptor expression and the loss of MPO suggest a strong activation and shedding reaction of circulating monocyte after CPB, further exacerbated by contact with coronary ischemic vessels. The changes of differentiation antigens may reflect the appearance of a partially immature population immediately after CPB. The reduced proinflammatory cytokine production, observed at 24 h after CPB, suggests a functional polarization of circulating monocytes.