David A. Rubenstein

Differences Between Mainstream and Sidestream Cigarette Smoke Extracts and Nicotine in the Activation of Platelets Under Static and Flow Conditions

Background—Cigarette smoke is a primary risk factor for cardiovascular diseases. Enhanced function of the hemostatic system, in which platelets play a major role, is a significant underlying mechanism in cardiovascular disease and its progression. Epidemiological studies, complemented by physiological and biochemical data, show that cigarette smoke adversely affects platelet function, both in smokers and in nonsmokers exposed to sidestream smoke. Methods and Results—The thrombogenic potential of platelets subjected to mainstream smoke extracts, sidestream extracts, and nicotine was measured in vitro under static and dynamic flow conditions. Platelet activation state was measured with a modified prothrombinase-based method. Mainstream and sidestream smoke extracts caused increased platelet activation. Although low-tar mainstream extracts activated platelets less than high-tar extracts, the sidestream extracts were almost equally potent. Modification of the filters of low-tar cigarettes, by blocking the air-bypass holes, raised activation rates by mainstream extracts to the level of high-tar extracts. Nicotine (50 nmol/L and 5 &mgr;mol/L) inhibited platelet activation under both flow and static conditions. Conclusions—Cigarette smoke extracts directly cause platelet activation but also markedly increase the susceptibility of platelets to activation by shear stress. In contrast, nicotine, although also a constituent of cigarette smoke, significantly reduces platelet susceptibility to shear stress.

The effect of physiologically relevant dynamic shear stress on platelet and endothelial cell activation.

INTRODUCTION Blood flow induced shear stress plays an important role in platelet and endothelial cell functions. The goal of this study was to investigate the effect of physiologically relevant dynamic shear stress on platelet and endothelial cells. MATERIALS AND METHODS Pulsatile shear stress waveforms mimicking the flow in a normal left coronary artery (0.1-1 Pa), at a 60% stenosis (0.2 - 6 Pa) and in the recirculation zone (0.01 - 0.5 Pa) behind a stenosis were used to stimulate platelets and endothelial cells in a cone and plate shearing device. Platelet activation was measured by CD62P expression and thrombogenicity. Meanwhile, endothelial cell activation and damage was measured by cell surface ICAM-1 and tissue factor expression using fluorescence microscopy. Endothelial tissue factor activity was measured using a commercial kit. RESULTS Results showed that for platelets, a short exposure to elevated shear stress at the stenosis throat did not induce significant increase in platelet activation or thrombogenicity. While the low pulsatile shear stress had a potential for enhanced thrombosis. Both low and high pulsatile shear stress led to a significant increase in ICAM-1 expression on endothelial cell surface, but only low shear stress caused tissue factor over expression and enhanced tissue factor activity. CONCLUSION These results suggest that low pulsatile shear stress may be more atherogenic, compared to elevated shear stress induced by stenosis.

Effect of Geometrical Assumptions on Numerical Modeling of Coronary Blood Flow Under Normal and Disease Conditions

Shear stress plays a pivotal role in pathogenesis of coronary heart disease. The spatial and temporal variation in hemodynamics of blood flow, especially shear stress, is dominated by the vessel geometry. The goal of the present study was to investigate the effect of 2D and 3D geometries on the numerical modeling of coronary blood flow and shear stress distribution. We developed physiologically realistic 2D and 3D models (with similar geometries) of the human left coronary artery under normal and stenosis conditions (30%, 60%, and 80%) using PROE (WF 3). Transient blood flows in these models were solved using laminar and turbulent (k-ω) models using a computational fluid dynamics solver, FLUENT (v6.3.26). As the stenosis severity increased, both models predicted a similar pattern of increased shear stress at the stenosis throat, and in recirculation zones formed downstream of the stenosis. The 2D model estimated a peak shear stress value of 0.91, 2.58, 5.21, and 10.09 Pa at the throat location under normal, 30%, 60%, and 80% stenosis severity. The peak shear stress values at the same location estimated by the 3D model were 1.41, 2.56, 3.15, and 13.31 Pa, respectively. The 2D model underestimated the shear stress distribution inside the recirculation zone compared with that of 3D model. The shear stress estimation between the models diverged as the stenosis severity increased. Hence, the 2D model could be sufficient for analyzing coronary blood flow under normal conditions, but under disease conditions (especially 80% stenosis) the 3D model was more suitable.

Glycated albumin modulates platelet susceptibility to flow induced activation and aggregation.

A diabetic vasculature promotes cardiovascular diseases via endothelial cell activation induced by advanced glycation end products. It has recently become clear that activated platelets are a hallmark of cardiovascular disease and diabetes progression, by initiating and/or perpetuating the endothelial cell response. However, the role that platelets play in diabetic cardiovascular diseases remains elusive. Our objective was to evaluate the effects of glycated serum albumin on flow induced platelet activation and platelet aggregation. Albumin was glycated for up to 8 weeks. Timed samples of glycated or non-glycated albumin were removed to determine the effects of the extent of glycation on platelet functional changes. Thrombin receptor agonist peptide 6 (TRAP6, residues 42-47 of the thrombin receptor) and collagen I induced platelet aggregation was measured as a time course of glycated albumin incubation. The thrombogenicity of platelets incubated with glycated albumin was also measured under static and dynamic flow conditions using the modified prothrombinase assay. CD41 and CD62P expression was examined using flow cytometry to validate aggregation and activation studies. Platelets subjected to glycated albumin were more susceptible to TRAP6- and collagen-induced aggregation and flow induced activation. The extent of albumin glycation modulates these changes. As the albumin glycation time increased, this enhancement in platelet function was more pronounced. These results indicate that under diabetic conditions activated platelets may act to promote cardiovascular disease progression.

In Vitro Biocompatibility of Sheath–Core Cellulose-Acetate-Based Electrospun Scaffolds Towards Endothelial Cells and Platelets

Typically, tissue-engineered scaffolds mimic the topographical properties of the native extracellular matrix. However, other physical properties, such as the scaffold mechanical stiffness, are not imitated. The purpose of this study was to fabricate scaffolds with improved mechanical properties and investigate their biocompatibility towards endothelial cells and platelets. To enhance mechanical properties, an electrospinning apparatus was developed that fabricates fibers with sheath–core morphologies. Different combinations of cellulose acetate and chitosan were chosen to modulate the mechanical properties of the formed fibers. We hypothesized that mechanically stiffer scaffolds would improve endothelial cell growth and that all scaffolds would be compatible towards endothelial cells and platelets. Endothelial cell-culture conditions were quantified up to 5 days. Migration onto scaffolds was monitored for 10 days. Platelet aggregation, antagonized by thrombin receptor agonist peptide 6, was measured after scaffold incubation. A platelet activation time-course was assessed with the modified prothrombinase assay. As scaffold mechanical stiffness increased, endothelial cell growth within and adhesion to and migration throughout the scaffolds was promoted. Also, scaffolds did not induce platelet aggregation or activation. These results indicate that the mechanical stiffness of engineered scaffolds regulates endothelial cell-culture parameters and that these sheath–core electrospun scaffolds are compatible towards endothelial cells and platelets.

Bioassay Chamber for Angiogenesis with Perfused Explanted Arteries and Electrospun Scaffolding

Objective: The purpose of this study was to test the hypothesis that explanted perfused arteries can serve as the initial endothelial cell culture source to evaluate the onset of angiogenesis in a cellulose acetate electrospun scaffold.

Tobacco and e-cigarette products initiate Kupffer cell inflammatory responses

Kupffer cells are liver resident macrophages that are responsible for screening and clearing blood of pathogens and foreign particles. It has recently been shown that Kupffer cells interact with platelets, through an adhesion based mechanism, to aid in pathogen clearance and then these platelets re-enter the general systemic circulation. Thus, a mechanism has been identified that relates liver inflammation to possible changes in the systemic circulation. However, the role that Kupffer cells play in cardiovascular disease initiation/progression has not been elucidated. Thus, our objective was to determine whether or not Kupffer cells are responsive to a classical cardiovascular risk factor and if these changes can be transmitted into the general systemic circulation. If Kupffer cells initiate inflammatory responses after exposure to classical cardiovascular risk factors, then this provides a potential alternative/synergistic pathway for cardiovascular disease initiation. We aimed to elucidate the prevalence of this potential pathway. We hypothesized that Kupffer cells would initiate a robust inflammatory response after exposure to tobacco cigarette or e-cigarette products and that the inflammatory response would have the potential to antagonize other salient cells for cardiovascular disease progression. To test this, Kupffer cells were incubated with tobacco smoke extracts, e-cigarette vapor extracts or pure nicotine. Complement deposition onto Kupffer cells, Kupffer cell complement receptor expression, oxidative stress production, cytokine release and viability and density were assessed after the exposure. We observed a robust inflammatory response, oxidative stress production and cytokine release after Kupffer cells were exposed to tobacco or e-cigarette extracts. We also observed a marginal decrease in cell viability coupled with a significant decrease in cell density. In general, this was not a function of the extract formulation (e.g. tobacco vs. e-cigarette products or the formulation of the cigarette product). These results indicate that Kupffer cells are responsive to classical cardiovascular risk factors and that an inflammatory response is initiated that may pass into the general systemic circulation.

Glycated Albumin Modulates Endothelial Cell Thrombogenic and Inflammatory Responses

Background: It has become established that a diabetic vasculature promotes cardiovascular disease progression via changes to endothelial cells, platelets, and the interactions of these cells. It is believed that the majority of these changes are induced by the presence of advanced glycation end products (AGEs), which permanently alter various functions. Studies have shown that platelets perpetuate endothelial cell responses under these conditions. However, the role of changes in endothelial cell thrombogenicity and inflammatory responses, after subjected to AGEs, has not been characterized. Our objective was to evaluate the effects of AGEs on these functions. Methods: To accomplish this, albumin was chemically modified by exposure to glucose for up to 8 weeks, and endothelial cells were subjected to glycated albumin for up to 5 days in a cell culture system. A time course for changes in endothelial cell viability, density, morphology, and metabolic activity were investigated, along with the surface expression of intercellular adhesion molecule-1, thrombomodulin, tissue factor, connexin-43, and caveolin-1. Results: Endothelial cells exposed to irreversibly glycated albumin were less viable, proliferated slower, and had a lower metabolic activity as compared to cells exposed to nonglycated albumin. Endothelial cells that were exposed to any glycated albumin were procoagulant and proinflammatory as compared with all other conditions. There were no overall trends in the expression of connexin-43 or caveolin-1. Conclusions: Our data suggest that the presence of irreversible glycated albumin is deleterious to endothelial cells, makes endothelial cells more procoagulant, and promotes inflammatory responses. It is therefore possible that endothelial cell activation may precede and promote platelet activation during diabetic conditions.

Platelet activation, adhesion, inflammation, and aggregation potential are altered in the presence of electronic cigarette extracts of variable nicotine concentrations.

Abstract Tobacco smoke extracts prepared from both mainstream and sidestream smoking have been associated with heightened platelet activation, aggregation, adhesion, and inflammation. Conversely, it has been shown that pure nicotine inhibits similar platelet functions. In this work, we 1) evaluated the effects of e-cigarette extracts on platelet activities and 2) elucidated the differences between the nicotine-dependent and non-nicotine dependent (e.g. fine particulate matter or toxic compounds) effects of tobacco and e-cigarette products on platelet activities. To accomplish these goals, platelets from healthy volunteers (n = 50) were exposed to tobacco smoke extracts, e-cigarette vapor extracts, and pure nicotine and changes in platelet activation, adhesion, aggregation, and inflammation were evaluated, using optical aggregation, flow cytometry, and ELISA methods. Interestingly, the exposure of platelets to e-vapor extracts induced a significant up-regulation in the expression of the pro-inflammatory gC1qR and cC1qR and induced a marked increase in the deposition of C3b as compared with traditional tobacco smoke extracts. Similarly, platelet activation, as measured by a prothrombinase based assay, and platelet aggregation were also significantly enhanced after exposure to e-vapor extracts. Finally, platelet adhesion potential toward fibrinogen, von Willebrand factor, and other platelets was also enhanced after exposure to e-cigarette vapor extracts. In the presence of pure nicotine, platelet functions were observed to be inhibited, which further suggests that other constituents of tobacco smoke and electronic vapor can antagonize platelet functions, however, the presence of nicotine in extracts somewhat perpetuated the platelet functional changes in a dose-dependent manner.

Quantifying the effects of shear stress and shear exposure duration regulation on flow induced platelet activation and aggregation

A heightened flow shear stress magnitude or increased exposure duration at any shear stress magnitude enhances platelet activation. It has been suggested that increased circulating activated platelets are an indication of enhanced cardiovascular risk and that activated platelets can perpetuate cardiovascular diseases. However, the combined effects that shear stress and shear exposure duration have on platelet activation and aggregation are not clear. Our objective was to identify a new parameter (“shear stress-exposure time”) to predict platelet functional changes better than the absolute magnitude of shear stress. Platelets were exposed to shear stress waveforms, with different combinations of shear stress magnitude and exposure duration, in a circulation flow loop. Timed samples were removed from the flow loop to quantify thrombogenicity (modified prothrombinase assay), aggregation potential (optical platelet aggregometry), and cell surface marker expression (flow cytometry). Flow induced platelet activation and aggregation was enhanced with increased shear stress-exposure time. Platelets that were exposed to waveforms with the same shear stress-exposure time had the same thrombogenicity and aggregation potential. This was true even for waveforms that exposed platelets to shear stress magnitudes varying from 4 to 40 dynes/cm2. These results indicate that shear stress-exposure time is a good predictor of platelet activation and aggregation levels and it may be able to predict the likelihood of cardiovascular disease onset.