Obesity and the Risk of Cryptogenic Ischemic Stroke in Young Adults.

OBJECTIVES: We examined the association between obesity and early-onset cryptogenic ischemic stroke (CIS) and whether fat distribution or sex altered this association. MATERIALS AND METHODS: This prospective, multi-center, case-control study included 345 patients, aged 18-49 years, with first-ever, acute CIS. The control group included 345 age- and sex-matched stroke-free individuals. We measured height, weight, waist circumference, and hip circumference. Obesity metrics analyzed included body mass index (BMI), waist-to-hip ratio (WHR), waist-to-stature ratio (WSR), and a body shape index (ABSI). Models were adjusted for age, level of education, vascular risk factors, and migraine with aura. RESULTS: After adjusting for demographics, vascular risk factors, and migraine with aura, the highest tertile of WHR was associated with CIS (OR for highest versus lowest WHR tertile 2.81, 95%CI 1.43-5.51; P=0.003). In sex-specific analyses, WHR tertiles were not associated with CIS. However, using WHO WHR cutoff values (>0.85 for women, >0.90 for men), abdominally obese women were at increased risk of CIS (OR 2.09, 95%CI 1.02-4.27; P=0.045). After adjusting for confounders, WC, BMI, WSR, or ABSI were not associated with CIS. CONCLUSIONS: Abdominal obesity measured with WHR was an independent risk factor for CIS in young adults after rigorous adjustment for concomitant risk factors.

Predicting the In Vivo Performance of Cardiovascular Biomaterials: Current Approaches In Vitro Evaluation of Blood-Biomaterial Interactions.

The therapeutic efficacy of a cardiovascular device after implantation is highly dependent on the host-initiated complement and coagulation cascade. Both can eventually trigger thrombosis and inflammation. Therefore, understanding these initial responses of the body is of great importance for newly developed biomaterials. Subtle modulation of the associated biological processes could optimize clinical outcomes. However, our failure to produce truly blood compatible materials may reflect our inability to properly understand the mechanisms of thrombosis and inflammation associated with biomaterials. In vitro models mimicking these processes provide valuable insights into the mechanisms of biomaterial-induced complement activation and coagulation. Here, we review (i) the influence of biomaterials on complement and coagulation cascades, (ii) the significance of complement-coagulation interactions for the clinical success of cardiovascular implants, (iii) the modulation of complement activation by surface modifications, and (iv) in vitro testing strategies.

The Role of Biodegradable Poly-(L-lactide)-Based Polymers in Blood Cell Activation and Platelet-Monocyte Interaction.

The main purpose of new stent technologies is to overcome unfavorable material-related incompatibilities by producing bio- and hemo-compatible polymers with anti-inflammatory and anti-thrombogenic properties. In this context, wettability is an important surface property, which has a major impact on the biological response of blood cells. However, the influence of local hemodynamic changes also influences blood cell activation. Therefore, we investigated biodegradable polymers with different wettability to identify possible aspects for a better prediction of blood compatibility. We applied shear rates of 100 s-1 and 1500 s-1 and assessed platelet and monocyte activation as well as the formation of CD62P+ monocyte-bound platelets via flow cytometry. Aggregation of circulating platelets induced by collagen was assessed by light transmission aggregometry. Via live cell imaging, leukocytes were tracked on biomaterial surfaces to assess their average velocity. Monocyte adhesion on biomaterials was determined by fluorescence microscopy. In response to low shear rates of 100 s-1, activation of circulating platelets and monocytes as well as the formation of CD62P+ monocyte-bound platelets corresponded to the wettability of the underlying material with the most favorable conditions on more hydrophilic surfaces. Under high shear rates, however, blood compatibility cannot only be predicted by the concept of wettability. We assume that the mechanisms of blood cell-polymer interactions do not allow for a rule-of-thumb prediction of the blood compatibility of a material, which makes extensive in vitro testing mandatory.

Methodology for comprehensive cell-level analysis of wound healing experiments using deep learning in MATLAB.

BACKGROUND: Endothelial healing after deployment of cardiovascular devices is particularly important in the context of clinical outcome. It is therefore of great interest to develop tools for a precise prediction of endothelial growth after injury in the process of implant deployment. For experimental investigation of re-endothelialization in vitro cell migration assays are routinely used. However, semi-automatic analyses of live cell images are often based on gray value distributions and are as such limited by image quality and user dependence. The rise of deep learning algorithms offers promising opportunities for application in medical image analysis. Here, we present an intelligent cell detection (iCD) approach for comprehensive assay analysis to obtain essential characteristics on cell and population scale. RESULTS: In an in vitro wound healing assay, we compared conventional analysis methods with our iCD approach. Therefore we determined cell density and cell velocity on cell scale and the movement of the cell layer as well as the gap closure between two cell monolayers on population scale. Our data demonstrate that cell density analysis based on deep learning algorithms is superior to an adaptive threshold method regarding robustness against image distortion. In addition, results on cell scale obtained with iCD are in agreement with manually velocity detection, while conventional methods, such as Cell Image Velocimetry (CIV), underestimate cell velocity by a factor of 0.5. Further, we found that iCD analysis of the monolayer movement gave results just as well as manual freehand detection, while conventional methods again shows more frayed leading edge detection compared to manual detection. Analysis of monolayer edge protrusion by ICD also produced results, which are close to manual estimation with an relative error of 11.7%. In comparison, the conventional Canny method gave a relative error of 76.4%. CONCLUSION: The results of our experiments indicate that deep learning algorithms such as our iCD have the ability to outperform conventional methods in the field of wound healing analysis. The combined analysis on cell and population scale using iCD is very well suited for timesaving and high quality wound healing analysis enabling the research community to gain detailed understanding of endothelial movement.

Platelet apelin receptor expression is reduced in patients with acute myocardial infarction.

BACKGROUND: The G-protein-coupled apelin receptor and its apelin ligand are an emerging regulatory system of the vascular homeostasis. To date, the implications of the apelin/apelin receptor system in athero-thrombosis are not completely clarified yet. This study determines the expression of the apelin receptor on human platelets, the effect of different apelin isoforms on platelet aggregation and the potential role of the apelin/apelin receptor system in acute myocardial infarction. METHODS: We applied immunofluorescence staining, Western Blot analysis, aggregometry, and flow cytometry to elucidate the role of the apelin receptor in activated platelets. Furthermore, in an observational pilot study, we assessed platelet apelin recpetor expression and apelin-17 plasma levels in patients with acute myocardial infarction (AMI, n = 27). RESULTS: Immunofluorescence staining indicates that the apelin receptor is located at the cell membrane in resting platelets and diminishes upon activation with a selective thrombin receptor-activating peptide (AP1, 3 to 100 µM). Western Blot analyses of AP1-activated platelets and their supernatants suggest that the apelin receptor is not predominantly internalized but is released from activated platelets. The isoform apelin-17 attenuated AP-1-induced platelet activation in-vitro, presumably via a NO-dependent mechanism. Furthermore, platelet apelin receptor expression was significantly reduced in patients with AMI (n = 27) compared to age-matched controls (n = 14; p < 0.05) and inversely correlated with troponin I plasma levels (r = -0.46; p = 0.03). Besides that, circulating apelin-17 was significantly reduced in MI patients compared to the control group. CONCLUSION: Taken together, our data support a crucial role of the platelet apelinergic system assuming an antithrombotic effect and therefore holding a potential diagnostic and therapeutic impact.

Acupuncture for Relief of Gag Reflex in Patients Undergoing Transoesophageal Echocardiography-A Protocol for a Randomized Placebo-Controlled Trial.

Background: Gagging during transesophageal echocardiography examination (TEE) can be distressing and even dangerous for patients. The needling of acupuncture point CV24 was described to be effective in reducing the gag reflex during TEE in patients with ischemic stroke or transient ischemic attack. Methods: We describe a proposal for a prospective, randomized, patient, practitioner and assessor-blinded, single-center trial with two arms/groups; real acupuncture will be compared to placebo acupuncture. A total of 60 (30 per group) patients scheduled for elective TEE in order to exclude a cardiac embolic source, endocarditis or for valve failure evaluation will be recruited according to patients' selection criteria and receive either indwelling fixed intradermal needles at acupoints CV24 and bilateral PC6 or placebo needles at the same areas. Patients, the practitioners who will perform the TEE procedure, and the assessor of the outcome measures will be unaware of the group's (real or placebo) allocation. Results: The primary outcome is the intensity of gagging, measured using verbal rating scale (VRS-11) from 0 = no gagging to 10 = intolerable gagging. Secondary outcomes include the incidence of gagging, the use of rescue medication, patients' satisfaction with relief of unwanted side effects during TEE procedure, success of patients' blinding (patients' opinion to group allocation), heart rate and oxygen saturation measured by pulse oxymetry. Conclusions: To study the effects of acupuncture against gagging during TEE, we test the needling of acupoints CV24 and PC6 bilaterally. A placebo acupuncture is used for the control group. Trial registration number: NCT NCT0382142.

The apelin receptor influences biomechanical and morphological properties of endothelial cells.

The adaption of endothelial cells to local flow conditions is a multifunctional process which leads to distinct alterations in cell shape, the subcellular distribution of structural proteins, and cellular function. G-protein-coupled receptors (GPCRs) have been identified to be fundamentally involved in such processes. Recently, we and others have shown that the expression of the endothelial GPCR apelin receptor (APJ) is regulated by fluid flow and that activation of APJ participates in signaling pathways which are related to processes of mechanotransduction. The present study aims to illuminate these findings by further visualization of APJ function. We show that APJ is located to the cellular junctions and might thus be associated with platelet endothelial cell adhesion molecule-1 (PECAM-1) in human umbilical vein endothelial cells (HUVEC). Furthermore, siRNA-mediated silencing of APJ expression influences the shear-induced adaption of HUVEC in terms of cytoskeletal remodeling, cellular elasticity, cellular motility, attachment, and distribution of adhesion complexes. Taken together, our results demonstrate that APJ is crucial for complemented endothelial adaption to local flow conditions.

Statins are related to impaired exercise capacity in males but not females.

BACKGROUND: Exercise and statins reduce cardiovascular disease (CVD). Exercise capacity may be assessed using cardiopulmonary exercise testing (CPET). Whether statin medication is associated with CPET parameters is unclear. We investigated if statins are related with exercise capacity during CPET in the general population. METHODS: Cross-sectional data of two independent cohorts of the Study of Health in Pomerania (SHIP) were merged (n = 3,500; 50% males). Oxygen consumption (VO2) at peak exercise (VO2peak) and anaerobic threshold (VO2@AT) was assessed during symptom-limited CPET. Two linear regression models related VO2peak with statin usage were calculated. Model 1 adjusted for age, sex, previous myocardial infarction, and physical inactivity and model 2 additionally for body mass index, smoking, hypertension, diabetes and estimated glomerular filtration rate. Propensity score matching was used for validation. RESULTS: Statin usage was associated with lower VO2peak (no statin: 2336; 95%-confidence interval [CI]: 2287-2,385 vs. statin 2090; 95%-CI: 2,031-2149 ml/min; P < .0001) and VO2@AT (no statin: 1,172; 95%-CI: 1,142-1,202 vs. statin: 1,111; 95%-CI: 1,075-1,147 ml/min; P = .0061) in males but not females (VO2peak: no statin: 1,467; 95%-CI: 1,417-1,517 vs. statin: 1,503; 95%-CI: 1,426-1,579 ml/min; P = 1.00 and VO2@AT: no statin: 854; 95%-CI: 824-885 vs. statin 864; 95%-CI: 817-911 ml/min; P = 1.00). Model 2 revealed similar results. Propensity scores analysis confirmed the results. CONCLUSION: In the general population present statin medication was related with impaired exercise capacity in males but not females. Sex specific effects of statins on cardiopulmonary exercise capacity deserve further research.

Impact of atrial fibrillation detected by extended monitoring-A population-based cohort study.

BACKGROUND: The clinical relevance of extended monitoring of AF in the general population is unclear. The study evaluated the detection of AF using transtelephonic electrocardiography and the clinical relevance of additional AF findings, especially with regard to stroke risk and mortality. METHODS: The data of 1678 volunteers participating in the tele-ECG-subproject of the Study of Health in Pomerania was evaluated. Occurrence of AF as revealed by tele-ECG and conventional ECG was evaluated. Associations with mortality, history of stroke, and other clinical parameters were analyzed. RESULTS: AF was detected in 21 subjects (1.3%) by conventional ECG (ECG-AF) and in 43 (2.6%) by tele-ECG. All individuals with AF revealed by conventional ECG were also diagnosed to have AF by tele-ECG; 22 were diagnosed by tele-ECG only (Tele-AF). During follow-up (median: 6.3 years) 42/1635, 1/22, and 5/21 participants died in the no-AF-, tele-AF-, and ECG-AF groups (p < .001). Whereas, in comparison to the no-AF group, the risk of death was higher in the ECG-AF group (HR 9.4; 3.7-23.8; p < .001), there was no significant increase in mortality in the tele-AF group (HR 1.9; 0.26-14.0; p = .52). Prevalence of stroke history was higher in the ECG-AF group (19%; 5.5-42%) than with the no-AF (1.9%; 1.3-2.7%; p = .001) and the tele-AF groups (0%; 0-15%; p = .05). CONCLUSIONS: Tele-ECG identifies significantly more AF cases in a population-based setting compared to conventional ECG. The impact of AF diagnosed only by extended monitoring differs from conventionally diagnosed AF. Additional studies are warranted, since this might have an impact on clinical management.

Biodegradable Polymers Influence the Effect of Atorvastatin on Human Coronary Artery Cells.

Drug-eluting stents (DES) have reduced in-stent-restenosis drastically. Yet, the stent surface material directly interacts with cascades of biological processes leading to an activation of cellular defense mechanisms. To prevent adverse clinical implications, to date almost every patient with a coronary artery disease is treated with statins. Besides their clinical benefit, statins exert a number of pleiotropic effects on endothelial cells (ECs). Since maintenance of EC function and reduction of uncontrolled smooth muscle cell (SMC) proliferation represents a challenge for new generation DES, we investigated the effect of atorvastatin (ATOR) on human coronary artery cells grown on biodegradable polymers. Our results show a cell type-dependent effect of ATOR on ECs and SMCs. We observed polymer-dependent changes in IC50 values and an altered ATOR-uptake leading to an attenuation of statin-mediated effects on SMC growth. We conclude that the selected biodegradable polymers negatively influence the anti-proliferative effect of ATOR on SMCs. Hence, the process of developing new polymers for DES coating should involve the characterization of material-related changes in mechanisms of drug actions.

The soluble guanylate cyclase stimulator riociguat and the soluble guanylate cyclase activator cinaciguat exert no direct effects on contractility and relaxation of cardiac myocytes from normal rats.

In cardiovascular diseases, reduced responsiveness of soluble guanylate cyclase (sGC) to nitric oxide (NO) upon long-term application has led to the development of NO-independent sGC stimulators (heme-dependent) and sGC activators (heme-independent). Any direct inotropic or lusitropic effects of these compounds on isolated cardiac myocytes, however, remain to be elucidated. Here, we analyzed the dose-dependent effects of clinical relevant concentrations (10(-10)-10(-5) M) of the sGC activator cinaciguat and the sGC stimulator riociguat on the contraction, relaxation, and calcium transients of isolated field-stimulated cardiac myocytes from healthy rats. For comparison, we used isoproterenol, which induced a dose-dependent significant increase in cell contractility, relaxation, and calcium transients, verapamil that significantly decreased these parameters (both at 10(-9)-10(-5) M) and 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) that induced a negative inotropic effect at 10(-5) M accompanied by a slight increase in relaxation. In contrast, neither cinaciguat nor riociguat significantly influenced any measured parameters. Furthermore, isoproterenol significantly increased intracellular cAMP levels that were not influenced by cinaciguat or riociguat (all at 10(-6) M). Otherwise, riociguat and cinaciguat (both at 10(-6) M) significantly enhanced intracellular cGMP generation. This accumulation was significantly augmented by cinaciguat in the presence of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 25 µM), whereas ODQ blocked cGMP generation by riociguat. However, blocking of sGC did not influence cell contractility. Our results demonstrate that, in isolated cardiac myocytes from healthy rats, the increase in cGMP levels induced by cinaciguat and riociguat at clinical relevant concentrations is not associated with acute direct effects on cell contraction and relaxation.

Regulation of the endothelial apelin/APJ system by hemodynamic fluid flow.

Although the apelin/APJ system is abundantly expressed in vascular endothelial cells (EC), it has not yet been considered to be regulated by fluid flow. The aim of this study was to explore the influence of shear stress on the expression of apelin/APJ in human EC. Therefore, gene and protein expression were assessed after flow exposure; cell supernatants were collected for measurements of NO and apelin; APJ or apelin knockdown were performed using siRNA. Our data show that gene and protein expression of apelin and APJ are modulated by fluid flow depending on the magnitude of shear stress. Moreover, apelin-12 activated NO production via PI3K/Akt signaling in human EC. In contrast, apelin-13 additionally activated Erk1/2 phosphorylation and enhanced EC proliferation. Knockdown of APJ inhibited phosphorylation of PI3K and impaired flow-induced eNOS and PECAM-1 expression. Knockdown of apelin had no influence on flow-induced APJ and PECAM-1 expression, but derogated eNOS expression under static and flow conditions. The present study reveals a flow-mediated adjustment of the apelin/APJ system in human EC in which APJ expression is induced by shear stress independently of its ligand. Furthermore, apelin-12 signaling is an essential regulatory element in endothelial NO synthesis.

Site-selective immobilization of anti-CD34 antibodies to poly(l-lactide) for endovascular implant surfaces.

Aiming at a speed up of the re-endothelialization process of biodegradable endovascular implants, novel approaches for the functionalization of poly(l-lactide) (PLLA) with anti-CD34 antibodies were established. We propose a three-step process involving PLLA surface activation with functional amino groups, attachment of a protein repelling peptide spacer, and covalent random or site-selective immobilization of the antibodies. Obtainable antibody surface densities and antigen binding capacities were thoroughly evaluated by means of enzyme-linked immunosorbent assay. Results indicate that a lower amount of anchoring sites on the antibody favors high coupling efficiency, while localization of the anchoring sites, facing the antigen binding moiety, strongly enhances the antigen capture capacity of the support. Besides minimization of physisorption and cell adhesion exemplarily shown with bovine serum albumin, avidin, and human umbilical vein endothelial cells, respectively, the inclusion of the protein-repelling spacer strengthened this effect, yielding antigen capture capacities exceeding values so far reported in literature. In contrast, the number of amino groups on the PLLA surfaces, which is indeed highly dependent on the applied activation procedure, does not seem to influence antibody coupling efficiency and antigen capture capacity considerably. This allows the choice of surface activation treatment, plasma or wet-chemical, regarding other processing parameters as for instance sterilizability or favored modification depth.

New stent surface materials: the impact of polymer-dependent interactions of human endothelial cells, smooth muscle cells, and platelets.

Despite the development of new coronary stent technologies, in-stent restenosis and stent thrombosis are still clinically relevant. Interactions of blood and tissue cells with the implanted material may represent an important cause of these side effects. We hypothesize material-dependent interaction of blood and tissue cells. The aim of this study is accordingly to investigate the impact of vascular endothelial cells, smooth muscle cells and platelets with various biodegradable polymers to identify a stent coating or platform material that demonstrates excellent endothelial-cell-supportive and non-thrombogenic properties. Human umbilical venous endothelial cells, human coronary arterial endothelial cells and human coronary arterial smooth muscle cells were cultivated on the surfaces of two established biostable polymers used for drug-eluting stents, namely poly(ethylene-co-vinylacetate) (PEVA) and poly(butyl methacrylate) (PBMA). We compared these polymers to new biodegradable polyesters poly(l-lactide) (PLLA), poly(3-hydroxybutyrate) (P(3HB)), poly(4-hydroxybutyrate) (P(4HB)) and a polymeric blend of PLLA/P(4HB) in a ratio of 78/22% (w/w). Biocompatibility tests were performed under static and dynamic conditions. Measurement of cell proliferation, viability, glycocalix width, eNOS and PECAM-1 mRNA expression revealed strong material dependency among the six polymer samples investigated. Only the polymeric blend of PLLA/P(4HB) achieved excellent endothelial markers of biocompatibility. Data show that PLLA and P(4HB) tend to a more thrombotic response, whereas the polymer blend is characterized by a lower thrombotic potential. These data demonstrate material-dependent endothelialization, smooth muscle cell growth and thrombogenicity. Although polymers such as PEVA and PBMA are already commonly used for vascular implants, they did not sufficiently meet the criteria for biocompatibility. The investigated biodegradable polymeric blend PLLA/P(4HB) evidently represents a promising material for vascular stents and stent coatings.

Evaluation of transmitral pressure gradients in the intraoperative echocardiographic diagnosis of mitral stenosis after mitral valve repair.

OBJECTIVE: Acute mitral stenosis (MS) following mitral valve (MV) repair is a rare but severe complication. We hypothesize that intraoperative echocardiography can be utilized to diagnose iatrogenic MS immediately after MV repair. METHODS: The medical records of 552 consecutive patients undergoing MV repair at a single institution were reviewed. Post-cardiopulmonary bypass peak and mean transmitral pressure gradients (TMPG), and pressure half time (PHT) were obtained from intraoperative transesophageal echocardiographic (TEE) examinations in each patient. RESULTS: Nine patients (9/552 = 1.6%) received a reoperation for primary MS, prior to hospital discharge. Interestingly, all of these patients already showed intraoperative post-CPB mean and peak TMPGs that were significantly higher compared to values for those who did not: 10.7±4.8 mmHg vs 2.9±1.6 mmHg; p<0.0001 and 22.9±7.9 mmHg vs 7.6±3.7 mmHg; p<0.0001, respectively. However, PHT varied considerably (87±37 ms; range: 20-439 ms) within the entire population, and only weakly predicted the requirement for reoperation (113±56 vs. 87±37 ms, p = 0.034). Receiver operating characteristic curves showed strong discriminating ability for mean gradients (AUC = 0.993) and peak gradients (area under the curve, AUC = 0.996), but poor performance for PHT (AUC = 0.640). A value of ≥7 mmHg for mean, and ≥17 mmHg for peak TMPG, best separated patients who required reoperation for MS from those who did not. CONCLUSIONS: Intraoperative TEE diagnosis of a peak TMPG ≥17 mmHg or mean TMPG ≥7 mmHg immediately following CPB are suggestive of clinically relevant MS after MV repair.