Sites Of Endothelial Injury Research Articles

Биохимические и иммунологические аспекты патофизиологии острого респираторного дистресс-синдрома

Aim of the study. To analyze publications of scientific studies devoted to the study of biochemical and immunological aspects of the pathophysiology of acute respiratory distress syndrome. Material and methods. An analysis of 54 sources was conducted. The search for articles was carried out in the abstract databases Scopus and Web of Science, as well as in the search engines PubMed, eLIBRARY.RU and Google Scholar, from 2000 to December 2023. Conclusions. The immunological aspect of the pathophysiology of acute respiratory distress syndrome is characterized by the activation of the innate immune system and antigen-presenting cells, which contributes to the initiation of the immune response. In turn, neutrophils release cytotoxic molecules, active oxygen metabolites, bioactive lipids and proinflammatory cytokines and trigger an inflammatory cascade. Cytotoxic molecules cause tissue necrosis, initiate apoptosis and autophagy, which maintains and enhances inflammatory reactions and lung injury, as well as the formation of a vicious circle. Damage to vascular endothelial cells initiates coagulation, promoting the activation of platelets and procoagulant cascades, which leads to the formation of microthrombi in the pulmonary microcirculatory network and fibrin deposition in the intraalveolar and interstitial compartments. The interaction of platelets and neutrophils at the site of endothelial injury is considered a humoral regulatory process. Uncontrolled immunothrombosis can cause concomitant tissue damage and contribute to organ dysfunction.

Identification of the Role of SEL1L in Platelet Function with Implications for Atypical Equine Thrombasthenia in Thoroughbred Horses

Atypical Equine Thrombasthenia (AET) is a recessive heritable platelet disorder in horses due to aberrant platelet signaling after thrombin stimulation, preventing platelets from fully activating or efficiently binding to fibrinogen, leading to prolonged bleeding. To date, AET has only been identified in the Thoroughbred horse breed. A prevalence study performed at one breeding farm found that one in every 150 Thoroughbreds was affected. Despite the negative effects on horse health and racing performance, the underlying etiology of this unique platelet disorder is unknown. Here we show that a variant in SEL1L, a gene which encodes a protein known to play a pivotal role in endoplasmic reticulum-associated protein degradation (ERAD) and not previously known to be involved in platelet function, leads to AET. In order to identify the underlying genetic mechanism, we performed whole genome sequencing on five affected, one equivocal, and eleven control Thoroughbred horses. A whole-genome association study was conducted, and associated variants were filtered by determining if they were in or near genes known to be expressed in platelets. This evaluation identified three putative loci: a missense variant in SEL1L (p.Ile604Val), a 1.6kb deletion in a long non-coding RNA ( AL355838.1) and a 73bp intronic deletion in VIPAS39. The long non-coding RNA AL355838.1 did not show expression in horse platelets, and there was no differential expression of SEL1L or VIPAS39 mRNA identified between affected and unaffected individuals. However, a significant difference in SEL1L protein expression was observed in AET-affected horses' platelets. Flow cytometry and immunofluorescence studies demonstrated that SEL1L is located intracellularly in equine platelets, but not in the α-granule, and moves to the surface of the platelet upon activation with thrombin. Although the template bleeding time was normal, platelets from horses that were homozygous for the SEL1L variant showed defective spreading on collagen. To support SEL1L as the causative gene and dissect the underlying mechanisms, we undertook studies in three additional models. Differentiation of human megakaryocytes revealed the presence of two SEL1L protein isoforms, p100 and p38, and both showed increased expression during megakaryopoiesis, although only p100 was delivered to mature platelets. We perfused whole blood from Mx1-Cre +; Sel1l fl/fl conditional knockouts and controls through a collagen-coated microfluidic chamber. We found that significantly fewer platelets from knockout mice bound to the collagen as compared to control siblings. Finally, a knockout zebrafish line was developed by deleting exons 3-20 using CRISPR/Cas9 mediated genome editing. Similar to the mouse, laser-mediated arterial endothelial injury of 5-day old mutant larvae resulted in significantly fewer thrombocytes (the fish platelet equivalent) adhering to the injured vessel wall as compared to wild-type siblings. Overall, these data support the conclusion that the SEL1L missense variant leads to AET, and that SEL1L has a previously undescribed and conserved role in platelet function, particularly the ability of platelets/thrombocytes to adhere to exposed collagen at sites of endothelial injury. These results will allow the Thoroughbred industry to easily diagnose AET-affected horses and make appropriate decisions for their racing and breeding careers. These data also provide an excellent introduction to further investigation into the understudied and potential role of ERAD proteins in human platelet disorders.

The impact of aging and atrial fibrillation on thrombus formation in-silico

Abstract Background Atrial fibrillation (AF) is an age-related cardiac arrythmia that underlies one third of all strokes, with thromboemboli commonly originating from the left atrial appendage (LAA). Personalised in-silico modelling of Virchow’s triad (blood stasis, hypercoagulability and endothelial damage) can improve stroke risk assessment metrics, such as the CHA2DS2-VASc score, by providing quantitative mechanistic information [1]. The ability to quantify the impact of aging may play a critical role in patient stratification and therapy selection to reduce the risk of AF-related stroke. Aim We aim to perform in-silico modelling to quantify the effect of aging and AF on thrombus formation in the LAA by using advanced computational tools to capture blood flow, clotting mechanisms and endothelial damage [2]. Methods A virtual physiological LA model was derived from cardiac Cine MRI from an adult AF patient imaged during sinus rhythm (SR). Biomarkers for a geriatric patient were obtained from literature and applied as: i) elevated plasma fibrinogen concentration and ii) longer thrombin time-to-peak (ttP) [3]. Blood flow was simulated in SR and AF conditions for 5 cardiac cycles in the LA for both ages (totalling 4 test cases). The catalytic conversion of fibrinogen to a fibrin clot by thrombin was modelled to mimic hypercoagulability [2]. The site of endothelial injury was prescribed in the middle of the LAA. The area under curve for the fibrin concentration (Fn AUC) was measured to quantify thrombus formation. Results In the two SR cases, LA blood flow velocity increased to a maximum of 1.8 m/s after 0.6 s with a mean of 0.63 m/s during the cardiac cycle, while the two AF cases showed 1.3 m/s and 0.39 m/s, respectively, corresponding to a 38% decrease in AF. Peak velocity in the LAA was 0.35 m/s during SR and 0.18 m/s in AF. The fibrin concentration in the adult-SR case rose to 1.1 mmol/m³ over the first cardiac cycle, but both thrombin and fibrin were washed out of the LA after 1.5 s, with a Fn AUC of 0.045mmol s/m³ (Fig. 1a). The geriatric-SR case sustained fibrin generation throughout the 5 cardiac cycles reaching 3.1 mmol/m³, an Fn AUC of 2.4 mmol s/m³, and the formation of an LAA thrombus (Fig. 1b). Similarly, the adult-AF and geriatric-AF cases also saw continuous growth to 5.0 and 7.4 mmol/m³, with Fn AUCs of 6.4 and 12.6 mmol s/m³, respectively, with formation of a thrombus in the LAA in both (Fig. 1c and d). The thrombus formed in the adult-AF case was 25% larger than the geriatric-SR case, while thrombus in the geriatric-AF case was 80% larger. Conclusions This investigation quantifies the increased risk of LAA thrombus formation in older patients, with an Fn AUC increase of 55% between adult and geriatric in SR and a 100% increase when combining geriatric biomarkers with AF conditions. With further validation on larger patient cohorts, this novel in-silico tool may improve our understanding of the risks in AF-related stroke.

Injury Site Specific Xenon Delivered by Platelet Membrane-Mimicking Hybrid Microbubbles to Protect Against Acute Kidney Injury via Inhibition of Cellular Senescence.

Inhalation of xenon gas improves acute kidney injury (AKI). However, xenon can only be delivered through inhalation, which causes non-specific distribution and low bioavailability of xenon, thus limiting its clinical application. In this study, xenon is loaded into platelet membrane-mimicking hybrid microbubbles (Xe-Pla-MBs). In ischemia-reperfusion-induced AKI, intravenously injected Xe-Pla-MBs adhere to the endothelial injury site in the kidney. Xe-Pla-MBs are then disrupted by ultrasound, and xenon is released to the injured site. This release of xenon reduced ischemia-reperfusion-induced renal fibrosis and improved renal function, which are associated with decreased protein expression of cellular senescence markers p53 and p16, as well as reduced beta-galactosidase in renal tubular epithelial cells. Together, platelet membrane-mimicking hybrid microbubble-delivered xenon to the injred site protects against ischemia-reperfusion-induced AKI, which likely reduces renal senescence. Thus, the delivery of xenon by platelet membrane-mimicking hybrid microbubbles is a potential therapeutic approach for AKI.

Collagen-Binding Nanoparticles: A Scoping Review of Methods and Outcomes

(1) Background: Collagen is the main component of the connective tissue, playing an important role in the histological architecture and function of living organisms. Targeted therapy and improved imaging diagnosis can be obtained through collagen-binding nanoparticles that concentrate in the extracellular matrix. (2) Methods: We performed a scoping review of studies that analyzed the binding capacity of collagen-targeting nanoparticles. The search algorithm and inclusion criteria were based on PRISMA and ARRIVE guidelines. (3) Results: Fourteen studies matched all the inclusion criteria. All studies analyzed the distribution of nanoparticles in the collagen matrix, either by using collagen-targeting nanoparticles or by using unmodified ones. Most studies used collagen-binding nanoparticles for vascular research to target sites of endothelial injury, atherosclerotic plaques, or myocardial infarction. Two studies targeted the exposed collagen in models of liver fibrosis. (4) Conclusions: Our review summarizes the current literature on the methods and outcomes of using nanoparticles to target collagen. The studies reveal that there is high applicability for collagen-binding nanoparticles in cardiac or hepatic pathology and they could prove useful for targeted therapy of neoplastic lesions, which show an abundance of stromal collagen.

Extracellular Mitochondrial Components and Effects on Cardiovascular Disease.

Besides being powerhouses of the cell, mitochondria released into extracellular space act as intercellular signaling. Mitochondria and their components mediate cell-to-cell communication in free form or embedded in a carrier. The pathogenesis of cardiovascular disease is complex, which shows close relationship with inflammation and metabolic abnormalities. Since mitochondria sustain optimal function of the heart, extracellular mitochondria are emerging as a key regulator in the development of cardiovascular disease. In this review, we provide recent findings in the presence and forms of mitochondria transfer between cells, as well as the effects of these mitochondria on vascular inflammation and ischemic myocardium. Mitochondrial transplantation is a novel treatment paradigm for patients suffering from acute cardiovascular accident and challenges the traditional methods of mitochondria isolation.

Quantitative 3D microscopy highlights altered von Willebrand factor α‐granule storage in patients with von Willebrand disease with distinct pathogenic mechanisms

BackgroundPlatelets play a key role in hemostasis through plug formation and secretion of their granule contents at sites of endothelial injury. Defects in von Willebrand factor (VWF), a platelet α‐granule protein, are implicated in von Willebrand disease (VWD), and may lead to defective platelet adhesion and/or aggregation. Studying VWF quantity and subcellular localization may help us better understand the pathophysiology of VWD. ObjectiveQuantitative analysis of the platelet α‐granule compartment and VWF storage in healthy individuals and VWD patients. Patients/MethodsStructured illumination microscopy (SIM) was used to study VWF content and organization in platelets of healthy individuals and patients with VWD in combination with established techniques. ResultsSIM capably quantified clear morphological and granular changes in platelets stimulated with proteinase‐activated receptor 1 (PAR‐1) activating peptide and revealed a large intra‐ and interdonor variability in VWF‐positive object numbers within healthy resting platelets, similar to variation in secreted protein acidic and rich in cysteine (SPARC). We subsequently characterized VWD platelets to identify changes in the α‐granule compartment of patients with different VWF defects, and were able to stratify two patients with type 3 VWD rising from different pathological mechanisms. We further analyzed VWF storage in α‐granules of a patient with homozygous p.C1190R using electron microscopy and found discrepant VWF levels and different degrees of multimerization in platelets of patients with heterozygous p.C1190 in comparison to VWF in plasma. ConclusionsOur findings highlight the utility of quantitative imaging approaches in assessing platelet granule content, which may help to better understand VWF storage in α‐granules and to gain new insights in the etiology of VWD.

Role of Purinergic Signalling in Endothelial Dysfunction and Thrombo-Inflammation in Ischaemic Stroke and Cerebral Small Vessel Disease.

The cerebral endothelium is an active interface between blood and the central nervous system. In addition to being a physical barrier between the blood and the brain, the endothelium also actively regulates metabolic homeostasis, vascular tone and permeability, coagulation, and movement of immune cells. Being part of the blood–brain barrier, endothelial cells of the brain have specialized morphology, physiology, and phenotypes due to their unique microenvironment. Known cardiovascular risk factors facilitate cerebral endothelial dysfunction, leading to impaired vasodilation, an aggravated inflammatory response, as well as increased oxidative stress and vascular proliferation. This culminates in the thrombo-inflammatory response, an underlying cause of ischemic stroke and cerebral small vessel disease (CSVD). These events are further exacerbated when blood flow is returned to the brain after a period of ischemia, a phenomenon termed ischemia-reperfusion injury. Purinergic signaling is an endogenous molecular pathway in which the enzymes CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia and CSVD, eATP is released from dying neurons as a damage molecule, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response. Evidently, therapies that promote adenosine generation or boost CD39 activity at the site of endothelial injury have promising benefits in the context of atherothrombotic stroke and can be extended to current CSVD known pathomechanisms. Here, we have reviewed the rationale and benefits of CD39 and CD39 therapies to treat endothelial dysfunction in the brain.

The great platelet paradox: evolution of platelet contribution to hemostasis, inflammation, and thrombosis after injury

AbstractIn the setting of severe injury, a rapid hemostatic response led by platelets at the site of endothelial injury is critical to achieve hemostasis. In a process intricately linked with hemostasis, platelets undertake the role of a sentinel immune cell, sensing and releasing danger-associated molecular pattern proteins (DAMPs) and proinflammatory ligands to conduct the initial inflammatory response. Although this serves as a well-adapted system for hemostasis at the local site of endothelial injury, initiation of this cascade at sites other than a point of active hemorrhage may have deleterious consequences. Severe trauma, including soft tissue injury even in the absence of hemorrhage, is characterized by a systemic surge of DAMPs and activating ligands that results in a series of changes in the morphology and function of the platelet that are linked to thrombotic complications, such as microvascular injury and venous thromboembolism. This talk explores some of the platelet signals and functions unique to the innate immune profile of severe trauma, as well as highlights major knowledge gaps in the field in the setting of an overarching hypothesis of how such a dual role for platelets may have evolved.

Rapid effects of aldosterone on platelets, coagulation, and fibrinolysis lead to experimental thrombosis augmentation

An increase in aldosterone levels positively correlates with an increased risk of acute cardiovascular thrombotic events. The aim of the study was to determine the mechanism of action of prothrombotic aldosterone focusing on the rapid effects of the hormone on platelets, coagulation, and fibrinolysis. A wide panel of advanced ex vivo and in vitro techniques was used for the evaluation of coagulation and fibrinolysis in aldosterone-treated rats. Additionally, two experimental mice models of thrombosis, which allowed for the intravital observation of the first stage of thrombus formation in real time, were used. Acute administration of aldosterone in rats increased the density of fibrin net and platelet aggregates in clots as well as reduced fibrinolysis. These effects were observed within 10 min and were partially suppressed by eplerenone. Moreover, acute administration of aldosterone in mice enhanced platelet accumulation at the site of endothelial injury induced by laser and increased the area of irreversibly activated platelets in FeCl3-induced thrombus. These results demonstrate that aldosterone acutely affects platelets, coagulation, and fibrinolysis, leading to an enhanced thrombosis. The aldosterone effects were mediated partially via a mineralocorticoid receptor. The mechanism seems to involve non-genomic signaling since the effects were observed within a few minutes of aldosterone administration.

P4637Elevated von Willebrand factor (VWF) and factor VIII are associated with higher clinical SYNTAX score in patients with stable coronary artery disease

Abstract Background Von Willebrand factor (VWF plays) central role in thrombogenesis, and circulates in a noncovalent complex with factor VIII (FVIII), acting as a transporter protein and stabilizer. VW factor protects FVIII from proteolytic inactivation and concentrate it at site of endothelial injury. Raised plasma VWF factor is detected in case of endothelial damage and it has been considered as a useful marker of endothelial dysfunction. Increased plasma FVIII has been found in venous thromboembolism and in some clinical conditions associated with chronic inflammation. The connection between FVIII and more incidence of arterial thrombosis is, partly, due to increased platelet adhesion/aggregation, induced by VWF at sites of arterial wall damage. The correlation between VWF factor, FVIII levels and acute coronary syndrome is well documented, but there are no available dates regarding relation between plasma levels of VW factor, FVIII and the severity of coronary artery disease according to SYNTAX I (SS) and Clinical SYNTAX score (CSS). Purpose The aim of this study was to determine the association between levels of VW factor and FVIII and the severity of coronary artery disease according to SS and CSS. Methods A total of 82 patients with symptoms of stable angina underwent coronary angiography and were divided into three groups according to SS and CSS: Group I (<22 points), Group II (23–32), Group III (>33). We calculated Clinical SYNTAX multiplying the value of SYNTAX score with the modified ACEF score, based on the patients' left ventricular ejection fraction, age and creatinine clearance derived using the Cockcroft–Gault equation. Results There were positive association between plasma levels of VW factor and severity of CAD according to SYNTAX I (Group I: 1.16±0.59, Group II: 1.52±0.62, Group III 1.49±0.80, Kruskal Wallis p=0.040) and Clinical SYNTAX score (Group I: 1.15±0.53, Group II: 1.38±0.72, Group III 1.57±0.75, Kruskal Wallis p=0.034). VW factor levels were significantly higher in Group II and Group III compared to Group I (SS: Mann- Whitney p=0.023 and 0.071, respectively), (CSS: p=0.251 and 0.009, respectively). We also found positive association between FVIII levels and severity of CAD according to SYNTAX I (Group I: 2.25±0.75, Group II: 2.21±0.53, Group III 2.97±0.95, Kruskal Wallis p=0.007) and Clinical SYNTAX score (Group I: 2.17±0.71, Group II: 2.26±0.68, Group III 2.89±0.87, Kruskal Wallis p=0.002). This study demonstrates that FVIII levels were significantly higher in Group III compared to Group I and Group II (SS: Mann- Whitney p=0.005 and 0.005, respectively), (CSS: p=0.001 and 0.014, respectively). The correlation between plasma levels of VW factor Conclusion Patients with stable angina pectoris and higher levels of VW and factor VIII had a higher Clinical SYNTAX and SYNTAX I score. Our study revealed that concomitant elevation in both VW and FVIII factors are a significant risk factor and predictor of more severe and extended CAD.

Tissue Factor-Negative Cell-Derived Microparticles Play a Distinctive Role in Hemostasis: A Viewpoint Review.

Circulating cell-derived microparticles (MPs) exhibit procoagulant activity and have been investigated for a possible role in some human pathologies. However, their potential role in hemostasis has been neglected and often denied. This review brings to attention a specific body of direct clinical evidence supporting an important but distinctive role of MPs in hemostasis. Evidence for a role of MPs in hemostasis includes: (1) two congenital bleeding disorders attributed to impaired release of MPs; (2) two recent studies of trauma patients relating naturally elevated endogenous MPs at admission to reduced transfusion requirements and better outcomes; (3) a study of coronary surgery patients showing that elevated MP before surgery reduces transfusion requirements during surgery; and (4) a clinical study of patients with immune thrombocytopenia demonstrating that those with high circulating MP have reduced bleeding compared to patients with similar platelet counts but lower MP levels. Mechanisms involving potentiating the contact factor pathway are thought to play a key role and are probably synergistic with polyphosphate released from activated platelets at sites of endothelial injury. Hemostatic defect of patients with deficient MP-mediated coagulation resembles deficiency of FXI (hemophilia C), distinct from hemophilia A or B, so can be termed type C hemostasis. A better understanding of this proposed hemostatic pathway may lead to improved methods for controlling excessive bleeding in surgery, trauma, and other clinical settings.

Potential different impact of inhibition of thrombin function and thrombin generation rate for the growth of thrombi formed at site of endothelial injury under blood flow condition

IntroductionThrombin inhibitor and anti-Xa are now widely used in clinical practice. However, the difference between thrombin inhibitor and anti-Xa in prevention of thrombosis is still to be elucidated. Materials and methodsComputer simulator implementing the function of platelet, coagulation, fibrinolysis and blood flow was developed. The function of thrombin is defined as to activated platelet at the rate of 0.01 s−1 and to produce fibrin at the rate of 0.1 s−1 in control. The effect of thrombin inhibitor was settled to reduce the rate of platelet activation and fibrin generation changed from 10 to 100% as compared to the control. The local thrombin generation rate on activated platelet was settled as 1.0 s−1 as a control. The effect of anti-Xa was settled to reduce to thrombin generation rate on activated platelet from 10% to 100% as compared to the control. The sizes of thrombi formed at site of endothelial injury in the presence and absence of thrombin inhibitor and anti-Xa were compared. Results and conclusionsThe size of thrombi formed by 30-s perfusion of blood at site of endothelial injury reduced both in the presence of thrombin inhibitor and anti-Xa. There was significant positive relationship between thrombin inhibitor effect and the size of formed thrombi with R value of 0.96. (p < 0.0001) However, the sizes of thrombi were not influence by anti-Xa until it decreased 30% or less as compared to control. There was no significant relationship between anti-Xa effect and the size of formed thrombi. (R = 0.39, p = 0.09) Our results suggest the different dose-dependent effects of thrombin inhibitor and anti-Xa on thrombus formation at least in specific conditions. Computer simulation may help to predict quantitative antithrombotic effects of various antithrombotic agents.

Complement Activation Contributes to the Pathophysiology of Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections have become a threat to public health globally because of the severe illnesses that they can trigger, such as hemorrhagic colitis and the post-diarrheal hemolytic uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney failure. Glomerular endothelial cells are primary targets of Stx which, after binding to its specific receptor globotriaosylceramide, upregulates proinflammatory proteins involved both in the recruitment and adhesion of leukocytes and thrombus formation at the site of endothelial injury. In this review, we discuss the role of complement activation in promoting glomerular microvascular dysfunction, providing evidence from experimental models and patients with STEC-HUS. Within the glomerulus, an important target for Stx-induced complement activation is the podocyte, a cell type that is in close contact with endothelial cells and participates in maintaining the filtration barrier. Recently, podocyte injury and loss have been indicated as potential risk factors for long-term renal sequelae in patients with STEC-HUS. Therapeutic approaches targeting the complement system, that may be useful options for patients with STEC-HUS, will also be discussed.

Increased platelet content of SDF-1alpha is associated with worse prognosis in patients with pulmonary prterial hypertension

Inflammatory processes and platelet activity play an important role in the pathophysiology of pulmonary arterial hypertension (PAH). Enhanced IL-6 signaling and higher concentration of stromal-derived factor alpha (SDF-1) have been previously shown to be linked with prognosis in PAH. We hypothesized that platelets of PAH patients have higher content of IL-6 and SDF-1 and thus are involved in disease progression.We enrolled into study 22 PAH patients and 18 healthy controls. Patients with PAH presented significantly higher plasma concentrations and platelet contents of IL-6, sIL-6R, and SDF-1 than healthy subjects (platelet content normalized to protein concentration: IL-6 (0.85*10–10 [0.29 – 1.37] vs. 0.45*10–10 [0.19–0.65], sIL-6R 1.54*10–7 [1.32–2.21] vs. 1.14*10–7 [1.01–1.28] and SDF-1 (2.72*10–7 [1.85–3.23] vs. 1.70*10–7 [1.43–2.60], all p < 0.05). Patients with disease progression (death, WHO class worsening, or therapy escalation, n = 10) had a significantly higher platelet SDF-1/total platelet protein ratio (3.68*10–7 [2.45–4.62] vs. 1.69*10–7 [1.04–2.28], p = 0.001), with no significant differences between plasma levels. Kaplan–Meier analysis revealed that patients with higher platelet SDF-1/total platelet protein ratio had more frequently deterioration of PAH in the follow-up (15.24 ± 4.26 months, log-rank test, p = 0.01).Concentrations of IL-6, sIL-6 receptor and SDF-1 in plasma and platelets are elevated in PAH patients. Higher content of SDF-1 in platelets is associated with poorer prognosis. Our study, despite of limitation due to small number of enrolled patients, suggests that activated platelets may be an important source of cytokines at the site of endothelial injury, but their exact role in the pathogenesis of PAH requires further investigation.

Posterior stromal cell apoptosis triggered by mechanical endothelial injury and basement membrane component nidogen-1 production in the cornea

This study was performed to determine whether cells in the posterior stroma undergo apoptosis in response to endothelial cell injury and to determine whether basement membrane component nidogen-1 was present in the cornea. New Zealand White rabbits had an olive tip cannula inserted into the anterior chamber to mechanically injure corneal endothelial cells over an 8 mm diameter area of central cornea with minimal injury to Descemet's membrane. At 1 h (6 rabbits) and 4 h (6 rabbits) after injury, three corneas at each time point were cryopreserved in OCT for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry (IHC) for vimentin and nidogen-1, and three corneas at each time point were fixed for transmission electron microscopy (TEM). Uninjured corneas were controls. Stromal cells over approximately the posterior 25% of the stroma overlying to the site of corneal endothelial injury underwent apoptosis detected by the TUNEL assay. Many of these apoptotic cells were vimentin+, suggesting they were likely keratocytes or corneal fibroblasts. Stromal cells peripheral to the site of endothelial injury and more anterior stromal cells overlying the site of endothelial injury did not undergo apoptosis. Stromal cell death was confirmed to be apoptosis by TEM. No apoptosis of stromal cells was detected in control, uninjured corneas. Nidogen-1 was detected in the stroma of unwounded corneas, with higher nidogen-1 in the posterior stroma than the anterior stroma. After endothelial scrape injury, concentrations of nidogen-1 appeared to be in the extracellular matrix of the posterior stroma and, possibly, within apoptotic bodies of stromal cells. Thus, posterior stromal cells, likely including keratocytes, undergo apoptosis in response to corneal endothelial injury, analogous to anterior keratocytes undergoing apoptosis in response to epithelial injury.

P4349A method for precision prediction of platelet adhesion at site of endothelial injury under blood flow conditions

P4349A method for precision prediction of platelet adhesion at site of endothelial injury under blood flow conditions

Endothelial progenitor cells and cardiovascular risk: does ageing trump all other factors?

Discovered nearly 20 years ago, endothelial progenitor cells (EPC) attract both basic and clinical researchers (1). As key regulators of vascular homeostasis in health and disease, circulating progenitor cell levels reflect endogenous regenerative potential. Upon endothelial damage, EPC are mobilized from the bone marrow and follow chemokine gradients to sites of endothelial injury where they assist in endothelial repair mainly via paracrine mechanisms. Next to the production of angiogenic growth factors, novel modes of paracrine regulation are being discovered, such as the release of endothelial cell-derived microparticles or microvesicles that contain pro-angiogenic microRNAs (2).

Identification and function probing of an antithrombin IIIβ conformation‐specific antibody

ESSENTIALS: Antithrombin III (AT)β binds heparin with higher affinity than ATα. A conformation-specific antibody against ATβ, TPP2009, was made to investigate ATβ in hemostasis. TPP2009 bound specifically to heparin-ATβ and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATβ in hemostasis. Antithrombin III (AT)β is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. To characterize a conformation-specific antibody against ATβ and begin to investigate the role of ATβ in maintaining hemostasis. Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATβ (ATβ*H) by the use of phage display. SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATβ*H and glycosaminoglycan-associated ATβ on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATβ and heparin. In purified systems with ATβ and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies. TPP2009 specifically targets a unique conformational epitope on ATβ*H and blocks ATβ-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATβ in hemostasis.

Role of Acid Sphingomyelinase in Shifting the Balance Between Proinflammatory and Reparative Bone Marrow Cells in Diabetic Retinopathy.

The metabolic insults associated with diabetes lead to low-grade chronic inflammation, retinal endothelial cell damage, and inadequate vascular repair. This is partly due to the increased activation of bone marrow (BM)-derived proinflammatory monocytes infiltrating the retina, and the compromised function of BM-derived reparative circulating angiogenic cells (CACs), which home to sites of endothelial injury and foster vascular repair. We now propose that a metabolic link leading to activated monocytes and dysfunctional CACs in diabetes involves upregulation of a central enzyme of sphingolipid signaling, acid sphingomyelinase (ASM). Selective inhibition of ASM in the BM prevented diabetes-induced activation of BM-derived microglia-like cells and normalized proinflammatory cytokine levels in the retina. ASM upregulation in diabetic CACs caused accumulation of ceramide on their cell membrane, thereby reducing membrane fluidity and impairing CAC migration. Replacing sphingomyelin with ceramide in synthetic membrane vesicles caused a similar decrease in membrane fluidity. Inhibition of ASM in diabetic CACs improved membrane fluidity and homing of these cells to damaged retinal vessels. Collectively, these findings indicate that selective modulation of sphingolipid metabolism in BM-derived cell populations in diabetes normalizes the reparative/proinflammatory cell balance and can be explored as a novel therapeutic strategy for treating diabetic retinopathy.