Coronavirus disease 2019-induced hypercoagulability and its clinical implications

Coronavirus disease 2019 is the disease produced by severe acute respiratory syndrome-coronavirus-2, which is introduced into the host’s cell thanks to the angiotensin-converting enzyme 2 receptor. Once there, it uses the cell’s machinery to multiply itself. In this process, it generates an immune response that stimulates the lymphocytes to produce cytokines and reactive oxygen species that begin to deteriorate the endothelial cell. Complement activation, through the complement attack complex and C5a, contributes to this endothelial damage. The different mediators further promote the expression of adhesion molecules on the endothelial surface, which encourages all blood cells to adhere to the endothelial surface to form small conglomerates, called clots, which obstruct the lumen of the small blood vessels. Furthermore, the mediators of clot lysis are inhibited. All this promotes a prothrombotic environment within the pulmonary capillaries that is reflected in the elevation of D-dimer. The only solution for this cascade of events seems to be the implementation of an effective anticoagulation protocol that early counteracts the changes induced by thrombi in the pulmonary circulation and reflected in the functioning of the right ventricle.

The Glomerular Endothelium Restricts Albumin Filtration

Inflammatory activation and/or dysfunction of the glomerular endothelium triggers proteinuria in many systemic and localized vascular disorders. Among them are the thrombotic microangiopathies, many forms of glomerulonephritis, and acute inflammatory episodes like sepsis and COVID-19 illness. Another example is the chronic endothelial dysfunction that develops in cardiovascular disease and in metabolic disorders like diabetes. While the glomerular endothelium is a porous sieve that filters prodigious amounts of water and small solutes, it also bars the bulk of albumin and large plasma proteins from passing into the glomerular filtrate. This endothelial barrier function is ascribed predominantly to the endothelial glycocalyx with its endothelial surface layer, that together form a relatively thick, mucinous coat composed of glycosaminoglycans, proteoglycans, glycolipids, sialomucins and other glycoproteins, as well as secreted and circulating proteins. The glycocalyx/endothelial surface layer not only covers the glomerular endothelium; it extends into the endothelial fenestrae. Some glycocalyx components span or are attached to the apical endothelial cell plasma membrane and form the formal glycocalyx. Other components, including small proteoglycans and circulating proteins like albumin and orosomucoid, form the endothelial surface layer and are bound to the glycocalyx due to weak intermolecular interactions. Indeed, bound plasma albumin is a major constituent of the endothelial surface layer and contributes to its barrier function. A role for glomerular endothelial cells in the barrier of the glomerular capillary wall to protein filtration has been demonstrated by many elegant studies. However, it can only be fully understood in the context of other components, including the glomerular basement membrane, the podocytes and reabsorption of proteins by tubule epithelial cells. Discovery of the precise mechanisms that lead to glycocalyx/endothelial surface layer disruption within glomerular capillaries will hopefully lead to pharmacological interventions that specifically target this important structure.

Neutrophil Extracellular Traps (NETs) Contribute to the Formation of Microvascular Thrombosis in Immune Thrombotic Thrombocytopenic Purpura

Introduction. Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal blood disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from immunoglobulin (Ig) G-mediated inhibition of plasma ADAMTS13 activity. However, severe ADAMTS13 deficiency is necessary but not sufficient to cause acute iTTP. An environmental factor such as infection or acute inflammation may be necessary to trigger the acute onset of the disease. We and others have previously reported that plasma markers of neutrophil activation and neutrophil extracellular traps (NETs) formation are significantly elevated in patients with acute iTTP, which returns to normal during remission. However, the pathogenetic role of NETs in acute iTTP is not fully understood. Methods and results. Using flow cytometry, microfluidic shear-based assay, and confocal imaging analysis, we determined the in vivo NETosis in blood samples obtained from patients with acute episode of iTTP and ex vivo NETs formation, as well as the therapeutic efficacy of DNase I on thrombus formation under flow. We showed that by flow cytometry that only very few CitH3+/MPO+ positive neutrophils were present in the healthy donor blood. This population of cells dramatically increased after being stimulated with a bacterial toxin (i.e., Shigatoxin-2) at ~100 ng/mL for 15 min. Importantly, the number of CitH3+/MPO+ positive neutrophils in the sample obtained from a patient with acute iTTP was ~1,000 times higher than that in the healthy controls (Fig. 1), suggesting a massive NETosis in patients with acute iTTP. Microfluidic shear-based assay and confocal imaging analysis further confirmed a dramatic increase in adhesion and aggregation of murine platelets (stained with Alexa647 anti-CD41) and neutrophil (stained with Hoechst), as well as formation of NETs (stained with Syto green) following a perfusion of an Adamts13 -/- murine whole blood (anti-coagulated with thrombin inhibitor, PPACK) under arterial shear (15 dyne/cm 2) over a stimulated murine endothelial surface. Interestingly, an addition of DNase I (100 U/mL) significantly reduced the overall surface coverage of platelets and neutrophils on the murine endothelial surface under the same conditions (Fig. 2). Conclusions. These results demonstrate for the first time NETosis and NETs formation are common in patients with acute iTTP and in Adamts13 -/- mice after being stimulated with shigatoxin; DNase I appears to be highly efficacious eliminating the NETs and platelet/neutrophil-dominant thrombosis under arterial flow. Our findings support the pathogenetic role of NETs in the onset and progression of iTTP, and the therapeutic potential of DNase I in such a fatal disease. Figure 1 Figure 1. Disclosures Zheng: Alexion: Speakers Bureau; Sanofi-Genzyme: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria; Clotsolution: Other: Co-founder; AJMC: Honoraria.

Flagellar Hook Protein FlgE Induces Microvascular Hyperpermeability via Ectopic ATP Synthase β on Endothelial Surface

We previously demonstrated the immunostimulatory efficacy of Pseudomonas aeruginosa flagellar hook protein FlgE on epithelial cells, presumably via ectopic ATP synthases or subunits ATP5B on cell membranes. Here, by using recombinant wild-type FlgE, mutant FlgE (FlgEM; bearing mutations on two postulated critical epitopes B and F), and a FlgE analog in pull-down assay, Western blotting, flow cytometry, and ELISA, actual bindings of FlgE proteins or epitope B/F peptides with ATP5B were all confirmed. Upon treatment with FlgE proteins, human umbilical vein endothelial cells (HUVECs) and SV40-immortalized murine vascular endothelial cells manifested decreased proliferation, migration, tube formation, and surface ATP production and increased apoptosis. FlgE proteins increased the permeability of HUVEC monolayers to soluble large molecules like dextran as well as to neutrophils. Immunofluorescence showed that FlgE induced clustering and conjugation of F-actin in HUVECs. In Balb/c-nude mice bearing transplanted solid tumors, FlgE proteins induced a microvascular hyperpermeability in pinna, lungs, tumor mass, and abdominal cavity. All effects observed in FlgE proteins were partially or completely impaired in FlgEM proteins or blocked by pretreatment with anti-ATP5B antibodies. Upon coculture of bacteria with HUVECs, FlgE was detectable in the membrane and cytosol of HUVECs. It was concluded that FlgE posed a pathogenic ligand of ectopic ATP5B that, upon FlgE–ATP5B coupling on endothelial cells, modulated properties and increased permeability of endothelial layers both in vitro and in vivo. The FlgE-ectopic ATP5B duo might contribute to the pathogenesis of disorders associated with bacterial infection or ectopic ATP5B-positive cells.

Enterococcal Endocarditis: Hiding in Plain Sight

Enterococcus faecalis is a major opportunistic bacterial pathogen of increasing clinical relevance. A substantial body of experimental evidence suggests that early biofilm formation plays a critical role in these infections, as well as in colonization and persistence in the GI tract as a commensal member of the microbiome in most terrestrial animals. Animal models of experimental endocarditis generally involve inducing mechanical valve damage by cardiac catheterization prior to infection, and it has long been presumed that endocarditis vegetation formation resulting from bacterial attachment to the endocardial endothelium requires some pre-existing tissue damage. Here we review both historical and contemporary animal model studies demonstrating the robust ability of E. faecalis to directly attach and form stable microcolony biofilms encased within a bacterially-derived extracellular matrix on the undamaged endovascular endothelial surface. We also discuss the morphological similarities when these biofilms form on other host tissues, including when E. faecalis colonizes the GI epithelium as a commensal member of the normal vertebrate microbiome - hiding in plain sight where it can serve as a source for systemic infection via translocation. We propose that these phenotypes may allow the organism to persist as an undetected infection in asymptomatic individuals and thus provide an infectious reservoir for later clinical endocarditis.

Links between Endothelial Glycocalyx Changes and Microcirculatory Parameters in Septic Patients

The glycocalyx is an endothelial surface layer that is essential for maintaining microvascular homeostasis. Impaired integrity of the endothelial glycocalyx may be directly related to the development of microvascular dysfunction. To explore this hypothesis, we conducted a prospective observational study on adult patients diagnosed with sepsis. The study aimed to evaluate the degree of damage to the glycocalyx and to identify correlations between microcirculatory parameters and glycocalyx thickness based on capillary diameter. Sublingual microcirculation was examined using a handheld Cytocam-incident dark field video microscope. A sidestream dark field video microscope attached to a GlycoCheck monitor was used to determine the perfused boundary regions (PBRs) of sublingual blood vessels grouped by diameter (5–9 μm, 10–19 μm, and 20–25 μm). We identified significant damage to the glycocalyx in sublingual blood vessels of all the aforementioned diameters in septic patients compared to healthy age-matched controls. Furthermore, we found that the PBRs of the smallest capillaries (diameter class 5–9µm) correlated moderately and inversely with both total and perfused blood vessel densities. Collectively, our data suggest that there may be a functional relationship between damage to the endothelial glycocalyx of the smallest capillaries and alterations in the microcirculation observed in response to sepsis.

Effects of controlled ovarian stimulation on vascular barrier and endothelial glycocalyx: a pilot study

Purpose Controlled ovarian stimulation significantly amplifies the number of maturing and ovulated follicles as well as ovarian steroid production. The ovarian hyperstimulation syndrome (OHSS) increases capillary permeability and fluid extravasation. Vascular integrity intensely is regulated by an endothelial glycocalyx (EGX) and we have shown that ovulatory cycles are associated with shedding of EGX components. This study investigates if controlled ovarian stimulation impacts on the integrity of the endothelial glycocalyx as this might explain key pathomechanisms of the OHSS. Methods Serum levels of endothelial glycocalyx components of infertility patients (n=18) undergoing controlled ovarian stimulation were compared to a control group of healthy women with regular ovulatory cycles (n=17). Results Patients during luteal phases of controlled ovarian stimulation cycles as compared to normal ovulatory cycles showed significantly increased Syndecan-1 serum concentrations (12.6 ng/ml 6.1125th–19.1375th to 13.9 ng/ml 9.625th–28.975th; p=0.026), indicating shedding and degradation of the EGX. Conclusion A shedding of EGX components during ovarian stimulation has not yet been described. Our study suggests that ovarian stimulation may affect the integrity of the endothelial surface layer and increasing vascular permeability. This could explain key features of the OHSS and provide new ways of prevention of this serious condition of assisted reproduction.

Co-growth of Stem Cells With Target Tissue Culture as an Easy and Effective Method of Directed Differentiation

The long-term co-culture of mouse embryonic stem cells (mESC) with rat endothelial cells (EC) was tested for contact differentiation into the endothelial lineage. Serial passaging of rat ECs mixed with mESC in ratio 10:1 resulted in the emergence of a homogeneous cell population expressing mouse endothelial surface markers CD102, CD29, CD31. Rat endothelial surface marker RECA-1 completely disappeared from the co-cultured population after 2 months of weekly passaging. Co-incubation of mESC with rat ECs without cell-to-cell contact did not result in the conversion of mESC into ECs. After co-cultivation of adult mesenchymal stem cells from human endometrium (eMSC) with pre-hepatocyte-like cells of human hepatocarcinoma Huh7 the resulting co-culture expressed mature liver markers (oval cell antigen and cytokeratin 7), none of which were expressed by any of co-cultivated cultures, thus proving that even an immature (proliferating) pre-hepatocyte-like line can induce hepatic differentiation of stem cells. In conclusion, we have developed conditions where long-term co-proliferation of embryonic or adult SC with fully or partially differentiated cells results in stem cell progeny expressing markers of target tissue. In the case of endothelial differentiation, the template population quickly disappeared from the resulted culture and the pure endothelial population of stem cell progeny emerged. This approach demonstrates the expected fate of stem cells during various in vivo SC-therapies and also might be used as an effective in vitro differentiation method to develop the pure endothelium and, potentially, other tissue types of desirable genetic background.

Scanning Electron Microscopic Studies on the Auricular and Atrio-ventricular Valvular Architecture of Pre-natal Non-descript Sheep

Background: Being the vital organ of circulatory system, the development of the heart before birth must be studied to safeguard the animal from the incidence of various developmental anomalies. The ultrastructural details of auricular and atrio-ventricular valvular architecture of heart especially in pre-natal sheep has not yet been reported. Methods: The collected foeti of sheep were divided into three age groups viz. early prenatal (up to 50 days), mid prenatal (51-100 days) and late prenatal (101 to 150 days). The samples of auricle, bicuspid and tricuspid valves were processed for scanning electron microscopic study (Scanning Electron Microscope, Make: Hitachi and Model: S-3400N) and subsequently, the samples were viewed and the photographs were taken in the facility available at Central Instrumentation Facility (CIF), OUAT, Bhubaneswar. The measurements of various parameters of auricles and valves were also taken at the ultrastructural level by the inbuilt software programming system. The recorded data were subjected to routine statistical analysis.Result: It was revealed that the endocardium was the inner layer of the auricles of heart. The endothelial surface of the auricles was occupied by the simple squamous epithelium. The endothelial cells were elongated in shape in all the age groups. The subendothelial layer was consisted of connective tissue fibers and conducting fibers. The pectinate muscles covered the inner side of the right and left atria and were interconnected having a network like appearance except in mid prenatal stage, i.e. at 96 days of gestation, where they were arranged linearly. There was presence of pores among the pectinate muscles in both the atria in all the age groups under study. The endothelial surface of the bicuspid or mitral and tricuspid valves was lined by simple squamous endothelium that projected into the lumen of the valve. There was presence of pores among the endothelial cells of the valves. Further, the subendothelial layer was present just below the endothelium and comprised of connective tissue fibers and conducting fibers in both the valves. The width of the fibers in bicuspid valve was not uniform and was further categorized into thick and thin fibers especially in the mid prenatal stage, i.e. at 96 days of gestation. There was presence of clusters of pores among the endothelial cells of the tricuspid valve in the heart especially in the late prenatal stage, i.e. at 120 days of gestation period.

Scanning Electron Microscopic Studies on the Ventricular Architecture of Pre-natal Non-descript Sheep

Background: Heart is the principal organ of circulatory system that pumps blood into the blood vessels and performs many vital functions. Studies on its development before birth of utmost important to safeguard the animal from the occurrence and consequences of various developmental anomalies. The ultrastructural details of ventricular architecture of heart especially in pre-natal sheep has not yet been reported. Methods: The collected foeti of sheep were divided into three age groups viz. early prenatal (up to 50 days), mid prenatal (51-100 days) and late prenatal (101 to 150 days). The samples from ventricles were processed for scanning electron microscopic study and subsequently, the samples were viewed and the photographs were taken in the facility available at Central Instrumentation Facility (CIF), OUAT, Bhubaneswar. The measurements of various parameters of ventricles were also taken at the ultrastructural level. The recorded data were subjected to routine statistical analysis. Result: It was observed that the endocardium of the ventricles consisted of a simple squamous epithelium. The endothelial cells were elongated in shape and arranged linearly on the surface of ventricles in 33 days prenatal non-descript sheep. The boundaries among the endothelial cells were indistinct in this age group. There was presence of pores among the endothelial cells on the surface of the ventricles. In late prenatal period, the endothelial cells of the ventricles became elongated in shape. The subendocardial layer consisted of connective tissue fibers and conducting purkinje fibers, which were quite predominant in the late prenatal period, i.e. at the age of 120 days of gestation. The endocardial fibers were distinctly coiled around the endothelial cells of the ventricles in this age group. The myocardium comprised of cylindrical and highly branched cardiac muscle fibers with pores all over the surface of the myocardium of the ventricles. Each muscle bundle in the myocardium consisted of muscle fibers extending parallel to each other longitudinally and that these fibers made collateral connections with each other in some regions. The papillary muscles were ridge like structures projecting into the lumen of the ventricles of heart. The chordae tendinae connected the papillary muscles and the bicuspid and tricuspid valves. The endothelial surface of the chordae tendinae consisted of elongated endothelial cells with pores among them. The pores of various sizes were arranged linearly in patches on the endothelial surface of the chordae tendinae of the ventricles especially in the late prenatal period, i.e. at about 105 days of gestation.