A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500µm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

Endothelialitis, Microischemia, and Intussusceptive Angiogenesis in COVID-19.

COVID-19 has been associated with a range of illness severity-from minimal symptoms to life-threatening multisystem organ failure. The severe forms of COVID-19 appear to be associated with an angiocentric or vascular phase of the disease. In studying autopsy patients succumbing to COVID-19, we found alveolar capillary microthrombi were 9 times more common in COVID-19 than in comparable patients with influenza. Corrosion casting of the COVID-19 microcirculation has revealed microvascular distortion, enhanced bronchial circulation, and striking increases in intussusceptive angiogenesis. In patients with severe COVID-19, endothelial cells commonly demonstrate significant ultrastructural injury. High-resolution imaging suggests that microcirculation perturbations are linked to ischemic changes in microanatomic compartments of the lung (secondary lobules). NanoString profiling of these regions has confirmed a transcriptional signature compatible with microischemia. We conclude that irreversible tissue ischemia provides an explanation for the cystic and fibrotic changes associated with long-haul COVID-19 symptoms.

Methamphetamine enhances caveolar transport of therapeutic agents across the rodent blood-brain barrier.

The blood-brain barrier (BBB) restricts clinically relevant accumulation of many therapeutics in the CNS. Low-dose methamphetamine (METH) induces fluid-phase transcytosis across BBB endothelial cells in vitro and could be used to enhance CNS drug delivery. Here, we show that low-dose METH induces significant BBB leakage in rodents ex vivo and in vivo. Notably, METH leaves tight junctions intact and induces transient leakage via caveolar transport, which is suppressed at 4°C and in caveolin-1 (CAV1) knockout mice. METH enhances brain penetration of both small therapeutic molecules, such as doxorubicin (DOX), and large proteins. Lastly, METH improves the therapeutic efficacy of DOX in a mouse model of glioblastoma, as measured by a 25% increase in median survival time and a significant reduction in satellite lesions. Collectively, our data indicate that caveolar transport at the adult BBB is agonist inducible and that METH can enhance drug delivery to the CNS.

Oxidative pentose phosphate pathway controls vascular mural cell coverage by regulating extracellular matrix composition.

Vascular mural cells (vMCs) play an essential role in the development and maturation of the vasculature by promoting vessel stabilization through their interactions with endothelial cells. Whether endothelial metabolism influences mural cell recruitment and differentiation is unknown. Here, we show that the oxidative pentose phosphate pathway (oxPPP) in endothelial cells is required for establishing vMC coverage of the dorsal aorta during early vertebrate development in zebrafish and mice. We demonstrate that laminar shear stress and blood flow maintain oxPPP activity, which in turn, promotes elastin expression in blood vessels through production of ribose-5-phosphate. Elastin is both necessary and sufficient to drive vMC recruitment and maintenance when the oxPPP is active. In summary, our work demonstrates that endothelial cell metabolism regulates blood vessel maturation by controlling vascular matrix composition and vMC recruitment.

IL-6-induced FOXO1 activity determines the dynamics of metabolism in CD8 T cells cross-primed by liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) are liver-resident antigen (cross)-presenting cells that generate memory CD8 T cells, but metabolic properties of LSECs and LSEC-primed CD8 T cells remain understudied. Here, we report that high-level mitochondrial respiration and constitutive low-level glycolysis support LSEC scavenger and sentinel functions. LSECs fail to increase glycolysis and co-stimulation after TLR4 activation, indicating absence of metabolic and functional maturation compared with immunogenic dendritic cells. LSEC-primed CD8 T cells show a transient burst of oxidative phosphorylation and glycolysis. Mechanistically, co-stimulatory IL-6 signaling ensures high FOXO1 expression in LSEC-primed CD8 T cells, curtails metabolic activity associated with T cell activation, and is indispensable for T cell functionality after re-activation. Thus, distinct immunometabolic features characterize non-immunogenic LSECs compared with immunogenic dendritic cells and LSEC-primed CD8 T cells with memory features compared with effector CD8 T cells. This reveals local features of metabolism and function of T cells in the liver.

The Role of Cytoskeletal Proteins in the Formation of a Functional In Vitro Blood-Brain Barrier Model.

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain's microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

Endothelial thrombomodulin downregulation caused by hypoxia contributes to severe infiltration and coagulopathy in COVID-19 patient lungs.

BACKGROUND: Thromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19. METHODS: We obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy. FINDINGS: The autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury. INTERPRETATION: Pulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2. FUNDING: This work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.

Effects of FW2 Nanoparticles Toxicity in a New In Vitro Pulmonary Vascular Cells Model Mimicking Endothelial Dysfunction.

Several epidemiological studies have revealed the involvement of nanoparticles (NPs) in respiratory and cardiovascular mortality. In this work, the focus will be on the effect of manufactured carbon black NPs for risk assessment of consumers and workers, as human exposure is likely to increase. Since the pulmonary circulation could be one of the primary targets of inhaled NPs, patients suffering from pulmonary hypertension (PH) could be a population at risk. To compare the toxic effect of carbon black NPs in the pulmonary circulation under physiologic and pathological conditions, we developed a new in vitro model mimicking the endothelial dysfunction and vascular dynamics observed in vascular pathology such as PH. Human pulmonary artery endothelial cells were cultured under physiological conditions (static and normoxia 21% O2) or under pathological conditions (20% cycle stretch and hypoxia 1% O2). Then, cells were treated for 4 or 6 h with carbon black FW2 NPs from 5 to 10 µg/cm2. Different endpoints were studied: (i) NPs internalization by transmission electronic microscopy; (ii) oxidative stress by CM-H2DCFDA probe and electron paramagnetic resonance; (iii) NO (nitrites and nitrates) production by Griess reaction; (iv) inflammation by ELISA assay; and (v) calcium signaling by confocal microscopy. The present study characterizes the in vitro model mimicking endothelial dysfunction in PH and indicates that, under such pathological conditions, oxidative stress and inflammation are increased along with calcium signaling alterations, as compared to the physiological conditions. Human exposure to carbon black NPs could produce greater deleterious effects in vulnerable patients suffering from cardiovascular diseases.

Angiokeratoma corporis diffusum with severe acroparesthesia, an endothelial abnormality, and inconspicuous genetic findings.

Angiokeratoma corporis diffusum (ACD) was long thought to be a specific dermal sign of Fabry disease (FD, X-linked alpha-galactosidase A [GLA] deficiency). However, other lysosomal storage diseases (LSDs) have also been identified as triggers of ACD. Generalized vasculopathy is an important pathogenetic factor in FD and may also lead to the acroparesthesia (AP) often predominant in FD. We report on an 85-year-old woman with ACD present since her youth and associated with severe AP. Ultrastructure of the dermal lesion showed no lysosomal involvement, but the absence of the basement membrane of the endothelial cells of the capillary vessels was noteworthy. Repeated analyses of the GLA gene revealed no evidence of FD. Whole-exome sequencing was negative for FD and other LSDs, and allowed us to also study FD-related intronic regions of the GLA gene. This is the first report of a patient with FD-like ACD with an endothelial abnormality, otherwise unexplained vasculopathy and severe AP, which are not due to FD or another LSD. Based on family history, another genetic, yet unidentified, defect may cause the disease in this patient. In unexplained ACD, extended genetic analysis is required to exclude particular pathogenic variants of the GLA gene and other genes.

Mild COVID-19 and Impaired Blood Cell-Endothelial Crosstalk: Considering Long-Term Use of Antithrombotics?

BACKGROUND: Current coronavirus disease 2019 (COVID-19) pandemic reveals thrombotic, vascular, and endothelial dysfunctions at peak disease. However, the duration, degree of damage, and appropriate long-term use of antithrombotic strategies are unclear. Most COVID data are yielded from random clinical observations or autopsy of postmortem samples, while precise blood cellular data in survivors are insufficient. METHODS: We analyzed erythrocytes, circulating endothelial cells, and echinocytes by electron microscopy and flow cytometry in patients with confirmed COVID-19 (n = 31) and matched healthy controls (n = 32) on admission and at hospital discharge. RESULTS: All patients experienced mild disease, none required pulmonary support, and all survived. Admission number of circulating endothelial cells was significantly (40-100 times) higher in COVID-19 patients. Cells were massively damaged by multiple fenestrae in membranes with diameter comparable to the size of supercapsid in SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus. COVID-19 also provoked formation of stacked aggregated erythrocytes capable of clogging microvascular bed and of diminishing oxygen supply. In some patients, such abnormalities persisted at hospital discharge revealing remaining intracellular penetration of SARS-CoV-2 where it may be replicated and returned to circulation. CONCLUSION: These observational and descriptive data suggest that persistent viral cell injury may cause blood vessel damage; their increased permeability resulted in tissue edema, inflammation, platelet activation, and augmented thrombosis. There is a residual blood cell damage following the acute phase in some COVID-19 survivors. Controlled outcome-driven trials are urgently needed for exploring optimal use of long-term antithrombotics and vascular protection strategies even after mild COVID-19.

DCA Protects against Oxidation Injury Attributed to Cerebral Ischemia-Reperfusion by Regulating Glycolysis through PDK2-PDH-Nrf2 Axis.

Cerebral ischemic stroke (IS) is still a difficult problem to be solved; energy metabolism failure is one of the main factors causing mitochondrion dysfunction and oxidation stress damage within the pathogenesis of cerebral ischemia, which produces considerable reactive oxygen species (ROS) and opens the blood-brain barrier. Dichloroacetic acid (DCA) can inhibit pyruvate dehydrogenase kinase (PDK). Moreover, DCA has been indicated with the capability of increasing mitochondrial pyruvate uptake and promoting oxidation of glucose in the course of glycolysis, thereby improving the activity of pyruvate dehydrogenase (PDH). As a result, pyruvate flow is promoted into the tricarboxylic acid cycle to expedite ATP production. DCA has a protective effect on IS and brain ischemia/reperfusion (I/R) injury, but the specific mechanism remains unclear. This study adopted a transient middle cerebral artery occlusion (MCAO) mouse model for simulating IS and I/R injury in mice. We investigated the mechanism by which DCA regulates glycolysis and protects the oxidative damage induced by I/R injury through the PDK2-PDH-Nrf2 axis. As indicated from the results of this study, DCA may improve glycolysis, reduce oxidative stress and neuronal death, damage the blood-brain barrier, and promote the recovery of oxidative metabolism through inhibiting PDK2 and activating PDH. Additionally, DCA noticeably elevated the neurological score and reduced the infarct volume, brain water content, and necrotic neurons. Moreover, as suggested from the results, DCA elevated the content of Nrf2 as well as HO-1, i.e., the downstream antioxidant proteins pertaining to Nrf2, while decreasing the damage of BBB and the degradation of tight junction proteins. To simulate the condition of hypoxia and ischemia in vitro, HBMEC cells received exposure to transient oxygen and glucose deprivation (OGD). The DCA treatment is capable of reducing the oxidative stress and blood-brain barrier of HBMEC cells after in vitro hypoxia and reperfusion (H/R). Furthermore, this study evidenced that HBMEC cells could exhibit higher susceptibility to H/R-induced oxidative stress after ML385 application, the specific inhibitor of Nrf2. Besides, the protection mediated by DCA disappeared after ML385 application. To sum up, as revealed from the mentioned results, DCA could exert the neuroprotective effect on oxidative stress and blood-brain barrier after brain I/R injury via PDK2-PDH-Nrf2 pathway activation. Accordingly, the PDK2-PDH-Nrf2 pathway may play a key role and provide a new pharmacology target in cerebral IS and I/R protection by DCA.

An adapted dorsal skinfold model used for 4D intravital followed by whole-mount imaging to reveal endothelial cell-pericyte association.

Endothelial cells and pericytes are highly dynamic vascular cells and several subtypes, based on their spatiotemporal dynamics or molecular expression, are believed to exist. The interaction between endothelial cells and pericytes is of importance in many aspects ranging from basic development to diseases like cancer. Identification of spatiotemporal dynamics is particularly interesting and methods to studies these are in demand. Here we describe the technical details of a method combining the benefits of high resolution intravital imaging and whole-mount histology. With intravital imaging using an adapted light weight dorsal skinfold chamber we identified blood flow patterns and spatiotemporal subtypes of endothelial cells and pericytes in a 4D (XYZ, spatial+T, time dimension) manner as representative examples for this model. Thereafter the tissue was extracted and stained as a whole-mount, by which the position and volumetric space of endothelial cells as well as pericytes were maintained, to identify molecular subtypes. Integration of the two imaging methods enabled 4D dissection of endothelial cell-pericyte association at the molecular level.

Single-cell nuclear architecture across cell types in the mouse brain.

Diverse cell types in tissues have distinct gene expression programs, chromatin states, and nuclear architectures. To correlate such multimodal information across thousands of single cells in mouse brain tissue sections, we use integrated spatial genomics, imaging thousands of genomic loci along with RNAs and epigenetic markers simultaneously in individual cells. We reveal that cell type­specific association and scaffolding of DNA loci around nuclear bodies organize the nuclear architecture and correlate with differential expression levels in different cell types. At the submegabase level, active and inactive X chromosomes access similar domain structures in single cells despite distinct epigenetic and expression states. This work represents a major step forward in linking single-cell three-dimensional nuclear architecture, gene expression, and epigenetic modifications in a native tissue context.

Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts.

Cryo-electron tomography is the highest resolution tool available for structural analysis of macromolecular complexes within their native cellular environments. At present, data acquisition suffers from low throughput, in part due to the low probability of positioning a cell such that the subcellular structure of interest is on a region of the electron microscopy (EM) grid that is suitable for imaging. Here, we photo-micropatterned EM grids to optimally position endothelial cells so as to enable high-throughput imaging of cell-cell contacts. Lattice micropatterned grids increased the average distance between intercellular contacts and thicker cell nuclei such that the regions of interest were sufficiently thin for direct imaging. We observed a diverse array of membranous and cytoskeletal structures at intercellular contacts, demonstrating the utility of this technique in enhancing the rate of data acquisition for cellular cryo-electron tomography studies.

Erythrocyte flow through the interendothelial slits of the splenic venous sinus.

The flow patterns of red blood cells through the spleen are intimately linked to clearance of senescent RBCs, with clearance principally occurring within the open flow through the red pulp and slits of the venous sinus system that exists in humans, rats, and dogs. Passage through interendothelial slits (IESs) of the sinus has been shown by MacDonald et al. (Microvasc Res 33:118-134, 1987) to be mediated by the caliber, i.e., slit opening width, of these slits. IES caliber within a given slit of a given sinus section has been shown to operate in an asynchronous manner. Here, we describe a model and simulation results that demonstrate how the supporting forces exerted on the sinus by the reticular meshwork of the red pulp, combined with asymmetrical contractility of stress fibers within the endothelial cells comprising the sinus, describe this vital and intriguing behavior. These results shed light on the function of the sinus slits in species such as humans, rats, and dogs that possess sinusoidal sinuses. Instead of assuming a passive mechanical filtering mechanism of the IESs, our proposed model provides a mechanically consistent explanation for the dynamically modulated IES opening/filtering mechanism observed in vivo. The overall perspective provided is also consistent with the view that IES passage serves as a self-protective mechanism in RBC vesiculation and inclusion removal.

Myocardial Damage by SARS-CoV-2: Emerging Mechanisms and Therapies.

Evidence is emerging that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect various organs of the body, including cardiomyocytes and cardiac endothelial cells in the heart. This review focuses on the effects of SARS-CoV-2 in the heart after direct infection that can lead to myocarditis and an outline of potential treatment options. The main points are: (1) Viral entry: SARS-CoV-2 uses specific receptors and proteases for docking and priming in cardiac cells. Thus, different receptors or protease inhibitors might be effective in SARS-CoV-2-infected cardiac cells. (2) Viral replication: SARS-CoV-2 uses RNA-dependent RNA polymerase for replication. Drugs acting against ssRNA(+) viral replication for cardiac cells can be effective. (3) Autophagy and double-membrane vesicles: SARS-CoV-2 manipulates autophagy to inhibit viral clearance and promote SARS-CoV-2 replication by creating double-membrane vesicles as replication sites. (4) Immune response: Host immune response is manipulated to evade host cell attacks against SARS-CoV-2 and increased inflammation by dysregulating immune cells. Efficiency of immunosuppressive therapy must be elucidated. (5) Programmed cell death: SARS-CoV-2 inhibits programmed cell death in early stages and induces apoptosis, necroptosis, and pyroptosis in later stages. (6) Energy metabolism: SARS-CoV-2 infection leads to disturbed energy metabolism that in turn leads to a decrease in ATP production and ROS production. (7) Viroporins: SARS-CoV-2 creates viroporins that lead to an imbalance of ion homeostasis. This causes apoptosis, altered action potential, and arrhythmia.

Aortic valve disease in diabetes: Molecular mechanisms and novel therapies.

Valve disease and particularly calcific aortic valve disease (CAVD) and diabetes (DM) are progressive diseases constituting a global health burden for all aging societies (Progress in Cardiovascular Diseases. 2014;56(6):565: Circulation Research. 2021;128(9):1344). Compared to non-diabetic individuals (The Lancet. 2008;371(9626):1800: The American Journal of Cardiology. 1983;51(3):403: Journal of the American College of Cardiology. 2017;69(12):1523), the diabetic patients have a significantly greater propensity for cardiovascular disorders and faster degeneration of implanted bioprosthetic aortic valves. Previously, using an original experimental model, the diabetic-hyperlipemic hamsters, we have shown that the earliest alterations induced by these conditions occur at the level of the aortic valves and, with time these changes lead to calcifications and CAVD. However, there are no pharmacological treatments available to reverse or retard the progression of aortic valve disease in diabetes, despite the significant advances in the field. Therefore, it is critical to uncover the mechanisms of valve disease progression, find biomarkers for diagnosis and new targets for therapies. This review aims at presenting an update on the basic research in CAVD in the context of diabetes. We provide an insight into the accumulated data including our results on diabetes-induced progressive cell and molecular alterations in the aortic valve, new potential biomarkers to assess the evolution and therapy of the disease, advancement in targeted nanotherapies, tissue engineering and the potential use of circulating endothelial progenitor cells in CAVD.

In Vivo Characterization of Endogenous Cardiovascular Extracellular Vesicles in Larval and Adult Zebrafish.

Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

Plasmalemmal Vesicle-Associated Protein Is Associated with Endothelial Cells Sprouting from the Peribiliary Capillary Plexus in Human Cirrhotic Liver.

INTRODUCTION: Plasmalemmal vesicle-associated protein (PLVAP) is an endothelial-specific integral membrane glycoprotein that localizes to caveolae and fenestrae in animal models; however, little is known about PLVAP in endothelial cells (ECs) in hepatic sinusoids during liver cirrhosis (LC). Here, we aimed to elucidate PLVAP localization and expression in the human liver during LC progression. METHODS: PLVAP protein expression was detected in specimens from normal control livers and hepatitis C-related cirrhotic livers using immunohistochemistry, Western blotting, and immunoelectron microscopy. RESULTS: PLVAP mainly localized to the peribiliary capillary plexus (PCP) and was rarely observed in hepatic artery branches and portal venules in control tissue, but was aberrantly expressed in capillarized sinusoids and proliferated capillaries in fibrotic septa within cirrhotic liver tissue. Ultrastructural analysis indicated that PLVAP localized to thin ECs in some caveolae, whereas PLVAP localized primarily to caveolae-like structures and proliferative sinusoid capillary EC vesicles in cirrhotic liver tissue. Western blot analysis confirmed that PLVAP was overexpressed at the protein level in advanced cirrhotic liver tissue. CONCLUSION: PLVAP was strongly expressed in the caveolae of proliferated capillaries directly connected with sinusoids linked with the PCP, suggesting that it plays a role in angiogenesis and sinusoidal remodeling in LC.

Ultrastructure of Endothelial Cells of Myocardial Capillaries in Burn Septicotoxemia.

We studied ultrastructure and vesicular structures in endothelial cells of myocardial micro-vessels in burn patients. Electron microscopy revealed a significant decrease in volume density of vesicular structures in the endotheliocytes of myocardial capillaries in patients with burn septicotoxemia. The observed structural signs of endothelial dysfunction revealed in this category of patients can be a promising area for further research and for the development of methods of pathogenetic correction of myocardial disorders in the case of burn injury.