Endothelial cell monolayer formation on a small-diameter vascular graft surface under pulsatile flow conditions

Objective: to create a cell-populated small-diameter vascular graft (SDVG) using autologous endothelial cells and extracellular matrix proteins, and to evaluate the efficiency of endothelial cell monolayer formation during shear stress preconditioning in a SDVG.Materials and methods. PHBV/PCL tubular scaffolds of vascular grafts were made by electrospinning from a mixture of polyhydroxybutyrate-valerate (PHBV) copolymer and polycaprolactone (PCL) and modified with fibrin. To populate the graft, an endothelial cell culture was isolated from the blood of patients with coronary heart disease. Phenotyping of endothelial colony-forming cell (ECFC) culture was performed by flow cytometry and immunofluorescence microscopy. Cell proliferative and angiogenic activity were also studied. Cell-populated vascular scaffolds were cultured in a pulsatile flow setup with a final shear stress of 2.85 dyne/cm2. The effect of pulsatile flow on monolayer formation was assessed by immunofluorescence, scanning electron microscopy, atomic force microscopy, and whole-transcriptome RNA sequencing.Results. Under the influence of pulsatile flow, endothelial cells that were seeded into the tubular scaffold showed an increase in the expression level of endothelial profile proteins, focal adhesion and cytoskeleton. In contrast to endothelial cell culture on a vascular graft surface under static conditions, when cultured under pulsatile flow with 2.85 dyne/ cm2 shear stress, endothelial lining cells have an increased ability to adhere and are oriented along the pulsatile flow path. Whole-transcriptome RNA sequencing showed that induced shear stress increased expression levels of differentially expressed genes encoding proteins that ensure vascular development, endothelial integrity, and endothelial metabolism. A protocol for fabrication of a personalized cell-populated biodegradable SDVG under pulsatile flow conditions was developed.Conclusion. The use of autologous fibrin and ECFC culture, as well as shear stress preconditioning, allow to obtain a personalized cell-populated SDVG with continuous functional endothelial monolayer adapted to the flow.

Challenges in corneal endothelial cell culture

Corneal endothelial cells (CECs) facilitate the function of maintaining the transparency of the cornea. Damage or dysfunction of CECs can lead to blindness, and the primary treatment is corneal transplantation. However, the shortage of cornea donors is a significant problem worldwide. Thus, cultured CEC therapy has been proposed and found to be a promising approach to overcome the lack of tissue supply. Unfortunately, CECs in humans rarely proliferate in vivo and, therefore, can be extremely challenging to culture in vitro. Several promising cell isolation and culture techniques have been proposed. Multiple factors affecting the success of cell expansion including donor characteristics, preservation and isolation methods, plating density, media preparation, trans-differentiation and biomarkers have been evaluated. However, there is no consensus on standard technique for CEC culture. This review aimed to determine the challenges and investigate potential options that would facilitate the standardization of CEC culture for research and therapeutic application.

Chemically Modified Biomimetic Carbon-Coated Iron Nanoparticles for Stent Coatings: In Vitro Cytocompatibility and In Vivo Structural Changes in Human Atherosclerotic Plaques

Atherosclerosis, a systematic degenerative disease related to the buildup of plaques in human vessels, remains the major cause of morbidity in the field of cardiovascular health problems, which are the number one cause of death globally. Novel atheroprotective HDL-mimicking chemically modified carbon-coated iron nanoparticles (Fe@C NPs) were produced by gas-phase synthesis and modified with organic functional groups of a lipophilic nature. Modified and non-modified Fe@C NPs, immobilized with polycaprolactone on stainless steel, showed high cytocompatibility in human endothelial cell culture. Furthermore, after ex vivo treatment of native atherosclerotic plaques obtained during open carotid endarterectomy surgery, Fe@C NPs penetrated the inner structures and caused structural changes of atherosclerotic plaques, depending on the period of implantation in Wistar rats, serving as a natural bioreactor. The high biocompatibility of the Fe@C NPs shows great potential in the treatment of atherosclerosis disease as an active substance of stent coatings to prevent restenosis and the formation of atherosclerotic plaques.

MO710GALECTIN-3, IS IT A NEW PLAYER IN PERITONEAL INFLAMMATION AMONG PATIENTS UNDERGOING PERITONEAL DIALYSIS?

Background and Aims Peritoneal dialysis (PD) is a common used method for renal replacement therapy. Prolonged PD treatment causes structural and functional changes in the peritoneal membrane which are attributed to local inflammatory process in the peritoneal cavity. Galectin-3 (Gal-3) is a galactoside-binding lectin with pro-inflammatory and pro-fibrotic effects. The aim of this study was to assess correlation between Gal-3 serum and dialysate effluent levels with peritoneal membrane transport characteristics. Method Gal-3 levels in serum and dialysate effluent were measured simultaneously in prevalent PD patients in morning visit or during peritoneal equilibration test (PET). Gal-3 levels were correlated with clinical and laboratory parameters. Interlukin (IL) -6 levels were measured in dialysate effluent. Gal-3 mRNA and protein expression were evaluated after exposure of primary endothelial cell culture to several dialysate solutions. Results 37 PD patients were included in the study; mean age was 65.7±13.1 years, mean dialysis vintage was 17.5±13 months. Gal-3 levels in dialysate effluent correlated with peritoneal equilibration test (PET) results (0.663, p=0.005) and effluent IL-6 levels (0.674, p=0.002) but not with serum Gal-3 levels or dialysis vintage. Patients with high PET results had higher effluent Gal-3 levels as compared average low PET results. In multivariate regression analysis effluent IL-6 level was the most dominant predictor of effluent Gal-3 levels. Gal-3 mRNA and protein expression in primary endothelial cell culture were not affected by stimulation with dialysate solutions. Conclusion Our study demonstrated presence of Gal-3 within the dialysate effluent in PD patients. Gal-3 levels correlated with peritoneal membrane transport characteristics and effluent IL-6 levels suggesting a role in the inflammatory process within the peritoneal cavity.

Development of an In Vitro Airway Epithelial–Endothelial Cell Culture Model on a Flexible Porous Poly(Trimethylene Carbonate) Membrane Based on Calu-3 Airway Epithelial Cells and Lung Microvascular Endothelial Cells

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial–endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air–liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial–endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial–endothelial and alveolar–endothelial models with primary lung epithelial cells.

Biomaterials for corneal endothelial cell culture and tissue engineering

The corneal endothelium is the posterior monolayer of cells that are responsible for maintaining overall transparency of the avascular corneal tissue via pump function. These cells are non-regenerative in vivo and therefore, approximately 40% of corneal transplants undertaken worldwide are a result of damage or dysfunction of endothelial cells. The number of available corneal donor tissues is limited worldwide, hence, cultivation of human corneal endothelial cells (hCECs) in vitro has been attempted in order to produce tissue engineered corneal endothelial grafts. Researchers have attempted to recreate the current gold standard treatment of replacing the endothelial layer with accompanying Descemet’s membrane or a small portion of stroma as support with tissue engineering strategies using various substrates of both biologically derived and synthetic origin. Here we review the potential biomaterials that are currently in development to support the transplantation of a cultured monolayer of hCECs.

The Anti-Inflammatory Effects of Anacardic Acid on a TNF-α - Induced Human Saphenous Vein Endothelial Cell Culture Model

Background and Objective: Coronary bypass operations are commonly performed for the treatment of ischemic heart diseases. Coronary artery bypass surgery with autologous human saphenous vein maintains its importance as a commonly used therapy for advanced atherosclerosis. Vascular inflammation-related intimal hyperplasia and atherosclerotic progress have major roles in the pathogenesis of saphenous vein graft disease. Methods: In our study, we investigated the effect of anacardic acid (AA), which is a bioactive phytochemical in the shell of Anacardium occidentale, on atherosclerosis considering its inhibitory effect on NF-κB. We observed relative ICAM-1 and NF-κB mRNA levels by qRT-PCR method in a TNF-α- induced inflammation model of saphenous vein endothelial cell culture after 0.1, 0.5, 1 and 5 μM of AA were applied to the cells. In addition, protein levels of ICAM-1 and NF-κB were evaluated by immunofluorescent staining. The results were compared between different concentrations of AA, and also with the control group. Results: It was found that 5 μM, 1 μM and 0.5 μM of AA had toxic effects, while cytotoxicity decreased when 0.1 μM of AA was applied both alone and with TNF-α. When AA was applied with TNF-α, there was a decrease and suppression in NF-κB expression compared with the TNF-α group. TNF-α-induced ICAM-1 expression was significantly reduced more in the AA-applied group than in the TNF-α group. Conclusion: In accordance with our results, it can be said that AA has a protective role in the pathogenesis of atherosclerosis and hence in saphenous vein graft disease.

Making cell culture more physiological: a call for a more comprehensive assessment of racial disparities in endothelial cell culture studies

In the United States, cardiovascular diseases (CVDs) are the leading cause of death and disproportionately affect ethnic and racial minority populations. Black individuals are more likely to develop advanced CVD and microvascular complications resulting in end-organ damage. Endothelial cell dysfunction leads to microvascular and macrovascular dysfunction and is predictive of the development of CVD. Black versus white racial disparities in in vivo and in vitro studies of endothelial cell function are well documented. However, race-related disparities in maternal environment and lifestyle may be a major unconsidered factor in racial differences in endothelial cell culture studies. Further, rates of hypertensive disorders of pregnancy are higher in black versus white women. These pregnancy complications may result in placental dysfunction, including excess production of inflammatory and antiangiogenic molecules that impair endothelial function. Therefore, studies that include other ethnic and racial minorities are needed, in addition to a more thorough characterization of endothelial cell donors and targeted cell culture studies (e.g., genotyping) to generate information that can be translated into effective preventive or treatment strategies for ethnic/racial disparities in CVD.