Endothelial Monolayers Research Articles

Extracellular Vesicles in Sickle Cell Disease: A Promising Tool

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide. It is characterized by an impairment of shear stress-mediated vasodilation, a pro-coagulant, and a pro-adhesive state orchestrated among others by the depletion of the vasodilator nitric oxide, by the increased phosphatidylserine exposure and tissue factor expression, and by the increased interactions of erythrocytes with endothelial cells that mediate the overexpression of adhesion molecules such as VCAM-1, respectively. Extracellular vesicles (EVs) have been shown to be novel actors involved in SCD pathophysiological processes. Medium-sized EVs, also called microparticles, which exhibit increased plasma levels in this pathology, were shown to induce the activation of endothelial cells, thereby increasing neutrophil adhesion, a key process potentially leading to the main complication associated with SCD, vaso-occlusive crises (VOCs). Small-sized EVs, also named exosomes, which have also been reported to be overrepresented in SCD, were shown to potentiate interactions between erythrocytes and platelets, and to trigger endothelial monolayer disruption, two processes also known to favor the occurrence of VOCs. In this review we provide an overview of the current knowledge about EVs concentration and role in SCD.

The neuronal protein Neuroligin 1 promotes colorectal cancer progression by modulating the APC/β-catenin pathway

BackgroundColorectal cancer (CRC) remains largely incurable when diagnosed at the metastatic stage. Despite some advances in precision medicine for this disease in recent years, new molecular targets, as well as prognostic/predictive markers, are highly needed. Neuroligin 1 (NLGN1) is a transmembrane protein that interacts at the synapse with the tumor suppressor adenomatous polyposis Coli (APC), which is heavily involved in the pathogenesis of CRC and is a key player in the WNT/β-catenin pathway.MethodsAfter performing expression studies of NLGN1 on human CRC samples, in this paper we used in vitro and in vivo approaches to study CRC cells extravasation and metastasis formation capabilities. At the molecular level, the functional link between APC and NLGN1 in the cancer context was studied.ResultsHere we show that NLGN1 is expressed in human colorectal tumors, including clusters of aggressive migrating (budding) single tumor cells and vascular emboli. We found that NLGN1 promotes CRC cells crossing of an endothelial monolayer (i.e. Trans-Endothelial Migration or TEM) in vitro, as well as cell extravasation/lung invasion and differential organ metastatization in two mouse models. Mechanistically, NLGN1 promotes APC localization to the cell membrane and co-immunoprecipitates with some isoforms of this protein stimulates β-catenin translocation to the nucleus, upregulates mesenchymal markers and WNT target genes and induces an “EMT phenotype” in CRC cell linesConclusionsIn conclusion, we have uncovered a novel modulator of CRC aggressiveness which impacts on a critical pathogenetic pathway of this disease, and may represent a novel therapeutic target, with the added benefit of carrying over substantial knowledge from the neurobiology field.

Emergence of synchronized multicellular mechanosensing from spatiotemporal integration of heterogeneous single-cell information transfer.

Multicellular synchronization is a ubiquitous phenomenon in living systems. However, how noisy and heterogeneous behaviors of individual cells are integrated across a population toward multicellular synchronization is unclear. Here, we study the process of multicellular calcium synchronization of the endothelial cell monolayer in response to mechanical stimuli. We applied information theory to quantify the asymmetric information transfer between pairs of cells and defined quantitative measures to how single cells receive or transmit information within a multicellular network. Our analysis revealed that multicellular synchronization was established by gradual enhancement of information spread from the single cell to the multicellular scale. Synchronization was associated with heterogeneity in the cells' communication properties, reinforcement of the cells' state, and information flow. Altogether, we suggest a phenomenological model where cells gradually learn their local environment, adjust, and reinforce their internal state to stabilize the multicellular network architecture to support information flow from local to global scales toward multicellular synchronization.

Bio-Mimicking Brain Vasculature to Investigate the Role of Heterogeneous Shear Stress in Regulating Barrier Integrity.

A continuous, sealed endothelial membrane is essential for the blood-brain barrier (BBB) to protect neurons from toxins present in systemic circulation. Endothelial cells are critical sensors of the capillary environment, where factors like fluid shear stress (FSS) and systemic signaling molecules activate intracellular pathways that either promote or disrupt the BBB. The brain vasculature exhibits complex heterogeneity across the bed, which is challenging to recapitulate in BBB microfluidic models with fixed dimensions and rectangular cross-section microchannels. Here, a Cayley-tree pattern, fabricated using lithography-less, fluid shaping technique in a modified Hele-Shaw cell is used to emulate the brain vasculature in a microfluidic chip. This geometry generates an inherent distribution of heterogeneous FSS, due to smooth variations in branch height and width. hCMEC/D3 endothelial cells cultured in the Cayley-tree designed chip generate a 3D monolayer of brain endothelium with branching hierarchy, enabling the study of the effect of heterogeneous FSS on the brain endothelium. The model is employed to study neuroinflammatory conditions by stimulating the brain endothelium with tumor necrosis factor-α under heterogeneous FSS conditions. The model has immense potential for studies involving drug transport across the BBB, which can be misrepresented in fixed dimension models.

Human T-bet+ B cell development is associated with BTK activity and suppressed by evobrutinib.

Recent clinical trials have shown promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti–VLA-4 antibody) treatment. Under in vitro T follicular helper–like conditions, BTK phosphorylation was enhanced by T-bet–inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet–associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

In Vivo Evaluation of PCL Vascular Grafts Implanted in Rat Abdominal Aorta.

Electrospun tissue-engineered grafts made of biodegradable materials have become a perspective search field in terms of vascular replacement, and more research is required to describe their in vivo transformation. This study aimed to give a detailed observation of hemodynamic and structural properties of electrospun, monolayered poly-ε-caprolactone (PCL) grafts in an in vivo experiment using a rat aorta replacement model at 10, 30, 60 and 90 implantation days. It was shown using ultrasound diagnostic and X-ray tomography that PCL grafts maintain patency throughout the entire follow-up period, without stenosis or thrombosis. Vascular compliance, assessed by the resistance index (RI), remains at the stable level from the 10th to the 90th day. A histological study using hematoxylin-eosin (H&E), von Kossa and Russell–Movat pentachrome staining demonstrated the dynamics of tissue response to the implant. By the 10th day, an endothelial monolayer was forming on the graft luminal surface, followed by the gradual growth and compaction of the neointima up to the 90th day. The intense inflammatory cellular reaction observed on the 10th day in the thickness of the scaffold was changed by the fibroblast and myofibroblast penetration by the 30th day. The cellularity maximum was reached on the 60th day, but by the 90th day the cellularity significantly (p = 0.02) decreased. From the 60th day, in some samples, the calcium phosphate depositions were revealed at the scaffold-neointima interface. Scanning electron microscopy showed that the scaffolds retained their fibrillar structure up to the 90th day. Thus, we have shown that the advantages of PCL scaffolds are excellent endothelialization and good surgical outcome. The disadvantages include their slow biodegradation, ineffective cellularization, and risks for mineralization and intimal hyperplasia.

Inhibition of HSP90 Preserves Blood-Brain Barrier Integrity after Cortical Spreading Depression.

Cortical spreading depression (CSD) is a pathophysiological mechanism underlying headache disorders, including migraine. Blood–brain barrier (BBB) permeability is increased during CSD. Recent papers have suggested that heat shock proteins (HSP) contribute to the integrity of the blood–brain barrier. In this study, the possible role of HSP90 in CSD-associated blood–brain barrier leak at the endothelial cell was investigated using an in vitro model, for the blood–endothelial barrier (BEB), and an in vivo model with an intact BBB. We measured barrier integrity using trans endothelial electric resistance (TEER) across a monolayer of rodent brain endothelial cells (bEnd.3), a sucrose uptake assay, and in situ brain perfusion using female Sprague Dawley rats. CSD was induced by application of 60 mM KCl for 5 min in in vitro experiments or cortical injection of KCl (1 M, 0.5 µL) through a dural cannula in vivo. HSP90 was selectively blocked by 17-AAG. Our data showed that preincubation with 17-AAG (1 µM) prevented the reduction of TEER values caused by the KCl pulse on the monolayer of bEnd.3 cells. The elevated uptake of 14C-sucrose across the same endothelial monolayer induced by the KCl pulse was significantly reduced after preincubation with HSP90 inhibitor. Pre-exposure to 17-AAG significantly mitigated the transient BBB leak after CSD induced by cortical KCl injection as determined by in situ brain perfusion in female rats. Our results demonstrated that inhibition of HSP90 with the selective agent 17-AAG reduced CSD-associated BEB/BBB paracellular leak. Overall, this novel observation supports HSP90 inhibition mitigates KCl-induced BBB permeability and suggests the development of new therapeutic approaches targeting HSP90 in headache disorders.

NF-κB-dependent repression of Sox18 transcription factor requires the epigenetic regulators histone deacetylases 1 and 2 in acute lung injury.

In acute lung injury (ALI), the NF-κB-mediated downregulation of Sox18 gene expression leads to the disruption of the pulmonary endothelial barrier. Previous studies have suggested that the action of NF-κB as a transcriptional repressor also requires the action of class I histone deacetylases (HDACs). Thus, the purpose of this study was to investigate and further delineate the mechanism of Sox18 repression during lipopolysaccharide (LPS) induced ALI. Using selective inhibitors and specific siRNA-driven depletion of HDACs 1-3 in human lung microvascular endothelial cells (HLMVEC) we were able to demonstrate a critical role for HDACs 1 and 2 in the LPS-mediated repression of Sox18 gene expression and the loss of endothelial monolayer integrity. Moreover, our data demonstrate that HDAC1 associates with a transcription-repressive complex within the NF-κB-binding site of Sox18 promoter. Further, we were able to show that the selective inhibitor of HDAC1, tacedinaline, significantly reduced the endothelial permeability and injury associated with LPS challenge in the mouse lung. Taken together, our data demonstrate, for the first time, that transcription repressors HDACs 1 and 2 are involved in pathological mechanism of ALI and can be considered as therapeutic targets.

Toxoplasma effector-induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endothelium.

The obligate intracellular parasite Toxoplasma gondii makes use of infected leukocytes for systemic dissemination. Yet, how infection impacts the processes of leukocyte diapedesis has remained unresolved. Here, we addressed the effects of T. gondii infection on the trans-endothelial migration (TEM) of dendritic cells (DCs) across polarised brain endothelial monolayers. We report that upregulated expression of leukocyte ICAM-1 is a feature of the enhanced TEM of parasitised DCs. The secreted parasite effector GRA15 induced an elevated expression of ICAM-1 in infected DCs that was associated with enhanced cell adhesion and TEM. Consequently, gene silencing of Icam-1 in primary DCs or deletion of parasite GRA15 reduced TEM. Further, the parasite effector TgWIP, which impacts the regulation of host actin dynamics, facilitated TEM across polarised endothelium. The data highlight that the concerted action of the secreted effectors GRA15 and TgWIP modulate the leukocyte-endothelial interactions of TEM in a parasite genotype-related fashion to promote dissemination. In addition to the canonical roles of endothelial ICAM-1, this study identifies a previously unappreciated role for leukocyte ICAM-1 in infection-related TEM.

Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation.

Off-the-shelf small diameter vascular grafts are an attractive alternative to eliminate the shortcomings of autologous tissues for vascular grafting. Bovine saphenous vein (SV) extracellular matrix (ECM) scaffolds are potentially ideal small diameter vascular grafts, due to their inherent architecture and signaling molecules capable of driving repopulating cell behavior and regeneration. However, harnessing this potential is predicated on the ability of the scaffold generation technique to maintain the delicate structure, composition, and associated functions of native vascular ECM. Previous de-cellularization methods have been uniformly demonstrated to disrupt the delicate basement membrane components of native vascular ECM. The antigen removal (AR) tissue processing method utilizes the protein chemistry principle of differential solubility to achieve a step-wise removal of antigens with similar physiochemical properties. Briefly, the cellular components of SV are permeabilized and the actomyosin crossbridges are relaxed, followed by lipophilic antigen removal, sarcomeric disassembly, hydrophilic antigen removal, nuclease digestion, and washout. Here, we demonstrate that bovine SV ECM scaffolds generated using the novel AR approach results in the retention of native basement membrane protein structure, composition (e.g., Collagen IV and laminin), and associated cell modulatory function. Presence of basement membrane proteins in AR vascular ECM scaffolds increases the rate of endothelial cell monolayer formation by enhancing cell migration and proliferation. Following monolayer formation, basement membrane proteins promote appropriate formation of adherence junction and apicobasal polarization, increasing the secretion of nitric oxide, and driving repopulating endothelial cells toward a quiescent phenotype. We conclude that the presence of an intact native vascular basement membrane in the AR SV ECM scaffolds modulates human endothelial cell quiescent monolayer formation which is essential for vessel homeostasis.

Microplastic particles induce endothelial activation

Background and Aims : Although microplastics being omnipresent in our environment and already detected in various organisms, less is known about the effects of microplastics on vascular biology in particular. Here we investigated the effects of carboxylated polystyrene (PS) microplastic particles (1 μm) on murine endothelial and immune cells, which are both crucially involved in vascular inflammation.Methods: Murine monocytic J774A.1 cells and endothelial MyEND cells were stimulated with PS particles (3/6 hrs, n=7-10). mRNA and protein levels were analyzed using real-time PCR and ELISA. Adhesion assays were performed incubating confluent MyEND cells with PS particles and Calcein-AM labelled J774A.1 (static/flow). Adherent J774A.1 were detected by fluorescence microscopy (n=6). C57BL/6J mice were injected with TRITC-conjugated PS i.v. (n=6/group). PS particles were localized in blood and liver using IHC staining.Results: In J774A.1 cells, PS particles induced Il1-β and Tnf-α expression and TNF-α release. In MyEND cells, PS particles up-regulated Icam-1 and Vcam-1 levels and sVCAM-1 release. PS particles induced adhesion of J774A.1 cells on endothelial monolayers under static as well as under flow conditions. In vivo, PS particles taken up by neutrophiles (Ly6G-positive) in the peripheral blood. PS particles accumulated in the liver, accompanied by a strong up-regulation of the acute phase proteins Saa1, Saa2 and Saa3. Moreover, Aortic tissue from mice injected with PS particles showed enhanced Il-1β and Vcam-1 expression.Conclusions: PS particles are able to activate the endothelium with subsequent monocyte adhesion, representing a hallmark of vascular inflammation. Thus, microplastics need to be evaluated as a potential novel environmental risk factor for endothelial inflammation. Background and Aims : Although microplastics being omnipresent in our environment and already detected in various organisms, less is known about the effects of microplastics on vascular biology in particular. Here we investigated the effects of carboxylated polystyrene (PS) microplastic particles (1 μm) on murine endothelial and immune cells, which are both crucially involved in vascular inflammation. Methods: Murine monocytic J774A.1 cells and endothelial MyEND cells were stimulated with PS particles (3/6 hrs, n=7-10). mRNA and protein levels were analyzed using real-time PCR and ELISA. Adhesion assays were performed incubating confluent MyEND cells with PS particles and Calcein-AM labelled J774A.1 (static/flow). Adherent J774A.1 were detected by fluorescence microscopy (n=6). C57BL/6J mice were injected with TRITC-conjugated PS i.v. (n=6/group). PS particles were localized in blood and liver using IHC staining. Results: In J774A.1 cells, PS particles induced Il1-β and Tnf-α expression and TNF-α release. In MyEND cells, PS particles up-regulated Icam-1 and Vcam-1 levels and sVCAM-1 release. PS particles induced adhesion of J774A.1 cells on endothelial monolayers under static as well as under flow conditions. In vivo, PS particles taken up by neutrophiles (Ly6G-positive) in the peripheral blood. PS particles accumulated in the liver, accompanied by a strong up-regulation of the acute phase proteins Saa1, Saa2 and Saa3. Moreover, Aortic tissue from mice injected with PS particles showed enhanced Il-1β and Vcam-1 expression. Conclusions: PS particles are able to activate the endothelium with subsequent monocyte adhesion, representing a hallmark of vascular inflammation. Thus, microplastics need to be evaluated as a potential novel environmental risk factor for endothelial inflammation.

Giant multinucleated endothelial cells are associated with local accumulation of LDL and CD45+ lymphocytes in the intima of human aorta

Background and Aims : At the early stages of atherogenesis, low-density lipoprotein (LDL) particles and immune cells penetrate into the intimal layer of the arterial wall through the endothelium. In adult humans, the luminal surface of the arterial wall is a heterogeneous monolayer of cells with varying morphology, including typical endothelial cells (ECs) and multinucleated variant endothelial cells (MVECs). We hypothesized that distribution of MVECs in the endothelial monolayer can explain the distribution pattern of early atherosclerotic lesions.

Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers.

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, the dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify the motion of the peripheral membrane of the cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large-length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, a stiffness consistent with a healthy pulmonary artery. Unlike these large-length scale morphological changes, the baseline of PMFs was largely insensitive to the substrate stiffness on which the cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond the control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel-a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate that 1) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; 2) PMFs increase in response to calcium influx through store-operated calcium entry channels; and 3) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

Endothelial progenitor cells overexpressing Grb2-associated binder 1 for in vitro-constructed tissue-engineered heart valves.

Aim Endothelial progenitor cells (EPCs) play important roles in heart valve replacement surgery. Up-regulation of Grb2‑associated binder 1 (Gab1) promotes hepatocyte growth factor (HGF) - induced endothelial progenitor cell proliferation and migration. This study aimed to investigate the effects of up-regulation of Gab1 in hepatocyte growth factor-induced EPCs in tissue-engineered heart valves (TEHV).Material and methods Fresh porcine aortic valves were placed in 1 % Triton X-100 and trypsin buffer for decellularization. EPCs in the control group were cultured normally, whereas those in the experimental group were both HGF stimulated and transfected with adenovirus containing the Gab1 gene. Cells in the two groups were seeded onto the decellularized valve scaffolds and cultured for 3 or 7 days. TEHV were analyzed by HE and AB-PAS staining.Results By day 3, the experimental group had formed confluent endothelial monolayers on top of the decellularized valves, on the basis of by HE staining and AB-PAS staining. One week later, the control group showed a imperfect endothelial layer.Conclusion HGF-induced EPCs overexpressing Gab1 can endothelialize the decellularized matrix and create functional TEHV, which may then be preconditioned in a bioreactor before clinical implantation.

Multi-Targeting Approach in Glioblastoma Using Computer-Assisted Drug Discovery Tools to Overcome the Blood-Brain Barrier and Target EGFR/PI3Kp110β Signaling.

Simple SummaryTreatment of glioblastoma is hampered by the activation of compensatory survival mechanisms by malignant cells that lead to drug resistance. Moreover, the blood–brain barrier (BBB) precludes the brain entrance of most drugs. We hypothesized that computer-assisted drug discovery tools would reveal novel multi-targeting drug candidates with BBB-permeant and favorable ADMET properties. We aimed to discover molecules with predicted ability to inhibit the EGFR/PI3Kp110β pathway and to validate their efficacy and safety in biological assays. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Moreover, we tested their anti-tumor efficacy and BBB safety and permeation in cell models. We found two EGFR, two PI3Kp110β, and, mostly, two dual inhibitors with anti-tumor effects. Among them, one EGFR and two PI3Kp110β inhibitors were able to cross the BBB endothelium without compromising it. These studies revealed novel drug candidates for glioblastoma treatment.The epidermal growth factor receptor (EGFR) is upregulated in glioblastoma, becoming an attractive therapeutic target. However, activation of compensatory pathways generates inputs to downstream PI3Kp110β signaling, leading to anti-EGFR therapeutic resistance. Moreover, the blood–brain barrier (BBB) limits drugs’ brain penetration. We aimed to discover EGFR/PI3Kp110β pathway inhibitors for a multi-targeting approach, with favorable ADMET and BBB-permeant properties. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Predictions were validated in in vitro models of the human BBB and BBB-glioma co-cultures. The results disclosed 27 molecules (18 EGFR, 6 PI3Kp110β, and 3 dual inhibitors) for biological validation, performed in two glioblastoma cell lines (U87MG and U87MG overexpressing EGFR). Six molecules (two EGFR, two PI3Kp110β, and two dual inhibitors) decreased cell viability by 40–99%, with the greatest effect observed for the dual inhibitors. The glioma cytotoxicity was confirmed by analysis of targets’ downregulation and increased apoptosis (15–85%). Safety to BBB endothelial cells was confirmed for three of those molecules (one EGFR and two PI3Kp110β inhibitors). These molecules crossed the endothelial monolayer in the BBB in vitro model and in the BBB-glioblastoma co-culture system. These results revealed novel drug candidates for glioblastoma treatment.

Sinomenine alleviates glomerular endothelial permeability by activating the C/EBP-α/claudin-5 signaling pathway.

Diabetic nephropathy (DN) is one of the main complications of diabetes. It is closely associated with the dysfunction of glomerular endothelial cells (GECs) under hyperglycemia. Severe inflammation is an important inducer for the development of GECs dysfunction, and it contributes to the disruption of tight junctions in GECs and the increased endothelial permeability. Sinomenine, an alkaloid monomer extracted from the rhizome of Sinomenium acutum, is recognized for its multiple pharmacological functions, including an anti-DN property. The present study aimed to explore the potential functional mechanism of Sinomenine against DN. Animals were randomly divided into Sham, DN, DN + Sinomenine (20mg/kg), and DN + Sinomenine (40mg/kg) groups. The Sinomenine or vehicle was administered every day for 6weeks, followed by collecting renal tissues for further detection. Increased body weights, elevated blood glucose levels and UAE values, aggravated renal tissue pathology, higher concentrations of IL-18 and IL-1β in renal tissues, and reduced claudin-5 expression were observed in DN rats. However, the administration of Sinomenine significantly alleviated all these DN-related changes. Furthermore, human renal glomerular endothelial cells (HrGECs) were treated with high glucose (HG, 30mM) with or without Sinomenine (50, 100μM) for 24h. We found that Sinomenine treatment ameliorated the elevated production of IL-18 and IL-1β, increased fluorescence intensity of FITC-dextran, declined trans-endothelial electrical resistance (TEER) value, and reduction of claudin-5 and C/EBP-α in HG-treated HrGECs. Moreover, the regulatory effect of Sinomenine on endothelial monolayer permeability in HG-treated HrGECs was abolished by the knockdown of C/EBP-α, indicating C/EBP-α is required for the effect of Sinomenine. We concluded that Sinomenine alleviated diabetic nephropathy-induced renal glomerular endothelial dysfunction via activating the C/EBP-α/claudin-5 axis.

The blockage of downstream P2Y2 receptor signaling inhibits the prostate cancer cell adhesion to endothelial cells

AimsProstate cancer is the second most frequently malignancy in men worldwide. Most deaths are caused by metastasis, and tumor cell dissemination involves the interaction with endothelial cells. However, the endothelial cell signaling involved in such interaction is not entirely understood. The tumor microenvironment contains extracellular ATP, an endogenous agonist of the purinergic P2Y2 receptor (P2Y2R). P2Y2R signaling changes endothelial cell phenotype, which may be relevant to cancer pathophysiology. Therefore, we hypothesized that P2Y2R activation could favor the metastatic prostate cancer cells adhesion to endothelial cells. Main methodsFor adhesion assays, confluent endothelial cells EA.hy926 were treated with P2Y2R agonists before adding and imaging stained DU-145 cells. Alternatively, fluorescent probes and antibodies were used to determine intracellular endothelial Ca2+, nitric oxide (NO), and flow cytometry assays. Key findingsEndothelial P2Y2R activation with ATP, UTP, or the selective agonist 2-thio-UTP increased DU-145 cell adhesion to EA.hy926 cells. This effect required endothelial cell Ca2+ mobilization and relied on the endothelial expression of VCAM-1 and ICAM-1. Conversely, inhibiting this proadhesive endothelial phenotype could impair DU-145 cell adhesion. To evaluate this, we chose atorvastatin based on its notable improvement of endothelial cell dysfunction. Atorvastatin blocked UTP-induced DU-145 cell adhesion to endothelial cell monolayer in a NO-dependent manner, unveiling a P2Y2R and NO signaling crosstalk. SignificanceEndothelial P2Y2R signaling contributes to the adhesion of metastatic prostate cancer cells suggesting that the downstream signaling blockade by statins could be a putative mechanism to reduce prostate cancer metastasis.

Tranexamic acid: Beyond antifibrinolysis.

Tranexamic acid (TXA) is a popular antifibrinolytic drug widely used in hemorrhagic trauma patients and cardiovascular, orthopedic, and gynecological surgical patients. TXA binds plasminogen and prevents its maturation to the fibrinolytic enzyme plasmin. A number of studies have demonstrated the broad life-saving effects of TXA in trauma, superior to those of other antifibrinolytic agents. Besides preventing fibrinolysis and blood loss, TXA has been reported to suppress posttraumatic inflammation and edema. Although the efficiency of TXA transcends simple inhibition of fibrinolysis, little is known about its mechanisms of action besides the suppression of plasmin maturation. Understanding the broader effects of TXA at the cell, organ, and organism levels are required to elucidate its potential mechanisms of action transcending antifibrinolytic activity. In this article, we provide a brief review of the current clinical use of TXA and then focus on the effects of TXA beyond antifibrinolytics such as its anti-inflammatory activity, protection of the endothelial and epithelial monolayers, stimulation of mitochondrial respiration, and suppression of melanogenesis.

ApoE-mediated systemic nanodelivery of granzyme B and CpG for enhanced glioma immunotherapy.

The response of malignant glioma to immunotherapy remains gloomy due to its discrete immunological environment and poor brain penetration of immunotherapeutic agents. Here, we disclose that ApoE peptide-mediated systemic nanodelivery of granzyme B (GrB) and CpG ODN co-stimulates enhanced immunotherapy of murine malignant LCPN glioma model. ApoE peptide-functionalized polymersomes encapsulating GrB (ApoE-PS-GrB) could effectively penetrate the blood-brain barrier-mimicking endothelial cell monolayer in vitro and further be taken up by LCPN cells, inducing strong immunogenic cell death (ICD). The co-administration of ApoE-PS-GrB and ApoE-PS-CpG in orthotopic LCPN glioma-bearing mice co-stimulated cytokine production, maturation of dendritic cells (DCs), infiltration of cytotoxic T lymphocytes (CTLs) while reduction of regulatory T lymphocytes (Treg) and M2 phenotype macrophages in the tumor microenvironment, leading to greatly delayed tumor progression and significantly prolonged survival time compared with all controls. The ApoE-mediated systemic nanodelivery of GrB and CpG ODN opens a new pathway for potent immunotherapy of malignant glioma.