Response Of Endothelial Cells Research Articles

Phosphatidylserine-binding receptor, CD300f, on macrophages mediates host invasion of pathogenic and non-pathogenic rickettsiae.

Some arthropod-borne obligate intracellular rickettsiae are among the most virulent human pathogens. Rickettsia species modulate immune (e.g., macrophages; MΦ) and non-immune cell (e.g., endothelial cells) responses to create a habitable environment for host colonization. In particular, MΦ play a crucial role in either terminating an infection at an early stage or succumbing to bacterial replication and colonization. However, our understanding on how Rickettsia species invade host cells, including MΦ, remain poorly defined. In this study, we describe a mechanism of host invasion by Rickettsia species, involving rickettsial phosphatidylserine (PS), as a ligand, and the CD300f receptor on MΦ. Using bone marrow-derived macrophages (BMDMΦ) from wild-type (WT) and CD300f-/- mice, we demonstrated that engulfment of both pathogenic R. typhi (the etiologic agent of murine typhus) and R. rickettsii (the etiologic agent of Rocky Mountain spotted fever) species as well as the non-pathogenic R. montanensis was significantly reduced in CD300f-/- BMDMΦ as compared to that of WT BMDMΦ. Furthermore, our mechanistic analysis suggests bacterial PS as the potential source for the CD300f-mediated rickettsiae engulfment by MΦ. In vivo infection studies using WT and CD300f-/- C57BL/6J mice showed that CD300f-/- animals were protected against R. typhi- or R. rickettsii-induced fatal rickettsiosis, which correlated with levels of bacterial burden detected in the spleens of mice. Adoptive transfer studies further revealed that CD300f-expressing MΦ are important mediators to control rickettsiosis in vivo. Collectively, our findings describe a previously unappreciated role for the efferocytic receptor, CD300f, to facilitate engulfment of rickettsiae within the host.

Absence of Functional Autoantibodies Targeting Angiotensin II Receptor Type 1 and Endothelin-1 Type A Receptor in Circulation and Purified IgG From Patients With Systemic Sclerosis.

Systemic sclerosis (SSc) is a rare but severe autoimmune disease characterized by immune dysregulation, fibrosis, and vasculopathy. Although previous studies have highlighted the presence of functional autoantibodies targeting the angiotensin II receptor type 1 (AT1) and endothelin-1 type A receptor (ETAR), leading to autoantibody-mediated receptor stimulation and subsequent activation of endothelial cells (ECs), a comprehensive understanding of the direct interaction between these autoantibodies and their receptors is currently lacking. Moreover, existing data confirming the presence of these autoantibodies in SSc often rely on similar methodologies and assays. Our aim was to replicate previous findings and to investigate the functional effects of IgG derived from patients with SSc (SSc IgG) on AT1 and ETAR signaling, the downstream EC response, and the presence of AT1-binding autoantibodies in circulation. Quantitative polymerase chain reaction and cytokine enzyme-linked immunosorbent assay, alongside a real-time cell analyzer, were used to assess receptor-specific functional characteristics of purified SSc IgG (n = 18). Additionally, a novel protein capture assay using solubilized epitope-tagged AT1 was developed to detect AT1-binding autoantibodies in plasma samples from patients with SSc (n = 28) and healthy donors (n = 14). No evidence for EC activation in an AT1- or ETAR-dependent manner was revealed. Furthermore, stimulation with SSc IgG did not induce receptor activation or alter G protein-coupled receptor signaling on agonist stimulation in a model with receptor overexpression. Lastly, no AT1-binding autoantibodies were detected in plasma samples from patients with SSc when using epitope-tagged solubilized AT1. Overall, our study did not provide evidence to support the presence of AT1- or ETAR-activating autoantibodies in purified SSc IgG or AT1-binding autoantibodies in the circulation of patients with SSc.

Diverse microtubule-destabilizing drugs induce equivalent molecular pathway responses in endothelial cells.

Microtubule (MT)-destabilizing drugs, despite their potential for toxicity, are used to treat a surprising range of diseases, including cancers, inflammatory disorders, and parasitic infections. To investigate how drugs with apparently similar actions on MTs achieve diverse clinical effects, we compared the molecular pathways that are activated by three drugs with different clinical profiles, vinblastine, combretastatin A4, and plinabulin. All three elicited similar gene expression responses via Rho GTPase activation. This finding suggests that their distinct clinical effects are not caused by different effects on MTs, but rather by differences in drug transport, pharmacokinetics or tubulin isotype affinity. Our findings provide insights into how plinabulin might protect the bone marrow and may help medicinal chemists design MT drugs for new applications.

Divergent endothelial mechanisms drive arteriovenous malformations in Alk1 and SMAD4 loss-of-function.

Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder caused by mutations in the bone morphogenetic protein signaling pathway, leading to arteriovenous malformations. While previously thought to share molecular and cellular dysregulation, this study reveals highly distinct mechanisms depending on whether mutations occur in Alk1 or SMAD4. Loss of SMAD4 enhances endothelial cell responses to flow, including flow-regulated transcription and cell migration against blood flow, causing excessive pruning of capillaries and the formation of single large shunts. Conversely, Alk1 deficiency disrupts endothelial flow responses, including cell polarization and directional migration, leading to a dense vascular network and the persistence of a malformation nidus. In vivo cell population tracking of mutant cells validates unique endothelial cell migration defects. Mosaic cell culture models further illustrate that mutant cells co-opt wild-type cells driving distinct Alk1 or SMAD4 mutant-like behavioral defects. These findings demonstrate that arteriovenous malformations develop through fundamentally different cellular mechanisms based on the specific genetic mutation emphasizing the need for tailored diagnostic and therapeutic strategies.

ENO2-Regulated Glycolysis in Endothelial Cells Contributes to FGF2-Induced Retinal Neovascularization.

Ocular neovascularization is a major cause of blindness. Although fibroblast growth factor-2 (FGF2) has been implicated in the pathophysiology of angiogenesis, the underlying mechanisms remain incompletely understood. The purpose of this study was to investigate the role of FGF2 in retinal neovascularization and elucidate its underlying mechanisms. The oxygen-induced retinopathy mouse model was used to study the pathogenesis of retinal neovascularization. Immunofluorescence was used to quantify the neovascularization in retina. Data-independent acquisition proteomics were performed to quantify differentially expressed proteins in human retinal microvascular endothelial cells stimulated with FGF2 and associated pathways were analyzed. We carried out qRT-PCR and Western Blot assays to detect the expression of genes at mRNA and protein levels. The angiogenesis abilities of human retinal microvascular endothelial cells were measured by transwell, EdU and tube formation assays. FGF2 was significantly upregulated in retinal tissues of the oxygen-induced retinopathy mouse model and it markedly enhanced tube formation, migration, and proliferation abilities of human retinal microvascular endothelial cells in vitro. The proteomic analysis identified 287 differentially expressed proteins in endothelial cells in response to FGF2 stimulation, characterized by a notable upregulation of the glycolysis pathway, among which we confirmed that the enolase 2 (ENO2) levels were elevated after FGF2 stimulation, and its knockdown resulted in diminished glycolytic activity and impaired angiogenic processes. Furthermore, the use of the ENO2 inhibitor AP-Ⅲ-a4 alleviated angiogenesis in vivo and in vitro. Our findings underscore the pivotal role of ENO2-mediated glycolysis in FGF2-induced angiogenesis, suggesting that ENO2 may serve as a promising therapeutic target for managing pathological neovascularization.

Disturbed shear stress promotes atherosclerosis through TRIM21-regulated MAPK6 degradation and consequent endothelial inflammation.

Coronary artery plaques often develop in regions subjected to disturbed shear stress (DSS), yet the mechanisms underlying this phenomenon remain poorly understood. Our study aimed to elucidate the unknown role of MAPK6 in shear stress and plaque formation. In vitro and in vivo experiments, RNA-seq, CO-IP and proteomic analysis, combined with single-cell RNA-seq datasets were used to reveal the upstream and downstream mechanisms involved. AAV-MAPK6, ApoE-/-MAPK6flox/floxTEKCre mice and the CXCL12 neutraligand were used to confirm the beneficial effects of MAPK6 against atherosclerosis. Our study revealed a substantial decrease in MAPK6 protein levels in endothelial cells in response to DSS, both in vivo and in vitro, which was contingent on the binding of the ubiquitin ligase TRIM21 to MAPK6. Endothelium-specific MAPK6 overexpression exerts antiatherosclerotic effects in ApoE-/- mice, elucidating the unexplored role of MAPK6 in atherosclerosis. Comprehensive RNA-seq, integrated single-cell mapping and further experiments unveiled the involvement of MAPK6 in inflammation through the EGR1/CXCL12 axis. ApoE-/-MAPK6flox/floxTEKCre mice finally confirmed that conditional MAPK6 knockout resulted in endothelial inflammation and significant increases in plaque areas. Notably, these effects could be reversed through the neutralization of CXCL12. Our study illuminates the advantages of MAPK6 in decelerating plaque progression, highlighting the potential of safeguarding MAPK6 as a novel therapeutic strategy against atherosclerosis. Disturbed flow activates the ubiquitin‒proteasome degradation pathway of MAPK6 in endothelial cells, which is contingent on the binding of the ubiquitin ligase TRIM21 to MAPK6. Endothelial MAPK6 has an advantageous impact on decelerating plaque progression. MAPK6 regulates endothelial inflammation via the EGR1/CXCL12 axis.

Sustained hypoxia but not intermittent hypoxia induces HIF-1α transcriptional response in human aortic endothelial cells.

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxic environments at the cellular level and is an independent risk factor for the development of cardiovascular disease. Endothelial cell (EC) dysfunction precedes the development of cardiovascular disease; however, the mechanisms by which ECs respond to these intermittent hypoxic events are poorly understood. To better understand EC responses to hypoxia, we examined the effects of sustained hypoxia (SH) and intermittent hypoxia (IH) on the activation of HIF-1α in ECs. While SH stabilized HIF-1α and led to its nuclear localization, IH did not activate HIF-1α and the expression of its target genes. Using RNA-sequencing, we evaluated transcriptional responses of ECs to hypoxia. SH induced the expression of HIF-1α and hypoxia response genes, while IH affected cell-cycle regulation genes. A cytoscape protein-protein interaction network for EC response to hypoxia was created with differentially expressed genes. The network comprises cell-cycle regulation, inflammatory signaling via NF-κB and response to VEGF stimulus subnetworks on which SH and IH had distinct activities. As OSA is associated with elevated catecholamines, we investigated the effect of epinephrine on the EC response to SH and IH. Transcriptomic responses under IH and epinephrine revealed protein-protein interaction networks emphasizing distinct subnetworks, including cytokine-mediated TNFα signaling via NF-κB, Wnt/LRP/DKK signaling and cell cycle regulation. This study reveals differential transcriptomic responses under SH and IH characterised by HIF-1α transcriptional response induced only by SH, but not by IH. The study also features the potential molecular events that may occur at the vascular level in OSA.

Vascularized tumor on a microfluidic chip to study mechanisms promoting tumor neovascularization and vascular targeted therapies.

The cascade of events leading to tumor formation includes induction of a tumor supporting neovasculature, as a primary hallmark of cancer. Developing vasculature is difficult to evaluate in vivo but can be captured using microfluidic chip technology and patient derived cells. Herein, we established an on chip approach to investigate the mechanisms promoting tumor vascularization and vascular targeted therapies via co-culture of cancer spheroids and endothelial cells in a three dimensional environment. Methods: We investigated both, tumor neovascularization and therapy, via co-culture of human derived endothelial cells and adjacently localized metastatic renal cell carcinoma spheroids on a commercially available microfluidic chip system. Metastatic renal cell carcinoma spheroids adjacent to primary vessels model tumor, and induce vessels to sprout neovasculature towards the tumor. We monitored real time changes in vessel formation, probed the interactions of tumor and endothelial cells, and evaluated the role of important effectors in tumor vasculature. In addition to wild type endothelial cells, we evaluated endothelial cells that overexpress Prostate Specific Membrane Antigen (PSMA), that has emerged as a marker of tumor associated neovasculature. We characterized the process of neovascularization on the microfluidic chip stimulated by enhanced culture medium and the investigated metastatic renal cell carcinomas, and assessed endothelial cells responses to vascular targeted therapy with bevacizumab via confocal microscopy imaging. To emphasize the potential clinical relevance of metastatic renal cell carcinomas on chip, we compared therapy with bevacizumab on chip with an in vivo model of the same tumor. Results: Our model permitted real-time, high-resolution observation and assessment of tumor-induced angiogenesis, where endothelial cells sprouted towards the tumor and mimicked a vascular network. Bevacizumab, an antiangiogenic agent, disrupted interactions between vessels and tumors, destroying the vascular network. The on chip approach enabled assessment of endothelial cell biology, vessel's functionality, drug delivery, and molecular expression of PSMA. Conclusion: Observations in the vascularized tumor on chip permitted direct and conclusive quantification of vascular targeted therapies in weeks as opposed to months in a comparable animal model, and bridged the gap between in vitro and in vivo models.

Endothelial cell (EC)-specific CTGF/CCN2 Expression Increases EC Reprogramming and Atherosclerosis.

Arterial endothelial cells (ECs) reside in a complex biomechanical environment. ECs sense and respond to wall shear stress. Low and oscillatory wall shear stress is characteristic of disturbed flow and commonly found at arterial bifurcations and around atherosclerotic plaques. Disturbed flow is pro-inflammatory to ECs. Arteries also stiffen with aging and/or the onset of vascular disease. ECs sense and respond to stiffening in a pro-fibrotic manner. Thus, flow and stiffening disturbances elicit EC responses that promote pathologic arterial remodeling. However, the pathways elicited by ECs under pathologic stiffening and disturbed flow are not well understood. The objective of this work was to discover and test the modifiability of key pathways in ECs. To do this we used the partial carotid ligation model to impose disturbed flow onto the precociously stiffened fibulin-5 knockout (Fbln5-/-) mouse carotid arteries. Biomechanical testing demonstrated that Fbln5-/- arteries under disturbed flow approximate the stiffness ratio of diseased human arteries, and the ECs in these Fbln5-/- arteries underwent rapid reprogramming via endothelial to mesenchymal transition (EndMT). Under atherogenic conditions, disturbed flow Fbln5-/- arteries developed more vulnerable plaques than the wild type (WT) mouse arteries. Connective tissue growth factor/cellular communication network factor 2 (Ctgf/Ccn2) was upregulated in vivo in ECs with aging, with stiffening in the Fbln5-/- arteries, and increased again by disturbed flow under stiffened conditions, supporting CTGF as a key biomarker for flow and stiffening. This was validated by immunohistochemistry, which demonstrated increased CTGF deposition in areas of disturbed flow in patient carotid endarterectomy and peripheral artery disease (PAD) specimens. Finally, to test the role of CTGF in regulating and combining these processes, we created an EC-specific Ctgf knockout (Ctgfecko). We identified that carotid arteries under disturbed flow and atherogenic conditions in male Ctgfecko, but not female, mice had decreased plaque area compared to WT control mice. We then tested the Ctgf expression in the carotid endothelium exposed to disturbed or stable flow in WT and Fbln5-/- mice. Here we found that under disturbed flow male mice had greater Ctgf expression than female mice. This work demonstrates that stiffened + disturbed flow conditions drive EC reprogramming, that CTGF is increased by these conditions, and that this increase is more prominent in male carotid arteries. Future exploration of sex-based differences in these fibrotic pathways are warranted to develop targeted therapeutics to limit pathologic arterial remodeling under pathologically stiffened + disturbed flow environments.

Endothelial Response to Blood-Brain Barrier Disruption in the Human Brain.

Cerebral endothelial cell (EC) injury and blood-brain barrier (BBB) permeability contribute to neuronal injury in acute neurological disease states. Preclinical experiments have used animal models to study this phenomenon, yet the response of human cerebral ECs to BBB disruption remains unclear. In our Phase 1 clinical trial (NCT04528680), we used low-intensity pulsed ultrasound with microbubbles (LIPU/MB) to induce transient BBB disruption of peri-tumoral brain in patients with recurrent glioblastoma. We found radiographic evidence that BBB integrity was mostly restored within 1-hour of this procedure. Using single-cell RNA sequencing and transmission electron microscopy, we analyzed the acute response of human brain ECs to ultrasound-mediated BBB disruption. Our analysis revealed distinct EC gene expression changes after LIPU/MB, particularly in genes related to neurovascular barrier function and structure, including changes to genes involved in the basement membrane, EC cytoskeleton, and junction complexes, as well as caveolar transcytosis and various solute transporters. Ultrastructural analysis showed that LIPU/MB led to a decrease in luminal caveolae, the emergence of cytoplasmic vacuoles, and the disruption of the basement membrane and tight junctions, among other things. These findings suggested that acute BBB disruption by LIPU/MB led to specific transcriptional and ultrastructural changes and could represent a conserved mechanism of BBB repair after neurovascular injury in humans.

Bortezomib induces Rho-dependent hyperpermeability of endothelial cells synergistically with inflammatory mediators

BackgroundBortezomib (BTZ), a selective 26 S proteasome inhibitor, is clinically useful in treating multiple myeloma and mantle cell lymphoma. BTZ exerts its antitumor effect by suppressing nuclear factor-B in myeloma cells, promoting endothelial cell apoptosis, and inhibiting angiogenesis. Despite its success, pulmonary complications, such as capillary leak syndrome of the vascular hyperpermeability type, were reported prior to its approval. Although the incidence of these complications has decreased with the use of steroids, the underlying mechanism remains unclear. This study aims to investigate how BTZ influences endothelial cell permeability.MethodsWe examined the impact of BTZ on vascular endothelial cells, focusing on its effects on RhoA and RhoC proteins. Stress fiber formation, a known indicator of increased permeability, was assessed through the Rho/ROCK pathway.ResultsBTZ was found to elevate the protein levels of RhoA and RhoC in vascular endothelial cells, leading to stress fiber formation via the Rho/ROCK pathway. This process resulted in enhanced vascular permeability in a Rho-dependent manner. Furthermore, the stress fiber formation induced by BTZ had synergistic effects with the inflammatory mediator histamine.ConclusionsOur findings suggest that BTZ accumulates RhoA and RhoC proteins in endothelial cells, amplifying the inflammatory mediator-induced increase in the active GTP-bound state of Rho, thereby exaggerating vascular permeability during pulmonary inflammation. This study provides novel insights into the molecular mechanism underlying the pulmonary complications of BTZ, suggesting that BTZ may enhance inflammatory responses in pulmonary endothelial cells by increasing RhoA and RhoC protein levels.

Endothelial Pim3 kinase protects the vascular barrier during lung metastasis

Endothelial cells (ECs) form a tissue-specific barrier for disseminating cancer cells in distant organs. However, the molecular regulation of the ECs in the metastatic niche remains unclear. Here, we analyze using scRNA-Seq, the transcriptional reprogramming of lung ECs six hours after the arrival of melanoma cells in mouse lungs. We discover a reactive capillary EC cluster (rCap) that increases from general capillary ECs in response to infiltrating cancer cells. rCap is enriched for angiogenic and inflammatory pathways and is also found in human lung datasets. The JAK-STAT activated oncogenic Pim3 kinase is a marker of rCap, being upregulated in spontaneous metastasis models. Notably, PIM inhibition increases vascular leakage and metastatic colonization and impairs the EC barrier by decreasing the junctional cadherin-5 and catenins α, β and δ. These results highlight the pulmonary endothelium’s plasticity and its protection by PIM3, which may impair the efficacy of PIM inhibitors in cancer therapies.

Cytoadhesion of Plasmodium falciparum-Infected Red Blood Cells Changes the Expression of Cytokine-, Histone- and Antiviral Protein-Encoding Genes in Brain Endothelial Cells.

Malaria remains a significant global health problem, mainly due to Plasmodium falciparum, which is responsible for most fatal infections. Infected red blood cells (iRBCs) evade spleen clearance by adhering to endothelial cells (ECs), triggering capillary blockage, inflammation, endothelial dysfunction and altered vascular permeability, prompting an endothelial transcriptional response. The iRBCIT4var04/HBEC-5i model, where iRBCs present IT4var04 (VAR2CSA) on their surface, was used to analyze the effects of iRBC binding on ECs at different temperature (37°C vs. 40°C). Binding of non-infected RBCs (niRBCs) and fever alone altered the expression of hundreds of genes in ECs. Comparing the expression profile of HBEC-5i cells cultured either in the presence of iRBCs or in the presence of niRBCs revealed significant upregulation of genes linked to immune response, nucleosome assembly, NF-kappa B signaling, angiogenesis, and antiviral immune response/interferon-alpha/beta signaling. Raising the temperature to 40°C, simulating fever, led to further upregulation of many genes, particularly those involved in cytokine production and angiogenesis. In summary, the presence of iRBCs stimulates ECs, activating several immunological pathways and affecting antiviral (-parasitic) mechanisms and angiogenesis. Our data uncovered the induction of the interferon-alpha/beta signaling pathway in ECs in response to iRBCs.

Endothelial KDM5B Regulated by Piezo1 Contributes to Disturbed Flow Induced Atherosclerotic Plaque Formation.

Epigenetic modifications play an important role in disturbed flow (d-flow) induced atherosclerotic plaque formation. By analysing a scRNA-seq dataset of the left carotid artery (LCA) under d-flow conditions, we found that Jarid1b (KDM5B) was upregulated primarily in a subcluster of endothelial cells in response to d-flow stimulation. We therefore investigated the mechanism of KDM5B expression and the role of KDM5B in endothelial cell. Intriguingly, activation of Piezo1, a major endothelial mechanosensor, was found to promote KDM5B expression, which was reversed by Piezo1 inhibition in HUVECs. Downstream of Piezo1, ETS1 expression and c-JUN phosphorylation were enhanced by d-flow or Piezo1 activation, leading to an increase in KDM5B expression. Furthermore, knockdown of either KDM5B or Piezo1 was found to prevent d-flow induced H3K4me3 demethylation, which was supported by the pharmacological inhibition of Piezo1 in HUVECs. RNA sequencing on shKdm5b HUVECs implied that KDM5B is associated with endothelial inflammation and atherosclerosis. Using partial carotid ligation surgery on Kdm5bf/f Cdh5cre mice with mAAV-PCSK9D377Y infected, we showed that endothelial KDM5B deficiency reduced atherosclerotic lesions in hypercholesterolemic mice. Our findings indicate that endothelial KDM5B expression induced by d-flow via the Piezo1 pathway promotes atherosclerotic plaque formation, providing targets for the prevention or therapeutic intervention of atherosclerosis.

Coronary Microvascular Dysfunction Is Associated With Augmented Lysosomal Signaling in Hypercholesterolemic Mice.

Accumulating evidence indicates that coronary microvascular dysfunction (CMD) caused by hypercholesterolemia can lead to myocardial ischemia, with or without obstructive atherosclerotic coronary artery disease. However, the molecular pathways associated with compromised coronary microvascular function before the development of myocardial ischemic injury remain poorly defined. In this study, we investigated the effects of hypercholesterolemia on the function and integrity of the coronary microcirculation in mice and the underlying mechanisms. Mice were fed a hypercholesterolemic Paigen's diet for 8 weeks. Echocardiography data showed that Paigen's diet caused CMD, characterized by significant reductions in coronary blood flow and coronary flow reserve, but did not affect cardiac remodeling or dysfunction. Immunofluorescence studies revealed that Paigen's diet-induced CMD was associated with activation of coronary arterioles inflammation and increased myocardial inflammatory cell infiltration. These pathological changes occurred in parallel with the upregulation of lysosomal signaling pathways in endothelial cells (ECs). Treating hypercholesterolemic mice with the cholesterol-lowering drug ezetimibe significantly ameliorated Paigen's diet-induced adverse effects, including hypercholesterolemia, steatohepatitis, reduced coronary flow reserve, coronary endothelial cell inflammation, and myocardial inflammatory cell infiltration. In cultured mouse cardiac ECs, 7-ketocholesterol increased mitochondrial reactive oxygen species and inflammatory responses. Meanwhile, 7-ketocholesterol induced the activation of transcriptional factor EB and lysosomal signaling in mouse cardiac ECs, whereas the lysosome inhibitor bafilomycin A1 blocked 7-ketocholesterol-induced transcriptional factor EB activation and exacerbated 7-ketocholesterol-induced inflammation and cell death. Interestingly, ezetimibe synergistically enhanced 7-ketocholesterol-induced transcriptional factor EB activation and attenuated 7-ketocholesterol-induced mitochondrial reactive oxygen species and inflammatory responses in mouse cardiac ECs. These results suggest that CMD can develop and precede detectable cardiac functional or structural changes in the setting of hypercholesterolemia and that upregulation of transcriptional factor EB-mediated lysosomal signaling in endothelial cells plays a protective role against CMD.

Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes.

Background/Objectives: The key components of the blood-brain barrier (BBB) are endothelial cells, pericytes, astrocytes, and the capillary basement membrane. The BBB serves as the main barrier for drug delivery to the brain and is the most restrictive endothelial barrier in the body. Nearly all large therapeutic molecules and over 90% of small-molecule drugs cannot cross the BBB. To overcome this challenge, nanotechnology, particularly drug delivery systems such as nanoparticles (NPs), have gained significant attention. Methods: Poly(lactide-co-glycolide) (PLGA) and albumin-based NPs (bovine/human), with or without transferrin (Tf) ligands (BSA, HSA, BSA-Tf, HSA-Tf), and nanolipid carriers (NLC) were synthesized. The interactions of these NPs with human brain microvascular endothelial cells (hBMECs), human brain vascular pericytes (hBVPs), and human astrocytes (hASTROs) were analyzed. Results: At doses of 15.62 µg/mL, 31.25 µg/mL, and 62.5 µg/mL, none of the NPs caused toxic effects on hBMECs, hBVPs, or hASTROs after 3 h of incubation. All NPs were internalized by the cells, but BSA-Tf and HSA-Tf showed significantly higher uptake in hBMECs in a dose-dependent manner. Ultrastructural analysis revealed notable differences between NP formulation and cell type. Conclusions: Our findings underscore the potential of ligand-targeted NPs to selectively interact with BBB endothelial cells. Ultrastructural analysis reveals distinct cellular processing pathways for various NP formulations across BBB-associated cell types, with autophagy emerging as a crucial mechanism for NP handling in pericytes and astrocytes. Changes in NP chemical properties upon biological exposure present significant challenges for nanomedicine design, emphasizing the need for further investigation into NP interactions at the cellular and subcellular levels.

Tumor-derived extracellular vesicles disrupt the blood–brain barrier endothelium following high-frequency irreversible electroporation

High-frequency irreversible electroporation (H-FIRE), a nonthermal brain tumor ablation therapeutic, generates a central tumor ablation zone while transiently disrupting the peritumoral blood–brain barrier (BBB). We hypothesized that bystander effects of H-FIRE tumor cell ablation, mediated by small tumor-derived extracellular vesicles (sTDEV), disrupt the BBB endothelium. Monolayers of bEnd.3 cerebral endothelial cells were exposed to supernatants of H-FIRE or radiation (RT)-treated LL/2 and F98 cancer cells. Endothelial cell response was evaluated microscopically and via flow cytometry for apoptosis. sTDEV were isolated following H-FIRE and RT, characterized via nanoparticle tracking analysis (NTA) and transmission electron microscopy, and applied to a Transwell BBB endothelium model to quantify permeability changes. Supernatants of H-FIRE-treated tumor cells, but not supernatants of sham- or RT-treated cells, disrupted endothelial cell monolayer integrity while maintaining viability. sTDEV released by glioma cells treated with 3000 V/cm H-FIRE increased permeability of the BBB endothelium model compared to sTDEV released after lower H-FIRE doses and RT. NTA revealed significantly decreased sTDEV release after the 3000 V/cm H-FIRE dose. Our results demonstrate that sTDEV increase permeability of the BBB endothelium after H-FIRE ablation in vitro. sTDEV-mediated mechanisms of BBB disruption may be exploited for drug delivery to infiltrative margins following H-FIRE ablation.

A Multichamber Pulsating-Flow Device With Optimized Spatial Shear Stress and Pressure for Endothelial Cell Testing.

Design and analysis are presented for a new device to test the response of endothelial cells to the simultaneous action of cyclic shear stresses and pressure fluctuations. The design consists of four pulsatile-flow chambers connected in series, where shear stress is identical in all four chambers and pressure amplitude decreases in successive chambers. Each flow chamber is bounded above and below by two parallel plates separated by a small gap. The design of the chamber planform must ensure that cells within the testing region experience spatially uniform time-periodic shear stress. For conditions typically encountered in applications, the viscous unsteady flow exhibits order-unity values of the associated Womersley number. The corresponding solution to the unsteady lubrication problem, with general nonsinusoidal flowrate, is formulated in terms of a stream function satisfying Laplace's equation, which can be integrated numerically to determine the spatial distribution of shear stresses for chambers of general planform. The results are used to optimize the design of a device with a hexagonal planform. Accompanying experiments using particle tracking velocimetry (PTV) in a fabricated chamber were conducted to validate theoretical predictions. Pressure readings indicate that intrachamber pressure variations associated with viscous pressure losses and acoustic fluctuations are relatively small, so that all cells in a given testing region experience nearly equal pressure forces.

MTOR maintains endothelial cell integrity to limit lung vascular injury

The functional and structural integrity of the endothelium is essential for vascular homeostasis. Loss of barrier function in quiescent and migratory capacity in proliferative endothelium causes exuberant vascular permeability, a cardinal feature of many inflammatory diseases including acute lung injury (ALI). However, the signals governing these fundamental endothelial cell (EC) functions are poorly understood. Here, we identify Mechanistic Target of Rapamycin (MTOR) as an important link in preserving the barrier integrity and migratory/angiogenic responses in EC and preventing lung vascular injury and mortality in mice. Knockdown of MTOR in EC altered cell morphology, impaired proliferation and migration, and increased endocytosis of cell surface VE-Cadherin leading to disrupted barrier function. MTOR-depleted EC also exhibited reduced VE-Cadherin and VEGFR2 levels mediated in part by autophagy. Similarly, lungs from mice with EC-specific MTOR deficiency displayed spontaneous vascular leakage marked by decreased VE-Cadherin and VEGFR2 levels, indicating that MTOR deficiency in EC is sufficient to disrupt lung vascular integrity and may be a key pathogenic mechanism of ALI. Indeed, MTOR as well as VEGFR2 and VE-Cadherin levels were markedly reduced in injured mouse lungs or EC. Importantly, EC-targeted gene transfer of MTOR cDNA, either prophylactically or therapeutically, mitigated inflammatory lung injury, and improved lung function and survival in mouse models of ALI. These findings reveal an essential role of MTOR in maintaining EC function, identify loss of endothelial MTOR as a key mechanism of lung vascular injury, and show the therapeutic potential of EC-targeted MTOR expression in combating ALI and mortality in mice.

Interleukin-17D accelerates atherosclerosis through promoting endothelial cells ferroptosis via CD93/miR-181a-5p/SLC7A11 signaling

IL-17D has been found to induce inflammatory cytokines in endothelial cells, but its exact role in atherosclerosis (AS) is unclear. This study aims to explore IL-17D’ function in AS development. The expression of IL-17D was examined in AS patients and mice, and its clinical significance was evaluated in patients with acute coronary syndrome (ACS). Apolipoprotein E and IL-17D deficient mice (ApoE−/−IL-17D−/−) were generated for this study. The inflammation response and ferroptosis status in vascular endothelial cells were assessed following IL-17D treatment. Flow cytometry was used to identify the functional receptor of IL-17D. Additionally, RNA-seq was utilized to analyze the miRNA expression profiles induced by IL-17D. Plasma levels of IL-17D were elevated in both AS patients and mice, and were correlated with an increased incidence of major adverse cardiovascular events (MACEs). ApoE−/−IL-17D−/− mice displayed reduced inflammation and fewer atherosclerotic lesions. Treatment with IL-17D resulted in elevated levels of IL-6, IL-8, and ROS, as well as impaired cell viability and GSH production in endothelial cells. Ferroptosis inhibitor (Fer-1) suppressed the proinflammatory effects by IL-17D. Furthermore, CD93 was identified as the functional receptor for IL-17D in endothelial cells. The inhibition of miR-181a-5p led to a significant increase in cell viability and GSH levels, alongside a reduction in ROS and IL-6/IL-8 levels, while the suppression of SLC7A11 abolished these effects. Our findings suggest that IL-17D promotes endothelial inflammation by causing ferroptosis via CD93/miR-181a-5p/SLC7A11 signaling pathway. These insights advance our understanding of the pathophysiology of AS and identify a potential target for therapeutic intervention.