Search Results (254)

Streptozotocin (STZ) is an alkylating agent that has neurotoxic effects when injected into the cerebral ventricles (ICV) and also models many other features of Alzheimer’s disease. However, the mechanisms of STZ neurotoxicity are not well understood. In this study, we hypothesized that some of the neurotoxic effects of STZ could be due to direct activities on brain endothelial cells and astrocytes, which are key in forming and supporting the functions of the blood–brain barrier (BBB), respectively. To test this hypothesis, we characterized the changes induced by STZ either in cultures of human-induced pluripotent stem cell (iPSC)-derived brain endothelial-like cells (iBECs), which form an in vitro BBB model, or in primary human astrocytes. We found that STZ at a dosage of 5 mM caused a delayed reduction in the transendothelial electrical resistance (TEER) of iBECs at 7–11 days post-treatment, indicating induction of BBB leakage. Additionally, we observed significant increases in albumin leakage across the monolayer, altered iBEC morphology, and reductions in tight junction proteins, suggesting that STZ causes BBB disruption. We further found that the BBB glucose transporter GLUT-1 was reduced in iBECs, as was the total number of iBECs. In astrocytes, the 5 mM dose of STZ reduced the GFAP signal and total number of cells, suggesting that STZ has anti-proliferative and/or toxic effects on astrocytes. Together, these data support that STZ’s neurotoxic effects could be due, in part, to its direct toxic activities on brain endothelial cells and astrocytes. Full article

Diabetic ulcers are among the most common and challenging complications of diabetes mellitus, and effective therapeutic strategies remain elusive. While stem cell secretome (SCS)-based therapy has attracted considerable attention due to its regenerative potential, its direct application is hindered by low bioavailability and rapid diffusion at the wound site. To address these limitations, we designed a bilayer bacterial cellulose–gelatin (Bi-BCG) scaffold inspired by the hierarchical structure of native skin. This scaffold features a compact bacterial cellulose (BC) upper layer with nanoscale porosity and a porous BCG lower layer with pore sizes of ~52 μm, optimized for SCS delivery. The Bi-BCG scaffold demonstrated a water vapor transmission rate of 2384 g/(m²·24 h) and exhibited significantly improved SCS retention capacity while maintaining high fluid absorption, outperforming monolayer BCG scaffolds. Functionally, human umbilical cord-derived mesenchymal stem cell (hUCMSCs)-derived secretomes significantly enhanced the proliferation (by up to 70.7%) and migration of skin fibroblasts under high-glucose conditions, promoted vascular endothelial cell proliferation (increasing Ki-67+ cells from 25.87% to 46.89%) and angiogenic network formation, and effectively suppressed macrophage-mediated inflammatory responses and oxidative stress. In vivo, the combination of SCSs with the Bi-BCG scaffold exhibited a clear synergistic effect, achieving a wound closure rate of 78.8% by day 10 and promoting superior structural restoration with well-organized collagen deposition, outperforming either treatment alone. These findings underscore the potential of the Bi-BCG scaffold combined with SCSs as an effective strategy for enhancing diabetic wound healing. Full article

Small-diameter vascular grafts often fail due to thrombosis and compliance mismatch. Decellularized plant scaffolds are a biocompatible, sustainable alternative. Leatherleaf viburnum leaves provide natural architecture and mechanical integrity suitable for tissue-engineered vessels. However, the persistence of immunogenic plant biomolecules and limited degradability remain barriers to clinical use. This study tested whether mild heat treatment improves scaffold biocompatibility without compromising mechanical performance. Decellularized leatherleaf viburnum scaffolds were treated at 30–40 °C in 5% NaOH for 15–60 min and then evaluated via tensile testing, burst pressure analysis, scanning electron microscopy, histology, and in vitro assays with white blood cells and endothelial cells. Scaffold properties were compared to those of untreated controls. Heat treatment did not significantly affect scaffold thickness but decreased fiber area fraction and diameter across all anatomical layers. Scaffolds treated at 30–35 °C for ≤30 min retained >90% of tensile strength and achieved burst pressures ≥820 mmHg, exceeding physiological arterial pressures. Heat treatment reduced surface fractal dimension while increasing entropy and lacunarity, producing a smoother but more heterogeneous microarchitecture. White blood cell viability increased up to 2.5-fold and endothelial cell seeding efficiency improved with treatment duration, with 60 min producing near-confluent monolayers. Mild alkaline heat treatment therefore improved immune compatibility and endothelialization while preserving mechanical integrity, offering a simple, scalable modification to advance plant-derived scaffolds for grafting. Full article

Background: Type 2 diabetes mellitus (DM) is a major cardiovascular risk factor that induces monocyte dysfunction and contributes to their accumulation in atherosclerotic lesions. Monocyte recruitment and accumulation in the tissues contribute to chronic inflammation and are essential to the pathobiology of diabetes-induced atherosclerosis. However, the mechanisms that drive the accumulation of monocytes in the diabetic environment are not clearly understood. Methods: Primary monocytes from type 2 (T2) DM and non-T2DM individuals were isolated using magnet-assisted cell sorting. To examine the influence of a diabetic milieu on monocyte function, monocytes from T2DM patients, db/db mice, or human monocytes subjected to hyperglycaemia were analysed for their responses to pro-atherogenic cytokines using Boyden chamber assays. Furthermore, the interactions of non-diabetic and diabetic monocytes with TNFα-inflamed endothelium were studied using live-cell imaging under physiological flow conditions. RT-qPCR and FACS were used to study the expression of relevant molecules involved in monocyte-endothelium interaction. Results: CD14⁺CD16⁻ monocytes isolated from T2DM patients or monocytes exposed to hyperglycaemic conditions showed reduced chemotactic responses towards atherosclerosis-promoting cytokines, CCL2 and CX3CL1, indicating monocyte dysfunction. Under flow conditions, the transendothelial migration (TEM) capacity of T2DM monocytes was significantly reduced. Even though these monocytes adhered to the endothelial monolayer, only a few transmigrated. Interestingly, the T2DM monocytes and monocytes exposed to hyperglycaemic conditions accumulated in the ablumen following transendothelial migration. The time period in the ablumen of T2DM cells was prolonged, as there was a significant impairment of the reverse transendothelial migration (rTEM). Mechanistically, the T2DM milieu specifically induced the activation of monocyte integrins, Macrophage-1 antigen (Mac-1; integrin αMβ2 consisting of CD11b and CD18), and Lymphocyte function-associated antigen 1 (LFA-1; αLβ2 consisting of CD11a and CD18). Furthermore, elevated levels of CD18 transcripts were detected in T2DM monocytes. Junctional Adhesion Molecule 3 (JAM-3)–MAC-1 interactions are known to impede rTEM and T2DM milieu-potentiated JAM-3 expression in human coronary artery endothelial cells (HCAEC). Finally, the overexpression of JAM-3 on HCAEC was sufficient to completely recapitulate the impaired rTEM phenotype. Conclusions: Our results revealed for the first time that the enhanced T2DM monocyte accumulation in the ablumen is not secondary to the elevated transmigration through the endothelium. Instead, the accumulation of monocytes is due to the direct consequence of a dysfunctional rTEM, potentially due to enhanced JAM3-MAC1 engagement. Our results highlight the importance of restoring the rTEM capacity of monocytes to reduce monocyte accumulation-dependent inflammation induction and atherogenesis in the T2DM environment. Full article

Organoid technology has emerged as a revolutionary tool in cancer research, offering physiologically accurate, three-dimensional models that preserve the histoarchitecture, genetic stability, and phenotypic complexity of primary tumors. These self-organizing structures, derived from adult stem cells, induced pluripotent stem cells, or patient tumor biopsies, recapitulate critical aspects of tumor heterogeneity, clonal evolution, and microenvironmental interactions. Organoids serve as powerful systems for modeling tumor progression, assessing drug sensitivity and resistance, and guiding precision oncology strategies. Recent innovations have extended organoid capabilities beyond static culture systems. Integration with microfluidic organoid-on-chip platforms, high-throughput CRISPR-based functional genomics, and AI-driven phenotypic analytics has enhanced mechanistic insight and translational relevance. Co-culture systems incorporating immune, stromal, and endothelial components now permit dynamic modeling of tumor–host interactions, immunotherapeutic responses, and metastatic behavior. Comparative analyses with conventional platforms, 2D monolayers, spheroids, and patient-derived xenografts emphasize the superior fidelity and clinical potential of organoids. Despite these advances, several challenges remain, such as protocol variability, incomplete recapitulation of systemic physiology, and limitations in scalability, standardization, and regulatory alignment. Addressing these gaps with unified workflows, synthetic matrices, vascularized and innervated co-cultures, and GMP-compliant manufacturing will be crucial for clinical integration. Proactive engagement with regulatory frameworks and ethical guidelines will be pivotal to ensuring safe, responsible, and equitable clinical translation. With the convergence of bioengineering, multi-omics, and computational modeling, organoids are poised to become indispensable tools in next-generation oncology, driving mechanistic discovery, predictive diagnostics, and personalized therapy optimization. Full article

The blood–brain barrier (BBB) is critical for central nervous system homeostasis, yet it is highly vulnerable to viral insults. While acute coronavirus infections are known to impair BBB integrity, the long-term impact of persistent infection on brain endothelial cells remains poorly understood. Using an in vitro BBB model, we examined the effects of a 12-week infection with the neurotropic murine coronavirus MHV-JHM. Structural and functional changes were assessed via fluorescein isothiocyanate (FITC)-dextran permeability assay, confocal imaging of mitochondria, actin cytoskeleton, reactive oxygen species (ROS), scanning electron microscopy (SEM) and RT-qPCR for viral RNA level. Long-term infection induced progressive mitochondrial fragmentation and sustained ROS overproduction. Permeability to 70 kDa dextran increased significantly at 48 h post-infection and exceeded control levels threefold by 168 h. SEM revealed gradual endothelial surface roughening, blebbing, and eventual monolayer collapse with extensive intercellular gaps by week 12. Our study demonstrates that long-term MHV-JHM infection profoundly alters brain endothelial cell structure and function, triggering a cascade of changes that culminate in the disintegration of the BBB model. Full article

Background/Objectives: The inner blood–retinal barrier (iBRB) is a specialized neurovascular interface essential for retinal homeostasis and visual function and is compromised in several vision-threating conditions. Therefore, the ability to model iBRB function and dysfunction in a controlled, reproducible and scalable manner is crucial for pharmaceutical research. However, the complex anatomy and physiology of the iBRB raise challenges for cell-based in vitro modeling. Methods/Results: This review follows the evolution of iBRB models—from simple monolayers of retinal endothelial cells (ECs) to sophisticated multicellular microphysiological systems (MPs). Advanced diverse microfluidic platforms aim to replicate key structural, biochemical and functional aspects of the iBRB, each incorporating distinct strategies regarding cell sourcing, device design, flow dynamics and functional readouts. Conclusions: Despite their limitations, these models are highly valuable for drug screening and mechanistic studies aimed at preserving or restoring barrier integrity while also helping to bridge the translational gap in ophthalmic drug discovery. Full article

Background: Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are critical conditions lacking effective pharmacologic therapies. Lipopolysaccharide (LPS), a bacterial endotoxin, is a well-established trigger of ALI. Emerging evidence suggests that heparin derivatives may attenuate lung injury, but their mechanisms remain unclear. Methods: This study evaluated the protective effects of 2-O, 3-O desulfated heparin (ODSH) in a murine model of LPS-induced ALI. Mice received LPS intratracheally with or without ODSH pre-treatment. Lung injury was assessed by bronchoalveolar lavage fluid (BALF) analysis, Evans blue dye albumin EBDA) extravasation, and histopathology. Results: ODSH treatment significantly reduced BALF protein concentration, inflammatory cell infiltration, and EBDA leakage. ODSH preserved endothelial barrier function in vitro, as evidenced by transendothelial electrical resistance (TER) measurements in human lung microvascular endothelial cell (HLMVEC) monolayers. Histological assessment (H&E staining) and myeloperoxidase (MPO) staining demonstrated reduced lung injury and neutrophil infiltration in the ODSH group. ODSH also downregulated pro-inflammatory mediators (NF-κB, IL-6, p38 MAPK) and upregulated the anti-inflammatory cytokine IL-10. Conclusions: ODSH mitigates LPS-induced ALI by reducing vascular permeability, neutrophilic inflammation, and pro-inflammatory signaling while enhancing IL-10 expression. These findings suggest ODSH may offer a novel therapeutic approach for treating ALI. Full article

The internal mammary arteries (IMAs) and coronary arteries share many common characteristics. The inner layer (tunica intima, or intima) of both arteries is lined with a smooth, longitudinally orientated monolayer of endothelial cells (ECs), connective tissue, and an internal elastic lamina that separates the tunica intima from the tunica media (middle layer). The intima of IMAs is lined with an additional protective layer, the neointima, containing vascular smooth muscle cells (VSMCs). The neointima, located between the intima and internal elastic lamina, protects IMAs from damage by assisting in the remodeling of VSMCs. Coarse longitudinal folds in the internal elastic lamina of IMAs partially prevent the infiltration of VSMCs into damaged IMAs, and intimal thickening is thus less likely to occur. Inflamed IMAs resist the migration of monocytes across the endothelial layer and prevent the formation of lipid-rich macrophages (foam cells) within the subintimal or medial layers of arteries. IMAs are thus less likely to form plaques and develop atherosclerosis (AS). Higher levels of prostacyclin (PGI2) in IMAs prevent blood clotting. The anti-thrombotic agents, and production of tumor necrosis factor α (TNF-α), interferon-γ (INF-γ), and visfatin render IMAs more resistant to inflammation. An increase in the production of nitric oxide (NO) by ECs of IMAs may be due to small ubiquitin-like modifier (SUMO) proteins that alter the nuclear factor kappa B (NF-κB) and TLR pathways. The production of reactive oxygen species (ROS) in IMAs is suppressed due to the inhibition of NADPH oxidase (NOX) by a pigment epithelium-derived factor (PEDF), which is a serine protease inhibitor (SERPIN). In this review, a comparison is drawn between the anatomy of IMAs and coronary arteries, with an emphasis on how ECs of IMAs react to immunological changes, rendering them more suited for coronary artery bypass grafts (CABGs). This narrative review covers the most recent findings published in PubMed and Crossref databases. Full article

The development of functional endothelial monolayers on synthetic vascular grafts remains challenging, particularly for small-diameter vessels (<6 mm) prone to thrombosis. Here, we present a pharmacological strategy combining 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate sodium salt (pCPT-cAMP, a tight junction promoter) with nitric oxide/cGMP pathway agonists 3-morpholinosydnonimine (SIN-1), captopril, and sildenafil) to enhance endothelialization. In human umbilical vein endothelial cells (HUVECs), this four-agent cocktail induced a flat, extended phenotype with a 3-fold increased cell area and 57.5% fewer cells required for surface coverage compared to controls. Immunofluorescence analysis revealed enhanced ZO-1 expression and continuous tight junction formation, while sustained nitric oxide (NO) production (3.9-fold increase) and restored prostacyclin (PGI₂) secretion demonstrated preserved endothelial functionality. Anticoagulation assays confirmed a significant reduction in thrombus formation (p < 0.01) via dual inhibition of platelet activation and thrombin binding. These findings establish a synergistic drug combination that promotes rapid endothelialization while maintaining antithrombogenic activity, offering a promising solution for small-diameter vascular grafts. Further studies should validate long-term stability and translational potential in preclinical models. Full article

In endothelial cells, high-dose irradiation induces numerous dysfunctions including alteration in junctional proteins such as VE-Cadherin, apoptosis and enhanced adhesiveness linked to overexpression of adhesion molecules like Intercellular Adhesion Molecule 1 (ICAM-1). Such endothelial dysfunctions can lead to altered tissue perfusion, development of tissue inflammation through increased endothelial permeability, and ultimately organ damage. As intracellular cyclic AMP (cAMP) levels are known to control intercellular junctions or apoptosis in the endothelium, we investigated here the effect of the Phosphodiesterase 4 inhibitor Roflumilast, a drug increasing cAMP levels, on irradiation-induced endothelial dysfunctions in human pulmonary microvascular endothelial cells (HPMECs). Using continuous impedance measurements in confluent endothelial cell monolayers, Roflumilast was found to rapidly reinforce the endothelial barrier and to prevent irradiation-induced barrier disruption. In accordance, irradiation-induced alteration in membrane VE-Cadherin-composed adherens junctions was prevented by Roflumilast treatment after irradiation, which was correlated with its protective effect of the actin cytoskeleton. Post-irradiation treatment with Roflumilast also protected HPMECs from irradiation-induced late apoptosis, but was without effect on irradiation-induced ICAM-1 overexpression. Overall, our results indicate that the beneficial effects of Roflumilast after irradiation are linked to the strengthening/protection of the endothelial barrier and reduced apoptosis, suggesting that this medicine may be useful for the treatment of endothelial damages after exposure to a high dose of radiation. Full article

Background/Objectives: Rho-associated coiled-coil-containing protein kinase inhibitors (ROCKis) have now become known as modulators of corneal endothelial wound repair and cell survival. However, evidence remains fragmented across laboratory and clinical reports. We performed a systematic review to synthesize preclinical and clinical data on ROCKis in corneal disease, assess their efficacy and safety, and identify research gaps. Methods: We searched PubMed, Web of Science, Scopus, and Google Scholar (until May 2025) for English-language original studies evaluating ROCKis in corneal models or patients. Inclusion criteria encompassed in vitro, ex vivo, in vivo, and clinical trials reporting functional outcomes (endothelial cell density, wound closure, visual acuity). Results: Thirty-one studies met criteria: 14 preclinical studies and 17 clinical studies. Preclinical models (rabbit, porcine, human explants) uniformly showed ROCKis (Y-27632, Ripasudil, Netarsudil, H-1152) accelerate corneal endothelial cell proliferation, migration, and restoration of a hexagonal monolayer with improved barrier and pump function over days to weeks. In 17 clinical investigations, topical Ripasudil or Netarsudil and cultured cell injections achieved significant corneal thinning, endothelial cell density and central corneal thickness changes, and visual acuity improvements (≥2 lines) with minimal adverse events. Overall bias was moderate in non-randomized studies and low in the RCTs. Conclusions: ROCKis demonstrate consistent pro-regenerative effects on corneal endothelium in multiple models and show promising clinical efficacy in Fuchs endothelial dystrophy and pseudophakic endothelial failure. Future work should explore novel delivery systems and larger controlled trials to optimize dosing, safety, and long-term outcomes. Full article

Compelling clinical evidence strongly indicates that anti-angiogenesis therapeutics including Bevacizumab, a humanised anti-VEGF mAb, can alleviate the resistance to immunotherapy. We explored the direct modulation of Bevacizumab on endothelial cell (EC) immune response including surface expression of adhesion and MHC molecules and EC-elicited proliferation of immune cells under inflammatory conditions. Flow cytometry showed that addition of VEGF inhibited TNF-α stimulation of expression of ICAM-1 and VCAM-1 on HUVECs, whereas Bevacizumab enhanced this TNF-α-stimulated expression. The presence of MHC Class I on HUVECs was decreased by VEGF and increased by TNF-α, respectively. Bevacizumab reversed VEGF downregulation and promoted TNF-α upregulation of MHC class I expression, suggesting that anti-VEGF treatment can boost the endothelial immunological reaction, a prerequisite for immune cell trafficking. Functionally, real-time monitoring of the proliferation of human PBMCs co-cultured on HUVEC monolayers over 3 days showed opposing effects on the proliferation of PBMCs between VEGF and TNF-α. Consistently, Bevacizumab antagonised VEGF suppression and sensitized TNF-α activation of PBMC growth over the time course. In line with these findings, Bevacizumab increased the surface expression of CD69 on VEGF-treated T cells collected from PBMCs after 3-day co-cultures with HUVECs. Furthermore, the proliferation of CD3+, CD8+ and CD4+ T cells was promoted via Bevacizumab. Collectively, this study demonstrates that targeting VEGF can enhance the immune response of ECs required for T cell recruitment. Our findings provide insights to a deeper understanding of increased vascular inflammatory response conferred by anti-VEGF treatment in addition to inhibiting angiogenesis, which supports its favourable dual role in the positive immunological synergism with immunotherapy. Full article

Ruxolitinib, a clinically approved JAK1/2 inhibitor used in the treatment of hematologic malignancies and inflammatory conditions, has been shown to interfere with the function of cytotoxic T lymphocytes (CTLs). Previous studies supported the involvement of the multidrug resistance transporter P-glycoprotein (Pgp/ABCB1) in CTL biology; however, the nature of its regulation remains unclear. To address this, we investigated the impact of ruxolitinib on Pgp expression and function in human CD8⁺ T cells. We demonstrate that CD8⁺ T lymphocytes express Pgp dynamically at both the mRNA and protein levels across naïve, short-term, and long-term activation states. Ruxolitinib increased the calcein accumulation in human Pgp-overexpressing NIH-3T3 cells and in CTLs and directly modulated Pgp function by increasing its basal ATPase activity in a concentration-dependent manner (10–100 μM), similar to the effect of the known Pgp substrate/modulator verapamil. Although measurable ATPase stimulation and transport inhibition were observed at supratherapeutic concentrations of ruxolitinib, its Pgp-mediated efflux may also occur at therapeutically relevant concentrations. In contrast, at therapeutically relevant plasma concentrations (1–3 μM), ruxolitinib significantly stabilized the mRNA expression of Pgp during early T-cell receptor (TCR) activation and inhibited the TCR-induced upregulation of Pgp, CD8, and PD-1 surface markers, suggesting its interference with activation-associated differentiation. At these same concentrations, ruxolitinib also impaired CCL19-directed transmigration of CTLs across human umbilical vein endothelial cell (HUVEC) monolayers, indicating disruption of lymphoid homing cues. Collectively, these findings demonstrate that ruxolitinib modulates Pgp at both the transcriptional and functional levels, with distinct concentration dependence. The ability of ruxolitinib to alter CTL activation and migration at clinically relevant plasma concentrations highlights the need for careful evaluation of JAK inhibitor–mediated immunomodulation and its implications for vaccination, transplantation, and T cell-based immunotherapies. Full article