Drug-Induced Mitochondrial Disruption in the Blood-Brain Barrier Cells: Overlooked Player in Drug Safety Evaluation.

The blood-brain barrier (BBB) is based on the unique pattern of the microvasculature of the central nervous system (CNS), which controls the transport of molecules between the CNS and the blood. The blood-brain barrier is mainly composed of endothelial cells, pericytes, and basement membrane, as well as the astrocytes and immune cells as perivascular macrophages and microglial cells. The dysfunction of this barrier can cause serious neuronal disorders due to the transport of hazardous molecules and immune cells to the CNS. Mitochondria plays a major role in cellular homeostasis in terms of health and disease. This review evaluated the published data about the effect of the drugs on the cells of BBB. Only seven articles were found that considered the effect of drugs on the barrier endothelial cells and mitochondria via different assays. Further studies are recommended to evaluate the impact of used medications on BBB cell bioenergetics. Also, the effect of the newly studied pharmaceutical agents on the BBB bioenergetics should be included within their safety profile studies.

Matrix Stiffness of GelMA Hydrogels Regulates Lymphatic Endothelial Cells toward Enhanced Lymphangiogenesis.

Lymphatic vessel regeneration is crucial for various tissue engineering strategies, particularly in resolving inflammation and restoring tissue homeostasis. In our study, we focused on investigating how hydrogel matrix stiffness influences lymphatic endothelial cells (LECs) in promoting lymphatic vessel regeneration. Gelatin methacrylate (GelMA) was chosen as our biomaterial due to its versatility in tissue engineering and biofabrication. We fabricated GelMA hydrogels at concentrations of 5, 7.5, and 15% (w/v) with corresponding Young's modulus values of 1.55 kPa (soft matrix), 12.02 kPa (medium matrix), and 48.50 kPa (stiff matrix). Among these, the 7.5% GelMA hydrogel exhibited optimal stiffness for promoting lymphangiogenesis. LECs seeded either on the hydrogel surface or within spontaneously formed a more stable lymphatic capillary network compared with other GelMA formulations. Furthermore, we investigated the enhancement of lymphangiogenesis by incorporating VEGF-C into the GelMA hydrogel, leveraging the synergistic effects of mechanical and chemical cues. Our results underscored the critical role of FAK-phosphorylation in this process; treatment with an FAK-specific inhibitor prevented the formation of tube-like structures by LECs and attenuated the expression of lymphatic markers. Overall, our findings highlight how the mechanical and chemical cues provided by GelMA hydrogels can effectively regulate LEC behavior toward enhanced lymphangiogenesis via the integrin/FAK mechanotransduction pathway. This study proposes a promising strategy for developing hydrogel-based scaffolds or bioinks tailored to promote lymphatic vessel regeneration in therapeutic applications.

Endothelial YAP/TAZ activation promotes atherosclerosis in a mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare disease caused by the expression of progerin, an aberrant protein produced by a point mutation in the LMNA gene. HGPS patients show accelerated aging and die prematurely mainly from complications of atherosclerosis such as myocardial infarction, heart failure, or stroke. However, the mechanisms underlying HGPS vascular pathology remain ill defined. We used single-cell RNA sequencing to characterize the aorta in progerin-expressing LmnaG609G/G609G mice and wild-type controls, with a special focus on endothelial cells (ECs). HGPS ECs showed gene expression changes associated with extracellular matrix alterations, increased leukocyte extravasation, and activation of the yes-associated protein 1/transcriptional activator with PDZ-binding domain (YAP/TAZ) mechanosensing pathway, all validated by different techniques. Atomic force microscopy experiments demonstrated stiffer subendothelial extracellular matrix in progeroid aortas, and ultrasound assessment of live HGPS mice revealed disturbed aortic blood flow, both key inducers of the YAP/TAZ pathway in ECs. YAP/TAZ inhibition with verteporfin reduced leukocyte accumulation in the aortic intimal layer and decreased atherosclerosis burden in progeroid mice. Our findings identify endothelial YAP/TAZ signaling as a key mechanism of HGPS vascular disease and open a new avenue for the development of YAP/TAZ targeting drugs to ameliorate progerin-induced atherosclerosis.

Angiogenic properties and intercellular communication of differentiated porcine endothelial cells in vascular therapy.

Endothelial cell dysfunction can lead to various vascular diseases. Blood flow disorder is a common symptom of vascular diseases. Regenerative angiogenesis, which involves transplanting vascular cells or stem cells into the body to shape new vasculature, can be a good therapeutic strategy. However, there are several limitations to using autologous cells from the patients themselves. We sought to investigate the new vascular cells that can play a role in the formation of angiogenesis in vivo using stem cells from alternative animals suitable for cellular therapy. Porcine is an optimal animal model for xenotransplantation owing to its physiological similarity to humans. We used differentiated porcine endothelial cells (pECs) as a therapeutic strategy to restore vessel function. Differentiated pECs formed vessel-like structures in mice, distinguishing them from stem cells. MMPs activity and migration assays indicated that differentiated pECs possessed angiogenic potential. Tube formation and 3D spheroid sprouting assays further confirmed the angiogenic phenotype of the differentiated pECs. Immunofluorescence and immunoprecipitation analyses revealed claudin-mediated tight junctions and connexin 43-mediated gap junctions between human ECs and differentiated pECs. Additionally, the movement of small RNA from human ECs to differentiated pECs was observed under co-culture conditions. Our findings demonstrated the in vivo viability and angiogenetic potential of differentiated pECs and highlighted the potential for intercellular communication between human and porcine ECs. These results suggest that transplanted cells in vascular regeneration completed after cell therapy have the potential to achieve intercellular communication within the body.

VEGF-dependent testicular vascularisation involves MEK1/2 signalling and the essential angiogenesis factors, SOX7 and SOX17.

BACKGROUND: Abnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4-eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor. RESULTS: RNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes, the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5. In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane. CONCLUSIONS: Together, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis.

Cerebral small vessel injury in mice with damage to ACE2-expressing cerebral vascular endothelial cells and post COVID-19 patients.

INTRODUCTION: The angiotensin-converting enzyme 2 (ACE2), which is expressed in cerebral vascular endothelial cells (CVECs), has been currently identified as a functional receptor for SARS-CoV-2. METHODS: We specifically induced injury to ACE2-expressing CVECs in mice and evaluated the effects of such targeted damage through magnetic resonance imaging (MRI) and cognitive behavioral tests. In parallel, we recruited a single-center cohort of COVID-19 survivors and further assessed their brain microvascular injury based on cognition and emotional scales, cranial MRI scans, and blood proteomic measurements. RESULTS: Here, we show an array of pathological and behavioral alterations characteristic of cerebral small vessel disease (CSVD) in mice that targeted damage to ACE2-expressing CVECs, and COVID-19 survivors. These CSVD-like manifestations persist for at least 7 months post-recovery from COVID-19. DISCUSSION: Our findings suggest that SARS-CoV-2 may induce cerebral small vessel damage with persistent sequelae, underscoring the imperative for heightened clinical vigilance in mitigating or treating SARS-CoV-2-mediated cerebral endothelial injury throughout infection and convalescence. HIGHLIGHTS: Cerebral small vessel disease-associated changes were observed after targeted damage to angiotensin-converting enzyme 2-expressing cerebral vascular endothelial cells. SARS-CoV-2 may induce cerebral small vessel damage with persistent sequelae. Clinical vigilance is needed in preventing SARS-CoV-2-induced cerebral endothelial damage during infection and recovery.

Sinusoidal communication in chronic liver disease.

The liver sinusoid, mainly composed of liver sinusoidal endothelial cells, hepatic macrophages and hepatic stellate cells, shapes the hepatic vasculature and is key maintaining liver homeostasis and function. During chronic liver disease (CLD), the function of sinusoidal cells is impaired, being directly involved in the progression of liver fibrosis, cirrhosis, and main clinical complications including portal hypertension and hepatocellular carcinoma. In addition to their roles in liver diseases pathobiology, sinusoidal cells' paracrine communication or cross-talk is being studied as a mechanism of disease but also as a remarkable target for treatment. The aim of this review is to gather current knowledge of intercellular signalling in the hepatic sinusoid during the progression of liver disease. We summarise studies developed in pre-clinical models of CLD, specially emphasizing those pathways characterized in human-based clinically relevant models. Finally, we describe pharmacological treatments targeting sinusoidal communication as promising options to treat CLD and its clinical complications.

Comprehensive combined analysis of physician-initiated phase II and III clinical trials on a cultured human corneal endothelial cell product for treating bullous keratopathy.

To evaluate the efficacy and safety of a cultured human corneal endothelial cell (cHCEC) product in eyes with bullous keratopathy (BK). Combined analysis of multicenter phase II and III clinical trials. This analysis involved 15 BK eyes in the phase II trial and 12 BK eyes in the phase III trial that underwent cHCEC transplant therapy. Safety was assessed in all the cases. Efficacy was assessed in 17 cases with exclusion of the low- and medium-dose groups in the phase II trial. The primary endpoint was a corneal endothelial cell density of 1000 cells/mm2 or more at 24 weeks post-transplant, which was attained in 94.1% of the eyes (16 of 17), with a 95% CI of 71.3-99.9%. Additionally, 82.4% of the eyes (14 of 17) met the secondary endpoint of reduction in corneal thickness to less than 630 µm without corneal epithelial edema within the same time frame, with a 95% CI of 56.6-96.2%. The mean decrease in corneal thickness from baseline to 24 weeks post-transplant was -187.4 µm (95% CI, -240.2 µm to -134.5 µm). Furthermore, all the eyes exhibited improvement in best-corrected visual acuity from baseline to 24 weeks post-transplant (95% CI, 80.5-100.0%). By 24 weeks post-transplant, 88.9% of the patients (24 of 27) had experienced adverse events, which were mostly local, mild, and transient. The cHCEC product of this study reconstitutes the corneal endothelial layer with high cellular density and restores corneal thickness and improves visual acuity.

In vitro vascularization of 3D cell aggregates in microwells with integrated vascular beds.

Most human tissues possess vascular networks supplying oxygen and nutrients. Engineering of functional tissue and organ models or equivalents often require the integration of artificial vascular networks. Several approaches, such as organs on chips and three-dimensional (3D) bioprinting, have been pursued to obtain vasculature and vascularized tissues in vitro. This technical feasibility study proposes a new approach for the in vitro vascularization of 3D microtissues. For this, we thermoform arrays of round-bottom microwells into thin non-porous and porous polymer films/membranes and culture vascular beds on them from which endothelial sprouting occurs in a Matrigel-based 3D extra cellular matrix. We present two possible culture configurations for the microwell-integrated vascular beds. In the first configuration, human umbilical vein endothelial cells (HUVECs) grow on and sprout from the inner wall of the non-porous microwells. In the second one, HUVECs grow on the outer surface of the porous microwells and sprout through the pores toward the inside. These approaches are extended to lymphatic endothelial cells. As a proof of concept, we demonstrate the in vitro vascularization of spheroids from human mesenchymal stem cells and MG-63 human osteosarcoma cells. Our results show the potential of this approach to provide the spheroids with an abundant outer vascular network and the indication of an inner vasculature.

TLR4/TRIF/Caspase-8/Caspase-1 Pathway in Choroidal Endothelial Cells Promotes Choroidal Neovascularization.

PURPOSE: The purpose of this study was to investigate the role and mechanism of caspase-8 in the development of choroidal neovascularization induced by age-related macular degeneration, with the aim of identifying a potential therapeutic target for neovascular age-related macular degeneration. METHODS: Mouse models of laser photocoagulation-induced choroidal neovascularization and hypoxic human choroidal endothelial cells were utilized to examine the involvement of caspase-8 in choroidal neovascularization development. The toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8 pathway was explored in hypoxic human choroidal endothelial cells to elucidate its contribution to pathological angiogenesis. Various experimental techniques, including inhibition assays and immunoblotting analysis, were employed to assess the effects and mechanisms of caspase-8 activation. RESULTS: Inhibition of caspase-8 demonstrated attenuated choroidal neovascularization development in mice subjected to laser photocoagulation. Activation of the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8 pathway was observed in hypoxic human choroidal endothelial cells. Upon activation by the toll-like receptor 4/TIR domain-containing adaptor molecule 1 axis, caspase-8 directly cleaved caspase-1, leading to the cleavage of interleukin-1ß and interleukin-18 by caspase-1. Consequently, activation of interleukin-1ß and interleukin-18 through the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8/caspase-1 pathway promoted the proliferative, migratory, and tube-forming abilities of hypoxic human choroidal endothelial cells. CONCLUSION: The findings of this study indicate that caspase-8 plays a crucial role in promoting choroidal neovascularization by activating interleukin-1ß and interleukin-18 through the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8/caspase-1 pathway in choroidal endothelial cells. Therefore, targeting caspase-8 may hold promise as a therapeutic approach for neovascular age-related macular degeneration.

An overview of the role of chemokine CX3CL1 (Fractalkine) and CX3C chemokine receptor 1 in systemic sclerosis.

INTRODUCTION: Systemic sclerosis (SSc) is a complex autoimmune disease characterized by fibrosis, vascular damage, and immune dysregulation. Fractalkine or chemokine (C-X3-C motif) ligand 1 (CX3CL1), a chemokine and adhesion molecule, along with its receptor CX3CR1, have been implicated in the inflammatory processes of SSc. CX3CL1 functions as both a chemoattractant and an adhesion molecule, guiding immune cell trafficking. This systematic review examines the role of CX3CL1 and its receptor CX3CR1 in the pathogenesis of SSc, with a focus on pulmonary and vascular complications. METHODS: A systematic literature search was conducted across databases including PubMed, Scopus, and Web of Science from inception to November 2020. The search focused on studies investigating the CX3CL1/CX3CR1 axis in the context of SSc. RESULTS: The review identified elevated CX3CL1 expression in SSc patients, particularly in the skin and lungs, where CX3CR1 is expressed on infiltrating immune cells. Higher levels of CX3CL1 were correlated with the severity of interstitial lung disease in SSc patients, indicating a potential predictive marker for disease progression. CX3CR1-positive monocytes and NK cells were recruited to inflamed tissues, contributing to fibrosis and tissue damage. Animal studies showed that inhibition of the CX3CL1/CX3CR1 axis reduced fibrosis and improved vascular function. CONCLUSION: The CX3CL1/CX3CR1 axis plays a key role in immune cell recruitment and fibrosis in SSc. Elevated CX3CL1 levels are associated with lung and vascular complications, making it a potential biomarker for disease progression and a promising therapeutic target.

SOX17 expression in tumor-penetrating vessels in relation to CD8+ T-cell infiltration in cancer stroma niches.

INTRODUCTION: Sex-determining region Y-related high-mobility group box 17 protein (SOX17), a proangiogenic transcription factor, is specifically expressed in tumor endothelial cells (TECs) of implanted Lewis lung carcinoma. However, the expression profile of SOX17 is largely unknown in human lung cancer. We aimed to elucidate SOX17 expression in cancer cells and the tumor microenvironment of lung adenocarcinoma. METHODS: In the present study, we examined SOX17 expression in whole-tissue specimens of 83 lung adenocarcinomas by immunohistochemistry. RESULTS: SOX17 immunoreactivity was minimal in lung adenocarcinoma cells, except in five non-mucinous adenocarcinomas in situ. SOX17 was also expressed in cultured A549 lung adenocarcinoma cells, which is widely used as a model of malignant alveolar type II epithelial cells. Notably, SOX17 immunoreactivity was found in endothelial cells of tumor-penetrating vessels in 19 of 83 lung adenocarcinoma tissue specimens, with statistical significance to stromal infiltration of CD8+ T cells (p < 0.01) but was not associated with the number of tertiary lymph nodes. Although not statistically significant, SOX17 immunoreactivity was related to favorable patient outcomes. CONCLUSION: Our findings indicate that SOX17 might play a pleiotropic role in lung adenocarcinoma in cancer cells and stromal niches. SOX17-mediated CD8+ T-cell-rich tumor microenvironment might attract interest in improving the effect of cancer immunotherapy.

VCAM-1 targeted nanocarriers of shRNA-Smad3 mitigate endothelial-to-mesenchymal transition triggered by high glucose concentrations and osteogenic factors in valvular endothelial cells.

Endothelial to mesenchymal transition (EndMT) of valvular endothelial cells (VEC) is a key process in the development and progression of calcific aortic valve disease (CAVD). High expression of the Smad3 transcription factor is crucial in the transition process. We hypothesize that silencing Smad3 could hinder EndMT and provide a novel treatment for CAVD. We aimed at developing nanoparticles encapsulating short-hairpin (sh)RNA sequences specific for Smad3 targeted to the aortic valve. We synthesized VCAM-1-targeted lipopolyplexes encapsulating shRNA-Smad3 plasmid (V-LPP/shSmad3) and investigated their potential to reduce the EndMT of human VEC. VEC incubation in a medium containing high glucose concentrations and osteogenic factors (HGOM) triggers EndMT and increased expression of Smad3. Exposed to lipopolyplexes, VEC took up efficiently the V-LPP/shSmad3. The latter reduced the EndMT process in VEC exposed to HGOM by downregulating the expression of αSMA and S100A4 mesenchymal markers and increasing the expression of the CD31 endothelial marker. In vivo, V-LPP/shSmad3 accumulated in the aortic root and aorta of a murine model of atherosclerosis complicated with diabetes, without affecting the liver and kidney function. The results suggest that targeting activated VEC with lipopolyplexes to silence Smad3 could be an effective, novel treatment for CAVD mediated by the EndMT process.

Acetaminophen Overdose Reveals PAR4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium in Mice.

BACKGROUND: The protease thrombin, which elicits multiple physiological and pathological effects on vascular endothelial cells (ECs), can signal through PARs (protease-activated receptors) 1 and 4. PAR1 is a high-affinity thrombin receptor known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generation and hepatic vascular dysfunction that occur during acetaminophen (APAP) overdose to determine (1) whether hepatic endothelial PAR4 is a functional receptor, and (2) the endothelial-specific functions for PAR1 and PAR4 in a high thrombin and pathological setting. METHODS: We generated mice with conditional deletion of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte accumulation in the liver, thrombin generation, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial translating ribosome affinity purification followed by next-generation sequencing. RESULTS: We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability, although mice deficient for both receptors had lower vascular permeability at an earlier timepoint after APAP overdose than either of the single mutants. Additionally, mice with loss of both endothelial Par1 and Par4 had reduced thrombin generation after APAP overdose, suggesting decreased hypercoagulability. Last, we found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. CONCLUSIONS: Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

The role of miR-134-5p in 7-ketocholesterol-induced human aortic endothelial dysfunction.

Atherosclerotic cardiovascular diseases are the leading causes of morbidity and mortality worldwide. In our previous study, a panel of miRNA including miR-134-5p was deregulated in young acute coronary syndrome (ACS) patients. However, the roles of these ACS-associated miRNAs in endothelial dysfunction, an early event preceding atherosclerosis, remain to be investigated. In the present study, human aortic endothelial cells (HAECs) were treated with 7-ketocholesterol (7-KC) to induce endothelial dysfunction. Following treatment with 20 µg/ml 7-KC, miR-134-5p was significantly up-regulated and endothelial nitric oxide synthase (eNOS) expression was suppressed. Endothelial barrier disruption was evidenced by the deregulation of adhesion molecules including the activation of focal adhesion kinase (FAK), down-regulation of VE-cadherin, up-regulation of adhesion molecules (E-selectin and ICAM-1), increased expression of inflammatory genes (IL1B, IL6 and COX2) and AKT activation. Knockdown of miR-134-5p in 7-KC-treated HAECs attenuated the suppression of eNOS, the activation of AKT, the down-regulation of VE-cadherin and the up-regulation of E-selectin. In addition, the interaction between miR-134-5p and FOXM1 mRNA was confirmed by the enrichment of FOXM1 transcripts in the pull-down miRNA-mRNA complex. Knockdown of miR-134-5p increased FOXM1 expression whereas transfection with mimic miR-134-5p decreased FOXM1 protein expression. In summary, the involvement of an ACS-associated miRNA, miR-134-5p in endothelial dysfunction was demonstrated. Findings from this study could pave future investigations into utilizing miRNAs as a supplementary tool in ACS diagnosis or as targets for the development of therapeutics.

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48 h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6 h, 24 h, and 30 h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

Tumour cell-released autophagosomes promote lung metastasis by upregulating PD-L1 expression in pulmonary vascular endothelial cells in breast cancer.

PURPOSE: Establishing an immunosuppressive premetastatic niche (PMN) in distant organs is crucial for breast cancer metastasis. Vascular endothelial cells (VECs) act as barriers to transendothelial cell migration. However, the immune functions of PMNs remain unclear. Tumour cell-released autophagosomes (TRAPs) are critical modulators of antitumour immune responses. Herein, we investigated the mechanism through which TRAPs modulate the immune function of pulmonary VECs in lung PMN in breast cancer. METHODS: Immortalised mouse pulmonary microvascular endothelial cells were incubated with TRAPs in vitro. RNA sequencing, flow cytometry, and western blotting were employed to assess immunosuppressive function and mechanism. In vivo, TRAP-trained and autophagy-deficient tumour mice were used to detect immunosuppression, and high-mobility group box 1 (HMGB1)-deficient TRAP-trained and TLR4 knockout mice were utilised to investigate the underlying mechanisms of pulmonary VECs. Additionally, the efficacy of anti-programmed cell death ligand-1 (PD-L1) immunotherapy was evaluated in early tumour-bearing mice. RESULTS: HMGB1 on TRAPs surfaces stimulated VECs to upregulate PD-L1 via a TLR4-MyD88-p38/STAT3 signalling cascade that depended on the cytoskeletal movement of VECs. Importantly, PD-L1 on TRAP-induced VECs can inhibit T cell function, promote lung PMN immunosuppression, and result in more pronounced lung metastasis. Treatment with anti-PD-L1 reduces lung metastasis in early stage tumour-bearing mice. CONCLUSIONS: These findings revealed a novel role and mechanism of TRAP-induced immunosuppression of pulmonary VECs in lung PMN. TRAPs and their surface HMGB1 are important therapeutic targets for reversing immunosuppression, providing a new theoretical basis for the treatment of early stage breast cancer using an anti-PD-L1 antibody.