Human Microvascular Endothelial Cells Research Articles

Ghrelin inhibits autophagy mediated by AKT/mTOR pathway to ameliorate retinal angiogenesis induced by high glucose stress

AIM: To observe the effect of ghrelin, a growth hormone-releasing peptide, on retinal angiogenesis in vitro under high glucose (HG) stress and to explore the possible mechanism of autophagy. METHODS: Human retinal microvascular endothelial cells (HRMECs) were treated with high concentration of glucose alone or in combination with ghrelin. The cell migration, tube formation and the expression of the autophagy-related proteins LC3-II/I, Beclin-1, p62, phosphorylated AKT (p-AKT)/AKT and phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR were detected. Then, to clarify the correlation between ghrelin effect and autophagy, AKT inhibitor VIII was adopted to treat HRMECs, and cell migration, tube formation as well as the protein expressions of LC3-II/I, Beclin-1 and p62 were observed. RESULTS: Under HG stress, ghrelin inhibited migration and tube formation of HRMECs. Ghrelin inhibited the increases in the protein levels of LC3-II/I, Beclin-1 and the decreases in the protein levels of p62, p-AKT/AKT and p-mTOR/mTOR induced by HG stress. Moreover, under the action of AKT/mTOR pathway inhibitors, the effects of ghrelin on migration and tube formation were both reduced. In addition, the expression of LC3-II/I and Beclin-1 were significantly up-regulated and the expression of p62 was down-regulated. CONCLUSION: Retinal angiogenesis under in vitro HG stress can be inhibited by ghrelin through activating AKT/mTOR pathway to inhibit autophagy.

MicroRNA-451 Regulates Angiogenesis in Intracerebral Hemorrhage by Targeting Macrophage Migration Inhibitory Factor.

Intracerebral hemorrhage (ICH) is a subtype of stroke with the highest fatality and disability rate. Up to now, commonly used first-line therapies have limited value in improving prognosis. Angiogenesis is essential to neurological recovery after ICH. Recent studies have shown that microRNA-451(miR-451) plays an important role in angiogenesis by regulating the function of vascular endothelial cells. We found miR-451 was significantly decreased in the peripheral blood of ICH patients in the acute stage. Based on the clinical findings, we conducted this study to investigate the potential regulatory effect of miR-451 on angiogenesis after ICH. The expression of miR-451 in ICH mouse model and in a hemin toxicity model of human brain microvascular endothelial cells (hBMECs) was decreased the same as in ICH patients. MiR-451 negatively regulated the proliferation, migration, and tube formation of hBMECs in vitro. MiR-451 negatively regulated the microvessel density in the perihematoma tissue and affected neural functional recovery of ICH mouse model. Knockdown of miR-451 could recovered tight junction and protect the integrity of blood-brain barrier after ICH. Based on bioinformatic programs, macrophage migration inhibitory factor (MIF) was predicted to be the target gene and identified to be regulated by miR-451 inhibiting the protein translation. And p-AKT and p-ERK were verified to be downstream of MIF in angiogenesis. These results all suggest that miR-451 will be a potential target for regulating angiogenesis in ICH.

Neovascularization by DPSC-ECs in a Tube Model for Pulp Regeneration Study.

The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies.

Inflammation mediated angiogenesis in chronic lymphocytic leukemia.

Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19+ cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19+ cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

BackgroundTriptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood–brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp).MethodsWe investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice.ResultsWe demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min−1·mg protein−1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice.ConclusionsWe have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.Graphical

Primary Human Breast Cancer-Associated Endothelial Cells Favor Interactions with Nanomedicines.

Cancer nanomedicines predominately rely on transport processes controlled by tumor-associated endothelial cells to deliver therapeutic and diagnostic payloads into solid tumors. While the dominant role of this class of endothelial cells for nanoparticle transport and tumor delivery is established in animal models, the translational potential in human cells needs exploration. Using primary human breast cancer as a model, the differential interactions of normal and tumor-associated endothelial cells with clinically relevant nanomedicine formulations are explored and quantified. Primary human breast cancer-associated endothelial cells exhibit up to ≈2times higher nanoparticle uptake than normal human mammary microvascular endothelial cells. Super-resolution imaging studies reveal a significantly higher intracellular vesicle number for tumor-associated endothelial cells, indicating a substantial increase in cellular transport activities. RNA sequencing and gene expression analysis indicate the upregulation of transport-related genes, especially motor protein genes, in tumor-associated endothelial cells. Collectively, the results demonstrate that primary human breast cancer-associated endothelial cells exhibit enhanced interactions with nanomedicines, suggesting a potentially significant role for these cellsin nanoparticle tumor delivery in human patients. Engineering nanoparticles that leverage the translational potential of tumor-associated endothelial cell-mediated transport into human solid tumors may lead to the development of safer and more effective clinical cancer nanomedicines.

Poricoic acid A promotes angiogenesis and myocardial regeneration by inducing autophagy in myocardial infarction

Myocardial infarction (MI) is a kind of cardiovascular diseases with high morbidity and mortality. Poricoic acid A (PAA) is the main active substance in Poria cocos, which has been discovered to exhibit an ameliorative role in the progression of many diseases. However, no report has been focused on the regulatory effects of PAA on MI progression. In this study, at first, oxygen glucose deprivation (OGD) treatment was performed in human cardiac microvascular endothelial cells (HCMECs) to mimic MI cell model. Our findings demonstrated that cell proliferation was reduced post OGD treatment, but which was reversed by PAA treatment. Moreover, PAA suppressed cell apoptosis in OGD-triggered HCMEC cells. Next, it revealed that PAA induced autophagy in OGD-treated HCMEC cells through enhancing LC3-II/LC3-I level and reducing P62 level. In addition, PAA strengthened the angiogenesis ability and migration ability in OGD-induced HCMEC cells. Lastly, it was uncovered that PAA modulated the AMPK/mTOR signaling pathway through affecting the p-mTOR/mTOR and p-AMPK/AMPK levels. In conclusion, PAA can promote angiogenesis and myocardial regeneration after MI by inducing autophagy through modulating the AMPK/mTOR pathway. This work suggested that PAA may be a potential and useful drug for MI treatment.

Ultrasmall copper nanodots eye drops for microenvironment regulation of diabetes retinopathy by relieving hypoxia and inhibiting HIF-1α/VEGF signaling pathway

Diabetic retinopathy (DR) is the main cause of visual loss among blinding diseases. The pathological microenvironment, characterized by hypoxia, reactive oxygen species (ROS) accumulation and oxidative stress, is critical to the onset and progression of DR. In this study, an ultrasmall (4–6 nm) copper nanodots was developed as nanozyme (Cu NZs) and dispersed in 2 % Methocel to formulate Cu NZs eye drops to relieve hypoxia and scavenge excrescent ROS for the effective treatment of DR. Due to its smaller size and higher specific surface area, Cu NZs exposes more active sites and can guarantee excellent antioxidant activity. The results of in vitro studies indicated that Cu NZs had biocompatible and protective effects on human retinal microvascular endothelial cells (HRMECs), alleviating hypoxia in HRMECs, reducing oxidative stress, and inhibiting hypoxia-inducing factor-1α (HIF-1α)/ vascular endotheliogenesis factor (VEGF) signaling pathway. In an animal model of streptozotocin (STZ)-induced DR, Cu NZs eye drops have been shown to greatly relieve the hypoxic microenvironment of DR and inhibit the expression of HIF-1α and VEGF. CuNZs eye drops reduced inflammatory factors in the DR microenvironment, significantly reducing the progression of neovascularization and vascular leakage. And the nano-enzyme eye drops have no toxic side effects on the eyes and important organs of the whole body. This treatment mode based on the nano enzyme cascade reaction from comprehensive microenvironment regulation (hypoxia relief, free radical scavenging, anti-inflammatory), has excellent ability to treat DR. Although the nanoparticle is effective against DR in the short term at the cellular and animal levels, the long-term efficacy and safety remains to be evaluated.

Erianin alleviates LPS-induced acute lung injury via antagonizing P-selectin-mediated neutrophil adhesion function

Ethnopharmacological relevanceDendrobium officinale Kimura et Migo, known as "Tiepi Shihu" in traditional Chinese medicine, boasts an extensive history of medicinal use documented in the Chinese Pharmacopoeia. "Shen Nong Ben Cao Jing" records D. officinale as a superior herbal medicine for fortifying "Yin" and invigorating the five viscera. Erianin, a benzidine compound, emerges as a prominent active constituent derived from D. officinale, with the pharmacological efficacy of D. officinale closely linked to the anti-inflammatory properties of erianin. Aim of the studyAcute lung injury (ALI) is a substantial threat to global public health, while P-selectin stands out as a promising novel target for treating acute inflammatory conditions. This investigation aims to explore the therapeutic potential of erianin in ALI treatment and elucidate the underlying mechanisms. Experimental designThe effectiveness of erianin in conferring protection against ALI was investigated through comprehensive histopathological and biochemical analyses of lung tissues and bronchoalveolar lavage fluid (BALF) in an in vivo model of LPS-induced ALI in mice. The impact of erianin on fMLP-induced neutrophil chemotaxis was quantitatively assessed using the Transwell and Zigmond chamber, respectively. To determine the therapeutic target of erianin and elucidate their binding capability, a series of sophisticated assays were employed, including drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and molecular docking analyses. ResultsErianin demonstrated a significant alleviation of LPS-induced acute lung injury, characterized by reduced total cell and neutrophil counts and diminished total protein contents in BALF. Moreover, erianin exhibited a capacity to decrease proinflammatory cytokine production in both lung tissues and BALF. Notably, erianin effectively suppressed the activation of NF-κB signaling in the lung tissues of LPS- challenged mice; however, it did not exhibit in vitro inhibitory effects on inflammation in LPS-induced human pulmonary microvascular endothelial cells (HPMECs). Additionally, erianin blocked the adhesion and rolling of neutrophils on HPMECs. While erianin did not influence endothelial P-selectin expression or cytomembrane translocation, it significantly reduced the ligand affinity between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). ConclusionsErianin inhibits P-selectin-mediated neutrophil adhesion to activated endothelium, thereby alleviating ALI. The present study highlights the potential of erianin as a promising lead for ALI treatment.

Microvesicles derived from dermal myofibroblasts modify the integrity of the blood and lymphatic barriers using distinct endocytosis pathways

AbstractMicrovesicles (MVs) are a subtype of extracellular vesicles that can transfer biological information from their producer cells to target cells. This communication can in turn affect both normal and pathological processes. Mounting evidence has revealed that dermal wound myofibroblasts (Wmyo) produce MVs, which can transfer biomolecules impacting receptor cells such as human dermal microvascular endothelial cells (HDMECs). While the effects of MVs on HDMECs are generally well described in the literature, little is known about the transport of MVs across the HDMEC barrier, and their potential effect on the barrier integrity remains unknown. Here, we investigated these roles of Wmyo‐derived MVs on two sub‐populations of HDMECs, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs). Using an in vitro model to mimic the endothelial barrier, we showed that MVs crossed the LEC barrier but not the BEC barrier. In addition, we demonstrated that MVs were able to influence the cell‐cell junctions of HDMECs. Specifically, we observed that after internalization via the predominantly caveolin‐dependent pathway, MVs induced the opening of junctions in BECs. Conversely, in LECs, MVs mainly use the macropinocytosis pathway and induce closure of these junctions. Moreover, proteins in the MV membrane were responsible for this effect, but not specifically those belonging to the VEGF family. Finally, we found that once the LEC barrier permeability was reduced by MV stimuli, MVs ceased to cross the barrier. Conversely, when the BEC barrier was rendered permeable following stimulation with MVs, they were subsequently able to cross the barrier via the paracellular pathway. Taken together, these results suggest that the study of Wmyo‐derived MVs offers valuable insights into their interaction with the HDMEC barrier in the context of wound healing. They highlight the potential significance of these MVs in the overall process.

Sphingosine-1-phosphate receptor 1/5 selective agonist alleviates ocular vascular pathologies

Ocular abnormal angiogenesis and edema are featured in several ocular diseases. S1P signaling via S1P1 likely is part of the negative feedback mechanism necessary to maintain vascular health. In this study, we conducted pharmacological experiments to determine whether ASP4058, a sphingosine 1-phosphate receptor 1/5 (S1P1/5) agonist, is useful in abnormal vascular pathology in the eye. First, human retinal microvascular endothelial cells (HRMECs) were examined using vascular endothelial growth factor (VEGF)-induced cell proliferation and hyperpermeability. ASP4058 showed high affinity and inhibited VEGF-induced proliferation and hyperpermeability of HRMECs. Furthermore, S1P1 expression and localization changes were examined in the murine laser-induced choroidal neovascularization (CNV) model, a mouse model of exudative age-related macular degeneration, and the efficacy of ASP4058 was verified. In the CNV model mice, S1P1 tended to decrease in expression immediately after laser irradiation and colocalized with endothelial cells and Müller glial cells. Oral administration of ASP4058 also suppressed vascular hyperpermeability and CNV, and the effect was comparable to that of the intravitreal administration of aflibercept, an anti-VEGF drug. Next, efficacy was also examined in a retinal vein occlusion (RVO) model in which retinal vascular permeability was increased. ASP4058 dose-dependently suppressed the intraretinal edema. In addition, it suppressed the expansion of the perfusion area observed in the RVO model. ASP4058 also suppressed the production of VEGF in the eye. Collectively, ASP4058 can be a potential therapeutic agent that normalizes abnormal vascular pathology, such as age-related macular degeneration and RVO, through its direct action on endothelial cells.

Unraveling the Impact of Extracellular Vesicle-Depleted Serum on Endothelial Cell Characteristics over Time.

Extracellular vesicles (EVs) are produced by all kinds of cells, including endothelial cells. It has been observed that EVs present in fetal bovine serum (FBS), broadly used in cell culture, can be a confounding factor and lead to misinterpretation of results. To investigate this phenomenon, human brain microvascular endothelial cells (HBMECs) were cultured for 2 or 24 h in the presence of EV-depleted FBS (EVdS). Cell death, gene and protein expression, and the presence of EVs isolated from these cells were evaluated. The uptake of EVs, intercellular adhesion molecule 1 (ICAM-1) expression, and monocyte adhesion to endothelial cells exposed to EVs were also evaluated. Our results revealed higher apoptosis rates in cells cultured with EVdS for 2 and 24 h. There was an increase in interleukin 8 (IL8) expression after 2 h and a decrease in interleukin 6 (IL6) and IL8 expression after 24 h of culture. Among the proteins identified in EVs isolated from cells cultured for 2 h (EV2h), several were related to ribosomes and carbon metabolism. EVs from cells cultured for 24 h (EV24h) presented a protein profile associated with cell adhesion and platelet activation. Additionally, HBMECs exhibited increased uptake of EV2h. Treatment of endothelial cells with EV2h resulted in greater ICAM-1 expression and greater adherence to monocytes than did treatment with EV24h. According to our data, HBMEC cultivated with EVdS produce EVs with different physical characteristics and protein levels that vary over time.

MiR-199a-5p modulates choroidal neovascularization by regulating Wnt7b/Wnt/β-catenin signaling pathway.

Choroidal neovascularization (CNV) can be seen in many fundus diseases, and lead to fundus exudation, bleeding, or vision loss. miRNAs are vital regulator in CNV. miR-199a-5p has been proved to be involved in regulating vascular formation of endothelial cells, but its role in CNV remains unclear. This study aims to study the role of miR-199a-5p in CNV. Laser irradiation was used to induce CNV model. The lesion area of CNV was calculated by high-resolution angiography with fluorescein isothiocyanate-dextran. Wnt family member 7b (Wnt7b), β-catenin, and Wnt pathway proteins was measured by western blot. Immunofluorescence was performed to test Wnt7b, β-catenin, CD31, and p-p65. miR-199a-5p and Wnt7b mRNA were tested by reverse transcription real-time polymerase chain reaction. Cell count kit-8, wound healing, Transwell, tube formation, and flow cytometry were used to detect the function of miR-199a-5p and Wnt7b on human retinal microvascular endothelial cells (HRMEC). TargetScan database and dual-luciferase reporter assay verified the interaction between miR-199a-5p and Wnt7b. The results revealed that Wnt7b increased in CNV rats. Knocking down Wnt7b repressed cell proliferation, migration, invasion, and angiogenesis, and accelerated cell apoptosis of HRMEC. Dual-luciferase reporter assay verified that miR-199a-5p targeted Wnt7b. Overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC and promoted cell apoptosis by inhibiting Wbt7b. In vivo experiment found that Wnt7b rescued the promotion of miR-199a-5p inhibition on CNV lesion of rats. In addition, Wnt7b positively regulated Wnt/β-catenin signaling pathway and promoted the angiogenesis of HRMEC. In conclusion, overexpression of miR-199a-5p inhibited the angiogenesis of HRMEC by regulating Wnt7b/Wnt/β-catenin signaling pathway, which may serve as a promising therapy target of CNV.

Endothelial lincRNA-p21 alleviates cerebral ischemia/reperfusion injury by maintaining blood-brain barrier integrity.

Blood-brain barrier (BBB) disruption is increasingly recognized as an early contributor to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury, and is also a key event in triggering secondary damage to the central nervous system. Recently, long non-coding RNA (lncRNA) have been found to be associated with ischemic stroke. However, the roles of lncRNA in BBB homeostasis remain largely unknown. Here, we report that long intergenic non-coding RNA-p21 (lincRNA-p21) was the most significantly down-regulated lncRNA in human brain microvascular endothelial cells (HBMECs) after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment among candidate lncRNA, which were both sensitive to hypoxia and involved in atherosclerosis. Exogenous brain-endothelium-specific overexpression of lincRNA-p21 could alleviate BBB disruption, diminish infarction volume and attenuate motor function deficits in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Further results showed that lincRNA-p21 was critical to maintain BBB integrity by inhibiting the degradation of junction proteins under MCAO/R and OGD/R conditions. Specifically, lincRNA-p21 could inhibit autophagy-dependent degradation of occludin by activating PI3K/AKT/mTOR signaling pathway. Besides, lincRNA-p21 could inhibit VE-cadherin degradation by binding with miR-101-3p. Together, we identify that lincRNA-p21 is critical for BBB integrity maintenance, and endothelial lincRNA-p21 overexpression could alleviate cerebral I/R injury in mice, pointing to a potential strategy to treat cerebral I/R injury.

Extracellular vesicles containing circMYBL1 induce CD44 in adenoid cystic carcinoma cells and pulmonary endothelial cells to promote lung metastasis.

Adenoid cystic carcinoma (ACC) is a rare malignant epithelial neoplasm that arises in secretory glands and commonly metastasizes to the lungs. MYBL1 is frequently overexpressed in ACC and has been suggested to be a driver of the disease. Here, we identified a circRNA derived from MYBL1 pre-mRNA that accompanied overexpression of MYBL1 in ACC. Overexpression of circMYBL1 was correlated with increased lung metastasis and poor overall survival in ACC patients. Ectopic circMYBL1 overexpression promoted malignant phenotypes and lung metastasis of ACC cells. Mechanistically, circMYBL1 formed a circRNA-protein complex with CCAAT enhancer binding protein beta (CEBPB), which inhibited ubiquitin-mediated degradation and promoted nuclear translocation of CEBPB. In the nucleus, circMYBL1 increased the binding of CEBPB to the CD44 promoter region and enhanced its transcription. In addition, circMYBL1 was enriched in small extracellular vesicles (sEVs) isolated from the plasma of ACC patients. Treatment with sEVs containing circMYBL1 in sEVs enhanced pro-metastatic phenotypes of ACC cells, elevated the expression of CD44 in human pulmonary microvascular endothelial cells (HPMECs), and enhanced the adhesion between HPMECs and ACC cells. Moreover, circMYBL1 encapsulated in sEVs increased the arrest of circulating ACC cells in the lung and enhanced the lung metastatic burden. This data suggests that circMYBL1 is a tumor-promoting circRNA that could serve as a potential biomarker and therapeutic target in ACC.

Genomic characterization of Cronobacter sakazakii strains in the neonatal meningitis clonal complex 4

AbstractCronobacter sakazakii clonal complex 4 (CC4) is strongly associated with neonatal meningitis. However, C. sakazakii CC4‐specific pathogenicity traits have not been determined. In this study, the comparative genomic analysis of 144 genomes of C. sakazakii CC4 strains and 376 non‐CC4 C. sakazakii strains was undertaken. Twenty‐four CC4 strains were previously undescribed clinical and nonclinical strains that had been collected from various regions in China (2006–2017). The remaining genomes were of multiple sequence types of C. sakazakii strains, which had been isolated worldwide (1973–2021). The pan‐genome of C. sakazakii comprised 32,332 genes, of which 2.58% (832 genes) constituted the core genome. More C. sakazakii CC4 strains have a complete cusABCFR efflux system with a significant difference compared with non‐CC4 strains, and the ibeB‐homologous cusC gene in the cusABCFR efflux system is probably associated with the invasion of human brain microvascular endothelial cells (BMEC). In addition, the isolates with K2:CA2 capsule or type IV pili‐associated genes associated with neonatal meningitis were statistically more present in CC4 strains than non‐CC4 strains (p &lt; .0001). The thorough description of VirB/VirD4 gene cluster for the type IV secretion system and the impB/impF and vasL/vasJ gene clusters for the type VI secretion system in C. sakazakii were provided. Overall, the specific and diverse virulence factors and genes may have led to C. sakazakii CC4 clone increasing its ability to invade human BMEC and leading to neonatal meningitis. Our findings should facilitate the development of novel strategies to prevent, diagnose, and treat C. sakazakii CC4 infections.

ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

The Role of NOX2-Derived Reactive Oxygen Species in the Induction of Endothelin-Converting Enzyme-1 by Angiotensin II.

Endothelin-1 is a key regulator of vascular tone and blood pressure in health and disease. We have recently found that ET-1 production in human microvascular endothelial cells (HMECs) can be promoted by angiotensin II (Ang II) through a novel mechanism involving octamer-binding transcription factor-1 (Oct-1), NADPH oxidase-2 (NOX2), and superoxide anions. As the formation of bioactive ET-1 also depends on endothelin-converting enzyme-1 (ECE-1), we investigated the transcriptional regulation of the ECE1 gene. We found that exposure of HMECs to Ang II resulted in a concentration- and time-dependent increase in ECE1 mRNA expression. Pharmacological inhibition of ECE-1 reduced Ang II-stimulated ET-1 release to baseline values. The effect of Ang II on ECE1 mRNA expression was associated with Oct-1 binding to the ECE1 promoter, resulting in its increased activity. Consequently, the Ang II-stimulated increase in ECE1 mRNA expression could be prevented by siRNA-mediated Oct-1 inhibition. It could also be abolished by silencing the NOX2 gene and neutralizing superoxide anions with superoxide dismutase. In mice fed a high-fat diet, cardiac expression of Ece1 mRNA increased in wild-type mice but not in Nox2-deficient animals. It can be concluded that Ang II engages Oct-1, NOX2, and superoxide anions to stimulate ECE1 expression in the endothelium.

Aquaporins enriched in endothelial vacuole membrane regulate the diameters of microvasculature in hyperglycemia.

In patients with diabetic microvascular complications, decreased perfusion or vascular occlusion, caused by reduced vascular diameter, is a common characteristic that will lead to insufficient blood supply. Yet, the regulatory mechanism and effective treatment approach remain elusive. Our initial findings revealed a notable decrease in the expression of human AQP1 in both diabetic human retina samples (49 healthy vs. 54 diabetic samples) and high-glucose-treated human retinal microvascular endothelial cells. Subsequently, our investigations unveiled a reduction in vascular diameter and compromised perfusion within zebrafish embryos subjected to high glucose treatment. Further analysis indicated a significant downregulation of two aquaporins, aqp1a.1 and aqp8a.1, which are highly enriched in ECs and are notably responsive to hyperglycemic conditions. Intriguingly, the loss of function of aqp1a.1 and/or aqp8a.1 resulted in a reduction of intersegmental vessel diameters, effectively mirroring the phenotype observed in the hyperglycemic zebrafish model.The overexpression of aqp1a.1/aqp8a.1 in zebrafish ECs led to notable enlargement of microvascular diameters. Moreover, the reduced vessel diameters resulting from high-glucose treatment were effectively rescued by the overexpression of these aquaporins. Additionally, both aqp1a.1 and apq8a.1 were localized in the intracellular vacuoles in cultured ECs as well as the ECs of sprouting ISVs, and the loss of Aqps caused the reduction of those vacuoles, which was required for lumenization. Notably, while the loss of AQP1 did not impact EC differentiation from human stem cells, it significantly inhibited vascular formation in differentiated ECs. EC-enriched aquaporins regulate the diameter of blood vessels through an intracellular vacuole-mediated process under hyperglycemic conditions. These findings collectively suggest that aquaporins expressed in ECs hold significant promise as potential targets for gene therapy aimed at addressing vascular perfusion defects associated with diabetes.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.