Adult Human Endothelial Cells Research Articles

PM2.5 Temporally Decreases Human Brain Endothelial Barrier Proteins and Concomitantly Increases Inflammation and Autophagy in a Dose Dependent Manner

Exposure to airborne particulate matter 2.5 (particle size <PM2.5 um) pollution is a significant co-morbidity for stroke and other cardio- and cerebrovascular diseases, causing premature mortality. However, mechanisms underlying PM2.5-mediated endothelial dysfunction and integrity loss are not known. Our goal was to elucidate how PM2.5 alters brain endothelial phenotype causing inflammation and loss of barrier integrity to provide better insight to the potential pathogenesis of PM2.5. We hypothesized that PM2.5 would alter integral endothelial barrier proteins as well as inflammatory mediators in a dose and temporal dependent manner, subsequently exacerbating the impact of acute ischemic-like injury. Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with PM2.5 (300, 75, or 15 μg/m3; 12, 24, or 36h) or vehicle, then exposed to normoxia (21% O2) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O2), for 3h. HBMEC barrier function was assessed via trans-endothelial electrical resistance (TEER). HBMEC levels or expression of barrier markers (claudin-5, occludin, ZO-1), adhesion molecules (ICAM-1 and PECAM-1), inflammatory proteins (iNOS, COX-2, LOX-1, TNF-α, IL-1β), and autophagic protein (Beclin-1) were examined using qRT-PCR, in cell western, and standard immunoblotting. Ischemia-like injury as well as PM2.5 decreased HBMEC barrier function. PM2.5 also induced a concomitant temporal and dose dependent decrease in claudin-5 and increase in iNOS as well as TNF-α levels. Additionally, PM2.5 increased protein levels of beclin-1 and LOX-1 like that of HGD alone; however, combination of PM2.5 and HGD decreased levels of beclin-1. HGD alone or in combination with PM2.5 decreased levels of PECAM-1, but PM2.5 alone did not alter PECAM-1. PM2.5 in the presence or absence of HGD induced differential alteration in claudin-5 isoform protein levels. Intriguingly, PM2.5 decreased claudin-5 mRNA levels like that observed in HBMECs exposed to HGD alone. PM2.5 plus HGD significantly increased mRNA levels of both ICAM-1 and IL-1β. In conclusion, PM2.5 mediated inflammatory and vascular pathogenesis involves a potential complex intracellular molecular framework associated with the brain endothelium which is compounded by ischemic like injury. These findings have revealed detrimental effects of PM2.5 on the brain vascular endothelium which will help to identify targets in the endothelium to restore or preserve cerebrovascular homeostasis. University of Arizona Valley Research Partnership Grant VRP37 P2 (RJG), American Heart Association 19AIREA34480018 (RJG), UA VRP Grant VRP55 P1a (TSW). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract TP307: oxLDL Preconditioning Intensifies Ischemic-Like Injury Mediated Alterations in Human Male Brain Endothelial Cell Tight Junction Protein Levels and Proinflammatory Mediators

Introduction: Acute ischemic stroke (AIS) triggers endothelial activation and induces cerebrovascular inflammation which can result in vascular function and integrity loss leading to worsened stroke outcome. A clinically correlated risk factor for stroke shown to mediate vascular dysfunction and injury is elevated levels of oxidized low density lipoprotein (oxLDL). We hypothesized that oxLDL would exacerbate acute ischemic injury mediated alteration of endothelial proinflammatory mediator and integral barrier protein levels. Methods: Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with human oxLDL (50 or 100ug/dL; 6, 12, 24, or 36h) or vehicle, using a serum dose reported in AIS patients. HBMECs were then exposed to normoxia (21% O 2 ) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O 2 ), for 3 or 6h. HBMEC levels or expression of barrier markers, adhesion molecules, and inflammatory mediators were examined using qRT-PCR, in cell western, and FIJI mediated immunocytochemical analysis. Secondary analysis was performed on RNA sequencing of adult male aortic endothelial cells (HAoECs) exposed to oxLDL (50ug/mL; 3, 6, 12, 24h) obtained from GEO dataset GSE13139. Results: HGD injury concomitantly decreased HBMEC claudin-5, occludin, and ZO-1 as well as increased IL-1b, ICAM-1, VCAM-1, iNOS, and TNF-a. OxLDL exacerbated an HGD mediated decrease in HBMEC barrier levels and increase in proinflammatory levels in a dose and time dependent manner. HAoEC barrier, adhesion, and proinflammatory levels were not altered by oxLDL. Conclusion: OxLDL may preferentially alter brain endothelial barrier and proinflammatory levels, as opposed to aortic endothelial cells in dose and time dependent manner. Mitigating the effect of oxLDL on brain endothelial permeability and inflammation, possibly via receptor inhibition may be a novel, viable therapeutic target in the treatment of AIS.

Paraquat disrupts the blood-brain barrier by increasing IL-6 expression and oxidative stress through the activation of PI3K/AKT signaling pathway.

Paraquat (PQ) is a frequently used herbicide with neurotoxic effects after acute or chronic exposure. Although in vitro evidence supports the PQ toxicity to dopamine cells, its in vivo effects (especially the chronic exposure) remain ambiguous. In this study, we investigated the effect of chronic PQ exposure on the blood-brain barrier (BBB) damage and the underlying mechanisms. Adult male Sprague Dawley rats and primary human brain microvascular endothelial (PHBME) cells were exposed to PQ as the animal and cell models. Evans Blue staining and hematoxylin & eosin staining were conducted to examine the BBB and brain tissue damages. The inflammatory cytokines were quantified via enzyme linked immunosorbent assay. The changes of PI3K/AKT signaling pathway were detected by western blot. PQ exposure can cause significant pathological lesions in the brain tissues and the BBB. IL-6 and reactive oxygen species levels were found to be significantly upregulated after PQ exposure in both the animal and cell models. PQ treatment could arrest the cell proliferation and migration in PHBME cells. PQ treatment promoted the phosphorylation of PI3K and AKT, and the application of PI3K inhibitor could attenuate PQ-induced IL-6 production, oxidative stress, BBB disruption, and brain tissue damage. Our study demonstrated that chronic PQ exposure could impair the BBB function and induce brain tissue damage. The overactivation of the PI3K/AKT pathway, consequent upregulation of IL-6 production, and increased oxidative stress appear to mediate the inflammatory damage resulting from PQ exposure.

Endothelial sensing of AHR ligands regulates intestinal homeostasis

Endothelial cells line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance and coordinate angiogenesis and lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric endothelial cells actively sense and integrate such signals is currently unknown. Here we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for endothelial cell sensing of dietary metabolites in adult mice and human primary endothelial cells. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic endothelial cells. Analyses of AHR-mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR deficiency in adult mice resulted in dysregulated inflammatory responses and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human endothelial cells, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the effect of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting endothelial cell quiescence and vascular normalcy.

Von Willebrand Factor and Angiopoietin-2 are Sensitive Biomarkers of Pulsatility in Continuous-Flow Ventricular Assist Device Patients.

Nonsurgical bleeding occurs in a significant proportion of patients implanted with continuous-flow ventricular assist devices (CF-VADs) and is associated with nonphysiologic flow with diminished pulsatility. An in vitro vascular pulse perfusion model seeded with adult human aortic endothelial cells (HAECs) was used to identify biomarkers sensitive to changes in pulsatility. Diminished pulsatility resulted in an ~45% decrease in von Willebrand factor (vWF) levels from 9.80 to 5.32 ng/ml (n = 5, p < 0.05) and a threefold increase in angiopoietin-2 (ANGPT-2) levels from 775.29 to 2471.93 pg/ml (n = 5, p < 0.05) in cultured HAECs. These changes are in agreement with evaluation of patient blood samples obtained pre-CF-VAD implant and 30-day postimplant: a decrease in plasma vWF level by 50% from ~45.59 to ~22.49 μg/ml (n = 15, p < 0.01) and a 64% increase in plasma ANGPT-2 level from 7,073 to 11,615 pg/ml (n = 8, p < 0.05). This study identified vWF and ANGPT-2 as highly sensitive to changes in pulsatility, in addition to interleukin-6 (IL-6), IL-8, and tumor necrosis-α (TNF-α). These biomarkers may help determine the optimal level of pulsatility and help identify patients at high risk of nonsurgical bleeding.

IPSC-Derived Corneal Endothelial Cells.

The corneal endothelium is the innermost monolayer of the cornea that maintains corneal transparency and thickness. However, adult human corneal endothelial cells (CECs) possess limited proliferative capacity, and injuries can only be repaired by migration and enlargement of resident cells. When corneal endothelial cell density is lower than the critical level (400-500 cells/mm2) due to disease or trauma, corneal endothelial dysfunction will occur and lead to corneal edema. Corneal transplantation remains the most effective clinical treatment therapy but is limited by the global shortage of healthy corneal donors. Recently, researchers have developed several alternative strategies for the treatment of corneal endothelial disease, including the transplantation of cultured human CECs and artificial corneal endothelial replacement. Early-stage results show that these strategies can effectively resolve corneal edema and restore corneal clarity and thickness, but the long-term efficacy and safety remain to be further validated. Induced pluripotent stem cells (iPSCs) represent an ideal cell source for the treatment and drug discovery of corneal endothelial diseases, which can avoid the ethical-related and immune-related problems of human embryonic stem cells (hESCs). At present, many approaches have been developed to induce the differentiation of corneal endothelial-like cells from human induced pluripotent stem cells (hiPSCs). Their safety and efficacy for the treatment of corneal endothelial dysfunction have been confirmed in rabbit and nonhuman primate animal models. Therefore, the iPSC-derived corneal endothelial cell model may provide a novel effective platform for basic and clinical research of disease modeling, drug screening, mechanistic investigation, and toxicology testing.

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi.

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.

SARS‐CoV‐2 Spike Protein Enhances Toxicant‐Induced Blood‐Brain Barrier Dysfunction

Exposure to toxicants such as benzo(a)pyrene (BaP), 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), or perfluorooctane sulfonate (PFOS), or viruses such as severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is associated with neuropathology and blood‐brain barrier (BBB) dysfunction. Recently, toxicant exposure has been reported to result in poorer health outcomes of (SARS‐CoV‐2) infection. Thus, we hypothesized that co‐exposure to toxicants and SARS‐CoV‐2 will cause greater BBB disruption than exposure to toxicants or SARS‐CoV‐2 alone.BBB permeability was assessed in vitro by measuring transendothelial electrical resistance (TEER; ohms cm2) of a monolayer of primary adult human brain microvascular endothelial cells (hBMECs). Cells were allowed to form a confluent monolayer on a 96‐well plate outfitted with circuit electrodes. Cells were treated with BaP (2, 10, and 50 uM), TCDD (1.5, 15, and 150 nM), PFOS (3, 30, and 300 uM), or recombinant SARS‐CoV‐2 spike protein (SP; 0.1, 1, and 10 nM). In experiments to test the synergistic effects of toxicants and SP, cells were treated with toxicants, then immediately were treated with SP (10 nM). Cell viability was assessed 24 hours after treatment using Cell Proliferation Kit I (Roche Diagnostics, Inc). Gene expression of tight junction protein 1 (TJP1) and vascular cell adhesion molecule‐1 (VCAM‐1) was measured by RT‐qPCR. One‐ and two‐way ANOVA followed by Holm‐Sidak post‐hoc comparisons were performed to detect differences in TEER (resistance, ohms cm2) and cell viability (absorbance, nm). T‐tests were performed to compare RT‐qPCR data. Statistical significance was reached at p < 0.05. Technical triplicates were averaged on 4 different days to obtain N = 4/group.Recombinant SARS‐CoV‐2 spike protein did not significantly affect the resistance or leakiness of the monolayer of hBMECs at any of the concentrations tested. However, when hBMECs were treated with SP (10 nM) in conjunction with BaP or PFOS, TEER was significantly reduced beyond treatment with the toxicants alone. When compared to BaP treatment alone, SP significantly lowered TEER in hBMECs treated with 10 uM or 50 uM of BaP at 9 to 24 hours after treatment (Holm‐Sidak, t’s > 2.507, p’s < 0.04), indicating a synergistic effect of SP and BaP in increasing leakiness of the hBMEC barrier. Similarly, SP caused a significant reduction in TEER of hBMECs treated with 3 uM PFOS starting at 15 hours post treatment when compared to PFOS treatment alone (Holm‐Sidak, t’s > 2.273, p’s < 0.04). TEER of TCDD‐treated hBMECs were not affected by SP treatment. At 24 hours after treatment, cell viability and expression of VCAM‐1 and TJP1 were not altered by treatment by SP alone, nor did SP affect cell viability or gene expression of toxicant‐treated hBMECs. These results suggest that disruption of the hBMEC monolayer was not due to cell death, or expression levels of VCAM‐1 and TJP1.To our knowledge, this is the first report of the synergistic effects of SARS‐CoV‐2 spike protein and environmental toxicants in an in vitro model of the BBB. These findings suggest that individuals who are exposed to environmental pollutants may suffer greater damage of their BBB if subsequently exposed to SARS‐CoV‐2 spike proteins or SARS‐CoV‐2.

Metabolic fingerprinting of HUVEC cells during phenotypic shifts using fluorescence lifetime image microscopy (FLIM)

Adult human vascular endothelial cells (ECs) change their phenotype in response to changes in vascular tumor microenvironments, oxygen concentration and during wound healing. During angiogenesis, the physiological process of creating new blood vessels, hundreds of genes are activated with one of the most important being VEGF (vascular endothelial growth factor). VEGF, the signaling protein, stimulates EC migration, proliferation, and vascularization. During angiogenesis, ECs undergo an ‘angiogenic switch’; they change from a resting (quiescent) phenotype to a more elongated and mobile (tip cells) or proliferative and mobile (stalk cells) phenotype.

Reduced Notch1 Cleavage Promotes the Development of Pulmonary Hypertension.

Clinical trials of Dll4 (Delta-like 4) neutralizing antibodies (Dll4nAbs) in cancer patients are ongoing. Surprisingly, pulmonary hypertension (PH) occurs in 14% to 18% of patients treated with Dll4nAbs, but the mechanisms have not been studied. Here, PH progression was measured in mice treated with Dll4nAbs. We detected Notch signaling in lung tissues and analyzed pulmonary vascular permeability and inflammation. Notch target gene array was performed on adult human pulmonary microvascular endothelial cells (ECs) after inhibiting Notch cleavage. Similar mechanisms were studied in PH mouse models and pulmonary arterial hypertension patients. The rescue effects of constitutively activated Notch1 in vivo were also measured. We observed that Dll4nAbs induced PH in mice as indicated by significantly increased right ventricular systolic pressure, as well as pulmonary vascular and right ventricular remodeling. Mechanistically, Dll4nAbs inhibited Notch1 cleavage and subsequently impaired lung endothelial barrier function and increased immune cell infiltration in vessel walls. In vitro, Notch targeted genes' expression related to cell growth and inflammation was decreased in human pulmonary microvascular ECs after the Notch1 inactivation. In lungs of PH mouse models and pulmonary arterial hypertension patients, Notch1 cleavage was inhibited. Consistently, EC cell-cell junction was leaky, and immune cell infiltration increased in PH mouse models. Overexpression activated Notch1-attenuated progression of PH in mice. In conclusion, Dll4nAbs led to PH development in mice by impaired EC barrier function and increased immune cell infiltration through inhibition of Notch1 cleavage in lung ECs. Reduced Notch1 cleavage in lung ECs could be an underlying mechanism of PH pathogenesis.

Differential Capability of Clinically Employed Dermal Regeneration Scaffolds to Support Vascularization for Tissue Bioengineering

The loss of skin integrity has always represented a major challenge for clinicians dealing with dermal defects, such as ulcers (diabetic, vascular and chronic), postoncologic resections (i.e., radical vulvectomy) or dermatologic disorders. The introduction in recent decades of acellular dermal matrices (ADMs) supporting the repair and restoration of skin functionality represented a significant step toward achieving clean wound repair before performing skin grafts. Hard-to-heal ulcers generally depend on local ischemia and nonadequate vascularization. In this context, one possible innovative approach could be the prevascularization of matrices with vessel-forming cells (inosculation). This paper presents a comparative analysis of the most widely used dermal templates, i.e., Integra® Bilayer Matrix Wound Dressing, PELNAC®, PriMatrix® Dermal Repair Scaffold, Endoform® Natural Dermal Template, and Myriad Matrix®, testing their ability to be colonized by human adult dermal microvascular endothelial cells (ADMECs) and to induce and support angiogenesis in vitro and in vivo. By in vitro studies, we demonstrated that Integra® and PELNAC® possess superior pro-adhesive and pro-angiogenetic properties. Animal models allowed us to demonstrate the ability of preseeded ADMECs on Integra® to promote the engraftment, integration and vascularization of ADMs at the site of application.

Conversion of Adult Human Endothelium to Lymphoid Competent Haematopoietic Stem Cells

We devised a tractable approach to fully reprogram adult human endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors.rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to adult haematopoietic stem cells, and can be used for clonal engraftment with serial primary and secondary multi-lineage reconstitution. rEC-HSCs give rise to fully mature lymphoid CD4+ and CD8+ T cells. These T cells exhibit a diverse TCR repertoire and are endowed with antigen-dependent adaptive immune function.These T cells are capable of anti-tumor response, and are amenable to chimeric antigen receptor (CAR) gene editing without losing their T lymphoid properties.Our combined approach of transcription factor and niche-mediated cell fate conversion produces haematopoietic stem cells from adult human endothelium and holds promise for modelling hematologic disease in humanized mice and for immune-therapeutic strategies in malignant blood disorders. DisclosuresNo relevant conflicts of interest to declare.

Loss of Primary Cilia Protein IFT20 Dysregulates Lymphatic Vessel Patterning in Development and Inflammation.

Microenvironmental signals produced during development or inflammation stimulate lymphatic endothelial cells to undergo lymphangiogenesis, in which they sprout, proliferate, and migrate to expand the vascular network. Many cell types detect changes in extracellular conditions via primary cilia, microtubule-based cellular protrusions that house specialized membrane receptors and signaling complexes. Primary cilia are critical for receipt of extracellular cues from both ligand-receptor pathways and physical forces such as fluid shear stress. Here, we report the presence of primary cilia on immortalized mouse and primary adult human dermal lymphatic endothelial cells in vitro and on both luminal and abluminal domains of mouse corneal, skin, and mesenteric lymphatic vessels in vivo. The purpose of this study was to determine the effects of disrupting primary cilia on lymphatic vessel patterning during development and inflammation. Intraflagellar transport protein 20 (IFT20) is part of the transport machinery required for ciliary assembly and function. To disrupt primary ciliary signaling, we generated global and lymphatic endothelium-specific IFT20 knockout mouse models and used immunofluorescence microscopy to quantify changes in lymphatic vessel patterning at E16.5 and in adult suture-mediated corneal lymphangiogenesis. Loss of IFT20 during development resulted in edema, increased and more variable lymphatic vessel caliber and branching, as well as red blood cell-filled lymphatics. We used a corneal suture model to determine ciliation status of lymphatic vessels during acute, recurrent, and tumor-associated inflammatory reactions and wound healing. Primary cilia were present on corneal lymphatics during all of the mechanistically distinct lymphatic patterning events of the model and assembled on lymphatic endothelial cells residing at the limbus, stalk, and vessel tip. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute corneal lymphangiogenesis resulting in increased lymphatic vessel density and branching. These data are the first functional studies of primary cilia on lymphatic endothelial cells and reveal a new dimension in regulation of lymphatic vascular biology.

The protective effect of gastrodin against the synergistic effect of HIV-Tat protein and METH on the blood-brain barrier via glucose transporter 1 and glucose transporter 3.

Many individuals infected with human immunodeficiency virus (HIV) are also afflicted with HIV-associated neurocognitive disorders (HANDs). Methamphetamine (METH) abuse puts HIV-1 patients at risk for HANDs because METH and HIV-1 proteins, such as trans-activator of transcription (Tat), can synergistically damage the blood-brain barrier (BBB). However, the underlying mechanism of METH- and HIV-Tat-induced BBB damage remains unclear. In this study, male adult tree shrews and human brain capillary endothelial cells were treated with HIV-Tat, METH, and gastrodin. We used western blotting to examine the expressions of glucose transporters (GLUT1 and GLUT3), tight junctions, and junctional adhesion molecule A (JAMA) and to evaluate the damage and detect Evans blue (EB) and fluorescein sodium in the brain to assess BBB permeability to study the effect of METH and the HIV-1 Tat protein on BBB function in vitro and in vivo. The results showed that the group treated with Tat and METH experienced a significant change at the ultrastructural level of the tree shrew cerebral cortex, decreased protein levels of occluding, claudin-5, Zonula occludens 1 (ZO1), and JAMA in vitro and in vivo, and increased levels of EB and fluorescein sodium in the tree shrew cerebral cortex. The protein levels of GLUT1 and GLUT3 was downregulated in patients with Tat- and METH-induced BBB damage. Pretreatment with gastrodin significantly increased the levels of EB and fluorescein sodium in the tree shrew cerebral cortex and increased the expressions of occluding, ZO1, JAMA, and GLUT1 and GLUT. These results indicate that gastrodin may offer a potential therapeutic option for patients with HANDs.

Kanglexin protects against cardiac fibrosis and dysfunction in mice by TGF-β1/ERK1/2 noncanonical pathway.

Cardiac fibrosis is a common pathological manifestation accompanied by various heart diseases, and antifibrotic therapy is an effective strategy to prevent diverse pathological processes of the cardiovascular system. We currently report the pharmacological evaluation of a novel anthraquinone compound (1,8-dihydroxy-6-methyl-9,10-anthraquinone-3-oxy ethyl succinate) named Kanglexin (KLX), as a potent cardioprotective agent with antifibrosis activity. Our results demonstrated that the administration of KLX by intragastric gavage alleviated cardiac dysfunction, hypertrophy, and fibrosis induced by transverse aortic constriction (TAC) surgical operation. Meanwhile, KLX administration relieved endothelial to mesenchymal transition of TAC mice. In TGF β1-treated primary cultured adult mouse cardiac fibroblasts (CFs) and human umbilical vein endothelial cells (HUVECs), KLX inhibited cell proliferation and collagen secretion. Also, KLX suppressed the transformation of fibroblasts to myofibroblasts in CFs. Further studies revealed that KLX-mediated cardiac protection was due to the inhibitory role of TGF-β1/ERK1/2 noncanonical pathway. In summary, our study indicates that KLX attenuated cardiac fibrosis and dysfunction of TAC mice, providing a potentially effective therapeutic strategy for heart pathological remodeling.

Involvement of Sigma Receptor-1 in Lymphatic Endothelial Barrier Integrity and Bioenergetic Regulation.

Background: Lymphatic endothelium plays significant roles in lymph transport and maintaining a barrier between the lymph and interstitial compartments. Lymphatic endothelial dysfunction is suspected to be a key factor in the pathogenesis of lymphatic diseases such as lymphedema. Sigma receptor-1 (σ1) was recently identified to promote endothelial-dependent production of nitric oxide and relaxation of collecting lymphatic vessels. In this study, we investigated the potential role of σ1 in lymphatic endothelial barrier function. Methods and Results: Cultured adult human dermal lymphatic endothelial cells (HDLEC) were grown into confluent monolayers. Transendothelial electrical resistance (TER) served as an index of barrier function. Glycolytic rate of HDLEC was determined with the Agilent Seahorse system. The σ1-selective agonist PRE-084 was used to test the impact of σ1 on HDLEC monolayer barrier function and endothelial bioenergetics, whereas the contribution of basal σ1 activity was assessed with small interfering RNA (siRNA)-mediated knockdown of σ1 expression. The ability of σ1 activation to counteract interleukin (IL)-1β-induced barrier dysfunction was also tested. The results show that PRE-084 increases HDLEC TER in a concentration-dependent manner, whereas reducing σ1 expression with siRNA decreases HDLEC TER. PRE-084 also enhances glycolytic rate parameters in HDLEC. Moreover, PRE-084 treatment partially counteracts IL-1β-induced HDLEC monolayer barrier dysfunction. Conclusions: Collectively, the results suggest that σ1 contributes to basal lymphatic endothelial barrier function, potentially through its ability to enhance glycolytic energy production. Our work also highlights the therapeutic potential of σ1 agonists for preventing lymphatic barrier dysfunction caused by inflammatory mediators.

Molecular Analysis of Fetal and Adult Primary Human Liver Sinusoidal Endothelial Cells: A Comparison to Other Endothelial Cells.

In humans, Factor VIII (F8) deficiency leads to hemophilia A and F8 is largely synthesized and secreted by the liver sinusoidal endothelial cells (LSECs). However, the specificity and characteristics of these cells in comparison to other endothelial cells is not well known. In this study, we performed genome wide expression and CpG methylation profiling of fetal and adult human primary LSECs together with other fetal primary endothelial cells from lung (micro-vascular and arterial), and heart (micro-vascular). Our results reveal expression and methylation markers distinguishing LSECs at both fetal and adult stages. Differential gene expression of fetal LSECs in comparison to other fetal endothelial cells pointed to several differentially regulated pathways and biofunctions in fetal LSECs. We used targeted bisulfite resequencing to confirm selected top differentially methylated regions. We further designed an assay where we used the selected methylation markers to test the degree of similarity of in-house iPS generated vascular endothelial cells to primary LSECs; a higher similarity was found to fetal than to adult LSECs. In this study, we provide a detailed molecular profile of LSECs and a guide to testing the effectiveness of production of in vitro differentiated LSECs.

Identification of Potential Hub Genes and Signal Pathways Promoting the Distinct Biological Features of Cord Blood-Derived Endothelial Progenitor Cells Via Bioinformatics.

Background: Numerous studies, ranging from the alleviation of tissue ischemia to the assessment of cancer prognosis, have demonstrated the fundamental biological differences between human umbilical cord blood-derived endothelial progenitor cells (CB-EPCs) and adult peripheral blood-derived endothelial progenitor cells (PB-EPCs). However, the underlying molecular mechanisms that produce these differences are not clear.The purpose of this study was to identify potential hub genes, key protein interactive networks, and correlated signal pathways unique to CB-EPC biology via bioinformatic methods. Materials and Methods: We selected the microarray dataset GSE39763 and identified the differentially expressed genes (DEGs) using the "limma" package in the RStudio software. These DEGs were annotated by gene ontology enrichment analyses and signal pathway analyses. A protein-protein interaction (PPI) analysis was then performed to construct PPI networks and identify a hub protein module. We further validated candidate DEGs from the selected module in the gene expression profiling interactive analysis (GEPIA) database because the DEGs were enriched in cancer pathways. Results: Setting an adjusted p-value <0.01 and |Log2 fold change (FC)| ≥ 2 as cutoff criteria, a total of 346 DEGs, including 314 upregulated genes and 32 downregulated genes in CB-EPCs, were identified. Expression of the genes encoding the AT-Hook Containing Transcription Factor 1 (AHCTF1), the Cancer Susceptibility Candidate 5 (CASC5), the Centromere Protein C (CENPC), the Centromere Protein E (CENPE), the Centromere Protein F (CENPF), the NUF2 Component of NDC80 Kinetochore Complex (NUF2), the RAN-Binding Protein 2 (RANBP2), the Shugoshin-like 2 (SGOL2), the Structural Maintenance of Chromosomes 3 (SMC3), and the Spindle Apparatus Coiled-Coil Protein 1 (SPDL1) proteins were specifically associated with CB-EPCs. Except for CENPC, the other nine genes' expression are all associated with a poorer overall survival rate in cancers. The expression levels of the CENPF and NUF2 genes in tumor patients were significantly higher than those in the controls. Conclusion: The CB-EPCs express genes with greater potential for proliferation and increased migration compared to PB-EPCs; in this regard they are similar to cancer cells.

Sigma‐1 Receptors Promote Lymphatic Endothelial Barrier Function

The lymphatic endothelium plays important roles in lymphatic contractile activity, lymph transport and maintaining a barrier between the lymph and interstitial compartments and its dysfunction is a key factor in the pathogenesis of lymphatic diseases such as lymphedema. Sigma‐1 (σ1) receptors are chaperone proteins located in the endoplasmic reticulum were recently identified to promote collecting lymphatic dilation through endothelial production of nitric oxide. However, the role of σ1 in other functions of lymphatic endothelial cells are not clear. The aim of the current study was to test the hypothesis that σ1 promotes barrier function of lymphatic endothelium. We confirmed expression of σ1 in adult and juvenile human dermal lymphatic endothelial cells (HDLEC) using the ProteinSimple Western (WES) system and immunofluorescence microscopy. To test whether activation of σ1 promotes lymphatic endothelial barrier function, we applied the selective agonist PRE‐084 to cultured monolayers of HDLEC and determined the transendothelial electrical resistance (TER) as an index of barrier function using electrical cell‐substrate impedance sensing. The contribution of basal σ1 activity to HDLEC TER was also assessed either with the σ1 antagonist BD1047 (200 nM) or by silencing σ1 expression with siRNA. Because σ1 has been previously described to be located in the ER‐mitochondrial membrane interface, we also evaluated its role in endothelial bioenergetics using the Agilent Seahorse system glycolytic rate assay. Lastly, we tested the ability of PRE‐084 to counteract IL‐1β‐induced barrier dysfunction in HDLEC monolayers. The results show that the activation of σ1 receptor with PRE‐084 increases HDLEC TER in a concentration‐dependent manner, with 50, 100, 150, and 200 μM PRE‐084 eliciting significant elevations in TER (100 μM was chosen for subsequent experiments). Blockade of σ1 either with the antagonist BD‐1047 or reducing σ1 expression with siRNA resulted in decreased HDLEC TER. PRE‐084 also enhanced basal and compensatory glycolysis as well as proton efflux rate in HDLEC. Moreover, PRE‐084 partially counteracted the IL‐1β‐induced TER drop indicating its potential anti‐inflammatory impact on lymphatic endothelial cells. Collectively our results suggest that σ1 contributes to basal lymphatic endothelial barrier function, potentially through its ability to enhance glycolytic ATP production. Our work also highlights the therapeutic potential of σ1 agonists for preventing lymphatic barrier dysfunction caused by inflammatory mediators.Support or Funding InformationThis work was supported by NIH Grant R01GM120774.

Early Vascular Cells Improve Microvascularization Within 3D Cardiac Spheroids.

Introduction: A key obstacle in the creation of engineered cardiac tissues of clinically relevant sizes is limited diffusion of oxygen and nutrients. Thus, there is a need for organized vascularization within a three-dimensional (3D) tissue environment. Human induced pluripotent stem cell (hiPSC)-derived early vascular cells (EVCs) have shown to improve organization of vascular networks within hydrogels. We hypothesize that introduction of EVCs into 3D microtissue spheroids will lead to increased microvascular formation and improve spheroid formation. Methods: HiPSC-derived cardiomyocytes (CMs) were cocultured with human adult ventricular cardiac fibroblasts (FB) and either human umbilical vein endothelial cells (HUVECs) or hiPSC-derived EVCs for 72 h to form mixed cell spheroids. Three different groups of cell ratios were tested: Group 1 (control) consisted of CM:FB:HUVEC 70:15:15, Group 2 consisted of CM:FB:EVC 70:15:15, and Group 3 consisted of CM:FB:EVC 40:15:45. Vascularization, cell distribution, and cardiac function were investigated. Results: Improved microvasculature was found in EVC spheroids with new morphologies of endothelial organization not found in Group 1 spheroids. CMs were found in a core-shell type distribution in Group 1 spheroids, but more uniformly distributed in EVC spheroids. Contraction rate increased into Group 2 spheroids compared to Group 1 spheroids. Conclusion: The triculture of CM, FB, and EVC within a multicellular cardiac spheroid promotes microvascular formation and cardiac spheroid contraction.