Endothelial Cell Tight Junctions Research Articles

Abstract 5110: Exploring the effects of Indirubins in pediatric diffuse high grade glioma models

Abstract Diffuse midline glioma (DMG) is an invasive pediatric brain tumor which grows in the pons of the brainstem. The current course of treatment is focal radiation however, DMG continues to have a very poor prognosis with a median survival of less than 10 months. A major barrier for chemotherapeutic treatment of DMG is the blood brain barrier (BBB) which tightly controls access of molecules to the brain. This limits the efficacy of drugs to treat brain cancer and other diseases of the central nervous system. Previous studies from the Lawler lab and others have shown that the indirubin derivative, 6-bromoindirubin-3′-acetoxime(BIO-acetoxime/BIA), has anti-invasive properties and can enhance survival in glioblastoma xenograft models. Here, we investigated the effects of BIA on pediatric glioma models using a range of relevant assays. In an in vitro collagen migration assay, BIA was observed to significantly slow cell migration at a concentration of 1µM in DIPG 36 cells, over 72 hours. Furthermore, we have also shown that BIA modulates the tumor vasculature, which could limit tumor growth, and improves drug delivery to tumors by targeting tight junctions in tumor associated endothelium. The effect on BBB drug penetration was assessed in a multicellular 3D in vitro BBB model and effects of BIA on endothelial barrier functions was determined using trans-endothelial resistance assays (TEER). BIA treatment increased dextran uptake into 3D BBB models, and also lowered endothelial cell permeability in vitro via a reduction in the expression of tight junction proteins as shown by staining and a decrease in TEER values. In order to assess potential synergistic interactions, a drug screen was performed on a panel of distinct pediatric glioma cell lines (DIPG 36 and KNS42). This identified several FDA approved drugs that combine well with BIA. From this screen, BIA was shown to synergize with DNA damaging chemotherapy to enhance toxicity and in combination with cisplatin promote DNA damage in pediatric glioma cell lines. Other drugs identified in the screen are currently under investigation and details will be reported. Thus, our hypothesis is that BIA may provide an effective approach for further development as a DMG therapeutic through targeting invasion and enhancing drug potency and delivery via modulation of endothelial cell tight junctions. Interrogating the mechanisms involved and developing drug formulations informed by the drug screen for in vivo studies to determine the translational potential of this approach is our focus for future studies. Citation Format: Jasmine S. Clark, Jorge Jimenez-Macias, Philippa Vaughn-Beaucaire, Shengliang Zhang, Wafik El-Deiry, Rishi Lulla, Sean Lawler. Exploring the effects of Indirubins in pediatric diffuse high grade glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5110.

CNS resident macrophages enhance dysfunctional angiogenesis and circulating monocytes infiltration in brain arteriovenous malformation

Myeloid immune cells are abundant in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. We hypothesize that CNS resident macrophages enhance bAVM development and hemorrhage. RNA sequencing using cultured endothelial cells (ECs) and mouse bAVM samples revealed that downregulation of two bAVM causative genes, activin-like kinase 1 (ALK1) or endoglin, increased inflammation and innate immune signaling. To understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor inhibitor to bAVM mice with brain focal Alk1 deletion. Transient depletion of CNS resident macrophages at an early stage of bAVM development mitigated the phenotype severity of bAVM, including a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages increased EC tight junction protein expression, reduced the number of dysplasia vessels and severe hemorrhage in established bAVMs. Thus, EC AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophage could be a therapeutic target to mitigate the development and severity of bAVMs.

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.

A novel human iPSC model of COL4A1/A2 small vessel disease unveils a key pathogenic role of matrix metalloproteinases

Cerebral small vessel disease (SVD) affects the small vessels in the brain and is a leading cause of stroke and dementia. Emerging evidence supports a role of the extracellular matrix (ECM), at the interface between blood and brain, in the progression of SVD pathology, but this remains poorly characterized. To address ECM role in SVD, we developed a co-culture model of mural and endothelial cells using human induced pluripotent stem cells from patients with COL4A1/A2 SVD-related mutations. This model revealed that these mutations induce apoptosis, migration defects, ECM remodeling, and transcriptome changes in mural cells. Importantly, these mural cell defects exert a detrimental effect on endothelial cell tight junctions through paracrine actions. COL4A1/A2 models also express high levels of matrix metalloproteinases (MMPs), and inhibiting MMP activity partially rescues the ECM abnormalities and mural cell phenotypic changes. These data provide a basis for targeting MMP as a therapeutic opportunity in SVD.

SURG-22. MECHANISMS OF BBB PERMEABILITY CHANGES TRIGGERED BY LASER THERAPY

Abstract The blood-brain barrier (BBB) severely limits access of systemically delivered drugs to the brain and brain tumors. Laser interstitial thermal therapy (LITT) has previously been shown to increase BBB permeability in both patients and in mice. The exact mechanisms of LITT-induced BBB permeability changes and their temporal features have yet to be elucidated. To answer this question, we used an established mouse model of LITT to evaluate changes in endothelial cell tight junction integrity and transcytosis over time in normal brain and demonstrate changes in transcytosis in human GBM tumor samples. Mean vesicular density (MVD) was calculated using electron microscopy to evaluate transcytosis. Immunohistochemistry (IHC) was used to assess tight junction integrity (Claudin 5 area/CD31 area) and Mfsd2a levels (Mfsd2a area/CD31 area) along endothelial cells in the laser penumbra. Tight junction integrity decreased 2.5 fold 3 days after LITT, with continued decrease 14 days post-LITT. Mean vesicular density (MVD) in endothelial cells within the laser penumbra increased 3-4 fold compared to sham treated mice, with a peak increase 14 days after LITT before returning to baseline levels 21 days after treatment. These changes in transcytosis correlated with a significant decrease in the intensity of Mfsd2a IHC in the laser penumbra at day 14. Finally, we found similar low levels of transcytosis in non-enhancing and enhancing human GBM tumor samples, confirming that transcytosis is potentially targetable to manipulate BBB permeability. CONCLUSION: Disruptions in tight junction integrity continue up to 14 days after LITT, while LITT-induced increases in transcytosis occur in a delayed fashion, peaking 14 days after laser treatment. Increased transcytosis is inversely correlated with decreased Mfsd2a expression, suggesting a role for Mfsd2a in regulation of LITT-induced BBB changes. Importantly, baseline levels of transcytosis in human GBM appear to be low, such that transcytosis could be targeted to increase BBB permeability.

DDEL-04. MODULATION OF THE BLOOD-TUMOR BARRIER FOR IMPROVED DRUG DELIVERY IN GLIOBLASTOMA

Abstract Effective drug delivery remains a major challenge in glioblastoma (GBM) treatment. Specialized intercellular endothelial cell tight junctions at the blood-brain and blood-tumor barriers (BBB/BTB) represent obstacles to therapeutic efficacy by limiting intratumoral drug uptake. To understand the factors at the BTB that limit drug accessibility we performed bioinformatics analysis of TCGA data which suggested enrichment of the tight junction protein VE-cadherin (CDH5). CDH5 expression at the BTB was confirmed by immunostaining of patient GBM specimens and spatial transcriptomics using the 10X Visium platform. We found that a broadly selective protein kinase inhibitor, 6-bromoindirubin-3’-acetoxime (BIA), potently down-regulated CDH5 in cultured human cerebrovascular endothelial cells (HCMEC/D3) at the transcriptional level and this correlated with decreased trans-endothelial electrical resistance (TEER) measurement. In vivo administration of BIA (20 mg/kg IP) to mice bearing intracranial GBM xenografts showed striking downregulation of CDH5 in tumor associated endothelium. Furthermore, increased BTB permeability was shown using fluorescent tracers as well as DCE-MRI, which demonstrated elevated Ktrans values 24 hours after BIA administration. Integrated phospho-proteomic and transcriptomic analysis of BIA treated HCMEC/D3 cells support a mechanism involving p38 MAP kinase regulation of Ets-family transcription factors that drive CDH5 expression. At least a 20-fold increase in intra-tumoral accumulation of cisplatin and temozolomide was observed using G9-pCDH GBM xenografts when administered 24 h after BIA treatment. No increased drug uptake was seen in healthy brain tissue, indicating specific targeting of tumor vasculature. Efficacy studies showed extended survival when using cisplatin in combination with BIA (median 27d, p=0.0016) when compared to cisplatin (18.5d), BIA (21.5d) and control (18d). Further efficacy studies are underway. These data therefore identify the GBM-associated vasculature as a target for small molecule inhibition of tight junction formation to enhance intratumoral drug uptake. Ongoing studies are investigating the potential of this approach with a range of agents.

Cranial venous-outflow obstruction promotes neuroinflammation via ADAM17/solTNF-α/NF-κB pathway following experimental TBI

Traumatic brain injury (TBI) is a global public health problem. As an important cause of secondary injury, cerebrovascular reaction can cause secondary bleeding, venous sinus thrombosis, and malignant brain swelling. Recent clinical studies have confirmed that intracranial venous return disorder is closely related to the prognosis of patients, yet the specific molecular mechanism involved in this process is still unclear. This study used an acute subdural hematoma (ASDH) model with cranial venous outflow obstruction (CVO) to explore how CVO aggravates the pathological process after TBI, especially for inflammation and tissue damage. The results suggest that intracranial venous return disorder exacerbates neurological deficits and brain edema in rats with ASDH by aggravating the destruction of endothelial cell tight junctions (TJs) proteins and promoting the expression of inflammatory factors, the activation of microglia and expression of recombinant A disintegrin and metalloprotease 17 (ADAM17) as well as the secretion of solTNF-α, a soluble form of tumor necrosis factor-alpha (TNFα), which in turn increase IκB-α ((inhibitor of the transcription factor nuclear factor-κB) and NF-κB p65. Our study revealed a molecular basis of how CVO aggravates inflammation and tissue damage.

TIMP-1 Protects Tight Junctions of Brain Endothelial Cells From MMP-Mediated Degradation.

The blood-brain barrier (BBB) plays a critical role in central nervous system homeostasis, and the integrity ofBBB is disrupted in many neurodegenerative diseases. Matrix metalloproteinases (MMPs) degrade the tight junctions (TJs) of endothelial cells and basement membrane components essential to BBB integrity, which leads to increased BBB permeability and allows inflammatory cells and neurotoxic substances to enter the brain. Tissue inhibitors of metalloproteinases (TIMPs), endogenous inhibitors of MMPs, regulate MMP activity, thereby maintaining BBB integrity. The disruptive impacts of MMP-3 and MMP-9 on BBB and protective effect of TIMP-1 were investigated in a simplified in vitro model of the BBB, which was generated using rat brain microvascular endothelial cells (RBMEC). The main features of BBB formation, including permeability and the trans-endothelial electrical resistance (TEER), were monitored over time after the addition of MMP-3 and MMP-9 and their complexes with TIMP-1 inhibitor. Our results indicated that MMP-3 and MMP-9 caused a dose-dependent disruption of the BBB, with 1.5µM MMPs resulting in an over threefold increase in permeability, while TIMP-1 inhibition protected the integrity of the BBB model and recovered TEER and permeability of RBMECs. The disruption and recovery of tight junction proteins of RBMECs after MMP and TIMP treatment were also detected using fluorescent microscopy. MMP-9 and MMP-3 disrupt the BBB bydegrading tight junctions in endothelial cells, and TIMP-1 could inhibit the disruptive effect of MMP-3 and MMP-9 by showing potential as therapeutic proteinagainst MMP-related diseases where BBB disruption plays a role.

Physiological oxygen conditions enhance the angiogenic properties of extracellular vesicles from human mesenchymal stem cells

BackgroundFollowing an ischemic injury to the brain, the induction of angiogenesis is critical to neurological recovery. The angiogenic benefits of mesenchymal stem cells (MSCs) have been attributed at least in part to the actions of extracellular vesicles (EVs) that they secrete. EVs are membrane-bound vesicles that contain various angiogenic biomolecules capable of eliciting therapeutic responses and are of relevance in cerebral applications due to their ability to cross the blood–brain barrier (BBB). Though MSCs are commonly cultured under oxygen levels present in injected air, when MSCs are cultured under physiologically relevant oxygen conditions (2–9% O2), they have been found to secrete higher amounts of survival and angiogenic factors. There is a need to determine the effects of MSC-EVs in models of cerebral angiogenesis and whether those from MSCs cultured under physiological oxygen provide greater functional effects.MethodsHuman adipose-derived MSCs were grown in clinically relevant serum-free medium and exposed to either headspace oxygen concentrations of 18.4% O2 (normoxic) or 3% O2 (physioxic). EVs were isolated from MSC cultures by differential ultracentrifugation and characterized by their size, concentration of EV specific markers, and their angiogenic protein content. Their functional angiogenic effects were evaluated in vitro by their induction of cerebral microvascular endothelial cell (CMEC) proliferation, tube formation, and angiogenic and tight junction gene expressions.ResultsCompared to normoxic conditions, culturing MSCs under physioxic conditions increased their expression of angiogenic genes SDF1 and VEGF, and subsequently elevated VEGF-A content in the EV fraction. MSC-EVs demonstrated an ability to induce CMEC angiogenesis by promoting tube formation, with the EV fraction from physioxic cultures having the greatest effect. The physioxic EV fraction further upregulated the expression of CMEC angiogenic genes FGF2, HIF1, VEGF and TGFB1, as well as genes (OCLN and TJP1) involved in BBB maintenance.ConclusionsEVs from physioxic MSC cultures hold promise in the generation of a cell-free therapy to induce angiogenesis. Their positive angiogenic effect on cerebral microvascular endothelial cells demonstrates that they may have utility in treating ischemic cerebral conditions, where the induction of angiogenesis is critical to improving recovery and neurological function.

Acetaldehyde dehydrogenase 2 ameliorates lung endothelial barrier and balances mitochondrial dynamics in mice with acute lung injury

To investigate the protective effects of acetaldehyde dehydrogenase 2 (ALDH2) against lipopolysaccharide (LPS)- induced acute lung injury (ALI) in mice and explore the possible mechanisms. Sixty C57BL/6J mice were equally randomized into Sham group, LPS group, LPS + Alda-1 (an ALDH2 agonist) group, and LPS + Daidzin (an ALDH2 inhibitor) group. After the treatment, the wet/dry lung mass ratio of the mice was measured, and the lung permeability was evaluated with Evans Blue (EB). The lung tissue pathologies were evaluated with HE staining and transmission electron microscopy. Serum levels of 4-hydroxynonenal (4-HNE) were measured with ELISA, and malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined to measure oxidative stress levels. The expressions of ALDH2, ZO-1, Occludin, Mfn2, OPA1, Drp1, Fis1, and nuclear Nrf2 and HO-1 proteins in the lung tissues were detected using Western blotting. The mice with LPS-induced ALI showed severe disruption of the lung tissue structure and endothelial cell tight junctions with significantly increased the lung permeability (P<0.01), increased levels of 4-HNE and MDA (P<0.01), decreased activities of CAT and SOD (P<0.01), lowered expressions of ALDH2, ZO-1, Occludin, Mfn2, and OPA1 proteins, and increased expressions of Drp1, Fis1, and nuclear Nrf2 and HO-1 proteins (P<0.05, P<0.01). Treatment with Alda-1 significantly improved lung tissue pathologies and mitochondrial damage in ALI mice (P<0.01), increased the expressions of ALDH2, ZO-1, Occludin, OPA1, Mfn2, and nuclear Nrf2 and HO-1 proteins, and lowered the expressions of Drp1 and Fis1 proteins (P<0.05, P<0.01). Compared with Alda-1, treatment with Daidzin significantly increased the lung permeability, exacerbated mitochondrial damage, decreased the expression of ALDH2, ZO-1, Occludin, Mfn2, OPA1, and nuclear Nrf2 and HO-1 proteins, and increased expressions of Drp1 and Fis1 proteins (P<0.05, P<0.01). ALDH2 can ameliorate LPSinduced lung endothelial barrier damage in ALI mice by maintaining the balance of mitochondrial dynamics and inhibiting oxidative stress, and the mechanism may be related to the Nrf2/HO-1 pathway.

Iguratimod promotes functional recovery after SCI by repairing endothelial cell tight junctions

Destruction of the blood-spinal cord barrier (BSCB) after spinal cord injury (SCI) is an important factor promoting the progression of the injury. This study addressed how to repair the BSCB in order to promote the repair of injured spinal cords. Iguratimod (IGU), an anti-rheumatic drug, has been approved for clinical use. A spinal cord injury mouse model and TNF-α-stimulated bEnd.3 cells were used to investigate the effect and mechanism of IGU on injured BSCB. An intracerebroventricular osmotic pump was used to administer drugs to the SCI mouse model. The results showed that the SCI mice in the treatment group had better recovery of neurological function than the control group. Examination of the tissue revealed better repair of the BSCB in injured spinal cords after medication. According to the results from the cell model, IGU promoted the expression of tight junction proteins and reduced cell permeability. Further research found that IGU repaired the barrier function by regulating glycolysis levels in the injured endothelial cells. In studying the mechanism, IGU was found to regulate HIF-1α expression through the NF-κB pathway, thereby regulating the expression of the glycolytic enzymes related to endothelial injury. In summary, IGU promoted functional recovery in vivo by repairing the BSCB. In vitro, IGU regulated the level of glycolysis in the damaged endothelium through the NF-κB pathway, thereby repairing the tight junctions between the endothelium. Therefore, IGU may become a potential drug for treating spinal cord injury.

TLR7/TLR8 activation and susceptibility genes synergize to breach gut barrier in a mouse model of lupus.

Mounting evidence suggests that increased gut permeability, or leaky gut, and the resulting translocation of pathobionts or their metabolites contributes to the pathogenesis of Systemic Lupus Erythematosus. However, the mechanisms underlying the induction of gut leakage remain unclear. In this study, we examined the effect of a treatment with a TLR7/8 agonist in the B6.Sle1.Sle2.Sle3 triple congenic (TC) mouse, a spontaneous mouse model of lupus without gut leakage. Lupus-prone mice (TC), TC.Rag1-/- mice that lack B and T cells, and congenic B6 healthy controls were treated with R848. Gut barrier integrity was assessed by measuring FITC-dextran in the serum following oral gavage. Claudin-1 and PECAM1 expression as well as the extent of CD45+ immune cells, B220+ B cells, CD3+ T cells and CD11b+ myeloid cells were measured in the ileum by immunofluorescence. NKp46+ cells were measured in the ileum and colon by immunofluorescence. Immune cells in the ileum were also analyzed by flow cytometry. R848 decreased gut barrier integrity in TC but not in congenic control B6 mice. Immunofluorescence staining of the ileum showed a reduced expression of the tight junction protein Claudin-1, endothelial cell tight junction PECAM1, as well as an increased infiltration of immune cells, including B cells and CD11b+ cells, in R848-treated TC as compared to untreated control mice. However, NKp46+ cells which play critical role in maintaining gut barrier integrity, had a lower frequency in treated TC mice. Flow cytometry showed an increased frequency of plasma cells, dendritic cells and macrophages along with a decreased frequency of NK cells in R848 treated TC mice lamina propria. In addition, we showed that the R848 treatment did not induce gut leakage in TC.Rag1-/- mice that lack mature T and B cells. These results demonstrate that TLR7/8 activation induces a leaky gut in lupus-prone mice, which is mediated by adaptive immune responses. TLR7/8 activation is however not sufficient to breach gut barrier integrity in non-autoimmune mice.

Glucuronoxylomannan intranasal challenge prior to Cryptococcus neoformans pulmonary infection enhances cerebral cryptococcosis in rodents

The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis, with the highest rate of disease in patients with AIDS or immunosuppression. This microbe enters the human body via inhalation of infectious particles. C. neoformans capsular polysaccharide, in which the major component is glucuronoxylomannan (GXM), extensively accumulates in tissues and compromises host immune responses. C. neoformans travels from the lungs to the bloodstream and crosses to the brain via transcytosis, paracytosis, or inside of phagocytes using a "Trojan horse" mechanism. The fungus causes life-threatening meningoencephalitis with high mortality rates. Hence, we investigated the impact of intranasal exogenous GXM administration on C. neoformans infection in C57BL/6 mice. GXM enhances cryptococcal pulmonary infection and facilitates fungal systemic dissemination and brain invasion. Pre-challenge of GXM results in detection of the polysaccharide in lungs, serum, and surprisingly brain, the latter likely reached through the nasal cavity. GXM significantly alters endothelial cell tight junction protein expression in vivo, suggesting significant implications for the C. neoformans mechanisms of brain invasion. Using a microtiter transwell system, we showed that GXM disrupts the trans-endothelial electrical resistance, weakening human brain endothelial cell monolayers co-cultured with pericytes, supportive cells of blood vessels/capillaries found in the blood-brain barrier (BBB) to promote C. neoformans BBB penetration. Our findings should be considered in the development of therapeutics to combat the devastating complications of cryptococcosis that results in an estimated ~200,000 deaths worldwide each year.

Hypergravity Increases Blood-Brain Barrier Permeability to Fluorescent Dextran and Antisense Oligonucleotide in Mice.

The earliest effect of spaceflight is an alteration in vestibular function due to microgravity. Hypergravity exposure induced by centrifugation is also able to provoke motion sickness. The blood-brain barrier (BBB) is the crucial interface between the vascular system and the brain to ensure efficient neuronal activity. We developed experimental protocols of hypergravity on C57Bl/6JRJ mice to induce motion sickness and reveal its effects on the BBB. Mice were centrifuged at 2× g for 24 h. Fluorescent dextrans with different sizes (40, 70 and 150 kDa) and fluorescent antisense oligonucleotides (AS) were injected into mice retro-orbitally. The presence of fluorescent molecules was revealed by epifluorescence and confocal microscopies in brain slices. Gene expression was evaluated by RT-qPCR from brain extracts. Only the 70 kDa dextran and AS were detected in the parenchyma of several brain regions, suggesting an alteration in the BBB. Moreover, Ctnnd1, Gja4 and Actn1 were upregulated, whereas Jup, Tjp2, Gja1, Actn2, Actn4, Cdh2 and Ocln genes were downregulated, specifically suggesting a dysregulation in the tight junctions of endothelial cells forming the BBB. Our results confirm the alteration in the BBB after a short period of hypergravity exposure.

Single-cell transcriptomics-based multidisease analysis revealing the molecular dynamics of retinal neurovascular units under inflammatory and hypoxic conditions

The retinal neurovascular unit (NVU) is paramount to maintaining the homeostasis of the retina and determines the progression of various diseases, including diabetic retinopathy (DR), glaucoma, and retinopathy of prematurity (ROP). Although some studies have investigated these diseases, a combined analysis of disease-wide etiology in the NUV at the single-cell level is lacking. Herein, we constructed an atlas of the NVU under inflammatory and hypoxic conditions by integrating single-cell transcriptome data from retinas from wild-type, AireKO, and NdpKO mice. Based on the heterogeneity of the NVU structure and transcriptome diversity under normal and pathological conditions, we discovered two subpopulations of Müller cells: Aqp4hi and Aqp4lo cells. Specifically, Aqp4lo cells expresses phototransduction genes and represent a special type of Müller cell distinct from Aqp4hi cells, classical Müller cells. AireKO mice exhibit experimental autoimmune uveitis (EAU) with severe damage to the NVU structure, mainly degeneration of Aqp4hi cells. NdpKO mice exhibited familial exudative vitreoretinopathy (FEVR), with damage to the endothelial barrier, endothelial cell tight junction destruction and basement membrane thickening, accompanied by the reactive secretion of proangiogenic factors by Aqp4hi cells. In both EAU and FEVR, Aqp4hi cells are a key factor leading to NVU damage, and the mechanism by which they are generated is regulated by different transcription factors. By studying the pattern of immune cell infiltration in AireKO mice, we constructed a regulatory loop of “inflammatory cells/NVU - monocytes - APCs - Ifng+ T cells”, providing a new target for blocking the inflammatory cascade. Our elucidation of the cell-specific molecular changes, cell–cell interactions and transcriptional mechanisms of the retinal NVU provides new insights to support the development of multipurpose drugs to block or even reverse NVU damage.

PEDF Protects Endothelial Barrier Integrity during Acute Myocardial Infarction via 67LR.

Maintaining the integrity and protecting the stability of tight junctions in endothelial cells is a potential therapeutic strategy against myocardial ischaemia. Laminin receptors (67LR) are highly expressed on endothelial cell membranes and are associated with endothelial barrier function. Herein, we sought to demonstrate the direct effects of pigment epithelial-derived factor (PEDF) on tight junctions between endothelial cells via 67LR during acute myocardial infarction (AMI) and elucidate its underlying mechanisms. We detected that PEDF directly increased the level of the tight junction protein zonula occludens protein 1 (ZO-1) after overexpression in vitro and in vivo using Western blotting. Evans Blue/TTC staining showed that PEDF significantly reduced the size of the infarcted myocardium. Immunofluorescence and the transwell cellular experiments suggested that PEDF significantly upregulated PI3K-AKT permeability and the distribution of ZO-1 between endothelial cells under OGD conditions. Interestingly, PEDF significantly upregulated the phosphorylation levels of PI3K-AKT-mTOR under oxygen and glucose deprivation conditions but had no significant effects on the total protein expression. The protective effect of PEDF on ZO-1 was significantly inhibited following the inhibition of PI3K-AKT-mTOR. The activation of phosphorylation of PI3K-AKT-mTOR by PEDF was blocked after silencing 67LR, as were the protective effects of PEDF on ZO-1. Therefore, we have reason to believe that PEDF increased ZO-1 expression through the 67LR-dependent PI3K-AKT-mTOR signaling pathway, thus maintaining tight junction stability and protecting cardiac function.

Neuroprotection by Preconditioning in Mice is Dependent on MyD88-Mediated CXCL10 Expression in Endothelial Cells.

The central nervous system (CNS) can be preconditioned to resist damage by peripheral pretreatment with low-dose gram-negative bacterial endotoxin lipopolysaccharide (LPS). Underlying mechanisms associated with transient protection of the cerebral cortex against traumatic brain injury include increased neuronal production of antiapoptotic and neurotrophic molecules, microglial-mediated displacement of inhibitory presynaptic terminals innervating the soma of cortical projection neurons, and synchronized firing of cortical projection neurons. However, the cell types and signaling responsible for these neuronal and microglial changes are unknown. A fundamental question is whether LPS penetrates the CNS or acts on the luminal surface of brain endothelial cells, thereby triggering an indirect parenchymal neuroprotective response. The present study shows that a low-dose intraperitoneal LPS treatment increases brain endothelial cell activation markers CD54, but does not open the blood-brain barrier or alter brain endothelial cell tight junctions as assessed by electron microscopy. NanoString nCounter transcript analyses of CD31-positive brain endothelial cells further revealed significant upregulation of Cxcl10, C3, Ccl2, Il1β, Cxcl2, and Cxcl1, consistent with identification of myeloid differentiation primary response 88 (MyD88) as a regulator of these transcripts by pathway analysis. Conditional genetic endothelial cell gene ablation approaches demonstrated that both MyD88-dependent Toll-like receptor 4 (TLR4) signaling and Cxcl10 expression are essential for LPS-induced neuroprotection and microglial activation. These results suggest that C-X-C motif chemokine ligand 10 (CXCL10) production by endothelial cells in response to circulating TLR ligands may directly or indirectly signal to CXCR3 on neurons and/or microglia. Targeted activation of brain endothelial receptors may thus provide an attractive approach for inducing transient neuroprotection.

Single-Cell Transcriptomic Profiles of Lung Pre-Metastatic Niche Reveal Neutrophil and Lymphatic Endothelial Cell Roles in Breast Cancer

The establishment of a pre-metastatic niche (PMN) is critical for cancer metastasis. However, it remains unclear as to which phenotypes induce changes in the PMN. Single-cell transcriptomic profiling of all cells of the lung in cancer-bearing MMTV-PyVT mice revealed an increased infiltration of N2-type neutrophils and classical monocytes associated with chronic inflammation; notably, lung neutrophils isolated from mice with primary cancer exhibited similar N2-type phenotypes and expressed high levels of inflammatory and angiogenic factors. We also discovered a new cluster of Ki67-upregulated lymphatic endothelial cells (ECs) that activated several cell division-related pathways. Receptor-ligand interactions within the lung potentially mediated PMN formation; these were exemplified by the cross talk of lymphatic EC-N2-type neutrophil via S100A6. In vitro study revealed S100A6 impaired EC tight junction and increased the transendothelial migration of neutrophils. Our results highlight the molecular mechanisms that shape lung PMN and inspire preventive strategies for lung metastasis in breast cancer.

Role of pericytes in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes of endothelial cells. It is characterized by pericyte deficiency. However, the role of pericytes in CCMs is not yet clarified. We found pericytes in Cdh5Cre ERT2 ;Ccm1 fl/fl (Ccm1 ECKO ) mice had a high expression of PDGFRβ. The inhibition of pericyte function by CP-673451 aggravated the CCM lesion development. RNA-sequencing analysis revealed the molecular traits of pericytes, such as highly expressed ECM-related genes, especially Fn1. Furthermore, KLF4 coupled with phosphorylated SMAD3 (pSMAD3) promoted the transcription of fibronectin in the pericytes of CCM lesions. RGDS peptide, an inhibitor of fibronectin, decreased the lesion area in the cerebella and retinas of Ccm1 ECKO mice. Also, human CCM lesions had abundant fibronectin deposition, and pSMAD3- and KLF4-positive pericytes. These findings indicate that pericytes are essential for CCM lesion development, and fibronectin intervention may provide a novel target for therapeutic intervention in such patients.

Contribution of astrocytic MMP9 toward progression of VCID pathology

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia. High levels of plasma homocysteine or hyperhomocysteinemia has been characterized as a risk factor for VCID however, the mechanism underlying the connection between hyperhomocysteinemia and development of VCID pathology remains elusive. I hypothesize that hyperhomocysteinemia initiates a pro‐inflammatory cascade that increases the activity of MMP9 causing both perivascular astrocytes to dissociate from their vessels and initiating the degradation of endothelial cell tight junction proteins, leading to blood brain barrier dysfunction and the progression toward VCID pathology.MethodFor in vivo studies, C57BL6 WT and MMP9KO mice were placed on a control diet or a diet deficient in folate, vitamins B6 and B12 and enriched in methionine to induce hyperhomocysteinemia for 4, 8, 12, and 16 weeks. For in vitro, experiments, primary astrocytes from WT and MMP9KO mice were treated with a 50μM homocysteine stimulus or an inflammatory stimulus (TNF‐a, IL‐1b and C1q) for 48 hours and the conditioned media was collected. This homocysteine or inflammatory astrocyte conditioned media was then used to treat WT endothelial cells. Immunohistochemistry and gene expression analysis were used to determine neuroinflammatory changes while histology was used to identify changes in astrocytic end‐feet proteins, tight junction proteins, vascular density and microhaemorrhages. Both gel and in situ zymography were used to assess proteinase activity of MMP9 and related gelatinases. Western blots were used to investigate substrates of MMP9 including claudin, occludin and β‐dystroglycan. Behaviour was assessed using rotarod, novel object recognition and spontaneous alternation testing. Transendothelial electrical resistance and sodium fluorescein assays were used to measure the integrity of endothelial cell tight junctions in vitro.ResultStudies are underway to examine changes in cognition, microhaemorrhages, neuroinflammation, astrocyte end‐foot integrity, tight junction protein integrity and activity of MMP9.ConclusionCollectively, our findings suggest that astrocytic MMP9 may play an integral role in the mechanism associating homocysteine induced neuroinflammation with vascular pathogenesis leading to VCID, highlighting this pathway as an important subject for future study.