Endothelial Junctions Research Articles

Expression of Collagen VI, Anticollagenase, Laminin, MM9, Claudins 1 and 5, N and E Cadherins in Choroid Plexus Tumors

Background: CPTs are rare intraventricular papillary neoplasms derived from the choroid plexus epithelium. Anti-collagenase and extracellular matrix which have not been expressed in brain tumors. Objective: The purpose of this study was to investigate the expression levels of collagen type VI, anti-collagenase, laminin, MM9, claudins 1 and 5, N and E cadherins, and collagen VII, tejido, and collagen degradation enzyme complexes in choroid plexus tumors. Materials and methods: We studied the expression of adhesion molecules, extracellular matrix, and anticollagenase with an immunohistochemistry approach and electron microscopy analysis in 42 choroid plexus tumors. Results: 28(67%) were choroid plexus papillomas, 8 (19%) were atypical choroid plexus papillomas and 6 (14%) were choroid plexus carcinomas. The Ki67-li and MVD increased from CPC to ACPP, being the highest in malignant tumors as well as a strong immunoexpression of anti-collagenase and were inverse correlation with claudin 5, E, and N cadherin and collagen IV immunoexpressions which added further significant information to the prognosis and varied according to the histologic classification. By ultrastructure, the loss of basal membrane and cilia, disorganization, and proliferation of ECM were observed in CPC. Cerebral homeostasis largely results from the ability of both the Blood–Brain Barrier (BBB) at the brain microvascular endothelium and the Blood–Cerebrospinal Fluid Barrier (BCSFB) at the epithelium of the Choroid Plexuses (CPs), to control the composition of the CSF and cerebral extracellular fluid. Under expression of the tight junction proteins occludin, claudin-1 and claudin-5 are key molecular abnormalities responsible for the increased permeability of tumor endothelial tight junctions. Conclusion: The loss of basement membrane and ECM overexpression could be considered as a poor prognosis predictor in CPT. Anti-collagenase and MMP9 overexpression could be related to basal membrane and BBB plasticity in CPTs.

ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood–brain barrier integrity after traumatic brain injury

BackgroundTraumatic brain injury (TBI) is a significant worldwide public health concern that necessitates attention. Apoptosis signal-regulating kinase 1 (ASK1), a key player in various central nervous system (CNS) diseases, has garnered interest for its potential neuroprotective effects against ischemic stroke and epilepsy when deleted. Nonetheless, the specific impact of ASK1 on TBI and its underlying mechanisms remain elusive. Notably, mutation of ATP-binding sites, such as lysine residues, can lead to catalytic inactivation of ASK1. To address these knowledge gaps, we generated transgenic mice harboring a site-specific mutant ASK1 Map3k5-e (K716R), enabling us to assess its effects and elucidate potential underlying mechanisms following TBI.MethodsWe employed the CRIPR/Cas9 system to generate a transgenic mouse model carrying the ASK1-K716R mutation, aming to investigate the functional implications of this specific mutant. The controlled cortical impact method was utilized to induce TBI. Expression and distribution of ASK1 were detected through Western blotting and immunofluorescence staining, respectively. The ASK1 kinase activity after TBI was detected by a specific ASK1 kinase activity kit. Cerebral microvessels were isolated by gradient centrifugation using dextran. Immunofluorescence staining was performed to evaluate blood–brain barrier (BBB) damage. BBB ultrastructure was visualized using transmission electron microscopy, while the expression levels of endothelial tight junction proteins and ASK1 signaling pathway proteins was detected by Western blotting. To investigate TBI-induced neuroinflammation, we conducted immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analyses. Additionally, immunofluorescence staining and electrophysiological compound action potentials were conducted to evaluate gray and white matter injury. Finally, sensorimotor function and cognitive function were assessed by a battery of behavioral tests.ResultsThe activity of ASK1-K716R was significantly decreased following TBI. Western blotting confirmed that ASK1-K716R effectively inhibited the phosphorylation of ASK1, JNKs, and p38 in response to TBI. Additionally, ASK1-K716R demonstrated a protective function in maintaining BBB integrity by suppressing ASK1/JNKs activity in endothelial cells, thereby reducing the degradation of tight junction proteins following TBI. Besides, ASK1-K716R effectively suppressed the infiltration of peripheral immune cells into the brain parenchyma, decreased the number of proinflammatory-like microglia/macrophages, increased the number of anti-inflammatory-like microglia/macrophages, and downregulated expression of several proinflammatory factors. Furthermore, ASK1-K716R attenuated white matter injury and improved the nerve conduction function of both myelinated and unmyelinated fibers after TBI. Finally, our findings demonstrated that ASK1-K716R exhibited favorable long-term functional and histological outcomes in the aftermath of TBI.ConclusionASK1-K716R preserves BBB integrity by inhibiting ASK1/JNKs pathway in endothelial cells, consequently reducing the degradation of tight junction proteins. Additionally, it alleviates early neuroinflammation by inhibiting the infiltration of peripheral immune cells into the brain parenchyma and modulating the polarization of microglia/macrophages. These beneficial effects of ASK1-K716R subsequently result in a reduction in white matter injury and promote the long-term recovery of neurological function following TBI.

Transvascular delivery of talaporfin sodium to subcutaneous tumors in mice by nanosecond pulsed laser-induced photomechanical waves

BackgroundWe previously developed a site-specific transvascular drug delivery system (DDS) based on photomechanical waves (PMWs) or laser-induced stress/shock waves (LISWs). In this study, we investigated the validity of this method to deliver a clinical photosensitizer, talaporfin sodium (TS), to subcutaneous tumors in mice and to enhance the efficacy of photodynamic therapy (PDT). MethodsTS solution (2.5 mg/kg) was intravenously injected into mice. Immediately thereafter, PMWs were applied to the tumor by irradiating a laser target with a Q-switched ruby laser pulse (0.8 J/cm2). Five hours after TS administration, some tumors were excised to evaluate the depth distribution of the delivered TS under a fluorescence microscope. Other tumors were subjected to PDT by irradiating the tissues with a 665 nm continuous-wave laser diode (75 mW/cm2, 667 s) at this timepoint. The effects of PDT were evaluated on the basis of the two primary therapeutic mechanisms of TS-mediated PDT: i) damage to tumor cells and ii) damage to endothelial cells of tumor vessels, i.e., the vascular shutdown effect on tumors. ResultsPMW application significantly increased the accumulation of TS in the tumor parenchyma but not in the tumor vessel walls; the endothelial cell junctions of tumor vessels should be the route of TS delivery enhanced by PMWs. Thus, as a result of PMW application followed by PDT, while the vascular shutdown effect on the tumors was not enhanced, direct damage to the tumor cells was increased, resulting in significant tumor growth retardation without body weight loss for 7 days after treatment.

A 3D-printed blood-brain barrier model with tunable topology and cell-matrix interactions

Recent developments in digital light processing (DLP) can advance the structural and biochemical complexity of perfusable in vitro models of the blood–brain barrier. Here, we describe a strategy to functionalize complex, DLP-printed vascular models with multiple peptide motifs in a single hydrogel. Different peptides can be clicked into the walls of distinct topologies, or the peptide motifs lining channel walls can differ from those in the bulk of the hydrogel. The flexibility of this approach is used to both characterize the effects of various bioactive domains on endothelial coverage and tight junction formation, in addition to facilitating astrocyte attachment in the hydrogel surrounding the endothelialized vessel to mimic endothelial–astrocyte interaction. Peptides derived from proteins mediating cell-extracellular matrix (e.g. RGD and IKVAV) and cell–cell (e.g. HAVDI) adhesions are used to mediate endothelial cell attachment and coverage. HAVDI and IKVAV-lined channels exhibit significantly greater endothelialization and increased zonula-occluden-1 (ZO-1) localization to cell–cell junctions of endothelial cells, indicative of tight junction formation. RGD is then used in the bulk hydrogel to create an endothelial–astrocyte co-culture model of the blood–brain barrier that overcomes the limitations of previous platforms incapable of complex topology or tunable bioactive domains. This approach yields an adjustable, biofabricated platform to interrogate the effects of cell-matrix interaction on blood–brain barrier mechanobiology.

Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions

Vascular endothelial cadherin (VE-cadherin) expressed at endothelial adherens junctions (AJs) is vital for vascular integrity and endothelial homeostasis. Here we identify the requirement of the ubiquitin E3-ligase CHFR as a key mechanism of ubiquitylation-dependent degradation of VE-cadherin. CHFR was essential for disrupting the endothelium through control of the VE-cadherin protein expression at AJs. We observe augmented expression of VE-cadherin in endothelial cell (EC)-restricted Chfr knockout (ChfrΔEC) mice. We also observe abrogation of LPS-induced degradation of VE-cadherin in ChfrΔEC mice, suggesting the pathophysiological relevance of CHFR in regulating the endothelial junctional barrier in inflammation. Lung endothelial barrier breakdown, inflammatory neutrophil extravasation, and mortality induced by LPS were all suppressed in ChfrΔEC mice. We find that the transcription factor FoxO1 is a key upstream regulator of CHFR expression. These findings demonstrate the requisite role of the endothelial cell-expressed E3-ligase CHFR in regulating the expression of VE-cadherin, and thereby endothelial junctional barrier integrity.

Endothelial VEGFR2-PLCγ signaling regulates vascular permeability and antitumor immunity through eNOS/Src.

Endothelial phospholipase Cγ (PLCγ) is essential for vascular development; however, its role in healthy, mature, or pathological vessels is unexplored. Here, we show that PLCγ was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLCγ expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLCγ was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2Y1173F/+ mice or mice lacking endothelial PLCγ (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B cells, helper T cells and regulatory T cells, and improved response to chemo- and immunotherapy. Mechanistically, pY1173/PLCγ signaling induced Ca2+/protein kinase C-dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLCγ/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLCγ and eNOS and suppressed vascular leakage. Thus, we believe that we have identified a clinically relevant endothelial PLCγ pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier and results in loss of antitumor immunity.

Early Progression of Abdominal Aortic Aneurysm is Decelerated by Improved Endothelial Barrier Function via ALDH2-LIN28B-ELK3 Signaling.

The involvement of endothelial barrier function in abdominal aortic aneurysm (AAA) and its upstream regulators remains unknown. Single-cell RNA sequencing shows that disrupted endothelial focal junction is an early (3 days) and persistent (28 days) event during Angiotensin II (Ang II)-induced AAA progression. Consistently, mRNA sequencing on human aortic dissection tissues confirmed downregulated expression of endothelial barrier-related genes. Aldehyde dehydrogenase 2 (ALDH2), a negative regulator of AAA, is found to be upregulated in the intimal media of AAA samples, leading to testing its role in early-stage AAA. ALDH2 knockdown/knockout specifically in endothelial cells (ECs) significantly increases expression of EC barrier markers related to focal adhesion and tight junction, restores endothelial barrier integrity, and suppresses early aortic dilation of AAA (7 and 14 days post-Ang II). Mechanically, ELK3acts as an ALDH2 downstream regulator for endothelial barrier function preservation. At the molecular level, ALDH2 directly binds to LIN28B, a regulator of ELK3 mRNA stability, hindering LIN28B binding to ELK3 mRNA, thereby depressing ELK3 expression and impairing endothelial barrier function. Therefore, preserving vascular endothelial barrier integrity via ALDH2-specific knockdown in ECs holds therapeutic potential in the early management of AAAs.

Cortistatin deficiency reveals a dysfunctional brain endothelium with impaired gene pathways, exacerbated immune activation, and disrupted barrier integrity

BackgroundBrain activity governing cognition and behaviour depends on the fine-tuned microenvironment provided by a tightly controlled blood–brain barrier (BBB). Brain endothelium dysfunction is a hallmark of BBB breakdown in most neurodegenerative/neuroinflammatory disorders. Therefore, the identification of new endogenous molecules involved in endothelial cell disruption is essential to better understand BBB dynamics. Cortistatin is a neuroimmune mediator with anti-inflammatory and neuroprotective properties that exerts beneficial effects on the peripheral endothelium. However, its role in the healthy and injured brain endothelium remains to be evaluated. Herein, this study aimed to investigate the potential function of endogenous and therapeutic cortistatin in regulating brain endothelium dysfunction in a neuroinflammatory/neurodegenerative environment.MethodsWild-type and cortistatin-deficient murine brain endothelium and human cells were used for an in vitro barrier model, where a simulated ischemia-like environment was mimicked. Endothelial permeability, junction integrity, and immune response in the presence and absence of cortistatin were evaluated using different size tracers, immunofluorescence labelling, qPCR, and ELISA. Cortistatin molecular mechanisms underlying brain endothelium dynamics were assessed by RNA-sequencing analysis. Cortistatin role in BBB leakage was evaluated in adult mice injected with LPS.ResultsThe endogenous lack of cortistatin predisposes endothelium weakening with increased permeability, tight-junctions breakdown, and dysregulated immune activity. We demonstrated that both damaged and uninjured brain endothelial cells isolated from cortistatin-deficient mice, present a dysregulated and/or deactivated genetic programming. These pathways, related to basic physiology but also crucial for the repair after damage (e.g., extracellular matrix remodelling, angiogenesis, response to oxygen, signalling, and metabolites transport), are dysfunctional and make brain endothelial barrier lacking cortistatin non-responsive to any further injury. Treatment with cortistatin reversed in vitro hyperpermeability, tight-junctions disruption, inflammatory response, and reduced in vivo BBB leakage.ConclusionsThe neuropeptide cortistatin has a key role in the physiology of the cerebral microvasculature and its presence is crucial to develop a canonical balanced response to damage. The reparative effects of cortistatin in the brain endothelium were accompanied by the modulation of the immune function and the rescue of barrier integrity. Cortistatin-based therapies could emerge as a novel pleiotropic strategy to ameliorate neuroinflammatory/neurodegenerative disorders with disrupted BBB.

Telomerase reverse transcriptase restores pancreatic microcirculation profiles and attenuates endothelial dysfunction by inhibiting mitochondrial superoxide production: A potential target for acute pancreatitis therapy

BackgroundAcute pancreatitis (AP) is a potentially lethal disease related to prominent microcirculation dysfunction. Pancreatic microvascular endothelial dysfunction enhances oxidative stress with tissue damage. Increased superoxide production disrupts endothelial junction integrity and increases endothelial permeability. Endothelial mitochondrial ROS (mtROS) represent a major intracellular source of superoxide anions. The non-canonical function of telomerase reverse transcriptase (TERT) involves the maintenance of cellular redox homeostasis in somatic tissues. MethodsWe investigated whether TERT restores microcirculation dysfunction and attenuates the endothelium injury by inhibiting superoxide production during AP progression. We established TERT transgenic and TERT knock-down mice and used cerulein (CER) and lipopolysaccharide (LPS) injections to induce AP models. In addition, we exposed HUVECs to LPS following TERT overexpression or silencing to explore the role of TERT in endothelial dysfunction. We also performed flow cytometry and confocal microscopy assays by using HUVECs. And a mtROS inhibitor, MitoTempo, was used to scavenge mitochondria superoxide and alkyl. ResultsTERT transgenic mice were found to have restored pancreatic microcirculation profiles and microvascular endothelial morphology compared with wild-type mice under cerulein injection. In contrast, TERT silencing displayed the opposite effect in response to cerulein. Subsequently, we showed that TERT overexpression attenuates mtROS production and mitochondrial dysfunction during LPS-stimulated endothelial dysfunction. Furthermore, we found that TERT overexpression maintains the balance between mitochondrial contents and ATP level during endothelial dysfunction. In addition, the protective trend of MitoTempo is impeded after TERT silencing. ConclusionTERT restores pancreatic microcirculation dysfunction and attenuates microvascular endothelium lesions by inhibiting the increase of superoxide production and mitochondrial dysfunction.

Skeletal muscle-derived FSTL1 starting up angiogenesis by regulating endothelial junction via activating Src pathway can be upregulated by hydrogen sulfide.

Hydrogen sulfide (H2S) promotes microangiogenesis and revascularization after ischemia. Neovascularization starts with the destruction of intercellular junctions and is accompanied by various endothelial cell angiogenic behaviors. Follistatin-like 1 (FSTL1) is a cardiovascular-protective myokine that works against ischemic injury. The present study examined whether FSTL1 was involved in H2S-induced angiogenesis and explored the underlying molecular mechanism. We observed that H2S accelerated blood perfusion after ischemia in the mouse hindlimb ischemia model. Western blot analysis showed that H2S stabilized FSTL1 transcript and increased FSTL1 and Human antigen R (HuR) levels in skeletal muscle. RNA-interference HuR significantly inhibited the H2S-promoted increase in FSTL1 levels. Exogenous FSTL1 promoted the wound-healing migration of human umbilical vein endothelial cells (HUVECs) and increased monolayer endothelial barrier permeability. Immunostaining showed that FSTL1 increased interendothelial gap formation and decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression. In addition, FSTL1 significantly increased the phosphorylation of Src and VEGFR2. However, the Src inhibitor, not the VEGFR2 inhibitor, could block FSTL1-induced effects in angiogenesis. In conclusion, we demonstrated that H2S could upregulate the expression of FSTL1 by increasing the HuR levels in skeletal muscle, and paracrine FSTL1 could initiate angiogenesis by opening intercellular junctions via the Src signaling pathway.NEW & NOTEWORTHY The myocyte-derived paracrine protein FSTL1 acts on vascular endothelial cells and initiates the process of angiogenesis by opening the intercellular junction via activating Src kinase. H2S can significantly upregulate FSTL1 protein levels in skeletal muscles by increasing HuR expression.

Asiaticoside Attenuates Blood-Spinal Cord Barrier Disruption by Inhibiting Endoplasmic Reticulum Stress in Pericytes After Spinal Cord Injury.

The blood-spinal cord barrier (BSCB) plays a vital role in the recovery of spinal cord function after spinal cord injury (SCI). Pericytes, pluripotent members of the neurovascular unit (NVU), receive signals from neighboring cells and are critical for maintaining CNS function. Therapeutic targets for the BSCB include endothelial cells (ECs) and glial cells, but few drugs target pericytes. This study was designed to explore whether asiaticoside has a positively effect on pericytes and the integrity of the BSCB. In this study, we found that asiaticoside could inhibit the loss of junction proteins just 1 day after SCI in vivo, but our in vitro study showed no significant differences in the expression of endothelial junction proteins between the control and asiaticoside treatment groups. We also found that asiaticoside could inhibit endoplasmic reticulum (ER) stress and pericyte apoptosis, which might be associated with the inhibition of junction protein reduction in ECs. Thus, we investigated the interactions between pericytes and ECs. Our results showed that asiaticoside could decrease the release of matrix metalloproteinase (MMP)-9 in pericytes and therefore upregulate the expression of junction proteins in ECs. Furthermore, the protective effect of asiaticoside on pericytes is related to the inhibition of ER stress via the MAPK signaling pathway. Taken together, our results demonstrate that asiaticoside treatment inhibits BSCB disruption and enhances functional recovery after SCI.

Virus-like Silica Nanoparticles Improve Permeability of Macromolecules across the Blood-Brain Barrier In Vitro.

The presence of the blood-brain barrier (BBB) limits the delivery of therapies into the brain. There has been significant interest in overcoming the BBB for the effective delivery of therapies to the brain. Inorganic nanomaterials, especially silica nanoparticles with varying surface chemistry and surface topology, have been recently used as permeation enhancers for oral protein delivery. In this context, nanoparticles with varying sizes and surface chemistries have been employed to overcome this barrier; however, there is no report examining the effect of nanoscale roughness on BBB permeability. This paper reports the influence of nanoscale surface roughness on the integrity and permeability of the BBB in vitro, using smooth surface Stöber silica nanoparticles (60 nm) compared to rough surface virus-like silica nanoparticles (VSNP, 60 nm). Our findings reveal that VSNP (1 mg/mL) with virus-mimicking-topology spiky surface have a greater effect on transiently opening endothelial tight junctions of the BBB than the same dose of Stöber silica nanoparticles (1 mg/mL) by increasing the FITC-Dextran (70 kDa) permeability 1.9-fold and by decreasing the trans-endothelial electrical resistance (TEER) by 2.7-fold. This proof-of-concept research paves the way for future studies to develop next-generation tailored surface-modified silica nanoparticles, enabling safe and efficient macromolecule transport across the BBB.

Early-life noise exposure causes cognitive impairment in a sex-dependent manner by disrupting homeostasis of the microbiota–gut–brain axis

Epidemiological investigations show that noise exposure in early life is associated with health and cognitive impairment. The gut microbiome established in early life plays a crucial role in modulating developmental processes that subsequently affect brain function and behavior. Here, we examined the impact of early-life exposure to noise on cognitive function in adolescent rats by analyzing the gut microbiome and metabolome to elucidate the underlying mechanisms. Chronic noise exposure during early life led to cognitive deficits, hippocampal injury, and neuroinflammation. Early-life noise exposure showed significant difference on the composition and function of the gut microbiome throughout adolescence, subsequently causing axis-series changes in fecal short-chain fatty acid (SCFA) metabolism and serum metabolome profiles, as well as dysregulation of endothelial tight junction proteins, in both intestine and brain. We also observed sex-dependent effects of microbiota depletion on SCFA-related beneficial bacteria in adolescence. Experiments on microbiota transplantation and SCFA supplementation further confirmed the role of intestinal bacteria and related SCFAs in early-life noise-exposure-induced impairments in cognition, epithelial integrity, and neuroinflammation. Overall, these results highlight the homeostatic imbalance of microbiota–gut–brain axis as an important physiological response toward environmental noise during early life and reveals subtle differences in molecular signaling processes between male and female rats.

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.

Metal-Free Peroxidase-Mimetic Nanocatalysts for Chemodynamic Vascular-Disrupting Cancer Therapy.

Metal ion-facilitated chemodynamic therapy (CDT) is an emerging method for treating cancer. However, its potential is hindered by its low catalytic performance in weakly acidic tumor microenvironments (TMEs) and the severe toxicity of free metal ions. We introduce a new approach to tumor therapy, chemodynamic vascular disruption (CVD), using metal-free, peroxidase (POD)-mimetic multihydroxylated [70] fullerene (MHF) nanocatalysts. Our research shows that MHF contains C···O active sites, as demonstrated by density functional theory (DFT) calculations, and converts H2 O2 into •OH across a pH range of 6.0-10.0. The generation of •OH and the dismantling of tumor blood vessels are observed in real-time using mouse dorsal skin-fold chamber (DSFC) models. Applying proteomics, we discover that the CVD mechanism involves the nanocatalytic MHF enhancing H2 O2 decomposition in the TME, producing •OH. This damages tumor vascular endothelial junction proteins, causing vascular leakage and subsequently cutting off the vascular supply to the tumor cells. This method deviates from the traditional CDT that targets tumor cells. Instead, our proficient MHF nanocatalysts aim to directly disrupt the tumor vasculature, enhancing anti-tumor efficiency without triggering harmful toxicity. The proposed CVD therapeutic strategy enhances the application of gentle carbon nanocatalysts in cancer therapy, offering new perspectives on nanocatalytic medicine. This article is protected by copyright. All rights reserved.

Molecular Mechanisms of Endothelialitis in SARS-CoV-2 Infection: Evidence for VE-Cadherin Cleavage by ACE2.

Long COVID-19 syndrome appears after Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection with acute damage to microcapillaries, microthrombi, and endothelialitis. However, the mechanisms involved in these processes remain to be elucidated. All blood vessels are lined with a monolayer of endothelial cells called vascular endothelium, which provides a the major function is to prevent coagulation. A component of endothelial cell junctions is VE-cadherin, which is responsible for maintaining the integrity of the vessels through homophilic interactions of its Ca++-dependent adhesive extracellular domain. Here we provide the first evidence that VE-cadherin is a target in vitro for ACE2 cleavage because its extracellular domain (hrVE-ED) contains two amino acid sequences for ACE2 substrate recognition at the positions 256P-F257 and 321PMKP-325L. Indeed, incubation of hrVE-ED with the active ectopeptidase hrACE2 for 16 hrs in the presence of 10 μM ZnCl2 showed a dose-dependent (from 0.2 ng/μL to 2 ng/μL) decrease of the VE-cadherin immunoreactive band. In vivo, in the blood from patients having severe COVID-19 we detected a circulating form of ACE2 with an apparent molecular mass of 70 kDa, which was barely detectable in patients with mild COVID-19. Of importance, in the patients with severe COVID-19 disease, the presence of three soluble fragments of VE-cadherin (70, 62, 54 kDa) were detected using the antiEC1 antibody while only the 54 kDa fragment was present in patients with mild disease. Altogether, these data clearly support a role for ACE2 to cleave VE-cadherin, which leads to potential biomarkers of SARS-CoV-2 infection related with the vascular disease in "Long COVID-19".

Structure of the Blood Brain Barrier and its Role in the Transporters for the Movement of Substrates across the Barriers.

For the brain to operate normally and to develop with structural integrity in addition to neuronal function, blood-brain barrier present in brain capillaries serves as a vital barrier mechanism. In addition to the transport barrier created by membranes, transporters, and vesicular processes, the structure and function of the BBB are summarised. The physical barrier is created by endothelial tight junctions. The permeability and transport of molecules between extracellular fluid and plasma are constrained by the presence of tight junctions between neighbouring endothelial cells. Each solute must pass through both membranes in the luminal and abluminal divisions. The functions of the neurovascular unit are described, with special emphasis on the pericytes, microglia, and astrocyte endfeet. The luminal membrane contains five separate facilitative transport mechanisms, each of which is exclusive to a few substrates. Nevertheless, the import of big-branched and aromatic neutral amino acids is facilitated by two key carriers (System L and y+) in the plasma membrane. It is asymmetrically present in both membranes. The sodium pump Na+/K+-ATPase is highly expressed in the abluminal membrane, where many Na+ dependent transport mechanisms move amino acids against its concentration gradient. The trojan horse strategy, which uses molecular tools to bind the medication and its formulations, is also preferred in drug delivery. The BBB's cellular structure, the transport systems unique to each substrate, and the necessity to identify transporters with changes that assist the transfer of various medications have all been changed in the current work. Nevertheless, to rule out the BBB passage for the new class of neuroactive medications, the mixing of traditional pharmacology and nanotechnology needs to be focused on outcomes that show promise.

Noninvasive disruption of the blood-brain barrier in the marmoset monkey

The common marmoset monkey (Callithrix jacchus) is a species of rising prominence in the neurosciences due to its small size, ease of handling, fast breeding, and its shared functional and structural brain characteristics with Old World primates. With increasing attention on modeling human brain diseases in marmosets, understanding how to deliver therapeutic or neurotropic agents to the marmoset brain noninvasively is of great preclinical importance. In other species, including humans, transcranial focused ultrasound (tFUS) aided by intravenously injected microbubbles has proven to be a transient, reliable, and safe method for disrupting the blood-brain barrier (BBB), allowing the focal passage of therapeutic agents that do not otherwise readily traverse the tight endothelial junctions of the BBB. The critical gap that we address here is to document parameters to disrupt the BBB reliably and safely in marmosets using tFUS. By integrating our marmoset brain atlases and the use of a marmoset-specific stereotactic targeting system, we conduct a series of systematic transcranial sonication experiments in nine marmosets. We demonstrate the effects of center frequency, acoustic pressure, burst period, and duration, establish a minimum microbubble dose, estimate microbubble clearance time, and estimate the duration that the BBB remains open to passage. Successful BBB disruption is reported in vivo with MRI-based contrast agents, as well as Evans blue staining assessed ex vivo. Histology (Hematoxylin and Eosin staining) and immunohistochemistry indicate that the BBB can be safely and reliably opened with the parameters derived from these experiments. The series of experiments presented here establish methods for safely, reproducibly, and focally perturbing the BBB using tFUS in the common marmoset monkey that can serve as a basis for noninvasive delivery of therapeutic or neurotropic agents.