Cerebrovascular Endothelial Cells Research Articles

The necessity of eliminating the interference of panaxatriol saponins to maximize the preventive effect of panaxadiol saponins against Parkinson's disease in rats

BackgroundThe effects of individual panaxadiol saponin and panaxatriol saponin on rodent models of Parkinson's disease (PD) have been recognized. However, it is not clear whether purified total ginsenosides as an entirety has effect against PD in rat model. This study compared the protective effects of a purified panaxadiol saponin fraction (PDSF), a purified panaxatriol saponin fraction (PTSF), and their mixtures against the rotenone (ROT)-induced PD in rats. MethodsPotential effects of PDSF, PTSF, and their mixtures against motor dysfunction and impairments of nigrostriatal dopaminergic neurons (DN), blood-brain barrier (BBB), cerebrovascular endothelial cells (CEC), and glial cells were measured in the models of ROT-induced PD rats and cell damage. Pro-inflammatory NF-kB p65 (p65) activation was localized in DN and other cells in the striatum. ResultsPDSF and PTSF had a dose-dependent effect against motor dysfunction with a larger effective dose range for PDSF. PDSF protected CEC, glial cells, and DN in models of PD rats and cell damage, while PTSF had no such protections. Chronic ROT exposure potently activated p65 in CEC with enhanced pro-inflammatory and decreased anti-inflammatory factors and impaired BBB in the striatum, PDSF almost completely blocked the ROT-induced p65 activation and maintained both anti- and pro-inflammatory factors at normal levels and BBB integrity, but PTSF aggravated the p65 activation with impaired BBB. Furthermore, PTSF nullified all the effects of PDSF when they were co-administrated. ConclusionPDSF had significant protective effect against the ROT-induced PD in rats by protecting CEC, glial cells, and DN, likely through inhibiting NF-κB p65 in CEC from triggering neuroinflammation, and also directly protecting glial cells and neurons against ROT-induced toxicity. PDSF has great potential for preventing and treating PD.

Gene Expression Profile of the Mitochondrial Calcium Uniporter and Antioxidant Enzymes throughout the Brain During Alzheimer’s Disease

In 2023, there were an estimated ~6.7 million Americans ≥65 years old living with Alzheimer’s disease (AD), a prominent form of dementia. Development of AD has been associated with impaired blood flow to and throughout the brain. In turn, there is diminished delivery of oxygen and nutrients in tandem with reduced clearance of neurotoxic cellular byproducts, notably reactive oxygen species (ROS; e.g., superoxide, peroxide/hydroxyl radicals). As a primary source of cerebrovascular ROS, endothelial mitochondria become progressively dysfunctional in producing ATP relative to ROS in response to aging and cell senescence. A primary mechanism is described by enhanced Ca2+ sequestration into the mitochondrial matrix through the mitochondrial Ca2+ uniporter (MCU) complex ( Mcu, Mcub, Mcur1, Micu1, Micu2, Micu3) and a resulting overproduction of ROS up to conditions of cell death. The primary antioxidants that eliminate ROS are superoxide dismutase ( Sod2), catalase ( Cat), and glutathione peroxidase ( Gpx1). We tested the hypothesis that upregulation of the MCU complex occurs in tandem with decreasing expression of antioxidant enzymes throughout the brain during progression of AD pathology. To investigate this hypothesis, we employed a triple mutation mouse model of AD ( 3xTg-AD) comprising young control (YC; 1-2 mo), mild cognitive impairment (MCI; 4-5 mo), presence of extracellular amyloid-β plaques (Aβ; 6-8 mo), and extracellular amyloid-β plaques with neurofibrillary tangles composed of tau (AβT; ≥12 mo) [n ≥3 per group, males & females]. Quantitative polymerase chain reaction (qPCR), Nanostring, and/or RNA-Seq have been performed on cortex, hippocampus, cerebral vessels, cerebrovascular endothelial cells (ECs), and peripheral blood. Primary highlights of our findings for each of the AD study groups are described below. With detectable presence of all target genes throughout tissue compartments, the YC group is particularly characterized by a higher MCU complex and antioxidant expressions in cortex relative to hippocampus. During the MCI phase, the MCU complex and Cat are upregulated in ECs relative to the YC group. Upon onset of AD (Aβ stage), there is a downregulation and upregulation of MCU complex in the brain and peripheral blood respectively. Further, Gpx1 expression is upregulated in ECs in the Aβ group. Relative to YC, the MCU complex and antioxidants are downregulated in cortex but generally stable in hippocampus during AβT conditions with exception of upregulation in peroxide scavenger mRNAs Cat and Gpx1. In contrast for ECs, the MCU complex increases, while Cat and Gpx1 decrease, in the AβT group relative to YC. Altogether, these findings support an intricate expression profile of the MCU complex and key antioxidant enzymes across brain tissue and cell compartments preceding and accompanying AD pathology. A remarkable observation is that expression profiles of cerebrovascular vessels and blood indicate MCI and early onset of AD, whereas those of the cortex and hippocampus best accompany late-stage AD pathology. This research was supported by the National Institutes of Health (R01AG073230). 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.

Influence of Cholesterol Dysregulation and Cell Senescence on Hippocampal Neurovascular Integrity in Alzheimer’s Disease

The neurovascular unit (NVU) is an essential construct of cerebral blood flow control and contains neurons, astrocytes, microglia, smooth muscle, pericytes, and endothelial cells (ECs). This cellular ensemble, among other roles, balances cerebrovascular blood flow control with the metabolic demand of active neurons. Recent studies underscore the pivotal role of the NVU in Alzheimer’s disease (AD), particularly with molecular, cellular, and integrative evidence that vascular dysfunction precedes amyloid-beta (Aβ) plaque deposition. Cerebrovascular ECs are particularly vulnerable in the aging brain while central to regulation of the blood-brain barrier, hemodynamic flow, angiogenesis, and immunity. As a common characteristic of an array of cardiovascular and neurodegenerative diseases, interactions among dyslipidemia and EC health may establish cerebrovascular injury that precedes and accompanies AD. Furthermore, aberrant vascularization in the hippocampus coincides with development of mild cognitive impairment (MCI) and AD due to compromised oxygen and energy provisions in tandem with accumulation of metabolic waste and toxins. Thus, we hypothesize that interaction among cholesterol dysregulation and EC senescence signaling in the hippocampus contribute to progressive AD pathology. To test this hypothesis, we examine transgenic AD mice ( 3xTg-AD) demonstrating MCI (4-5 mo), presence of extracellular Aβ plaques (6-8 mo), and extracellular Aβ plaques with neurofibrillary tau tangles (≥12 mo) compared to young controls (1-2 mo). Our investigation employs an innovative tissue clearing method (iDISCO+), adapted for imaging on a confocal microscope, to probe three-dimensional vascular structure (CD31, Podocalyxin) and senescent cell aggregation (p16, β-Galactosidase) in the hippocampus. Second, Filipin III, selective blood vessel, and senescence markers are imaged in 25 μm coronal sections to examine broader spatial distribution of cholesterol around the hippocampus and cortex. Third, ex vivo blood vessel staining enables an in-depth analysis among membrane cholesterol inclusions and endothelial inward-rectifying K+ (KIR2.1) channels, an ion channel marker particularly sensitive to the distribution of membrane cholesterol while integral to cerebral blood flow control. As cholesterol dysregulation poses a significant risk to the integrity of the NVU including EC senescence and diminishment of KIR2.1 channel function, we anticipate that our results will ultimately highlight its potential role in the early to late stages of AD pathogenesis. This work was supported by the National Institutes of Health (R01AG073230). Imaging was performed in the LLUSM Advanced Imaging and Microscopy Core that is supported by NSF Grant No. MRI-DBI 0923559 and the Loma Linda University School of Medicine. 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.

Generation of Slco1a4-CreERT2-tdTomato Knock-in Mice for Specific Cerebrovascular Endothelial Cell Targeting.

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood-brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells.

Two Signaling Modes Are Better than One: Flux-Independent Signaling by Ionotropic Glutamate Receptors Is Coming of Age.

Glutamate is the major excitatory neurotransmitter in the central nervous system. Glutamatergic transmission can be mediated by ionotropic glutamate receptors (iGluRs), which mediate rapid synaptic depolarization that can be associated with Ca2+ entry and activity-dependent change in the strength of synaptic transmission, as well as by metabotropic glutamate receptors (mGluRs), which mediate slower postsynaptic responses through the recruitment of second messenger systems. A wealth of evidence reported over the last three decades has shown that this dogmatic subdivision between iGluRs and mGluRs may not reflect the actual physiological signaling mode of the iGluRs, i.e., α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA) receptors (AMPAR), kainate receptors (KARs), and N-methyl-D-aspartate (NMDA) receptors (NMDARs). Herein, we review the evidence available supporting the notion that the canonical iGluRs can recruit flux-independent signaling pathways not only in neurons, but also in brain astrocytes and cerebrovascular endothelial cells. Understanding the signaling versatility of iGluRs can exert a profound impact on our understanding of glutamatergic synapses. Furthermore, it may shed light on novel neuroprotective strategies against brain disorders.

X-irradiated umbilical cord blood cells retain their regenerative effect in experimental stroke

Although regenerative therapy with stem cells is believed to be affected by their proliferation and differentiation potential, there is insufficient evidence regarding the molecular and cellular mechanisms underlying this regenerative effect. We recently found that gap junction-mediated cell–cell transfer of small metabolites occurred very rapidly after stem cell treatment in a mouse model of experimental stroke. This study aimed to investigate whether the tissue repair ability of umbilical cord blood cells is affected by X-irradiation at 15 Gy or more, which suppresses their proliferative ability. In this study, X-irradiated mononuclear (XR) cells were prepared from umbilical cord blood. Even though hematopoietic stem/progenitor cell activity was diminished in the XR cells, the regenerative activity was surprisingly conserved and promoted recovery from experimental stroke in mice. Thus, our study provides evidence regarding the possible therapeutic mechanism by which damaged cerebrovascular endothelial cells or perivascular astrocytes may be rescued by low-molecular-weight metabolites supplied by injected XR cells in 10 min as energy sources, resulting in improved blood flow and neurogenesis in the infarction area. Thus, XR cells may exert their tissue repair capabilities by triggering neo-neuro-angiogenesis, rather than via cell-autonomous effects.

Plasma-derived exosomes contributes to endothelial-to-mesenchymal transition in Moyamoya disease

BackgroundMoyamoya disease (MMD) is a cerebrovascular disease with a high disability rate; however, its pathogenesis remains unknown. Endothelial-mesenchymal transition (EndMT) is the pathological basis of many vascular diseases; however, the key role of EndMT in MMD has not yet been reported. MethodWe collected vascular tissues from three control samples and six patients with MMD to detect the expression of EndMT-related genes. To elucidate the mechanism of EndMT in MMD, we performed in vitro cell experiments. Plasma-derived exosomes (PDEs) can transmit information between cells and tissues and are of considerable importance in several disease studies. PDEs were used to stimulate EndMT phenotype in cerebrovascular endothelial cells. ResultsMultiplex fluorescent immunohistochemistry staining confirmed that CD31, VE-cadherin and E-cadherin down-regulated, whereas α-SMA and vimentin were significantly up-regulated in moyamoya vascular endothelial cells than in control samples. PDEs from MMD patients significantly promoted cell proliferation and migration, resulting in slender cells. PDEs induce EndMT-related phenotype changes in cerebral vascular endothelial cells, including decreased endothelial cell marker expression and increased mesenchymal cell marker expression. We demonstrated that EndMT phenotypic alterations are mediated, in part, by microRNA(miRNAs). ConclusionThis study was the first to propose that EndMT may exist in the vessels of patients with MMD. PDEs induce the EndMT phenotype to promote the development of MMD. This study aimed to provide a new theoretical basis for elucidating the pathogenesis of MMD.

Abstract WMP23: Tobacco Cigarette Smoking Causes Cognitive Impairment Through Cerebrovascular Oxidative Damage

Introduction: Tobacco cigarette smoking (TCS) has been established as a risk factor for cardiovascular disease. Recent epidemiological studies have identified an association between TCS and cognitive impairment. However, the underlying mechanisms are yet to be established. We hypothesize that chronic TCS induces systemic and cerebrovascular oxidative stress that leads to cognitive impairment. Methods: Male C57BL6 mice were exposed to cigarette smoke or air (two hours per day, five days per week) for up to 60 weeks, n =10/group. The vascular reactivity of the carotid artery (CA) was assessed using a wire myograph. The CA blood flow was measured by Doppler ultrasonography. Dihydroethidium and diaminofluorescein were used to measure superoxide and nitric oxide. Endothelial nitric oxide synthase (eNOS), tight junction proteins (occludin and caudine-5), β-amyloid, and phosphorylated Tau were measured by immunoblotting and immunofluorescence microscopy. We assessed the cognitive function using Barnes Maze and fear conditioning testing. Results: Vascular endothelial dysfunction occurred in the CA after 16 weeks of TCS exposure, followed by a 23% reduction in CA blood flow (P=0.0006). Superoxide was significantly elevated in the CA and subsequently in the brain of TCS-exposed mice by 181% (P<0.0001), compared to air-exposed mice. Reduction in eNOS and tight junction proteins were observed in the cerebrovascular endothelial cells. At 60 weeks of exposure, the brain of TCS-exposed mice showed higher levels of β-amyloid and phosphorylated-tau (352% and 260%, P=0.0032 and P<0.0001, respectively), compared to air-exposed mice. At this time, TCS-exposed mice exhibited impaired spatial learning and memory. Conclusions: TCS causes progressive systemic and cerebrovascular oxidative damage, leading to cognitive impairment. Targeting vascular oxidative stress is a potential strategy to mitigate cognitive function impairment.

Biomimetic nanodecoys deliver cholesterol-modified heteroduplex oligonucleotide to target dopaminergic neurons for the treatment of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of α-synuclein (α-syn) aggregates called Lewy bodies leading to the gradual loss of dopaminergic (DA) neurons in the substantia nigra. Although α-syn expression can be attenuated by antisense oligonucleotides (ASOs) and heteroduplex oligonucleotide (HDO) by intracerebroventricular (ICV) injection, the challenge to peripheral targeted delivery of oligonucleotide safely and effectively into DA neurons remains unresolved. Here, we designed a new DNA/DNA double-stranded (complementary DNA, coDNA) molecule with cholesterol conjugation (Chol-HDO (coDNA)) based on an α-syn-ASO sequence and evaluated its silence efficiency. Further, Chol-HDO@LMNPs, Chol-HDO-loaded, cerebrovascular endothelial cell membrane with DSPE-PEG2000-levodopa modification (L-DOPA-CECm)-coated nanoparticles (NPs), were developed for the targeted treatment of PD by tail intravenous injection. CECm facilitated the blood-brain barrier (BBB) penetration of NPs, together with cholesterol escaped from reticuloendothelial system uptake, as well as L-DOPA was decarboxylated into dopamine which promoted the NPs toward the PD site for DA neuron regeneration. The behavioral tests demonstrated that the nanodecoys improved the efficacy of HDO on PD mice. These findings provide insights into the development of biomimetic nanodecoys loading HDO for precise therapy of PD. Statement of significanceThe accumulation of α-synuclein (α-syn) aggregates is a hallmark of PD. Our previous study designed a specific antisense oligonucleotide (ASO) targeting human SNCA, but the traumatic intracerebroventricular (ICV) is not conducive to clinical application. Here, we further optimize the ASO by creating a DNA/DNA double-stranded molecule with cholesterol-conjugated, named Chol-HDO (coDNA), and develop a DA-targeted biomimetic nanodecoy Chol-HDO@LMNPs by engineering cerebrovascular endothelial cells membranes (CECm) with DSPE-PEG2000 and L-DOPA. The in vivo results demonstrated that tail vein injection of Chol-HDO@LMNPs could target DA neurons in the brain and ameliorate motor deficits in a PD mouse model. This investigation provides a promising peripheral delivery platform of L-DOPA-CECm nanodecoy loaded with a new Chol-HDO (coDNA) targeting DA neurons in PD therapy.

Catalpol attenuates ischemic stroke by promoting neurogenesis and angiogenesis via the SDF–1α/CXCR4 pathway

BackgroundStroke is a leading cause of disability and death worldwide. Currently, there is a lack of clinically effective treatments for the brain damage following ischemic stroke. Catalpol is a bioactive compound derived from the traditional Chinese medicine Rehmannia glutinosa and shown to be protective in various neurological diseases. However, the potential roles of catalpol against ischemic stroke are still not completely clear. PurposeThis study aimed to further elucidate the protective effects of catalpol against ischemic stroke. MethodsA rat permanent middle cerebral artery occlusion (pMCAO) and oxygen–glucose deprivation (OGD) model was established to assess the effect of catalpol in vivo and in vitro, respectively. Behavioral tests were used to examine the effects of catalpol on neurological function of ischemic rats. Immunostaining was performed to evaluate the proliferation, migration and differentiation of neural stem cells (NSCs) as well as the angiogenesis in each group. The protein level of related molecules was detected by western–blot. The effects of catalpol on cultured NSCs as well as brain microvascular endothelial cells (BMECs) subjected to OGD in vitro were also examined by similar methods. ResultsCatalpol attenuated the neurological deficits and improved neurological function of ischemic rats. It stimulated the proliferation of NSCs in the subventricular zone (SVZ), promoted their migration to the ischemic cortex and differentiation into neurons or glial cells. At the same time, catalpol increased the cerebral vessels density and the number of proliferating cerebrovascular endothelial cells in the infracted cortex of ischemic rats. The level of SDF–1α and CXCR4 in the ischemic cortex was found to be enhanced by catalpol treatment. Catalpol was also shown to promote the proliferation and migration of cultured NSCs as well as the proliferation of BMECs subjected to OGD insult in vitro. Interestingly, the impact of catalpol on cultured cells was inhibited by CXCR4 inhibitor AMD3100. Moreover, the culture medium of BMECs containing catalpol promoted the proliferation of NSCs, which was also suppressed by AMD3100. ConclusionOur data demonstrate that catalpol exerts neuroprotective effects by promoting neurogenesis and angiogenesis via the SDF–1α/CXCR4 pathway, suggesting the therapeutic potential of catalpol in treating cerebral ischemia.

Inhibition of mitochondrial carbonic anhydrases prevents Amyloid β‐induced cell death and blood brain barrier permeability in cerebrovascular endothelial cells

AbstractBackgroundThe Neurovascular Unit (NVU) is an important multicellular structure of the central nervous system (CNS) which has been observed to become dysfunctional in Alzheimer Disease (AD) and cerebral amyloid angiopathy (CAA). Amyloidosis disrupts the function of cerebrovascular endothelial cells (ECs) through the activation of the intrinsic apoptotic pathway, and the induction of blood brain barrier (BBB) permeability. Previous findings in our lab demonstrate that Acetazolamide (ATZ) and methazolamide (MTZ), pan‐Carbonic Anhydrase (CA) inhibitors, prevent mitochondria mediated apoptosis in cerebrovascular ECs through the reduction of Aβ induced cytochrome C release from the mitochondria, caspase‐9 activation, and mitochondrial H2O2 production. Due to the reduction of mitochondria mediated cell death via pan‐CA inhibitors we hypothesize that the mitochondrial isoforms, CA‐VA/B, are involved in cell death and other deleterious mechanisms induced by Aβ. Thus, we anticipate using selective CA‐V inhibitors will reduce cerebrovascular EC apoptosis and prevent the loss of BBB resistance.MethodHuman cerebral microvascular ECs (hCMECs) were treated with Aβ40‐Q22 Dutch mutant alone and in combination with a CA‐V inhibitor. We measured outcomes of mitochondria mediated apoptosis and BBB integrity. Apoptosis was evaluated by measuring the changes in the protein expression of caspase‐9, BAX, and BCL‐2 with western blot. We used live‐cell imaging to measure active caspase‐3. The release of cytochrome C and the mitochondrial membrane potential was detected using immunocytochemistry (ICC). Mitochondria H2O2 production was measured by isolating mitochondria and using a commercially available kit, Amplex Red (Invitrogen). We assessed the permeability of hCMECs using the ECIS ZΘ technology. The changes in protein expression of EC activation markers, VCAM‐1 and ICAM‐1, were determined with western blot.ResultInhibition of CA‐VA/B prevented Aβ40‐Q22 induced DNA fragmentation, caspase‐9 and ‐3 activation, cytochrome C release from the mitochondria, loss of mitochondria membrane potential, and mitochondrial H2O2 production in human cerebrovascular ECs. The inhibition of CA‐VA/B also reduced Aβ40‐Q22 induced BBB permeability and increase in vascular adhesion protein VCAM‐1 in hCMECs.ConclusionThe inhibition of CA‐V is protective to cerebrovascular ECs in models of amyloidosis, through the prevention of mitochondria‐mediated cell death and loss of cerebral EC barrier function.

GPR124 induces NLRP3 inflammasome-mediated pyroptosis in endothelial cells during ischemic injury

ObjectiveG protein-coupled receptor 124 (GPR124) regulates central nervous system angiogenesis and blood-brain barrier (BBB) integrity, and its deficiency aggravates BBB breakdown and hemorrhagic transformation in ischemic mice. However, excessive GPR124 expression promotes inflammation in atherosclerotic mice. In this study, we aimed to elucidate the role of GPR124 in hypoxia/ischemia-induced cerebrovascular endothelial cell injury. MethodsbEnd.3 cells were exposed to oxygen-glucose deprivation (OGD), and time-dependent changes in GPR124 mRNA and protein expression were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The effects of GPR124 overexpression or knockdown on the expression of pyroptosis-related genes were assessed at the mRNA and protein levels. Tadehaginoside (TA) was screened as a potential small molecule targeting GPR124, and its effects on pyroptosis-related signaling pathways were investigated. Finally, the therapeutic efficacy of TA was evaluated using a rat model of transient middle cerebral artery occlusion/reperfusion (tMCAO/R). ResultsDuring OGD, the expression of GPR124 initially increased and then decreased over time, with the highest levels observed 1 h after OGD. The overexpression of GPR124 enhanced the OGD-induced expression of NLRP3, Caspase-1, and Gasdermin D (GSDMD) in bEnd.3 cells, whereas GPR124 knockdown reduced pyroptosis. Additionally, TA exhibited a high targeting ability to GPR124, significantly inhibiting its function and expression and suppressing the expression of pyroptosis-related proteins during OGD. Furthermore, TA treatment significantly reduced the cerebral infarct volume and pyroptotic signaling in tMCAO/R rats. ConclusionsOur findings suggest that GPR124 mediates pyroptotic signaling in endothelial cells during the early stages of hypoxia/ischemia, thereby exacerbating ischemic injury.

Navigating the Blood-Brain Barrier: An Anti-CD19 Modified-Exosome Delivery System for Precise Targeting of CNS Lymphoma

High-dose methotrexate (HDMTX) serves as the cornerstone of central nervous system lymphoma (CNSL) treatment, but its efficacy is limited due to low Blood-Brain Barrier (BBB) penetration (2% to 20%) and adverse effects like myelosuppression and hepatotoxicity. Thus, this study focuses on exosome-based drug delivery to enhance BBB permeability, reducing methotrexate (MTX) dosage while targeting CNSL specifically (Figure 1A). Considering the recipient's compatibility and reduced risk of adverse reactions or rejection, exosomes derived from human adipose tissue-derived stem cells (ADSCs) were selected as drug delivery vehicles. ADSCs-Exo underwent purification through ultracentrifugation and were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscope (TEM). CD19 is considered an excellent target for immunotherapy in DLBCL. To confer targeting capabilities, ADSCs were infected with a lentiviral vector encoding the anti-CD19 prior to the purification of exosomes. The anti-CD19 (patent number: ZL201811653085.8) was strongly expressed in ADSCs, integrated into ADSCs-derived exosomes, and localized on the external exosomal surface, as evidenced by western blots, colloidal gold immunoelectron microscopy, and flow cytometry. Based on the properties of MTX, we employed co-incubation to load MTX into anti-CD19-modified exosomes. The resulting exosomes, loaded with MTX, were then quantified for the encapsulated MTX using High Performance Liquid Chromatography. The anti-CD19-modified exosome-loaded methotrexate (anti-CD19-Exo-MTX) interacted with cerebrovascular endothelial cells and astrocytes of the BBB, leading to endocytosis and facilitating MTX transportation across the barrier. Additionally, anti-CD19-Exo-MTX crossed the BBB model without compromising its structure, as assessed by dextran's luminescent signal assay. Compared to drug-free formulations, anti-CD19-Exo-MTX exhibited enhanced BBB permeability, thereby improving its capability to inhibit CNSL cell proliferation. Liquid chromatography-tandem mass spectrometry identified and quantified proteins in exosomes, revealing significant enrichment of neuron projection and axon development-associated proteins. Subsequent neurite outgrowth assays, Nissl staining, and modified neurological severity scores confirmed the beneficial effects of ADSCs-Exo in mitigating MTX-induced neuron damage. To evaluate the efficacy of anti-CD19-Exo-MTX in vivo, intracranial orthotopic CNSL models in mice were established. Magnetic resonance imaging (MRI) analysis revealed a reduced disease burden in the anti-CD19-Exo-MTX group compared to both the MTX and Exo-MTX groups (Figure 1B), along with prolonged overall survival. Cerebrospinal fluid (CSF) drug concentration analysis demonstrated enhanced stability and longer-lasting drug levels for anti-CD19-Exo-MTX in the CSF, while showing no toxicity to vital organs, as evidenced by the absence of significant histological damage in the heart, liver, spleen, lung, and kidney tissues. To assess biodistribution and targeting of anti-CD19-Exo-MTX, exosomes were labeled with VivoTrack Dil (Fluorescence). Intracranial fluorescence signal was observed for anti-CD19-Exo-MTX, while Exo-MTX exhibited systemic distribution. Two hours post-injection, organs removal revealed weaker fluorescence signals in the liver and spleen for anti-CD19-Exo-MTX, confirming its specific targeting of MTX to CNSL in vivo. In conclusion, we developed anti-CD19-Exo-MTX, an exosome-based drug delivery platform, demonstrating enhanced BBB permeability and specific targeting for CNSL. This promising approach offers potential for effective CNSL treatment with reduced adverse effects.

SARS-CoV-2 Spike Protein Intensifies Cerebrovascular Complications in Diabetic hACE2 Mice through RAAS and TLR Signaling Activation.

Diabetics are more vulnerable to SARS-CoV-2 neurological manifestations. The molecular mechanisms of SARS-CoV-2-induced cerebrovascular dysfunction in diabetes are unclear. We hypothesize that SARS-CoV-2 exacerbates diabetes-induced cerebrovascular oxidative stress and inflammation via activation of the destructive arm of the renin-angiotensin-aldosterone system (RAAS) and Toll-like receptor (TLR) signaling. SARS-CoV-2 spike protein was injected in humanized ACE2 transgenic knock-in mice. Cognitive functions, cerebral blood flow, cerebrovascular architecture, RAAS, and TLR signaling were used to determine the effect of SARS-CoV-2 spike protein in diabetes. Studies were mirrored in vitro using human brain microvascular endothelial cells treated with high glucose-conditioned media to mimic diabetic conditions. Spike protein exacerbated diabetes-induced cerebrovascular oxidative stress, inflammation, and endothelial cell death resulting in an increase in vascular rarefaction and diminished cerebral blood flow. SARS-CoV-2 spike protein worsened cognitive dysfunction in diabetes compared to control mice. Spike protein enhanced the destructive RAAS arm at the expense of the RAAS protective arm. In parallel, spike protein significantly exacerbated TLR signaling in diabetes, aggravating inflammation and cellular apoptosis vicious circle. Our study illustrated that SAR-CoV-2 spike protein intensified RAAS and TLR signaling in diabetes, increasing cerebrovascular damage and cognitive dysfunction.

Whole brain irradiation–induced endothelial dysfunction in the mouse brain

Whole brain irradiation (WBI), also known as whole brain radiation therapy (WBRT), is a well-established treatment for multiple brain metastases and as a preventive measure to reduce the risk of recurrence after surgical removal of a cerebral metastasis. However, WBI has been found to lead to a gradual decline in neurocognitive function in approximately 50% of patients who survive the treatment, significantly impacting their overall quality of life. Recent preclinical investigations have shed light on the underlying mechanisms of this adverse effect, revealing a complex cerebrovascular injury that involves the induction of cellular senescence in various components of the neurovascular unit, including endothelial cells. The emergence of cellular senescence following WBI has been implicated in the disruption of the blood-brain barrier and impairment of neurovascular coupling responses following irradiation. Building upon these findings, the present study aims to test the hypothesis that WBI-induced endothelial injury promotes endothelial dysfunction, which mimics the aging phenotype. To investigate this hypothesis, we employed a clinically relevant fractionated WBI protocol (5 Gy twice weekly for 4 weeks) on young mice. Both the WBI-treated and control mice were fitted with a cranial window, enabling the assessment of microvascular endothelial function. In order to evaluate the endothelium-dependent, NO-mediated cerebral blood flow (CBF) responses, we topically administered acetylcholine and ATP, and measured the resulting changes using laser Doppler flowmetry. We found that the increases in regional CBF induced by acetylcholine and ATP were significantly diminished in mice subjected to WBI. These findings provide additional preclinical evidence supporting the notion that WBI induces dysfunction in cerebrovascular endothelial cells, which in turn likely contributes to the detrimental long-term effects of the treatment. This endothelial dysfunction resembles an accelerated aging phenotype in the cerebrovascular system and is likely causally linked to the development of cognitive impairment. By integrating these findings with our previous results, we have deepened our understanding of the lasting consequences of WBI. Moreover, our study underscores the critical role of cerebromicrovascular health in safeguarding cognitive function over the long term. This enhanced understanding highlights the importance of prioritizing cerebromicrovascular health in the context of preserving cognitive abilities.

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.

Escin avoids hemorrhagic transformation in ischemic stroke by protecting BBB through the AMPK/Cav-1/MMP-9 pathway.

Hemorrhagic transformation (HT) seriously affects the clinical application of recombinant tissue plasminogen activator (rt-PA). The main strategy for combating HT is to keep the blood-brain barrier (BBB) stable. Escin is the active ingredient of Aesculus hippocastanum and a natural mixture of triterpene saponins, and may play a part in mitigation of HT. This study sought to investigate the effect of Escin in improving rt-PA-induced HT, explore possible mechanisms, and provide new ideas for the treatment of clinical HT. In in vivo experiments, transient middle cerebral artery occlusion (tMCAO) was undertaken in 6-week-old and 12-month-old mice, and rt-PA was administered to induce HT injury. The inhibitory effect of Escin on HT and its protective effect on neurobehavior, the BBB, and cerebrovascular endothelial cells was determined. In in vitro experiments, bEnd.3 cells were injured by oxygen-glucose deprivation/reperfusion (OGD/R) and rt-PA. The protective effect of Escin was measured by the CCK8 assay, release of lactate dehydrogenase (LDH), and expression of tight junction (TJ) proteins. In mechanistic studies, the effect of Escin on the adenosine monophosphate-activated kinase / caveolin-1 / matrix metalloprotease-9 (AMPK/Cav-1/MMP-9) pathway was investigated by employing AMPK inhibitor and Cav-1 siRNA. In mice suffering from ischemia, rt-PA caused HT as well as damage to the BBB and cerebrovascular endothelial cells. Escin reduced the infarct volume, cerebral hemorrhage, improved neurobehavioral deficits, and maintained BBB integrity in rt-PA-treated tMCAO mice while attenuating bEnd.3 cells damage caused by rt-PA and OGD/R injury. Under physiological and pathological conditions, Escin increased the expression of p-AMPK and Cav-1, leading to decreased expression of MMP-9, which further attenuated damage to cerebrovascular endothelial cells, and these effects were verified with AMPK inhibitor and Cav-1 siRNA. We revealed important details of how Escin protects cerebrovascular endothelial cells from HT, these effects were associated with the AMPK/Cav-1/MMP-9 pathway. This study provides experimental foundation for the development of new drugs to mitigate rt-PA-induced HT and the discovery of new clinical application for Escin.

Role of microRNA-34a in blood-brain barrier permeability and mitochondrial function in ischemic stroke.

Over the past decade, there has been an uptick in the number of studies conducting research on the role of microRNA (miRNA) molecules in stroke. Among these molecules, miR-34a has emerged as a significant player, as its levels have been observed to exhibit a substantial rise following ischemic events. Elevated levels of miR-34a have been found to have multiple effects, including the modulation of inflammatory molecules involved in the post-stroke recovery process, as well as negative effects on the blood-brain barrier (BBB) permeability. Interestingly, the increase of miR-34a appears to increase BBB permeability post stroke, through the negative effect on mitochondrial function. The strength of mitochondrial function is crucial for limiting para-cellular permeability and maintaining the structural integrity of the BBB. Furthermore, the activation of ischemic repair mechanisms and the reduction of ischemic event damage depend on healthy mitochondrial activity. This review aims to emphasize the involvement of miR-34a in ischemic stroke, specifically its interaction with mitochondrial genes in cerebrovascular endothelial cells, the effect on mitochondrial function, and lastly its regulatory role in BBB permeability. A comprehensive understanding of the role of miR-34a in maintaining BBB integrity and its contribution to the pathogenesis of stroke holds significant value in establishing a foundation for the development of future therapeutics and diagnostic markers.

PFT-α protects the blood-brain barrier through the Wnt/β-catenin pathway after acute ischemic stroke.

The dysfunction of blood-brain barrier (BBB) plays a pivotal role in brain injury and subsequent neurological deficits of ischemic stroke. The current study aimed to examine the potential correlation between p53 inhibition and the neuroprotective effect of on the BBB. Rat middle cerebral artery occlusion and reperfusion model (MCAO/R) and oxygen-glucose deprivation/re-oxygenation model (OGD/R) were employed to simulate cerebral ischemia-reperfusion (CI/R) injury occurrence in vivo and in vitro. mNSS and TTC staining were applied to evaluate neurological deficits and brain infarct volumes. Evans blue (EB) staining was carried out to examine the permeability of BBB. RT-qPCR and Western blot to examine the mRNA and protein levels. Cell viabilities were detected by CCK-8. Flow cytometry and ELISA assaywere employed to examine apoptosis and neuroinflammation levels. TEER value and sodium fluorescein were carried out to explore the permeability of HBMEC cells. PFT-α inhibited P53 and promoted the expression of β-catenin and cyclin D1, which were reversed by DKK1. PFT-α inhibited neurological deficits, brain infarct volume, neuroinflammation, apoptosis, and BBB integrity than the MCAO/R rats; however, this inhibition was reversed by DKK1. PFT-α promoted OGD/R-induced cell viability in NSCs, and suppressed inflammation and apoptosis, but DKK1 weakened the effect of PFT-α. PFT-α increased OGD/R-induced TEER values in cerebrovascular endothelial cells, inhibited sodium fluorescein permeability, and increased the mRNA levels of tight junction protein, but they were all attenuated by DKK1. PFT-α protects the BBB after acute ischemic stroke via the Wnt/β-catenin pathway, which in turn improves neurological function.

Gut microbiome of Crocodylus porosus and cellular stress: inhibition of nitric oxide, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells.

Crocodiles are renowned for their resilience and capacity to withstand environmental stressors, likely influenced by their unique gut microbiome. In this study, we determined whether selected gut bacteria of Crocodylus porosus exhibit anti-inflammatory effects in response to stress, by measuring nitric oxide release, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells. Using the Griess assay, the findings revealed that among several C. porosus gut bacterial isolates, the conditioned media containing the metabolites of two bacterial strains (CP27 and CP36) inhibited nitric oxide production significantly, in response to the positive control, i.e., taxol-treatment. Notably, CP27 and CP36 were more potent at reducing nitric oxide production than senloytic compounds (fisetin, quercetin). Using enzyme linked immunosorbent assays, the production of pro-inflammatory cytokines (IL-1β, TNF-α, PGE2), was markedly reduced by treatment with CP27 and CP36, in response to stress. Both CP27 and CP36 contain a plethora of metabolites to exact their effects [(3,4-dihydroxyphenylglycol, 5-methoxytryptophan, nifedipine, 4-chlorotestosterone-17-acetate, 3-phenoxypropionic acid, lactic acid,f-Honaucin A,l,l-Cyclo(leucylprolyl), 3-hydroxy-decanoic acid etc.], indicative of their potential in providing protection against cellular stress. Further high-throughput bioassay-guided testing of gut microbial metabolites from crocodiles, individually as well as in combination, together with the underlying molecular mechanisms, in vitro and in vivo will elucidate their value in the rational development of innovative therapies against cellular stress/gut dysbiosis.