Brain Microvessels Research Articles

Data-independent acquisition proteomic analysis of the brain microvasculature in Alzheimer’s disease identifies major pathways of dysfunction and upregulation of cytoprotective responses

Brain microvascular dysfunction is an important feature of Alzheimer’s disease (AD). To better understand the brain microvascular molecular signatures of AD, we processed and analyzed isolated human brain microvessels by data-independent acquisition liquid chromatography with tandem mass spectrometry (DIA LC–MS/MS) to generate a quantitative dataset at the peptide and protein level. Brain microvessels were isolated from parietal cortex grey matter using protocols that preserve viability for downstream functional studies. Our cohort included 23 subjects with clinical and neuropathologic concordance for Alzheimer’s disease, and 21 age-matched controls. In our analysis, we identified 168 proteins whose abundance was significantly increased, and no proteins that were significantly decreased in AD. The most highly increased proteins included amyloid beta, tau, midkine, SPARC related modular calcium binding 1 (SMOC1), and fatty acid binding protein 7 (FABP7). Additionally, Gene Ontology (GO) enrichment analysis identified the enrichment of increased proteins involved in cellular detoxification and antioxidative responses. A systematic evaluation of protein functions using the UniProt database identified groupings into common functional themes including the regulation of cellular proliferation, cellular differentiation and survival, inflammation, extracellular matrix, cell stress responses, metabolism, coagulation and heme breakdown, protein degradation, cytoskeleton, subcellular trafficking, cell motility, and cell signaling. This suggests that AD brain microvessels exist in a stressed state of increased energy demand, and mount a compensatory response to ongoing oxidative and cellular damage that is associated with AD. We also used public RNAseq databases to identify cell-type enriched genes that were detected at the protein level and found no changes in abundance of these proteins between control and AD groups, indicating that changes in cellular composition of the isolated microvessels were minimal between AD and no-AD groups. Using public data, we additionally found that under half of the proteins that were significantly increased in AD microvessels had concordant changes in brain microvascular mRNA, implying substantial discordance between gene and protein levels. Together, our results offer novel insights into the molecular underpinnings of brain microvascular dysfunction in AD.

RiboTag RNA Sequencing Identifies Local Translation of HSP70 In Astrocyte Endfeet After Cerebral Ischemia.

Brain ischemia causes disruption in cerebral blood flow and blood-brain barrier (BBB) integrity which are normally maintained by the astrocyte endfeet. Emerging evidence points to dysregulation of the astrocyte translatome during ischemia, but its effects on the endfoot translatome are unknown. In this study, we aimed to investigate the early effects of ischemia on the astrocyte endfoot translatome in a rodent model of cerebral ischemia-reperfusion. To do so, we immunoprecipitated astrocyte-specific tagged ribosomes (RiboTag IP) from mechanically isolated brain microvessels. In mice subjected to middle cerebral artery occlusion and reperfusion and contralateral controls, we sequenced ribosome-bound RNAs from perivascular astrocyte endfeet and identified 205 genes that were differentially expressed in the translatome after ischemia. Pathways associated with the differential expressions included proteostasis, inflammation, cell cycle, and metabolism. Transcription factors whose targets were enriched amongst upregulated translating genes included HSF1, the master regulator of the heat shock response. The most highly upregulated genes in the translatome were HSF1-dependent Hspa1a and Hspa1b , which encode the inducible HSP70. We found that HSP70 is upregulated in astrocyte endfeet after ischemia, coinciding with an increase in ubiquitination across the proteome. These findings suggest a robust proteostasis response to proteotoxic stress in the endfoot translatome after ischemia. Modulating proteostasis in endfeet may be a strategy to preserve endfeet function and BBB integrity after ischemic stroke.

Neuroinflammatory responses and blood–brain barrier injury in chronic alcohol exposure: role of purinergic P2 × 7 Receptor signaling

Alcohol consumption leads to neuroinflammation and blood‒brain barrier (BBB) damage, resulting in neurological impairment. We previously demonstrated that ethanol-induced disruption of barrier function in human brain endothelial cells was associated with mitochondrial injury, increased ATP and extracellular vesicle (EV) release, and purinergic receptor P2 × 7R activation. Therefore, we aimed to evaluate the effect of P2 × 7R blockade on peripheral and neuro-inflammation in ethanol-exposed mice. In a chronic intermittent ethanol (CIE)-exposed mouse model, P2 × 7R was inhibited by two different methods: Brilliant Blue G (BBG) or gene knockout. We assessed blood ethanol concentration (BEC), brain microvessel gene expression by using RT2 PCR array, plasma P2 × 7R and P-gp, serum ATP, EV-ATP, number of EVs, and EV mtDNA copy numbers. An RT2 PCR array of brain microvessels revealed significant upregulation of proinflammatory genes involved in apoptosis, vasodilation, and platelet activation in CIE-exposed wild-type animals, which were decreased 15–50-fold in BBG-treated–CIE-exposed animals. Plasma P-gp levels and serum P2 × 7R shedding were significantly increased in CIE-exposed animals. Pharmacological or genetic suppression of P2 × 7R decreased receptor shedding to levels equivalent to those in control group. The increase in EV number and EV-ATP content in the CIE-exposed mice was significantly reduced by P2 × 7R inhibition. CIE mice showed augmented EV-mtDNA copy numbers which were reduced in EVs after P2 × 7R inhibition or receptor knockout. These observations suggested that P2 × 7R signaling plays a critical role in ethanol-induced brain injury. Increased extracellular ATP, EV-ATP, EV numbers, and EV-mtDNA copy numbers highlight a new mechanism of brain injury during alcohol exposure via P2 × 7R and biomarkers of such damage. In this study, for the first time, we report the in vivo involvement of P2 × 7R signaling in CIE-induced brain injury.

Increased SPARC in brain microvessels of ob/ob mice accelerates molecular transport into the brain accompany with albumin

Cytotoxic metabolites originating from the peripheral circulation can induce central nervous system complications associated with diabetes. Since a large proportion of these metabolites bind to plasma albumin, mechanisms for transporting albumin-metabolite complexes into the brain exist under diabetic conditions. Secreted protein acidic and rich in cysteine (SPARC) is one of the vesicular transport receptors responsible for albumin transport. This study aimed to investigate the changes in SPARC expression and cellular albumin transfer under high-glucose conditions and evaluate the permeability of molecules with high protein-bound properties to the brain tissue. Glucose (30 mM) increased SPARC expression, and intracellular albumin accumulation in NIH3T3 cells. In addition, these changes were observed in the brain of ob/ob mice. Brain microvessels function as a physiological barrier to limit the penetration of molecules from the peripheral blood circulation into the brain by forming tight junctions. Although protein expression of molecules involved in tight junction formation and cell adhesion was increased in the brain microvessels of ob/ob mice, molecular transfer into the brain through cellular junctions was not enhanced. However, Evans blue dye injected into the peripheral vein and endogenous advanced glycation end-products, exerted a high protein-binding property and accumulated in their brains. These observations indicate that peripheral molecules with high protein-binding properties invade the brain tissue and bind to albumin through transcytosis mediated by SPARC.

Angiopoietin-like 4 protects against endothelial dysfunction during bacterial sepsis.

Loss of endothelial integrity and vascular leakage are central features of sepsis pathogenesis; however, no effective therapeutic mechanisms for preserving endothelial integrity are available. Here we show that, compared to dermal microvessels, brain microvessels resist infection by Neisseria meningitidis, a bacterial pathogen that causes sepsis and meningitis. By comparing the transcriptional responses to infection in dermal and brain endothelial cells, we identified angiopoietin-like 4 as a key factor produced by the brain endothelium that preserves blood-brain barrier integrity during bacterial sepsis. Conversely, angiopoietin-like 4 is produced at lower levels in the peripheral endothelium. Treatment with recombinant angiopoietin-like 4 reduced vascular leakage, organ failure and death in mouse models of lethal sepsis and N. meningitidis infection. Protection was conferred by a previously uncharacterized domain of angiopoietin-like 4, through binding to the heparan proteoglycan, syndecan-4. These findings reveal a potential strategy to prevent endothelial dysfunction and improve outcomes in patients with sepsis.

Development of an in vitro model of the neurovascular unit for BBB permeability-linked neuroactivity screening

Many potential neurotherapeutic agents fail in the later stages during development due to insufficient blood-brain barrier (BBB) permeability or neurotoxic effects. To address this, we developed an in vitro model incorporating the neurovascular unit (NVU) — astrocytes, pericytes, neurons, and brain microvessel endothelial cells — designed to simulate the in vivo BBB and improve early drug screening. This model uses a direct contact triculture system enhanced by integrating SH-SY5Y neuron-like cells, enabling the study of permeability-linked neuronal responses. Our results show that this expanded NVU model, employing a Transwell® system, enhances the BBB’s restrictive properties and neuronal viability, potentially due to improved cell-cell signaling. Additionally, the model demonstrated increased efflux transporter expression, providing a more physiologically relevant assessment of neuroactivity in relation to BBB permeability. This innovative NVU model offers a predictive and robust tool for evaluating neurotherapeutic agents, facilitating the prioritization of candidates in large compound libraries and potentially reducing attrition rates in drug development. It represents a significant advancement in the methodology for early-stage neurotherapeutic screening, aligning in vitro findings more closely with in vivo responses.

Proteomic Profiling Reveals Age-Related Changes in Transporter Proteins in the Human Blood-Brain Barrier.

The Blood-Brain Barrier (BBB) is a crucial, selective barrier that regulates the entry of molecules including nutrients, environmental toxins, and therapeutic medications into the brain. This function relies heavily on brain endothelial cell proteins, particularly transporters and tight junction proteins. The BBB continues to develop postnatally, adapting its selective barrier function across different developmental phases, and alters with aging and disease. Here we present a global proteomics analysis focused on the ontogeny and aging of proteins in human brain microvessels (BMVs), predominantly composed of brain endothelial cells. Our proteomic profiling quantified 6,223 proteins and revealed possible age-related alteration in BBB permeability due to basement membrane component changes through the early developmental stage and age-dependent changes in transporter expression. Notable changes in expression levels were observed with development and age in nutrient transporters and transporters that play critical roles in drug disposition. This research 1) provides important information on the mechanisms that drive changes in the metabolic content of the brain with age and 2) enables the creation of physiologically based pharmacokinetic models for CNS drug distribution across different life stages.

Cortical plasticity is associated with blood-brain barrier modulation.

Brain microvessels possess the unique properties of a blood-brain barrier (BBB), tightly regulating the passage of molecules from the blood to the brain neuropil and vice versa. In models of brain injury, BBB dysfunction and the associated leakage of serum albumin to the neuropil have been shown to induce pathological plasticity, neuronal hyper-excitability, and seizures. The effect of neuronal activity on BBB function and whether it plays a role in plasticity in the healthy brain remain unclear. Here we show that neuronal activity induces modulation of microvascular permeability in the healthy brain and that it has a role in local network reorganization. Combining simultaneous electrophysiological recording and vascular imaging with transcriptomic analysis in rats, and functional and BBB-mapping MRI in human subjects, we show that prolonged stimulation of the limb induces a focal increase in BBB permeability in the corresponding somatosensory cortex that is associated with long-term synaptic plasticity. We further show that the increased microvascular permeability depends on neuronal activity and involves caveolae-mediated transcytosis and transforming growth factor β signaling. Our results reveal a role of BBB modulation in cortical plasticity in the healthy brain, highlighting the importance of neurovascular interactions for sensory experience and learning.

Neutrophil migration participates in the side effect of recombinant human tissue plasminogen activator.

Ischemic stroke remains a challenge in medical research because of the limited treatment options. Recombinant human tissue plasminogen activator (rtPA) is the primary treatment for recanalization. However, nearly 50% of the patients experience complications that result in ineffective reperfusion. The precise factors contributing to ineffective reperfusion remain unclear; however, recent studies have suggested that immune cells, notably neutrophils, may influence the outcome of rtPA thrombolysis via mechanisms such as the formation of neutrophil extracellular traps. This study aimed to explore the nonthrombolytic effects of rtPA on neutrophils and highlight their contribution to ineffective reperfusion. We evaluated the effects of rtPA treatment on middle cerebral artery occlusion in rats. We also assessed neutrophil infiltration and activation after rtPA treatment invitro and invivo in a small cohort of patients with massive cerebral ischemia (MCI). rtPA increased neutrophil infiltration into the brain microvessels and worsened blood-brain barrier damage during ischemia. It also increased the neutrophil counts of the patients with MCI. Neutrophils play a crucial role in promoting ischemic injury and blood-brain barrier disruption, making them potential therapeutic targets.

Modified histological staining for the identification of arterial and venous segments of brain microvessels

BackgroundThis study aimed to develop a modified histochemical staining technique to successfully identify arterial and venous segments of brain microvessels. New methodGelatin/red ink-alkaline phosphatase-oil red O (GIAO) staining was developed from the traditional gelatin–ink perfusion method. Oil red Chinese ink for brush writing and painting mixed with gelatin was used to label cerebral vascular lumens. Subsequently, alkaline phosphatase staining was used to label endothelial cells on the arterial segments of cerebral microvessels. Thereafter, the red ink color in vessel lumens was highlighted with oil red O staining. ResultsThe arterial segments of the brain microvessels exhibited red lumens surrounded by dark blue walls, while the venous segments were bright red following GIAO staining. Meanwhile, the nerve fiber bundles were stained brownish-yellow, and the nuclei appeared light green under light microscope. After cerebral infarction, we used GIAO staining to determine angiogenesis features and detected notable vein proliferation inside the infarct core. Moreover, GIAO staining in conjunction with hematoxylin staining was performed to assess the infiltration of foamy macrophages. Comparison with existing methodRed Chinese ink enabled subsequent multiple color staining on brain section. Oil red O was introduced to improved the resolution and contrast between arterial and venous segments of microvessels. ConclusionWith excellent resolution, GIAO staining effectively distinguished arterial and venous segments of microvessels in both normal and ischemic brain tissue. GIAO staining, as described in the present study, will be useful for histological investigations of microvascular bed alterations in a variety of brain disorders.

Aquaporin-4 deletion leads to reduced infarct volume and increased peri-infarct astrocyte reactivity in a mouse model of cortical stroke.

Aquaporin-4 (AQP4) is the main water channel in brain and is enriched in perivascular astrocyte processes abutting brain microvessels. There is a rich literature on the role of AQP4 in experimental stroke. While its role in oedema formation following middle cerebral artery occlusion (MCAO) has been studied extensively, its specific impact on infarct volume remains unclear. This study investigated the effects of total and partial AQP4 deletion on infarct volume in mice subjected to distal medial cerebral artery (dMCAO) occlusion. Compared to MCAO, this model induces smaller infarcts confined to neocortex, and less oedema. We show that AQP4 deletion significantly reduced infarct volume as assessed 1week after dMCAO, suggesting that the role of AQP4 in stroke goes beyond its effect on oedema formation and dissolution. The reduction in infarct volume was associated with increased astrocyte reactivity in the peri-infarct areas. No significant differences were observed in the number of microglia among the genotypes. These findings provide new insights in the role of AQP4 in ischaemic injury indicating that AQP4 affects both infarct volume and astrocyte reactivity in the peri-infarct zone. KEY POINTS: Aquaporin-4 (AQP4) is the main water channel in brain and is enriched in perivascular astrocyte processes abutting microvessels. A rich literature exists on the role of AQP4 in oedema formation following middle cerebral artery occlusion (MCAO). We investigated the effects of total and partial AQP4 deletion on infarct volume in mice subjected to distal medial cerebral artery occlusion (dMCAO), a model inducing smaller infarcts confined to neocortex and less oedema compared to MCAO. AQP4 deletion significantly reduced infarct volume 1week after dMCAO, suggesting a broader role for AQP4 in stroke beyond oedema formation. The reduction in infarct volume was associated with increased astrocyte reactivity in the peri-infarct areas, while no significant differences were observed in the number of microglia among the genotypes. These findings provide new insights into the role of AQP4 in stroke, indicating that AQP4 affects both infarct volume and astrocyte reactivity in the peri-infarct zone.

Plasma from older children in Malawi inhibits Plasmodium falciparum binding in 3D brain microvessels.

A hallmark of cerebral malaria is sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain microcirculation. Antibodies contribute to malaria immunity, but it remains unclear whether functional antibodies targeting parasite-expressed ligand can block cytoadhesion in the brain. Here, we screened the plasma of older children and young adults in Malawi to characterize the antibody response against the P. falciparum-IE surface and used a bioengineered 3D human brain microvessel model incorporating variable flow dynamics to measure adhesion blocking responses. We found a strong correlation between surface antibody reactivity by flow cytometry and reduced P. falciparum-IE binding in 3D microvessels. Moreover, there was a threshold of surface antibody reactivity necessary to achieve robust inhibitory activity. Our findings provide evidence of the acquisition of adhesion blocking antibodies against cerebral binding variants in people exposed to stable P. falciparum transmission and suggest the quality of the inhibitory response can be influenced by flow dynamics.

Морфологічні структури циркумвентрикулярного комплексу (Morphological structures of the circumvetricular complex)

Brain homeostasis requires the maintenance of barriers between the brain and the periphery, which are provided by brain microvessels in the blood-brain barrier and epithelial cells in the choroid plexus. Circumventricular complex (CVC) – structures located around the third and fourth ventricles, lining the cavity of the third ventricle (neurohypophysis, vascular organ of the end plate, epiphysis, subvault and subcommissural organs) and the fourth ventricle (posterior region), different from other structures of the brain due to the maximum vascularization and the absence of a typical blood-brain barrier. The subcommissural organ and the area postrema are located at the confluence between the ventricles, while the neurohypophysis, the vascular organ of the terminal plate, and the pineal gland line the ventricular depressions. All structures of the central nervous system are divided into sensory and secretory. Vessels in the CVC branch into a network of fenestrated capillaries with loosely connected astrocytic ends, which allows them to be considered as gates» to the brain; substances are transported by blood and freely leave the capillary lumen. Neurons and glial cells of the CVC form a unique symbiosis of receptors and ion channels, receiving chemical signals from the bloodstream. CVCs are described as the «windows of the brain» that form the blood-CSF barrier on the ventricular wall, which is composed of tanycyte-like cells that line the ventricular ependyma. Astrocytes and tanycytes form a dense barrier in the distal part of the CVC, preventing the free diffusion of the molecules obtained. from the blood to the neighboring areas of the brain. The barrier in front of the fenestrated vessels of the CVC may limit molecules carried by the blood through these «windows of the brain» and prevent their diffusion into the cerebrospinal fluid. In the central nervous system, connections between the central nervous system and peripheral blood flow serve as an alternative route for peptides and hormones of nervous tissue into the bloodstream, primarily performing neuroimmune-endocrine functions, as well as the role of an «immune watchman». Key words: circumventricular complex, cerebrospinal fluid, brain, blood-brain barrier.

Brain Vascular Expression of Monomeric C-Reactive Protein Is Blocked by C10M Following Intraperitoneal Injection in an ApoE-/- Murine Model of Dyslipidemia: An Immunohistochemical Analysis.

Introduction The neurovascular unit (NVU), comprising vascular and glial cells along with neurons, is vital for maintaining the blood-brain barrier (BBB) and cerebral homeostasis. Dysfunction of the NVU is implicated in key neurodegenerative disorders such as Alzheimer's disease (AD). Monomeric C-reactive protein (mCRP), the dissociated form of native, pentameric C-reactive protein (pCRP), is associated with enhanced pro-inflammatory responses in the vascular system, leading to increased permeability and potential NVU disruption. Methods This study utilized ApoE-/- mice receiving a high-fat diet which were injected intraperitoneally with either mCRP or mCRP together with a small molecule inhibitor (C10M) and investigated the deposition of mCRP and CD105 expression in the brain parenchyma and its localization within the microvasculature. Results Histological analysis revealed significant mCRP deposition in brain microvessels and neurons, indicating potential disruption of the BBB and neuronal damage. Moreover, co-administration of C10M effectively blocked mCRP accumulation in the brain parenchyma, suggesting its potential as a therapeutic agent for effectively inhibiting inflammation-associated degenerative changes. Immunohistochemical staining demonstrated co-localization of mCRP with CD105, indicating potential angiogenic activation and increased susceptibility to inflammatory insult. Discussion These findings provide evidence supporting the potential role of mCRP as a contributor to neuroinflammation in individuals with chronic systemic inflammation. Conclusion Further studies in human subjects should help validate the efficacy of C10M in preventing or halting neurodegeneration in conditions such as AD and stroke-associated dementia.

Angiotensin II Directly Increases Endothelial Calcium and Nitric Oxide in Kidney and Brain Microvessels InVivo With Reduced Efficacy in Hypertension.

The vasoconstrictor effects of angiotensin II via type 1 angiotensin II receptors in vascular smooth muscle cells are well established, but the direct effects of angiotensin II on vascular endothelial cells (VECs) invivo and the mechanisms how VECs may mitigate angiotensin II-mediated vasoconstriction are not fully understood. The present study aimed to explore the molecular mechanisms and pathophysiological relevance of the direct actions of angiotensin II on VECs in kidney and brain microvessels invivo. Changes in VEC intracellular calcium ([Ca2+]i) and nitric oxide (NO) production were visualized by intravital multiphoton microscopy of cadherin 5-Salsa6f mice or the endothelial uptake of NO-sensitive dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, respectively. Kidney fibrosis by unilateral ureteral obstruction and Ready-to-use adeno-associated virus expressing Mouse Renin 1 gene (Ren1-AAV) hypertension were used as disease models. Acute systemic angiotensin II injections triggered >4-fold increases in VEC [Ca2+]i in brain and kidney resistance arterioles and capillaries that were blocked by pretreatment with the type 1 angiotensin II receptor inhibitor losartan, but not by the type 2 angiotensin II receptor inhibitor PD123319. VEC responded to acute angiotensin II by increased NO production as indicated by >1.5-fold increase in 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence intensity. In mice with kidney fibrosis or hypertension, the angiotensin II-induced VEC [Ca2+]i and NO responses were significantly reduced, which was associated with more robust vasoconstrictions, VEC shedding, and microthrombi formation. The present study directly visualized angiotensin II-induced increases in VEC [Ca2+]i and NO production that serve to counterbalance agonist-induced vasoconstriction and maintain residual organ blood flow. These direct and endothelium-specific angiotensin II effects were blunted in disease conditions and linked to endothelial dysfunction and the development of vascular pathologies.

Characterization of Sex-Dependent Differences in Brain Microvascular Bioenergetics

Background: Women experience increased incidence of stroke-related deaths and lifetime risk of Alzheimer's disease (AD). Microvascular dysfunction has been implicated in the pathogenesis of ischemic stroke and AD. Our objective is to investigate the sex-dependent differences in bioenergetics of young mouse brain microvessels (BMVs). Methods: BMVs were isolated from young male and female mice (3-4 months), using a combination of gradient centrifugation and filtration (300μm and 40μm filters) methods. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMVs were measured utilizing the Agilent Seahorse XFe 24 analyzer. The levels of proteins and mRNAs of mitochondrial respiratory complexes were measured by western blot and RT-PCR, respectively. Citrate synthase (CS) enzyme activity and ATP levels were determined in lysates of BMVs. Results: Real-Time ATP Rate assay demonstrated a decreased total ATP production rate and ATP contribution from both glycolysis and oxidative phosphorylation (OxPhos) in BMVs from females compared to males. Mitochondrial stress test assay revealed significantly lower values of several mitochondrial respiratory parameters, including basal respiration, ATP production, maximal respiration, spare respiratory capacity, and proton leak in BMVs from females compared to males. Glycolytic Rate assay uncovered reduced basal glycolysis, proton efflux rate (PER) originating from basal glycolysis, and the mitoOCR/glycoPER ratio in BMVs from females compared to males. Interestingly, no sex-dependent differences were observed in basal PER and post-2-DG acidification rates in BMVs. Mito Fuel Flex Test assay showed no sex-dependent difference in the utilization of glucose, glutamine, and long-chain fatty acids as fuel substrates in BMVs. ATP measurements confirmed the reduced ATP levels in BMVs from females compared to males. Measurements of CS activity revealed no sex-dependent difference in the BMVs. Lastly, Western blot and RT-PCR experiments indicated no differences in either protein or mRNA levels of various mitochondrial complex protein levels.Conclusions: Female BMVs showed reduced mitochondrial respiratory and glycolytic parameters compared to male BMVs although relative contribution of various substrates for energy production were not different. These sex-dependent differences involving glycolysis and OxPhos make the microvasculature vulnerable to injury in females. Funding sources: NIH grants AG074489 and NS114286 (PVGK and RM). 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.

Novel, engineered fusogenic liposome-based anti-oxidant delivery system improves the blood-brain barrier integrity in aging

Vascular dysfunction plays a pivotal role in age-related cognitive decline and neurodegeneration. The aging process often leads to a loss of integrity in the blood-brain barrier (BBB), initiating neuroinflammation and contributing to a decline in cognitive function. In previous research, we demonstrated the potential of resveratrol (RSV), a natural polyphenol, to target cerebromicrovascular endothelial cells (CMVECs), effectively countering age-related oxidative stress and improving vascular function, both in vitro and in vivo. However, the limited bioavailability of resveratrol raised questions about its effectiveness concerning the BBB and neuroinflammation. Our study aimed to address this challenge by introducing a novel drug delivery system designed to enhance the effciency of polyphenol delivery. This innovative approach involved the use of fusogenic liposomes (FL) integrated with a bioengineered protein corona (PC) featuring specific apolipoprotein E (ApoE). Our central hypothesis posited that PC formation could be harnessed to directly target CMVECs and enhance liposomal uptake, with the ultimate objective of effectively delivering RSV to the brain's microvessels in aging subjects (ApoE-FL-RSV). Our investigation encompassed the characterization of ApoE-FL uptake by CMVECs, along with an analysis of its biodistribution and pharmacokinetics in vivo. Remarkably, our findings revealed a significant increase in ApoE-FL-RSV accumulation within CMVECs in vivo, compared to control FL uptake. We employed advanced in vivo multiphoton imaging to longitudinally monitor the effects of ApoE-FL-RSV on the BBB, which demonstrated a significant rejuvenation of the endothelial barrier function in aged mice treated with ApoE-FL-RSV. In light of these results, we concluded that the fusogenic liposomal delivery system holds promise as a viable pharmacological intervention for addressing age-related vascular cognitive impairment. AHA834339, RF1AG072295, R01AG055395, R01AG068295; R01AG070915, K01AG073614, R01NS100782, R01CA255840, and Presbyterian Health Foundation. 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.

Fasting upregulates the monocarboxylate transporter MCT1 at the rat blood-brain barrier through PPAR δ activation

BackgroundThe blood-brain barrier (BBB) is pivotal for the maintenance of brain homeostasis and it strictly regulates the cerebral transport of a wide range of endogenous compounds and drugs. While fasting is increasingly recognized as a potential therapeutic intervention in neurology and psychiatry, its impact upon the BBB has not been studied. This study was designed to assess the global impact of fasting upon the repertoire of BBB transporters.MethodsWe used a combination of in vivo and in vitro experiments to assess the response of the brain endothelium in male rats that were fed ad libitum or fasted for one to three days. Brain endothelial cells were acutely purified and transcriptionaly profiled using RNA-Seq. Isolated brain microvessels were used to assess the protein expression of selected BBB transporters through western blot. The molecular mechanisms involved in the adaptation to fasting were investigated in primary cultured rat brain endothelial cells. MCT1 activity was probed by in situ brain perfusion.ResultsFasting did not change the expression of the main drug efflux ATP-binding cassette transporters or P-glycoprotein activity at the BBB but modulated a restrictive set of solute carrier transporters. These included the ketone bodies transporter MCT1, which is pivotal for the brain adaptation to fasting. Our findings in vivo suggested that PPAR δ, a major lipid sensor, was selectively activated in brain endothelial cells in response to fasting. This was confirmed in vitro where pharmacological agonists and free fatty acids selectively activated PPAR δ, resulting in the upregulation of MCT1 expression. Moreover, dosing rats with a specific PPAR δ antagonist blocked the upregulation of MCT1 expression and activity induced by fasting.ConclusionsAltogether, our study shows that fasting affects a selected set of BBB transporters which does not include the main drug efflux transporters. Moreover, we describe a previously unknown selective adaptive response of the brain vasculature to fasting which involves PPAR δ and is responsible for the up-regulation of MCT1 expression and activity. Our study opens new perspectives for the metabolic manipulation of the BBB in the healthy or diseased brain.

The proteome of the blood–brain barrier in rat and mouse: highly specific identification of proteins on the luminal surface of brain microvessels by in vivo glycocapture

BackgroundThe active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the blood–brain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins.MethodsHere we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice.ResultsUsing the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture.ConclusionsThe proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.