BACKGROUND: Cerebrovascular dysfunction is a pathological hallmark of Alzheimer\'s disease (AD). Nevertheless, detecting cerebrovascular changes within bulk tissues has limited our ability to characterize proteomic alterations from less abundant cell types.
METHODS: We conducted quantitative proteomics on bulk brain tissues and isolated cerebrovasculature from the same individuals, encompassing control (N = 28), progressive supranuclear palsy (PSP) (N = 18), and AD (N = 21) cases.
RESULTS: Protein co-expression network analysis identified unique cerebrovascular modules significantly correlated with amyloid plaques, cerebrovascular amyloid angiopathy (CAA), and/or tau pathology. The protein products within AD genetic risk loci were concentrated within cerebrovascular modules. The overlap between differentially abundant proteins in AD cerebrospinal fluid (CSF) and plasma with cerebrovascular network highlighted a significant increase of matrisome proteins, SMOC1 and SMOC2, in CSF, plasma, and brain.
CONCLUSIONS: These findings enhance our understanding of cerebrovascular deficits in AD, shedding light on potential biomarkers associated with CAA and vascular dysfunction in neurodegenerative diseases.

Alzheimer\'s disease (AD) is a debilitating neurodegenerative disease characterized by the accumulation of extracellular amyloid-β peptides (Aβ) within the cerebral parenchyma and vasculature, which is known as cerebral amyloid angiopathy (CAA). This study utilized confocal imaging to investigate heparan sulfate (HS) expression within the cerebrovasculature and its associations with Aβ, gender, and ApoE4 genotype in AD. Our investigation revealed elevated levels of HS in the cerebrovasculature of AD patients with severe CAA. Additionally, these patients exhibited higher HS colocalization with Aβ in the cerebrovasculature, including both endothelial and vascular smooth muscle cell compartments. Intriguingly, a reversal in the polarized expression of HS within the cerebrovasculature was detected in AD patients with severe CAA. Furthermore, male patients exhibited lower levels of both parenchymal and cerebrovascular HS. Additionally, ApoE4 carriers displayed heightened cerebrovascular Aβ expression and a tendency of elevated cerebrovascular HS levels in AD patients with severe CAA. Overall, these findings reveal potential intricate interplay between HS, Aβ, ApoE, and vascular pathology in AD, thereby underscoring the potential roles of cerebrovascular HS in CAA development and AD pathology. Further study of the underlying mechanisms may present novel therapeutic avenues for AD treatment.

OBJECTIVE: A hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier.
METHODS: sEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors.
RESULTS: Using intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell-cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state.
CONCLUSIONS: Diabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.

Dysfunction of the neurovascular unit stands as a significant pathological hallmark of Alzheimer\'s disease (AD) and age-related neurodegenerative diseases. Nevertheless, detecting vascular changes in the brain within bulk tissues has proven challenging, limiting our ability to characterize proteomic alterations from less abundant cell types. To address this challenge, we conducted quantitative proteomic analyses on both bulk brain tissues and cerebrovascular-enriched fractions from the same individuals, encompassing cognitively unimpaired control, progressive supranuclear palsy (PSP), and AD cases. Protein co-expression network analysis identified modules unique to the cerebrovascular fractions, specifically enriched with pericytes, endothelial cells, and smooth muscle cells. Many of these modules also exhibited significant correlations with amyloid plaques, cerebral amyloid angiopathy (CAA), and/or tau pathology in the brain. Notably, the protein products within AD genetic risk loci were found concentrated within modules unique to the vascular fractions, consistent with a role of cerebrovascular deficits in the etiology of AD. To prioritize peripheral AD biomarkers associated with vascular dysfunction, we assessed the overlap between differentially abundant proteins in AD cerebrospinal fluid (CSF) and plasma with a vascular-enriched network modules in the brain. This analysis highlighted matrisome proteins, SMOC1 and SMOC2, as being increased in CSF, plasma, and brain. Immunohistochemical analysis revealed SMOC1 deposition in both parenchymal plaques and CAA in the AD brain, whereas SMOC2 was predominantly localized to CAA. Collectively, these findings significantly enhance our understanding of the involvement of cerebrovascular abnormalities in AD, shedding light on potential biomarkers and molecular pathways associated with CAA and vascular dysfunction in neurodegenerative diseases.

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.

Mild traumatic brain injury (mTBI) is an independent risk factor for ischemic stroke and can result in poorer outcomes- an effect presumed to involve the cerebral vasculature. Here we tested the hypothesis that mTBI-induced pericyte detachment from the cerebrovascular endothelium is responsible for worsened stroke outcomes. We performed a mild closed-head injury and/or treated C57/bl6 mice with imatinib mesylate, a tyrosine kinase inhibitor that induces pericyte detachment. The time course of pericyte detachment was assessed 7, 14, and 28 days post injury (DPI). To test the role of pericytes in TBI-induced exacerbation of ischemic stroke outcomes, we induced mTBI and/or treated mice with imatinib for one week prior to transient middle cerebral artery occlusion. We found that injury promoted pericyte detachment from the vasculature commensurate with the levels of detachment seen in imatinib-only treated animals, and that the detachment persisted for at least 14DPI, but recovered to sham levels by 28DPI. Further, mTBI, but not imatinib-induced pericyte detachment, increased infarct volume. Thus, we conclude that the transient detachment of pericytes caused by mTBI may not be sufficient to exacerbate subsequent ischemic stroke damage. These data have important implications for understanding cerebrovascular dysfunction following mTBI and potential mechanisms of increased risk for future ischemic strokes.

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Aging is associated with increased risk for cognitive decline and dementia due in part to increases in systolic blood pressure (SBP) and cerebrovascular dysfunction. High-resistance inspiratory muscle strength training (IMST) is a time-efficient, intensive respiratory training protocol (30 resisted inspirations/day) that lowers SBP and improves peripheral vascular function in midlife/older adults with above-normal SBP. However, whether, and by what mechanisms, IMST can improve cerebrovascular function is unknown. We hypothesized that IMST would increase cerebrovascular reactivity to hypercapnia (CVR to CO2), which would coincide with changes to the plasma milieu that improve brain endothelial cell function and enhance cognitive performance (NIH Toolbox). We conducted a 6-wk double-blind, randomized, controlled clinical trial investigating high-resistance IMST [75% maximal inspiratory pressure (PImax); 6×/wk; 4 females, 5 males] vs. low-resistance sham training (15% PImax; 6×/wk; 2 females, 5 males) in midlife/older adults (age 50-79 yr) with initial above-normal SBP. Human brain endothelial cells (HBECs) were exposed to participant plasma and assessed for acetylcholine-stimulated nitric oxide (NO) production. CVR to CO2 increased after high-resistance IMST (pre: 1.38 ± 0.66 cm/s/mmHg; post: 2.31 ± 1.02 cm/s/mmHg, P = 0.020). Acetylcholine-stimulated NO production increased in HBECs exposed to plasma from after vs. before the IMST intervention [pre: 1.49 ± 0.33; post: 1.73 ± 0.35 arbitrary units (AU); P < 0.001]. Episodic memory increased modestly after the IMST intervention (pre: 95 ± 13; post: 103 ± 17 AU; P = 0.045). Cerebrovascular and cognitive function were unchanged in the sham control group. High-resistance IMST may be a promising strategy to improve cerebrovascular and cognitive function in midlife/older adults with above-normal SBP, a population at risk for future cognitive decline and dementia.NEW & NOTEWORTHY Midlife/older adults with above-normal blood pressure are at increased risk of developing cognitive decline and dementia. Our findings suggest that high-resistance inspiratory muscle strength training (IMST), a novel, time-efficient (5-10 min/day) form of physical training, may increase cerebrovascular reactivity to CO2 and episodic memory in midlife/older adults with initial above-normal blood pressure.

Human stroke serum (HSS) has been shown to impair cerebrovascular function, likely by factors released into the circulation after ischemia. 20 nm gold nanoparticles (GNPs) have demonstrated anti-inflammatory properties, with evidence that they decrease pathologic markers of ischemic severity. Whether GNPs affect cerebrovascular function, and potentially protect against the damaging effects of HSS on the cerebral circulation remains unclear. HSS obtained 24 h poststroke was perfused through the lumen of isolated and pressurized third-order posterior cerebral arteries (PCAs) from male Wistar rats with and without GNPs (~2 × 109 GNP/ml), or GNPs in vehicle, in an arteriograph chamber (n = 8/group). All vessels were myogenically reactive ≥60 mmHg intravascular pressure; however, vessels containing GNPs had significantly less myogenic tone. GNPs increased vasoreactivity to small and intermediate conductance calcium activated potassium channel activation via NS309; however, reduced vasoconstriction to nitric oxide synthase inhibition. Hydraulic conductivity and transvascular filtration, were decreased by GNPs, suggesting a protective effect on the blood-brain barrier. The stress-strain curves of PCAs exposed to GNPs were shifted leftward, indicating increased vessel stiffness. This study provides the first evidence that GNPs affect the structure and function of the cerebrovasculature, which may be important for their development and use in biomedical applications.

UNASSIGNED: The 5xFAD mouse is a popular model of familial Alzheimer\'s disease (AD) that is characterized by early beta-amyloid (Aβ) deposition and cognitive decrements. Despite numerous studies, the 5xFAD mouse has not been comprehensively phenotyped for vascular and metabolic perturbations over its lifespan.
UNASSIGNED: Male and female 5xFAD and wild type (WT) littermates underwent in vivo 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging at 4, 6, and 12 months of age to assess regional glucose metabolism. A separate cohort of mice (4, 8, 12 months) underwent \"vessel painting\" which labels all cerebral vessels and were analyzed for vascular characteristics such as vessel density, junction density, vessel length, network complexity, number of collaterals, and vessel diameter.
UNASSIGNED: With increasing age, vessels on the cortical surface in both 5xFAD and WT mice showed increased vessel length, vessel and junction densities. The number of collateral vessels between the middle cerebral artery (MCA) and the anterior and posterior cerebral arteries decreased with age but collateral diameters were significantly increased only in 5xFAD mice. MCA total vessel length and junction density were decreased in 5xFAD mice compared to WT at 4 months. Analysis of 18F-FDG cortical uptake revealed significant differences between WT and 5xFAD mice spanning 4-12 months. Broadly, 5xFAD males had significantly increased 18F-FDG uptake at 12 months compared to WT mice. In most cortical regions, female 5xFAD mice had reduced 18F-FDG uptake compared to WT across their lifespan.
UNASSIGNED: While the 5xFAD mouse exhibits AD-like cognitive deficits as early as 4 months of age that are associated with increasing Aβ deposition, we only found significant differences in cortical vascular features in males, not in females. Interestingly, 5xFAD male and female mice exhibited opposite effects in 18F-FDG uptake. The MCA supplies blood to large portions of the somatosensory cortex and portions of motor and visual cortex and increased vessel length alongside decreased collaterals which coincided with higher metabolic rates in 5xFAD mice. Thus, a potential mismatch between metabolic demand and vascular delivery of nutrients in the face of increasing Aβ deposition could contribute to the progressive cognitive deficits seen in the 5xFAD mouse model.