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Cited by 350Neurological Disease Mechanisms and TreatmentsBarrier Structure and Function StudiesNeuroinflammation and Neurodegeneration Mechanisms

Perivascular macrophages mediate the neurovascular and cognitive dysfunction associated with hypertension

Open access
Faraco, Giuseppe, Sugiyama, Yukio, Lane, Diane, Garcia-Bonilla, Lidia, Chang, Haejoo, Santisteban, Monica M., Racchumi, Gianfranco, Murphy, Michelle +3 more·2016·Journal of Clinical Investigation
DOI 10.1172/jci86950PMC PMC5127678
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Abstract

Hypertension is a leading risk factor for dementia, but the mechanisms underlying its damaging effects on the brain are poorly understood. Due to a lack of energy reserves, the brain relies on continuous delivery of blood flow to its active regions in accordance with their dynamic metabolic needs. Hypertension disrupts these vital regulatory mechanisms, leading to the neuronal dysfunction and damage underlying cognitive impairment. Elucidating the cellular bases of these impairments is essential for developing new therapies. Perivascular macrophages (PVMs) represent a distinct population of resident brain macrophages that serves key homeostatic roles but also has the potential to generate large amounts of reactive oxygen species (ROS). Here, we report that PVMs are critical in driving the alterations in neurovascular regulation and attendant cognitive impairment in mouse models of hypertension. This effect was mediated by an increase in blood-brain barrier permeability that allowed angiotensin II to enter the perivascular space and activate angiotensin type 1 receptors in PVMs, leading to production of ROS through the superoxide-producing enzyme NOX2. These findings unveil a pathogenic role of PVMs in the neurovascular and cognitive dysfunction associated with hypertension and identify these cells as a putative therapeutic target for diseases associated with cerebrovascular oxidative stress.

AI snapshot

Written from the paper’s full text.

TL;DR

Perivascular macrophages (PVMs) are required for the neurovascular and cognitive dysfunction caused by hypertension in mouse models. Circulating angiotensin II reaches the perivascular space after blood-brain barrier opening, activates PVM AT1 receptors, and drives NOX2-dependent reactive oxygen species production.

Key findings

  • In slow-pressor ANGII hypertension, chronic ANGII infusion increased BBB permeability to sodium fluorescein and FITC-dextran but not Evans blue, and nearly 60% of PVMs were associated with biotinylated ANGII after 14 days; acute ANGII did not produce this BBB effect.
  • Intracerebroventricular clodronate depleted PVMs by 60%–65%, did not alter blood pressure, and restored neurovascular coupling and endothelium-dependent CBF responses in ANGII-treated mice; it also rescued CBF dysfunction in chronically hypertensive BPH/2J mice.
  • Bone marrow chimeras showed that AT1R or NOX2 deletion in PVMs attenuated ANGII-induced vascular oxidative stress and neurovascular dysfunction; by 14 weeks after transplant, 78% ± 2% of CD206+ cells were replaced by donor cells, and At1r−/−→WT chimerism was greater than 95%.
  • PVM depletion improved cognitive deficits in BPH/2J mice: it rescued novel object recognition memory and improved Barnes maze spatial memory, while not affecting normotensive controls.

Methods

  • Male C57BL/6 mice, male BPH/2J mice, and BPN/3J controls were used; hypertension was induced by osmotic minipumps delivering ANGII at 600 ng/kg/min for 2 weeks, and PVMs were depleted with intracerebroventricular liposome-encapsulated clodronate.
  • Bone marrow chimeras were generated by irradiating mice and transplanting At1r−/−, Nox2−/−, or GFP+ marrow; cerebrovascular function was assessed by laser-Doppler CBF responses to whisker stimulation, acetylcholine, and adenosine, with BBB permeability measured using sodium fluorescein, FITC-dextran, and Evans blue.
  • Vascular oxidative stress was assessed by hydroethidine microfluorography; cognition was tested with novel object recognition and Barnes maze; data were analyzed by Student’s t test or ANOVA with Bonferroni/Tukey tests, with P < 0.05 considered significant.

Limitations

  • The paper states that the molecular interactions of PVMs with cells of the neurovascular unit and their role in BBB alteration remain to be defined, and that the mechanisms of ANGII-induced BBB permeability increase remain to be established.
  • The paper states that the rescue by Nox2 deletion in PVMs was not complete, possibly implicating ROS sources other than NOX2 or the 20%–30% of PVMs not replaced by transplant.
  • The paper states that bone marrow chimera experiments could not be performed in BPH/2J mice because of their complex genetic background, and that cognitive function was not assessed in ANGII-treated mice because longer ANGII exposure is needed and is not compatible with the time course of PVM depletion by clodronate.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1172/jci86950

Cites

69 of 69 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 39 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Chronic ANGII administration does not alter blood-brain barrier permeability to Evans blue, indicating a size restriction in the barrier dysfunction.

    “In contrast to FITC-dextran and sodium fluorescein, the BBB permeability to Evans blue, which binds to serum albumin (MW ~69 kDa), was not altered by chronic ANGII administration (Supplemental Figure 2E), indicating a previously unappreciated size restriction in the BBB dysfunction.”
    Quote in the paperSubstantial claim
  • Findingown data

    The ANGII-induced increase in blood-brain barrier permeability is not solely due to blood pressure elevation, because phenylephrine increased blood pressure comparably but did not alter FITC-dextran permeability.

    “The effect of ANGII on the BBB could not be attributed solely to the elevation in BP, since 2 weeks of phenylephrine infusion increased BP comparably to ANGII, but did not alter BBB permeability to FITC-dextran (Supplemental Figure 2, G and H).”
    Quote in the paperSubstantial claim
  • Findingown data

    Infusion of low concentrations of ANGII (600 ng/kg/min) induced a delayed increase in blood pressure that was observed at day 7 and leveled off by day 14.

    “Infusion of low concentrations of ANGII (600 ng/kg/min) induced a delayed increase in BP that was observed at day 7 and leveled off by day 14 (Figure 2A).”
    Quote in the paperSubstantial claim
  • Findingown data

    In the slow pressor hypertension model, angiotensin II crosses the blood-brain barrier and reaches perivascular macrophages in the perivascular space.

    “First, we showed that in the slow pressor model of hypertension ANGII crosses the BBB and reaches PVMs in the perivascular space, demonstrating that these cells have access to circulating ANGII.”
    • Also stated in: § Discussion · boosted
      “Experiments with biotinylated ANGII demonstrated more directly that ANGII reaches the perivascular space and enters in contact with PVMs.”
    Quote in the paperSubstantial claim
  • Findingown data

    In both At1r-/-→WT and Nox2-/-→WT chimeras, the ANGII-induced increase in vascular ROS was attenuated.

    “In both At1r -/-→WT and Nox2 -/-→WT chimeras, the increase in ROS induced by ANGII was attenuated (Figure 7, C and D)”
    Quote in the paperSubstantial claim
  • May combine two claims. 49
  • The quote only partly states this claim. 3
  • Classified as probably not a substantial claim. 25

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    In mice treated with slow-pressor ANGII for 14 days, nearly 60% of PVMs were closely associated with biotinylated ANGII, while little or no labeling was seen in saline-treated or acutely ANGII-treated mice.

    “As shown in Figure 2, D-F, nearly 60% of the PVMs were closely associated with biotinylated ANGII in mice treated with ANGII for 14 days (Figure 2, D-F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Chronic ANGII infusion increases blood-brain barrier permeability to sodium fluorescein and FITC-dextran at 14 days, whereas acute ANGII at a comparable pressor dose does not.

    “We found that chronic ANGII infusion increased the permeability of the BBB to both markers at 14 days, whereas acute administration of ANGII (250 ng/kg/min) produced a comparable elevation in mean BP, but failed to do so (Figure 2C and Supplemental Figure 2E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BPH/2J mice show a recognition memory deficit, spending equal time exploring novel and familiar objects, unlike normotensive mice.

    “In contrast, BPH/2J mice spent equal time exploring the novel and the familiar object, reflecting a deficit in recognition memory (Figure 9A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BPH/2J mice exhibit elevated blood pressure from 4 weeks of age, and the elevation remains stable until they are studied at 4-6 months of age.

    “BPH/2J mice exhibited elevated BP at 4 weeks of age (Supplemental Figure 5A), compared with normotensive controls, and the BP elevation remained stable until the mice were studied at 4-6 months of age.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neocortical application of the AT1 receptor antagonist losartan or the ROS scavenger MnTBAP counteracts CBF dysfunction in BPH/2J mice.

    “The CBF dysfunction was counteracted by neocortical application of the AT1R antagonist losartan or the ROS scavenger MnTBAP (Figure 8B)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In BPH/2J mice, ANGII levels were elevated in plasma but not in brain.

    “At this time, levels of ANGII were elevated in plasma but not brain (Supplemental Figure 5, B and C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    CLOD treatment does not affect the performance of normotensive controls or total exploration time, but ameliorates the recognition memory deficit in BPH/2J mice.

    “CLOD treatment did not affect the performance of the normotensive controls and the total exploration time in both groups, but greatly ameliorated the memory deficit in BPH/2J mice (Figure 9A).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    At1r-/-→WT chimeras showed markedly improved CBF responses to neural activation and acetylcholine compared with WT→WT chimeras.

    “However, in At1r -/-→WT chimeras CBF responses to neural activation and ACh were markedly improved (Figure 5D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In ANGII-treated mice, CLOD counteracts the attenuation of CBF responses to whisker stimulation and acetylcholine, implicating PVMs in the neurovascular dysfunction.

    “However, in ANGII-treated mice CLOD counteracted the attenuation of the vascular responses (Figure 3D), implicating PVMs in the mechanisms of the dysfunction.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Brain PVMs are myeloid cells closely associated with the abluminal side of cerebral blood vessels, express the mannose receptor CD206, express low levels of IBA1 relative to microglia, and are phagocytic.

    “Brain PVMs were identified as myeloid cells closely associated with the abluminal side of cerebral blood vessels and (a) expressing the mannose receptor CD206 (Figure 1 1D); (c) expressing low levels of IBA1, compared with microglial cells (Figure 1C); and (d) endowed of phagocytic activity assessed by injection of dextran into the lateral ventricles (Figure 1, B-D, and ref. 26).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    At day 14 of ANGII infusion, ANGII concentration was increased in plasma but not in brain.

    “At this time, ANGII concentration was increased in plasma (Figure 2B), but not in brain (Supplemental Figure 2, A-C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Angiotensin II hypertension or chronic blood pressure elevation in BPH/2J mice does not increase the number of perivascular macrophages.

    “ANGII hypertension or chronic elevation of BP in BPH/2J mice does not increase the number of PVMs or alter their homing from the circulation to the perivascular space.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CLOD administration in BPH/2J mice depletes PVMs in cortex and hippocampus and rescues CBF responses to whisker stimulation and acetylcholine.

    “CLOD administration (i.c.v.) in BPH/2J mice depleted PVMs in both cortex and hippocampus (Supplemental Figure 5, D and E), and rescued the response to both whisker stimulation and ACh (Figure 8D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In bone marrow chimeras, replacing wild-type perivascular macrophages with cells lacking NOX2 reduces angiotensin II-induced vascular oxidative stress and counteracts the associated neurovascular dysfunction.

    “Using BM chimeras we provided evidence that replacement of WT PVMs with PVMs lacking AT1Rs or NOX2 abrogates the vascular oxidative stress induced by ANGII and counteracts the associated neurovascular dysfunction.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: In bone marrow chimeras, the donor genotype in perivascular macrophages is responsible for the observed effects, rather than other donor-derived immune cells or indirect effects.

  • May combine two claims.

    Findingcitation

    Blood-brain barrier permeability tended to rise before hypertension onset, similar to the cerebrovascular dysfunction first seen 7 days after angiotensin II administration.

    “BBB permeability tended to increase before the onset of hypertension, like the cerebrovascular dysfunction, which is first observed 7 days after ANGII administration (54).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In bone marrow chimeras, replacing wild-type perivascular macrophages with cells lacking AT1 receptors reduces angiotensin II-induced vascular oxidative stress and counteracts the associated neurovascular dysfunction.

    “Using BM chimeras we provided evidence that replacement of WT PVMs with PVMs lacking AT1Rs or NOX2 abrogates the vascular oxidative stress induced by ANGII and counteracts the associated neurovascular dysfunction.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: In bone marrow chimeras, the donor genotype in perivascular macrophages is responsible for the observed effects, rather than other donor-derived immune cells or indirect effects.

  • May combine two claims.

    Findingcitation

    PVMs are most abundant on pial and parenchymal vessels larger than 20 μm in diameter, especially pial arterioles, and cover 70%-80% of their length.

    “PVMs were found most abundantly in association with pial and parenchymal vessels larger than 20 μm in diameter, particularly pial arterioles, a key site for flow regulation (7), the length of which was covered 70%-80% by PVMs (Supplemental Figure 1, A-E; supplemental material available online with this article; doi:10.1172/JCI86950DS1).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In BPH/2J mice, CBF responses to functional hyperemia and acetylcholine are attenuated, while the CBF response to adenosine is normal.

    “The increase in CBF induced by functional hyperemia or ACh was attenuated, whereas the CBF response to adenosine was normal (Figure 8B and Supplemental Table 1), attesting to the integrity of the vasorelaxing function of the smooth muscle.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At1r-/-→WT chimeras were protected from cerebrovascular dysfunction induced by neocortical ANGII but not from dysfunction induced by amyloid-β.

    “We found that At1r -/-→WT chimeras were protected from the cerebrovascular dysfunction induced by neocortical application of ANGII, but not of amyloid-β (Figure 6, A and B)”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Perivascular macrophages show weak positivity for the microglial marker IBA1.

    “weakly positive for the microglia marker IBA1 (C)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Perivascular macrophages express the lymphatic endothelial marker LYVE1.

    “expressing the endothelial lymphatic vessel marker LYVE1 (D)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Antagonism of AT1 receptors with losartan or scavenging of reactive oxygen species with MnTBAP counteracts the neurovascular dysfunction in BPH/2J mice.

    “(B) Antagonism of AT1Rs with losartan or ROS scavenging with MnTBAP counteracts the neurovascular dysfunction in BPH/2J mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Slow-pressor hypertension, but not acute intravenous angiotensin II, raises blood-brain barrier permeability to 3-kDa FITC-dextran.

    “(C) Slow pressor hypertension but not acute i.v.ANGII administration increases BBB permeability to FITC-dextran (MW 3 kDa).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    In BPH/2J mice, CLOD shows a tendency to decrease the distance traveled, but the result is not statistically significant.

    “(C) CLOD tends to reduce the distance traveled in BPH/2J mice, but the effect does not reach statistical significance.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Low-dose ANGII given for two weeks produces a slow-developing rise in blood pressure and profound changes in neurovascular coupling and endothelium-dependent vasodilation.

    “Administration of low doses of ANGII for 2 weeks, which results in a slow-developing rise in BP (“slow pressor” hypertension) (14), induces profound alterations in neurovascular coupling and endothelium-dependent vasodilation (10,15).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [14] source

      Not checked in the free run.

    • cites [10] source

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    • cites [15] source

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  • May combine two claims.

    Premisecitation

    The cerebrovascular dysfunction caused by slow-pressor ANGII is mediated by AT1 receptor activation and by vascular oxidative stress from a NOX2-containing NADPH oxidase.

    “The cerebrovascular dysfunction is mediated by activation of ANGII type 1 receptors (AT1Rs) and vascular oxidative stress produced by a NOX2-containing NADPH oxidase (10,15).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [10] source

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    • cites [15] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    PVMs are resident brain macrophages located in the intracerebral perivascular space, closely apposed to the outer vessel wall, and derived from hematopoietic precursors.

    “Residing in the intracerebral perivascular space, delimited by the glia limitans and the vascular basement membrane (Virchow-Robin space), PVMs are closely apposed to the outer vessel wall and originate from hematopoietic precursors (18,20,21).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [18] source

      Not checked in the free run.

    • cites [20] source

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    • cites [21] Vallières, 2003, Bone marrow-derived cells that populate the adult mouse brain preserve their hematopoietic identity, J Neurosci source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    PVMs express AT1 receptors and can produce large amounts of ROS through NOX2.

    “As macrophages, PVMs express AT1Rs and have the potential to produce large amounts of ROS through NOX2 (23,24).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [23] source

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    • cites [24] Yanagitani, 1999, Angiotensin II type 1 receptor-mediated peroxide production in human macrophages, Hypertension source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The Barnes maze examines spatial learning and memory by testing the ability to learn and remember the location of a target zone.

    “This test is similar to the water maze navigation task in that it examines the ability of the mouse to learn and remember the location of a target zone (spatial learning and memory) (42).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Later studies with more moderate and long-lasting blood pressure increases or chronic hypertension found regional angiotensin II-dependent increases in blood-brain barrier permeability in some cases, but it remained unclear whether angiotensin II itself crosses the blood-brain barrier in slow pressor angiotensin II hypertension.

    “Subsequent studies with more moderate and long-lasting increases in BP or in models of chronic hypertension demonstrated in some cases regional ANGII-dependent increases in BBB permeability (e.g., ref. 55), but it remained unclear whether ANGII itself was able to cross the BBB in slow pressor ANGII hypertension.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [55] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Innate immune cells, particularly macrophages, have been implicated in vascular remodeling and atherosclerosis through production of reactive oxygen species and cytokines.

    “In addition, innate immune cells, particularly macrophages, have been implicated in vascular remodeling and atherosclerosis through the production of ROS and cytokines (19).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] source

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  • May combine two claims.

    Study design and conductown data

    CLOD depletes PVMs by 60%-65% in vehicle- or ANGII-treated mice and does not alter the ANGII-induced increase in blood pressure.

    “CLOD depleted PVMs by 60%-65% in mice receiving vehicle or ANGII (Figure 3B), but did not alter the increase in BP (Figure 3C and Supplemental Figure 3E).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pioneering studies in lymphocyte-deficient mice established that T lymphocytes are required for blood pressure elevations in several hypertension models.

    “Pioneering studies in lymphocyte-deficient mice (Rag1 -/-mice) established that T lymphocytes are required for the BP elevations in several hypertension models (61).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [61] source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Although plasma angiotensin II levels are not consistently elevated in essential hypertension, activation of tissue-specific renin-angiotensin systems, including in brain and blood vessels, is thought to play a role.

    “Although plasma levels of ANGII are not consistently elevated in essential hypertension (13), activation of tissue-specific renin-angiotensin systems, including in brain and blood vessels, is thought to play a role (62).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [13] Romero, 1999, State-of-the-Art lecture. Role of angiotensin and oxidative stress in essential hypertension, Hypertension source

      Not checked in the free run.

    • cites [62] source

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  • May combine two claims.

    Study design and conductown data

    At1r deletion or ANGII administration did not affect PVM numbers.

    “No effect of At1r deletion or ANGII administration on PVM numbers was found (Supplemental Figure 4E).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The effect is mediated by increased blood-brain barrier permeability that allows ANGII to enter the perivascular space and activate AT1 receptors on PVMs, leading to NOX2-dependent ROS production.

    “This effect was mediated by an increase in blood-brain barrier permeability that allowed angiotensin II to enter the perivascular space and activate angiotensin type 1 receptors in PVMs, leading to production of ROS through the superoxide-producing enzyme NOX2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Resident brain perivascular macrophages are a novel component of the neurovascular unit that is highly relevant to brain pathophysiology.

    “Our data implicate brain PVMs in the cognitive deficits induced by hypertension, and identify resident brain PVMs as a novel constituent of the NVU highly relevant to brain pathophysiology.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Perivascular macrophages are a novel source of vascular oxidative stress in the cerebral microcirculation and have a strong impact on neurovascular regulation and cognitive function.

    “Thus, PVMs emerge as a novel source of vascular oxidative stress in the cerebral microcirculation with a profound impact on neurovascular regulation and cognitive function.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The novel object recognition task is a test of recognition memory that relies on mice exploring novel objects more than familiar ones.

    “To examine the role of PVMs in the cognitive deficits in BPH/2J mice, we first used the novel object recognition task, a test of recognition memory that relies on the propensity of mice to explore novel objects more than familiar ones (41).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    BPH/2J mice are a genetic model of lifelong hypertension and show some cardinal features of essential hypertension, including neurohumoral dysfunction, sympathetic activation, cardiac hypertrophy, and kidney atrophy.

    “BPH/2J mice are a model of lifelong hypertension on a genetic basis and manifest some of the cardinal features of essential hypertension, i.e., neurohumoral dysfunction, sympathetic activation, cardiac hypertrophy, and kidney atrophy (58,59).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [58] source

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    • cites [59] source

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  • May combine two claims.

    Premisecitation

    Early studies described blood-brain barrier opening after sudden blood pressure increases caused by high angiotensin II or other pressor agents, but those studies used massive blood pressure increases that produced acute vascular damage and total vasoparalysis.

    “Although early studies described BBB opening by sudden increases in BP induced by high concentrations of ANGII or other pressor agents, these investigations used massive increases in BP that caused acute vascular damage and total vasoparalysis (57).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [57] Mayhan, 1986, Disruption of the blood-brain barrier in cerebrum and brain stem during acute hypertension, Am J Physiol

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The Barnes maze avoids the strong aversive stimulus of swimming that may affect performance.

    “However, the Barnes maze has the advantage of avoiding the strong aversive stimulus of swimming that may affect performance (43).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] source

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  • May combine two claims.

    Premisecitation

    BPH/2J mice are a model of spontaneous lifelong hypertension with well-documented and reproducible cognitive deficits.

    “In this study, we elected to use middle-aged BPH/2J mice, a model of spontaneous lifelong hypertension resulting in well-documented and reproducible cognitive deficits (38).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The rescue of ANGII-induced cerebrovascular dysfunction by Nox2 deletion in PVMs was incomplete, possibly implicating ROS sources other than NOX2.

    “However, the rescue of the dysfunction was not complete, possibly implicating ROS sources other than NOX2 (33).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The neurovascular alterations in BPH/2J mice are mediated by AT1 receptors and ROS.

    “indicating that the neurovascular alterations are mediated by AT1Rs and ROS.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    AT1 receptors and reactive oxygen species are involved in the mechanisms of cerebral blood flow dysfunction in BPH/2J mice.

    “The CBF dysfunction was counteracted by neocortical superfusion with the AT1R antagonist losartan or the ROS scavenger MnTBAP, implicating AT1Rs and ROS in its mechanisms.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The effect is mediated by infiltration of the vascular wall by T lymphocytes and macrophages, leading to production of deleterious cytokines such as IL-17 and TNF-alpha, which contribute to increased vascular resistance driving hypertension and end-organ damage.

    “The effect is mediated by infiltration of the vascular wall by T lymphocytes and macrophages leading to production of deleterious cytokines, such as IL-17 and TNF-α, which, in turn, contribute to the increased vascular resistance driving the hypertension, as well as end-organ damage (61).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [61] source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Increasing evidence indicates that adaptive and innate immunity play a key role in the pathogenesis of hypertension and in associated end-organ damage.

    “Increasing evidence indicates that adaptive immunity and innate immunity play a key role in the pathogenesis of hypertension and in the associated end-organ damage (60).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    WT→WT chimeras exhibited normal cerebrovascular reactivity and ANGII-induced attenuation, indicating that irradiation and bone marrow transplantation did not alter basic CBF responses or their susceptibility to ANGII.

    “WT mice engrafted with WT BM (WT→WT) exhibited normal cerebrovascular reactivity and attenuation by ANGII (Figure 5D), indicating that the irradiation and BM transplantation did not alter basic CBF responses and their susceptibility to ANGII.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Perivascular macrophages become targets of circulating angiotensin II, which leads to AT1 receptor engagement, NOX2-dependent reactive oxygen species formation, and neurovascular and cognitive dysfunction.

    “However, PVMs become the target of circulating ANGII, leading to engagement of AT1Rs, NOX2-dependent ROS formation, and neurovascular and cognitive dysfunction.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: In bone marrow chimeras, the donor genotype in perivascular macrophages is responsible for the observed effects, rather than other donor-derived immune cells or indirect effects.

  • May combine two claims.

    Study design and conductown data

    In vehicle-treated mice, CLOD did not affect CBF increases evoked by whisker stimulation, acetylcholine, or adenosine.

    “Similarly, in vehicle-treated mice CLOD did not affect the CBF increases evoked by whisker stimulation, ACh, or adenosine (Figure 3D and Supplemental Table 1).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Observational studies indicate that angiotensin II antagonists can protect the brain from hypertension-induced cognitive dysfunction.

    “Neurovascular coupling and BBB are altered in essential hypertension (63), and observational studies indicate that ANGII antagonists can protect the brain from hypertension-induced cognitive dysfunction (64).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [64] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Perivascular macrophages are a new cellular component of the neurovascular unit that is critical for neurovascular diseases associated with vascular oxidative stress.

    “The findings unveil a previously unappreciated role of PVMs in the full expression of the neurovascular and neurocognitive dysfunction of hypertension, and identify PVMs as a new cellular component of the NVU of critical importance for neurovascular diseases associated with vascular oxidative stress.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    The specificity of the immunofluorescence for biotinylated angiotensin II was verified by omitting the FITC-conjugated antibody or by using the anti-biotin antibody without biotinylated angiotensin II.

    “The specificity of the immunofluorescence was verified by omission of the FITC-conjugated antibody or by use of the anti-biotin antibody in the absence of biotinylated ANGII.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Evidence from randomized clinical trials that treating hypertension reduces dementia risk and that drugs targeting angiotensin II and its receptors have a class-specific effect is lacking.

    “However, evidence from randomized clinical trials that treatment of hypertension reduces dementia risk and that there is a class-specific effect of drugs targeting ANGII and its receptors is lacking (3).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Circulating angiotensin II reaches the perivascular space through a breach in the blood-brain barrier and acts on AT1 receptors on perivascular macrophages, leading to NOX2 activation and reactive oxygen species production.

    “circulating ANGII reaches the perivascular space through a breach of the BBB and acts on ANGII type 1 receptors (AT1R) on PVMs, resulting in the activation of NOX2 and ROS production.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Bone marrow-derived cells that replace CD206+ cells in the brain after transplantation are perivascular macrophages and not other cell types.

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    Exploratory behavior in the novel object recognition test was assessed manually by an experimenter blinded to treatment group.

    “Exploratory behavior was later assessed manually by an experimenter blinded to the treatment group.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Neurovascular coupling and the blood-brain barrier are altered in essential hypertension.

    “Neurovascular coupling and BBB are altered in essential hypertension (63), and observational studies indicate that ANGII antagonists can protect the brain from hypertension-induced cognitive dysfunction (64).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [63] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The studies were conducted according to ARRIVE guidelines.

    “Studies were conducted, according to ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines), in male C57BL/6 mice (age 3-5 months, weight 25-30 g; JAX, The Jackson Laboratory), in male BPH/2J mice (age 5 months, weight 20-25 g; JAX stock 003005), and in their respective controls, male BPN/3J mice (age 5 months, weight 25-30 g; JAX stock 003004).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Analysis was performed in a blinded fashion.

    “No animals were excluded, animals were randomly assigned to treatment and control groups, and analysis was performed in a blinded fashion.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    CD206-positive cells were counted by an investigator blinded to treatment.

    “CD206 + cells were counted by an investigator blinded to the treatment on 10 randomly selected fields (×20 objective) within the somatosensory cortex and the hippocampus.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Bone marrow reconstitution was verified 5 weeks after irradiation by measuring the percentage of positive At1r or Nox2 genomic DNA in isolated blood leukocytes.

    “Reconstitution of BM cells was verified 5 weeks after irradiation by testing of the percentage of positive At1r or Nox2 genomic DNA in isolated blood leukocytes (65).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [65] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The dye hydroethidine is membrane-permeable and is oxidized by superoxide.

    “HE is a cell-permeant dye that is oxidized by superoxide (68).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [68] source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Sample size was determined according to power analysis.

    “Sample size was determined according to power analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    No animals were excluded from the analysis.

    “No animals were excluded, animals were randomly assigned to treatment and control groups, and analysis was performed in a blinded fashion.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    CD206-positive cells associated with biotinylated angiotensin II were counted by an investigator blinded to treatment.

    “CD206 + cells associated with biotinylated ANGII were counted by an investigator blinded to the treatment on randomly selected fields (×40 objective) within the somatosensory cortex.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Isotype control antibodies, fluorescence-minus-one staining, and negative-population staining were employed to set sorting gates.

    “Appropriate isotype control antibodies, “fluorescence minus one” staining, and staining of negative populations were used to establish sorting parameters.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Animals were randomly assigned to treatment and control groups.

    “No animals were excluded, animals were randomly assigned to treatment and control groups, and analysis was performed in a blinded fashion.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In chimeric mice, cerebral blood flow experiments were performed 14 weeks after bone marrow transplant, when most host perivascular macrophages had been replaced by donor macrophages.

    “In chimeric mice, CBF experiments were performed 14 weeks after the BM transplant when most of the host PVMs have been replaced by donor macrophages.”
    Quote in the paperSubstantial claim
69 of 69

  • The paper relies on this without support.

    The factors responsible for the functional alterations of the neurovascular unit in hypertension are poorly understood.

    “The factors responsible for these functional alterations of the NVU are poorly understood, and their exploration is essential to develop preventative or therapeutic approaches to mitigate the impact of hypertension on brain health.”

  • The paper relies on this without support.

    The vascular cell type that produces ROS and initiates the cerebrovascular dysfunction in ANGII hypertension remains unknown.

    “However, the vascular cell type(s) that produces reactive oxygen species (ROS) and initiates the dysfunction remains to be elucidated.”

  • The paper relies on this without support.

    It is unclear whether neurovascular dysfunction is required for the development of cognitive deficits.

    “Furthermore, it is unclear whether the neurovascular dysfunction is required for the development of cognitive deficits.”

  • The paper relies on this without support.

    BPH/2J mice are a more representative model of chronic hypertension in humans than the slow-pressor ANGII model.

    “To overcome these limitations, we also examined the role of PVMs in a model of spontaneous lifelong hypertension more representative of chronic hypertension in humans.”

  • The paper relies on this without support.

    Clodronate liposome treatment selectively depletes perivascular macrophages without affecting other cell types or processes that could rescue neurovascular or cognitive function.

    “To deplete cerebral CD206 + macrophages, clodronate-or PBS-loaded liposomes were prepared as previously described (27).”

  • The paper relies on this without support.

    In bone marrow chimeras, the donor genotype in perivascular macrophages is responsible for the observed effects, rather than other donor-derived immune cells or indirect effects.

    “Using BM chimeras we provided evidence that replacement of WT PVMs with PVMs lacking AT1Rs or NOX2 abrogates the vascular oxidative stress induced by ANGII and counteracts the associated neurovascular dysfunction.”

  • The paper relies on this without support.

    The cerebrovascular and cognitive deficits in BPH/2J mice are representative of hypertension-induced dysfunction rather than unrelated genetic traits.

    “BPH/2J mice are a model of lifelong hypertension on a genetic basis and manifest some of the cardinal features of essential hypertension, i.e., neurohumoral dysfunction, sympathetic activation, cardiac hypertrophy, and kidney atrophy (58,59).”

  • The paper relies on this without support.

    CLOD selectively depletes perivascular macrophages without affecting other brain cells.

    “(A and B) Intracerebroventricular CLOD administration induces depletion of cells expressing the PVM marker CD206 in the somatosensory cortex assessed by histology.”

  • The paper relies on this without support.

    Bone marrow-derived cells that replace CD206+ cells in the brain after transplantation are perivascular macrophages and not other cell types.

    “(B) By 14 weeks after the transplant, approximately 80% of CD206 + cells in the somatosensory cortex are replaced by GFP + bone marrow cells.”
9 of 9

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Lipid droplet accumulation in macrophages elevates ROS production, promoting neurovascular dysfunction.

    “LD accumulation in macrophages can elevate their ROS production (9, 11), promoting neurovascular dysfunction (67).”

    From Reducing microglial lipid load enhances β amyloid phagocytosis in an Alzheimer’s disease mouse model (2025) · cites this paper as [67]

  • CAMs can respond to inflammatory and chemical cues, including signals from microbiota and mechanical cues.

    “Being directly exposed to peripheral stimuli, they can respond to inflammatory and chemical cues, including signals from microbiota [369] and mechanical cues [370].”

    From Revisiting the Pathogenesis of X-Linked Adrenoleukodystrophy (2025) · cites this paper as [370]

    Citation [370] is attached to “mechanical cues,” not the microbiota signals attributed to [369]. The paper’s Discussion describes perivascular macrophage activation by angiotensin II, a chemical signal, but the passages searched for mechanical stimuli, pressure sensing, and shear stress do not establish that these cells respond to mechanical cues. (LLM, conf=0.73)

    Experimental — accuracy not yet measured

  • Hypertension is a leading risk factor for depression incidence in obesity and causes neurovascular changes that involve the actions of perivascular macrophages to stimulate oxidative stress and inflammation that lead to cognitive dysfunction.

    “Hypertension is a leading risk factor for depression incidence in obesity and causes neurovascular changes that involve the actions of perivascular macrophages to stimulate oxidative stress and inflammation that lead to cognitive dysfunction [166].”

    From The menace of obesity to depression and anxiety prevalence (2022) · cites this paper as [166]

Cites

Flagged first, then by citations

2 of the 71 references couldn’t be matched to a known paper.

Research works printed but not linked: 2

  1. 10.1006/exnr.2000.7618 · DOI · Paper page
  2. 10.1152/ajpheart.00679.2010 · DOI · Paper page

Cited by

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Related

Authors
Faraco, G., Sugiyama, Y., Lane, D., Garcia-Bonilla, L., Chang, H., Santisteban, M. M., Racchumi, G., Murphy, M., Van Rooijen, N., Anrather, J., & Iadecola, C.
Affiliations
Weill Cornell Medicine (US)
Corresponding author
Iadecola, Costantino
ORCID
Venue
Journal of Clinical Investigation
Publisher
American Society for Clinical Investigation
Published
2016-11-14
Volume
126
Issue
12
Pages
4674-4689
ISSN
  • 0021-9738 (ISSN-L)
  • 1558-8238
DOI
10.1172/jci86950
PMC
PMC5127678
OpenAlex
W2552399604
Semantic Scholar
0d40c3885f611fa0a162a7d2b301ccd4c8c8722e
Cited by
350
References
71
Citations by year
2026: 34 · 2025: 39 · 2024: 47 · 2023: 39 · 2022: 43 · 2021: 31 · 2020: 34 · 2019: 36 · 2018: 26 · 2017: 20 · 2016: 1
FWCI
15.16
Type
journal article
Language
en
Open access
Locations
  • Journal of Clinical Investigation (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Pure Amsterdam UMC (publishedVersion, other-oa)
  • Amsterdam UMC (VU Amsterdam) - Institutional Repository (publishedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
Funders
  • American Heart Association (15SDG22760007)
  • Fondation Leducq
  • National Institutes of Health
  • College of Veterinary Medicine, Cornell University
  • National Institute of Neurological Disorders and Stroke (R37NS089323, R01NS095441)
Topics
  • Life Sciences › Neuroscience › Neurology › Neurological Disease Mechanisms and Treatments
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
Keywords
perivascular macrophages, hypertension, cognitive impairment, neurovascular regulation, blood-brain barrier permeability, angiotensin II, angiotensin II type 1 receptor, reactive oxygen species, NADPH oxidase 2, oxidative stress, cerebrovascular disease, dementia risk
MeSH terms
NADPH Oxidase 2, Angiotensin II, Animals, Blood-Brain Barrier, Disease Models, Animal, Hypertension, Macrophages, Male, Membrane Glycoproteins, Reactive Oxygen Species, Oxidative Stress, NADPH Oxidases, Receptor, Angiotensin, Type 1, Mice, Cognitive Dysfunction

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

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