DISCUSSION
There are several novel findings of this study. First,
neuroinflammation, IgG deposition, transcriptomic changes and cognitive
impairment during hypertension are dependent on the degree of elevated
blood pressure. Second, treatment of hypertensive mice with hydralazine
for two weeks does not reverse brain inflammation or cognitive
impairment. Our findings are consistent with the concept that chronic
hypertension promotes brain inflammation and memory impairment, but
these effects may not be readily reversible by lowering blood pressure.
Chronic hypertension is known to cause brain injury, and indeed infusion
with angiotensin II or aldosterone can promote cerebrovascular
dysfunction (Chrissobolis, Drummond, Faraci, & Sobey, 2014) and
oxidative stress (Dinh et al., 2016). Hypertension may also promote
leukocyte infiltration in blood pressure-regulating organs including the
kidneys (Dinh et al., 2021; Krishnan et al., 2019) and blood vessels
(Chan et al., 2015; Wei et al., 2014). We and others have reported that
angiotensin II infusion promotes T cell infiltration in the brain
(Don-Doncow, Vanherle, Zhang, & Meissner, 2019; Wei et al., 2014), and
here we show that there are increases in several immune cell subsets in
additional to T cells in the brain during two models of experimental
hypertension. Under normotensive conditions, circulating cells and
numerous molecules are prevented from entering the brain by the
blood-brain barrier, but chronic hypertension can cause blood-brain
barrier breakdown, allowing entry to the brain of a variety of factors
such as immune cells. IgG is one such class of circulating proteins that
do not normally penetrate the blood-brain barrier, but our finding of
significant IgG deposition in the hippocampus and cortex during
hypertension is indicative of hypertension-induced blood-brain barrier
dysfunction. These findings are analogous to reports that peripheral
immune infiltrate the brain stroke (Qian et al., 2018) and in
Alzheimer’s disease (Zenaro et al., 2015). In our study, oral
administration of the anti-hypertensive agent hydralazine prevented
angiotensin II-induced hypertension as well as IgG deposition and immune
cell infiltration, suggesting that these latter changes were also blood
pressure-dependent. Hydralazine also prevented angiotensin II-induced
expression of Ccr2 , and its ligands Ccl2 and Ccl8 ,
consistent with Ccr2 -mediated immune cell infiltration as a key
aspect of brain inflammation during hypertension.
Hypertension is a major risk factor for cognitive impairment in humans,
and our data are consistent with such a relationship in mouse models
(Csiszar et al., 2013; Foulquier et al., 2018). Importantly, our study
provides direct evidence that angiotensin II-induced cognitive
impairment is indeed blood pressure-dependent. There are a number of
ways in which hypertension could cause brain injury and cognitive
impairment, such as through disruption of cerebral artery structure and
function which could ultimately result in cerebral small-vessel disease,
ischemic white matter lesions and vascular dementia (Iadecola et al.,
2016; Verhaaren et al., 2013). Indeed, higher systolic blood pressure is
associated with faster decline in cognition (Levine et al., 2019), and
poorer cognition is associated with more severe hypertension (Muela et
al., 2017). While results from the Systolic Blood Pressure Intervention
Trial (SPRINT) substudy Memory and Cognition in Decreased Hypertension
(SPRINT MIND) suggest that intensive reduction in systolic blood
pressure (<120 mmHg) decreases the risk of developing mild
cognitive impairment but not dementia in patients compared to standard
reduction in systolic blood pressure (<140 mmHg) (Group et
al., 2019), there were no apparent effects on brain inflammation and
cognitive deficit after just two weeks of hypertension reversal in our
study in mice.
Accumulating evidence has suggested inflammation to be a mediator of
cognitive impairment (Skelly et al., 2018). Vascular inflammation is one
mechanism by which hypertension may alter the structure and function of
cerebral blood vessels through processes such as vascular remodelling
(Shenoy et al., 2018) and fibrosis (Aldrich & Kielian, 2011).
Furthermore, inflammation and immunity are features of cerebral small
vessel disease (Fu & Yan, 2018). Since anti-hypertensive therapy with
hydralazine prevented both angiotensin II-induced brain inflammation and
cognitive impairment, our data are consistent with the concept that
hypertension-induced brain inflammation leads to cognitive impairment.
Indeed, targeting inflammation is reported to reduce cognitive
impairment during hypertension (Faraco et al., 2016; Jalal, Yang,
Thompson, Roitbak, & Rosenberg, 2015) and other cognitive disorders (Fu
et al., 2019; Ni et al., 2018).
Bulk RNA sequencing showed that angiotensin II infusion upregulated
genes in the brain involved in biological processes such as inflammation
(Gdf7, Elf3 ), immune cell migration (Ccl8, Ccr1 ),
macrophage activity (Cd163l1, Cd209 ), T cell signalling and
proliferation (Cd3d, Trbjj2-3, H2-q6, Hsh2d, Il6 ), PANoptosis
(Zbp1 ), fibrosis (Fgf23 ) and thrombosis (F12 ). This
supports our flow cytometry data that angiotensin II activated pathways
that promote immune cell migration and neuroinflammation. Angiotensin II
downregulated genes involved in biological processes such as memory
consolidation (Arc ), anti-inflammatory (Dusp1 ), T
regulatory cell development (Junb ), macrophage differentiation
(Egr2 ), negative regulation of microglia proliferation
(Btg2 ), inhibition of apoptosis (Nr4a1 ) and the complement
pathway (C7, Cr2 ). Arc is a master regulator of long-term
memory formation (Plath et al., 2006), hence, downregulation ofArc may have contributed to the cognitive decline observed in
angiotensin II-infused mice.
In the hippocampus, a key brain region for learning and memory,
angiotensin II promoted upregulation of genes involved in inflammation
(Igsf6, Hyal1 ), T cell differentiation and activity
(Il12rb2, Ifitm7, Il2rb, Tnfrsf25 ), microglia regulation
(Kdm4a, Dok3 ), cell adhesion (Pcdhgb4, Icam1 ) and
apoptosis (Anxa11, Bcl9 ). Immune cells specifically in the
hippocampus were not analysed in our study but the data are suggestive
of increased immune cell infiltration as there was upregulation of the
cell adhesion molecule, Icam1, and evidence of BBB injury.
Il12rb2, Ifitm7, Il2rb and Tnfrsf25 are involved in T cell
differentiation and proliferation, consistent with a T cell response in
the hippocampus. We showed an increased brain infiltration of T cells,
which have been reported to promote cognitive impairment in a mouse
model of Alzheimer’s disease (Machhi et al., 2021). Hence, the presence
of T cells in the hippocampus may have contributed to the development of
cognitive impairment.
Angiotensin II downregulated genes involved in anti-inflammatory
pathways such as Dusp1 and Btg2 . Overexpression ofDusp1 attenuates inflammation in a mouse model of cardiomyopathy
(Tan et al., 2022) while deficiency of Dusp1 has been shown to
exacerbate inflammation in a mouse model of septic peritonitis (Hammer
et al., 2010). Deficiency of Btg2 has been found to increase
activation of microglia and impair spatial learning ability in a mouse
model of chronic cerebral hypoperfusion (Suzuki et al., 2021).
Downregulation of these genes thus likely contributed to a
pro-inflammatory response to angiotensin II. Co-treatment with
angiotensin II + hydralazine caused upregulation of Dusp1 andBtg2 suggesting that hydralazine reduced neuroinflammation partly
through these pathways. Co-administration of angiotensin II and
hydralazine also promoted downregulation of markers of immune cells and
their activity (Sh2d2a, Ly6g ), immune cell migration
(Ccl8 ) and immunoglobulin binding (Umod, Pigr) consistent
with protective effects of hydralazine on neuroinflammation.
Interestingly, administration of hydralazine with angiotensin II
reversed the upregulation of Il12rb2 in the hippocampus caused by
angiotensin II alone. As mentioned, Il12rb2 is involved in T cell
differentiation and proliferation and so the resulting reduction in
hippocampal neuroinflammation by hydralazine may have contributed to the
preservation of cognitive function during angiotensin II infusion,
potentially as an indirect effect of blood pressure reduction.
It is noteworthy that, while we found that development of brain
inflammation and cognitive impairment during hypertension were blood
pressure-dependent, intervention with hydralazine to lower blood
pressure in established hypertension did not readily reverse immune cell
infiltration in the brain or cognitive impairment. However, it is
plausible that a period of just 2 weeks of hydralazine administration is
insufficient to resolve the brain inflammation that develops in response
to angiotensin II-induced hypertension which may persist after the
initial hypertensive stimulus has abated, consistent with several
clinical effects of anti-hypertensive therapy on cognitive dysfunction
(Stuhec, Keuschler, Serra-Mestres, & Isetta, 2017). New strategies that
target both blood pressure and inflammation may be a more effective
approach to treat cognitive impairment.
It seems likely that breakdown of the blood-brain barrier was an
important factor in hypertension-induced cognitive impairment.
Blood-brain barrier dysfunction is associated with early cognitive
decline in vascular cognitive impairment (Li, Li, Zuo, Hu, & Jiang,
2021) and Alzheimer’s disease patients (Bowman et al., 2018) even in the
absence of amyloid-β and tau changes (Nation et al., 2019). Our data
reveal that in hypertensive mice the blood-brain barrier was compromised
in the hippocampus and cortex, key brain regions involved in cognition.
Besides facilitating immune cell infiltration, a leaky blood-brain
barrier is also associated with pathological events such as white matter
hyperintensities (Zhang et al., 2019) which can contribute to the
development of cognitive impairment.
The present findings indicate that hypertensive stimuli can promote
brain inflammation, cognitive impairment and changes to the brain
transcriptome in a blood pressure-dependent manner. The processes
involved in neuroinflammation during hypertension are complex and our
findings suggest that blood-brain barrier breakdown and CCR2 expression
are likely to be involved, and may contribute to the development of
cognitive impairment. As we found no evidence that drug-induced
attenuation of established hypertension could reduce brain inflammation
or cognitive dysfunction, our study highlights the importance of
managing hypertension to reduce the risk of developing neuroinflammation
and cognitive impairment.