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.