Frank M. Faraci

BACKGROUND: Hypertension is a leading risk factor for negative health outcomes due to end-organ effects that include small vessel disease in the brain. Low-renin hypertension is understudied at the blood pressure (BP), microvascular, and mechanistic level, and in relation to biological sex. This study examined the effects of low-renin hypertension, produced by activation of the brain renin-angiotensin system in a deoxycorticosterone acetate (DOCA) salt model. METHODS: C57BL/6J mice were treated with DOCA (or sham) and given tap H ₂ O and H ₂ O with 0.15 mol/L NaCl for 3 to 4 weeks followed by assessment of the microvasculature. Mean arterial pressure and BP variability were measured using radiotelemetry. RESULTS: Baseline and diurnal changes in mean arterial pressure, increases in mean arterial pressure, and BP variability during DOCA salt, were greater in male than female mice. Compared with sham treatment, endothelial function of cerebral arterioles in vivo was reduced by >70% by DOCA salt in males, dysfunction that could be reversed by local inhibition of AT1R (angiotensin II type 1 receptor), MR (mineralocorticoid receptor), or Rho kinase. DOCA salt increased arteriolar cross-sectional area and wall stiffness in male, but not female mice. In males (but not females), performance on a novel object recognition test was selectively impaired. CONCLUSIONS: Activation of the central renin-angiotensin system has sex-specific effects on BP, diurnal changes in BP, BP variability, arteriolar structure, and stiffness. Marked endothelial dysfunction was present in males (with several contributing mechanisms). These findings provide new insight into BP-related and small vessel disease–related phenotypes, mechanisms that contribute to endothelial dysfunction, and sex-specific differences in BP traits in a preclinical model of low-renin hypertension.

Hypertension is extremely common, affecting approximately 1 in every 2 adults globally. Chronic hypertension is the leading modifiable risk factor for cardiovascular disease and premature mortality worldwide. Despite considerable efforts to define mechanisms that underlie hypertension, a potentially major component of the disease, the role of circadian biology has been relatively overlooked in both preclinical models and humans. Although the presence of daily and circadian patterns has been observed from the level of the genome to the whole organism, the functional and structural impact of biological rhythms, including mechanisms such as circadian misalignment, remains relatively poorly defined. Here, we review the impact of daily rhythms and circadian systems in regulating blood pressure and the onset, progression, and consequences of hypertension. There is an emphasis on the impact of circadian biology in relation to vascular disease and end-organ effects that, individually or in combination, contribute to complex phenotypes such as cognitive decline and the loss of cardiac and brain health. Despite effective treatment options for some individuals, control of blood pressure remains inadequate in a substantial portion of the hypertensive population. Greater insight into circadian biology may form a foundation for novel and more widely effective molecular therapies or interventions to help in the prevention, treatment, and management of hypertension and its related pathophysiology.

Brain endothelial cells (BECs) have unique roles in controlling cerebral blood flow (CBF) and as a cornerstone of the blood–brain barrier (BBB). For example, they influence baseline CBF and act as sensors of moment-to-moment changes in neural activity. They participate in vascular remodelling and regulate immune cell interactions, thereby contributing to the maintenance of brain homoeostasis. Despite such advances, we have only scratched the surface in understanding the contribution of BECs to vascular control and subsequently brain health. Brain endothelial cells are a signalling focal point, integrating cellular, blood-borne, and mechanical stimuli. Acutely, this input is translated into electrical (ionic) or molecular signals that control vascular resistance, re-directing local CBF to regions of increased cellular activity.¹ Brain endothelial cells also exert chronic effects on mural cells (pericytes and vascular muscle), glia, and neurons. Single-cell RNA sequencing (scRNAseq) studies in humans and mice have uncovered a largely unanticipated zonation- and organ-specific heterogeneity of endothelial cells,² the impact of which is relatively poorly defined in relation to brain health. For instance, capillary ECs show enrichment in transport, metabolism, and O₂-response genes in accord with their positioning at the BBB, whereas ECs from arteries, arterioles, and venules are enriched in genes related to remodelling, structural organization, and inflammation, respectively.^2–4 Accounting for this heterogeneity is critical when studying cerebrovascular disease and for therapeutic targeting. As an example, the cell type expressing the most Alzheimer’s disease (AD) and AD-related trait genes [based on genome-wide association studies (GWAS) in humans] is BECs.⁵

The brain renin-angiotensin-aldosterone system (RAAS) regulates many physiological processes including fluid and electrolyte balance, vascular structure and function, blood pressure, cognition, and other aspects of brain function. Treatment with the mineralocorticoid deoxycorticosterone acetate and salt stimulates the local RAAS within the brain. In this chapter, we describe the surgical procedures used to induce activation of the brain RAAS with deoxycorticosterone acetate and salt. This technique can be used for studies of hypertension, cerebrovascular biology and dysfunction, and other diseases that impact brain health. Key words Cerebral artery Deoxycorticosterone acetate Renin-angiotensin-aldosterone system Angiotensin II

Changes in vascular structure contribute to vascular events and loss of brain health. We examined the inherent structural plasticity of cerebral arterioles during and after a period of hypertension. Arteriolar wall thickness, diameter, wall-to-lumen ratio, and biological stiffness changed rapidly during hypertension and recovered with blood pressure. In contrast, inward remodeling developed slowly and did not recover. This lack of recovery of arteriolar diameter has implications for the long-term impact of hypertension on vascular determinants of brain health.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that controls the transactivation of hundreds of target genes including RHOBTB1. Both genetic and pharmacological evidence suggested that PPARγ exerts cardioprotection and can lower arterial blood pressure but the target genes that mediate the anti-hypertensive effect of PPARγ remain unclear. Rho-related BTB domain-containing protein 1 (RhoBTB1) is an atypical Rho GTPase whose function is obscure. Notably, two genome-wide association studies (GWAS) reported association between RHOBTB1 with blood pressure. To understand whether RhoBTB1 mediates the anti-hypertensive effect of PPARγ, we first utilized S-P467L mice which express a dominant-negative PPARγ mutation (P467L) specifically in smooth muscle. The P467L mutation in PPARγ causes human hypertension. As a transcriptional target of PPARγ, RhoBTB1 was significantly lowered in the aorta of S-P467L mice. Remarkably, restoration of RhoBTB1 in the vasculature by genetic complementation reversed hypertension, aortic stiffness, and impaired vasodilation in the S-P467L mice. Further mechanistic studies showed that RhoBTB1 downregulated the activity of phosphodiesterase 5 (PDE5) which is a negative regulator of the vasodilation pathway mediated by nitric oxide/cGMP. Moreover, RhoBTB1 increased the ubiquitination of PDE5 in a Cullin-3-dependent manner, suggesting that RhoBTB1 might serve as a substrate adaptor for Cullin-3-RING E3 ubiquitin ligase. Next, we studied the role of RhoBTB1 in angiotensin II (Ang-II)-mediated hypertension because the renin-angiotensin system is an important determinant in clinical hypertension and inhibitors of the renin-angiotensin system are widely used in human patients to control their hypertension. Systemic infusion of Ang-II in mice led to hypertension, increased aortic stiffness, impaired vascular reactivity, and decreased RhoBTB1 expression in the aorta. Restoring RhoBTB1 in the vasculature in mice with pre-established Ang-II hypertension attenuated the aortic stiffness but did not lower the blood pressure or improve vascular function. To understand how RhoBTB1 lowered aortic stiffness, we employed RNA-seq along with various biochemical assays to interrogate individual mechanistic contributors to aortic stiffness. Interestingly, RhoBTB1 showed no profound effect on several classical aortic stiffness contributors such as collagen deposition, elastin abundance, vascular wall area, among others. Instead, restoration of RhoBTB1 abolished the increase of actin polymerization caused by Ang-II. Additionally, in response to RhoBTB1 restoration, changes in the protein levels of two actin polymerization regulators, Cofilin and vasodilator-stimulated phosphoprotein (VASP), were consistent with the changes in actin polymerization. Collectively, our studies revealed distinct physiological and molecular functions of RhoBTB1 in the vasculature.