A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.

Male and female high-fat diet-fed Dahl SS rats are largely protected from vascular dysfunctions: PVAT contributions reveal sex differences.

Vascular dysfunctions are observed in the arteries from hypertensive subjects. The establishment of the Dahl salt-sensitive (SS) male and female rat models to develop a reproducible hypertension with high-fat (HF) diet feeding from weaning allows addressing the question of whether HF diet-associated hypertension results in vascular dysfunction similar to that of essential hypertension in both sexes. We hypothesized that dysfunction of three distinct vascular layers, i.e., endothelial, smooth muscle, and perivascular adipose tissue (PVAT), would be present in the aorta from HF diet-fed versus control diet-fed male and female rats. Dahl SS rats were fed a control (10% kcal of fat) or HF (60%) diet from weaning for 24 wk. Male and female Dahl SS rats became equally hypertensive when placed on a HF diet. For male and female rats, the thoracic aorta exhibited medial hypertrophy in HF diet-induced hypertension versus control, but neither displayed a hyperresponsive contraction to the α-adrenergic agonist phenylephrine nor an endothelial cell dysfunction as measured by acetylcholine-induced relaxation. A beneficial PVAT function, support of stress relaxation, was reduced in the male versus female rats fed a HF diet. PVAT in the aorta of males but not in females retained the anticontractile activity. We conclude that this HF model does not display the same vascular dysfunctions observed in essential hypertension. Moreover, both male and female show significantly different vascular dysfunctions in this HF feeding model.NEW & NOTEWORTHY Although the aorta exhibits medial hypertrophy in response to HF diet-induced hypertension, it did not exhibit hyperresponsive contraction to an α-adrenergic agonist nor endothelial cell dysfunction; this was true for both sexes. Unlike other hypertension models, PVAT around aorta from (male) rats on the HF diet retained significant anticontractile activity. PVAT around aorta of the male on a HF diet was modestly more fibrotic and lost the ability to assist in arterial stress relaxation.

Deep transcriptomic profiling of Dahl salt-sensitive rat kidneys with mutant form of Resp18.

Expression of Regulated endocrine specific protein 18 (Resp18) is localized in numerous tissues and cell types; however, its exact cellular function is unknown. We previously showed that targeted disruption of the Resp18 locus in the Dahl SS (SS) rat (Resp18mutant) results in higher blood pressure (BP), increased renal fibrosis, increased urinary protein excretion, and decreased mean survival time following a chronic (6 weeks) 2% high salt (HS) diet compared with the SS rat. Based on this prominent renal injury phenotype, we hypothesized that targeted disruption of Resp18 in the SS rat promotes an early onset hypertensive-signaling event through altered signatures of the renal transcriptome in response to HS. To test this hypothesis, both SS and Resp18mutant rats were exposed to a 7-day 2% HS diet and BP was recorded by radiotelemetry. After a 7-day exposure to the HS diet, systolic BP was significantly increased in the Resp18mutant rat compared with the SS rat throughout the circadian cycle. Therefore, we sought to investigate the renal transcriptomic response to HS in the Resp18mutant rat. Using RNA sequencing, Resp18mutant rats showed a differential expression of 25 renal genes, including upregulation of Ren. Upregulation of renal Ren and other differentially expressed genes were confirmed via qRT-PCR. Moreover, circulating renin activity was significantly higher in the Resp18mutant rat compared with the WT SS rat after 7 days on HS. Collectively, these observations demonstrate that disruption of the Resp18 gene in the SS rat is associated with an altered renal transcriptomics signature as an early response to salt load.

CACNB2 is associated with aberrant RAS-MAPK signaling in hypertensive Dahl Salt-Sensitive rats.

Several independent genome-wide association studies (GWAS) have indicated that calcium (Ca2+) voltage-gated channel auxiliary subunit beta 2 (CACNB2) an L-type Ca2+ channel (LTCC) associated protein has strong association with hypertension. However, the molecular mechanism of CACNB2 and its role in the pathophysiology of hypertension is not clear. To address this knowledge gap, we utilized in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt-Sensitive (SS) rats. We demonstrated that CACNB2 over-expression in HEK293 cells triggers cell proliferation via an up-regulation of the RAS-MAPK pathway compared to non-transfected cells. These effects were likely independent of LTCC activity as treatment with nifedipine, a well-known LTCC blocker, in CACNB2 overexpressing cells failed to inhibit the RAS-MAPK pathway gene expressions or show an effect on apoptosis marker gene expression. Furthermore, the expression level of CACNB2 was up-regulated in the high salt (HS) diet fed SS rat kidneys compared to low salt diet (LS) fed group. Similar to our in vitro observation the RAS-MAPK mRNA levels were increased in HS fed SS rat kidneys, compared to LS fed group. Collectively, our data suggest that CACNB2 is associated with the increase in RAS-MAPK gene expressions and lead us to speculate that in addition to its role in regulating LTCC α1-subunit trafficking, CACNB2 might lead to aberrant RAS activation, which is one of the key cascade associated with hypertension.

Targeted disruption of regulated endocrine-specific protein ( Resp18) in Dahl SS/Mcw rats aggravates salt-induced hypertension and renal injury.

Hypertension is a classic example of a complex polygenic trait, impacted by quantitative trait loci (QTL) containing candidate genes thought to be responsible for blood pressure (BP) control in mammals. One such mapped locus is on rat chromosome 9, wherein the proof for a positional candidate gene, regulated endocrine-specific protein-18 ( Resp18) is currently inadequate. To ascertain the status of Resp18 as a BP QTL, a custom targeted gene disruption model of Resp18 was developed on the Dahl salt-sensitive (SS) background. As a result of this zinc-finger nuclease (ZFN)-mediated disruption, a 7 bp deletion occurred within exon 3 of the Resp18 locus. Targeted disruption of Resp18 gene locus in SS rats decreases its gene expression in both heart and kidney tissues regardless of their dietary salt level. Under a high-salt dietary regimen, both systolic and diastolic BP of Resp18mutant rats were significantly increased compared with SS rats. Resp18mutant rats demonstrated increased renal damage, as evidenced by higher proteinuria and increased renal fibrosis compared with SS rats. Furthermore, under a high-salt diet regimen, the mean survival time of Resp18mutant rats was significantly reduced compared with SS rats. These findings serve as evidence in support of Resp18 as a gene associated with the development of hypertension and renal disease.

Reactive oxygen species as important determinants of medullary flow, sodium excretion, and hypertension.

The physiological evidence linking the production of superoxide, hydrogen peroxide, and nitric oxide in the renal medullary thick ascending limb of Henle (mTAL) to regulation of medullary blood flow, sodium homeostasis, and long-term control of blood pressure is summarized in this review. Data obtained largely from rats indicate that experimentally induced elevations of either superoxide or hydrogen peroxide in the renal medulla result in reduction of medullary blood flow, enhanced Na(+) reabsorption, and hypertension. A shift in the redox balance between nitric oxide and reactive oxygen species (ROS) is found to occur naturally in the Dahl salt-sensitive (SS) rat model, where selective reduction of ROS production in the renal medulla reduces salt-induced hypertension. Excess medullary production of ROS in SS rats emanates from the medullary thick ascending limbs of Henle [from both the mitochondria and membrane NAD(P)H oxidases] in response to increased delivery and reabsorption of excess sodium and water. There is evidence that ROS and perhaps other mediators such as ATP diffuse from the mTAL to surrounding vasa recta capillaries, resulting in medullary ischemia, which thereby contributes to hypertension.

A Cell Atlas of Thoracic Aortic Perivascular Adipose Tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. To examine the cell-specificity of recognized mechanotransducers we used single nuclei RNA sequencing (snRNAseq) of the thoracic aorta PVAT (taPVAT) from male Dahl SS rats compared to subscapular brown adipose tissue (BAT). Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNAseq, identifying 8 major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. Presence of PIEZO1 in the PVAT was confirmed by RNAscope® and IHC; antagonism of PIEZO1 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.

Genetic Modifications to Alter Blood Pressure Level.

Genetic manipulation is one of the indispensable techniques to examine gene functions both in vitro and in vivo. In particular, cardiovascular phenotypes such as blood pressure cannot be evaluated in vitro system, necessitating the creation of transgenic or gene-targeted knock-out and knock-in experimental animals to understand the pathophysiological roles of specific genes on the disease conditions. Although genome-wide association studies (GWAS) in various human populations have identified multiple genetic variations associated with increased risk for hypertension and/or its complications, the causal links remain unresolved. Genome-editing technologies can be applied to many different types of cells and organisms for creation of knock-out/knock-in models. In the post-GWAS era, it may be more worthwhile to validate pathophysiological implications of the risk variants and/or candidate genes by creating genome-edited organisms.

Effects of zamicastat treatment in a genetic model of salt-sensitive hypertension and heart failure.

Hyperactivity of sympathetic nervous system plays an important role in the development and progression of cardiovascular diseases. An approach to mitigate the enhanced sympathetic nervous system drive is restricting the biosynthesis of noradrenaline via inhibition of the enzyme dopamine ß-hydroxylase (DßH), that catalyzes the hydroxylation of dopamine to noradrenaline in sympathetic nerves. The aim of the present study was to evaluate the effects of zamicastat, a novel DßH inhibitor that decreases noradrenaline and increases dopamine levels in peripheral sympathetically innervated tissues, on the hemodynamic and cardiometabolic parameters in salt-induced hypertension and heart failure in the Dahl salt-sensitive (SS) rat. Zamicastat (10, 30 and 100 mg/kg body weight) was tested acutely against salt-induced hypertension in the Dahl SS rat. Chronic zamicastat treatment (30 mg/kg/day) was evaluated against salt-induced cardiac hypertrophy and biomarkers of cardiometabolic risk and inflammation in Dahl SS rats and upon the survival rate in aged Dahl SS rats fed a high-salt diet. The reduction in the sympathetic tone attained with zamicastat shaped a dose- and time-dependent effect on blood pressure. Prolonged treatment with zamicastat ameliorated end-organ damage, metabolic syndrome and inflammation hallmarks in hypertensive Dahl SS rats. Survival rate of Dahl SS rats fed a high-salt diet demonstrated that zamicastat increased median survival of Dahl SS rats fed a high-salt diet. The use of DßH inhibitors, like zamicastat, is a promising approach to treat hypertension, heart failure and cardiovascular diseases where a reduction in the sympathetic tone has beneficial effects.

Progression of glomerular filtration rate reduction determined in conscious Dahl salt-sensitive hypertensive rats.

Sequential changes in glomerular filtration rate during development of hypertension in the conscious Dahl salt-sensitive rats were determined using a new method for measurement. Using a miniaturized device, disappearance curves of fluorescein isothiocyanate-sinistrin were measured by transcutaneous excitation and real-time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure by telemetry) were studied, while maintained on a 0.4% NaCl diet and on days 2, 5, 7, 14, and 21 after switching to 4.0% (high-salt [HS]) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. Mean arterial pressure rose progressively from the last day of 0.4% (130±2 mm Hg) reaching significance by day 5 of HS and averaged 162±7 mm Hg by day 21. Urine albumin excretion was significantly elevated (×3) by day 7 of HS in Dahl salt-sensitive rats. Glomerular filtration rate reduced on day 14 of HS falling from 1.53±0.06 mL/min per 100 g body weight to 1.27±0.04. By day 21, glomerular filtration rate had fallen 28% to 1.1±0.04 mL/min per 100 g (t(1/2) 28.4±1.1 minute.) No significant reductions of creatinine clearance were observed throughout the study in response to HS demonstrating the insensitivity of creatinine clearance measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of glomerular filtration rate was a consequence and not a cause of the hypertension and that this noninvasive approach could be used in these conscious Dahl salt-sensitive rats for a longitudinal assessment of renal function.