Integrins play a role in stress relaxation of perivascular adipose tissue.

Perivascular adipose tissue (PVAT) is known for being anti-contractile in healthy tissues. We discovered a new function of PVAT, the ability to stress relax and maintain a tone in response to a stretch. This is of note because stress relaxation has been attributed to smooth muscle, of which PVAT has none that is organized in a functional layer. We test the hypothesis the interactions of integrins with collagen play a role in stress relaxation. Our model is the thoracic aorta of the male Dahl SS rat. The PVAT and aorta were physically separated for most assays. Results from single nuclei RNA sequencing (snRNAseq) experiments, histochemistry and isometric contractility were also used. Masson Trichrome staining made evident the expression of collagen in PVAT. From snRNA seq experiments of the PVAT, mRNA for multiple collagen and integrin isoforms were detected: the α1 and ß1 integrin were most highly expressed. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1ß1 distintegrin obtustatin or general integrin inhibitor RGD peptide. RGD peptide but not obtustatin increased the stress relaxation. Cell-cell communication inference identified integrins αv and α5, two major RGD motif containing isoforms, as potential signaling partners of collagens. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so in part through integrin-collagen interactions that may not include α1ß1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.

Innervation of adipocytes is limited in mouse perivascular adipose tissue.

Perivascular adipose tissue (PVAT) regulates vascular tone by releasing anticontractile factors. These anticontractile factors are driven by processes downstream of adipocyte stimulation by norepinephrine; however, whether norepinephrine originates from neural innervation or other sources is unknown. The goal of this study was to test the hypothesis that neurons innervating PVAT provide the adrenergic drive to stimulate adipocytes in aortic and mesenteric perivascular adipose tissue (aPVAT and mPVAT), and white adipose tissue (WAT). Healthy male and female mice (8-13 wk) were used in all experiments. Expression of genes associated with synaptic transmission were quantified by qPCR and adipocyte activity in response to neurotransmitters and neuron depolarization was assessed in AdipoqCre+;GCaMP5g-tdTf/WT mice. Immunostaining, tissue clearing, and transgenic reporter lines were used to assess anatomical relationships between nerves and adipocytes. Although synaptic transmission component genes are expressed in adipose tissues (aPVAT, mPVAT, and WAT), strong nerve stimulation with electrical field stimulation does not significantly trigger calcium responses in adipocytes. However, norepinephrine consistently elicits strong calcium responses in adipocytes from all adipose tissues studied. Bethanechol induces minimal adipocyte responses. Imaging neural innervation using various techniques reveals that nerve fibers primarily run alongside blood vessels and rarely branch into the adipose tissue. Although nerve fibers are associated with blood vessels in adipose tissue, they demonstrate limited anatomical and functional interactions with adjacent adipocytes, challenging the concept of classical innervation. These findings dispute the significant involvement of neural input in regulating PVAT adipocyte function and emphasize alternative mechanisms governing adrenergic-driven anticontractile functions of PVAT.NEW & NOTEWORTHY This study challenges prevailing views on neural innervation in perivascular adipose tissue (PVAT) and its role in adrenergic-driven anticontractile effects on vasculature. Contrary to existing paradigms, limited anatomical and functional connections were found between PVAT nerve fibers and adipocytes, underscoring the importance of exploring alternative mechanistic pathways. Understanding the mechanisms involved in PVAT's anticontractile effects is critical for developing potential therapeutic interventions against dysregulated vascular tone, hypertension, and cardiovascular disease.

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.

How New Developments in Pharmacology Receptor Theory Are Changing (Our Understanding of) Hypertension Therapy.

BACKGROUND: Many hypertension therapeutics were developed prior to major advances in drug receptor theory. Moreover, newer drugs may take advantage of some of the newly understood modalities of receptor function. GOAL: The goal of this review is to provide an up-to-date summary of drug receptor theory. This is followed by a discussion of the drug classes recognized for treating hypertension to which new concepts in receptor theory apply. RESULTS: We raise ideas for mechanisms of potential new antihypertensive drugs and whether they may take advantage of new theories in drug-receptor interaction.

Perivascular Adipose Tissue Remodels Only after Elevation of Blood Pressure in the Dahl SS Rat Fed a High-Fat Diet.

INTRODUCTION: Tunica media extracellular matrix (ECM) remodeling is well understood to occur in response to elevated blood pressure, unlike the remodeling of other tunicas. We hypothesize that perivascular adipose tissue (PVAT) is responsive to hypertension and remodels as a protective measure. METHODS: The adventitia and PVAT of the thoracic aorta were used in measuring ECM genes from 5 pairs of Dahl SS male rats on 8 or 24 weeks of feeding from weaning on a control (10% Kcal fat) or high-fat (HF; 60%) diet. A PCR array of ECM genes was performed with cDNA from adventitia and PVAT after 8 and 24 weeks. A gene regulatory network of the differentially expressed genes (DEGs) (HF 2-fold > con) was created using Cytoscape. RESULTS: After 8 weeks, 29 adventitia but 0 PVAT DEGs were found. By contrast, at 24 weeks, PVAT possessed 47 DEGs while adventitia had 3. Top DEGs at 8 weeks in adventitia were thrombospondin 1 and collagen 8a1. At 24 weeks, thrombospondin 1 was also a top DEG in PVAT. The transcription factor Adarb1 was identified as a regulator of DEGs in 8-week adventitia and 24-week PVAT. CONCLUSION: These data support that PVAT responds biologically once blood pressure is elevated.

ß-arrestin biased signaling is not involved in the hypotensive actions of 5-HT7 receptor stimulation: use of Serodolin.

The 5-hydroxytryptamine 7 receptor (5-HT7) is necessary for 5-HT to cause a concentration-dependent vascular relaxation and hypotension. 5-HT7 is recognized as having biased signaling, transduced through either Gs or ß -arrestin. It is unknown whether 5-HT7 signals in a biased manner to cause vasorelaxation/hypotension. We used the recently described ß-arrestin selective 5-HT7 receptor agonist serodolin to test the hypothesis that 5-HT7 activation does not cause vascular relaxation or hypotension via the ß -arrestin pathway. Isolated abdominal aorta (no functional 5-HT7) and vena cava (functional 5-HT7) from male Sprague Dawley rats were used in isometric contractility studies. Serodolin (1 nM - 10 µM) did not change baseline tone of isolated tissues and did not relax the endothelin-1 (ET-1)-contracted vena cava or aorta. In the aorta, serodolin acted as a 5-HT2A receptor antagonist, evidenced by a rightward shift in 5-HT-induced concentration response curve [pEC50 5-HT [M]: Veh = 5.2 +/- 0.15; Ser (100 nM) = 4.49 +/- 0.08; p < 0.05]. In the vena cava, serodolin acted as a 5-HT7 receptor antagonist, shifting the concentration response curve to 5-HT left and upward (%10 µM NE contraction; Veh = 3.2 +/- 1.7; Ser (10 nM) = 58 +/- 11; p < 0.05) and blocking relaxation of pre-contracted tissue to the 5-HT1A/7 agonist 5-carboxamidotryptamine. In anesthetized rats, 5-HT or serodolin was infused at 5, 25 and 75 µg/kg/min, iv. Though 5-HT caused concentration-dependent depressor responses, serodolin caused an insignificant small depressor responses at all three infusion rates. With the final dose of serodolin on board, 5-HT was unable to reduce blood pressure. Collectively the data indicate that serodolin functions as a 5-HT7 antagonist with additional 5-HT2A blocking properties. 5-HT7 activation does not cause vascular relaxation or hypotension via the ß -arrestin pathway.

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.

Chemerin is resident to vascular tunicas and contributes to vascular tone.

The adipokine chemerin may support blood pressure, evidenced by a fall in mean arterial pressure after whole body antisense oligonucleotide (ASO)-mediated knockdown of chemerin protein in rat models of normal and elevated blood pressure. Although the liver is the greatest contributor of circulating chemerin, liver-specific ASOs that abolished hepatic-derived chemerin did not change blood pressure. Thus, other sites must produce the chemerin that supports blood pressure. We hypothesize that the vasculature is a source of chemerin independent of the liver that supports arterial tone. RNAScope, PCR, Western blot analyses, ASOs, isometric contractility, and radiotelemetry were used in the Dahl salt-sensitive (SS) rat (male and female) on a normal diet. Retinoic acid receptor responder 2 (Rarres2) mRNA was detected in the smooth muscle, adventitia, and perivascular adipose tissue of the thoracic aorta. Chemerin protein was detected immunohistochemically in the endothelium, smooth muscle cells, adventitia, and perivascular adipose tissue. Chemerin colocalized with the vascular smooth muscle marker α-actin and the adipocyte marker perilipin. Importantly, chemerin protein in the thoracic aorta was not reduced when liver-derived chemerin was abolished by a liver-specific ASO against chemerin. Chemerin protein was similarly absent in arteries from a newly created global chemerin knockout in Dahl SS rats. Inhibition of the receptor Chemerin1 by the receptor antagonist CCX832 resulted in the loss of vascular tone that supports potential contributions of chemerin by both perivascular adipose tissue and the media. These data suggest that vessel-derived chemerin may support vascular tone locally through constitutive activation of Chemerin1. This posits chemerin as a potential therapeutic target in blood pressure regulation.NEW & NOTEWORTHY Vascular tunicas synthesizing chemerin is a new finding. Vascular chemerin is independent of hepatic-derived chemerin. Vasculature from both males and females have resident chemerin. Chemerin1 receptor activity supports vascular tone.

5-HT7 receptors mediate dilation of rat cremaster muscle arterioles in vivo.

OBJECTIVE: Serotonin (5-HT) infusion in vivo causes hypotension and a fall in total peripheral resistance. However, the vascular segment and the receptors that mediate this response remain in question. We hypothesized that 5-HT7 receptors mediate arteriolar dilation to 5-HT in skeletal muscle microcirculation. METHODS: Cremaster muscles of isoflurane-anesthetized male Sprague-Dawley rats were prepared for in vivo microscopy of third- and fourth-order arterioles and superfused with physiological salt solution at 34°C. Quantitative real-time PCR (RT-PCR) was applied to pooled samples of first- to third-order cremaster arterioles (2-4 rats/sample) to evaluate 5-HT7 receptor expression. RESULTS: Topical 5-HT (1-10 nmols) or the 5-HT1/7 receptor agonist, 5-carboxamidotryptamine (10-30 nM), dilated third- and fourth-order arterioles, responses that were abolished by 1 µM SB269970, a selective 5-HT7 receptor antagonist. In contrast, dilation induced by the muscarinic agonist, methacholine (100 nmols) was not inhibited by SB269970. Serotonin (10 nmols) failed to dilate cremaster arterioles in 5-HT7 receptor knockout rats whereas arterioles in wild-type litter mates dilated to 1 nmol 5-HT, a response blocked by 1 µM SB269970. Quantitative RT-PCR revealed that cremaster arterioles expressed mRNA for 5-HT7 receptors. CONCLUSIONS: 5-HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to 5-HT-induced hypotension, in vivo.

Development of a 5-HT7 receptor antibody for the rat: the good, the bad, and the ugly.

Our laboratory has a vested interest in measuring the location and expression of the 5-hydroxytryptamine (5-HT, serotonin) 7 (5-HT7) receptor in the rat. Determining tissue-specific receptor expression would aid in validating understood and potentially new tissues that support the 5-HT7 receptor-mediated fall in blood pressure, an event we are committed to understand. We contracted with 7TM Antibodies to develop deliberately and rigorously a rat 5-HT7 (r5-HT7) receptor specific antibody. Three antigens, two targeting the third internal loop and one the C terminus, were used in three rabbits to generate antibodies. As a positive control, HEK293(T or AD) cells were transfected with a plasmid for the r5-HT7 receptor also expressing a C terminus 3xFLAG tag. Naïve rat tissues were also used in Western and immunohistochemical analyses. Nine antibodies (3 from three different rabbits) detected a ~ 75 kDa protein absent in homogenates of vector control HEK293T cells. Only antibodies that recognized the C terminus of the 5-HT7 receptor [ERPERSEFVLQNSDH(Abu)GKKGHDT; antibodies 3, 6, and 9] positively and concentration-dependently identified the r5-HT7 receptor expressed in Westerns of transfected HEK293T cells. These same C terminus antibodies also successfully detected the r5-HT7 receptor in immunocytochemical test of the transfected HEK293AD cells, colocalizing with the detected FLAG sequence. In naive tissue, antibody 6 performed the best, identifying specific bands in the brain cortex in Western analysis. These same antibodies produced a more diverse band profile in the vena cava, identifying 6 major proteins. In immunohistochemical experiments, the same C-terminus antibodies, with antibody 3 performing the best, detected the 5-HT7 receptor in rat veins. This deliberate work has given rise to at least three antibodies that can be used with good confidence in r5-HT7 transfected cells, two antibodies that can be used in immunohistochemical analyses of rat tissues and in Westerns of rat brain; we are less confident of the use of these same antibodies in rat veins.

Skin Vascular Resistance Decreases during 5-HT-Induced Hypotension in the Rat.

A recognized vasodilator, the infusion of 5-hydroxytryptamine (5-HT, serotonin) decreases blood pressure through the reduction of total peripheral resistance in the rat. It is not clear which vascular beds/tissues are responsible for this fall. We hypothesized that an increase in blood flow within the skin, measured as an elevated temperature (T) in the thermoregulatory tail and paws, enables at least part of 5-HT-induced reduction in blood pressure through active vasodilation. The temperature of thermoregulatory regions of the skin of an anesthetized male, Sprague Dawley rats were measured using a Optris PI640 thermal camera. The blood pressure of the animal and the temperature of each paw and four locations along the tail (TL1-4) were recorded before, during, and after the infusion of 5-HT at a rate of 25 mg/min into a femoral vein. Contrary to our hypothesis, the temperature of the paws and tail was stable before and during 5-HT infusion and actually increased during the 15-min recovery period. This finding suggests that hyperemia of the skin circulation is not necessary for the fall in blood pressure observed with infused 5-HT, but that a reduction in cutaneous vascular resistance plays a part in the fall in total peripheral resistance.

PIEZO1 mechanoreceptor activation reduces adipogenesis in perivascular adipose tissue preadipocytes.

During hypertension, vascular remodeling allows the blood vessel to withstand mechanical forces induced by high blood pressure (BP). This process is well characterized in the media and intima layers of the vessel but not in the perivascular adipose tissue (PVAT). In PVAT, there is evidence for fibrosis development during hypertension; however, PVAT remodeling is poorly understood. In non-PVAT depots, mechanical forces can affect adipogenesis and lipogenic stages in preadipocytes. In tissues exposed to high magnitudes of pressure like bone, the activation of the mechanosensor PIEZO1 induces differentiation of progenitor cells towards osteogenic lineages. PVAT's anatomical location continuously exposes it to forces generated by blood flow that could affect adipogenesis in normotensive and hypertensive states. In this study, we hypothesize that activation of PIEZO1 reduces adipogenesis in PVAT preadipocytes. The hypothesis was tested using pharmacological and mechanical activation of PIEZO1. Thoracic aorta PVAT (APVAT) was collected from 10-wk old male SD rats (n=15) to harvest preadipocytes that were differentiated to adipocytes in the presence of the PIEZO1 agonist Yoda1 (10 µM). Mechanical stretch was applied with the FlexCell System at 12% elongation, half-sine at 1 Hz simultaneously during the 4 d of adipogenesis (MS+, mechanical force applied; MS-, no mechanical force used). Yoda1 reduced adipogenesis by 33% compared with CON and, as expected, increased cytoplasmic Ca2+ flux. MS+ reduced adipogenesis efficiency compared with MS-. When Piezo1 expression was blocked with siRNA [siPiezo1; NC=non-coding siRNA], the anti-adipogenic effect of Yoda1 was reversed in siPiezo1 cells but not in NC; in contrast, siPiezo1 did not alter the inhibitory effect of MS+ on adipogenesis. These data demonstrate that PIEZO1 activation in PVAT reduces adipogenesis and lipogenesis and provides initial evidence for an adaptive response to excessive mechanical forces in PVAT during hypertension.

Is the 5-hydroxytryptamine 7 Receptor Constitutively Active in the Vasculature? A Study in Veins/Vein.

ABSTRACT: The 5-hydroxytryptamine 7 (5-HT 7 ) receptor is reported to have considerable constitutive activity when transfected into cells. Constitutive activity-receptor activity in the absence of known agonist-is important for understanding the contributions of a receptor to (patho)physiology. We test the hypothesis that the 5-HT 7 receptor possesses constitutive activity in a physiological situation. Isolated veins from male and female Sprague Dawley rats were used as models for measuring isometric force; the abdominal vena cava possesses a functional 5-HT 7 receptor that mediates relaxation, whereas the small mesenteric vein does not. Compounds reported to act as inverse agonists were investigated for their ability to cause contraction (moving a constitutively active relaxant receptor to an inactive state, removing relaxation). Compared with a vehicle control, clozapine, risperidone, ketanserin, and SB269970 caused no contraction in the isolated male abdominal vena cava. By contrast, methiothepin caused a concentration-dependent contraction of the male but not female abdominal vena cava, although with low potency (-log EC 50 [M] = 5.50 ± 0.45) and efficacy (∼12% of contraction to endothelin-1). Methiothepin-induced contraction was not reduced by the 5-HT 7 receptor antagonist (SB269970, 1 µM, not active in the vena cava). These same compounds showed little to no effect in the isolated mesenteric vein. We conclude that the 5-HT 7 receptor in the isolated veins of the Sprague Dawley rat does not possess constitutive activity. We raise the question of the physiological relevance of constitutive activity of this receptor important to such diverse physiological functions as sleep, circadian rhythm, temperature, and blood pressure regulation.

Divergence of Chemerin Reduction by an ATS9R Nanoparticle Targeting Adipose Tissue In Vitro vs. In Vivo in the Rat.

Nanoparticles (NPs) can enable delivery of a drug to a targeted tissue. Previous studies have shown that an NP utilizing an adipose targeting sequence (ATS) peptide in conjunction with a drug can selectively deliver the drug to mouse adipose tissues, using the prohibitin protein expressed in adipose tissue as the target of the ATS. Adipose tissue is a major source of the adipokine chemerin, a prohypertensive protein. Liver-derived chemerin, the largest source of circulating chemerin, is biologically inactive in blood pressure regulation. Our goal is to understand if chemerin produced in adipose tissue contributes to blood pressure/hypertension. We hypothesize the ATS drug delivery system could be used specifically to reduce the levels of adipose tissue-derived chemerin. We created an NP consisting of an antisense oligonucleotide (ASO) against chemerin and a FITC-labeled ATS with a nine arginine sequence (ATS9R). In vitro studies showed that the ASO is functional when incorporated into an NP with ATS9R as it reduced chemerin mRNA expression in isolated epidydimal (Epi) and retroperitoneal (RP) fat adipocytes from Dahl SS rats. This same NP reduced chemerin in isolated whole fats. However, this NP was unable to selectively deliver the ASO to adipose tissue in vivo; liver delivery was dominant. Varying NP doses, administration route, and the concentration of components constituting the NP showed no improvement in ASO delivery to fats vs. the liver. Further studies are therefore needed to develop the ATS9R system to deliver an ASO to adipose beds in rats.

Aortic stiffness is lower when PVAT is included: a novel ex vivo mechanics study.

Perivascular adipose tissue (PVAT) is increasingly recognized as an essential layer of the functional vasculature, being responsible for producing vasoactive substances and assisting arterial stress relaxation. Here, we test the hypothesis that PVAT reduces aortic stiffness. Our model was the thoracic aorta of the male Sprague-Dawley rat. Uniaxial mechanical tests for three groups of tissue were performed: aorta with PVAT attached (+PVAT) or removed (-PVAT), and isolated PVAT (PVAT only). The output of the mechanical test is reported in the form of a Cauchy stress-stretch curve. This work presents a novel, physiologically relevant approach to measure mechanical stiffness ex vivo in isolated PVAT. Low-stress stiffness (E0), high-stress stiffness (E1), and the stress corresponding to a stretch of 1.2 (σ1.2) were measured as metrics of distensibility. The low-stress stiffness was largest in the -PVAT samples and smallest in PVAT only samples. Both the high-stress stiffness and the stress at 1.2 stretch were significantly higher in -PVAT samples when compared with +PVAT samples. Taken together, these results suggest that -PVAT samples are stiffer (less distensible) both at low stress (not significant) as well as at high stress (significant) when compared with +PVAT samples. These conclusions are supported by the results of the continuum mechanics material model that we also used to interpret the same experimental data. Thus, tissue stiffness is significantly lower when considering PVAT as part of the aortic wall. As such, PVAT should be considered as a target for improving vascular function in diseases with elevated aortic stiffness, including hypertension.NEW & NOTEWORTHY We introduce a novel and physiologically relevant way of measuring perivascular adipose tissue (PVAT) mechanical stiffness which shows that PVAT's low, yet measurable, stiffness is linearly correlated with the amount of collagen fibers present within the tissue. Including PVAT in the measurement of the aortic wall's mechanical behavior is important, and it significantly affects the resulting metrics by decreasing aortic stiffness.

Identification of Piezo1 channels in perivascular adipose tissue (PVAT) and their potential role in vascular function.

The vasculature constantly experiences distension/pressure exerted by blood flow and responds to maintain homeostasis. We hypothesized that activation of the stretch sensitive, non-selective cation channel Piezo1 would directly increase vascular contraction in a way that might be modified by perivascular adipose tissue (PVAT). The presence and function of Piezo1 was investigated by RT-PCR, immunohistochemistry, and isolated tissue bath contractility. Superior and mesenteric resistance arteries, aortae, and their PVATs from male Sprague Dawley rats were used. Piezo1 mRNA was detected in aortic vessels, aortic PVAT, mesenteric vessels, and mesenteric PVAT. Both adipocytes and stromal vascular fraction of mesenteric PVAT expressed Piezo1 mRNA. In PVAT, expression of Piezo1 mRNA was greater in magnitude than that of Piezo2, transient receptor potential cation channel, subfamily V, member 4 (TRPV4), anoctamin 1, calcium activated chloride channel (TMEM16), and Pannexin1 (Panx1). Piezo1 protein was present in endothelium and PVAT of rat aortic and in PVAT of mesenteric artery. The Piezo1 agonists Yoda1 and Jedi2 (1 nM - 10 µM) did not stimulate aortic contraction [max < 10% phenylephrine (PE) 10 µM contraction] or relaxation in tissues + or -PVAT. Depolarizing the aorta by modestly elevated extracellular K+ did not unmask aortic contraction to Yoda1 (max <10% PE 10 µM contraction). Finally, the Piezo1 antagonist Dooku1 did not modify PE-induced aorta contraction + or -PVAT. Surprisingly, Dooku1 directly caused aortic contraction in the absence (Dooku1 =26 ± 11; Vehicle = 11 ± 11%PE contraction) but not in the presence of PVAT (Dooku1 = 2 ± 1; Vehicle = 8 ± 5% PE contraction). Thus, Piezo1 is present and functional in the isolated rat aorta but does not serve direct vascular contraction with or without PVAT. We reaffirmed the isolated mouse aorta relaxation to Yoda1, indicating a species difference in Piezo1 activity between mouse and rat.

A high fat diet does not stimulate blood pressure dependence on chemerin in the Sprague-Dawley rat.

The adipokine chemerin is a candidate for connecting obesity to hypertension. Study objective: To test the hypothesis that a high fat (HF) diet stimulates dependence on chemerin for blood pressure regulation. Design: Blood pressure in male Sprague Dawley rats fed a control (10 % fat) or HF (60 % fat) diet from weaning was measured using radiotelemetry. Antisense oligonucleotides (ASOs), administered after 17 weeks of feeding, were used to abolish chemerin production. Results: The HF diet did not increase blood pressure (mm Hg; control = 117.0 ± 2.5; HF = 122.0 ± 2.2). An ASO against chemerin (dosed 1×/week, 4 weeks) similarly reduced blood pressure in the control (-14.0 ± 2.7 mmHg) and HF rat (-12.4 ± 2.3). Chemerin mRNA was abolished in the liver and fats (primary producers of chemerin) from rats given the ASO chemerin vs control. Conclusion: A HF diet alone is insufficient to stimulate the dependence of blood pressure in the rat on chemerin.