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.

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.

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.

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.

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.

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.

5-HT7 Receptor Restrains 5-HT-induced 5-HT2A Mediated Contraction in the Isolated Abdominal Vena Cava.

ABSTRACT: Although discovered as a vasoconstrictor, 5-hydroxytryptamine (5-HT, serotonin) infused into man and rodent reduces blood pressure. This occurs primarily through activation of 5-HT7 receptors and, at least in part, venodilation. Vascular mechanisms by which this could occur include direct receptor activation leading to vasodilation and/or suppression of contractile 5-HT receptor activation. This study tests the hypothesis that the 5-HT7 receptor restrains activation of the 5-HT2A receptor. A subhypothesis is whether agonist-induced activation-independent of constitutive activity-of the 5-HT7 receptor is necessary for this restraint. The isolated abdominal aorta and vena cava from the normal male Sprague-Dawley rat was our model. Studies used real-time PCR and a pharmacological approach in the isolated tissue bath for measurement of isometric tone. Although 5-HT2A receptor mRNA expression in both aorta and vena cava was significantly larger than that of the 5-HT7 receptor mRNA, the 5-HT7/5-HT2A receptor mRNA ratio was greater in the vena cava (0.30) than in the aorta (0.067). 5-HT7 receptor antagonism by SB266970 and DR 4458 increased maximum contraction to 5-HT in the isolated vein by over 50% versus control. The 5-HT2A receptor agonists TCB-2 and NBOH were more potent in the aorta compared with 5-HT but less efficacious, serving as partial agonists. By contrast, these same three agonists caused no contraction in the vena cava isolated from the same rats up to 10 µM agonist. Antagonism of the 5-HT7 receptor by SB269970 did not increase either the potency or efficacy of TCB-2 or NBOH. These data support that the 5-HT7 receptor itself needs to be stimulated to reduce contraction and suggest there is little constitutive activity of the 5-HT7 receptor in the isolate abdominal vena cava.

Transglutaminases Are Active in Perivascular Adipose Tissue.

Transglutaminases (TGs) are crosslinking enzymes best known for their vascular remodeling in hypertension. They require calcium to form an isopeptide bond, connecting a glutamine to a protein bound lysine residue or a free amine donor such as norepinephrine (NE) or serotonin (5-HT). We discovered that perivascular adipose tissue (PVAT) contains significant amounts of these amines, making PVAT an ideal model to test interactions of amines and TGs. We hypothesized that transglutaminases are active in PVAT. Real time RT-PCR determined that Sprague Dawley rat aortic, superior mesenteric artery (SMA), and mesenteric resistance vessel (MR) PVATs express TG2 and blood coagulation Factor-XIII (FXIII) mRNA. Consistent with this, immunohistochemical analyses support that these PVATs all express TG2 and FXIII protein. The activity of TG2 and FXIII was investigated in tissue sections using substrate peptides that label active TGs when in a catalyzing calcium solution. Both TG2 and FXIII were active in rat aortic PVAT, SMAPVAT, and MRPVAT. Western blot analysis determined that the known TG inhibitor cystamine reduced incorporation of experimentally added amine donor 5-(biotinamido)pentylamine (BAP) into MRPVAT. Finally, experimentally added NE competitively inhibited incorporation of BAP into MRPVAT adipocytes. Further studies to determine the identity of amidated proteins will give insight into how these enzymes contribute to functions of PVAT and, ultimately, blood pressure.

Creation of the 5-hydroxytryptamine receptor 7 knockout rat as a tool for cardiovascular research.

Using CRISPR-Cas9 technology, we created a 5-HT7 receptor global knockout (KO) rat, on a Sprague-Dawley background, for use in cardiovascular physiology studies focused on blood pressure regulation. A stable line carrying indels in exons 1 and 2 of the rat Htr7 locus was established and validated. Surprisingly, 5-HT7 receptor mRNA was still present in the KO rat. However, extensive cDNA and genomic sequencing of KO tissues confirmed an 11 bp deletion in exon 1 and 4 bp deletion in exon 2. The exon 1 deletion resulted in a frameshifted mRNA sequence coding for a nonfunctional protein. While the Htr1B locus was a potential off-target for the guide RNAs designed for exon 2 of Htr7, there were no off-target sequence changes at this locus in the originating founder. When the F2 generation of KO was compared with wild-type (WT) counterparts, neither the male nor female KO rats were different in body size, fat weights, or mass of organs (kidney, heart, and brain) important to blood pressure. Females were smaller in mass than their counterpart males. Clinical measures of plasma from nonfasted rats revealed largely similar values, comparing WT and KO, of glucose, blood urea nitrogen, creatinine, phosphate, calcium, and albumin to name a few. Loss of a functional 5-HT7 receptor was validated by the complete loss of relaxation to the 5-HT1/7 receptor agonist 5-carboxamidotryptamine in the isolated abdominal vena cava. This newly created 5-HT7 receptor KO rat will be of use to investigate the importance of the 5-HT7 receptor in blood pressure regulation.

Fenfluramine-induced PVAT-dependent contraction depends on norepinephrine and not serotonin.

Perivascular adipose tissue (PVAT) modulates vascular tone and altered PVAT function is observed in vascular diseases such as hypertension and atherosclerosis. We discovered that the PVAT surrounding rat thoracic aorta (RA) and the superior mesenteric artery (SMA) contain significant amounts of 5-hydroxytryptamine (5-HT). We hypothesized that the 5-HT contained within the PVAT is functional and vasoactive. Isolated tissue baths were used for isometric contractility studies and high performance liquid chromatography was used to quantitatively measure amines in the PVAT and release studies. The 5-HT releaser fenfluramine (10 nM-100 µM) was tested for its ability to contract arteries with and without PVAT. Contraction was reported as a percentage of the initial contraction to 10 µM phenylephrine. The RA with PVAT contracted to fenfluramine to a greater maximum (98 ± 10%) than RA without PVAT (24 ± 4%), while no difference in contraction of SMA to maximum fenfluramine with (78 ± 2%) and without (75 ± 6%) PVAT was observed. Contradicting our hypothesis, the maximum contraction of RA with PVAT to fenfluramine was diminished by the alpha-1 adrenoreceptor antagonist prazosin (100 nM; vehicle: 71 ± 4%, prazosin: 24 ± 2%) and the norepinephrine transporter (NET) inhibitor nisoxetine (1 µM; vehicle: 71 ± 4%, nisoxetine: 25 ± 4%) but not the 5-HT2A/2C receptor antagonist ketanserin (10 nM) or serotonin specific reuptake inhibitor fluoxetine (10 µM). To test if fenfluramine caused release of 5-HT or NE from PVAT, PVAT from RA was incubated with vehicle or fenfluramine (10 µM-10 mM), and amines released into the incubating buffer were quantified. A pronounced concentration-dependent NE-release (more than 5-HT) was observed. Collectively, this research illustrates the pharmacology of fenfluramine to primarily stimulate NE release (better than 5-HT) in a NET-dependent manner, leading to vasoconstriction. This adds additional support to PVAT as being an important reservoir of amines.

Whole-Body but Not Hepatic Knockdown of Chemerin by Antisense Oligonucleotide Decreases Blood Pressure in Rats.

Chemerin is an inflammatory adipokine positively associated with hypertension and obesity. The majority of chemerin derives from the liver and adipose tissue, however, their individual contributions to blood pressure are unknown. We began studying chemerin in the normal rat using antisense oligonucleotides (ASO) with whole-body activity (Gen 2.5 chemerin ASO) or liver-restricted activity (GalNAc chemerin ASO). We hypothesized that in normotensive male Sprague-Dawley rats, circulating chemerin is predominately liver-derived and regulates blood pressure. A dosing study of the Gen 2.5 chemerin ASO (with a scrambled control ASO) supported 25 mg/kg as the appropriate dose. GalNAc chemerin ASO was also assessed and used at 10 mg/kg. Radiotelemetry monitored mean arterial pressure (MAP) for a 1-week baseline and weekly subcutaneous ASO injections for 4 weeks. Two days after the final injection, animals were euthanized for tissue reverse transcription-polymerase chain reaction and chemerin Western analysis. Gen 2.5 chemerin ASO treatments reduced chemerin mRNA and protein in liver, retroperitoneal fat (RP), and mesenteric perivascular adipose tissue (mPVAT), as well as reducing protein in plasma. GalNAc chemerin ASO treatments reduced chemerin mRNA and protein in liver and chemerin protein in plasma but had no effect on expression in RP fat or mPVAT. Gen 2.5 chemerin ASO treatment reduced MAP compared with control ASO but was unchanged in animals receiving the GalNAc chemerin ASO. Although circulating chemerin is liver-derived, it does not play a major role in blood pressure regulation. Local effects of chemerin from fat may explain this discrepancy and support chemerin's association with hypertension and obesity.

5-HT causes splanchnic venodilation.

Serotonin [5-hydroxytryptamine (5-HT)] causes relaxation of the isolated superior mesenteric vein, a splanchnic blood vessel, through activation of the 5-HT7 receptor. As part of studies designed to identify the mechanism(s) through which chronic (≥24 h) infusion of 5-HT lowers blood pressure, we tested the hypothesis that 5-HT causes in vitro and in vivo splanchnic venodilation that is 5-HT7 receptor dependent. In tissue baths for measurement of isometric contraction, the portal vein and abdominal inferior vena cava relaxed to 5-HT and the 5-HT1/7 receptor agonist 5-carboxamidotryptamine; relaxation was abolished by the 5-HT7 receptor antagonist SB-269970. Western blot analyses showed that the abdominal inferior vena cava and portal vein express 5-HT7 receptor protein. In contrast, the thoracic vena cava, outside the splanchnic circulation, did not relax to serotonergic agonists and exhibited minimal expression of the 5-HT7 receptor. Male Sprague-Dawley rats with chronically implanted radiotelemetry transmitters underwent repeated ultrasound imaging of abdominal vessels. After baseline imaging, minipumps containing vehicle (saline) or 5-HT (25 µg·kg-1·min-1) were implanted. Twenty-four hours later, venous diameters were increased in rats with 5-HT-infusion (percent increase from baseline: superior mesenteric vein, 17.5 ± 1.9; portal vein, 17.7 ± 1.8; and abdominal inferior vena cava, 46.9 ± 8.0) while arterial pressure was decreased (~13 mmHg). Measures returned to baseline after infusion termination. In a separate group of animals, treatment with SB-269970 (3 mg/kg iv) prevented the splanchnic venodilation and fall in blood pressure during 24 h of 5-HT infusion. Thus, 5-HT causes 5-HT7 receptor-dependent splanchnic venous dilation associated with a fall in blood pressure.NEW & NOTEWORTHY This research is noteworthy because it combines and links, through the 5-HT7 receptor, an in vitro observation (venorelaxation) with in vivo events (venodilation and fall in blood pressure). This supports the idea that splanchnic venodilation plays a role in blood pressure regulation.

Organic cation transporter 3 contributes to norepinephrine uptake into perivascular adipose tissue.

Perivascular adipose tissue (PVAT) reduces vasoconstriction to norepinephrine (NE). A mechanism by which PVAT could function to reduce vascular contraction is by decreasing the amount of NE to which the vessel is exposed. PVATs from male Sprague-Dawley rats were used to test the hypothesis that PVAT has a NE uptake mechanism. NE was detected by HPLC in mesenteric PVAT and isolated adipocytes. Uptake of NE (10 µM) in mesenteric PVAT was reduced by the NE transporter (NET) inhibitor nisoxetine (1 µM, 73.68 ± 7.62%, all values reported as percentages of vehicle), the 5-hydroxytryptamine transporter (SERT) inhibitor citalopram (100 nM) with the organic cation transporter 3 (OCT3) inhibitor corticosterone (100 µM, 56.18 ± 5.21%), and the NET inhibitor desipramine (10 µM) with corticosterone (100 µM, 61.18 ± 6.82%). Aortic PVAT NE uptake was reduced by corticosterone (100 µM, 53.01 ± 10.96%). Confocal imaging of mesenteric PVAT stained with 4-[4-(dimethylamino)-styrl]-N-methylpyridinium iodide (ASP(+)), a fluorescent substrate of cationic transporters, detected ASP(+) uptake into adipocytes. ASP(+) (2 µM) uptake was reduced by citalopram (100 nM, 66.68 ± 6.43%), corticosterone (100 µM, 43.49 ± 10.17%), nisoxetine (100 nM, 84.12 ± 4.24%), citalopram with corticosterone (100 nM and 100 µM, respectively, 35.75 ± 4.21%), and desipramine with corticosterone (10 and 100 µM, respectively, 50.47 ± 5.78%). NET protein was not detected in mesenteric PVAT adipocytes. Expression of Slc22a3 (OCT3 gene) mRNA and protein in PVAT adipocytes was detected by RT-PCR and immunocytochemistry, respectively. These end points support the presence of a transporter-mediated NE uptake system within PVAT with a potential mediator being OCT3.

Transglutaminase activity is decreased in large arteries from hypertensive rats compared with normotensive controls.

Transglutaminases (TGs) catalyze the formation of covalent cross-links between glutamine residues and amine groups. This cross-linking activity has been implicated in arterial remodeling. Because hypertension is characterized by arterial remodeling, we hypothesized that TG activity, expression, and functionality would be increased in the aorta, but not in the vena cava (which does not undergo remodeling), from hypertensive rats relative to normotensive rats. Spontaneously hypertensive stroke-prone rats (SHRSP) and DOCA-salt rats as well as their respective normotensive Wistar-Kyoto or Sprague-Dawley counterparts were used. Immunohistochemistry and Western blot analysis measured the presence and expression of TG1 and TG2, in situ activity assays quantified active TGs, and isometric contractility was used to measure TG functionality. Contrary to our hypothesis, the activity (52% DOCA-salt vs. control rats and 56% SHRSP vs. control rats, P < 0.05), expression (TG1: 54% DOCA-salt vs. control rats, P > 0.05, and TG2: 77% DOCA-salt vs. control rats, P < 0.05), and functionality of TG1 and TG2 were decreased in the aorta, but not in the vena cava, from hypertensive rats. Mass spectrometry identified proteins uniquely amidated by TGs in the aorta that play roles in cytoskeletal regulation, redox regulation, and DNA/RNA/protein synthesis and regulation and in the vena cava that play roles in cytoskeletal regulation, coagulation regulation, and cell metabolism. Consistent with the idea that growing cells lose TG2 expression, vascular smooth muscle cells placed in culture lost TG2 expression. We conclude that the expression, activity, and functionality of TG1 and TG2 are decreased in the aorta, but not in the vena cava, from hypertensive rats compared with control rats.

Chemerin connects fat to arterial contraction.

OBJECTIVE: Obesity and hypertension are comorbid in epidemic proportion, yet their biological connection is largely a mystery. The peptide chemerin is a candidate for connecting fat deposits around the blood vessel (perivascular adipose tissue) to arterial contraction. We presently tested the hypothesis that chemerin is expressed in perivascular adipose tissue and is vasoactive, supporting the existence of a chemerin axis in the vasculature. APPROACH AND RESULTS: Real-time polymerase chain reaction, immunohistochemistry, and Western analyses supported the synthesis and expression of chemerin in perivascular adipose tissue, whereas the primary chemerin receptor ChemR23 was expressed both in the tunica media and endothelial layer. The ChemR23 agonist chemerin-9 caused receptor, concentration-dependent contraction in the isolated rat thoracic aorta, superior mesenteric artery, and mesenteric resistance artery, and contraction was significantly amplified (more than 100%) when nitric oxide synthase was inhibited and the endothelial cell mechanically removed or tone was placed on the arteries. The novel ChemR23 antagonist CCX832 inhibited phenylephrine-induced and prostaglandin F2α-induced contraction (+perivascular adipose tissue), suggesting that endogenous chemerin contributes to contraction. Arteries from animals with dysfunctional endothelium (obese or hypertensive) demonstrated a pronounced contraction to chemerin-9. Finally, mesenteric arteries from obese humans demonstrate amplified contraction to chemerin-9. CONCLUSIONS: These data support a new role for chemerin as an endogenous vasoconstrictor that operates through a receptor typically attributed to function only in immune cells.

JAK inhibition with tofacitinib suppresses arthritic joint structural damage through decreased RANKL production.

OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.

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.

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.

Vena cava and aortic smooth muscle cells express transglutaminases 1 and 4 in addition to transglutaminase 2.

Transglutaminase (TG) function facilitates several vascular processes and diseases. Although many of these TG-dependent vascular processes have been ascribed to the function of TG2, TG2 knockout mice have a mild vascular phenotype. We hypothesized that TGs besides TG2 exist and function in the vasculature. Biotin-pentylamide incorporation, a measure of general TG activity, was similar in wild-type and TG2 knockout mouse aortae, and the general TG inhibitor cystamine reduced biotin-pentylamine incorporation to a greater extent than the TG2-specific inhibitor Z-DON, indicating the presence of other functional TGs. Additionally, 5-hydroxytryptamine-induced aortic contraction, a TG-activity-dependent process, was decreased to a greater extent by general TG inhibitors vs. Z-DON (maximum contraction: cystamine = abolished, monodansylcadaverine = 28.6 ± 14.9%, and Z-DON = 60.2 ± 15.2% vehicle), providing evidence for the importance of TG2-independent activity in the vasculature. TG1, TG2, TG4, and Factor XIII (FXIII) mRNA in rat aortae and vena cavae was detected by RT-PCR. Western analysis detected TG1 and TG4, but not FXIII, in rat aortae and vena cavae and in TG2 knockout and wild-type mouse aortae. Immunostaining confirmed the presence of TG1, TG2, and TG4 in rat aortae and vena cavae, notably in smooth muscle cells; FXIII was absent. K5 and T26, FITC-labeled peptide substrates specific for active TG1 and TG2, respectively, were incorporated into rat aortae and vena cavae and wild-type, but not TG2 knockout, mouse aortae. These studies demonstrate that TG2-independent TG activity exists in the vasculature and that TG1 and TG4 are expressed in vascular tissues.