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. While 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 the vasculature as a source of chemerin independent of the liver that supports arterial tone. RNAScope®, PCR, Western analyses, ASOs, isometric contractility, and radiotelemetry were used in the Dahl salt sensitive (SS) rat (male and female) on a normal diet. 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 a-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 loss of vascular tone that supports potential contributions of chemerin by both PVAT 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.

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.

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.

Various humoral factors produced by adipose tissue are suggested to contribute to obesity associated hypertension and cardiovascular pathology. The adipokine chemerin is a frontrunner candidate. Male Dahl SS rats fed a high fat (HF; 60% kCal) diet from weaning develop severe hypertension that is profoundly reduced by weekly treatment with an antisense oligonucleotide (ASO) that disables chemerin mRNA. Here we hypothesized that feeding a HF diet from weaning to male Sprague Dawley (SD) rats would similarly increase the dependence of blood pressure (BP) regulation on chemerin. Over 17 weeks of feeding, HF fed SD rats gained significantly more weight and body fat than those fed control (10% kCal) diet. Radiotelemeters were implanted at 17 weeks of age to measure BP. Two weeks of basal BP was collected, and as we and others have shown previously, the HF diet did not increase BP in SD rats (control = 117±2.5 mm Hg; HF = 122±2.2 mm Hg). Vehicle or Gen 2.5 ASO chemerin (25 mg/kg, sc) were then given once a week for four weeks. Gen 2.5 ASO chemerin caused a slowly developing but significant reduction in BP in control rats (-14.0±2.7 mm Hg) that was not significantly different from the BP fall in HF rats (-12.4±2.3 mm Hg). RT-PCR analyses validated complete loss of chemerin mRNA in the liver and fat (primary producers of chemerin) from rats given the Gen 2.5 ASO chemerin vs control. These data show that in normal, normotensive rats a HF diet alone is insufficient to increase BP dependence on chemerin