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
Evidence supports that Antisense oligonucleotides (ASO) against the adipokine chemerin reduce the blood pressure of the normal male Sprague Dawley rat and, to a greater magnitude, the hypertensive Dahl SS rat fed a high fat diet. In the Dahl SS rat, chemerin protein concentration in white adipose tissue [mesenteric perivascular adipose tissue (Mes PVAT)] was significantly higher in females vs males. We hypothesized that female Dahl SS rats would be more dependent on chemerin for blood pressure maintenance than males because of the Mes PVAT’s proximity to blood vessels that control total peripheral resistance. Age-matched male and female Dahl SS rats on a standard chow (0.2% NaCl, 6.2% fat) diet were used. Radiotelemeters were implanted to measure blood pressure (MAP) and heart rate (bpm). Following a two-week recovery period after implantation, one week of baseline measures were collected. Knockdown of whole body chemerin was achieved by subcutaneous injections of scrambled control ASO or whole-body ASO against chemerin (both 25 mg/kg) on days 0, 7, 14, and 21. On day 23, the rats were euthanized, and samples were collected for western blot and qPCR of the chemerin gene Rarres2 . Plasma chemerin was abolished in both male and female rats given the ASO against chemerin vs that of rats receiving the control ASO. In males and females, respectively, chemerin mRNA expression (2 -ΔCT ) was reduced from 0.53 to 0.001 ± 0.002 and 0.23 to 0.001 ± 0.02 in liver, 0.05 to 0.01 ± 0.005 and 0.07 to 0.005 ± 0.02 in RP fat, and 0.09 to 0.01 ± 0.003 and 0.05 to 0.001 ± 0.02 in epididymal/uterine fat. MAP in control ASO rats showed no significant change from baseline measurements (M:137.4 ± 3.0 /F: 130.6 ± 1.5 mmHg), while males and females treated with whole-body ASO dropped by 10.7 ± 1.6 and 10.9 ± 2.1 mmHg respectively after four injections, with no significant change in heart rate. Pulse pressure also decreased by 5.6 ± 1.3 and 6.8 ± 1.3 mmHg in whole-body ASO males and females, respectively, with no significant changes from baseline in the control animals. These data suggest, contrary to our hypothesis, that chemerin plays a similar role in basal blood pressure regulation in males and females. This indicates that males and females may be equally dependent on chemerin for high fat diet-induced hypertension.
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 in which to test interactions of amines and TGs. We hypothesized that TG2 and FXIII are active in PVAT. Sprague-Dawley rat aortic, superior mesenteric (SMA), and mesenteric resistance artery (MR) PVAT express TG2 and blood coagulation factor XIII (FXIII) mRNA (Figure 1A). Consistent with this, immunohistochemical analyses support that 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 and a catalyzing calcium solution, visualized with TRITC fluorescence (Figure 1B,C). Both TG2 and FXIII are 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 by 6.14% of total normalized signal (p<0.0001, N=7). Further Western blot analysis proved that experimentally added 5-HT competitively inhibits incorporation of experimentally added BAP into MRPVAT adipocytes, reducing total normalized signal by 10.75% (p=0.001, N=4). Further studies to determine what proteins TGs are amidating will give insight into how these enzymes contribute to the development of hypertension.
Nanoparticles (NPs) are a drug delivery system that can enable delivery of a drug to a targeted tissue. Previous studies have shown that the use of an adipose targeting sequence (ATS) peptide in conjunction with a drug in the form of a NP has the ability to selectively deliver the drug to mouse adipose tissues. The prohibitin protein located in adipose tissue is the target of ATS. Adipose tissue is a major source of the adipokine chemerin, a prohypertensive protein. Our laboratory has proven that liver derived chemerin, the largest source of circulating chemerin, is biologically inactive. Our goal is to understand if chemerin produced by adipose tissue contributes to hypertension. We hypothesize an ATS drug delivery system could be applied in rats to specifically reduce the levels of adipose tissue associated chemerin. We created a NP consisting of an antisense oligonucleotide (ASO) against chemerin and a FITC labeled ATS with 9 arginine sequence (ATS9R). In vitro studies showed that the ASO is functional when incorporated into NPs with ATS9R as it reduced chemerin mRNA expression in isolated epidydimal (Epi) adipocytes by 43‐fold compared to vehicle incubated cells (Veh mean 2‐ΔCT=0.039 ± 0.007; NP mean 2‐ΔCT=0.0009 ± 0.0001), and 13‐fold in isolated retroperitoneal (RP) adipocytes (Veh mean 2‐ΔCT=0.041 ± 0.007; NP mean 2‐ΔCT=0.0031 ± 0.0009) of Dahl SS rats. Ex vivo studies support the ability of the ATS9R‐ASO NP to reduce chemerin mRNA expression in tissues. In Epi fat incubated with the NP overnight at 37 oC, chemerin expression was reduced by 2.7‐fold (p<0.05) (Veh mean 2‐ΔCT=0.213 ± 0.088; NP mean 2‐ΔCT=0.079 ± 0.013). The NP also reduced chemerin expression in RP fat by 2.1‐fold (Veh mean 2‐ΔCT=0.159 ± 0.027; NP mean 2‐ΔCT=0.071 ± 0.011), although the changes did not reach statistical significance. However, this same NP was unable to deliver the ASO selectively to adipose tissue in vivo. Two days after subcutaneous injections in Dahl SS rats, the NP caused a significant (p<0.05) 2.4‐fold reduction in liver chemerin mRNA expression compared to vehicle treated animals (Veh 2‐ΔCT=0.753; NP mean 2‐ΔCT=0.311 ± 0.007), but not in Epi (1.8‐fold reduction; Veh 2‐ΔCT=0.175; NP mean 2‐ΔCT=0.095 ± 0.013) or RP fat (1.9‐fold reduction; Veh 2‐ΔCT=0.120; NP mean 2‐ΔCT=0.061 ± 0.015). Varying NP dose, administration route, and concentration of the components constituting the NP did not show any improvement in ASO delivery to fats vs liver. Further studies are needed to develop the ATS9R system to deliver ASO to adipose tissue beds in rats.
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