Abstract P071: Transglutaminases Are Active In Perivascular Adipose Tissue

Abstract

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.