Miller, V. M.

Increased shear, pressure, and oxygen tension in vein grafts may alter production of endothelium-derived vasoactive and anti-mitogenic factors such as nitric oxide which subsequently affect development of neointimal hyperplasia. This study was designed to determine whether or not nitric oxide mediates endothelium-dependent responses in femoral in situ vein grafts. Non-reversed, canine femoral vein grafts were placed bilaterally to bypass a ligated segment of the femoral artery in dogs. After 6 weeks, the grafts were removed, cut into rings, and suspended in organ chambers for measurement of isometric force. In some rings the endothelium was removed deliberately. In the presence of indomethacin, the synthetic analog of L-arginine, L-NG-monomethyl-arginine (10−4 M: L-NMMA) did not cause a significant change in baseline tension of rings with endothelium. L-NMMA reduced only contractions of rings with endothelium to the alpha-adrenergic agonist UK 14,304. The analog also reduced maximal relaxations to the calcium-ionophore, A23187 in rings with endothelium. In addition, L-NMMA reduced relaxations of rings without endothelium to adenosine diphosphate by 35%. Positive immunostaining for nitric oxide synthase was present in both the myointima and media of histological sections of grafts. In conclusion, these results suggest that in situ vein grafts exhibit two unique properties which are unlike unoperated arteries or veins: (i) alpha2-adrenergic receptors may be coupled to the release of contractile endothelium-derived factors associated with production of nitric oxide; and (ii) nitric oxide may be released by the smooth muscle in response to purinergic stimulation. The presence of nitric oxide synthase throughout the wall of the graft may result in production of nitric oxide in response to adenosine diphosphate released by platelets and to circulating catecholamines.

Menopausal hormone treatments (MHT) may limit progression of CIMT, a measure of atherosclerosis, but pose a risk for ischemic stroke. To test candidate gene variation for an association with CIMT, DNA from 714 women participating in KEEPS, a clinical trial of MHT to prevent progression of atherosclerosis, was genotyped using custom Illumina Infinium iSelect chemistry. Single nucleotide polymorphisms (SNPs; 13,229) were genotyped within 769 genes within the anticoagulant, procoagulant, fibrinolytic or innate immunity pathways. Of these SNPs, 75 were removed due to call rate<0.95, relatedness, duplication, and misidentification; 1275 were removed for quality control. There were 504 ancestry informative markers (AIM). Percent of European ancestry using AIM, age at enrollment and pulse pressure correlated positively with CIMT. Using logistic regression to adjust for these, one SNP (rs2236935 of MAP4K4 on chromosome 2) associated positively with CIMT (p‐value = 5.771e‐06). This gene encodes a protein of the serine/threonine protein kinase family involved in cytokine signaling pathways related to innate immunity, development of type II diabetes, and response to environmental stress. Whether this SNP remains associated with changes in CIMT in women randomized to hormonal treatment in KEEPS remains to be determined. (Supported by grants from the Aurora Foundation, NIH HL90639 and the Mayo Foundation)

Experiments were designed to evaluate endothelium-dependent responses of pulmonary arteries following prolonged exposure to oxygen-derived free radicals. Rings of canine pulmonary arteries with and without endothelium were suspended for measurement of isometric force in organ chambers and incubated with xanthine (10(-4)M) plus xanthine oxidase (0.015 U/ml) for 1 h in the absence and presence of either superoxide dismutase (SOD, 150 U/ml), catalase (1,200 U/ml), deferoxamine (10(-3)M), or a combination of all three scavengers. Xanthine plus xanthine oxidase caused significantly greater contractions of rings without compared with those with endothelium. In rings with endothelium, contractions were reduced by SOD or catalase but not by deferoxamine. Following 1 h of exposure to xanthine plus xanthine oxidase, endothelium-dependent relaxations to ADP were reduced but not those to bradykinin or the calcium ionophore A-23187 (calcimycin). Relaxations to ADP were not corrected by incubation with the antioxidants used singly or in combination during the exposure to xanthine plus xanthine oxidase. These results suggest that oxygen-derived free radicals generated from exogenously applied xanthine plus xanthine oxidase cause contractions of canine pulmonary arteries. In addition, even when contractions of rings with endothelium were prevented by SOD and catalase, subsequent expression of some but not all endothelium-dependent relaxations were reduced. Therefore, scavenging of oxygen-derived free radicals may prevent some but not all of the vascular injury caused by oxygen-derived free radicals.

Experiments were designed to characterize endothelin receptors in canine femoral veins and to determine whether their distribution or sensitivity could be altered by chronic changes in blood flow and oxygen tension in veins proximal to an arteriovenous fistula. Endothelium was removed from unoperated or fistula-operated femoral veins of anesthetized dogs. Veins were cut into rings and suspended in organ chambers for the measurement of isometric force or frozen for isolation of membrane proteins. Endothelin-1, endothelin-3, and sarafotoxin S6c caused concentration-dependent increases in tension in all rings. In rings of unoperated veins, maximal tensions were significantly less to endothelin-3 and sarafotoxin S6c than to endothelin-1. In rings of fistula-operated veins, maximal tensions to endothelin-1 and endothelin-3 were the same. Contractions to endothelin-1 or endothelin-3 in unoperated veins were not inhibited by an antagonist of endothelin-A receptors, BQ-123. Binding of 125I-labeled endothelin-1 (125I-endothelin-1) to membranes from veins without endothelium increased as a function of membrane protein. Affinity of receptors, as determined by competitive inhibition of 125I-endothelin-1 was as follows: endothelin-1 > endothelin-3 > sarafotoxin S6c. Competitive inhibition of 125I-endothelin-1 by endothelin-3 and sarafotoxin S6c was significant for a two-site binding model in all veins. The total number of binding sites was reduced significantly in fistula-operated veins; the relative proportions of high- and low-affinity binding sites did not change. Affinity of high- and low-affinity receptors increased in fistula-operated veins.(ABSTRACT TRUNCATED AT 250 WORDS)