A novel evaluation of endothelial dysfunction ex vivo: "Teaching an Old Drug a New Trick"

Abstract

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Many CVDs begin with endothelium dysfunction (ED), including hypertension, thrombosis, and atherosclerosis. Our assay evaluated ED in isolated murine aorta by quantifying phenylephrine-induced contractions (PE) in the presence of L-NAME, which blocked acetylcholine-induced relaxation (ACh %; >99%). The "L-NAME PE Contraction Ratio" (PECR) was defined as: "PE Tension post-L-NAME" divided by "PE Tension pre-L-NAME." We hypothesized that our novel PE Contraction Ratio would strongly correlate with alterations in endothelium function. Validation 1: PECR and ACh % values of naïve aortas were strongly and positively correlated (PECR vs. ACh %, r² = 0.91, n = 7). Validation 2: Retrospective analyses of published aortic PECR and ACh % data of female mice exposed to filtered air, propylene glycol:vegetable glycerin (PG:VG), formaldehyde (FA), or acetaldehyde (AA) for 4d showed that the PECR in air-exposed mice (PECR = 1.43 ± 0.05, n = 16) correlated positively with the ACh % (r² = 0.40) as seen in naïve aortas. Similarly, PECR values were significantly decreased in aortas with ED yet retained positive regression coefficients with ACh % (PG:VG r² = 0.54; FA r² = 0.55). Unlike other toxicants, inhaled AA significantly increased both PECR and ACh % values yet diminished their correlation (r² = 0.09). Validation 3: To assess species-specific dependence, we tested PECR in rat aorta, and found PECR correlated with ACh % relaxation albeit less well in this aged and dyslipidemic model. Because the PECR reflects NOS function directly, it is a robust measure of both ED and vascular dysfunction. Therefore, it is a complementary index of existing tests of ED that also provides insight into mechanisms of vascular toxicity.

Keywords: L-NAME; aldehydes; cardiovascular disease; eNOS; endothelium.

FIGURE 1

Protocol for development of the Phenylephrine Contraction Ratio (PECR) assay in isolated murine aorta. (a) All aortas were stimulated with High K⁺ followed by washout (i.e. 3 bath exchanges of fresh PSS; ///), and restimulated with High K⁺ followed by washout. Uncontracted vessels were then exposed to cumulative concentrations of PE (0.1 nM–10 µM) before addition of cumulative concentrations of acetylcholine (ACh; 0.1 nM–10 µM). Following washout, aortas were pre‐contracted with PE (10 µM) until a tension plateau occurred, after which, L‐NAME (100 µM) was added to the bath. After a second plateau in tension, ACh (10 µM) was added to confirm NOS inhibition. Finally, aortas were relaxed with cumulative concentrations of sodium nitroprusside (SNP; 0.01 nM–100 µM). (b) Representative traces of contractions induced by PE alone or PE+L‐NAME. Both cumulative concentrations of PE (up to 10 µM) and PE alone at 10 µM produced similar levels of tension. L‐NAME addition significantly increased tension in a time dependent manner. (c) The PECR was calculated as the PE+L‐NAME tension divided by the PE alone tension

FIGURE 2

Relationship between Phenylephrine (PE) Contraction Ratio and ACh‐induced endothelium‐dependent relaxation in aortas of naïve mice. (a) Representative myograph trace of isometric tension of a naïve aortic segment contracted by PE and relaxed by acetylcholine (ACh % relaxation). After 3× washout over 30 min, the aorta was contracted again with PE followed by addition of L‐NAME. After a plateau in tension, ACh was added to confirm L‐NAME‐induced inhibition of endothelium‐dependent relaxation (EDRF; NO). The PE Contraction Ratio (PECR) was calculated as the total tension of PE+L‐NAME divided by the total tension generated by PE alone (1st addition; see Figure 1a). (b) The values of PECR (x‐axis) were regressed against the values of % ACh relaxation (y‐axis) with a regression coefficient of r ² = 0.91. (c) Air perfusion‐induced endothelial dysfunction (ED) in aorta led to significantly increased PE‐stimulated contractility reflected by an increased PE+ED/HI K⁺ ratio and by an increased PE efficacy (mN/mm) without altering High K⁺ efficacy (mN/mm)

FIGURE 3

Relationship between Phenylephrine (PE) Contraction Ratio and ACh‐induced endothelium relaxation in aortas of mice exposed to constituents of electronic cigarette aerosols. Retrospective analyses of published PECR and ACh % data of ex vivo aortic function of female mice exposed to filtered air control, propylene glycol: vegetable glycerin (PG:VG), formaldehyde (FA), or acetaldehyde (AA) for 4 days, 6 h/day. (a) Representative myograph trace of isometric tension of an aortic segment contracted by PE and relaxed by acetylcholine (ACh % relaxation). After 3× washout over 30 min, the aorta was contracted again with PE followed by addition of L‐NAME. After a plateau in tension, ACh was added to confirm L‐NAME‐induced inhibition of endothelium‐dependent relaxation (EDRF; NO). The PE Contraction Ratio (PECR) was calculated as the total tension of PE+L‐NAME divided by the total tension generated by PE alone (1st addition; see Figure 1a). (b) The values of PECR (x‐axis) were regressed against the values of ACh % relaxation (y‐axis) for filtered air control PG:VG, FA, and AA with regression coefficients of r ² = 0.40, r ² = 0.54, r ² = 0.55, and, r ² = 0.09, respectively

FIGURE 4

Relationship between Phenylephrine (PE) Contraction Ratio and ACh‐induced endothelium‐dependent relaxation in aortas of mice exposed to concentrated ambient particulate matter (CAP). Retrospective analyses of published PECR and ACh % data of ex vivo aortic function (without and with perivascular adipose tissue (PVAT) intact) of male mice exposed to filtered air control or CAP for 9 days, 6 h/day. (a) Representative myograph traces of isometric tension of aortic segments without (clean) and with PVAT intact contracted by PE and relaxed by acetylcholine (ACh % relaxation). After 3× washout over 30 min, the aortas were contracted again with PE followed by addition of L‐NAME. After a plateau in tension, ACh was added to confirm L‐NAME‐induced inhibition of endothelium‐dependent relaxation (EDRF; NO). The PE Contraction Ratio (PECR) was calculated as the total tension of PE+L‐NAME divided by the total tension generated by PE alone (1st addition; see Figure 1a). (b) The values of PECR (x‐axis) were regressed against the values of % ACh relaxation (y‐axis) for filtered air control (HEPA) and CAP (clean and PVAT) with regression coefficients of HEPA‐Clean (r ² = 0.26), HEPA‐PVAT (r ² = 0.44), CAP‐clean (r ² = 0.02), and, CAP‐PVAT (r ² = 0.70), respectively

FIGURE 5

Relationship between Phenylephrine (PE) Contraction Ratio and ACh‐induced endothelium relaxation in aortas of 1‐year‐old male WT and LDLR‐KO rats exposed to water or nicotine in water. (a) Graph summary of the aortic PE Contraction Ratio (PECR) across four groups of rats. (b) The values of PECR (x‐axis) were regressed against the values of % ACh relaxation (y‐axis) for WT (water control), WT+nicotine, LDLR‐KO and LDLR‐KO+nicotine with regression coefficients of WT (r ² = 0.28), WT+nicotine (r ² = 0.13), LDLR‐KO (r ² = 0.06), and, LDLR‐KO+nicotine (r ² = 0.21), respectively. Values are means ± SE of 5–6 aortas per group. #, 0.05 < p < 0.10 versus WT water control