Impact of chronic anticholesterol therapy on development of microvascular rarefaction in the metabolic syndrome

Abstract

Objective: The obese Zucker rat (OZR) model of the metabolic syndrome is partly characterized by moderate hypercholesterolemia, in addition to other contributing comorbidities. Previous results suggest that vascular dysfunction in OZR is associated with chronic reduction in vascular nitric-oxide (NO) bioavailability and chronic inflammation, both frequently associated with hypercholesterolemia. As such, we evaluated the impact of chronic cholesterol-reducing therapy on the development of impaired skeletal muscle arteriolar reactivity and microvessel density in OZR and its impact on chronic inflammation and NO bioavailability.

Materials and methods: Beginning at seven weeks of age, male OZR were treated with gemfibrozil, probucol, atorvastatin, or simvastatin (in chow) for 10 weeks. Subsequently, plasma and vascular samples were collected for biochemical/molecular analyses, while arteriolar reactivity and microvessel network structure were assessed by using established methodologies after 3, 6, and 10 weeks of drug therapy.

Results: All interventions were equally effective at reducing total cholesterol, although only the statins also blunted the progressive reductions to vascular NO bioavailability, evidenced by greater maintenance of acetylcholine-induced dilator responses, an attenuation of adrenergic constrictor reactivity, and an improvement in agonist-induced NO production. Comparably, while minimal improvements to arteriolar wall mechanics were identified with any of the interventions, chronic statin treatment reduced the rate of microvessel rarefaction in OZR. Associated with these improvements was a striking statin-induced reduction in inflammation in OZR, such that numerous markers of inflammation were correlated with improved microvascular reactivity and density. However, using multivariate discriminant analyses, plasma RANTES (regulated on activation, normal T-cell expressed and secreted), interleukin-10, monocyte chemoattractant protein-1, and tumor necrosis factor alpha were determined to be the strongest contributors to differences between groups, although their relative importance varied with time.

Conclusions: While the positive impact of chronic statin treatment on vascular outcomes in the metabolic syndrome are independent of changes to total cholesterol, and are more strongly associated with improvements to vascular NO bioavailability and attenuated inflammation, these results provide both a spatial and temporal framework for targeted investigation into mechanistic determinants of vasculopathy in the metabolic syndrome.

Figure 1

Data (mean±SEM) microvessel density within skeletal muscle of LZR and OZR at 7 weeks (Panel A), 10 weeks (Panel B), 13 weeks (Panel C) and 17 weeks (Panel D) of age. Microvessel density data are presented under control conditions and following chronic treatment of OZR with the anti-cholesterol therapies: gemfibrozil, probucol, simvastatin or atorvastatin. Microvessel density was determined using fluorescence microscopy following labeling of microvessel with Griffonia simplicifolia I lectin (please see text for details). * p<0.05 vs. LZR; † p<0.05 vs. OZR.

Figure 2

Data (mean±SEM) describing incremental distensibility and the slope (β) coefficients from circumferential stress versus strain relationships (inset panels) of skeletal muscle arterioles of LZR and OZR at 7 weeks (Panel A), 10 weeks (Panel B), 13 weeks (Panel C) and 17 weeks (Panel D) of age. Arteriolar wall mechanics data are presented control conditions and following chronic treatment of OZR with the anti-cholesterol therapies: gemfibrozil, probucol, simvastatin or atorvastatin. * p<0.05 vs. LZR; † p<0.05 vs. OZR.

Figure 3

Data (mean±SEM) describing skeletal muscle arteriolar dilation in response to increasing concentrations of acetylcholine of LZR and OZR under control conditions and following chronic treatment of OZR with gemfibrozil, probucol, simvastatin or atorvastatin. Data area presented as paired panels, with the left panels summarizing the concentration-response relationship, and the right panel presenting the contribution of oxidant stress in terms of impacting acetylcholine-induced dilation where the change in the upper bound of this relationship is shown following treatment of the arteriole with TEMPOL. Data are presented for animals at 7 weeks (Panels A/B), 10 weeks (Panels C/D), 13 weeks (Panels E/F) and 17 weeks (Panels G/H) of age. * p<0.05 vs. LZR; † p<0.05 vs. OZR.

Figure 4

Vascular eNOS activity (Panel A; presented as the % arginine conversion), and methacholine-induced NO bioavailability (Panel B; where data present the slope of the relationship between vascular NO production and methacholine concentration, nM/log M methacholine) in LZR and OZR at 17 weeks of age. Data (presented as mean±SEM) are summarized for LZR and OZR under control conditions and following chronic treatment of OZR with gemfibrozil, probucol, simvastatin or atorvastatin. * p<0.05 vs. LZR; † p<0.05 vs. OZR.

Figure 5

Data (mean±SEM) describing skeletal muscle arteriolar constriction in response to increasing concentrations of phenylephrine in LZR and OZR under control conditions and following chronic treatment of OZR with gemfibrozil, probucol, simvastatin or atorvastatin. Data area presented as paired panels, with the left panels summarizing the concentration-response relationship, and the right panel presenting the contribution of vascular nitric oxide production in terms of impacting phenylephrine-induced constriction where the change in the upper bound of this relationship is shown following treatment of the arteriole with L-NAME. Data are presented for animals at 7 weeks (Panels A/B), 10 weeks (Panels C/D), 13 weeks (Panels E/F) and 17 weeks (Panels G/H) of age. * p<0.05 vs. LZR; † p<0.05 vs. OZR.

Figure 6

The relation between plasma total cholesterol level (Panel A), or a proxy variable for NO bioavailability (upper bound of the acetylcholine dose-response relationship; Panel B), and microvessel density from the different animals in the present study. Each animal used in the study, across the experimental groups is presented in this figure. The inset text presents the linear regression equation that best fits these data and the degree to which that equation explains the variability in the data.

Figure 7

Summary plot for the results of the discriminant analyses in the present study. These results provide the functions 1 and 2 (presented in Table 3) which contribute the majority (>90%) of the differences between the experimental groups at each age. Specifically, RANTES, IL-10, MCP-1 and TNF-α play the greatest role in terms of establishing differences between LZR (light blue), OZR (green), and OZR under the four treatment conditions of the present study; gemfibrozil (grey), probucol (purple), simvastatin (orange) and atorvastatin (red). The centroids for each group are presented in the dark blue squares.

Figure 8

Relationships between the four most significant markers of inflammation (identified using discriminant analyses; please see text for details), vascular NO bioavailability, and gastrocnemius muscle microvessel density for animals in the present study. Data are presented for RANTES (Panel A), IL-10 (Panel B), MCP-1 (Panel C), and TNF-α (Panel D), and the same color coding is used as in Figure 4; LZR (light blue), OZR (green), and OZR+GEM (grey), OZR+PRO (purple), OZR+SIM (orange) and OZR+ATOR (red).