Acute localized administration of tetrahydrobiopterin and chronic systemic atorvastatin treatment restore cutaneous microvascular function in hypercholesterolaemic humans

Abstract

Elevated oxidized low-density lipoproteins (LDL) are associated with vascular dysfunction in the cutaneous microvasculature, induced in part by upregulated arginase activity and increased globalized oxidant stress. Since tetrahydrobiopterin (BH(4)) is an essential cofactor for endothelial nitric oxide synthase (NOS3), decreased bioavailability of the substrate l-arginine and/or BH(4) may contribute to decreased NO production with hypercholesterolaemia. We hypothesized that (1) localized administration of BH(4) would augment NO-dependent vasodilatation in hypercholesterolaemic human skin, which would be further increased when combined with arginase inhibition and (2) the improvement induced by localized BH(4) would be attenuated after a 3 month oral atorvastatin intervention (10 mg). Four microdialysis fibres were placed in the skin of nine normocholesterolaemic (NC: LDL = 95 ± 4 mg dl(-1)) and nine hypercholesterolaemic (HC: LDL = 177 ± 6 mg dl(-1)) men and women before and after 3 months of systemic atorvastatin. Sites served as control, NOS inhibited, BH(4), and arginase inhibited + BH(4) (combo). Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilatation. After the established plateau l-NAME was perfused in all sites to quantify NO-dependent vasodilatation (NO). Data were normalized to maximum cutaneous vascular conductance (CVC). Vasodilatation at the plateau and NO-dependent vasodilatation were reduced in HC subjects (plateau HC: 70 ± 5% CVC(max) vs. NC: 95 ± 2% CVC(max); NO HC: 45 ± 5% CVC(max) vs. NC: 64 ± 5% CVC(max); both P < 0.001). Localized BH(4) alone or combo augmented the plateau (BH(4): 93 ± 3% CVC(max); combo 89 ± 3% CVC(max), both P < 0.001) and NO-dependent vasodilatation in HC (BH(4): 74 ± 3% CVC(max); combo 76 ± 3% CVC(max), both P < 0.001), but there was no effect in NC subjects (plateau BH(4): 90 ± 2% CVC(max); combo 95 ± 3% CVC(max); NO-dependent vasodilatation BH(4): 68 ± 3% CVC(max); combo 58 ± 4% CVC(max), all P > 0.05 vs. control site). After the atorvastatin intervention (LDL = 98 ± mg * dl(-1)) there was an increase in the plateau in HC (96 ± 4% CVC(max), P < 0.001) and NO-dependent vasodilatation (68 ± 3% CVC(max), P < 0.001). Localized BH(4) alone or combo was less effective at increasing NO-dependent vasodilatation after the drug intervention (BH(4): 60 ± 5% CVC(max); combo 58 ± 2% CVC(max), both P < 0.001). These data suggest that decreased BH(4) bioavailability contributes in part to cutaneous microvascular dysfunction in hypercholesterolaemic humans and that atorvastatin is an effective systemic treatment for improving NOS coupling mechanisms in the microvasculature.

Figure 1. A protocol schematic

A, schematic to illustrate the local heating protocol with each microdialysis treatment site. Sites served as: (1) control for a normative reference, (2) nitric oxide synthase inhibited (NOS-I) throughout the protocol, (3) localized tetrahydrobiopterin (BH₄) administered to supplement the essential NOS cofactor, and (4) arginase inhibited (A-I) combined with BH₄ to supplement the essential NOS cofactor and to increase NOS substrate (l-arginine) availability through inhibiting arginase. The non-specific NOS inhibitor l-NAME was perfused after the established plateau to quantify NO-dependent vasodilatation.

Figure 2. A representative tracing

Cutaneous vascular conductance (% max) throughout the time course of a local heating response in a normocholesterolaemic subject's control site. Baseline, initial peak, nadir, plateau, the per cent decrease with NOS inhibition (20 mm l-NAME) and the post-l-NAME plateau are illustrated.

Figure 3. Mean skin blood flow

Cutaneous vascular conductance (% max) at the plateau in skin blood flow during local warming and after NOS inhibition with l-NAME in normocholesterolaemic (Normo) control subjects, hypercholesterolaemic subjects and after the oral atorvastatin intervention in the control site (A), BH₄site (B), BH₄+ arginase-inhibited site (C) and l-NAME throughout local heating (D). *P < 0.05 different to the normocholersterolaemic group; †P < 0.05 different compared to the control site due to the localized microdialysis drug treatment; ‡P < 0.05 different due to the atorvastatin intervention.

Figure 4

The reduction in cutaneous vascular conductance with NOS inhibition in normocholesterolaemic control subjects, hypercholesterolaemic subjects and after the oral atorvastatin intervention in the control site (A), BH₄ site (B) and BH₄+ arginase-inhibited site (C). *P < 0.05 different from the normocholersterolaemic group; †P < 0.05 different compared to the control site due to the localized microdialysis drug treatment; ‡P < 0.05 difference due to the atorvastatin intervention §P < 0.05 difference from the control site with the atorvastatin intervention.

Figure 5. Maximal cutaneous vascular conductance

Absolute cutaneous vascular conductance in all microdialysis treatment sites for the normocholesterolaemic group and the hypercholesterolaemic group before and after the atorvastatin intervention. There was no difference due to localized drug treatment, between groups, or with the intervention.