PGC-1α (Peroxisome Proliferator-Activated Receptor γ Coactivator 1-α) Overexpression in Coronary Artery Disease Recruits NO and Hydrogen Peroxide During Flow-Mediated Dilation and Protects Against Increased Intraluminal Pressure

Abstract

Blood flow through healthy human vessels releases NO to produce vasodilation, whereas in patients with coronary artery disease (CAD), the mediator of dilation transitions to mitochondria-derived hydrogen peroxide (_mtH₂O₂). Excessive _mtH₂O₂ production contributes to a proatherosclerotic vascular milieu. Loss of PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) is implicated in the pathogenesis of CAD. We hypothesized that PGC-1α suppresses _mtH₂O₂ production to reestablish NO-mediated dilation in isolated vessels from patients with CAD. Isolated human adipose arterioles were cannulated, and changes in lumen diameter in response to graded increases in flow were recorded in the presence of PEG (polyethylene glycol)-catalase (H₂O₂ scavenger) or L-NAME (N^G-nitro-l-arginine methyl ester; NOS inhibitor). In contrast to the exclusively NO- or H₂O₂-mediated dilation seen in either non-CAD or CAD conditions, respectively, flow-mediated dilation in CAD vessels was sensitive to both L-NAME and PEG-catalase after PGC-1α upregulation using ZLN005 and α-lipoic acid. PGC-1α overexpression in CAD vessels protected against the vascular dysfunction induced by an acute increase in intraluminal pressure. In contrast, downregulation of PGC-1α in non-CAD vessels produces a CAD-like phenotype characterized by _mtH₂O₂-mediated dilation (no contribution of NO). Loss of PGC-1α may contribute to the shift toward the _mtH₂O₂-mediated dilation observed in vessels from subjects with CAD. Strategies to boost PGC-1α levels may provide a therapeutic option in patients with CAD by shifting away from _mtH₂O₂-mediated dilation, increasing NO bioavailability, and reducing levels of _mtH₂O₂ Furthermore, increased expression of PGC-1α allows for simultaneous contributions of both NO and H₂O₂ to flow-mediated dilation.

Keywords: arterioles; catalase; coronary artery disease; microcirculation; nitric oxide.

Figure 1

Effect of PGC-1α downregulation on flow-mediated dilation (FMD) in non-CAD vessels. A) The magnitude of FMD is preserved in human arterioles following 48-hour treatment with lentiviral GFP. L-NAME (eNOS inhibitor) acts to inhibit FMD. B) Confirmation of lentiviral GFP uptake in non-CAD vessel via immunohistochemistry. C) The magnitude of FMD is preserved in human arterioles following 48-hour treatment with PGC-1α siRNA. PEG-catalase (H2O2 scavenger) acts to inhibit FMD, whereas L-NAME has no effect. D) To determine the source of H2O2 following PGC-1α knockdown, vessels were incubated with mitochondria-targeted inhibitors rotenone (1 μM) and mitoPBA (5 μM) for 30 minutes prior to initiation of flow. n=4-7 per treatment condition. *P < 0.05 vs control curves at specific pressure gradients.

Figure 2

Effect of PGC-1α overexpression on flow-mediated dilation (FMD) in CAD vessels. Mechanism of FMD in CAD vessels following A) relies on H₂O₂ at baseline, n=4 (control) and 5 (PEG-catalase), *P < 0.05 Peg-CAT vs control curve at specific pressure gradients; both NO and H₂O₂ following treatment with B) 250 μM ALA (16-24 hr) and C) 15 μM ZLN005 (16-24 hr). n=5-8 per curve. *P < 0.05 Peg-CAT+L-NAME vs control curve at specific pressure gradients. D) Effect of Peg-CAT on FMD between different treatment groups. *P < 0.05 ALA/ZLN+Peg-CAT vs control+Peg-CAT curve at specific pressure gradients.

Figure 3

Source of H2O2 after PGC-1α overexpression on flow-mediated dilation (FMD) in CAD vessels. A) Reduction in MitoPY1 fluorescence following 24-hour ALA treatment (250 μM). Changes in fluorescence intensity in response to shear stress were evaluated in untreated or ALA-treated CAD vessels. n=6-7 per treatment group. *P < 0.05 ALA vs control; B) mitoPBA had no additional effect on dilation after incubation with the eNOS inhibitor L-NAME following 24-hour ALA treatment (250 μM) in CAD vessels. P=NS vs control between flow curves.

Figure 4

Assessment of NO and H2O2 production after shear and antioxidant levels following PGC-1α overexpression in HUVECS. A) PGC-1α overexpression using 250 μM ALA treatment increased NO production during 1 hour shear stress (15 dynes/cm2), as determined by DAF-2A/HPLC; B) PGC-1α overexpression using 250 μM ALA treatment increased global H2O2 production during 1 hour shear stress (15 dynes/cm2), as determined by Amplex Red fluorescence. Inhibition of NO production (L-NAME) did not further increase H2O2 release. N=4-6 per treatment group, *P < 0.05 ALA vs vehicle control, Data reported as mean + SEM. C&D) Western blot evaluation of the antioxidant levels of MnSOD and catalase following overnight treatment with ALA and ZLN in HUVECs. P=NS ALA/ZLN versus control.

Figure 5

Effect of increased intraluminal pressure following PGC-1α overexpression in CAD vessels. A) PGC-1α overexpression using 250 μM ALA preserved the magnitude of FMD in response to acute increases in intraluminal pressure (IILP: 150 mmHg, 30 min), whereas acute hypertension severely impaired the overall magnitude of dilation in untreated CAD vessels; *P<0.05 IILP vs control at specific pressure gradients. B) Endothelium-independent, papaverine-induced dilation is not altered by overnight treatment with lipoic acid. N=4 per treatment group. P=NS vs control.

Figure 6

Proposed schematic of the regulation of FMD by PGC-1α. A) Basal PGC-1α in non-CAD vessels allows for NO-mediated dilation (inhibitable by L-NAME alone). B) PGC-1α overexpression provides plasticity in non-CAD and CAD vessels, resulting in contributions of both NO and H2O2 to dilation.