Location: Exhibit/Poster Hall A-B - Pennsylvania Convention Center
Poster Board Number: E602
Rosstin Ahmadian (University of New Mexico Health Sciences Center), Jay Naik (University of New Mexico Health Sciences Center), Benjimen Walker (University of New Mexico Health Sciences Center), Nikki Jernigan (University of New Mexico Health Sciences Center), Thomas Resta (University of New Mexico Health Sciences Center)
Presenting Author University of New Mexico Health Sciences Center
Introduction: Chronic hypoxia (CH) leads to increased pulmonary vascular resistance and resultant pulmonary hypertension (PH) in patients with chronic obstructive pulmonary disease, sleep apnea, or in residents at high altitude. Recent studies from our laboratory have demonstrated that CH augments vasoconstrictor reactivity to a variety of stimuli, including pulmonary arterial smooth muscle cell (PASMC) stretch, membrane depolarization, and stimulation of G protein-coupled receptors. This effect of CH is mediated by a switch in PASMC signaling from Ca2+-dependent mechanisms towards a novel epidermal growth factor receptor (EGFR)-NADPH oxidase 2 (NOX2)-Rho kinase (ROK) Ca2+ sensitization pathway. The overall objective of this study is to understand the mechanism by which CH mediates this switch in signaling. We have recently demonstrated that CH decreases membrane cholesterol, a component of lipid membrane domains that can regulate both EGFR and NOX2 activity in other cell types. Therefore, we hypothesized that this EGFR-NOX2-ROK signaling pathway is intact in PASMC from control animals but quiescent due to inhibition by membrane cholesterol.
Methods: We first validated our ability to manipulate membrane cholesterol levels by treating primary cultures of PASMCs from Sprague Dawley rats with either cholesterol (2 mM), epicholesterol (an epimeric form of cholesterol; 2 mM), or MβCD (cholesterol chelator; 10 mM). Membrane cholesterol content was quantified by filipin (50 mg/ml) fluorescence using confocal microscopy. Subsequently, we examined vasoconstrictor responses to increasing concentrations of epidermal growth factor (EGF) (10-10 – 10-6 M) in pressurized, endothelium-disrupted rat pulmonary arteries (~150 mm diameter). The effect of diminished membrane cholesterol on vasomotor responses was tested by substituting native cholesterol with epicholesterol (2 mM), which lacks the ability participate in protein-sterol interactions while maintaining normal membrane fluidity. Vessels were additionally treated with the selective NOX2 inhibitor gp91ds-tat (50 mM), the selective Rho kinase inhibitor fasudil (10 mM), or their respective vehicles.
Results: Cholesterol supplementation significantly increased PASMC membrane cholesterol as indicated by filipin fluorescence. Conversely, treatment with either MβCD or epicholesterol significantly decreased membrane cholesterol compared to vehicle-treated cells (Figure 1). In agreement with our hypothesis, epicholesterol-dependent substitution of cholesterol revealed concentration-dependent vasoconstriction to EGF in pulmonary arteries (Figure 2A). Treatment with either fasudil (Figure 2A) or gp91ds-tat (Figure 2B) attenuated this effect.
Conclusions: The EGFR-NOX2-RhoA signaling cascade is present in arteries from normoxic animals but is normally inhibited by membrane cholesterol. The results of this study will allow for improved mechanistic analysis of how cholesterol regulates this pathway that is central to enhanced vasoconstrictor reactivity in PH.
Support or Funding Information
NIH RO1 HL132883 and T32 HL007736 to T.C. Resta
Figure 1: Epicholesterol Treatment Diminishes Filipin Fluorescence in Rat PASMCs. Mean filipin fluorescence in primary cultures of PASMC after treatment with cholesterol, the cholesterol substitution agent epicholesterol, or the cholesterol chelator MβCD. Values are mean ± SE. n = 8-9. *P < 0.05 vs. vehicle. Analyzed by 1-way ANOVA followed by Tukey’s post hoc comparison. AU, arbitrary units.; Figure 2: Epicholesterol Unmasks EGFR-Dependent Vasoconstriction through NOX2 and ROK Signaling in Pulmonary Arteries. Vasoconstriction (% baseline inner diameter) to EGF was assessed in pressurized, endothelium-disrupted, pulmonary arteries treated with epicholesterol (epichol), fasudil (A), gp91ds-tat (gp91) (B), or vehicle (Veh). Values are mean ± SE. n = 5-6. *P < 0.05 vs. Veh-Veh. #P < 0.05 vs. Epichol-Veh. Analyzed by 1-way ANOVA followed by Tukey’s post hoc comparison (A) or t-test (B).
