HO-1 and CO decrease platelet-derived growth factor-induced vascular smooth muscle cell migration via inhibition of Nox1

Abstract

Objective: Heme oxygenase-1 (HO-1), via its enzymatic degradation products, exhibits cell and tissue protective effects in models of vascular injury and disease. The migration of vascular smooth muscle cells (VSMC) from the medial to the intimal layer of blood vessels plays an integral role in the development of a neointima in these models. Despite this, there are no studies addressing the effect of increased HO-1 expression on VSMC migration. Results and Methods- The effects of increased HO-1 expression, as well as biliverdin, bilirubin, and carbon monoxide (CO), were studied in in vitro models of VSMC migration. Induction of HO-1 or CO, but not biliverdin or bilirubin, inhibited VSMC migration. This effect was mediated by the inhibition of Nox1 as determined by a range of approaches, including detection of intracellular superoxide, nicotinamide adenine dinucleotide phosphate oxidase activity measurements, and siRNA experiments. Furthermore, CO decreased platelet-derived growth factor-stimulated, redox-sensitive signaling pathways.

Conclusions: Herein, we demonstrate that increased HO-1 expression and CO decreases platelet-derived growth factor-stimulated VSMC migration via inhibition of Nox1 enzymatic activity. These studies reveal a novel mechanism by which HO-1 and CO may mediate their beneficial effects in arterial inflammation and injury.

Figure 1. Inhibition of PDGF-induced RASMC migration by HO-1 and CO

(A) Protein expression levels and HO activity in control, AdLacZ or AdHO-1 treated cells. (B,C,D) PDGF (10ng/ml)-induced RASMC migration was measured by the wound assay. Cells were incubated with AdHO-1 or AdLacZ (MOI 100) for 24h prior to PDGF stimulation. CO gas (250 ppm), CORM2, RuCl₃ (100μM), biliverdin (30μM) or bilirubin (10μM) was added 30min prior to stimulation with PDGF. Data represent the mean +/− S.E.M. of three individual experiments. *= Statistically different (p<0.05) from PDGF alone.

Figure 2. CO inhibits PDGF-stimulated increases in O₂⁻

RASMC were starved for 48h and treated as follows: (A) pretreated for 30 min with DPI (10μM) or peg-SOD (10U/ml ) and then stimulated with PDGF (25ng/ml) for 45 min or (B) pretreated for 30 min with CORM-2 (100μM), RuCl₃ (100μM) or 1 hour with CO gas (250 ppm) and then stimulated with PDGF (25ng/ml) for 45 min. Intracellular O₂⁻ production was then assayed as described in Methods. Data represent the mean +/− S.E.M. of at least 5 individual experiments. *= Statistically different (p<0.05) from PDGF alone.

Figure 3. Increased HO-1 expression leads to inhibition of PDGF-stimulated NADPH oxidase activity

PDGF-induced NADPH oxidase activity in RASMC treated with DPI (10μM) or peg-SOD (10U/ml) as assessed by (A) lucigenin chemiluminescence or (B) cytochrome c reduction. In (A) RASMC were also treated with L-NAME (100μM) or Allopurinol (60μM). (C) PDGF-induced NADPH oxidase activity in RASMC treated with AdHO-1 or AdLacZ (MOI 100). Data represent the mean +/− S.E.M. of 6 individual experiments. *= Statistically different (p<0.05) from PDGF alone.

Figure 4. CO inhibits PDGF-stimulated NADPH oxidase activity

(A) Effect of CO gas (100 and 250 ppm), CORM-2 (100μM) or RuCl₃ (100μM) on NADPH oxidase activity in RASMC. (B) Effect of CORM-2 (100μM) or peg-SOD (10U/ml) on PDGF-induced NADPH oxidase activity in the medial layer of the aorta. Data represent the mean +/− S.E.M. of 6 individual experiments. *=Statistically different (p<0.05) from PDGF alone. (C) Tracings of real time chemiluminescence measurements of NADPH oxidase activity in membrane fractions from control or PDGF-stimulated RASMC. Arrows indicate time of addition of NADPH (100μM) or CO (~9μM) saturated buffer. Data show mean +/− S.D. of 4 replicates and are representative of 3 individual experiments. *= Statistically different (p<0.05) from PDGF alone.

Figure 5. Inhibition of RASMC migration by CO is mediated by inhibition of Nox1

(A) Basal NADPH oxidase in membrane fractions from RASMC treated with Nox1, Nox4 or NT siRNA. (B) PDGF-stimulated NADPH oxidase activity measured in membrane fractions of RASMC treated with Nox1, Nox4 or NT siRNA with or without CORM-2 (100μM) treatment. Data represent the mean +/− S.E.M. of 6 individual experiments. (C) PDGF-induced migration in Nox1, Nox4 or NT siRNA transfected RASMC with or without CORM-2 (100μM) treatment. Data represent the mean +/− S.E.M. of 3 individual experiments. *= Statistically different (p<0.05) from NT siRNA w/o CORM-2 treatment. # = Statistically different (p<0.05) from Nox4 siRNA w/o CORM-2 treatment

Figure 6. CO inhibits redox-dependent signaling events in RASMC

(A) Serum starved RASMC were stimulated with 10ng/ml PDGF for the indicated times with or without treatment with CORM-2 (100μM) and analyzed by Western analysis using antibodies against phospho- or total AKT, p38, JNK, or ERK1/2. (B,C,D,E & F) Protein bands were quantitated by densitometric analysis, for p-p38, p-JNK, p-ERK1, p-ERK 2 & p-AKT respectively. Data are representative of mean +/− S.E.M. of 3 individual experiments. *= Statistically different (p<0.05) from control.