Prostacyclin prevents pulmonary endothelial cell apoptosis induced by cigarette smoke

Abstract

Rationale: Impaired endothelial cell-dependent vasodilation, inflammation, apoptosis, and proliferation are manifestations of endothelial dysfunction in chronic obstructive pulmonary disease (COPD). Prostacyclin (PGI(2)) is a major product of the cyclooxygenase pathway with potent vasodilatory and antimitogenic properties and may be relevant to endothelial dysfunction in COPD.

Objectives: To determine if PGI(2) expression is altered in smoking-related lung disease and if it may be protective in COPD-associated endothelial dysfunction.

Methods: We evaluated, by immunohistochemistry, Western blotting, and polymerase chain reaction, human emphysema tissue compared with normal tissue for expression of prostacyclin synthase (PGI(2)S). We examined the effects of cigarette smoke extract (CSE) and aldehyde components on eicosanoid expression in primary human pulmonary microvascular endothelial cells. Finally, we used a murine model of lung-specific PGI(2)S overexpression and in vitro studies to determine if PGI(2) expression has protective effects on cigarette smoke-induced endothelial apoptosis.

Measurements and main results: Human emphysema lung tissue exhibited lower PGI(2)S expression within the pulmonary endothelium than in normal lung. In vitro studies demonstrated that CSE, and in particular the alpha,beta unsaturated aldehyde acrolein, suppressed PGI(2)S gene expression, whereas CSE significantly induced the upstream mediators COX-2 and cytosolic phospholipase A2 in human pulmonary microvascular endothelial cells. Mice with lung-specific PGI(2)S overexpression exhibited less endothelial apoptosis after chronic smoke exposure. In vitro, iloprost exhibited protective effects on CSE-induced apoptosis.

Conclusions: PGI(2) has protective effects in the pulmonary vasculature after acute and chronic cigarette smoke exposure. An imbalance in eicosanoid expression may be important to COPD-associated endothelial dysfunction.

Figures

Figure 1.

Prostacyclin synthase (PGI2S) expression in human emphysema and nondiseased lung. We performed immunohistochemistry for PGI2S in emphysema (n = 4) and nondiseased lung (n = 3). There is a reduction in PGI2S staining (arrow) of arteriolar pulmonary endothelium from a patient with emphysema (A) compared with nondiseased lung (B). In small and medium-sized vessels, staining was similar in the endothelium from a patient with emphysema (C) compared with nondiseased lung (D) (arrow) (original magnification: ×40). Sections were scored by a pathologist in blinded fashion (number of PGI2S-positive endothelial cells/100 endothelial cells per case) for PGI2S staining and expressed as a ratio in capillaries, small/medium arteries, and arterioles. The differences in PGI2S staining were statistically significant in pulmonary arteriolar endothelium (E) (0.53 ± 0.086 vs. 0.97 ± 0.015; p < 0.01) but not in small/medium-sized vessels (F) (0.74 ± 0.12 vs. 0.97 ± 0.005; p = 0.16).

Figure 2.

Prostacyclin synthase (PGI2S) expression and 6-keto-PGF1α measurements in whole lung lysates from emphysema (n = 4) and nondiseased lung (n = 4). The expression of PGI2S measured by Western blotting is decreased in patients with emphysema (A). 6-Keto-PGF1α metabolite level assessed by ELISA is decreased in patients with emphysema (B) (p < 0.05). PGI2S gene expression measured by reverse transcriptase–polymerase chain reaction is decreased in patients with emphysema (C) (p < 0.05).

Figure 3.

Effect of cigarette smoke extract (CSE) on human pulmonary microvascular endothelial cell eicosanoid expression. Pulmonary microvascular endothelial cells were treated with two concentrations (0.5% and 1%) of CSE and then examined at 24 hours for eicosanoid gene expression. Both 0.5% and 1.0% CSE decreased prostacyclin synthase (PGI2S) at 24 hours (A) (p < 0.05), and cyclooxygenase (COX)-1 gene expression was decreased at 24 hours (B) (p < 0.05). CSE increased COX-2 and cytosolic phospholipase A2 (cPLA2) (p < 0.05) gene expression 24 hours after CSE exposure (C, D). All studies were conducted in triplicate on separate days.

Figure 4.

Effect of components of cigarette smoke (saturated and unsaturated aldehydes) on prostacyclin synthase (PGI2S) and COX-2 expression. Human pulmonary microvascular endothelial cells were exposed to acrolein, acetaldehyde, crotonaldehyde, and propionaldehyde at concentrations of 1 and 10 μM and assessed at 24 hours for eicosanoid expression. Acrolein exposure suppressed PGI2S mRNA at 24 hours and protein expression at 48 hours (A, B) (p < 0.01) while not altering COX-2 mRNA expression (C). Administration of other aldehydes (acetaldehyde, crotonaldehyde, and propionaldehyde) did not result in suppression of PGI2S mRNA expression. Propionaldehyde resulted in a statistically significant induction in PGI2S expression (D) (p < 0.001). All studies were conducted in triplicate on separate days. A = acrolein; C = control.

Figure 5.

Effect of pretreatment with N-acetylcysteine (NAC). Human pulmonary microvascular endothelial cells were treated with 1% cigarette smoke extract (CSE) after 4 hours of preincubation with 1 mM of NAC and examined at 24 hours for gene expression. NAC preincubation did not prevent down-regulation of prostacyclin synthase (PGI2S) by CSE (A) but did prevent up-regulation of COX-2 (p < 0.05) (B). All studies were conducted in triplicate on separate days.

Figure 6.

Effect of pretreatment with antioxidants. Human pulmonary microvascular endothelial cells were preincubated with superoxide dismutase (SOD) mimetic (100 U/ml) (A, B), catalase (100 U/ml) (C, D), or diphenyleneiodonium chloride (DPI) (0.1 μM) (E, F) for 4 hours followed by 1% cigarette smoke extract (CSE) exposure. Gene expression at 24 hours revealed that SOD mimetic, catalase, or DPI did not prevent down-regulation of PGI2S or up-regulation of COX-2 gene expression by CSE. All studies were conducted in triplicate on separate days.

Figure 7.

Effect of pretreatment with Nω-nitro-l-arginine methyl ester (l-NAME). Human pulmonary microvascular endothelial cells were preincubated with l-NAME 1 mM for 4 hours followed by 1% cigarette smoke extract (CSE) exposure. l-NAME pretreatment did not prevent down-regulation of prostacyclin synthase (PGI2S) at 24 hours (A). There was a trend toward the reversal of induction of COX-2 expression (B). This was not statistically significant. All studies were conducted in triplicate on separate days.

Figure 8.

Apoptosis in human pulmonary microvascular endothelial cells (HPMVECs) exposed to cigarette smoke extract (CSE) and effect of pretreatment with prostacyclin analog iloprost. HPMVECs exposed to CSE exhibited a concentration-dependent increase in apoptosis rate (by annexin V) from 3 to 9% (A) (control vs. 2% CSE after 24 h) (p < 0.05). To confirm, HPMVECs exposed to 2% CSE increased apoptosis at 24 hours by twofold by caspase 3/7 analysis (B) (p < 0.05). Thirty-minute preincubation of HPMVECs with iloprost (1 μM and 10 μM) resulted in a statistically significant reduction in CSE-induced apoptosis (C) (p < 0.05).

Figure 9.

Immunohistochemically cleaved caspase 3 staining of the pulmonary endothelium (arrow) of wild-type (A) and transgenic lung-specific prostacyclin synthase (PGI2S) overexpressing mice (B) after 6 months of cigarette smoke exposure (original magnification: ×200). Results represent the number of caspase-positive endothelial cells per total number of endothelial cells as a ratio. Quantification of cleaved caspase staining of the pulmonary endothelium in PGI2S-overexpressing transgenic (PGI2S OE) (0.3946 ± 0.06583) was significantly decreased in comparison to wild-type (WT) (0.6521 ± 0.07224) littermates after 6 months of cigarette smoke exposure (C). Vessel scoring was conducted in blinded fashion in six vessels per mouse (total, 18 vessels) in each experimental group (p < 0.05). OE = overexpressor.