Pulmonary prostacyclin synthase overexpression in transgenic mice protects against development of hypoxic pulmonary hypertension

Abstract

Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway leading to prostacyclin (PGI2) production. Patients with severe pulmonary hypertension have a PGIS deficiency of their precapillary vessels, but the importance of this deficiency for lung vascular remodeling remains unclear. We hypothesized that selective pulmonary overexpression of PGIS may prevent the development of pulmonary hypertension. To study this hypothesis, transgenic mice were created with selective pulmonary PGIS overexpression using a construct of the 3.7-kb human surfactant protein-C (SP-C) promoter and the rat PGIS cDNA. Transgenic mice (Tg+) and nontransgenic littermates (Tg-) were subjected to a simulated altitude of 17,000 ft for 5 weeks, and right ventricular systolic pressure (RVSP) was measured. Histology was performed on the lungs. The Tg+ mice produced 2-fold more pulmonary 6-keto prostaglandin F1alpha (PGF1alpha) levels than did Tg- mice. After exposure to chronic hypobaric hypoxia, Tg+ mice have lower RVSP than do Tg- mice. Histologic examination of the lungs revealed nearly normal arteriolar vessels in the Tg+ mice in comparison with vessel wall hypertrophy in the Tg- mice. These studies demonstrate that Tg+ mice were protected from the development of pulmonary hypertension after exposure to chronic hypobaric hypoxia. We conclude that PGIS plays a major role in modifying the pulmonary vascular response to chronic hypoxia. This has important implications for the pathogenesis and treatment of severe pulmonary hypertension.

Figure 1

Human SP-C/rat PGIS transgene construct. Pertinent restriction sites used in cloning are noted. SV-40 small T intron is used as a gene-specific probe for Northern blots, Southern blots, and in situ hybridization studies.

Figure 2

Southern (a) and Northern (b) blot analysis of transgenic mice and nontransgenic littermates. Representative autoradiograms are shown. (a) For Southern analysis, each lane was loaded with equal amounts of genomic DNA digested with EcoRI and probed with the 0.4-kb SV-40 small T intron as described in Methods. Because the cDNA for PGIS has an internal EcoRI site, the position of the transgene signal cannot be predicted. Lines 2 and 8 represent nontransgenic animals, and no signal is detected. (b) Total RNA was prepared from the F2 offspring of the founders in lines 4 and 9. Twenty micrograms of RNA was loaded in each lane, and the blot was probed with both SV-40 small T intron as a gene-specific probe and β-actin to demonstrate equal loading. Line 4 represents a low-expressing line, and line 9 represents a high-expressing line. There is no transgene expression in the nontransgenic littermates for each of these lines.

Figure 3

Detection of transgene expression by in situ hybridization. Sections from a transgenic mouse lung were hybridized to a digoxigenin-labeled gene-specific probe (SV-40 small T intron). (a) Antisense probe, showing transgene expression in the distal lung epithelium. b, bronchial lumen; a, alveolar spaces. Arrows indicate cells with positive signal. (b) Control sense probe. Minimal staining is shown.

Figure 4

Pulmonary PGIS activity. Whole lungs were homogenized in a buffer containing AA. The stable breakdown product of PGI₂, 6-keto PGF_1α, was assayed as a direct measure of PGIS enzyme activity. Transgenic animals (n = 5) had significantly higher 6-keto PGF_1α production than did their nontransgenic (n = 5) littermates (3,107 vs. 1,159 ng/g tissue; P = 0.01).

Figure 5

Hematocrits of animals at baseline and after hypobaric hypoxia exposure. Transgenic animals (n = 5) and nontransgenic littermates (n = 5) did not differ in hematocrits at baseline (32.2% vs. 33.2%; P = ns). After prolonged exposure to hypobaric hypoxia, both transgenic (n = 5) and nontransgenic (n = 5) animals developed significantly elevated hematocrits when compared with room air controls (32.0 vs. 47.8 for Tg⁺ [*P = 0.002]; 33.2 vs. 46.4 for Tg^– [*P = 0.005]). The hematocrit difference between transgenic and nontransgenic littermates was not significant at altitude (47.8 vs. 46.4; P = ns).

Figure 6

RVSP at baseline, with acute hypoxia, and after exposure to hypobaric hypoxia. Direct right ventricular pressure was determined by a transthoracic cannulation technique as described in Methods. At baseline (Control, Denver altitude), transgenic (n = 5) and nontransgenic (n = 5) animals show similar RVSP (30.0 vs. 31.1 mmHg; P = 0.68, ns). With acute hypoxia, transgenic mice (n = 10) did not show vasoconstriction, compared with their nontransgenic littermates (n = 14) (30.1 vs. 35.4 mmHg; *P = 0.02). After 5 weeks of exposure to hypobaric hypoxia, nontransgenic animals (n = 5) had significantly higher RVSP than did PGIS transgenic animals (n = 5) (46.6 vs. 29.2 mmHg; **P = 0.001).

Figure 7

Histology of lungs from high altitude–exposed animals. (a) Comparison of the vessel wall thickness of small- to medium-sized (30–50 μm) pulmonary arteries. Vessel wall thickness was calculated by external diameter (Ed) minus internal diameter (Id) divided by the external diameter: (Ed–Id)/Ed. At baseline (Control, Denver altitude), transgenic (n = 5) and nontransgenic (n = 5) animals have similar vessel wall thickness (0.165 vs. 0.167; P = 0.85, ns). After chronic hypoxia, nontransgenic animals (n = 5) had significantly more vessel wall hypertrophy of small- to medium-sized vessels than did transgenic mice (n = 5) (0.21 vs. 0.16; *P = 0.0015). (b) Vascular histology (×400). Vessels of comparable diameters, showing the hypertrophy of the vessel wall in nontransgenic animals compared with transgenic animals. Tg^–, nontransgenic littermate; Tg⁺, transgenic animal. Arrows indicate the measured vessels. Along with vessel wall hypertrophy, the Tg^– vessels show the prominence of endothelial cells.