Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia

Abstract

Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. The proinflammatory action of hypoxia is mediated by the induction of distinct cytokines and chemokines and is independent of tumor necrosis factor-alpha signaling. We have previously proposed a crucial role for heme oxygenase-1 (HO-1) in protecting cardiomyocytes from hypoxic stress, and potent anti-inflammatory properties of HO-1 have been reported in models of tissue injury. We thus established transgenic mice that constitutively express HO-1 in the lung and exposed them to chronic hypoxia. HO-1 transgenic mice were protected from the development of both pulmonary inflammation as well as hypertension and vessel wall hypertrophy induced by hypoxia. Significantly, the hypoxic induction of proinflammatory cytokines and chemokines was suppressed in HO-1 transgenic mice. Our findings suggest an important protective function of enzymatic products of HO-1 activity as inhibitors of hypoxia-induced vasoconstrictive and proinflammatory pathways.

Figure 1

Expression of HO-1 in transgenic mice. (a) Northern blot analysis for HO-1 and HO-2. HO-1 (Top) and HO-2 mRNA levels (Middle) were examined in total RNA (30 μg) extracted from the lungs of HO-1 transgenic mice (Tg) or nontransgenic littermates (non-Tg). β-actin mRNA levels were examined to confirm equal loading (Bottom). This blot is representative of three independent experiments, six mice per group. (b) Western blot analysis for HO-1 in the lung. Lung whole-cell lysates (30 μg) from five to six HO-1 Tg and a corresponding number of non-Tg littermates were analyzed for HO-1 protein levels using an anti-HO-1 antibody. The same blot was reprobed with an anti-β-actin antibody to verify equal loading (Lower). Note that the anti-HO-1 antibody used here is raised against the N-terminal amino acid sequence of the protein, a region conserved between mouse and human HO-1. (c) Western blot analysis for HO-1 in nonlung tissues. Tissue distribution of HO-1 was analyzed in whole cell lysates (30 μg) of heart, liver, spleen, and kidney from Tg mice (+) or non-Tg littermates (−). This blot represents identical patterns of expression observed in three Tg and three non-Tg animals.

Figure 2

Immunostaining for HO-1 in the lung of Tg mice. Paraffin-embedded sections of the lungs were analyzed for HO-1 expression by immunohistochemistry. (a) High levels of HO-1 expression are evident in the lung of Tg mice (brown, arrow). (b) High power photograph of the rectangle area indicated in a. (c) The section adjacent to that in a incubated with preimmune rabbit serum. (d) Absence of detectable HO-1 immunoreactivity in the lung of non-Tg mice. Original magnification: ×100 for a, c, and d and ×400 for b. Staining was performed in lung sections of four Tg and four non-Tg control mice.

Figure 3

Time course of hypoxic right ventricular hypertrophy: protection by HO-1 overexpression. (a) Non-Tg mice were exposed to hypoxia (8–10% O₂) for 18 h, 5 days (d), or 2–3 weeks (w). Development of RVH after hypoxic exposure was determined by the RV/(LV + S) ratio as described in Materials and Methods. *, P < 0.01, **, P < 0.001 vs. time point 0 (n = 6 for each group, one-way ANOVA). (b) Tg homozygous, heterozygous, and non-Tg control mice from founder 1 and homozygous Tg and non-Tg mice from line 2 were exposed to normoxia or hypoxia for 3 weeks. Under normoxia, line 1 homozygous Tg (n = 19), heterozygous Tg (n = 6), and non-Tg controls (n = 14) as well as line 2 homozygous Tg (n = 5) or non-Tg (n = 5) did not differ in baseline RV/(LV + S) (P > 0.05). After hypoxic exposure, line 1 homozygous Tg (n = 26) and heterozygous Tg mice (n = 8) show significant reduction in RV/(LV + S) as compared with non-Tg controls (n = 22). Similarly, homozygous line 2 Tg mice (n = 5) manifest significantly lower RV/(LV + S) values as compared with their non-Tg controls (n = 5) exposed to hypoxia. (*, P < 0.05, **, P < 0.005 vs. hypoxic controls; †P < 0.01 vs. normoxic controls, Mann–Whitney U test). Data are mean ± SEM.

Figure 4

HO-1 overexpressing mice are protected from pulmonary hypertension induced by hypoxia. (a) RVSP measurements were performed in Tg and non-Tg mice exposed to 2–3 weeks of hypoxia. RVSP did not differ between Tg (n = 13) and non-Tg (n = 8) mice at baseline normoxic conditions. Under hypoxia, Tg mice (n = 25) had significantly lower pressure measurements than non-Tg controls (n = 8) (*, P < 0.05, Mann–Whitney U test). Data are means ± SEM. (b) Hematoxylin and eosin staining of paraffin-embedded lung sections of Tg (Right) or non-Tg control mice (Left) exposed to hypoxia for 2–3 weeks. Original magnification: ×400. Percent wall thickness in arterioles of comparable size was estimated based on analysis of area as described in Materials and Methods (lower graph). Significant reduction in the wall thickness is observed in Tg mice as compared with non-Tg control. Data are expressed as mean ± SEM (n = 5–7). *, P < 0.05 vs. non-Tg control (Mann–Whitney U test).

Figure 5

HO-1 overexpressing mice are protected against pulmonary inflammation induced by hypoxia. (a) Immunostaining for neutrophils and macrophages. Frozen lung sections from Tg or non-Tg control mice that had been exposed to hypoxia for 48 h were stained with anti-Ly-6G antibody for neutrophils and anti-Mac-3 antibody for macrophages (brown staining). Original magnification: ×100. (b) The number of neutrophils and macrophages in BAL fluid after hypoxia. Differential cell counts for neutrophils (Neut, Upper) and macrophages (Mac, Lower) were performed in the BAL fluid of Tg (closed bars) and non-Tg control mice (open bars) after 48 h of hypoxia. Data are expressed as mean ± SEM (n = 4). *, P < 0.05 vs. normoxic non-Tg control. †, P < 0.05 vs. hypoxic non-Tg control (Mann–Whitney U test).

Figure 6

HO-1 overexpression inhibits hypoxic induction of cytokines in the lung. HO-1 Tg and non-Tg control mice were exposed to hypoxia (8–10% O₂) for the times indicated [48 h, 5 days (d), or 2 weeks (w)]. After exposure, total lung RNA (10 μg) was examined for cytokine expression by RNase protection assay. Levels of L32, a ribosomal protein mRNA, were unaffected by hypoxia and served as an internal control. Expression of TNFα 3 h after hypoxia in non-Tg mice is also shown (Lower). Similar results were observed in four independent experiments.