Sustained hypoxia promotes the development of a pulmonary artery-specific chronic inflammatory microenvironment

Abstract

Recent studies demonstrate that sustained hypoxia induces the robust accumulation of leukocytes and mesenchymal progenitor cells in pulmonary arteries (PAs). Since the factors orchestrating hypoxia-induced vascular inflammation are not well-defined, the goal of this study was to identify mediators potentially responsible for recruitment to and retention and differentiation of circulating cells within the hypoxic PA. We analyzed mRNA expression of 44 different chemokine/chemokine receptor, cytokine, adhesion, and growth and differentiation genes in PAs obtained via laser capture microdissection in adjacent lung parenchyma and in systemic arteries by RT-PCR at several time points of hypoxic exposure (1, 7, and 28 days) in Wistar-Kyoto rats. Analysis of inflammatory cell accumulation and protein expression of selected genes was concomitantly assessed by immunochemistry. We found that hypoxia induced progressive accumulation of monocytes and dendritic cells in the vessel wall with few T cells and no B cells or neutrophils. Upregulation of stromal cell-derived factor-1 (SDF-1), VEGF, growth-related oncogene protein-alpha (GRO-alpha), C5, ICAM-1, osteopontin (OPN), and transforming growth factor-beta (TGF-beta) preceded mononuclear cell influx. With time, a more complex pattern of gene expression developed with persistent upregulation of adhesion molecules (ICAM-1, VCAM-1, and OPN) and monocyte/fibrocyte growth and differentiation factors (TGF-beta, endothelin-1, and 5-lipoxygenase). On return to normoxia, expression of many genes (including SDF-1, monocyte chemoattractant protein-1, C5, ICAM-1, and TGF-beta) rapidly returned to control levels, changes that preceded the disappearance of monocytes and reversal of vascular remodeling. In conclusion, sustained hypoxia leads to the development of a complex, PA-specific, proinflammatory microenvironment capable of promoting recruitment, retention, and differentiation of circulating monocytic cell populations that contribute to vascular remodeling.

Fig. 1.

Hypoxia induces a progressive accumulation of leukocytic cells expressing CD11b in the rat pulmonary artery (PA) adventitia. Cryosections of PAs from normoxic (A), 7-day hypoxic (B), and 28-day hypoxic (C) rats were stained with antibodies against CD11b (red) and cell nuclei [4′,6′-diamidino-2-phenylindole (DAPI), blue]. Scale bar is 100 μm; all pictures were taken at ×10 magnification. D: quantification of the number of leukocytic cells expressing CD11b (CD11b+ cell index) in the rat PA over time. One-day hypoxic rats show no difference in the number of CD11b+ cells compared with controls, whereas 7- and 28-day hypoxic show a progressive increase in the number of CD11b+ cells. In addition, leukocytic cells are found further away from the external elastic lamina in the 7- and 28-day hypoxic rats due to the increased thickness of the adventitia.

Fig. 2.

Hypoxia induces accumulation of dendritic cells but not of neutrophils and lymphocytes in the PA adventitia. Cryosections of PAs from normoxic rats (left column images: A, C, E, and G) and 28-day hypoxic rats (right column images: B, D, F, and H) were stained with Alexa 594 (red)-conjugated antibodies against neutrophils (A and B), CD3 (T cells; C and D), CD45RA (B cells; E and F), and OX62 (dendritic cells; G and H). Cell nuclei were labeled with DAPI (blue). Green autofluorescence of PA elastic lamellae provide the boundaries of the medial layer (m). The thickened PA adventitia (adv) in samples from hypoxic rats is marked with a bar. Accumulation of neutrophils was clearly observed in the lung parenchyma (L.p.) of hypoxic rats (B) but not within the adventitial layer. T and B lymphocytes did not accumulate in the adventitia (for positive internal control, see insets in D and F) demonstrating bright staining of lymph nodes with the respective antibodies. Chronic (28-day) hypoxia induced significant accumulation of dendritic cells in the periadventitial areas (H).

Fig. 3.

Hypoxia induces increases in mRNA levels of cytokines and receptors that may control the recruitment of cells in rat PAs. A and B: distribution of the log-transformed relative expression levels of cytokines (A) and their receptors (B) in PAs isolated by laser capture microdissection (LCM) from hypoxic animals (dark gray) and regression animals (light gray). Bars represent the 25th and 75th percentiles, and whiskers designate 1.5 interquartile ranges. The lines between measurements connect the median values. Stars above the dark gray bars indicate significant deviation of the mean values from the reference line at 0. Stars above the 2- and 28-day regression time points (light gray bars) relate to significant differences between the means for those time points and the 28-day hypoxic values. *P = 0.01–0.05, **P < 0.01. SDF-1, stromal cell-derived factor-1; MCP-1, monocyte chemoattractant protein-1.

Fig. 4.

Hypoxia induces increases in mRNA levels of genes that may control the adhesion, differentiation, and growth of cells in rat PAs. A–C: distribution of the log-transformed relative expression levels of adhesion molecules (A), cell differentiation molecules (B), and growth factors (C) in PAs isolated by LCM from hypoxic animals (dark gray) and regression animals (light gray). Bars represent the 25th and 75th percentiles, and whiskers designate 1.5 interquartile ranges. The lines between measurements connect the median values. Stars above the dark gray bars indicate significant deviation of the mean values from the reference line at 0. Stars above the 28- and 2-day regression time points (light gray bars) relate to the significant difference between the means for those 2 time points and the 28-day hypoxic values. *P = 0.01–0.05, **P < 0.01. TGF-β₁, transforming growth factor-β₁.

Fig. 5.

Chronic hypoxia induces a proinflammatory and profibrotic microenvironment in rat PAs. Lung sections from normoxic (lowercase letters) and 28-day hypoxic (uppercase letters) rats were stained with antibodies against CXCL12/SDF-1 (a/A; red), osteopontin (b/B; red), VCAM-1 (c/C; red), TGF-β₁ (d/D; green), VEGF (e/E; brown), heat shock protein HSP-47 (f/F; green), cellular (ED-A isoform) fibronectin (ED-A-FN; g/G; green) and cell nuclei (DAPI, blue). Scale bar is 100 μm; all pictures were taken at ×10 magnification.

Fig. 6.

Removal of hypoxic stimulus reverses PA inflammation and remodeling. Cryosections of PAs from normoxic (a/A), 28-day hypoxic (b/B), 48-h regression (c/C), and 8-wk regression (d/D) rats stained with CD11b (uppercase letters, red) and ED-A-FN (lowercase letters, green) and cell nuclei (DAPI, blue) are shown. Scale bar is 100 μm; all pictures were taken at ×10 magnification.