Hypoxia exerts dualistic effects on inflammatory and proliferative responses of healthy and asthmatic primary human bronchial smooth muscle cells

Abstract

Background: For oxygen supply, airway wall cells depend on diffusion though the basement membrane, as well as on delivery by micro-vessels. In the asthmatic lung, local hypoxic conditions may occur due to increased thickness and altered composition of the basement membrane, as well as due to edema of the inflamed airway wall.

Objective: In our study we investigated the effect of hypoxia on proliferation and pro-inflammatory and pro-angiogenic parameter production by human bronchial smooth muscle cells (BSMC). Furthermore, conditioned media of hypoxia-exposed BSMC was tested for its ability to induce sprout outgrowth from endothelial cells spheroids.

Methods: BSMC were cultured in RPMI1640 (5% FCS) under normoxic (21% O₂) and hypoxic (1% and 5% O₂) conditions. Proliferation was determined by cell count and Western blot analysis for cyclin E and Proliferating Cell Nuclear Antigen (PCNA). Secretion of IL-6, IL-8, ENA-78 and VEGF-A was analyzed by ELISA. BSMC conditioned medium was tested for its angiogenic capacity by endothelial cell (EC)-spheroid in vitro angiogenesis assay.

Results: Proliferation of BSMC obtained from asthmatic and non-asthmatic patients was significantly reduced in the presence of 1% O₂, whereas 5% O₂ reduced proliferation of asthmatic BSMC only. Hypoxia induced HIF-1α expression in asthmatic and non-asthmatic BSMC, which coincided with significantly increased release of IL-6, IL-8 and VEGF-A, but not ENA-78. Finally, endothelial sprout outgrowth from EC spheroids was increased when exposed to hypoxia conditioned BSMC medium.

Conclusion: Hypoxia had dualistic effects on proliferative and inflammatory responses of asthmatic and non-asthmatic BSMC. First, hypoxia reduced BSMC proliferation. Second, hypoxia induced a pro-inflammatory, pro-angiogenic response. BSMC and EC may thus be promising new targets to counteract and/or alleviate airway wall remodeling.

Figure 1. Light microscopic photographic images of airway tissue sections obtained from a non-asthmatic (A) and an asthmatic (B) patient.

Images (magnification 60×) are representative of tissues obtained from 3 non-asthmatic and 7 asthmatic patients stained with Haematoxylin-Eosin. Note the increased thickening of the basement membrane in the asthmatic airways. E = epithelium, arrows are indicating the basement membrane.

Figure 2. Proliferation characteristics of BSMC from asthmatic (A) and non-asthmatic (NA) subjects in the presence of 1%, 5% and 21% O₂ (72 h).

BSMC were cultured for 72 hours in presence (A) and absence (B) of 5% FCS. The data presented in A and B are shown as one group (6A+6NA for 21% O2 and 4A+3NA for 1% and 5% O₂), as well as two separate groups (6A and 6NA for 21% O_2; 4A and 3NA for 1% and 5% O2). C and D, densitometric analysis of PCNA (9 independent experiments in 5 subjects) and cyclin E (6 independent experiments in 5 subjects) under hypoxic (1% O₂) and normoxic conditions (21% O₂). E and F, representative Western blots for PCNA and cyclin E protein expression. Densitometric values are given as mean ± SEM (*p-value ≤0.05, **p-value ≤0.01, ***p-value ≤0.001).

Figure 3. Western blot analysis detecting HIF-1α and GAPDH (loading control) in lysates of BSMC.

Cells were incubated under 1% and 21% O₂ for 4, 24 and 48 h (indicated at top). CoCl₂ was used as positive control to stabilize HIF-1α protein. In contrast to CoCl₂, which has a transient effect only, hypoxia induced a prolonged expression of HIF-1α. The experiments shown are representative for 5 independent experiments. The right hand panel (showing the 24 h and 48 h expression levels of HIF-1α) was obtained after longer exposure of the same blot, which contained all samples.

Figure 4. Normoxia (21% O2) versus hypoxia (1% O2). Effects on cytokine release from BSMC grown in the presence of 5%FCS (72 h).

Concentrations of VEGF (A), IL-6 (B) and IL-8 (C) in CM collected from BSMC of 5 non-asthmatic and 5 asthmatic subjects were determined by ELISA. Values are given as mean ± SEM. *p-value ≤0.05, *p-value ≤0.01, ***p-value ≤0.001 (all relative to 21% O₂). Abbreviations: A = asthmatics, NA = non-asthmatics.”

Figure 5. In vitro angiogenesis assay.

A, Representative images of the in vitro angiogenesis assay showing sprout outgrowth from EC-spheroids incubated with CM from BSMC cultured in presence of 21% O₂ (normoxia) or 1% O₂ (hypoxia). B, lengths of sprouts outgrowing from every spheroid were measured and the mean of the longest 10 sprouts of 7 randomly chosen spheroids was plotted. C, effects of the CXCR2-blocker SB 265610, anti-VEGF, and the combination of SB 265610 plus anti-VEGF on EC-sprouting using the in vitro angiogenesis assay. EC-sprouting was induced by pooled CM of BSMC (n = 5) cultured under hypoxic conditions (1% O₂, 72 h). Values are given as mean of the mean ± SEM (*p-value ≤0.05, **p-value ≤0.01, *** p-value ≤0.001).