Pulmonary Macrophages Attenuate Hypoxic Pulmonary Vasoconstriction via beta(3)AR/iNOS Pathway in Rats Exposed to Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (IH) induces activation of the sympathoadrenal system, which plays a pivotal role in attenuating hypoxic pulmonary vasoconstriction (HPV) via central beta(1)-adrenergic receptors (AR) (brain) and peripheral beta(2)AR (pulmonary arteries). Prolonged hypercatecholemia has been shown to upregulate beta(3)AR. However, the relationship between IH and beta(3)AR in the modification of HPV is unknown. It has been observed that chronic stimulation of beta(3)AR upregulates inducible nitric oxide synthase (iNOS) in cardiomyocytes and that IH exposure causes expression of iNOS in RAW264.7 macrophages. iNOS has been shown to have the ability to dilate pulmonary vessels. Hence, we hypothesized that chronic IH activates beta(3)AR/iNOS signaling in pulmonary macrophages, leading to the promotion of NO secretion and attenuated HPV. Sprague-Dawley rats were exposed to IH (3-min periods of 4-21% O-2) for 8 h/d for 6 weeks. The urinary catecholamine concentrations of IH rats were high compared with those of controls, indicating activation of the sympathoadrenal system following chronic IH. Interestingly, chronic IH induced the migration of circulating monocytes into the lungs and the predominant increase in the number of proinflammatory pulmonary macrophages. In these macrophages, both beta(3)AR and iNOS were upregulated and stimulation of the beta(3)AR/iNOS pathway in vitro caused them to promote NO secretion. Furthermore, in vivo synchrotron radiation microangiography showed that HPV was significantly attenuated in IH rats and the attenuated HPV was fully restored by blockade of beta(3)AR/iNOS pathway or depletion of pulmonary macrophages. These results suggest that circulating monocyte-derived pulmonary macrophages attenuate HPV via activation of beta(3)AR/iNOS signaling in chronic IH

Pulmonary Macrophages Attenuate Hypoxic Pulmonary Vasoconstriction via beta3AR/iNOS Pathway in Rats Exposed to Chronic Intermittent Hypoxia

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Pulmonary M1 macrophages release NO after activation of the β₃AR/iNOS pathway in IH rats.

<p>Nitrite synthesis from BALF-derived pulmonary macrophages that had been treated with or without CL316243 (CL), isoproterenol (ISO), and CL+L-NIL. A quantitative analysis of nitrite secretion was performed 30 hours after the addition of the drugs using the Griess reaction. The number of rats in each group is shown within each column. Data are presented as mean ± S.D. values.</p

Pulmonary macrophages in IH rats expressed pro-inflammatory markers including iNOS.

<p>The pulmonary macrophages were obtained from BALF after 6 weeks of IH or normoxic exposure. (A) Immunocytochemical staining of pro-inflammatory markers iNOS, CD11c, and IL-6 was performed. (B) Western blot analysis of iNOS, IL-6, and TNFα in BALF-derived macrophages (n = 5 each, mean ± S.D.). *Significant difference between the N and IH rats (*<i>P</i><0.05).</p

IH causes the accumulation of macrophages and upregulates β₃AR expression in the lungs.

<p>(A) Representative bright-field images of lung sections from the N and IH rats and images of immunofluorescent staining of such sections with anti-ED-1 antibody, anti-β_<b>3</b>AR antibody, or both (merged images). Calibration bar = 200 μm for 40 x, 50 μm for 200 x. (B, C) The numbers of ED-1- and β_<b>3</b>AR-positive cells per field (200 x) were counted using Image Pro Plus ver. 4.1 (n = 6 each, mean ± S.D.) (D) Ratio of the percentage of β_<b>3</b>AR-positive cells to the percentage of ED-1-positive cells (n = 6 each, mean ± S.D.) (E) Representative images of double immunocytochemical staining using anti-ED-1 and β_<b>3</b>AR antibody in BALF-derived macrophages. Calibration bar = 50 μm. (F) Western blot analysis of β_<b>3</b>AR in lung homogenate solutions from the N and IH rats (n = 6 each, mean ± S.D.) (G) The expression level of β_<b>3</b>AR mRNA in lung tissue samples from the N and IH rats (n = 6 each, mean ± S.E.M.) (H) Western blot analysis of β_<b>3</b>AR in BALF-derived pulmonary macrophages obtained after 6 weeks of IH or normoxic exposure (n = 5 each, mean ± S.D.) *Significant difference between the N and IH rats (*<i>P</i><0.05, **<i>P</i><0.01).</p

Acute intratracheal administration of clodronate restores HPV in IH rats.

<p>(A) Representative bright-field images and images of immunofluorescent staining using anti-ED-1 antibody of lung sections with or without clodronate. Clodronate (500 μg of clodronate in 100 μL of saline) was injected intratracheally just after the end of the 6-week IH/normoxia exposure period. Calibration bar = 200 μm. (B) Representative microangiographic images of the small pulmonary arteries in the N and IH rats obtained 3 days after the i.t. administration of clodronate. The black arrows point to branches that underwent vasoconstriction. (C) Relationship between vessel size and the extent of the pulmonary vasoconstriction induced in response to acute hypoxia. Data are presented as mean ± S.E.M. values. *Significant change in vessel diameter compared with the baseline conditions (**<i>P</i><0.01).</p

Blockade of iNOS completely restores attenuated HPV in IH rats.

<p>(A) Representative images of the branching pattern of the small pulmonary arteries at the baseline and after the administration of L-NIL (selective iNOS inhibitor). The black arrows point to constricted pulmonary arteries. (B, C) Relationship between vessel size and the extent of the pulmonary vasoconstriction induced in response to acute hypoxia with or without selective L-NIL treatment. Data are presented as mean ± S.E.M. values. *Significant change in vessel diameter compared with the baseline conditions (**<i>P</i><0.01). ^†Significant difference between the N and IH rats (^†<i>P</i><0.05; ^††<i>P</i><0.01). ^‡Significant difference compared with the no drug conditions (^‡<i>P</i><0.05, ^‡‡<i>P</i><0.01).</p

Blockade of β₃AR completely restores attenuated HPV in IH rats.

<p>(A) Representative microangiographic images showing the branching pattern of the small pulmonary arteries during normoxia and in response to hypoxia with or without SR59230A (selective β_<b>3</b>-blocker). Images were recorded after 5 min exposure to A: normoxia, B: hypoxia (10% O_<b>2</b>), C: SR59230A + normoxia, and D: SR59230A + hypoxia in the same lung field. The black arrows point to branches of the pulmonary arteries that constricted in response to acute hypoxia. The tungsten wire in the lower right corner of each image is a reference wire with a diameter of 50 μm. (B, C) Relationship between vessel size and the extent of the pulmonary vasoconstriction (% decrease in vessel diameter) induced in response to acute hypoxia in the N and IH rats treated with or without SR59230A. The data are presented as mean ± S.E.M. values. *Significant reduction in vessel diameter compared with the normoxic conditions (**<i>P</i><0.01). ^†Significant difference between the N and IH rats (^††<i>P</i><0.01). ^‡Significant difference compared with the no drug conditions. (^‡‡<i>P</i><0.01).</p