Hypoxic vasoconstriction of partial muscular intra-acinar pulmonary arteries in murine precision cut lung slices

Abstract

Background: Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) which serves to match lung perfusion to ventilation. The underlying mechanisms are not fully resolved yet. The major vascular segment contributing to HPV, the intra-acinar artery, is mostly located in that part of the lung that cannot be selectively reached by the presently available techniques, e.g. hemodynamic studies of isolated perfused lungs, recordings from dissected proximal arterial segments or analysis of subpleural vessels. The aim of the present study was to establish a model which allows the investigation of HPV and its underlying mechanisms in small intra-acinar arteries.

Methods: Intra-acinar arteries of the mouse lung were studied in 200 mum thick precision-cut lung slices (PCLS). The organisation of the muscle coat of these vessels was characterized by alpha-smooth muscle actin immunohistochemistry. Basic features of intra-acinar HPV were characterized, and then the impact of reactive oxygen species (ROS) scavengers, inhibitors of the respiratory chain and Krebs cycle metabolites was analysed.

Results: Intra-acinar arteries are equipped with a discontinuous spiral of alpha-smooth muscle actin-immunoreactive cells. They exhibit a monophasic HPV (medium gassed with 1% O2) that started to fade after 40 min and was lost after 80 min. This HPV, but not vasoconstriction induced by the thromboxane analogue U46619, was effectively blocked by nitro blue tetrazolium and diphenyleniodonium, indicating the involvement of ROS and flavoproteins. Inhibition of mitochondrial complexes II (3-nitropropionic acid, thenoyltrifluoroacetone) and III (antimycin A) specifically interfered with HPV, whereas blockade of complex IV (sodium azide) unspecifically inhibited both HPV and U46619-induced constriction. Succinate blocked HPV whereas fumarate had minor effects on vasoconstriction.

Conclusion: This study establishes the first model for investigation of basic characteristics of HPV directly in intra-acinar murine pulmonary vessels. The data are consistent with a critical involvement of ROS, flavoproteins, and of mitochondrial complexes II and III in intra-acinar HPV. In view of the lack of specificity of any of the classical inhibitors used in such types of experiments, validation awaits the use of appropriate knockout strains and siRNA interference, for which the present model represents a well-suited approach.

Figure 1

Distribution of α SMA-immunoreactivity in pulmonary veins. (A) Course of a pulmonary vein from the proximal cardiac muscle covered part (upper left) to the pleural surface (arrows). (B) Magnification of the vessel shown in upper boxed area in A. α SMA-immunoreactive cells are branched and build a loose mesh. (C) Magnification of the vessel shown in lower boxed area in A. Few immunoreactive cells are present in the distal part of the vein. α SMA-immunoreactive cells are predominantly located close to the branch point (arrows). (D) A singular, branched α SMA-immunoreactive cell in a distal intra-acinar vein. (D') Differential interference contrast image of D. Bars in A, B, C = 100 μm; in D, D' = 50 μm.

Figure 2

Distribution of α SMA-immunoreactivity in pulmonary arteries. (A) Course of a pulmonary artery from the bronchus (left) to the pleural surface (right). (B) A supernumerary branch (arrow) leaves a pulmonary artery that lies close to a bronchus (Br). (C) Magnification of the boxed area in A. The artery displays a dense mesh of α SMA-immunoreactive cells that becomes wider in distal direction and finally vanishes. To the left, a branch with almost no α SMA-immunoreactive cells leaves the artery. (D) Higher magnification from C that clearly depicts the branched morphology of α SMA-immunoreactive cells. (E) Transition of circular into mesh-like organization of α SMA-immunoreactive cells in the arterial wall. (F) At a branch point, the organization pattern of α SMA-immunoreactive cells changes. Only few if any α SMA-immunoreactive cells are visible in the branches (arrows). (G) Intra-acinar artery devoid of α SMA-immunoreactive cells. (G') Differential interference contrast image of G. Bars in A, B = 100 μm; in C-G' = 50 μm.

Figure 3

Difference between arteries and veins in PCLS. Arteries in cross sections (A, A') can be easily distinguished from veins (B, B') by their accompanying alveolar ducts (AD) in the bright field mode here shown in differential interference contrast (A', B'). The muscular coat of arteries is thicker than in veins as revealed by α SMA-immunohistochemistry (A, B). Bars = 100 μm.

Figure 4

HPV of a partial muscular intra-acinar artery. All panels (A)-(I) refer to the same intra-acinar artery. (A) Employing phase contrast optics the cross section of an intra-acinar artery was localized in a PLCS. The vessel responded to U46619 (U) with vasoconstriction (B), which was reversed by washing the PCLS with normoxic gassed medium (flow rate 6 ml/min) and application of Nipruss. The NO donor was removed by a normoxic wash followed by exposure to normoxic gassed medium (flow rate 0.7 ml/min) (C). Perfusion with hypoxic gassed medium induced constriction of the vessel (D) which was abolished by washing the PCLS with normoxic gassed medium (E). At the end of the experiment, the viability of the artery was validated by application of U46619 (F). (I) Changes in the luminal areas are expressed as relative values, setting the luminal area at the beginning of the experiment as 100%. Time points for which original micrographs are presented in panels A-F are indicated in the curve. U = U46619; W = wash; Ni = Nipruss; No = normoxia. After completion of the videomorphometric experiment, the PLCS was stained for αSMA, and the vessel from which luminal diameters were recorded was reidentified (G, H). Conventional epifluorescence microscopy (G) of the 200 μm thick PCLS suggests the presence of a continuous αSMA-immunoreactive muscular coat, but CLSM analysis and three-dimensional reconstruction of the course of this vessel within the PCLS clearly depicts the discontinuous, spiral arrangement of αSMA-immunoreactive smooth muscle cells. Bars = 20 μm.

Figure 5

HPV of intra-acinar arteries requires ROS and flavoproteins. Videomorphometric analysis of HPV was performed in absence or presence of the ROS scavenger NBT (A) and of the flavoprotein inhibitor DPI (B), respectively. In both cases, HPV was inhibited whereas U46619-induced vasoconstriction was unaffected. The changes in the luminal areas of the vessels are given as relative values, setting the luminal area immediately before exposure to hypoxia as 100%. Data are presented as means ± SEM. *p = 0.05, ** p = 0.01, n.s. = not significant.

Figure 6

Involvement of mitochondrial complex II in HPV. HPV of intra-acinar arteries was analyzed in absence or presence of the complex II inhibitors 3-NPA (A) and TTFA (B). The response to hypoxia was blocked and decelerated, respectively, in presence of the inhibitors. U46619-induced vasoconstriction was unaffected. Data are presented as means ± SEM. *p = 0.05, ** p = 0.01, n.s. = not significant.

Figure 7

Effect of the citrate cycle intermediates succinate, fumarate and malate on HPV. (A) Addition of succinate nearly completely blocked HPV. Significant differences between the succinate and the control group in their response to U46619 were noted when the data were standardized to the luminal areas recorded at the beginning of hypoxic exposure, as depicted here, but not when data were standardized to the luminal areas recorded at the end of the post-hypoxic wash period. (B) Addition of fumarate delayed the hypoxic response and was without significant effect on U46619-induced vasoconstriction. (C) Malate inhibited both HPV and U46619-induced vasoconstriction. Data are presented as means ± SEM. *p = 0.05, ** p = 0.01, n.s. = not significant.

Figure 8

Specific requirement of mitochondrial complex III, but not of complex IV, for HPV. Videomorphometric analysis of HPV was performed in absence or presence of the complex III inhibitor antimycin A (A) and of the complex IV inhibitor NaN₃ (B), respectively. (A) Antimycin A induced distinct vasodilation during hypoxic exposure whereas the response to U46619 was unaffected. (B) NaN₃ inhibited both HPV and U46619-induced vasoconstriction. Data are presented as means ± SEM. *p = 0.05, ** p = 0.01, n.s. = not significant.