Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung‐to‐gas bladder evolutionary transition in ray‐finned fishes

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray‐finned fishes is the gas bladder, an air‐filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe‐finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral‐to‐dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray‐finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.     

The adrenomedullin receptor acts as clearance receptor in pulmonary circulation

Adrenomedullin (AM) is a powerful pulmonary vasodilator with antimitogenic properties. We investigated the role of the AM receptor (AMR) and the calcitonin gene-related peptide type-1 receptor (CGRP1R) in regulating pulmonary vascular AM levels. The AMR antagonist hAM(22-52) (120 nmol/L) significantly elevated AM release compared with controls to 250% after 2 h in isolated rat lungs and to 830% after 4 h in pulmonary artery endothelial cells (PAEC). CGRP1R blockade had no effect. AMR blockade did not influence prepro-AM mRNA levels nor did inhibition of protein synthesis by cycloheximide (0.01 mg/mL) abolish the effect of the AMR antagonist. Radioligand-binding studies with PAEC membranes revealed a decrease by 44% of the AMR density in response to AMR antagonism. Altogether, the pulmonary vascular AMR represents not only a functionally active, but also a clearance receptor; its expression is constitutively stimulated by basal AM. This identifies a novel mechanism for controlling pulmonary AM levels.     

Development of respiratory structures in embryos and first and second instars of the bark scorpion,Centruroides gracilis (Scorpiones: Buthidae)

The SEM was used to study the development of respiratory structures in successive stages in relation to the overall changes occurring in the scorpions. Book lung development is a slow process, starting with spiracles and a sac‐like atrium in the early embryo and continuing lamellar formation to 150 or more in the adult. In the embryo, the primordial epithelial cells become aligned in a planar pattern as they secrete granules of material that aggregate spontaneously to form the cuticular walls of the lamellae. A blade‐like structure is formed consisting of cells sandwiched within the two cuticle walls they secreted. These cells are in the primordial air channel. The adjacent hemolymph channel is nearly devoid of cells, but cross‐bridges develop and help stabilize the cuticle walls and maintain the width of the channel. The cells in the primordial air channel undergo cytolysis, leaving it open for air except for cuticular cross‐bridges. Development continues in the newborn (first instars); the air channels of some lamellae still contain cells and are not yet functional for gas exchange. The first instars are weak and relatively inactive. They climb up on the mother's dorsum until the first molt (about 8 days). With the cuticular walls of the lamellae in place, cells adhering to the wall in the hemolymph channel produce a thin, new tissue layer (epithelium) on the lamellar wall facing the hemolymph channel. This layer has many discontinuities as though it is slowly developing. Formation of the tissue layer and cytolysis of the cells in the air channels continue through the first molt in which little book lung cuticle is shed as exuvium. The air channels of the second instars (foraging nymphs) are now cell free and open for air passage except for the cross‐bridges. The tissue layer is still incomplete and continues to be formed. It may provide the hypodermal primordium for cuticle replacement in later molts, but development was not studied beyond the second instar except for comparison with book lungs in the adult. The blade‐like lamellae in the adult are larger and more numerous than in the second instar, but in the anterior book lung the shape of the cuticle wall and cross‐bridges and the widths of the air and hemolymph channels are about the same as in the second instar. The air channels in the posterior part of the lamellae have distinctive, vein‐like space‐holders. The similarity of the adult anterior lamellae with those in the second instar suggests retention of this part through the 4–5 molts to maturation, and/or cell processes like those in the embryo are repeated, but this needs to be examined in further studies of cell and cuticle changes before and during the molts. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Inhibition of ozone-induced SP-A oxidation by plant polyphenols

Surfactant protein-A (SP-A) is the best studied and most abundant of the protein components of lung surfactant and plays an important role in host defense of the lung. It has been shown that ozone-induced oxidation of SP-A protein changes its functional and biochemical properties. In the present study, eight plant polyphenols (three flavonoids, three hydroxycinnamic acids, and two hydroxybenzoic acids) known as strong antioxidants, were tested for their ability to inhibit ozone-induced SP-A oxidation as a mechanism for chemoprevention against lung damage. SP-A isolated from alveolar proteinosis patients was exposed to ozone (1 ppm) for 4 h. The flavonoids protected SP-A from oxidation in a dose dependent manner. ( - )-Epicatechin was the most potent flavonoid and exhibited inhibition of ozone-induced formation of carbonyls by 35% at a concentration as low as 5 μM. Hydroxybenzoic acids inhibited SP-A oxidation in a dose-dependent manner although they were less potent than flavonoids. On the other hand, hydroxycinnamic acids exhibited a different inhibitory pattern. Inhibition was observed only at medium concentrations. The results indicate that inhibition of SP-A oxidation by plant polyphenols may be a mechanism accounting for the protective activity of natural antioxidants against the effects of ozone exposure on lungs.     

Characters in the book lungs of Scorpiones (Chelicerata,Arachnida) revealed by scanning electron microscopy

The fine structure of the book lungs in 29 species representing ten monophyletic taxa of the Scorpiones (Arachnida) was investigated using scanning electron microscopy (SEM). Scorpion lungs are not homogeneous across the group. Here we describe and score three sets of phylogenetically informative characters: (1) the surface ornament of the lung lamellae, (2) the distal margins of the lamellae and (3) the fine structure of the spiracle margin. Provisional results suggest that reticulation on the surface of the lung lamellae is characteristic of the Buthidae. By contrast, non-buthid scorpions maintain the air space between adjacent lamellae using projecting trabeculae. Typically they are simple struts, but the trabeculae are distally branched in all investigated Scorpionidae, plus at least one species belonging to the Liochelidae. Simple thorns on the lamellar margins probably represent the plesiomorphic condition, while more complex, branched, arcuate morphologies appear to be homoplastic, occurring sporadically in numerous scorpion sub-groups. The tightly packed, hexagonal pillars around the posterior margin of the spiracle support a close relationship between Scorpionidae and Liochelidae, to the exclusion of the Urodacidae.     

Niacinamide mitigated the acute lung injury induced by phorbol myristate acetate in isolated rat's lungs

Background

Phorbol myristate acetate (PMA) is a strong neutrophil activator and has been used to induce acute lung injury (ALI). Niacinamide (NAC) is a compound of B complex. It exerts protective effects on the ALI caused by various challenges. The purpose was to evaluate the protective effects of niacinamide (NAC) on the PMA-induced ALI and associated changes.

Methods

The rat''s lungs were isolated in situ and perfused with constant flow. A total of 60 isolated lungs were randomized into 6 groups to received Vehicle (DMSO 100 μg/g), PMA 4 μg/g (lung weight), cotreated with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. We measured the lung weight and parameters related to ALI. The pulmonary arterial pressure and capillary filtration coefficient (K_fc) were determined in isolated lungs. ATP (adenotriphosphate) and PARP [poly(adenosine diphophate-ribose) polymerase] contents in lung tissues were detected. Real-time PCR was employed to display the expression of inducible and endothelial NO synthases (iNOS and eNOS). The neutrophil-derived mediators in lung perfusate were determined.

Results

PMA caused increases in lung weight parameters. This agent produced pulmonary hypertension and increased microvascular permeability. It resulted in decrease in ATP and increase in PARP. The expression of iNOS and eNOS was upregulated following PMA. PMA increased the neutrophil-derived mediators. Pathological examination revealed lung edema and hemorrhage with inflammatory cell infiltration. Immunohistochemical stain disclosed the presence of iNOS-positive cells in macrophages and endothelial cells. These pathophysiological and biochemical changes were diminished by NAC treatment. The NAC effects were dose-dependent.

Conclusions

Our results suggest that neutrophil activation and release of neutrophil-derived mediators by PMA cause ALI and associated changes. NO production through the iNOS-producing cells plays a detrimental role in the PMA-induced lung injury. ATP is beneficial, while PARP plays a deteriorative effect on the PMA-induced ALI. NAC exerts protective effects on the inflammatory cascade leading to pulmonary injury. This B complex compound may be applied for clinical usage and therapeutic regimen.     

Differential tissular distribution of Litomosoides sigmodontis microfilariae between microfilaremic and amicrofilaremic mice following experimental infection

Filariases are caused by onchocercid nematodes that are transmitted by arthropod vectors. More than 180 million people are infected worldwide. Mass drug administration has been set up in many endemic areas to control the parasite burden. Although very successful in limiting microfilarial load, transmission has not been completely interrupted in such areas. A proportion of infected patients with lymphatic filariasis or loiasis are known to be amicrofilaremic, as they do not present microfilariae in their bloodstream despite the presence of adult worms. A mirror status also exists in CBA/Ca mice infected with Litomosoides sigmodontis, the well-established model of filariasis. Using this model, the goal of this study was to determine if the kinetics of blood clearance of microfilariae differed between amicrofilaremic CBA/Ca mice and microfilaremic BALB/c mice. For this purpose, a qPCR approach was devised to detect microfilariae in different tissues, after a controlled inoculation of microfilariae. We showed that the rapid clearance of microfilariae from the pleural cavity or from the bloodstream of CBA/Ca mice was associated with a massive accumulation of first stage larvae in the lungs, liver and spleen.     

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of ∼7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 μg porcine aprotinin inhibited 6 μg trypsin whereas 1 μg commercial soybean inhibitor inhibited only 1 μg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.     

Mitigating the squash effect: sloths breathe easily upside down

Sloths are mammals renowned for spending a large proportion of time hanging inverted. In this position, the weight of the abdominal contents is expected to act on the lungs and increase the energetic costs of inspiration. Here, we show that three-fingered sloths Bradypus variegatus possess unique fibrinous adhesions that anchor the abdominal organs, particularly the liver and glandular stomach, to the lower ribs. The key locations of these adhesions, close to the diaphragm, prevent the weight of the abdominal contents from acting on the lungs when the sloth is inverted. Using ventilation rate and body orientation data collected from captive and wild sloths, we use an energetics-based model to estimate that these small adhesions could reduce the energy expenditure of a sloth at any time it is fully inverted by almost 13%. Given body angle preferences for individual sloths in our study over time, this equates to mean energy saving of 0.8–1.5% across individuals (with individual values ranging between 0.01 and 8.6%) per day. Given the sloth''s reduced metabolic rate compared with other mammals and extremely low energy diet, these seemingly innocuous adhesions are likely to be important in the animal''s energy budget and survival.     

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments^1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 µm in diameter³.We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 µm inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 µm which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 µm which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O₂ and the response fades after 30-40 min at hypoxia.