Simple model of pulmonary hypertension secondary to left heart pressure overload induced by partial intravascular occlusion of the ascending aorta.

Pulmonary hypertension is a group of diseases characterized by elevated pulmonary artery pressure and pulmonary vascular resistance with significant morbidity and mortality. The most prevalent type is pulmonary hypertension secondary to left heart disease (PH-LHD). The available experimental models of PH-LHD use partial pulmonary clamping by technically nontrivial open chest surgery with lengthy recovery. We present a simple model in which reduction of the cross-sectional area of the ascending aorta is achieved not by external clamping, but by partial intravascular obstruction without opening the chest. In anesthetized rats, a blind polyethylene tubing was advanced from the right carotid artery to just above the aortic valve. The procedure is quick and easy to learn. Three weeks after the procedure, left heart pressure overload was confirmed by measuring left ventricular end diastolic pressure by puncture (1.3±0.2 vs. 0.4±0.3 mmHg in controls, mean±sd, P<0.0001). The presence of pulmonary hypertension was documented by measuring pulmonary artery pressure by catheterization (22.3±2.3 vs. 16.9±2.7 mmHg, P=0.0282) and by detecting right ventricular hypertrophy and increased muscularization of peripheral pulmonary vessels. Contributions of precapillary vascular segment and of vasoconstriction to the increased pulmonary vascular resistance were demonstrated, respectively, by arterial occlusion technique and by normalization of resistance by a vasodilator, sodium nitroprusside, in isolated lungs. These changes were comparable, but not additive, to those induced by an established pulmonary hypertension model, chronic hypoxic exposure. Intravascular partial aortic obstruction offers an easy model of pulmonary hypertension induced by left heart disease that has a vasoconstrictor and precapillary component.

The effect of exposure to hypoxia on superoxide formation by alveolar macrophages is indirect.

AIMS: To elucidate the role of alveolar macrophages (AM) in the pathogenesis of hypoxic pulmonary hypertension (HPH), we tested the effects of sustained hypoxia on AM polarization and on the formation of superoxide by AM in vivo and in vitro. MAIN METHODS: Rat AM were obtained by bronchoalveolar lavage. 4-day exposure to hypoxia (10% O2) was carried out in vivo (rats in isobaric hypoxic chamber, controls kept in air) or in vitro (control AM in 21% O2 and 5% CO2). Superoxide production was measured by luminol-orthovanadate chemiluminescence, AM polarization was detected immunocytochemically. To ascertain the effect of substances contained in the alveolar environment, we cultivated cells also in the presence of non-cellular components of the bronchoalveolar lavage fluid (BALF) either from controls or from rats exposed to 4 days of hypoxia. KEY FINDINGS: In vivo, but not in vitro, hypoxia increased AM superoxide production. Both types of hypoxia polarized AM into M2 (pro-proliferative) type. While the presence of control BALF attenuated superoxide production in AM cultivated in normoxia, BALF from the hypoxia-exposed rats had no effect. In AM cultivated in hypoxia, superoxide production was not altered by control BALF and elevated by BALF obtained from hypoxic rats. SIGNIFICANCE: Hypoxia does not influence superoxide production by AM directly but rather by modulating their milieu and their sensitivity to external influences.

Decreased collagen VI in the tunica media of pulmonary vessels during exposure to hypoxia: a novel step in pulmonary arterial remodeling.

The development of hypoxic pulmonary hypertension is characterized by the structural remodeling of pulmonary arteries. However, the relationship between changes of arterial cells and the extracellular matrix remains unclear. We focused on the evaluation of the non-fibrillar collagen changes in tunica media induced by a four-day exposure to hypoxia and the correlation of these changes with the pulmonary arterial wall structure modifications. We used 20 adult male Wistar rats. The amount and localization of collagen VI, collagen IV, matrix metalloproteinase (MMP) 2, and MMP9 were tested in pulmonary arteries immunohistochemically. Two-dimensional electrophoresis and messenger RNA (mRNA) expression were used for the subsequent comparison of protein changes in arterial tunica media cells (normoxia/hypoxia). Collagen VI was significantly reduced strictly in the tunica media of conduit arteries of hypoxia-exposed rats; however, its mRNA increased. The amount of collagen IV and its mRNA were not altered. We detected a significant increase of MMP9 strictly in the tunica media. In addition, a significantly increased number of MMP9-positive cells surrounded the arteries. MMP2 and the expression of its mRNA were decreased in tunica media. We conclude that the loss of collagen VI is an important step characterizing the remodeling of pulmonary arteries. It could influence the phenotypic status and behavior of smooth muscle cells and modify their proliferation and migration.

Novel contribution to clubfoot pathogenesis: The possible role of extracellular matrix proteins.

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Role of Kv7 channels in responses of the pulmonary circulation to hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K(+) channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3-5 days in an isobaric hypoxic chamber (FiO2 = 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease.

Airway wall remodeling in young and adult rats with experimentally provoked bronchial asthma.

BACKGROUND: Airway wall remodeling is a typical finding in patients suffering from bronchial asthma. While morphological changes have been thoroughly described in adults, less is known about such changes in children because of the limited accessibility of relevant material. To overcome this constraint, animal asthma models may be used instead of human specimens. This study examined rats with artificially stimulated chronic asthma-like symptoms. METHODS: Brown Norway rats of two age categories (young and adult) were sensitized by ovalbumin (OA), and their intrapulmonary airways (IA) were studied using morphometric and histochemical methods. RESULTS: OA administration induced a significant increase in lung resistance in young animals but not in adults. The total IA wall area was significantly increased in both young and adult OA rats. In young animals, thickening of the adventitia played a more crucial role in this increase than it did in adults, in which the mucosa and the submucosa participated to a higher degree. The IA walls of young OA rats had significantly higher levels of infiltrating eosinophils than those of adult OA animals. The multiplication of goblet cells was more pronounced in adult rats, which was associated with a tendency to produce a higher proportion of acidic glycoconjugates. CONCLUSIONS: OA stimulation affected the IA of young rats differently than those of adult animals. Changes in the outer IA layer of young rats can be triggered by activated eosinophils; however, stimulated airway epithelium can be a source of factors that influence the inner IA layers in adult rats.