Implication of in vivo circulating fibrocytes ablation in experimental pulmonary hypertension murine model.

BACKGROUND AND PURPOSE: Recruitment and involvement of bone-/blood-derived circulating fibrocytes (CF) in the promotion of fibrotic tissue remodelling processes have been shown. However, their direct contribution to pathological changes is not clear. The present study investigates the causal role of CF in the pathogenesis of pulmonary hypertension (PH). EXPERIMENTAL APPROACH: For selective ablation of CF, we applied the suicidal gene strategy with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir. The transgenic mice were generated, having HSV-TK-GFP transgene under the collagen 1 promoter. To selectively target CF, HSV-TK-GFP+ bone marrow transplanted into irradiated wild type mice. These chimera mice were subjected to hypoxia for PH induction and ganciclovir for CF ablation. KEY RESULTS: In vivo CF ablation reduced right ventricular hypertrophy and vascular remodelling with reduced total collagen content. We quantified the CF recruited in the perivascular area and arterial wall of small pulmonary arteries. There was significant recruitment of CF in the lung in response to hypoxia. The characterization of CF showed the expression of CD45 and collagen1 (GFP) along with α-smooth muscle actin (αSMA). CONCLUSION AND IMPLICATIONS: Our data demonstrated that CF ablation has a potential impact on right ventricular hypertrophy and vascular remodelling in the setting of experimental pulmonary hypertension induced by hypoxia. The beneficial effects may be related to the direct contribution of fibrocytes or its paracrine effect on other resident cell types. Thus, clinical manipulation of CF may represent a novel therapeutic approach to ameliorate the disease state in pulmonary hypertension.

The number of cardiac myocytes in the hypertrophic and hypotrophic left ventricle of the obese and calorie-restricted mouse heart.

Changes in body mass due to varying amounts of calorie intake occur frequently with obesity and anorexia/cachexia being at opposite sides of the scale. Here, we tested whether a high-fat diet or calorie restriction (CR) decreases the number of cardiac myocytes and affects their volume. Ten 6-8-week-old mice were randomly assigned to a normal (control group, n = 5) or high-fat diet (obesity group, n = 5) for 28 weeks. Ten 8-week-old mice were randomly assigned to a normal (control group, n = 5) or CR diet (CR group, n = 5) for 7 days. The left ventricles of the hearts were prepared for light and electron microscopy, and analysed by design-based stereology. In CR, neither the number of cardiac myocytes, the relationship between one- and multinucleate myocytes nor their mean volume were significantly different between the groups. In contrast, in the obese mice we observed a significant increase in cell size combined with a lower number of cardiomyocytes (P < 0.05 in the one-sided U-test) and an increase in the mean number of nuclei per myocyte. The mean volume of myofibrils and mitochondria per cardiac myocyte reflected the hypertrophic and hypotrophic remodelling in obesity and CR, respectively, but were only significant in the obese mice, indicating a more profound effect of the obesity protocol than in the CR experiments. Taken together, our data indicate that long-lasting obesity is associated with a loss of cardiomyocytes of the left ventricle, but that short-term CR does not alter the number of cardiomyocytes.

Myocardial remodelling in left ventricular atrophy induced by caloric restriction.

Changes in body weight due to changes in food intake are reflected by corresponding changes in the cardiac phenotype. Despite a growing body of literature on cardiac hypertrophy associated with obesity, little is known on the atrophic remodelling of the heart associated with calorie restriction. We hypothesized that, besides the cardiomyocyte compartment, capillaries and nerve fibres are involved in the atrophic process. C57Bl6 mice were kept on normal diet (control group) or at a calorie-restricted diet for 3 or 7 days (n = 5 each). At the end of the protocol, mice were killed and the hearts were processed for light and electron microscopic stereological analysis of cardiomyocytes, capillaries and nerve fibres. Body, heart and left ventricular weight were significantly reduced in the calorie-restricted animals at 7 days. Most morphological parameters were not significantly different at 3 days compared with the control group, but at 7 days most of them were significantly reduced. Specifically, the total length of capillaries, the volume of cardiomyocytes as well as their subcellular compartments and the interstitium were proportionally reduced during caloric restriction. No differences were observed in the total length or the mean diameter of axons between the cardiomyocytes. Our data indicate that diet-induced left ventricular atrophy leads to a proportional atrophic process of cardiomyocytes and capillaries. The innervation is not involved in the atrophic process.

Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice.

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.

Lung endothelial monocyte-activating protein 2 is a mediator of cigarette smoke-induced emphysema in mice.

Pulmonary emphysema is a disease characterized by alveolar cellular loss and inflammation. Recently, excessive apoptosis of structural alveolar cells has emerged as a major mechanism in the development of emphysema. Here, we investigated the proapoptotic and monocyte chemoattractant cytokine endothelial monocyte-activating protein 2 (EMAPII). Lung-specific overexpression of EMAPII in mice caused simplification of alveolar structures, apoptosis, and macrophage accumulation, compared with that in control transgenic mice. Additionally, in a mouse model of cigarette smoke-induced (CS-induced) emphysema, EMAPII levels were significantly increased in murine lungs. This upregulation was necessary for emphysema development, as neutralizing antibodies to EMAPII resulted in reduced alveolar cell apoptosis, inflammation, and emphysema-associated structural changes in alveoli and small airways and improved lung function. The mechanism of EMAPII upregulation involved an apoptosis-dependent feed-forward loop, since caspase-3 instillation in the lung markedly increased EMAPII expression, while caspase inhibition decreased its production, even in transgenic EMAPII mice. These findings may have clinical significance, as both current smokers and ex-smoker chronic obstructive pulmonary disease (COPD) patients had increased levels of secreted EMAPII in the bronchoalveolar lavage fluid compared with that of nonsmokers. In conclusion, we suggest that EMAPII perpetuates the mechanism of CS-induced lung emphysema in mice and, given its secretory nature, is a suitable target for neutralization antibody therapy.