Background: Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin expression and leads to severe ambulatory and cardiac function decline. However, the dystrophin-deficient mdx murine model of DMD only develops a very mild form of the disease. Our group and others have shown vascular abnormalities in animal models of MD, a likely consequence of the fact that blood vessels express the same dystrophin-associated glycoprotein complex (DGC) proteins as skeletal muscles. Methods: To test the blood vessel contribution to muscle damage in DMD, mdx 4cv mice were given elevated lipid levels via apolipoprotein E (ApoE) gene knockout combined with normal chow or lipid-rich Western diets. Ambulatory function and heart function (via echocardiogram) were assessed at 4 and 7 months of age. After sacrifice, muscle histology and aortic staining were used to assess muscle pathology and atherosclerosis development, respectively. Plasma levels of total cholesterol, high-density lipoprotein (HDL), triglycerides, and creatine kinase (CK) were also measured. Results: Although there was an increase in left ventricular heart volume in mdx-ApoE mice compared to that in mdx mice, parameters of heart function were not affected. Compared with wild-type and ApoE-null, only mdx-ApoE KO mice showed significant ambulatory dysfunction. Despite no significant difference in plasma CK, histological analyses revealed that elevated plasma lipids in chow-and Western diet-fed mdx-ApoE mice was associated with severe exacerbation of muscle pathology compared to mdx mice: significant increase in myofiber damage and fibrofatty replacement in the gastrocnemius and triceps brachii muscles, more reminiscent of human DMD pathology. Finally, although both ApoE and mdx-ApoE groups displayed increased plasma lipids, mdx-ApoE exhibited atherosclerotic plaque deposition equal to or less than that of ApoE mice. Conclusions: Since others have shown that lipid abnormalities correlate with DMD severity, our data suggest that plasma lipids could be primary contributors to human DMD severity and that the notoriously mild phenotype of mdx mice might be attributable in part to their endogenously low plasma lipid profiles. Hence, DMD patients may benefit from lipid-lowering and vascular-targeted therapies.

Progressive limb and girdle muscle atrophy leading to loss of ambulation is a hallmark of dysferlinopathies, which include limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. However, animal models fail to fully reproduce the disease severity observed in humans, with dysferlin-null (Dysf) mice exhibiting minor muscle damage and weakness without dramatic ambulatory dysfunction. As we have previously reported significant Dysf expression in blood vessels, we investigated the role of vascular function in development of muscle pathology by generating a Dysf-deficient mouse model with vascular disease. This was achieved by crossing Dysf mice with ApoE mice, which have high levels of nonHDL-associated cholesterol. Double-knockout DysfApoE mice exhibited severe ambulatory dysfunction by 11 months of age. In limb-girdle muscles, histology confirmed dramatic muscle wasting, fibrofatty replacement, and myofiber damage in DysfApoE mice without affecting the ratio of centrally nucleated myofibers. Although there were no major changes in ex vivo diaphragm and soleus muscle function, histological analyses revealed these muscles to be untouched by damage and remodelling. In all, these data suggest that cholesterol may be deleterious to dysferlinopathic muscle and lead to ambulatory dysfunction. Moreover, differences in plasma lipid handling between mice and humans could be a key factor affecting dysferlinopathy severity.

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Pulmonary surfactant is a crucial and dynamic lung structure whose primary functions are to reduce alveolar surface tension and facilitate breathing. Though disruptions in surfactant homeostasis are typically thought of in the context of respiratory distress and premature infants, many lung diseases have been noted to have significant surfactant abnormalities. Nevertheless, preclinical and clinical studies of pulmonary disease too often overlook the potential contribution of surfactant alterations – whether in quantity, quality or composition – to disease pathogenesis and symptoms. In inflammatory lung diseases, whether these changes are cause or consequence remains a subject of debate. This review will outline 1) the importance of pulmonary surfactant in the maintenance of respiratory health, 2) the diseases associated with primary surfactant dysregulation, 3) the surfactant abnormalities observed in inflammatory pulmonary diseases and, finally, 4) the available research on the interplay between surfactant homeostasis and smoking-associated lung disease. From these published studies, we posit that changes in surfactant integrity and composition contribute more considerably to chronic inflammatory pulmonary diseases and that more work is required to determine the mechanisms underlying these alterations and their potential treatability.

Marfan syndrome (MFS) is a genetic disorder that frequently leads to aortic root dissection and aneurysm. Despite promising preclinical and pilot clinical data, a recent large-scale study using antihypertensive angiotensin II (AngII) receptor type 1 (ATR1) blocker losartan has failed to meet expectations at preventing MFS-associated aortic root dilation, casting doubts about optimal therapy. To study the deleterious role of normal ATR1 signaling in aortic root widening, we generated MFS mice lacking ATR1a expression in an attempt to preserve protective ATR2 signaling. Despite being hypotensive and resistant to AngII vasopressor effects, MFS/ATR1a-null mice showed unabated aortic root enlargement and remained fully responsive to losartan, confirming that blood pressure lowering is of minor therapeutic value in MFS and that losartan's antiremodeling properties may be ATR1 independent. Having shown that MFS causes endothelial dysfunction and that losartan can activate endothelial function in mice and patients, we found that nitric oxide synthase (NOS) inhibition renders losartan therapeutically inactive, whereas multiple transgenic and pharmacologic models of endothelial NOS activation block aortic root dilation by correcting extracellular signal-regulated kinase signaling. In vitro, losartan can increase endothelial NO release in the absence of AngII and correct MFS NO levels in vivo. Our data suggest that increased protective endothelial function, rather than ATR1 inhibition or blood pressure lowering, might be of therapeutic significance in preventing aortic root disease in MFS.

Smoking alters pulmonary reverse lipid transport and leads to intracellular lipid accumulation in alveolar macrophages. We investigated whether stimulating reverse lipid transport with an agonist of the liver X receptor (LXR) would help alveolar macrophages limit lipid accumulation and dampen lung inflammation in response to cigarette smoke. Mice were exposed to cigarette smoke and treated intraperitoneally with the LXR agonist T0901317. Expression of lipid capture and lipid export genes was assessed in lung tissue and alveolar macrophages. Pulmonary inflammation was assessed in the bronchoalveolar lavage (BAL). Finally, cholesterol efflux capacity and pulmonary surfactant levels were determined. In room air-exposed mice, T0901317 increased the expression of lipid export genes in macrophages and the whole lung and increased cholesterol efflux capacity without inducing inflammation or affecting the pulmonary surfactant. However, cigarette smoke-exposed mice treated with T0901317 showed a marked increase in BAL neutrophils, IL-1α, C-C motif chemokine ligand 2, and granulocyte-colony-stimulating factor levels. T0901317 treatment in cigarette smoke-exposed mice failed to increase the ability of alveolar macrophages to export cholesterol and markedly exacerbated IL-1α release. Finally, T0901317 led to pulmonary surfactant depletion only in cigarette smoke-exposed mice. This study shows that hyperactivation of LXR and the associated lipid capture/export mechanisms only have minor pulmonary effects on the normal lung. However, in the context of cigarette smoke exposure, where the pulmonary surfactant is constantly oxidized, hyperactivation of LXR has dramatic adverse effects, once again showing the central role of lipid homeostasis in the pulmonary response to cigarette smoke exposure.

Cigarette smoke exposure induces inflammation marked by rapid and sustained neutrophil infiltration, IL-1α, release and altered surfactant homeostasis. However, the extent to which neutrophils and IL-1α contribute to the maintenance of pulmonary surfactant homeostasis is not well understood. We sought to investigate whether neutrophils play a role in surfactant clearance as well as the effect of neutrophil depletion and IL-1α blockade on the response to cigarette smoke exposure. In vitro and in vivo administration of fluorescently labeled surfactant phosphatidylcholine was used to assess internalization of surfactant by lung neutrophils and macrophages during or following cigarette smoke exposure in mice. We also depleted neutrophils using anti–Ly-6G or anti–Gr-1 Abs, or we neutralized IL-1α using a blocking Ab to determine their respective roles in regulating surfactant homeostasis during cigarette smoke exposure. We observed that neutrophils actively internalize labeled surfactant both in vitro and in vivo and that IL-1α is required for smoke-induced elevation of surfactant protein (SP)–A and SP-D levels. Neutrophil depletion during cigarette smoke exposure led to a further increase in SP-A levels in the bronchoalveolar lavage and increased IL-1α, CCL2, GM-CSF, and G-CSF release. Finally, macrophage expression of Mmp12, a protease linked to emphysema, was increased in neutrophil-depleted groups and decreased following IL-1α blockade. Taken together, our results indicate that neutrophils and IL-1α signaling are actively involved in surfactant homeostasis and that the absence of neutrophils in the lungs during cigarette smoke exposure leads to an IL-1α–dependent exacerbation of the inflammatory response.

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