Characterization of the Pathogenesis of Lymphangioleiomyomatosis (LAM)

Heart, Lung, And Blood Institute

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Abstract

LAM cells appear to be capable of metastatic spread, in which LAM cells may travel to the lungs from elsewhere in the body (perhaps from angiomyolipomas or the lymphatic system). To metastasize in the lung, the LAM cell must be able to travel via the blood or lymphatic vessels, traverse the vessel wall, and invade the pulmonary interstitum. In the lungs, LAM cells are thought to be responsible for destruction of the parenchyma, leading to cyst formation. Both processes involve degradation of the extracellular matrix. By immunohistochemistry, LAM lung nodules contain matrix metalloproteinases (MMPs)-1, -2, -9, and -14, which degrade collagen, as well as areas of disrupted/degraded collagen and elastin. MMP-1 degrades fibrillar collagen, while MMPs-2 and -9 degrade denatured collagen or gelatin. Collagens I and III are major interstitial components of the lung and provide architectural framework for the alveolar wall. Type I collagen is a triple-helix comprising two alpha 1 chains, encoded by the COL1A1 gene, and one alpha 2 chain, encoded by the COL1A2 gene. Type III collagen is a homotrimer of polypeptides encoded by the COL3A1 gene. Mutations in genes encoding extracellular matrix (ECM) proteins are associated with diseases characterized by weakness of the ECM (e.g., Ehlers-Danlos, osteogenesis imperfecta), and polymorphisms in these genes have been associated with disease. Pneumothorax is found in patients with Ehlers-Danlos type IV and is correlated with a reduced amount of type III collagen in the lungs, while spontaneous pneumothorax also occurs in Marfan patients, possibly due to irregular elastin fibers that have been deposited on microfibrils containing mutated fibrillin-1. Thus, genetic differences in the ECM may be critical to the development of pneumothoraces. [unreadable] [unreadable] To identify factors involved in the susceptibility to and predictive of pneumothorax, we initially compared clinical data on patients with a history of pneumothorax to those who had never had a pneumothorax, focusing on a possible relationship of pneumothoraces to disease progression and severity. Based on the hypothesis that the occurrence of pneumothoraces might be related to differences in genes involved in ECM formation, collagen, elastin, and MMP-1 polymorphisms were compared in LAM patients and matched healthy volunteers. Patients were genotyped for polymorphisms in genes of extracellular matrix proteins collagen, elastin, and matrix metalloproteinase-1 to assess their association with pneumothoraces. Clinical data and polymorphisms in the genes for Types I and III collagen, elastin, and matrix metalloproteinase-1 were compared with the prevalence of pneumothorax. Of 227 patients, 57% reported having had at least one pneumothorax. Cyst size on high resolution computed tomography scans was associated with pneumothorax; patients with a history of pneumothorax were more likely to have larger cysts than patients who had no pneumothoraces. In patients with mild disease, those with a history of pneumothorax had a faster rate of decline in FEV1 (P=0.001, adjusted for age) than those without. Genotype frequencies differed between patients with and without pneumothorax for polymorphisms in the Types I and III collagen and matrix metalloproteinase-1 genes. Thus, larger cysts may predispose lymphangioleiomyomatosis patients to pneumothorax, which, in early stages of disease, correlates with a more rapid rate of decline in FEV1. Polymorphisms in Types I and III collagen and matrix metalloproteinase-1 genes may cause differences in lung extracellular matrix that result in greater susceptibility to pneumothorax. These studies have identified both predictive clinical features associated with the risk of pneumothorax as well as genetic factors that may predispose LAM patients to pneumothorax. [unreadable] [unreadable] Identification of LAM cells in donor lungs, their isolation from blood, and presence in urine, chylous ascites and pleural effusions is consistent with their ability to metastasize. We investigated the presence on LAM cells of the hyaluronic acid receptor CD44 and its splice variants associated with metastasis. The heterogeneous populations of cells grown from lungs of twelve LAM patients contain cells expressing mRNA for the variant CD44v6. Histologically, CD44v6 was present in LAM lung nodules, but not in normal vascular smooth muscle cells. CD44v6-positive sorted cells showed loss of heterozygosity at the TSC2 locus; binding of CD44v6 antibody resulted in loss of cell viability. Levels of CD44 were higher in cultured Eker rat (Tsc2 -/-) cells than in Tsc2 (+/+) cells, but unlike human LAM cells, the TSC2 (-/-) Eker rat cells did not contain CD44v6 splice variant mRNA. CD44 splicing and signalling is regulated by osteopontin. Plasma from LAM patients contained higher concentrations of osteopontin than plasma of healthy, age- and sex-matched volunteers (p=0.00003). The cell surface receptor CD44 and its splice variant CD44v6 may contribute to the metastatic potential of LAM cells. [unreadable] [unreadable] These studies have enabled us to define better predictors of disease severity and biomarkers of disease for diagnostic and prognostic applications. The size of the cysts and occurrence of pneumothoraces appear to be predictors of future morbidity. Similarly, a marker of metastatic disease, osteopontin, may serve as a biomarker of disease. Biomarker discovery has led us to the identification of LAM-associated proteins as well as LAM cells in blood, urine, and chyle, which may be useful for their predictive and diagnostic value.