Characterization of the Pathogenesis of Lymphangioleiomyomatosis (LAM)

National Heart, Lung, And Blood Institute

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Abstract

A. A mixed blood-lymphatic endothelial cell phenotype is Lymphangioleiomyomatosis and Idiopathic Pulmonary Fibrosis but not in Kaposi's Sarcoma and Tuberous Sclerosis Complex:In LAM lungs, the walls of the cystic lesions contain LAM lung nodules, which are composed of neoplastic smooth muscle-like LAM cells, having mutant or absent TSC genes,TSC1, or TSC2, leading to activation of the mechanistic Target of Rapamycin (mTOR). LAM lung nodules contain multiple cell types, including mast cells, and are infiltrated by lymphatic capillaries lined by lymphatic endothelial cells (LECs). Chylous effusions isolated from patients with LAM contain LAM cell clusters (LCCs), which are aggregates of monoclonal antibody HMB-45-positive LAM cells, encircled by LECs. Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease characterized by the formation of fibrotic tissue resulting from injury, inflammation, and scarring. IPF fibroblastic areas are infiltrated with lymphatic vessels. Kaposis sarcoma is a vascular tumor, containing both blood vascular endothelial cells (BVECs) and LECs. In Kaposis sarcoma, infection with Kaposi sarcoma-associated herpesvirus causes a reprograming of endothelial cells. LECs are phenotypically different from BVECs, which are characterized by the presence of CD34 and von Willebrand growth factor. Mature LECs contain podoplanin, which is recognized by the monoclonal antibody D240. Because LECs are found in several lung diseases, we hypothesized that there may be subsets of lymphatic endothelial cells involved in pathogenesis. In LAM, lymphatic vessels (D240) were diffusely distributed within LAM nodules. In IPF, lymphatic structures were observed within fibrotic areas. In normal lung tissue, the majority of lymphatic vessels were located in proximity to bronchioles. Lung tissue sections from patients with LAM were coimmunostained with anti-podoplanin and anti-CD34 antibodies. Interestingly, four cases from LAM lung contained shunts, connecting blood and lymphatic vessels, and erythrocytes were observed within lymphatic vessels in LAM lung nodules. To define a lymphatic structure, we examined the capillary structure and histopathology as well as immunoreactivity with specific antibodies (e.g., podoplanin, CD34, PROX1). PROX1 is a critical transcription factor that participates in the terminal differentiation of lymphatics/lymphatic determination. Approximately 90% of endothelial cells lining vessels in the LAM lung nodules reacted with both D240 and anti-CD34 antibodies. In addition, endothelial cells in three lymphangioleiomyoma specimens from patients with LAM showed immunoreactivity with both anti-CD34 and D240 antibodies. To determine the colocalization of BVEC and LEC markers in LAM vessels, we reacted lung tissue from patients with LAM and IPF and healthy volunteers with anti-podoplanin, anti-CD31, and anti-CD34 antibodies and visualized the specimens by laser-scanning confocal microscopy. The frequency (97%) and intensity (threefold higher in LAM) of CD34 was greater in LAM tissue sections than in normal and IPF tissue sections. Microscopic fields of lung sections from LAM (97%), IPF (84%), and healthy volunteer (48%) were reactive to anti-podoplanin and anti-CD34 antibodies. However, D240 and anti-CD31 reactive cells in lymphatic structures of normal lung tissue were seen in 4% of normal tissue sections compared with 46% in IPF and 62% in LAM lung tissue sections. Tissue sections of TSC skin tumors and nodular Kaposis sarcoma were not co-reactive with D240 antibody and anti-CD34. Data did not support a viral etiology of LAM as is seen in Kaposis tumors. We investigated the presence of eight known herpesviruses in 10 explanted LAM lungs, and the results were negative. Thus, the mixed-endothelial phenotype in LAM does not appear to be a consequence of KSHV infection. Thus, not all LEC-containing vessels possessed a mixed endothelial cell phenotype, and this phenotype appeared to be more common in LAM. D240 positive cells lining LAM lymphatic vessels reacted with anti-PROX1 antibodies, a transcription factor involved in lymphatic determination, and anti-pS6 (235/236) antibodies. These data are consistent with hyperactivation of the mTOR pathway in LECs of LAM lung nodules as well as the smooth muscle-like cells of the lung nodules. LAM cell clusters may function as a vehicle for dissemination and metastasis of LAM cells. They were observed in lymphatic vessels of the lung and in 16% of chylous effusions from patients with LAM. LCCs from chylous effusions contained internal LAM cells expressing Pmel17, a melanosomal protein. LECs surrounding the LCC and some single cells contained podoplanin and CD34, supporting a mixed endothelial phenotype, although some encapsulated LECs of LCC did not have a mixed endothelial phenotype. Endothelial cells seen within the LCC were not reactive to anti-CD31 antibodies, consistent with different phenotypes of LECs within the same disease. LCCs were seen in dilated lymphatic channels. HMB-45positive LAM cells coated by LECs or fragments of nodules were identified in the marginal sinus, consistent with nodal metastasis in LAM. We identified endothelial cells with a mixed blood-lymphatic phenotype in vessels within LAM lung nodules, lymph nodes, LCCs, healthy lungs, and IPF lungs. The mixed endothelial phenotype was not observed in TSC skin lesions and nodal Kaposis sarcoma. It has been postulated that TSC2-null cells in LAM are derived from LECs. However, we did not observe cellular colocalization of podoplanin and the LAM cell biomarker HMB-45 in LCCs. Our results support hematogenous and lymphatic metastasis in LAM with histopathological evidence of nodal metastasis of LAM cells via circulating LCCs. We show a possible lymphatic/blood vascular structure that provides LAM cells with a conduit from blood to lymphatic vessels and eventually lymph nodes. Our data also support the existence of LEC/BVEC reprograming in LAM, consistent with the identification of a population of endothelial cells expressing both blood and LEC markers in LAM lesions, independent of herpes viral infection and perhaps independent of TSC2 loss, as supported by the absence of a mixed endothelial phenotype in TSC skin lesions. B. COVID 19-related studies: LAM and TSC patients may be treated with mTOR inhibitors, which are immunosuppressive drugs. We are currently comparing the vaccination responses of patients being treated or not being treated with these drugs by comparing the levels of anti-Spike protein antibodies.