ALTERAZIONI ISTOPATOLOGICHE INDOTTE DA CELLULE UMANE LAM/TSC IN TOPI NUDI: UN MODELLO DI LINFANGIOLEIOMIOMATOSI. REVERSIONE DEL DANNO CON ANTICORPO ANTI-EGFR

LAM is a rare and progressive disease characterized by widespread proliferation of abnormal smooth muscle-like cells (LAM cells). LAM cells cause cystic destruction of lung parenchyma, abdominal tumours (angiomyolipoma, AML) and infiltration of axial lymphatics in torax and abdomen (adenopathy and lymphangioleiomyoma). LAM occurs sporadically or in association with tuberous sclerosis complex (TSC), an inherited disorder with variable penetrance, which results from mutations in TSC1 or TSC2 genes. TSC1 or TSC2 genes, encoding hamartin and tuberin respectively, regulate mammalian target of rapamycin (mTOR). LAM affects primarly women of child-bearing age and the mechanisms causing the disease are not yet clarified. To explain the multisystemic clinical manifestations of LAM an experimental model is needed to study the pathological mechanism causing LAM. It may help to explain how LAM cells migrate from tissue to tissue and to develop a pharmacological approach. We recently isolated and characterized α-actin positive smooth muscle cells from chylous of a patient affected by LAM/TSC (LAM/TSC cells). These circulating cells showed reactivity to HMB45 and CD44v6 antibodies, markers of TSC and LAM, and bear a germline TSC2 mutation in exon 21. Like TSC2 smooth muscle cells previously isolated (TSC2-/- and TSC2-/meth ASM cells), LAM/TSC cells from chylous required epidermal growth factor (EGF) to proliferate and the blockade of EGF receptor (EGFR) caused progressive cell death. To better study LAM pathogenesis we developed a procedure for a quick invasion of the respiratory system by endonasally administrating LAM/TSC cells. LAM/TSC cells were administrated in immunodeficient female nude mice (nu/nu Hsd: athymic nude mice, 3 weeks old) and after 26 weeks anti-EGFR antibody and rapamycin were intraperitoneally injected 2 times a week for 4 weeks. 30 weeks after endonasal administration LAM/TSC cells were detected in lungs, lymph nodes and uterus. In lung parenchyma, LAM/TSC cells proliferated and caused cystic destruction with emphysematous-like picture such as in LAM patient lungs. This lesion and the proliferating rate were reverted by anti-EGFR antibody, while rapamycin was less effective and caused hemoptysis. In lungs blood vessel number was increased and, using LYVE-1 antibody, a significant increase of lymphatic vessel density (LVD) was observed in animals that received LAM/TSC cells suggesting a possible correlation between LAM/TSC cells and lymphangiogenesis. LVD decreased following anti-EGFR antibody and rapamycin treatments. When treatments were stopped, hemoptysis caused by rapamycin was reverted. Anti-EGFR antibody was more effective than rapamycin in reducing lung injury caused by LAM/TSC cells administration and in decreasing lymphangiogenesis. In some lung parenchyma noduli were detected. Such in lungs of LAM patients they were positive to human COX IV, ER, PR and phosphoS6. In lymph nodes, LAM/TSC cells promoted a lymphatic vessel invasion, as showed by PROX-1-reactivity. Anti-EGFR antibody and rapamycin treatments decreased lymphatic vessels. Differently from lungs, blood vessels in lymph nodes were not altered after LAM/TSC cells administration, as shown by CD31 immunoreactivity. LAM/TSC cells were also detected in uteri where they promoted a significant increase of cells with high levels of estrogen (ER) and progesterone receptors (PR). In uteri afterLAM/TSC cells administration the morphological structure was unchanged. Pharmacological treatments decreased ER and PR expressions. Our data show that endonasal administration of cells isolated from chylous of LAM/TSC patient developed a mouse LAM model. LAM/TSC cells invaded lungs, lymph nodes and uteri causing LAM-like lesions. Anti-EGFR antibody is more effective than rapamycin in promoting lung restauration and reducing lymphangiogenesis; its efficacy persists also when treatment is stopped. These data suggest that anti-EGFR antibody treatment may represent a useful pharmacological approach for LAM therapy.