Characterization of the Pathogenesis of Lymphangioleiomyomatosis (LAM)

National Heart, Lung, And Blood Institute

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Abstract

A.Lymphangioleiomyomatosis (LAM) is characterized in the lung by proliferation of LAM cells, abnormal smooth muscle-like cells with dysfunctional tuberous sclerosis complex genes. This dysfunction results in activation of mechanistic target of rapamycin (mTOR), leading to LAM cell proliferation. Sirolimus (rapamycin) is the only FDA-approved treatment for pulmonary LAM, resulting in decreased LAM cell growth/size and stabilized lung function. LAM is characterized by lung destruction, with cysts lined by nodules containing LAM cells and immune cells. LAM patients may exhibit partially reversible airflow obstruction, and patients with more LAM nodules have higher frequencies of bronchodilator response (BDR), suggesting that airflow obstruction results from an increase in airways resistance due to the proliferation of LAM cells surrounding the airways. A greater frequency of BDR was a clinical marker of worse lung function and a greater rate of decline in FEV1 and DLCO in patients without sirolimus treatment. Airway hyperresponsiveness may stem from increased smooth muscle(SM)-like LAM cells. an increase in contractile mediators (i.e., mast cell products, and/or increased intracellular calcium that enhance SM contractility (i.e., ryanodine receptor activation) among other factors. Since sirolimus stabilizes LAM disease, we predicted that BDR frequency in patients receiving sirolimus would decrease as compared to BDR frequency of patients pretreatment, and thus be indicative of treatment success. To test this idea, we examined the BDR of 133 patients (105 with both before and during sirolimus treatment visits, 28 with visits only during treatment), with 1528 visits. Patients were diagnosed based on clinical, radiological, physiological, and pathological criteria, and examined under protocols 95-H-0186 and 96-H-0100. BDR was defined as an albuterol-induced increase of FEV1 of >12% over baseline. As 48.0% of visits on sirolimus had a baseline FEV1 of less than 1.5L, this definition of BDR using a percentage-based difference over baseline as opposed to a volume-based cut-off is warranted, to avoid missing BDR in patients with low baseline FEV1 who cannot produce large changes in volume. Unexpectedly, a BDR was seen at 34.9% (310/887) of visits during treatment as compared to 25% (160/641) of pretreatment visits (P<0.001). To determine if this result was due to the association of worse lung function and BDR, we defined severe pulmonary disease as having percent-predicted FEV1 or DLCO <40, with 14.4% of pretreatment and 36.4% of during treatment visits falling into this category. Regardless of sirolimus treatment, the frequency of BDR was increased in those with severe disease as compared to those with normal/mild/moderate disease (P<0.001). However, in those with normal/mild/moderate disease, there was an increase in BDR frequency during sirolimus treatment (27.9%) as compared to pretreatment (21.4%) (P=0.013) that was not seen in those with severe disease (46.7% during treatment versus 45.7% pretreatment). This increase in BDR frequency in patients with normal/mild/moderate disease during treatment is opposite to our prediction that sirolimus treatment would decrease BDR frequency. To control for the inherent variability in BDR, we examined BDR in patients over multiple visits, grouping them as never having a BDR, having a BDR <50% of the time, or having a BDR >50% of the time, followed for at least 5 visits. As determined previously, patients without sirolimus treatment were more likely to have BDR when they had worse lung function and also showed a trend to faster decline in pulmonary function as compared to those without BDR. For patients during sirolimus treatment, there was a trend toward worse pulmonary function values in those patients with BDR. Interestingly, patients receiving sirolimus who showed a BDR more than 50% of the time had significantly slower rates of decline of pulmonary function than those with BDR less than 50% of the time, suggesting that those with more stable disease show more frequent BDR during sirolimus treatment than those with declining disease. When we examine BDR over time, we find that pretreatment, the longer time since the first visit, the higher the probability of BDR (P=0.002), suggesting that BDR frequency increases as disease worsens, as expected. Surprisingly, during treatment, increasing the time on sirolimus decreases the probability of BDR (P<0.001), despite more severe disease. This may be due to the inability of the destroyed lung to support an increase in FEV1 of 12% upon beta-agonist stimulation. Examination of transplanted lungs by ex-vivo computed tomography indicated that LAM lungs have at least a three-fold reduction in airway number, with collapse of airways due to cysts and filling of airways with exudate. The increase in BDR frequency in patients with normal/mild/moderate disease during sirolimus treatment as compared to the BDR frequency in the same patients before treatment is unexpected as treatment would be expected to slow LAM disease, relieving airway obstruction. A possible explanation may be activation of the ryanodine receptor, a channel responsible for changes in calcium concentrations in the cell. It is stabilized in a closed position by FKBP12. Sirolimus binds to FKBP12 and removes it from the ryanodine receptor, resulting in its activation. Stimulation of the ryanodine receptor in normal cells by sirolimus may explain some side effects seen with sirolimus treatment, since the ryanodine receptor is linked to airway hyperreactivity, lymphedema, and hypertension, as airway smooth muscle, lymphatic, and endothelial cells express ryanodine receptors. We immunostained LAM lung tissue from three patients with polyclonal antibodies to FKBP12 and ryanodine receptor type 2 (RyR2) using an antigen retrieval procedure and peroxidase/DAB method. Proliferative LAM cells, epithelial cells from bronchioles, and alveoli were immunoreactive with anti-FKBP12 antibodies. RyR2 was present in alveoli, LAM lung nodules, bronchioles, and endothelial cells. Interestingly, RyR2 was detected in LAM cells, in addition to its expected presence in normal cells. In patients before treatment with sirolimus, mTOR would be active and the FKBP12/ryanodine receptor complex would be stabilized in a closed state, suggesting that airway hyperreactivity is due to LAM cell hypertrophy/hyperplasia or other factors, e.g., mast cell recruitment. When patients are treated with sirolimus, FKBP12/sirolimus/mTOR would be inactive and the ryanodine receptor would be in an open state, suggesting that airway hyperreactivity is due to increases in intracellular free Ca2+ in normal airway cells. Thus, in LAM, the clinical marker of a BDR during sirolimus treatment may not be indicative of worsening disease, but may be indicative of adverse events due to ryanodine receptor activation in non-targeted normal cells. Ryanodine receptor activation by sirolimus explains airway hyperreactivity seen with treatment via effects on normal cells, while also allowing for LAM disease stabilization by sirolimus. Patients with BDRs may be treated with steroids; in the case of those with sirolimus treatment, perhaps therapy targeting the ryanodine receptor may be a better choice. B. COVID 19-related studies: LAM and TSC patients may be treated with mTOR inhibitors, which are immunosuppressive drugs. We are comparing the vaccination responses of LAM patients treated with sirolimus compared to those not receiving the drug. Both groups of patients appeared to respond to the Pfizer and Moderna vaccines, with two exceptions. These data suggested that vaccination may generate serological responses in LAM patients receiving sirolimus.