Lymphangioleiomyomatosis: circulating levels of FGF23 and pulmonary diffusion.

OBJECTIVE: Lymphangioleiomyomatosis (LAM) is a rare, destructive disease of the lungs with a limited number of determinants of disease activity, which are a critical need for clinical trials. FGF23 has been implicated in several chronic pulmonary diseases. We aimed to determine the association between serum FGF23 levels and pulmonary function in a cohort of patients with LAM. METHODS: This was a descriptive single-center study in which subjects with LAM and controls with unreported lung disease were recruited. Serum FGF23 levels were measured in all subjects. Clinical data, including pulmonary function testing, were retrospectively obtained from electronic medical records of LAM subjects. Associations between FGF23 levels and clinical features of LAM were explored via nonparametric hypothesis testing. RESULTS: The sample comprised 37 subjects with LAM and 16 controls. FGF23 levels were higher in the LAM group than in the control group. In the LAM group, FGF23 levels above the optimal cutoff point distinguished 33% of the subjects who had nondiagnostic VEGF-D levels. Lower FGF23 levels were associated with impaired DLCO (p = 0.04), particularly for those with isolated diffusion impairment with no other spirometric abnormalities (p = 0.04). CONCLUSIONS: Our results suggest that FGF23 is associated with pulmonary diffusion abnormalities in LAM patients and elicit novel mechanisms of LAM pathogenesis. FGF23 alone or in combination with other molecules needs to be validated as a biomarker of LAM activity in future clinical research.

Lymphangioleiomyomatosis: circulating levels of FGF23 and pulmonary diffusion / Linfangioleiomiomatose: níveis circulantes de FGF23 e difusão pulmonar

ABSTRACT Objective: Lymphangioleiomyomatosis (LAM) is a rare, destructive disease of the lungs with a limited number of determinants of disease activity, which are a critical need for clinical trials. FGF23 has been implicated in several chronic pulmonary diseases. We aimed to determine the association between serum FGF23 levels and pulmonary function in a cohort of patients with LAM. Methods: This was a descriptive single-center study in which subjects with LAM and controls with unreported lung disease were recruited. Serum FGF23 levels were measured in all subjects. Clinical data, including pulmonary function testing, were retrospectively obtained from electronic medical records of LAM subjects. Associations between FGF23 levels and clinical features of LAM were explored via nonparametric hypothesis testing. Results: The sample comprised 37 subjects with LAM and 16 controls. FGF23 levels were higher in the LAM group than in the control group. In the LAM group, FGF23 levels above the optimal cutoff point distinguished 33% of the subjects who had nondiagnostic VEGF-D levels. Lower FGF23 levels were associated with impaired DLCO (p = 0.04), particularly for those with isolated diffusion impairment with no other spirometric abnormalities (p = 0.04). Conclusions: Our results suggest that FGF23 is associated with pulmonary diffusion abnormalities in LAM patients and elicit novel mechanisms of LAM pathogenesis. FGF23 alone or in combination with other molecules needs to be validated as a biomarker of LAM activity in future clinical research.

RESUMO Objetivo: A linfangioleiomiomatose (LAM) é uma doença rara e destrutiva dos pulmões com um número limitado de determinantes da atividade da doença, que são uma necessidade crítica para ensaios clínicos. O FGF23 já foi implicado em várias doenças pulmonares crônicas. O nosso objetivo foi determinar a associação entre os níveis séricos de FGF23 e a função pulmonar em uma coorte de pacientes com LAM. Métodos: Estudo descritivo unicêntrico no qual foram recrutados indivíduos com LAM e controles com doenças pulmonares não declaradas. Os níveis séricos de FGF23 foram medidos em todos os indivíduos. Os dados clínicos, incluindo testes de função pulmonar, foram obtidos retrospectivamente a partir dos prontuários eletrônicos dos indivíduos com LAM. As associações entre os níveis de FGF23 e as características clínicas da LAM foram exploradas por meio do teste de hipóteses não paramétrico. Resultados: A amostra incluiu 37 indivíduos com LAM e 16 controles. Os níveis de FGF23 foram mais altos no grupo LAM do que no grupo controle. No grupo LAM, níveis de FGF23 acima do ponto de corte ideal distinguiram 33% dos indivíduos com níveis não diagnósticos de VEGF-D. Níveis mais baixos de FGF23 estavam associados à DLCO comprometida (p = 0,04), particularmente naqueles com comprometimento isolado da difusão e sem outras alterações espirométricas (p = 0,04). Conclusões: Nossos resultados sugerem que o FGF23 está associado a alterações na difusão pulmonar em pacientes com LAM e potencialmente indicam novos mecanismos de patogênese da LAM. O FGF23 isoladamente ou em combinação com outras moléculas precisa ser validado como um biomarcador da atividade da LAM em futuras pesquisas clínicas.

Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death.

Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth muscle-like "LAM" cells in the lungs. LAM cells bear loss-of-function mutations in tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2, causing hyperactivation of the proliferation promoting mammalian/mechanistic target of Rapamycin complex 1 pathway. Additionally, LAM-specific active renin-angiotensin system (RAS) has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear. Here, we report that TSC2-deficient cells are sensitive to the blockade of angiotensin II receptor type 1 (Agtr1). We show that treatment of these cells with the AGTR1 inhibitor losartan or silencing of the Agtr1 gene leads to increased cell death in vitro and attenuates tumor progression in vivo. Notably, we found the effect of Agtr1 blockade is specific to TSC2-deficient cells. Mechanistically, we demonstrate that cell death induced by Agtr1 inhibition is mediated by an increased expression of Klotho. In TSC2-deficient cells, we showed overexpression of Klotho or treatment with recombinant (soluble) Klotho mirrored the cytocidal effect of angiotensin blockade. Furthermore, Klotho treatment decreased the phosphorylation of AKT, potentially leading to this cytocidal effect. Conversely, silencing of Klotho rescued TSC2-deficient cells from cell death induced by Agtr1 inhibition. Therefore, we conclude that Agtr1 and Klotho are important for TSC2-deficient cell survival. These findings further illuminate the role of the RAS in LAM and the potential of targeting Agtr1 inhibition in TSC2-deficient cells.

Dysregulated myosin in Hermansky-Pudlak syndrome lung fibroblasts is associated with increased cell motility.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by improper biogenesis of lysosome-related organelles (LROs). Lung fibrosis is the leading cause of death among adults with HPS-1 and HPS-4 genetic types, which are associated with defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3), a guanine exchange factor (GEF) for a small GTPase, Rab32. LROs are not ubiquitously present in all cell types, and specific cells utilize LROs to accomplish dedicated functions. Fibroblasts are not known to contain LROs, and the function of BLOC-3 in fibroblasts is unclear. Here, we report that lung fibroblasts isolated from patients with HPS-1 have increased migration capacity. Silencing HPS-1 in normal lung fibroblasts similarly leads to increased migration. We also show that the increased migration is driven by elevated levels of Myosin IIB. Silencing HPS1 or RAB32 in normal lung fibroblasts leads to increased MYOSIN IIB levels. MYOSIN IIB is downstream of p38-MAPK, which is a known target of angiotensin receptor signaling. Treatment with losartan, an angiotensin receptor inhibitor, decreases MYOSIN IIB levels and impedes HPS lung fibroblast migration in vitro. Furthermore, pharmacologic inhibition of angiotensin receptor with losartan seemed to decrease migration of HPS lung fibroblasts in vivo in a zebrafish xenotransplantation model. Taken together, we demonstrate that BLOC-3 plays an important role in MYOSIN IIB regulation within lung fibroblasts and contributes to fibroblast migration.

ETV2 regulates PARP-1 binding protein to induce ER stress-mediated death in tuberin-deficient cells.

Lymphangioleiomyomatosis (LAM) is a rare progressive disease, characterized by mutations in the tuberous sclerosis complex genes (TSC1 or TSC2) and hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). Here, we report that E26 transformation-specific (ETS) variant transcription factor 2 (ETV2) is a critical regulator of Tsc2-deficient cell survival. ETV2 nuclear localization in Tsc2-deficient cells is mTORC1-independent and is enhanced by spleen tyrosine kinase (Syk) inhibition. In the nucleus, ETV2 transcriptionally regulates poly(ADP-ribose) polymerase 1 binding protein (PARPBP) mRNA and protein expression, partially reversing the observed down-regulation of PARPBP expression induced by mTORC1 blockade during treatment with both Syk and mTORC1 inhibitors. In addition, silencing Etv2 or Parpbp in Tsc2-deficient cells induced ER stress and increased cell death in vitro and in vivo. We also found ETV2 expression in human cells with loss of heterozygosity for TSC2, lending support to the translational relevance of our findings. In conclusion, we report a novel ETV2 signaling axis unique to Syk inhibition that promotes a cytocidal response in Tsc2-deficient cells and therefore maybe a potential alternative therapeutic target in LAM.

Blocking hyaluronan synthesis alleviates acute lung allograft rejection.

Lung allograft rejection results in the accumulation of low-molecular weight hyaluronic acid (LMW-HA), which further propagates inflammation and tissue injury. We have previously shown that therapeutic lymphangiogenesis in a murine model of lung allograft rejection reduced tissue LMW-HA and was associated with improved transplant outcomes. Herein, we investigated the use of 4-Methylumbelliferone (4MU), a known inhibitor of HA synthesis, to alleviate acute allograft rejection in a murine model of lung transplantation. We found that treating mice with 4MU from days 20 to 30 after transplant was sufficient to significantly improve outcomes, characterized by a reduction in T cell-mediated lung inflammation and LMW-HA content and in improved pathology scores. In vitro, 4MU directly attenuated activation, proliferation, and differentiation of naive CD4+ T cells into Th1 cells. As 4MU has already been demonstrated to be safe for human use, we believe examining 4MU for the treatment of acute lung allograft rejection may be of clinical significance.

FK506 induces lung lymphatic endothelial cell senescence and downregulates LYVE-1 expression, with associated decreased hyaluronan uptake.

BACKGROUND: Therapeutic lymphangiogenesis in an orthotopic lung transplant model has been shown to improve acute allograft rejection that is mediated at least in part through hyaluronan drainage. Lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expressed on the surface of lymphatic endothelial cells plays important roles in hyaluronan uptake. The impact of current immunosuppressive therapies on lung lymphatic endothelial cells is largely unknown. We tested the hypothesis that FK506, the most commonly used immunosuppressant after lung transplantation, induces lung lymphatic endothelial cell dysfunction. METHODS: Lung lymphatic endothelial cells were cultured in vitro and treated with FK506. Telomerase activity was measured using the TRAP assay. Protein expression of LYVE-1 and senescence markers p21 and ß-galactosidase was assessed with western blotting. Matrigel tubulation assay were used to investigate the effects of FK506 on TNF-α-induced lymphangiogenesis. Dual luciferase reporter assay was used to confirm NFAT-dependent transcriptional regulation of LYVE-1. Flow cytometry was used to examine the effects of FK506 on LYVE-1 in precision-cut-lung-slices ex vivo and on hyaluronan uptake in vitro. RESULTS: In vitro, FK506 downregulated telomerase reverse transcriptase expression, resulting in decreased telomerase activity and subsequent induction of p21 expression and cell senescence. Treatment with FK506 decreased LYVE-1 mRNA and protein levels and resulted in decreased LEC HA uptake. Similar result showing reduction of LYVE-1 expression when treated with FK506 was observed ex vivo. We identified a putative NFAT binding site on the LYVE-1 promoter and cloned this region of the promoter in a luciferase-based reporter construct. We showed that this NFAT binding site regulates LYVE-1 transcription, and mutation of this binding site blunted FK506-dependent downregulation of LYVE-1 promoter-dependent transcription. Finally, FK506-treated lymphatic endothelial cells show a blunted response to TNF-α-mediated lymphangiogenesis. CONCLUSION: FK506 alters lymphatic endothelial cell molecular characteristics and causes lymphatic endothelial cell dysfunction in vitro and ex vivo. These effects of FK506 on lymphatic endothelial cell may impair the ability of the transplanted lung to drain hyaluronan macromolecules in vivo. The implications of our findings on the long-term health of lung allografts merit more investigation.

Hyaluronan and LYVE-1 and allograft function in lung transplantation recipients.

Hyaluronan (HA) is associated with innate immune response activation and may be a marker of allograft dysfunction in lung transplant recipients. This was a prospective, single center study comparing levels of bronchioalveolar lavage (BAL) and serum HA and the HA immobilizer LYVE-1 in lung transplant recipients with and without acute cellular rejection (ACR). Chronic lung allograft dysfunction (CLAD)-free survival was also evaluated based on HA and LYVE-1 levels. 78 recipients were enrolled with a total of 115 diagnostic biopsies and 1.5 years of median follow-up. Serum HA was correlated with BAL HA (r = 0.25, p = 0.01) and with serum LYVE-1 (r = 0.32, p = 0.002). There was significant variation in HA and LYVE-1 over time, regardless of ACR status. Levels of serum HA (median 74.7 vs 82.7, p = 0.69), BAL HA (median 149.4 vs 134.5, p = 0.39), and LYVE-1 (mean 190.2 vs 183.8, p = 0.72) were not associated with ACR. CLAD-free survival was not different in recipients with any episode of elevated serum HA (HR = 1.5, 95% CI = 0.3-7.7, p = 0.61) or BAL HA (HR = 0.94, 95% CI = 0.2-3.6, p = 0.93). These results did not differ when stratified by bilateral transplant status. In this small cohort, serum HA, BAL HA, and LYVE-1 levels are not associated with ACR or CLAD-free survival in lung transplant recipients.

Glycogen synthase kinase 3-ß inhibition induces lymphangiogenesis through ß-catenin-dependent and mTOR-independent pathways.

Lymphatic vessels play an important role in health and in disease. In this study, we evaluated the effects of GSK3-ß inhibition on lung lymphatic endothelial cells in vitro. Pharmacological inhibition and silencing of GSK3-ß resulted in increased lymphangiogenesis of lung lymphatic endothelial cells. To investigate mechanisms of GSK3-ß-mediated lymphangiogenesis, we interrogated the mammalian/mechanistic target of rapamycin pathway and found that inhibition of GSK3-ß resulted in PTEN activation and subsequent decreased activation of AKT, leading to decreased p-P70S6kinase levels, indicating inhibition of the mTOR pathway. In addition, consistent with a negative role of GSK3-ß in ß-catenin stability through protein phosphorylation, we found that GSK3-ß inhibition resulted in an increase in ß-catenin levels. Simultaneous silencing of ß-catenin and inhibition of GSK3-ß demonstrated that ß-catenin is required for GSK3-ß-induced lymphangiogenesis.

Serum endostatin levels are associated with diffusion capacity and with tuberous sclerosis- associated lymphangioleiomyomatosis.

Endostatin is a naturally occurring collagen fragment with anti-angiogenic properties. We investigated the association between serum endostatin levels and DLCO in a cohort of patients with lymphangioleiomyomatosis (LAM). Associations of endostatin levels to clinical features of LAM were explored using logistic regression models. Endostatin levels were associated with DLCO and were higher in subjects with TSC-associated LAM compared to sporadic LAM. These data suggest that endostatin could be a predictive biomarker of decline in DLCO and that germline mutational inactivation of the TSC1 or TSC2 gene is associated with higher endostatin levels. These findings could offer novel insights into the pathogenesis of LAM.

Gene-Specific Variant Classifier (DPYD-Varifier) to Identify Deleterious Alleles of Dihydropyrimidine Dehydrogenase.

Deleterious variants in dihydropyrimidine dehydrogenase (DPD, DPYD gene) can be highly predictive of clinical toxicity to the widely prescribed chemotherapeutic 5-fluorouracil (5-FU). However, there are very limited data pertaining to the functional consequences of the >450 reported no-synonymous DPYD variants. We developed a DPYD-specific variant classifier (DPYD-Varifier) using machine learning and in vitro functional data for 156 missense DPYD variants. The developed model showed 85% accuracy and outperformed other in silico prediction tools. An examination of feature importance within the model provided additional insight into functional aspects of the DPD protein relevant to 5-FU toxicity. In the absence of clinical data for unstudied variants, prediction tools like DPYD-Varifier have great potential to individualize medicine and improve the clinical decision-making process.

Atlantoaxial rotatory displacement in children.

AIM: To correlate the Pang and Lee class with the clinical course in a consecutive series of patients presenting with painful torticollis. METHODS: Forty-seven dynamic rotational computed tomography (CT) scans in 35 patients were classified into one of the five types defined by Pang and Li, including types I (atlantoaxial rotatopry fixation), II ("pathologic stickiness" without crossover of C1 on C2), III ("pathologic stickiness" with crossover of C1 on C2), IV (normal or muscular torticollis), and V (diagnostic grey zone). The Pang and Li class was then compared with the radiologist's report, which was graded abnormal, diagnosis of rotatory subluxation or fixation, or non-diagnostic. Medical records were reviewed and the clinical course was compared among the five sub-types. RESULTS: We reviewed 47 CT scans in 35 patients, and the majority were performed without sedation. The average age was 7.7 years (4-14 years old) and associated conditions included minor trauma (20%), surgical procedures around the head and neck (29%), and Grisels syndrome (20%). Twenty-six percent of our studies fell within the pathologic spectrum (5% type 1 or rotatory fixation, 21% types 2 and 3 or rotatory subluxation), while 45% were classified as muscular torticollis (45%) and 28% fell within the diagnostic grey zone. Seven radiologists interpreted these studies, and their interpretation was discordant in 45% of cases. Clinical resolution occurred in 27 of 29 cases for which follow-up was available. One of two patients with fixed rotatory subluxation required a C1-C2 arthrodesis. CONCLUSION: The Pang and Li classification characterizes a spectrum of abnormalities in rotation to facilitate communication, although the indications for dynamic CT scan should be further defined.

Histone H3K27 Trimethylation Modulates 5-Fluorouracil Resistance by Inhibiting PU.1 Binding to the DPYD Promoter.

The antimetabolite 5-fluorouracil (5-FU) is one of the most widely used chemotherapy drugs. Dihydropyrimidine dehydrogenase (DPD) is a major determinant of 5-FU response and toxicity. Although DPYD variants may affect 5-FU metabolism, they do not completely explain the reported variability in DPD function or the resultant differences in treatment response. Here, we report that H3K27 trimethylation (H3K27me3) at the DPYD promoter regulated by Ezh2 and UTX suppresses DPYD expression by inhibiting transcription factor PU.1 binding, leading to increased resistance to 5-FU. Enrichment of H3K27me3 at the DPYD promoter was negatively correlated with both DPYD expression and DPD enzyme activity in peripheral blood specimens from healthy volunteers. Lastly, tumor expression data suggest that DPYD repression by Ezh2 predicts poor survival in 5-FU-treated cancers. Collectively, the findings of the present article suggest that a previously uncharacterized mechanism regulates DPD expression and may contribute to tumor resistance to 5-FU. Cancer Res; 76(21); 6362-73. ©2016 AACR.

Hip arthrodesis in children: A review of 28 patients.

BACKGROUND: The best method of treating intractable hip pain in an unsalvageable hip joint in a child is still a subject open to debate. We believe that hip arthrodesis in such patients provides a painless and stable hip for most activities of daily living in our challenging rural terrain. Therefore, we conducted this study to assess the functional ability of children with painful hip arthrosis treated by arthrodesis of the hip. MATERIALS AND METHODS: A retrospective evaluation of 28 children (out of 35) who had an arthrodesis of the hip performed between 1994 and 2008 was carried out. The average age was 14 years, with 12 males and 16 females. There was involvement of the right hip in 13 and left in 15 cases. The average duration of follow-up was 4.87 years. The preferred position of the hip for arthrodesis was 20-30 degrees of flexion, neutral abduction-adduction, and neutral rotation, irrespective of the method of fixation. RESULTS: The average duration of clinical and radiological arthrodesis was found to be 4 months (2-6 months). At the last follow-up, all patients were painfree and had good ambulatory capacity. The average Modified Harris Hip Score increased from 53 to 84 and the average post-surgical limb length discrepancy was 1.3 cm, which was well tolerated in all cases. Patients, however, had difficulty in squatting and had to modify their posture for foot care, putting on shoes, etc. Also, some patients complained of ipsilateral knee, contralateral hip, or low back pain with prolonged activity, but this was not severe enough to restrict activity except in one case that was known to have juvenile rheumatoid arthritis and needed ambulatory aid. CONCLUSION: In an environment where pathology generally presents very late and often in a dramatic manner, where the patient's socioeconomic status, understanding, compliance, and the logistics of follow-up are consistently a challenge in management, hip arthrodesis has been an important procedure for our patient group, with good short-term results and promising midterm, and, hopefully, long-term prospects. In our series of patients, we have been successful in restoring painfree mobility.

Open reduction for neglected traumatic hip dislocation in children and adolescents.

Neglected traumatic dislocation of the hip is extremely rare in children, and the preferred treatment remains unclear. This retrospective case series includes 8 children treated by open reduction. The mean age was 7.5 years (range, 2-16 years), and the mean follow-up was 7 years and 7 months (range, 4 month-16 years). Presenting complaints included pain (5/8) and gait disturbance (8/8). Traction failed to achieve a reduction in all cases. At follow-up, 6 hips remained reduced, and 2 achieved a non concentric reduction. All patients had evidence of avascular necrosis. Two patients, in whom a non concentric reduction was achieved, developed progressive flattening and joint space narrowing. Two patients had mild pain at follow-up, and 6 patients were able to squat. Range of motion was restricted both before and after open reduction, most notably in abduction and rotation. Postoperative improvement was seen in abduction (4 cases). Leg lengths were within 2 cm in 7 of 8 cases, and only 1 patient had a discrepancy greater than 2 cm. The results according to Garrett et al were good in 3, fair in 3, and poor in 2. The mean Harris hip score was 89. Patients with a concentric reduction had an adequate functional outcome despite evidence of avascular necrosis. The prognosis remains guarded, and we expect that a subset of patients will develop premature degenerative joint disease. However, we continue to offer patients an operative reduction, which we feel is preferable to other methods. A failed open reduction does not preclude options for salvage.