Lymphangioleiomyomatosis Nodules Research Articles

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) genesTSC1and/orTSC2, 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 theAgtr1gene leads to increased cell deathinvitroand attenuates tumor progressioninvivo. 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.

Mast-Cell Tryptase Release Contributes to Disease Progression in Lymphangioleiomyomatosis.

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

Evolution of lung pathology in lymphangioleiomyomatosis: associations with disease course and treatment response.

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease with a variable clinical course. The lungs are infiltrated by nodules of LAM cells, stromal cells and inflammatory cells, causing lung cysts and respiratory failure. We used immunohistochemical markers in lung biopsy and transplant samples from a national cohort of women with LAM with linked clinical data to understand how LAM nodule cell populations changed with disease progression. Marker distribution was examined qualitatively by dual immunohistochemistry, and markers for LAM cells, fibroblasts, lymphatics, mast cells, proliferation, cathepsin K and mTOR pathway activity were quantitated in LAM nodules and compared with clinical features and prospective lung function loss. The LAM cell marker PNL2 was more extensively expressed in those with higher forced expiratory volume in one second (FEV1), higher diffusion in the lung for carbon monoxide (DLCO) and less extensive disease involvement whilst the converse was true for the protease cathepsin K. Each percentage increase in cathepsin K reactivity was associated with a 0.65% decrease in FEV1 (95% CI −1.11 to −0.18) and a 0.50% decrease in DLCO (95% CI −0.96 to −0.05). Higher reactivity to the mTOR complex 1 activation marker, phospho‐ribosomal protein S6, was associated with a better lung function response to rapamycin (p = 0.0001). We conclude that LAM nodules evolve with disease progression, with LAM cells becoming outnumbered by fibroblasts. Increasing cathepsin K expression is associated with more severe disease and lung function loss. Markers of mTOR activation predict the response to rapamycin, suggesting that more advanced LAM may be less mTOR responsive and treatments specifically targeted towards LAM associated fibroblasts may have roles as adjuncts to mTOR inhibition.

Immunotherapy for Lymphangioleiomyomatosis and Tuberous Sclerosis: Progress and Future Directions

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic multisystem disease characterized by the nodular proliferation of smooth muscle-like LAM cells, progressive cystic changes of the lung, lymphatic abnormalities, and renal angiomyolipomas (AMLs). LAM can arise sporadically or in women with the autosomal dominant disorder, tuberous sclerosis complex (TSC), in which hamartomatous tumors of brain, heart, skin, kidney, and lung are found. LAM and TSC are caused by mutations in the TSC1 or TSC2 tumor suppressor genes leading to elevated mechanistic/mammalian target of rapamycin complex activity. Recent data indicate that Tcells within LAM nodules and renal AMLs exhibit features of T-cell exhaustion, with coinhibitory receptor programmed cell death protein 1 (PD-1) expression on tumor-infiltrating Tcells. Treatment of animal models of TSC and LAM with anti-PD-1 antibodies or with the combination of anti-PD-1 and anti-CTLA4 antibodies has led to remarkable results, suppressing TSC2-null tumor growth and inducing tumor rejection. Here we review our current knowledge about the potential for immunotherapy for the treatment of LAM and TSC and highlight critical unknowns and key next steps.

Oncogenic effects of urotensin-II in cells lacking tuberous sclerosis complex-2.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease that can arise sporadically or in adults suffering from the tumor syndrome tuberous sclerosis complex (TSC). Microscopic tumors (‘LAM nodules’) in the lung interstitium arise from lymphatic invasion and metastasis. These consist of smooth muscle-like cells (LAM cells) that exhibit markers of neural crest differentiation and loss of the tumor suppressor protein ‘tuberous sclerosis complex-2’ (TSC2). Consistent with a neural phenotype, expression of the neuropeptide urotensin-II and its receptor was detected in LAM nodules. We hypothesized that loss of TSC2 sensitizes cells to the oncogenic effects of urotensin-II. TSC2-deficient Eker rat uterine leiomyoma ELT3 cells were stably transfected with empty vector or plasmid for the expression of TSC2. Urotensin-II increased cell viability and proliferation in TSC2-deficient cells, but not in TSC2-reconstituted cells. When exposed to urotensin-II, TSC2-deficient cells exhibited greater migration, anchorage-independent cell growth, and matrix invasion. The effects of urotensin-II on TSC2-deficient cells were blocked by the urotensin receptor antagonist SB657510, and accompanied by activation of Erk mitogen-activated protein kinase and focal adhesion kinase. Urotensin-II-induced proliferation and migration were reproduced in TSC2-deficient human angiomyolipoma cells, but not in those stably expressing TSC2. In a mouse xenograft model, SB657510 blocked the growth of established ELT3 tumors, reduced the number of circulating tumor cells, and attenuated the production of VEGF-D, a clinical biomarker of LAM. Urotensin receptor antagonists may be selective therapeutic agents for the treatment of LAM or other neural crest-derived neoplasms featuring loss of TSC2 or increased expression of the urotensin receptor.

Wild type mesenchymal cells contribute to the lung pathology of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.

Rapamycin-resistant poly (ADP-ribose) polymerase-1 overexpression is a potential therapeutic target in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-predominant cystic lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis complex 2 (TSC2) mutations and mammalian target of rapamycin (mTOR) complex (mTORC) 1 activation. Clinical response to the mTORC1 inhibitors has been limited, prompting a search for additional therapy for LAM. In this study, we investigated the impact of TSC2 on the expression of poly (ADP-ribose) polymerase (PARP)-1 that initiates the DNA repair pathway, and tested the efficacy of PARP1 inhibitors in the survival of TSC2-deficient (TSC2(-)) cells. We analyzed publicly available expression arrays of TSC2(-) cells and validated the findings using real-time RT-PCR, immunoblotting, and immunohistochemistry. We examined the impact of rapamycin and Torin 1 on PARP1 expression. We also tested the effect of PARP1 inhibitors, 8-hydroxy-2-methylquinazoline-4-one and 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline, on the survival of TSC2(-) cells. We identified the up-regulation of PARP1 in TSC2(-) cells relative to cells in which wild-type TSC2 has been reintroduced (TSC2-addback [TSC2(+)] cells). The transcript levels of PARP1 in TSC2(-) cells were not affected by rapamycin. PARP1 levels were increased in TSC2(-) cells, xenograft tumors of rat-derived TSC2(-) cells, renal cystadenomas from Tsc2(+/-) mice, and human LAM nodules. RNA interference of mTOR failed to reduce PARP1 levels. Proliferation and survival of TSC2(-) cells was reduced in response to PARP1 inhibitor treatment, more so than TSC2(+) cells. TSC2(-) cells exhibit higher levels of PARP1 relative to TSC2(+) cells in an mTOR-insensitive manner. PARP1 inhibitors selectively suppress the growth and induce apoptosis of TSC2(-) cells from patients with LAM. Targeting PARP1 may be beneficial in the treatment of LAM and other neoplasm with mTORC1 activation.

Rapamycin-insensitive up-regulation of adipocyte phospholipase A2 in tuberous sclerosis and lymphangioleiomyomatosis.

Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/− mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2 −/−MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived “mesenchymal” cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role in promoting tumorigenesis and disease progression by modulating the production of prostaglandins and may serve as a potential therapeutic target in TSC and LAM.

LAM cells biology and lymphangioleiomyomatosis

Progressive lung tissue destruction in lymphangioleiomyomatosis (LAM) occurs as a result of excessive proliferation of LAM cells caused by a mutation in one of the tuberous sclerosis complex suppressor genes, TSC1 or TSC2. These cells show constitutive activation of the mammalian target of rapamycin (mTOR) pathway and many of the mTOR-related kinases such as Akt, Erk, S6K1 and S6. Phenotype of LAM cells differs considerably depending on their microenvironment. LAM cells show differences in morphology, size and expression of various factors depending on their location in the tumor or body fluids. The presence of LAM cells in blood, urine, bronchoalveolar lavage fluid (BALF), and chyle proves their ability to metastasis. Antigens of smooth muscle cells are expressed in most LAM cells. Some of these cells are immunoreactive with HMB-45 antibody, which is used for the immunohistochemical diagnosis of LAM. Receptors for estrogen and progesterone may also be expressed in these cells, which probably is associated with the fact that LAM occurs almost exclusively in women of childbearing age. LAM cells via increased production of metalloproteinases are involved in the destruction of the extracellular matrix, as well as the remodeling and damage of lung tissue. Sporadic LAM occurs extremely rarely. Therefore a good experimental model of this disease is necessary. To date, several animal and human cell lines, which both genetically and phenotypically resemble LAM cells, have been obtained. These cell lines, derived from LAM nodule or an angiomyolipoma, are usually characterized by a mutation of the TSC2 gene, expression of smooth muscle cell antigens such as a-smooth muscle actin (aSMA) or S6K1 and S6 protein hyperphosphorylation. Presently, there is no commercially available cell line representing a good model of LAM. A better understanding of LAM cell biology is necessary for creating a useful model in vitro for further exploration of both LAM pathomechanisms and more general mechanisms of carcinogenesis.

Exonic Mutations of TSC2/TSC1 Are Common but Not Seen in All Sporadic Pulmonary Lymphangioleiomyomatosis

Exonic Mutations of <i>TSC2/TSC1</i> Are Common but Not Seen in All Sporadic Pulmonary Lymphangioleiomyomatosis

Treatment Option(s) for Pulmonary Lymphangioleiomyomatosis: Progress and Current Challenges

Pulmonary lymphangioleiomyomatosis (LAM) is a rare progressive cystic lung disease affecting primarily women of childbearing age (1, 2). LAM occurs sporadically (LAM-S) with prevalence of 2.6 per 1 million women or in 34% of women with tuberous sclerosis (TS) (LAM-TS) (3), an autosomal dominant hamartoma syndrome that occurs in 1 of 5,800 live births (2). Clinical manifestations of LAM are pneumothorax from cyst rupture, chylothorax from obstruction of lymphatics, and progressive decline of pulmonary function (1, 2). About 40% of patients with LAM-S and about 80% of patients with LAM-TS also develop in kidney an angiomyolipoma (AML), a benign tumor of smooth muscle (SM), blood vessels, and fat cells (1, 2). Pathological changes in the LAM lung are associated with growth throughout the lung parenchyma of LAM nodules that consist of SM-like spindle-shaped cells and epithelioid-like polygonal cells positive for melanocytic cell marker HMB45 (human melanoma black 45) (4, 5) (Figure 1). SM-like LAM cells show high immunoreactivity for PCNA (proliferating cell nuclear antigen), a marker of DNA synthesis and cell proliferation, compared with the epithelioid-like HMB45-positive cells (2), suggesting that SM-like LAM cells represent the proliferative component of the LAM nodules. The role of melanocyte-specific markers in LAM and whether they could be targeted therapeutically have been explored (6) and were reviewed in the January issue (7).

High levels of extracellular matrix metalloproteinase inducer are expressed in lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis is pathologically characterized by the proliferation of abnormal smooth muscle-like cells (lymphangioleiomyomatosis cells) that synthesize excess matrix metalloproteinases. Extracellular matrix metalloproteinase inducer is minimally expressed in the healthy lung, but is up-regulated in various lung injuries that are apparently associated with matrix metalloproteinases. We therefore immunohistochemically stained extracellular matrix metalloproteinase inducer in lymphangioleiomyomatosis cells and measured extracellular matrix metalloproteinase inducer in bronchoalveolar lavage fluid using an enzyme-linked immunosorbent assay. We also quantified extracellular matrix metalloproteinase inducer messenger RNA expression in the lung using quantitative reverse transcriptase-polymerase chain reaction. Intense staining for extracellular matrix metalloproteinase inducer in lymphangioleiomyomatosis cells indicated high immunoreactivity. Dual immunofluorescence studies revealed diffuse colocalization between extracellular matrix metalloproteinase inducer and alpha smooth muscle actin, matrix metalloproteinase-2, or matrix metalloproteinase-9. Although levels of extracellular matrix metalloproteinase inducer messenger RNA did not differ in whole lung homogenates from patients with lymphangioleiomyomatosis and healthy controls (1.7 +/- 0.1 versus 1.5 +/- 0.2 SE, not significant; both n = 4), lymphangioleiomyomatosis nodules retrieved by laser capture microdissection expressed 5.1 (+/-1.0 SE)-fold higher levels of extracellular matrix metalloproteinase inducer messenger RNA than lung homogenates (P = .0077, n = 4). Levels of extracellular matrix metalloproteinase inducer were significantly elevated in bronchoalveolar lavage fluid from lymphangioleiomyomatosis patients compared with controls (82.7 +/- 19.5 versus 38.6 +/- 9.0 SE pg/mL, P = .0497; n = 8 and 9, respectively). Increased levels of extracellular matrix metalloproteinase inducer colocalized with increased matrix metalloproteinases in lymphangioleiomyomatosis cells indicate that it potentially functions in pulmonary lymphangioleiomyomatosis.

Vascular Endothelial Growth Factors C and D Induces Proliferation of Lymphangioleiomyomatosis Cells through Autocrine Crosstalk with Endothelium

Lymphangioleiomyomatosis (LAM) is a potentially fatal lung disease characterized by nodules of proliferative smooth muscle-like cells. The exact nature of these LAM cells and their proliferative stimuli are poorly characterized. Herein we report the novel findings that the lymphangiogenic vascular endothelial growth factors (VEGF) C and D induce LAM cell proliferation through activation of their cognate receptor VEGF-R3 and activation of the signaling intermediates Akt/mTOR/S6. Furthermore, we identify expression of the proteoglycan NG2, a marker of immature smooth muscle cells, as a characteristic of LAM cells both in vitro and in human lung tissue. VEGF-C-induced LAM cell proliferation was in part a result of autocrine stimulation that resulted from cross talk with lymphatic endothelial cells. Ultimately, these findings identify the lymphangiogenic VEGF proteins as pathogenic growth factors in LAM disease and at the same time provide a novel pharmacotherapeutic target for a lung disease that to date has no known effective treatment.

Tissue-Specific Renin–Angiotensin System in Pulmonary Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM), a multisystem disease found in middle-aged women, is characterized by cystic lung destruction and abdominal tumors (e.g., angiomyolipomas, lymphangioleimyomas), resulting from proliferation of abnormal-appearing, smooth muscle-like cells (LAM cells). The LAM cells, in combination with other cells, form nodular structures within the lung interstitium and in the walls of the cysts. LAM cells contain mutations in the tuberous sclerosis complex TSC1 and/or TSC2 genes, which lead to dysregulation of the mammalian target of rapamycin, affecting cell growth and proliferation. Proliferation and migration of vascular smooth muscle cells and production of angiogenic factors are regulated, in part, by angiotensin II. To determine whether a LAM-specific renin-angiotensin system might play a role in the pathogenesis of LAM, we investigated the expression of genes and gene products of this system in LAM nodules. mRNA for angiotensinogen was present in RNA isolated by laser-captured microdissection from LAM nodules. Angiotensin I-converting enzyme and chymase-producing mast cells were present within the LAM nodules. We detected renin in LAM cells, as determined by the presence of mRNA and immunohistochemistry. Angiotensin II type 1 and type II receptors were identified in LAM cells by immunohistochemistry and immunoblotting of microdissected LAM nodules. Angiotensin II is localized in cells containing alpha-smooth muscle actin (LAM cells). A LAM-specific renin-angiotensin system appears to function within the LAM nodule as an autocrine system that could promote LAM cell proliferation and migration, and could represent a pharmacologic target.

Abnormal Growth of Smooth Muscle–Like Cells in Lymphangioleiomyomatosis

The TSC1 and TSC2 proteins, which function as a TSC1/TSC2 tumor suppressor complex, are associated with lymphangioleiomyomatosis (LAM), a genetic disorder characterized by the abnormal growth of smooth muscle-like cells in the lungs. The precise molecular mechanisms that modulate LAM cell growth remain unknown. We demonstrate that TSC2 regulates LAM cell growth. Cells dissociated from LAM nodules from the lungs of five different patients with LAM have constitutively activated S6K1, hyperphosphorylated ribosomal protein S6, activated Erk, and increased DNA synthesis compared with normal cells from the same patients. These effects were augmented by PDGF stimulation. Akt activity was unchanged in LAM cells. Rapamycin, a specific S6K1 inhibitor, abolished increased LAM cell growth. The full-length TSC2 was necessary for inhibition of S6 hyperphosphorylation and DNA synthesis in LAM cells, as demonstrated by co-microinjection of the C-terminus, which contains the GTPase activating protein homology domain, and the N-terminus, which binds TSC1. Our data demonstrate that increased LAM cell growth is associated with constitutive S6K1 activation, which is extinguishable by TSC2 expression. Loss of TSC2 GAP activity or disruption of the TSC1/TSC2 complex dysregulates S6K1 activation, which leads to abnormal cell proliferation associated with LAM disease.

Role of Discoidin Domain Receptors 1 and 2 in Human Smooth Muscle Cell-Mediated Collagen Remodeling: Potential Implications in Atherosclerosis and Lymphangioleiomyomatosis

Obstructive diseases of blood vessels and the lung are characterized by degradation and synthesis of new extracellular matrix (ECM) components. Regulated remodeling of the ECM in diseases such as atherosclerosis and lymphangioleiomyomatosis (LAM), both characterized by excessive accumulation of smooth muscle cells (SMCs), is thought to be controlled in part by cell surface receptors for specific ECM components. Discoidin domain receptors (DDR) 1 and 2 represent a family of tyrosine kinase collagen receptors that are activated by fibrillar collagens. To test the hypothesis that DDR may be involved in ECM remodeling by SMCs in vivo, we analyzed DDR expression by reverse transcriptase-polymerase chain reaction and immunohistochemistry and demonstrate that both DDR1 and DDR2 are up-regulated in nodules of LAM as compared to normal controls, and are expressed in lesions of atherosclerosis. In vitro, retroviral overexpression of DDR1 or DDR2 in human SMCs cultured on polymerized collagen gels leads to a reduction of collagen expression and induces matrix metalloproteinase (MMP) 1 at both mRNA and protein levels, but only DDR2 enhances MMP2 activation. Moreover, DDR2 overexpression increases SMC-mediated collagen and elastin degradation in vitro. Using laser microdissection, we extend our studies to the analysis of SMCs from LAM nodules where we observe higher MMP1 expression and MMP2 activation. Taken together, these data provide evidence for the potential roles of DDR1 and DDR2 in the regulation of collagen turnover mediated by SMCs in obstructive diseases of blood vessels and the lung.

Lymphangioleiomyomatosis (LAM): a review of clinical and morphological features.

A review is presented of the clinical and morphological manifestations of lymphangioleiomyomatosis (LAM), a systemic disorder of unknown etiology that affects women. The clinical features include dyspnea, hemoptysis, recurrent pneumothorax, chylothorax, and chylous ascites. It is characterized by: 1) proliferation of abnormal smooth muscle cells (LAM cells) in pulmonary interstitium and along the axial lymphatics of the thorax and abdomen; 2) thin-walled pulmonary cysts, and 3) a high incidence of angiomyolipomas. The pulmonary cystic lesions have a characteristic appearance on high resolution computed tomography. The most specific method for diagnosing LAM is lung biopsy to demonstrate the presence of LAM cells, either by their characteristic histological appearance or by specific immunostaining with HMB-45 antibody. LAM cells differ in several important respects from the types of smooth muscle cells normally present in lung. Their reactivity with HMB-45 antibody is localized in stage I and stage II melanosomes. LAM cells show additional evidence of incomplete melanogenesis, and the significance of these observations remains to be determined. Two types of LAM cells are recognized: 1) small, spindle-shaped cells that are centrally located in the LAM nodules and are highly immunoreactive for matrix metalloproteinase-2 (MMP-2), its activating enzyme (MT-1-MMP), and proliferating cell nuclear antigen (PCNA), and 2) large, epithelioid cells that are distributed along the periphery of the nodules and show a high degree of immunoreactivity with HMB-45 antibody and with antibodies against estrogen and progesterone receptors. Types of treatment used for LAM include oophorectomy, administration of Lupron or progesterone and in very severe cases, pulmonary transplantation (following the onset of respiratory insufficiency, not relieved by O(2)).