Lymphangioleiomyoma Cells and Lymphatic Endothelial Cells
2010; Elsevier BV; Volume: 176; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.091239
ISSN1525-2191
AutoresKuniaki Seyama, Keiko Mitani, Toshio Kumasaka, Shiv K. Gupta, Saji Oommen, Gang Liu, Jay H. Ryu, Nicholas E. Vlahakis,
Tópico(s)Corporate Governance and Law
ResumoThis Correspondence addresses the use of LAM and LAM-derived cells in Issaka et al. (Am J Pathol 2009,175: 1410–1420) This Correspondence addresses the use of LAM and LAM-derived cells in Issaka et al. (Am J Pathol 2009,175: 1410–1420) We have read the article by Issaka et al1Issaka RB Oommen S Gupta SK Liu G Myers JL Ryu JH Vlahakis NE Vascular endothelial growth factors C and D induces proliferation of lymphangioleiomyomatosis cells through autocrine crosstalk with endothelium.Am J Pathol. 2009; 175: 1410-1420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar with great interest as well as deep concern. They reported that lymphangioleiomyoma (LAM) cells express vascular endothelial growth factor (VEGF) receptor (R)-3 and, in turn, that autocrine and paracrine signaling via the VEGF-C/VEGF-D/VEGFR-3 axis plays a role in LAM cell biology in vivo. The source of their LAM-derived cells (LDCs)2Goncharova EA Goncharov DA Eszterhas A Hunter DS Glassberg MK Yeung RS Walker CL Noonan D Kwiatkowski DJ Chou MM Panettieri Jr, RA Krymskaya VP Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM).J Biol Chem. 2002; 277: 30958-30967Crossref PubMed Scopus (372) Google Scholar, 3Goncharova EA Goncharov DA Spaits M Noonan DJ Talovskaya E Eszterhas A Krymskaya VP Abnormal growth of smooth muscle-like cells in lymphangioleiomyomatosis: role for tumor suppressor TSC2.Am J Respir Cell Mol Biol. 2006; 34: 561-572Crossref PubMed Scopus (64) Google Scholar was a clone established from LAM nodules in patients' lungs. We commend the authors' enthusiasm for their results, but have found several inaccuracies. First, although they admit that the LDCs they tested were not LAM cells in vivo, they state in the text as well as the article's title that LAM cells in vivo grow through autocrine cross talk with lymphatic endothelial cells (LECs). Second, they kindly cite our studies reporting that LAM tissues consist of abundant VEGFR-3-positive LECs and that LAM is characterized by an association with lymphangiogenesis.4Kumasaka T Seyama K Mitani K Sato T Souma S Kondo T Hayashi S Minami M Uekusa T Fukuchi Y Suda K Lymphangiogenesis in lymphangioleiomyomatosis: its implication in the progression of lymphangioleiomyomatosis.Am J Surg Pathol. 2004; 28: 1007-1016Crossref PubMed Scopus (122) Google Scholar, 5Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (137) Google Scholar VEGFR-3 is one of the most well-known markers of LECs.6Lohela M Bry M Tammela T Alitalo K VEGFs and receptors involved in angiogenesis versus lymphangiogenesis.Curr Opin Cell Biol. 2009; 21: 154-165Crossref PubMed Scopus (577) Google Scholar Although we provided concrete evidence that our VEGFR-3-positive cells were LECs, we have never stated that LAM cells are VEGFR-3 positive.4Kumasaka T Seyama K Mitani K Sato T Souma S Kondo T Hayashi S Minami M Uekusa T Fukuchi Y Suda K Lymphangiogenesis in lymphangioleiomyomatosis: its implication in the progression of lymphangioleiomyomatosis.Am J Surg Pathol. 2004; 28: 1007-1016Crossref PubMed Scopus (122) Google Scholar, 5Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (137) Google Scholar In Figure 4 of their study, Issaka et al state that LAM cells are positive for VEGFR-3. However, the lung tissues illustrated in Figure 4 do not have VEGFR-3-positive-LECs. Despite the fact that many LAM cells stained positive for VEGFR-3 in this figure, no LECs stained positively for VEGFR-3. We have documented immunostaining of LAM tissue with anti-VEGFR-3 numerous times4Kumasaka T Seyama K Mitani K Sato T Souma S Kondo T Hayashi S Minami M Uekusa T Fukuchi Y Suda K Lymphangiogenesis in lymphangioleiomyomatosis: its implication in the progression of lymphangioleiomyomatosis.Am J Surg Pathol. 2004; 28: 1007-1016Crossref PubMed Scopus (122) Google Scholar, 5Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (137) Google Scholar, 7Mitani K Kumasaka T Takemura H Hayashi T Gunji Y Kunogi M Akiyoshi T Takahashi K Suda K Seyama K Cytologic, immunocytochemical and ultrastructural characterization of lymphangioleiomyomatosis cell clusters in chylous effusions of patients with lymphangioleiomyomatosis.Acta Cytol. 2009; 53: 402-409Crossref PubMed Scopus (37) Google Scholar but have never obtained results like those reported by Issaka et al. Our immunostaining in LAM tissues in vivo using the same methods as described previously demonstrates that LECs are positive for VEGFR-3, whereas LAM cells are negative.4Kumasaka T Seyama K Mitani K Sato T Souma S Kondo T Hayashi S Minami M Uekusa T Fukuchi Y Suda K Lymphangiogenesis in lymphangioleiomyomatosis: its implication in the progression of lymphangioleiomyomatosis.Am J Surg Pathol. 2004; 28: 1007-1016Crossref PubMed Scopus (122) Google Scholar, 5Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (137) Google Scholar, 7Mitani K Kumasaka T Takemura H Hayashi T Gunji Y Kunogi M Akiyoshi T Takahashi K Suda K Seyama K Cytologic, immunocytochemical and ultrastructural characterization of lymphangioleiomyomatosis cell clusters in chylous effusions of patients with lymphangioleiomyomatosis.Acta Cytol. 2009; 53: 402-409Crossref PubMed Scopus (37) Google Scholar Double immunostaining with α-smooth muscle actin (SMA) as a marker of LAM cells and VEGFR-3 as a marker of LEC as well as immunofluorescence using SMA and VEGFR-3 clearly indicate a separation between SMA positive and VEGFR-3 positive cells, with little overlap (unpublished data). Therefore, we question their use of prox-1 immunostaining to show the existence of LECs in LAM tissue and their claim that perilymphatically-located LAM cells were positive for VEGFR-3. In our hands, double immunostaining for prox-1, a transcription factor specific for LEC, and VEGFR-3 revealed VEGFR-3-positive cytoplasm with prox-1-positive nuclei in only a subset of VEGFR-3 cells (unpublished data). These data suggest that not all LECs are transcriptionally active; that is, some LECs are VEGFR-3-positive but prox-1-negative, whereas some are both VEGFR-3- and prox-1-positive. When LAM cell clusters (LCCs), isolated from chylous effusion, are cultured, they break into LAM cells and LECs in 5 to 7 days.5Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (137) Google Scholar Using this model, polygonally shaped LECs are positive for VEGFR-3, whereas spindle-shaped and SMA-positive LAM cells are negative for VEGFR-3 (unpublished data). Importantly, LCCs are genuine LAM cells5Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (137) Google Scholar, 7Mitani K Kumasaka T Takemura H Hayashi T Gunji Y Kunogi M Akiyoshi T Takahashi K Suda K Seyama K Cytologic, immunocytochemical and ultrastructural characterization of lymphangioleiomyomatosis cell clusters in chylous effusions of patients with lymphangioleiomyomatosis.Acta Cytol. 2009; 53: 402-409Crossref PubMed Scopus (37) Google Scholar and differ from LDCs, where clones are isolated from lung tissue after multiple procedural steps and 3 to 10 rounds of passages.1Issaka RB Oommen S Gupta SK Liu G Myers JL Ryu JH Vlahakis NE Vascular endothelial growth factors C and D induces proliferation of lymphangioleiomyomatosis cells through autocrine crosstalk with endothelium.Am J Pathol. 2009; 175: 1410-1420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Indeed, LCC-derived cells are maintained in vitro for a very short period and are thus are highly likely to retain the physiological properties of their status in vivo, in contrast to the cloned LDCs that Issaka et al used throughout their experiments.1Issaka RB Oommen S Gupta SK Liu G Myers JL Ryu JH Vlahakis NE Vascular endothelial growth factors C and D induces proliferation of lymphangioleiomyomatosis cells through autocrine crosstalk with endothelium.Am J Pathol. 2009; 175: 1410-1420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar If the results of the immunohistochemical analysis of LAM tissues (Figure 4, A and B) by Issaka et al were truly correct, we would have to admit that a great difference must exist in the pathogenesis of LAM between the patient populations examined in our studies. It is now widely accepted that LAM is a neoplastic disease and progresses through metastasis, although LAM cells appear histopathologically benign. However, LAM is a "chronic and slowly progressive neoplasm," leading to destruction of the lungs in a course that is quite distinct from, for example, that of lung cancer, a representative malignant neoplasm of the lungs. Patients with LAM have about a 70% survival rate at 10 years after diagnosis,8Hayashida M Seyama K Inoue Y Fujimoto K Kubo K The epidemiology of lymphangioleiomyomatosis in Japan: a nationwide cross-sectional study of presenting features and prognostic factors.Respirology. 2007; 12: 523-530Crossref PubMed Scopus (84) Google Scholar, 9Matsui K Beasley MB Nelson WK Barnes PM Bechtle J Falk R Ferrans VJ Moss J Travis WD Prognostic significance of pulmonary lymphangioleiomyomatosis histologic score.Am J Surg Pathol. 2001; 25: 479-484Crossref PubMed Scopus (106) Google Scholar which is much longer than that of individuals with lung cancers. Accordingly, LAM patients are candidates for lung transplantation, although LAM can recur in the donor lung. This point seems to have been missed in research on LAM cell biology. LAM cells do not proliferate in vivo as fast as in vitro; therefore, the gap between results gained under circumstances most closely duplicating fresh living cells versus those in longer-term culture would be much bigger for LAM cell biology than for cancer cells. In addition, the precise identity of LAM cells remains unclear.10Finlay G The LAM cell: what is it, where does it come from, and why does it grow?.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L690-L693Crossref PubMed Scopus (48) Google Scholar LAM tissues are composed of heterogeneous cells with different morphologies and immunoreactivities.10Finlay G The LAM cell: what is it, where does it come from, and why does it grow?.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L690-L693Crossref PubMed Scopus (48) Google Scholar Accordingly, the differing appearances of cells in LAM lesions are associated with cell-specific functions, which may help to explain why LAM patients have variable clinical courses.10Finlay G The LAM cell: what is it, where does it come from, and why does it grow?.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L690-L693Crossref PubMed Scopus (48) Google Scholar Conceivably, the clones Issaka et al used may be just one phenotype of LAM cells consisting of LAM lung tissues. However, that type must be a small minority of the overall content in LAM lesions judging from our consecutive results of repeated immunohistochemical analyses.4Kumasaka T Seyama K Mitani K Sato T Souma S Kondo T Hayashi S Minami M Uekusa T Fukuchi Y Suda K Lymphangiogenesis in lymphangioleiomyomatosis: its implication in the progression of lymphangioleiomyomatosis.Am J Surg Pathol. 2004; 28: 1007-1016Crossref PubMed Scopus (122) Google Scholar, 5Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (137) Google Scholar, 7Mitani K Kumasaka T Takemura H Hayashi T Gunji Y Kunogi M Akiyoshi T Takahashi K Suda K Seyama K Cytologic, immunocytochemical and ultrastructural characterization of lymphangioleiomyomatosis cell clusters in chylous effusions of patients with lymphangioleiomyomatosis.Acta Cytol. 2009; 53: 402-409Crossref PubMed Scopus (37) Google Scholar From the viewpoint of physician-scientists caring for LAM patients, we believe that LAM researchers must bear in mind whether the choice of cells used in experiments and the role those cells play in vivo are truly relevant for human application. We thank Seyama et al for their interest in our recent publication1Issaka RB Oommen S Gupta SK Liu G Myers JL Ryu JH Vlahakis NE Vascular endothelial growth factors C and D induces proliferation of lymphangioleiomyomatosis cells through autocrine crosstalk with endothelium.Am J Pathol. 2009; 175: 1410-1420Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar in the Journal and commend their group on the extensive and important work they have contributed to the field of LAM research. Despite their quite lengthy correspondence regarding our publication, we have identified only two points of concern, which we address in our response below. Foremost is the apparent discrepancy in the cell-type expression of VEGF-R3. Seyama et al highlight an apparent conflict in findings with their previously published studies2Kumasaka T Seyama K Mitani K Sato T Souma S Kondo T Hayashi S Minami M Uekusa T Fukuchi Y Suda K Lymphangiogenesis in lymphangioleiomyomatosis: its implication in the progression of lymphangioleiomyomatosis.Am J Surg Pathol. 2004; 28: 1007-1016Crossref PubMed Scopus (67) Google Scholar, 3Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (70) Google Scholar and a significant amount of non-peer reviewed work referred to in their letter. The majority of their work is based on immunohistochemistry, a technique that although helpful has significant limitations when trying to draw conclusions on cellular biology. This is in part why we went to great lengths to use an in vitro LAM cell model that was as close to a physiologically relevant model available: cells from human lung LAM nodules. This allowed us to more thoroughly develop an understanding of VEGF/VEGF-R3 biology in LAM that would not be possible with immunohistochemistry alone. Thus, we believe that although the criticisms of our immunohistochemistry findings are reasonable given the apparent difference in findings, by applying the same critical logic Seyama et al have used, their previous work can be as easily called into question. To address Seyama et al's concern more directly, we found that human LAM nodule cells with an endothelial cell morphology that lined irregularly shaped channels all expressed both Prox-1 and VEGF-R3 and, as such, were clearly identified as LECs. However, the surrounding cells within LAM nodules expressed VEGF-R3 alone without Prox-1 staining. We agree that VEGF-R3 is a well known marker of LECs, however, compared to Prox-1 it is significantly less specific4Wilting J Papoutsi M Christ B Nicolaides KH von Kaisenberg CS Borges J Stark GB Alitalo K Tomarev SI Niemeyer C Rössler J The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues.FASEB J. 2002; 16: 1271-1273PubMed Google Scholar, 5Johnson NC Dillard ME Baluk P McDonald DM Harvey NL Frase SL Oliver G Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity.Genes Dev. 2008; 22: 3282-3291Crossref PubMed Scopus (252) Google Scholar since VEGF-R3 has clearly been shown to be expressed in other cell types, including corneal epithelium,6Cursiefen C Chen L Saint-Geniez M Hamrah P Jin Y Rashid S Pytowski B Persaud K Wu Y Streilein JW Dana R Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.Proc Natl Acad Sci USA. 2006; 103: 11405-11410Crossref PubMed Scopus (226) Google Scholar osteoblasts,7Orlandini M Spreafico A Bardelli M Rocchigiani M Salameh A Nucciotti S Capperucci C Frediani B Oliviero S Vascular endothelial growth factor-D activates VEGFR-3 expressed in osteoblasts inducing their differentiation.J Biol Chem. 2006; 281: 17961-17967Crossref PubMed Scopus (73) Google Scholar hepatocytes,8Lian Z Liu J Wu M Wang HY Arbuthnot P Kew M Feitelson MA Hepatitis Bx antigen up-regulates vascular endothelial growth factor receptor 3 in hepatocarcinogenesis.Hepatology. 2007; 45: 1390-1399Crossref PubMed Scopus (28) Google Scholar and renal podocytes.9Foster RR Satchell SC Seckley J Emmett MS Joory K Xing CY Saleem MA Mathieson PW Bates DO Harper SJ VEGF-C promotes survival in podocytes.Am J Physiol Renal Physiol. 2006; 291: F196-F207Crossref PubMed Scopus (41) Google Scholar Also, in our experience and that of others, including Kumasaka et al,2Kumasaka T Seyama K Mitani K Sato T Souma S Kondo T Hayashi S Minami M Uekusa T Fukuchi Y Suda K Lymphangiogenesis in lymphangioleiomyomatosis: its implication in the progression of lymphangioleiomyomatosis.Am J Surg Pathol. 2004; 28: 1007-1016Crossref PubMed Scopus (67) Google Scholar variability in the pattern of VEGF-R3 expression and lymphangiogenesis in LAM tissues is not uncommon. In fact, this point is well highlighted by Seyama et al in the description of their unpublished work where they report an apparent discrepancy in distribution of Prox-1 and VEGF-R3 expression: a finding consistent with our publication. However, we respectfully disagree with their interpretation that the cells expressing VEGF-R3 without nuclear Prox-1 were "transcriptionally inactive" since expression of Prox-1 is necessary to maintain the lymphatic identity of endothelial cells.5Johnson NC Dillard ME Baluk P McDonald DM Harvey NL Frase SL Oliver G Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity.Genes Dev. 2008; 22: 3282-3291Crossref PubMed Scopus (252) Google Scholar Therefore, our interpretation of those cells expressing VEGF-R3 without nuclear Prox-1 as LAM cells appears to be quite plausible. Furthermore, Seyama et al point out that LAM can be considered a "neoplastic process," and we believe the cancer literature contains biological evidence that further supports our findings and the plausibility of an important biological role for VEGF-R3 in LAM. First, cancer cells may demonstrate plasticity resulting in "vasculogenic mimicry."10Hendrix MJ Seftor EA Hess AR Seftor RE Vasculogenic mimicry and tumour-cell plasticity: lessons from melanoma.Nat Rev Cancer. 2003; 3: 411-421Crossref PubMed Scopus (705) Google Scholar In this setting, cancer cells are able to mimic the activities of endothelial cells, resulting in a process similar to neovascularization with the formation of a fluid-conducting, matrix-rich meshwork. Second, VEGFR-3 expression in non-endothelial cells such as corneal epithelium6Cursiefen C Chen L Saint-Geniez M Hamrah P Jin Y Rashid S Pytowski B Persaud K Wu Y Streilein JW Dana R Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.Proc Natl Acad Sci USA. 2006; 103: 11405-11410Crossref PubMed Scopus (226) Google Scholar and osteoblasts7Orlandini M Spreafico A Bardelli M Rocchigiani M Salameh A Nucciotti S Capperucci C Frediani B Oliviero S Vascular endothelial growth factor-D activates VEGFR-3 expressed in osteoblasts inducing their differentiation.J Biol Chem. 2006; 281: 17961-17967Crossref PubMed Scopus (73) Google Scholar has been implicated in cell differentiation. Third, VEGFR-3 expression has been implicated as a pathogenic factor during hepatocarcinogenesis8Lian Z Liu J Wu M Wang HY Arbuthnot P Kew M Feitelson MA Hepatitis Bx antigen up-regulates vascular endothelial growth factor receptor 3 in hepatocarcinogenesis.Hepatology. 2007; 45: 1390-1399Crossref PubMed Scopus (28) Google Scholar and in lung cancers,11Su JL Chen PS Chien MH Chen PB Chen YH Lai CC Hung MC Kuo ML Further evidence for expression and function of the VEGF-C/VEGFR-3 axis in cancer cells.Cancer Cell. 2008; 13: 57-60Abstract Full Text Full Text PDF Scopus (43) Google Scholar, 12Saintigny P Kambouchner M Ly M Gomes N Sainte-Catherine O Vassy R Czernichow S Letoumelin P Breau JL Bernaudin JF Kraemer M Vascular endothelial growth factor-C and its receptor VEGFR-3 in non-small-cell lung cancer: concurrent expression in cancer cells from primary tumour and metastatic lymph node.Lung Cancer. 2007; 58: 205-213Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar wherein VEGF-R3 contributes to cell proliferation and migration via autocrine signaling involving VEGF-C/ VEGFR-3. The second point of concern for Seyama et al addresses the nature of a LAM cell and the biological plausibility of in vitro models in a disease where the pathogenic cell is poorly characterized. This is a key quandary faced by all LAM researchers and has greatly hindered our collective understanding of LAM. As previously stated, we used an in vitro, physiologically relevant LAM cell model13Goncharova EA Goncharov DA Spaits M Noonan DJ Talovskaya E Eszterhas A Krymskaya VP Abnormal growth of smooth muscle-like cells in lymphangioleiomyomatosis: role for tumor suppressor TSC2.Am J Respir Cell Mol Biol. 2006; 34: 561-572Crossref PubMed Scopus (46) Google Scholar from human lung LAM nodules to more closely study LAM biology in a way that is not possible with immunohistochemistry alone. Seyama et al rightfully highlight the inherent limitations of such cell models; however, we are puzzled by how the authors can then use the data obtained from their own rudimentary in vitro human LAM lung cells3Kumasaka T Seyama K Mitani K Souma S Kashiwagi S Hebisawa A Sato T Kubo H Gomi K Shibuya K Fukuchi Y Suda K Lymphangiogenesis-mediated shedding of LAM cell clusters as a mechanism for dissemination in lymphangioleiomyomatosis.Am J Surg Pathol. 2005; 29: 1356-1366Crossref PubMed Scopus (70) Google Scholar to further question the validity of our findings. In fact, their short term cultures of LAM cell clusters showing co-immunostaining for VEGF-R3 and smooth muscle actin in a subpopulation of peripheral polygonal-shaped cells is not compelling, since morphologically all of the cells looked similar and were not two distinct cell populations. Compared with previously used cells models such as rat renal leiomyoma14Astrinidis A Cash TP Hunter DS Walker CL Chernoff J Henske EP Tuberin, the tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell adhesion and migration.Oncogene. 2002; 21: 8470-8476Crossref PubMed Scopus (114) Google Scholar cells and fibroblasts,15Zhe X Yang Y Jakkaraju S Schuger L Tissue inhibitor of metalloproteinase-3 downregulation in lymphangioleiomyomatosis: potential consequence of abnormal serum response factor expression.Am J Respir Cell Mol Biol. 2003; 28: 504-511Crossref PubMed Scopus (64) Google Scholar we believe our use of cells described in numerous high quality publications13Goncharova EA Goncharov DA Spaits M Noonan DJ Talovskaya E Eszterhas A Krymskaya VP Abnormal growth of smooth muscle-like cells in lymphangioleiomyomatosis: role for tumor suppressor TSC2.Am J Respir Cell Mol Biol. 2006; 34: 561-572Crossref PubMed Scopus (46) Google Scholar, 16Goncharova EA Goncharov DA Eszterhas A Hunter DS Glassberg MK Yeung RS Walker CL Noonan D Kwiatkowski DJ Chou MM Panettieri Jr, RA Krymskaya VP Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM).J Biol Chem. 2002; 277: 30958-30967Crossref PubMed Scopus (270) Google Scholar from different laboratories is a reasonable and biologically pertinent approach. We also believe that if data derived from non-human non-LAM tissue can be used to spearhead clinical human trials with sirolimus,17Bissler JJ McCormack FX Young LR Elwing JM Chuck G Leonard JM Schmithorst VJ Laor T Brody AS Bean J Salisbury S Franz DN Sirolimus for Angiomyolipoma in Tuberous Sclerosis Complex or Lymphangioleiomyomatosis.N Engl J Med. 2008; 358: 140-151Crossref PubMed Scopus (990) Google Scholar it seems reasonable that cell-based biological findings from our experiments might serve as important clues to find alternative therapeutic targets. Lastly, we respectfully disagree with Seyama et al's assertion that we interpreted our findings as proof of the extent or importance of LAM/VEGF-R3 functioning in vivo, but rather we clearly suggest that VEGF may play an important role and deserves further in vitro and in vivo study. The letter from Seyama et al appears overly concerned that their previous results are brought in to question by our findings. Rather we believe that our bodies of work are complimentary and have served to foster healthy debate in a field of research sorely lacking in an understanding of biological mechanism and treatment "breakthroughs." We also believe that further studies must focus on developing a more disease-relevant in vitro cell for detailed study of LAM biology so that we do not have to rely so heavily on immunohistochemistry findings and physiologically less relevant cell types and that real steps should be taken toward assessing the safety and efficacy of an anti-VEGF-R3 strategy for treatment of LAM.
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