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Laboratory models for the investigation of lymphangiomatosis
YMVRE-03470; No. of pages: 4; 4C: Microvascular Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
Laboratory models for the investigation of lymphangiomatosis Stanley G. Rockson ⁎,1 Stanford Center for Lymphatic and Venous Disorders, Stanford University School of Medicine, Stanford, CA 94305, USA
a r t i c l e
i n f o
Article history: Accepted 21 August 2014 Available online xxxx Keywords: Lymphangioma Lymphatic malformation Gorham–Stout disease Lymphangiectasia Wnt5A VEGF-C
a b s t r a c t Lymphangiomatosis is an uncommon proliferative disorder of the lymphatic vasculature whose etiology remains poorly understood. The lymphangiomatosis spectrum encompasses a remarkable heterogeneity in its potential presentation, including micro- and macrocystic isolated lymphatic malformations, thoracic and intraabdominal diffuse lymphangiomatosis, and osseous and soft-tissue presentations known as Gorham–Stout disease. Recent therapeutic advances are empirical in nature or, at best, inferential, reflecting the scanty availability of laboratory-based model systems for the mechanistic study of this disease. Several promising model systems are reviewed here. The laboratory investigation of lymphangiomatosis will likely continue to benefit from the remarkable growth of insights into the mechanisms of lymphangiogenesis and vascular development. © 2014 Elsevier Inc. All rights reserved.
Introduction Lymphangiomatosis is an uncommon proliferative disorder of the lymphatic vasculature, whose etiology remains poorly understood. This disease spectrum affects a patient population that is small in number, but the consequences of this pathology are potentially devastating (Rockson, 2011b). The disease can be considered a failure of normal lymphatic development (Gordon and Mortimer, 2011; Lee et al., 2005). Sites of involvement can include soft tissue, viscera, and bone (Gordon and Mortimer, 2011; Venkatramani et al., 2011). Visceral involvement carries a particularly poor prognosis. Furthermore, lymphangiomatosis can be limited to a defined organ-delimited structure (e.g., spleen, liver, and thoracic cavity) or can reflect a more generalized process (Blei, 2011).
The spectrum of human clinical lymphangiomatosis The lymphangiomatosis spectrum encompasses a remarkable heterogeneity in its potential presentation. These various features should be considered in attempts to recapitulate the human pathology in suitable animal models of disease. Isolated lymphatic malformations, also known as lymphangiomas, are low-flow vascular malformations (Perkins et al., 2010). Seventyfive percent of these lesions occur in the head and neck region. The clinical presentation is related to the size of the malformation and to the impact of the lesion on the structure and function of adjacent tissues ⁎ Stanford Center for Lymphatic and Venous Disorders, Stanford University School of Medicine, Stanford, California 94305. Fax: +1 650 725 1599. E-mail address: [email protected]. 1 Allan and Tina Neill Professor of Lymphatic Research and Medicine.
(Perkins et al., 2010). Thus, these lesions are benign but have the capacity to be life-threatening. These lesions are conventionally classified as either macro- or microcystic. Malformations with cysts larger than 2 cm in diameter are macrocystic, while those below this threshold are microcystic (Chen et al., 2009). The two entities differ significantly in clinical behavior (Lei et al., 2007; Perkins et al., 2007). Microcystic lesions are generally more difficult to treat, while macrocystic lesions are treatment responsive and can, in fact, spontaneously resolve (Perkins et al., 2008). Interestingly, there is no histological or immunohistological distinction between these two disease categories (Chen et al., 2009). Lymphatic endothelial cells within both subtypes of malformation appear histologically normal. Both microcystic and macrocystic LM tissues express podoplanin and smooth muscle actin in a similar pattern (Fig. 1). It is therefore conjectured that the pathological alteration in lymphatic malformation may not reside in the lymphatic endothelium but, rather, in an extracellular matrix or stromal component that compromises the fluid exchange function of lymphatic vessels (Chen et al., 2009). Diffuse pulmonary lymphangiomatosis (Tazelaar et al., 1993) has been variously described (Faul et al., 2000) as pulmonary lymphangiectasis (Kirchner et al., 1997); generalized lymphangiectasis (White et al., 1996), intrathoracic lymphangiomatosis (Swank et al., 1989), thoracic lymphangiomatosis (Margraf, 1996), and diffuse pulmonary angiomatosis (Canny et al., 1991). In an adult, the presentation may mimic that of lymphangioleiomyomatosis (Swank et al., 1989). Single or multiple lymphangiomas are located within the mediastinum, the chest wall, or adherent to the pleura. There may be diffuse infiltration of the mediastinal fat with anastamosing, pathologically dilated lymphatics (Faul et al., 2000). Diffuse pulmonary lymphangiomatosis involves both lungs. There are prominent spindle-shaped cells and collagen deposition surrounding the endothelially lined lumens that are
http://dx.doi.org/10.1016/j.mvr.2014.08.007 0026-2862/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Rockson, S.G., Laboratory models for the investigation of lymphangiomatosis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.08.007
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S.G. Rockson / Microvascular Research xxx (2014) xxx–xxx
Fig. 1. Cystic lymphangioma, demonstrating a strong immunoexpression for D2-40. Reprinted with permission from (Fukunaga, 2005).
oriented along the normal lymphatic routes within the lung (Tazelaar et al., 1993). Pleural effusion is common and surrounding lung parenchyma is preserved (Faul et al., 2000; Satria et al., 2011). In lymphangiomatosis of the abdomen, endothelial-lined cysts containing smooth muscle, small lymphatic spaces, abundant lymphoid tissue, and foamy macrophage infiltration are observed (Cutler et al., 2010). Immunohistochemical characterization of podoplanin expression with the D2-40 antibody is often helpful (Kalof and Cooper, 2009). Patients with bony involvement with lymphangiomatosis might be designated as affected by Gorham–Stout syndrome or disease (GSD). However, it is possible that the bony involvement in some of these patients is simply a manifestation of the diffuse process of lymphangiomatosis. It is likely that these two discrete presentations represent the clinical spectrum of the pathological process of lymphatic proliferation (Gordon and Mortimer, 2011). The etiology of Gorham's disease remains unknown. Immunohistochemical analysis with antibodies to D2-40 and LYVE-1 confirm that, while lymphatic vessels are not present in normal bone (Edwards et al., 2008), they can be detected in the soft tissues (Brodszki et al., 2011) and the bony cortex and medulla of GSD (Lala et al., 2013). GSD is considered to be a malformation and not a neoplasm (Dellinger et al., 2014). Proposed etiologies (Venkatramani et al., 2011) include trauma-induced vascular proliferation (Gorham and Stout, 1955), interleukin-6 (IL-6)-mediated osteoclast stimulation (Devlin et al., 1996), and VEGF-mediated pathological lymphangiogenesis (Dupond et al., 2010).
kinase inhibitors, and, in the case of bony involvement, biphosponates (Ozeki et al., 2007). Of late, there has been growing enthusiasm for newer molecular interventions that have the potential for improving the outcome of these often devastating developmental disorders. The putative role of vascular endothelial growth factor (VEGF) overexpression in lymphangiomatosis (Dupond et al., 2010) has led to an observed favorable response to bevacizumab-induced VEGF antagonism (Aman et al., 2012; Grunewald et al., 2010). In Gorham's disease, a subset of this condition predominated by osseous manifestations, the suggestion that increased osteoclastic activity is driven by interleukin-6 (IL-6) has led to the proposal of therapeutic administration of anti-IL-6 antibodies such as tocilizumab (Venkatramani et al., 2011). Most recently, through extrapolation from the experience with lymphangioleiomyomatosis (LAM) (Cai et al., 2014; McCormack et al. 2011), great promise surrounds the therapeutic potential of mTOR inhibition with rapamycin to limit morbidity in diffuse lymphangiomatosis (Reinglas et al., 2011; Venkatramani et al., 2011). In localized lesions, there is great promise for the use of sildenafil (Danial et al., 2014; Gandhi et al., 2013), a vasodilator that selectively inhibits cGMP-specific phosphodiesterase type 5, as there may be for propranolol, a sympatholytic non-selective betaadrenergic antagonist, in the diffuse form of the disease (Ozeki et al., 2011). While the application of either of these latter two agents in therapy is not based on prospective mechanistic insights, it can be surmised that their relative efficacy derives, within each form of the pathology, from the potential to alter flow through the malformation(s). Regrettably, most of these therapeutic advances are empirical in nature or, at best, inferential. This reflects the scanty availability of laboratory-based model systems for the mechanistic study of this disease (Rockson, 2011a). The available models can be readily summarized here. Existing laboratory models relevant to lymphangiomatosis Both in vitro and in vivo model systems have been described In vitro models
Clinical therapeutic observations
Three-dimensional lymphatic ring assay. This is a model that attempts to recapitulate in vivo findings within an in vitro model of lymphangiogenesis. The lymphatic ring assay recapitulates the different steps of cell sprouting from a pre-existing lymphatic vessel in a collagen environment (Bruyere et al., 2008). Thoracic ducts used for lymphatic ring cultures were collected from male and female C57BL/6 mice and 3-dimensional lymphatic ring cultures are undertaken. The ring-shaped explants embedded in rat tail interstitial collagen-I gel are cultured in medium at 37 °C under hypoxic conditions. In these cultures, LYVE-1positive endothelial sprouts first appear after 5 days. Confocal microscopy reveals that neovessel tips are composed of migrating cells that probe the surrounding matrix. Ultimately, these neovessels develop a visible lumen and degradation products of collagen are visible in intracellular vesicles, in inter- and extracellular spaces (Detry et al., 2011). While this model is not a model of lymphangioma growth per se, inasmuch as it recapitulates much of the structural and ultrastructural characteristics, it may represent a suitable model for the study of therapeutic inhibition of vascular growth. Further study of this model in both BALB/c and nonobese diabetic mice has led to the observation that these induced lesions express mRNA for Prox-1, VEGF-A, VEGF-C, VEGF-D, and VEGFR2 and VEGFR3 (Ji et al., 2010). This expression profile underscores the applicability of the model for the study of lymphatic pathology, and the ready availability of these relevant biomarkers extends its utility for future investigations.
Conventional non-surgical options for patients with lymphangiomatosis have been limited. In isolated cases or limited case series, some disease stabilization has been accomplished through treatment with pegylated (PEG) interferons, glucocorticoids, tyrosine
Endothelial cell tube formation in cultured murine lymphatic endothelial cells. The mammalian target of rapamycin (mTOR) has increasingly become a therapeutic target for the inhibition of lymphangiogenesis in cancer (Luo et al., 2012). Early successes for rapamycin administration
Please cite this article as: Rockson, S.G., Laboratory models for the investigation of lymphangiomatosis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.08.007
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have been reported in lymphatic malformations (Venkatramani et al., 2011). Thus, it is of interest that, in cultured murine LECs, rapamycin can suppress stimulated tube formation (Luo et al., 2012). Furthermore, rapamycin inhibits both proliferation and motility of LECs, as well as the protein expression of VEGFR3. In vivo models Acquired lymphatic endothelial hyperplasia. This murine model (Fig. 2) attempts to recapitulate the histological characteristics of the human lesion in lymphangiomatosis (Bruyere et al., 2010; Mancardi et al., 1999; Rockson, 2011a). The in vivo pathology is created in mice (Mancardi et al., 1999) or rats (Short et al., 2007) through sequential intraperitoneal injection of incomplete Freund's adjuvant (Bruyere et al., 2010; Detry et al., 2011; Mancardi et al., 1999). Within one month after the first injection, the lesions begin to appear on the abdominal surface of the diaphragm. Hematoxylin-eosin staining discloses the typical features of the human lesion and the lymphatic nature of the endothelial cells that line the cystic structures is confirmed through LYVE-1 immunohistochemistry (Detry et al., 2011). With transmission electron microscopy, there is evidence of lymphatic endothelial cell alignment into cords. There is a thin, irregular lumen and fragmented matrix materials can be seen in the lumen and within inter-endothelial junctions (Detry et al., 2011). Maldevelopment of dermal lymphatics in Wnt5a-knockout-mic. In a first attempt to comprehend the molecular etiology of human lymphangioma, Norgall et al. characterized the LECs derived from dermal lesions of two affected pediatric subjects (Norgall et al., 2007). Comparison of the isolated cells with those from normal dermis and with blood vascular endothelial was performed by microarray analysis. Lymphoendothelial markers were highly expressed but, in addition, there was a notable differential expression of wingless-type MMTV integration site family, member 5A (WNT5A). Recent studies suggest that Wnts may play an essential role in angiogenic processes (Cirone et al., 2008). Accordingly, based upon their human studies of differential gene expression, the authors explored the lymphatic vasculature of Wnt5Aknockout mouse embryos in comparison to heterozygous and wild type mice (Buttler et al., 2013). While the knockout mice have a significant reduction in the number of dermal lymphatic capillaries, the mean size of individual vessels and the number of LECs/vessel are greater. They conclude that Wnt5A-knockout mice display a sprouting defect and suggest that this gene serves as a regulator of normal lymphangiogenesis. Thus, further study of this family of genes may be fruitful in relationship to the disease process. An animal model of human microcystic lymphatic malformation. Isolated microcystic lymphatic malformations represent an element of the lymphangiomatosis spectrum (Rockson, 2010). Hou et al. have elaborated an interesting murine model within which to study the biology of
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these tissues (Hou et al., 2011). Fresh microcystic lymphatic malformations were harvested from human subjects and xenografted into immunologically naive nude mice (athymic nude-Foxn1nu) Nearly complete (81%) xenograft survival of the engrafted malformations was achieved in the mouse. Immunohistochemical analysis disclosed that 77% of the surviving xenografts were D2-40-positive, 69% were positive for human-specific nuclear antigen, 62% were positive for Ki-67. Inasmuch as the histological and immunohistochemical attributes of the human malformations were preserved; suggesting that this model may be useful for future mechanistic and therapeutic investigations. Pulmonary lymphangiectesia induced by murine developmental VEGF-C overexpression. In transgenic mice that have inducible VEGF-C overexpression within the period of e15.5 to postnatal day 14, overexpression produces respiratory distress, chylothorax, pulmonary lymphangiectesia, and failure to survive (Yao et al., 2014). The effect is both VEGFR-2- and VEGFR-3-dependent in neonates and is exquisitely dependent on the critical period of overexpression. The histopathology conforms strongly to the pattern observed in neonatal pulmonary lymphangiectasia. While lymphangiectesia is a form of dysregulated pulmonary lymphatic development distinct from lymphangiomatosis, there are certainly overlapping features (Fishman, 2013) that render this model interesting in the current discussion. Spheroid-based engineering of a human lymphatic vasculature in mice. By analogy to the previously described in vitro lymphatic ring model (Bruyere et al., 2008) as a platform to study the native steps of lymphatic vascular development, Alajati et al. have described a method in which endothelial cells are engrafted as spheroids into a matrix, thereby producing a complex three-dimensional network of human neovessels that develop in vivo in mice (Alajati et al., 2008). The grafted vasculature matures and is connected to the host murine circulation. The authors have demonstrated that this technique is useful for the specific study of lymphatic development: coimplantation of LECs with human fibroblasts resulted in the formation of more than 90% podoplanin-positive vessels. Of note, however, is the fact that this engineered neovasculature does not demonstrate functional integration into the host lymphatic circulation. Lymphangiomatosis is part of the lymphatic continuum Despite the continued absence of an ideal laboratory model for lymphangiomatosis, it is apparent that lymphatic maldevelopment is central to the pathogenesis of the disorder. Thus, it is plausible to imagine that laboratory investigation of lymphangiomatosis will be the beneficiary, directly or indirectly, of the remarkable insights into the mechanisms of lymphangiogenesis that continue to expand and to be translated into therapeutic approaches to lymphatic pathology (Zheng et al., 2014). Furthermore, substantial discoveries have been made that relate to
Fig. 2. Mouse lymphangiomata induced by intraperitoneal injection of incomplete Freund's adjuvant. (Left) Lymphangiomas are visible as white masses on the surface of the diaphragm (white arrowheads). (Middle) Hematoxylin-eosin stain of a histological section of diaphragm (black arrowheads delineate lymph vessels). (Right) Lymphatic vessels are decorated by specific immunostaining of LYVE-1. The arrow shows the process of fusion that leads to increased lumen size. Reproduced with permission from (Detry et al., 2011). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
Please cite this article as: Rockson, S.G., Laboratory models for the investigation of lymphangiomatosis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.08.007
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the genetics of lymphatic anomalies that encompass, among other pathologies, chylothorax, chylous ascites, and a variety of lymphatic malformations (Brouillard et al., 2014). At least eight such genes have been identified and, for most, there is a corresponding animal model (Brouillard et al., 2014). While none of these models directly reflects the human pathology of lymphangiomatosis, the biological overlap is substantial, suggesting that much can be learned from continued exploration of the identified pathways.
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Grunewald, T.G., et al., 2010. First report of effective and feasible treatment of multifocal lymphangiomatosis (Gorham–Stout) with bevacizumab in a child. Ann. Oncol. 21, 1733–1734. Hou, F., et al., 2011. A pilot in vivo model of human microcystic lymphatic malformations. Arch. Otolaryngol. Head Neck Surg. 137, 1280–1285. Ji, R.C., et al., 2010. Multiple expressions of lymphatic markers and morphological evolution of newly formed lymphatics in lymphangioma and lymph node lymphangiogenesis. Microvasc. Res. 80, 195–201. Kalof, A.N., Cooper, K., 2009. D2-40 immunohistochemistry—so far! Adv. Anat. Pathol. 16, 62–64. Kirchner, J., et al., 1997. Primary congenital pulmonary lymphangiectasia—a case report. Wien. Klin. Wochenschr. 109, 922–924. Lala, S., et al., 2013. Gorham-Stout disease and generalized lymphatic anomaly—clinical, radiologic, and histologic differentiation. Skelet. Radiol. 42, 917–924. Lee, B.B., et al., 2005. Current concepts in lymphatic malformation. Vasc Endovascular Surg. 39, 67–81. Lei, Z.M., et al., 2007. Surgery of lymphatic malformations in oral and cervicofacial regions in children. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 104, 338–344. Luo, Y., et al., 2012. Rapamycin inhibits lymphatic endothelial cell tube formation by downregulating vascular endothelial growth factor receptor 3 protein expression. Neoplasia 14, 228–237. Mancardi, S., et al., 1999. Lymphatic endothelial tumors induced by intraperitoneal injection of incomplete Freund's adjuvant. Exp. Cell Res. 246, 368–375. Margraf, L.R., 1996. Thoracic lymphangiomatosis. Pediatr Pathol Lab Med. 16, 155–160. McCormack, F.X., et al., 2011. Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N Engl J Med. 364, 1595–1606. Norgall, S., et al., 2007. Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas. BMC Cancer 7, 105. Ozeki, M., et al., 2007. Clinical improvement of diffuse lymphangiomatosis with pegylated interferon alfa-2b therapy: case report and review of the literature. Pediatr. Hematol. Oncol. 24, 513–524. Ozeki, M., et al., 2011. Propranolol for intractable diffuse lymphangiomatosis. N. Engl. J. Med. 364, 1380–1382. Perkins, J.A., et al., 2007. Clinical outcomes in lymphocytopenic lymphatic malformation patients. Lymphat. Res. Biol. 5, 169–174. Perkins, J.A., et al., 2008. Clinical and radiographic findings in children with spontaneous lymphatic malformation regression. Otolaryngol. Head Neck Surg. 138, 772–777. Perkins, J.A., et al., 2010. Lymphatic malformations: current cellular and clinical investigations. Otolaryngol. Head Neck Surg. 142, 789–794. Reinglas, J., et al., 2011. The successful management of diffuse lymphangiomatosis using sirolimus: a case report. Laryngoscope 121, 1851–1854. Rockson, S.G., 2010. Causes and consequences of lymphatic disease. Ann. N. Y. Acad. Sci. 1207 (Suppl. 1), E2–E6. Rockson, S.G., 2011a. Animal models for the mechanistic study of systemic lymphangiomatosis. Lymphat. Res. Biol. 9, 195–199. Rockson, S.G., 2011b. Lymphangiomatosis and Gorham's disease. Lymphat. Res. Biol. 9, 181. Satria, M.N., et al., 2011. Pulmonary lymphangiomatosis. Lymphat. Res. Biol. 9, 191–193. Short, R.F., et al., 2007. Site-specific induction of lymphatic malformations in a rat model for image-guided therapy. Pediatr. Radiol. 37, 530–534. Swank, D.W., et al., 1989. Intrathoracic lymphangiomatosis mimicking lymphangioleiomyomatosis in a young woman. Mayo Clin. Proc. 64, 1264–1268. Tazelaar, H.D., et al., 1993. Diffuse pulmonary lymphangiomatosis. Hum. Pathol. 24, 1313–1322. Venkatramani, R., et al., 2011. Gorham's disease and diffuse lymphangiomatosis in children and adolescents. Pediatr. Blood Cancer 56, 667–670. White, J.E., et al., 1996. Generalised lymphangiectasia: pulmonary presentation in an adult. Thorax 51, 767–768. Yao, L.C., et al., 2014. Pulmonary lymphangiectasia resulting from vascular endothelial growth factor-C overexpression during a critical period. Circ. Res. 114, 806–822. Zheng, W., et al., 2014. Lymphangiogenic factors, mechanisms, and applications. J. Clin. Invest. 124, 878–887.
Please cite this article as: Rockson, S.G., Laboratory models for the investigation of lymphangiomatosis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.08.007
Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
Laboratory models for the investigation of lymphangiomatosis Stanley G. Rockson ⁎,1 Stanford Center for Lymphatic and Venous Disorders, Stanford University School of Medicine, Stanford, CA 94305, USA
a r t i c l e
i n f o
Article history: Accepted 21 August 2014 Available online xxxx Keywords: Lymphangioma Lymphatic malformation Gorham–Stout disease Lymphangiectasia Wnt5A VEGF-C
a b s t r a c t Lymphangiomatosis is an uncommon proliferative disorder of the lymphatic vasculature whose etiology remains poorly understood. The lymphangiomatosis spectrum encompasses a remarkable heterogeneity in its potential presentation, including micro- and macrocystic isolated lymphatic malformations, thoracic and intraabdominal diffuse lymphangiomatosis, and osseous and soft-tissue presentations known as Gorham–Stout disease. Recent therapeutic advances are empirical in nature or, at best, inferential, reflecting the scanty availability of laboratory-based model systems for the mechanistic study of this disease. Several promising model systems are reviewed here. The laboratory investigation of lymphangiomatosis will likely continue to benefit from the remarkable growth of insights into the mechanisms of lymphangiogenesis and vascular development. © 2014 Elsevier Inc. All rights reserved.
Introduction Lymphangiomatosis is an uncommon proliferative disorder of the lymphatic vasculature, whose etiology remains poorly understood. This disease spectrum affects a patient population that is small in number, but the consequences of this pathology are potentially devastating (Rockson, 2011b). The disease can be considered a failure of normal lymphatic development (Gordon and Mortimer, 2011; Lee et al., 2005). Sites of involvement can include soft tissue, viscera, and bone (Gordon and Mortimer, 2011; Venkatramani et al., 2011). Visceral involvement carries a particularly poor prognosis. Furthermore, lymphangiomatosis can be limited to a defined organ-delimited structure (e.g., spleen, liver, and thoracic cavity) or can reflect a more generalized process (Blei, 2011).
The spectrum of human clinical lymphangiomatosis The lymphangiomatosis spectrum encompasses a remarkable heterogeneity in its potential presentation. These various features should be considered in attempts to recapitulate the human pathology in suitable animal models of disease. Isolated lymphatic malformations, also known as lymphangiomas, are low-flow vascular malformations (Perkins et al., 2010). Seventyfive percent of these lesions occur in the head and neck region. The clinical presentation is related to the size of the malformation and to the impact of the lesion on the structure and function of adjacent tissues ⁎ Stanford Center for Lymphatic and Venous Disorders, Stanford University School of Medicine, Stanford, California 94305. Fax: +1 650 725 1599. E-mail address: [email protected]. 1 Allan and Tina Neill Professor of Lymphatic Research and Medicine.
(Perkins et al., 2010). Thus, these lesions are benign but have the capacity to be life-threatening. These lesions are conventionally classified as either macro- or microcystic. Malformations with cysts larger than 2 cm in diameter are macrocystic, while those below this threshold are microcystic (Chen et al., 2009). The two entities differ significantly in clinical behavior (Lei et al., 2007; Perkins et al., 2007). Microcystic lesions are generally more difficult to treat, while macrocystic lesions are treatment responsive and can, in fact, spontaneously resolve (Perkins et al., 2008). Interestingly, there is no histological or immunohistological distinction between these two disease categories (Chen et al., 2009). Lymphatic endothelial cells within both subtypes of malformation appear histologically normal. Both microcystic and macrocystic LM tissues express podoplanin and smooth muscle actin in a similar pattern (Fig. 1). It is therefore conjectured that the pathological alteration in lymphatic malformation may not reside in the lymphatic endothelium but, rather, in an extracellular matrix or stromal component that compromises the fluid exchange function of lymphatic vessels (Chen et al., 2009). Diffuse pulmonary lymphangiomatosis (Tazelaar et al., 1993) has been variously described (Faul et al., 2000) as pulmonary lymphangiectasis (Kirchner et al., 1997); generalized lymphangiectasis (White et al., 1996), intrathoracic lymphangiomatosis (Swank et al., 1989), thoracic lymphangiomatosis (Margraf, 1996), and diffuse pulmonary angiomatosis (Canny et al., 1991). In an adult, the presentation may mimic that of lymphangioleiomyomatosis (Swank et al., 1989). Single or multiple lymphangiomas are located within the mediastinum, the chest wall, or adherent to the pleura. There may be diffuse infiltration of the mediastinal fat with anastamosing, pathologically dilated lymphatics (Faul et al., 2000). Diffuse pulmonary lymphangiomatosis involves both lungs. There are prominent spindle-shaped cells and collagen deposition surrounding the endothelially lined lumens that are
http://dx.doi.org/10.1016/j.mvr.2014.08.007 0026-2862/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Rockson, S.G., Laboratory models for the investigation of lymphangiomatosis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.08.007
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S.G. Rockson / Microvascular Research xxx (2014) xxx–xxx
Fig. 1. Cystic lymphangioma, demonstrating a strong immunoexpression for D2-40. Reprinted with permission from (Fukunaga, 2005).
oriented along the normal lymphatic routes within the lung (Tazelaar et al., 1993). Pleural effusion is common and surrounding lung parenchyma is preserved (Faul et al., 2000; Satria et al., 2011). In lymphangiomatosis of the abdomen, endothelial-lined cysts containing smooth muscle, small lymphatic spaces, abundant lymphoid tissue, and foamy macrophage infiltration are observed (Cutler et al., 2010). Immunohistochemical characterization of podoplanin expression with the D2-40 antibody is often helpful (Kalof and Cooper, 2009). Patients with bony involvement with lymphangiomatosis might be designated as affected by Gorham–Stout syndrome or disease (GSD). However, it is possible that the bony involvement in some of these patients is simply a manifestation of the diffuse process of lymphangiomatosis. It is likely that these two discrete presentations represent the clinical spectrum of the pathological process of lymphatic proliferation (Gordon and Mortimer, 2011). The etiology of Gorham's disease remains unknown. Immunohistochemical analysis with antibodies to D2-40 and LYVE-1 confirm that, while lymphatic vessels are not present in normal bone (Edwards et al., 2008), they can be detected in the soft tissues (Brodszki et al., 2011) and the bony cortex and medulla of GSD (Lala et al., 2013). GSD is considered to be a malformation and not a neoplasm (Dellinger et al., 2014). Proposed etiologies (Venkatramani et al., 2011) include trauma-induced vascular proliferation (Gorham and Stout, 1955), interleukin-6 (IL-6)-mediated osteoclast stimulation (Devlin et al., 1996), and VEGF-mediated pathological lymphangiogenesis (Dupond et al., 2010).
kinase inhibitors, and, in the case of bony involvement, biphosponates (Ozeki et al., 2007). Of late, there has been growing enthusiasm for newer molecular interventions that have the potential for improving the outcome of these often devastating developmental disorders. The putative role of vascular endothelial growth factor (VEGF) overexpression in lymphangiomatosis (Dupond et al., 2010) has led to an observed favorable response to bevacizumab-induced VEGF antagonism (Aman et al., 2012; Grunewald et al., 2010). In Gorham's disease, a subset of this condition predominated by osseous manifestations, the suggestion that increased osteoclastic activity is driven by interleukin-6 (IL-6) has led to the proposal of therapeutic administration of anti-IL-6 antibodies such as tocilizumab (Venkatramani et al., 2011). Most recently, through extrapolation from the experience with lymphangioleiomyomatosis (LAM) (Cai et al., 2014; McCormack et al. 2011), great promise surrounds the therapeutic potential of mTOR inhibition with rapamycin to limit morbidity in diffuse lymphangiomatosis (Reinglas et al., 2011; Venkatramani et al., 2011). In localized lesions, there is great promise for the use of sildenafil (Danial et al., 2014; Gandhi et al., 2013), a vasodilator that selectively inhibits cGMP-specific phosphodiesterase type 5, as there may be for propranolol, a sympatholytic non-selective betaadrenergic antagonist, in the diffuse form of the disease (Ozeki et al., 2011). While the application of either of these latter two agents in therapy is not based on prospective mechanistic insights, it can be surmised that their relative efficacy derives, within each form of the pathology, from the potential to alter flow through the malformation(s). Regrettably, most of these therapeutic advances are empirical in nature or, at best, inferential. This reflects the scanty availability of laboratory-based model systems for the mechanistic study of this disease (Rockson, 2011a). The available models can be readily summarized here. Existing laboratory models relevant to lymphangiomatosis Both in vitro and in vivo model systems have been described In vitro models
Clinical therapeutic observations
Three-dimensional lymphatic ring assay. This is a model that attempts to recapitulate in vivo findings within an in vitro model of lymphangiogenesis. The lymphatic ring assay recapitulates the different steps of cell sprouting from a pre-existing lymphatic vessel in a collagen environment (Bruyere et al., 2008). Thoracic ducts used for lymphatic ring cultures were collected from male and female C57BL/6 mice and 3-dimensional lymphatic ring cultures are undertaken. The ring-shaped explants embedded in rat tail interstitial collagen-I gel are cultured in medium at 37 °C under hypoxic conditions. In these cultures, LYVE-1positive endothelial sprouts first appear after 5 days. Confocal microscopy reveals that neovessel tips are composed of migrating cells that probe the surrounding matrix. Ultimately, these neovessels develop a visible lumen and degradation products of collagen are visible in intracellular vesicles, in inter- and extracellular spaces (Detry et al., 2011). While this model is not a model of lymphangioma growth per se, inasmuch as it recapitulates much of the structural and ultrastructural characteristics, it may represent a suitable model for the study of therapeutic inhibition of vascular growth. Further study of this model in both BALB/c and nonobese diabetic mice has led to the observation that these induced lesions express mRNA for Prox-1, VEGF-A, VEGF-C, VEGF-D, and VEGFR2 and VEGFR3 (Ji et al., 2010). This expression profile underscores the applicability of the model for the study of lymphatic pathology, and the ready availability of these relevant biomarkers extends its utility for future investigations.
Conventional non-surgical options for patients with lymphangiomatosis have been limited. In isolated cases or limited case series, some disease stabilization has been accomplished through treatment with pegylated (PEG) interferons, glucocorticoids, tyrosine
Endothelial cell tube formation in cultured murine lymphatic endothelial cells. The mammalian target of rapamycin (mTOR) has increasingly become a therapeutic target for the inhibition of lymphangiogenesis in cancer (Luo et al., 2012). Early successes for rapamycin administration
Please cite this article as: Rockson, S.G., Laboratory models for the investigation of lymphangiomatosis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.08.007
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have been reported in lymphatic malformations (Venkatramani et al., 2011). Thus, it is of interest that, in cultured murine LECs, rapamycin can suppress stimulated tube formation (Luo et al., 2012). Furthermore, rapamycin inhibits both proliferation and motility of LECs, as well as the protein expression of VEGFR3. In vivo models Acquired lymphatic endothelial hyperplasia. This murine model (Fig. 2) attempts to recapitulate the histological characteristics of the human lesion in lymphangiomatosis (Bruyere et al., 2010; Mancardi et al., 1999; Rockson, 2011a). The in vivo pathology is created in mice (Mancardi et al., 1999) or rats (Short et al., 2007) through sequential intraperitoneal injection of incomplete Freund's adjuvant (Bruyere et al., 2010; Detry et al., 2011; Mancardi et al., 1999). Within one month after the first injection, the lesions begin to appear on the abdominal surface of the diaphragm. Hematoxylin-eosin staining discloses the typical features of the human lesion and the lymphatic nature of the endothelial cells that line the cystic structures is confirmed through LYVE-1 immunohistochemistry (Detry et al., 2011). With transmission electron microscopy, there is evidence of lymphatic endothelial cell alignment into cords. There is a thin, irregular lumen and fragmented matrix materials can be seen in the lumen and within inter-endothelial junctions (Detry et al., 2011). Maldevelopment of dermal lymphatics in Wnt5a-knockout-mic. In a first attempt to comprehend the molecular etiology of human lymphangioma, Norgall et al. characterized the LECs derived from dermal lesions of two affected pediatric subjects (Norgall et al., 2007). Comparison of the isolated cells with those from normal dermis and with blood vascular endothelial was performed by microarray analysis. Lymphoendothelial markers were highly expressed but, in addition, there was a notable differential expression of wingless-type MMTV integration site family, member 5A (WNT5A). Recent studies suggest that Wnts may play an essential role in angiogenic processes (Cirone et al., 2008). Accordingly, based upon their human studies of differential gene expression, the authors explored the lymphatic vasculature of Wnt5Aknockout mouse embryos in comparison to heterozygous and wild type mice (Buttler et al., 2013). While the knockout mice have a significant reduction in the number of dermal lymphatic capillaries, the mean size of individual vessels and the number of LECs/vessel are greater. They conclude that Wnt5A-knockout mice display a sprouting defect and suggest that this gene serves as a regulator of normal lymphangiogenesis. Thus, further study of this family of genes may be fruitful in relationship to the disease process. An animal model of human microcystic lymphatic malformation. Isolated microcystic lymphatic malformations represent an element of the lymphangiomatosis spectrum (Rockson, 2010). Hou et al. have elaborated an interesting murine model within which to study the biology of
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these tissues (Hou et al., 2011). Fresh microcystic lymphatic malformations were harvested from human subjects and xenografted into immunologically naive nude mice (athymic nude-Foxn1nu) Nearly complete (81%) xenograft survival of the engrafted malformations was achieved in the mouse. Immunohistochemical analysis disclosed that 77% of the surviving xenografts were D2-40-positive, 69% were positive for human-specific nuclear antigen, 62% were positive for Ki-67. Inasmuch as the histological and immunohistochemical attributes of the human malformations were preserved; suggesting that this model may be useful for future mechanistic and therapeutic investigations. Pulmonary lymphangiectesia induced by murine developmental VEGF-C overexpression. In transgenic mice that have inducible VEGF-C overexpression within the period of e15.5 to postnatal day 14, overexpression produces respiratory distress, chylothorax, pulmonary lymphangiectesia, and failure to survive (Yao et al., 2014). The effect is both VEGFR-2- and VEGFR-3-dependent in neonates and is exquisitely dependent on the critical period of overexpression. The histopathology conforms strongly to the pattern observed in neonatal pulmonary lymphangiectasia. While lymphangiectesia is a form of dysregulated pulmonary lymphatic development distinct from lymphangiomatosis, there are certainly overlapping features (Fishman, 2013) that render this model interesting in the current discussion. Spheroid-based engineering of a human lymphatic vasculature in mice. By analogy to the previously described in vitro lymphatic ring model (Bruyere et al., 2008) as a platform to study the native steps of lymphatic vascular development, Alajati et al. have described a method in which endothelial cells are engrafted as spheroids into a matrix, thereby producing a complex three-dimensional network of human neovessels that develop in vivo in mice (Alajati et al., 2008). The grafted vasculature matures and is connected to the host murine circulation. The authors have demonstrated that this technique is useful for the specific study of lymphatic development: coimplantation of LECs with human fibroblasts resulted in the formation of more than 90% podoplanin-positive vessels. Of note, however, is the fact that this engineered neovasculature does not demonstrate functional integration into the host lymphatic circulation. Lymphangiomatosis is part of the lymphatic continuum Despite the continued absence of an ideal laboratory model for lymphangiomatosis, it is apparent that lymphatic maldevelopment is central to the pathogenesis of the disorder. Thus, it is plausible to imagine that laboratory investigation of lymphangiomatosis will be the beneficiary, directly or indirectly, of the remarkable insights into the mechanisms of lymphangiogenesis that continue to expand and to be translated into therapeutic approaches to lymphatic pathology (Zheng et al., 2014). Furthermore, substantial discoveries have been made that relate to
Fig. 2. Mouse lymphangiomata induced by intraperitoneal injection of incomplete Freund's adjuvant. (Left) Lymphangiomas are visible as white masses on the surface of the diaphragm (white arrowheads). (Middle) Hematoxylin-eosin stain of a histological section of diaphragm (black arrowheads delineate lymph vessels). (Right) Lymphatic vessels are decorated by specific immunostaining of LYVE-1. The arrow shows the process of fusion that leads to increased lumen size. Reproduced with permission from (Detry et al., 2011). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
Please cite this article as: Rockson, S.G., Laboratory models for the investigation of lymphangiomatosis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.08.007
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the genetics of lymphatic anomalies that encompass, among other pathologies, chylothorax, chylous ascites, and a variety of lymphatic malformations (Brouillard et al., 2014). At least eight such genes have been identified and, for most, there is a corresponding animal model (Brouillard et al., 2014). While none of these models directly reflects the human pathology of lymphangiomatosis, the biological overlap is substantial, suggesting that much can be learned from continued exploration of the identified pathways.
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Please cite this article as: Rockson, S.G., Laboratory models for the investigation of lymphangiomatosis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.08.007