BJD
British Journal of Dermatology
C U T A N E O U S B I OL O G Y
Phenotypic modulation of smooth muscle cells in lymphoedema F. Ogata,1,2 K. Fujiu,1,3 I. Koshima,2 R. Nagai4 and I. Manabe1 Departments of 1Cardiovascular Medicine, 2Plastic, Reconstructive and Aesthetic Surgery and 3Translational Systems Biology and Medicine Initiative, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan 4 Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi-ken 329-0498, Japan
Summary Correspondence Ichiro Manabe. E-mail: [email protected]
Accepted for publication 11 October 2014
Funding sources This study was supported by the ‘Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)’, initiated by the Council for Science and Technology Policy (to R.N.); Grants-in-Aid for Scientific Research (S) and (B) from the Japanese Society for the Promotion Science (to R.N. and I.M.); a grant for Translational Systems Biology and Medicine Initiative (to R.N.) from the Japanese Science and Technology Agency; a Grant-in-Aid for Scientific Research on Innovative Areas ‘Homeostatic Inflammation’ (to I.M.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and research grants from the Japan Science and Technology Institute, Sumitomo Foundation, Takeda Science Foundation, SENSHIN Medical Research Foundation, and Mochida Memorial Foundation for Medical and Pharmaceutical Research (to I.M.).
Background Lymphoedema is a debilitating progressive condition that is frequently observed following cancer surgery and severely restricts quality of life. Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood. Smooth muscle cells (SMCs) play pivotal roles in the pathogenesis of various vascular diseases, including atherosclerosis. Objectives We analysed SMCs in lymphatic vessels from the lymphoedematous legs of 29 patients. Methods Expression of smooth muscle a-actin (SMaA) and smooth muscle myosin heavy chain (SM-MHC) isoforms SM1 and SM2 was investigated using immunohistochemistry. Results Compared with normal lymphatic vessels, all affected lymphatic vessels in chronic lymphoedema showed marked wall thickening. In addition to increases in the numbers of rows of SMaA+ SM1+ SMCs in the tunica media, SMCs were also observed in the subendothelial region (tunica intima). While most intimal and medial cells were positive for SMaA and SM1, staining for SM1 and particularly SM2, a marker of mature SMCs, progressively declined in lymphatic vessels in increasingly severe lymphoedema lesions. Consequently, the SM1+ and SM2+ cell fractions were significantly reduced in the tunica media and intima of lymphatic vessels. Conclusions These observations indicate that the lymphatic tunica media and tunica intima consist mainly of phenotypically modulated SMCs, and that SMCs play a key role in the development of lymphoedema.
Conflicts of interest None declared. DOI 10.1111/bjd.13482
What’s already known about this topic?
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Lymphoedema is a debilitating progressive condition that is frequently observed following cancer surgery and severely restricts quality of life. Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood.
What does this study add?
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1286
Lymphatic smooth muscle cells play a key role in the development of lymphoedema.
British Journal of Dermatology (2015) 172, pp1286–1293
© 2014 British Association of Dermatologists
Phenotypic modulation of SMCs in lymphoedema, F. Ogata et al. 1287
Lymphoedema is a debilitating progressive condition that is frequently observed after cancer surgery and/or irradiation, trauma and infectious diseases.1 Patients with even mild lymphoedema suffer disabilities such as conspicuous enlargement of one or more extremities, recurrent cellulitis and lymphorrhea, which adversely affect their everyday activity. Once these symptoms occur, it is difficult to cure them completely, even when treated at an early stage; and once the disease reaches the chronic stage, it is difficult to even mitigate the symptoms. In addition, 007–045% of patients with long-standing chronic lymphoedema develop Stewart–Treves syndrome, a rare deadly cutaneous angiosarcoma.2,3 The incidences of lymphoedema secondary to breast and gynaecological cancers are reported to be between 12% and 47%.4–6 Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood, and effective therapeutic and preventive strategies have not been established.7 Previous studies have shown that lymphoedema is associated with various pathological changes, including the accumulation of lipids in the tissue and infiltration of the tissue by inflammatory cells.8,9 Also seen are various degenerative changes to the walls of the lymphatic vessels; these include endothelial cell hyperplasia, thickening of the subendothelial collagen layer and reduction of the number of muscle fibres.3,10–12 The processes underlying the observed pathological changes within the lymphatic vessel wall are not clear. However, we have been using a surgical procedure, lymphaticovenous anastomosis, to treat patients with severe lymphoedema who respond poorly to conservative therapy,13,14 and during that operation we always notice sclerosis of the lymphatic vessels. We therefore hypothesize that the ‘lymphaticosclerosis’ that occurs in lymphoedema is analogous to arteriosclerosis, in which smooth muscle cells (SMCs) play a central role. Smooth muscle cells are required for the rhythmic contraction of lymphangions that provides the main propelling forces for lymph flow. The abnormal lymphatic contractions and flow seen in lymphoedema suggest SMC dysfunction and/or obstruction of the lymphatic vessel lumen,15 but little is known about the changes in SMCs in the affected lymphatic vessels in lymphoedema.16,17 In mature arteries, the primary function of SMCs is contraction, which is required to maintain vessel wall tone and integrity.18 These fully differentiated cells proliferate at extremely low rates and produce only small amounts of extracellular matrix (ECM). However, during development, SMCs play a key role in matrix deposition and vessel morphogenesis. Moreover, even fully differentiated SMCs remain highly plastic, enabling them to modify their phenotype in response to environmental cues. For example, intimal SMCs within atherosclerotic lesions exhibit phenotypes that clearly differ from those of mature medial cells. Such changes in phenotype are collectively referred to as ‘phenotypic modulation’. The phenotype of mature SMCs is referred to as ‘contractile’; that of intimal SMCs as ‘synthetic’. Synthetic SMCs play a central role in vascular pathology.19 A number of © 2014 British Association of Dermatologists
markers that report the phenotypes of SMCs have been identified. Smooth muscle a-actin (SMaA) has been used extensively as a marker for SMCs, although it is actually not a definitive lineage marker and is known to be expressed in a wide variety of non-SMC types, including myofibroblasts.20 Expression of smooth muscle myosin heavy chain (SM-MHC) is more tightly restricted to SMCs. The SM1 isoform of SM-MHC is widely expressed in SMCs, while the SM2 isoform is expressed in differentiated SMCs. In phenotypically modulated SMC in arterial disease, expression of these SMC differentiation markers is downregulated.21 In the present study we analysed the involvement of lymphatic SMCs in lymphoedema. We found that the walls of lymphatic vessels were thickened, mainly due to hyperplasia of SMCs exhibiting phenotypic modulation. These changes in SMCs appear to underlie the obstruction and dysfunction of lymphatic vessels seen in lymphoedema.
Methods Pathological samples We collected 45 lymphatic vessels from 29 randomly chosen patients with lymphoedema who were refractory to conventional conservative therapy and underwent lymphaticovenous anastomosis between 2007 and 2011 (25 women and four men; 29–86 years of age, mean 583 years). All of these patients had developed secondary leg lymphoedema after surgery: 11 had been treated for uterine cervical cancer, nine for uterine body cancer, three for ovarian cancer, two for hernias, two for ureteral cancer, one for bladder cancer and one for Paget disease. The patterns of oedema varied widely with respect to the affected area, the onset of symptoms and progression. Eighteen of the 29 patients first noticed swelling of the entire leg, while eight noticed swelling of the entire thigh, and three noticed swelling of the lower thigh. In addition, eight of the patients suffered from bilateral leg lymphoedema. In two of these patients, the lymphoedema appeared on both sides at the same time; in the others the lymphoedema broadened to affect both sides over periods ranging from 2 months to 6 years (mean 277 months) after the appearance of lymphoedema on one side. The mean latent duration (from surgical treatment to the onset of oedema) was 415 months. The mean duration of oedema (from onset to start of our treatment) was 440 months. Prior to surgery, lymphangiography was performed to identify lymphatic vessels and to determine the sites for skin incision. After skin incision, lymphatic vessels were identified using a surgical microscope. The lymphatic vessels examined for this study were collected from the foot or ankle. Three lymphatic vessels were obtained from normal legs (an ankle and dorsal side of the foot in two persons) as normal controls. These patients had no symptoms of lymphoedema and were operated on for cervical cancer, after which they underwent prophylactic lymphaticovenous anastomosis in our hospital. British Journal of Dermatology (2015) 172, pp1286–1293
1288 Phenotypic modulation of SMCs in lymphoedema, F. Ogata et al.
Written informed consent was obtained from each subject after a full explanation of the study, which was approved by the Ethics Committee of the University of Tokyo Hospital. Clinical and pathological grading of lymphoedema The clinical severity of lymphoedema was graded according to International Society of Lymphology (ISL) staging (Table S1; see Supporting Information).22 Because the clinical manifestations of lymphoedema (e.g. skin changes and oedema) are often milder in the feet and ankles than in the thighs (Supplementary Fig. S1), we also used ISL staging to grade the severity of local clinical manifestations at the sites of sample collection, based on the skin changes and the level of oedema. We graded the macroscopic pathological changes at the sites of sample collection into three groups. The skin of grade 1 lesions was soft, and subcutaneous tissue was not fibrotic. The subcutaneous collector lymphatic vessel was transparent, the vessel wall was pliant, and lymph within the vessel was easily detectable when the proximal portion of the vessel was closed using forceps. The skin of grade 2 lesions was harder than unaffected skin. While the subcutaneous tissue was apparently fibrotic, the subcutaneous fibrotic layers could be easily cut with a forceps, and the subcutaneous lymphatic vessel could be separated from the fibrotic subcutaneous tissue. The lymphatic vessel was less translucent than grade 1 vessels, but the lumen was more visible than in grade 1 vessels because the vessel wall was stiff and not elastic. Lymph could be identified. The skin of grade 3 lesions was thickened and verrucous with lymphorrhea and/or ulcer formation. The subcutaneous tissue was markedly fibrotic, and firm white fibrotic septa had formed. The lymphatic vessel was opaque and white, and the lumen was invisible in most cases. Although these severely affected lymphatic vessels may resemble peripheral nerves, they lack the transverse stripe pattern of myelin sheaths (Fig. 1g–i). Histological and immunohistochemical analysis Paraffin-embedded sections (5-lm thick) were deparaffinized and blocked with 2% bovine serum albumin. To evaluate histopathological changes and tissue fibrosis, elastica van Gieson and Masson’s trichrome staining were performed. For immunohistochemical staining, anti-SM1 antibody (Kyowa Medex, Shizuoka, Japan), anti-SM2 antibody (Yamasa, Tokyo, Japan), anti-SMaA antibody (Sigma-Aldrich, St Louis, MO, U.S.A.), anti-CD31 antibody (BD Biosciences, San Diego, CA, U.S.A.) and anticollagen type 1 antibody (Abcam, Cambridge, U.K.) were used with the avidin–biotin complex technique and Vector Red substrate (Vector Laboratories, Burlingame, CA, U.S.A.). Sections were counterstained with haematoxylin. For each section, the area of immunoreactive lymphatic SMCs and collagen type I in the lymphatic vessels was quantified by imaging software (WinROOF, Mitani, Tokyo, Japan). British Journal of Dermatology (2015) 172, pp1286–1293
Transmission electron microscopy Samples were fixed with 25% glutaraldehyde in 01 mol L 1 cacodylate buffer (pH 74) for 1 h at 4 °C and then postfixed with 1% osmium tetroxide in cacodylate buffer (pH 74) for 1 h at 4 °C. After staining with 1% aqueous uranyl acetate solution for 10 min at room temperature, the samples were dehydrated in a graded series of ethanol solutions. For transmission electron microscopy (TEM), the blocks were embedded in Epon after dehydration. Ultrathin sections were cut at 50 nm with a diamond knife, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (H-7000; Hitachi, Tokyo, Japan) at an acceleration voltage of 75 kV. Statistical analysis Data are shown as means SEM. Differences among multiple groups were analysed using one-way ANOVA followed by posthoc Tukey–Kramer tests. Values of P < 005 were considered significant.
Results Pathological grading of lymphoedema tissues Lymphatic samples were collected from the dorsal area of the foot and the ankle of patients who underwent lymphaticovenous anastomosis. We graded the macroscopic pathology of the sites of sample collection into three groups based on changes in the skin, subcutaneous tissue and lymphatic vessels (Fig. 1). The collector lymphatic vessels in grade 1 samples were translucent and pliant. The skin in the region from which the tissue was collected was soft, and the subcutaneous tissue was not fibrotic. The lymphatic vessels in grade 2 samples were less translucent than those in grade 1. The skin was clearly harder than in unaffected regions, and the subcutaneous tissue was moderately fibrotic but was soft and easily cut with a forceps. The lymphatic vessels in grade 3 samples were white and opaque. The vessels were stiff, and the lumens were mostly invisible. The skin was clearly thickened and the subcutaneous tissues were markedly fibrotic. For ISL staging, the clinical severity of lymphoedema is graded based on oedematous and trophic changes in the affected limb (Supplementary Table S1).22 However, the severity of skin changes and oedema at the site of sample collection may differ from the clinical staging of the entire leg. In fact, oedema and skin changes were often milder in the feet and ankles than in the thighs. For that reason, we graded the local manifestation of lymphoedema at the sites of sample collection based on the state of the skin and oedema, as defined in the ISL staging. We found that the macroscopic pathological grades were well correlated with the stages of local clinical manifestation of lymphoedema (Fig. S1; see Supporting Information), which suggests the progression of pathology from grade 1 to grade 3. © 2014 British Association of Dermatologists
Phenotypic modulation of SMCs in lymphoedema, F. Ogata et al. 1289
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Lymphatic smooth muscle cells in lymphoedema Like the walls of blood vessels, the walls of normal collector lymphatic vessels in the legs consist of three layers. The tunica intima is comprised of CD31+ endothelial cells (Fig. 2). Unlike arterial walls, normal collector lymphatic vessels do not have a well-defined internal elastic lamina. The tunica media contains one to three rows of SMCs that are positive for SMaA and SM1 and SM2 SM-MHC (Fig. 2). Compared with normal lymphatic vessels, all lymphatic vessels from patients with chronic lymphoedema showed marked wall thickening (Fig. 3a). Most of the medial cells were positive for both SMaA and SM1, indicating these cells were SMCs (Fig. 2). The medial layer was thickened and consisted of 3– 10 or more rows of SMCs. More strikingly, SMaA+ SM1+ SMCs were also observed in the subendothelial region. The subendothelial SM layer was particularly thick in greatly obstructed lymphatic vessels, and between the medial and subendothelial SM layers there was a fibrous intermediate region that contained far fewer cells than the adjacent SMC-rich regions. Within this region, most of the cells were negative for SMC markers, suggesting they were either not © 2014 British Association of Dermatologists
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Fig 1. Gross pathological changes in lymphoedema. Lymphoedema lesions were graded into three groups based on the macroscopic pathological changes. Shown are representative lesions. The sites of surgery and sample collection are marked with bars in panels (a), (d) and (g). The photographs shown in (b), (e) and (h) were taken during lymphaticovenous anastomosis surgery, when the affected collector lymphatic vessels were identified. Panels (c), (f) and (i) provide schematic representations of the gross appearance of the images in (b), (e) and (h). (a–c) A representative grade 1 lesion. Although the patient suffered from clinical stage 2 lymphoedema in the affected (left) thigh, the tissue sample was collected from the foot, which showed only mild oedema and minimal skin changes that were considered to indicate local stage 1 lymphoedema. Note that the blue nylon suture is clearly visible behind the lymphatic vessel (b). (d–f) A representative grade 2 lesion. The patient suffered from stage 2 lymphoedema in the entire affected (left) leg, including the foot. Note that the nylon suture behind the lymphatic vessel is barely visible (e). (g–i) A representative grade 3 lesion. The affected leg exhibited lymphostatic elephantiasis in which pitting was absent. The lumen of the affected lymphatic vessel (arrow heads) was obstructed. The areas shown in grey in panel (i) indicate firm white septa. Scale bars, 1 mm (b, e, h).
(b)
SMCs or they were highly modulated SMCs. As mentioned, the normal lymphatic wall does not have a well-defined internal elastic lamina but, based on the clear distinction between the two SM layers, we will hereafter refer to the subendothelial SMCs as intimal SMCs. Phenotypic modulation of lymphatic SMCs appeared to be more pronounced in advanced lymphoedema. In grade 1 lesions, SMC layers were clearly thickened, but most SMCs stained intensely for SMaA, SM1 and SM2 (Fig. 2). In grade 2 and 3 lymphatic vessels, levels of SM1 were clearly reduced in intimal and medial SMCs, although most mural cells still weakly stained for SM1 (Figs 2, 3b). While SM1 is widely expressed in SMCs, expression of SM2 is known to be more restricted to differentiated SMCs.18,21 SM2 levels were markedly reduced in grade 2 and 3 lymphatic vessels, and the mural cells were nearly negative for SM2 in grade 3 (Figs 2, 3c). By contrast, the cells were clearly positive for SMaA, even in grade 3 lymphatic vessels. Smooth muscle cells in affected lymphatic vessels were smaller than those in normal vessels, and they appeared to be misaligned. On electron microscopy, SMCs in normal collector lymphatic vessels were long and fusiform and were rich in British Journal of Dermatology (2015) 172, pp1286–1293
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Grade 1
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Fig 2. Histopathology of lymphatic vessels in lymphoedema. Histopathological features of representative lymphatic vessels at each pathological group. Serial sections were immunostained for CD31, SMaA, SM1, SM2 and collagen type 1. The sections were also stained with Masson’s trichrome (MTC). Scale bars, 100 lm. SMaA, smooth muscle a actin.
myofilaments and dense bodies, but contained few organelles such as Golgi complexes, rough endoplasmic reticulum or mitochondria (Fig. 4a). In sharp contrast, the SMCs in lymphatic vessels from a lymphoedematous leg had lost their normal fusiform shape, and the cell membranes appeared to be more convoluted. The cells also contained more intracellular vesicles and mitochondria than SMCs in normal lymphatic vessels (Fig. 4b,c). These features indicate that lymphatic SMCs are phenotypically modulated in lymphoedema. There were also British Journal of Dermatology (2015) 172, pp1286–1293
large amounts of irregularly disposed, extracellular collagen fibres between the SMCs (Fig. 4b,c). In addition, some of the SMCs were apoptotic, and apoptotic bodies were observed (Fig. 4d). Vessel wall fibrosis in lymphoedema The walls of lymphatic vessels from lymphoedematous legs showed clear fibrosis spreading between the SMCs in the intima, media and adventitia, particularly in grade 2 and grade © 2014 British Association of Dermatologists
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Fig 3. Phenotypic modulation of SMCs and collagen deposition in lymphatic vessels. (a) Total area of the intimal + medial layers. Normal, n = 3; grade 1, n = 12; grade 2, n = 16; grade 3, n = 17. (b, c) Fractional SM1+ (b) and SM2+ (c) areas within SMaA+ areas. Normal, n = 3; grade 1, n = 11 (b), 4 (c); grade 2, n = 14 (b), 6 (c); grade 3, n = 16 (b), 5 (c). (d) Fractional collagen type I+ area within the intimal + medial area; n = 3, each. *P < 005 vs. normal. #P < 005. SMCs, smooth muscle cells; SMaA, smooth muscle a actin.
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Fig 4. Transmission electron micrographs of lymphatic SMCs. (a) SMCs in a normal lymphatic vessel. The cytoplasm is predominantly occupied by myofilaments (m) and a few synthetic organelles can be seen. (b) SMCs in a grade 1 lesion. Although the cells contain abundant myofilaments, Golgi vesicles (G), rough endoplasmic reticulum (rER) and mitochondria (mt) are also prominent. (c) SMCs in a grade 2 lymphatic vessel. The cells show a characteristic synthetic phenotype, with numerous synthetic organelles and few myofilaments. (d) An apoptotic SMC in a grade 3 lymphatic vessel. The cell contains residual myofilaments, and condensed chromatin. Scale bars, 5 lm. SMCs, smooth muscle cells.
mt G
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3 lesions (Figs 2, 5). The area of collagen type I+ grew significantly with the progression of lymphoedema (Fig. 3d).
Discussion In the present study, we showed that within lymphatic oedematous lesions the walls of collecting lymphatic vessels were © 2014 British Association of Dermatologists
G mt
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thickened, mainly due to hyperplasia of SMCs exhibiting modulated phenotypes. Expression of two markers of differentiated SMCs, SM1 and SM2, was downregulated, and TEM revealed features of the SMC synthetic phenotype, including increased numbers of synthetic organelles, such as endoplasmic reticulum and Golgi complex, and decreased numbers of contractile elements.23 Lymphatic vessel wall fibrosis was also prominent British Journal of Dermatology (2015) 172, pp1286–1293
1292 Phenotypic modulation of SMCs in lymphoedema, F. Ogata et al.
Normal
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Fig 5. Collagen deposition in lymphatic vessel walls. Transmission electron micrograph of normal and grade 2 lymphatic vessels. Scale bars, 5 lm.
in advanced cases. And in highly advanced cases, the vessel lumen was severely obstructed. These results strongly suggest that lymphatic contractility is greatly diminished in patients with grade 2 or 3 pathology. Even in grade 1 lesions, the vessel walls were thickened with misaligned SMCs. Although oedema may not be apparent with grade 1 lesions, these observations suggest that lymphatic function is already impaired. Indeed, even in cases of latent lymphoedema, lymphoscintigraphy showed clear evidence of deficient lymphatic flow.9,24,25 Lymphography using indocyanine green dye to detect subcutaneous lymphatic vessels also showed retarded lymphatic flow at stage 0 and dermal backflow at stage 1.25,26 These results strongly suggest that SMC proliferation and phenotypic modulation make a critical contribution to lymphatic dysfunction. The results of the present study demonstrate that SMCs undergo phenotypic modulation in lymphoedema. Similar changes are commonly observed in arterial disease, such as atherosclerosis, but we noted several differences between SMCs in lymphoedema and arterial disease. For instance, the SMCs in lymphatic vessels did not show foam cell-like phenotypes in lymphoedema, and we found only a few CD68+ macrophages in our lymphatic samples (data not shown). Given that macrophages are integral to atherogenesis, this suggests the processes underlying the lymphatic vessel pathology seen in lymphoedema differ somewhat from those involved in atherogenesis. In addition to macrophages, endothelial cells (ECs) as well as other immune cells, including lymphocytes, have been shown to affect SMC proliferation, migration and death.27 It will be important to assess the changes in those cells in future studies. A related question that remains to be addressed is how SMC phenotypic modulation and proliferation are induced during the development of lymphoedema. An attractive hypothesis is that disturbance of the lymphatic flow modulates lymphatic EC function, which in turn promotes lymphatic SMC growth. Within arteries, laminar flow shear stress is known to increase nitric oxide (NO) production by ECs and maintain healthy EC function. Disruption of the laminar flow reduces NO production and induces expression of adhesion molecules and proinflammatory cytokines in ECs, promoting vascular inflammation and SMC proliferation.27 Recent studies have shown that flow-mediated signalling also British Journal of Dermatology (2015) 172, pp1286–1293
controls lymphatic EC function,28 and that lymphatic flow plays a key role in lymphatic vessel development and regeneration. It is tempting to speculate that disturbance of the normal lymphatic flow contributes to lymphatic EC dysfunction and SMC phenotypic modulation. Consistent with that idea, lymphatic flow is disturbed in lymphoedema, even at very early stages of the condition.25,26 Although our results strongly suggest proliferation of lymphatic SMCs during development of lymphoedema, we observed very few SMCs that stained positively for Ki-67 in our lymphoedema samples (data not shown). One possible explanation for the small Ki-67+ SMC fraction is that SMCs in lymphatic vessels were replicating at very low rates when the samples were collected. In fact, a previous study of human coronary artery plaques showed that although human coronary plaques clearly have more layers of intimal and medial SMCs than healthy arteries, proliferating cell nuclear antigen positive and Ki-67+ cells were very rare (< 1%).29 Given the long period of time needed for development of atherosclerotic plaques, this low proliferation rate is not surprising. Development of lymphoedema also takes a long period of time. In fact, the mean time from surgical treatment to lymphaticovenous anastomosis surgery, during which the samples were collected, was 887 months (range 10–264 months). We suggest this reflects the very low rates of SMC proliferation at the time the samples were collected. We found apoptotic SMCs in samples of advanced lymphoedema (Fig. 4d). SMC apoptosis is reportedly induced during both physiological and pathological vessel remodelling. For instance, forced induction of low-level SMC apoptosis promotes neointima formation and medial repopulation by increasing SMC proliferation, migration and ECM production in a mouse carotid artery ligation model.30 Chronic low-level SMC apoptosis has also been shown to promote medial expansion and degeneration, plaque calcification and fibrous cap thinning.31 These findings suggest that SMC apoptosis actively contributes to vessel wall remodelling. It is currently unknown whether SMC apoptosis is also involved in lymphatic vessel remodelling in lymphoedema. In addition, cellular senescence has been noted among SMCs in several arterial disease models.32 Given that cell senescence promotes tissue remodelling by activating inflammatory processes and clearance of the senescent © 2014 British Association of Dermatologists
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cells, it would be interesting to assess the involvement of SMC senescence in lymphatic vessel remodelling. It is presently difficult to predict a patient’s risk of developing lymphoedema after a surgical procedure, and the therapeutic options are limited for those who do develop the condition. To stratify high-risk patients and provide them with effective prevention and therapy, it will be essential to further clarify the molecular mechanisms underlying lymphoedema. The results of the present study demonstrate that phenotypic modulation and proliferation of SMCs are crucially involved in the development of lymphoedema. However, a limitation of this study is the small number of samples, particularly from early-stage lymphoedema. To better understand the mechanisms leading to SMC phenotypic modulation and proliferation, it will be important to analyse pathological processes going on earlier during lymphoedema development. It is anticipated that further analysis of the mechanisms controlling lymphatic SMCs (e.g. identification of the stimuli and signals inducing phenotypic modulation of lymphatic SMCs) will not only provide a better understanding of the pathogenesis lymphoedema, but also enable identification of therapeutic targets.
Acknowledgments We thank N. Yamanaka, M. Hayashi, Y. Xiao and A. Ono for their excellent technical assistance and S. Fukuda at Electron Microscopic Research Room, Graduated School of Medicine, University of Tokyo for assistance with electron microscopic techniques. We also thank the patients who agreed to take part in this study.
References 1 Szuba A, Rockson SG. Lymphedema: classification, diagnosis and therapy. Vasc Med 1998; 3:145–56. 2 Fitzpatrick PJ. Lymphangiosarcoma and breast cancer. Can J Surg 1969; 12:172–7. 3 Schirger A, Harrison EG Jr, Janes JM. Idiopathic lymphedema. Review of 131 cases. J Am Med Assoc 1962; 182:14–22. 4 Williams AF, Franks PJ, Moffatt CJ. Lymphoedema: estimating the size of the problem. Palliat Med 2005; 19:300–13. 5 Ryan M, Stainton MC, Slaytor EK et al. Aetiology and prevalence of lower limb lymphoedema following treatment for gynaecological cancer. Aust N Z J Obstet Gynaecol 2003; 43:148–51. 6 Carvalho JP, Souen JS, Carram~ao Sda S et al. Wertheim-Meigs radical hysterectomy. Sao Paulo Med J 1994; 112:539–42. 7 Rockson SG. Lymphedema. Am J Med 2001; 110:288–95. 8 Zampell JC, Aschen S, Weitman ES et al. Regulation of adipogenesis by lymphatic fluid stasis: part I. Adipogenesis, fibrosis, and inflammation. Plast Reconstr Surg 2012; 129:825–34. 9 Lee BB, Laredo J. Contemporary role of lymphoscintigraphy: we can no longer afford to ignore!. Phlebology 2011; 26:177–8. 10 Alitalo K, Tammela T, Petrova TV. Lymphangiogenesis in development and human disease. Nature 2005; 438:946–53. 11 Daroczy J. Pathology of lymphedema. Clin Dermatol 1995; 13:433–44. 12 Olszewski WL, Engeset A, Romaniuk A et al. Immune cells in peripheral lymph and skin of patients with obstructive lymphedema. Lymphology 1990; 23:23–33.
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13 Koshima I, Nanba Y, Tsutsui T et al. Minimal invasive lymphaticovenular anastomosis under local anesthesia for leg lymphedema: is it effective for stage III and IV? Ann Plast Surg 2004; 53:261–6. 14 Koshima I, Inagawa K, Urushibara K et al. Supermicrosurgical lymphaticovenular anastomosis for the treatment of lymphedema in the upper extremities. J Reconstr Microsurg 2000; 16:437–42. 15 Zawieja DC. Contractile physiology of lymphatics. Lymphat Res Biol 2009; 7:87–96. 16 Ikomi F, Yokoyama Y, Ogiwara N et al. Recanalization of the collecting lymphatics in rabbit hind leg. Microcirculation 2006; 13:365–76. 17 Koshima I, Kawada S, Moriguchi T, Kajiwara Y. Ultrastructural observations of lymphatic vessels in lymphedema in human extremities. Plast Reconstr Surg 1996; 97:397–405. 18 Manabe I, Nagai R. Regulation of smooth muscle phenotype. Curr Atheroscler Rep 2003; 5:214–22. 19 Ross R. Atherosclerosis – an inflammatory disease. N Engl J Med 1999; 340:115–26. 20 Darby I, Skalli O, Gabbiani G. Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990; 63:21–9. 21 Aikawa M, Sivam PN, Kuro-o M et al. Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis. Circ Res 1993; 73:1000–12. 22 International Society of Lymphology. The diagnosis and treatment of peripheral lymphedema. 2009 Concensus Document of the International Society of Lymphology. Lymphology 2009; 42:51–60. 23 Perrotta I. Ultrastructural features of human atherosclerosis. Ultrastruct Pathol 2013; 37:43–51. 24 Gebousky P, Karny M, Krizova H et al. Staging of upper limb lymphedema from routine lymphoscintigraphic examinations. Comput Biol Med 2009; 39:1–7. 25 Szuba A, Strauss W, Sirsikar SP et al. Quantitative radionuclide lymphoscintigraphy predicts outcome of manual lymphatic therapy in breast cancer-related lymphedema of the upper extremity. Nucl Med Commun 2002; 23:1171–5. 26 Ogata F, Narushima M, Mihara M et al. Intraoperative lymphography using indocyanine green dye for near-infrared fluorescence labeling in lymphedema. Ann Plast Surg 2007; 59:180–4. 27 Iwata H, Manabe I, Nagai R. Lineage of bone marrow-derived cells in atherosclerosis. Circ Res 2013; 112:1634–47. 28 Wang Y, Simons M. Flow-regulated lymphatic vasculature development and signaling. Vasc Cell 2014; 6:14. 29 Gordon D, Reidy MA, Benditt EP et al. Cell proliferation in human coronary arteries. Proc Natl Acad Sci USA 1990; 87:4600–4. 30 Yu H, Clarke MC, Figg N et al. Smooth muscle cell apoptosis promotes vessel remodeling and repair via activation of cell migration, proliferation, and collagen synthesis. Arterioscler Thromb Vasc Biol 2011; 31:2402–9. 31 Clarke MC, Littlewood TD, Figg N et al. Chronic apoptosis of vascular smooth muscle cells accelerates atherosclerosis and promotes calcification and medial degeneration. Circ Res 2008; 102:1529–38. 32 Munoz-Espin D, Serrano M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol 2014; 15:482–96.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Fig S1. Relationship between clinical stage, local manifestation and pathological grade. Table S1. Staging of lymphoedema.
British Journal of Dermatology (2015) 172, pp1286–1293
British Journal of Dermatology
C U T A N E O U S B I OL O G Y
Phenotypic modulation of smooth muscle cells in lymphoedema F. Ogata,1,2 K. Fujiu,1,3 I. Koshima,2 R. Nagai4 and I. Manabe1 Departments of 1Cardiovascular Medicine, 2Plastic, Reconstructive and Aesthetic Surgery and 3Translational Systems Biology and Medicine Initiative, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan 4 Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi-ken 329-0498, Japan
Summary Correspondence Ichiro Manabe. E-mail: [email protected]
Accepted for publication 11 October 2014
Funding sources This study was supported by the ‘Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)’, initiated by the Council for Science and Technology Policy (to R.N.); Grants-in-Aid for Scientific Research (S) and (B) from the Japanese Society for the Promotion Science (to R.N. and I.M.); a grant for Translational Systems Biology and Medicine Initiative (to R.N.) from the Japanese Science and Technology Agency; a Grant-in-Aid for Scientific Research on Innovative Areas ‘Homeostatic Inflammation’ (to I.M.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and research grants from the Japan Science and Technology Institute, Sumitomo Foundation, Takeda Science Foundation, SENSHIN Medical Research Foundation, and Mochida Memorial Foundation for Medical and Pharmaceutical Research (to I.M.).
Background Lymphoedema is a debilitating progressive condition that is frequently observed following cancer surgery and severely restricts quality of life. Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood. Smooth muscle cells (SMCs) play pivotal roles in the pathogenesis of various vascular diseases, including atherosclerosis. Objectives We analysed SMCs in lymphatic vessels from the lymphoedematous legs of 29 patients. Methods Expression of smooth muscle a-actin (SMaA) and smooth muscle myosin heavy chain (SM-MHC) isoforms SM1 and SM2 was investigated using immunohistochemistry. Results Compared with normal lymphatic vessels, all affected lymphatic vessels in chronic lymphoedema showed marked wall thickening. In addition to increases in the numbers of rows of SMaA+ SM1+ SMCs in the tunica media, SMCs were also observed in the subendothelial region (tunica intima). While most intimal and medial cells were positive for SMaA and SM1, staining for SM1 and particularly SM2, a marker of mature SMCs, progressively declined in lymphatic vessels in increasingly severe lymphoedema lesions. Consequently, the SM1+ and SM2+ cell fractions were significantly reduced in the tunica media and intima of lymphatic vessels. Conclusions These observations indicate that the lymphatic tunica media and tunica intima consist mainly of phenotypically modulated SMCs, and that SMCs play a key role in the development of lymphoedema.
Conflicts of interest None declared. DOI 10.1111/bjd.13482
What’s already known about this topic?
• •
Lymphoedema is a debilitating progressive condition that is frequently observed following cancer surgery and severely restricts quality of life. Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood.
What does this study add?
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1286
Lymphatic smooth muscle cells play a key role in the development of lymphoedema.
British Journal of Dermatology (2015) 172, pp1286–1293
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Phenotypic modulation of SMCs in lymphoedema, F. Ogata et al. 1287
Lymphoedema is a debilitating progressive condition that is frequently observed after cancer surgery and/or irradiation, trauma and infectious diseases.1 Patients with even mild lymphoedema suffer disabilities such as conspicuous enlargement of one or more extremities, recurrent cellulitis and lymphorrhea, which adversely affect their everyday activity. Once these symptoms occur, it is difficult to cure them completely, even when treated at an early stage; and once the disease reaches the chronic stage, it is difficult to even mitigate the symptoms. In addition, 007–045% of patients with long-standing chronic lymphoedema develop Stewart–Treves syndrome, a rare deadly cutaneous angiosarcoma.2,3 The incidences of lymphoedema secondary to breast and gynaecological cancers are reported to be between 12% and 47%.4–6 Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood, and effective therapeutic and preventive strategies have not been established.7 Previous studies have shown that lymphoedema is associated with various pathological changes, including the accumulation of lipids in the tissue and infiltration of the tissue by inflammatory cells.8,9 Also seen are various degenerative changes to the walls of the lymphatic vessels; these include endothelial cell hyperplasia, thickening of the subendothelial collagen layer and reduction of the number of muscle fibres.3,10–12 The processes underlying the observed pathological changes within the lymphatic vessel wall are not clear. However, we have been using a surgical procedure, lymphaticovenous anastomosis, to treat patients with severe lymphoedema who respond poorly to conservative therapy,13,14 and during that operation we always notice sclerosis of the lymphatic vessels. We therefore hypothesize that the ‘lymphaticosclerosis’ that occurs in lymphoedema is analogous to arteriosclerosis, in which smooth muscle cells (SMCs) play a central role. Smooth muscle cells are required for the rhythmic contraction of lymphangions that provides the main propelling forces for lymph flow. The abnormal lymphatic contractions and flow seen in lymphoedema suggest SMC dysfunction and/or obstruction of the lymphatic vessel lumen,15 but little is known about the changes in SMCs in the affected lymphatic vessels in lymphoedema.16,17 In mature arteries, the primary function of SMCs is contraction, which is required to maintain vessel wall tone and integrity.18 These fully differentiated cells proliferate at extremely low rates and produce only small amounts of extracellular matrix (ECM). However, during development, SMCs play a key role in matrix deposition and vessel morphogenesis. Moreover, even fully differentiated SMCs remain highly plastic, enabling them to modify their phenotype in response to environmental cues. For example, intimal SMCs within atherosclerotic lesions exhibit phenotypes that clearly differ from those of mature medial cells. Such changes in phenotype are collectively referred to as ‘phenotypic modulation’. The phenotype of mature SMCs is referred to as ‘contractile’; that of intimal SMCs as ‘synthetic’. Synthetic SMCs play a central role in vascular pathology.19 A number of © 2014 British Association of Dermatologists
markers that report the phenotypes of SMCs have been identified. Smooth muscle a-actin (SMaA) has been used extensively as a marker for SMCs, although it is actually not a definitive lineage marker and is known to be expressed in a wide variety of non-SMC types, including myofibroblasts.20 Expression of smooth muscle myosin heavy chain (SM-MHC) is more tightly restricted to SMCs. The SM1 isoform of SM-MHC is widely expressed in SMCs, while the SM2 isoform is expressed in differentiated SMCs. In phenotypically modulated SMC in arterial disease, expression of these SMC differentiation markers is downregulated.21 In the present study we analysed the involvement of lymphatic SMCs in lymphoedema. We found that the walls of lymphatic vessels were thickened, mainly due to hyperplasia of SMCs exhibiting phenotypic modulation. These changes in SMCs appear to underlie the obstruction and dysfunction of lymphatic vessels seen in lymphoedema.
Methods Pathological samples We collected 45 lymphatic vessels from 29 randomly chosen patients with lymphoedema who were refractory to conventional conservative therapy and underwent lymphaticovenous anastomosis between 2007 and 2011 (25 women and four men; 29–86 years of age, mean 583 years). All of these patients had developed secondary leg lymphoedema after surgery: 11 had been treated for uterine cervical cancer, nine for uterine body cancer, three for ovarian cancer, two for hernias, two for ureteral cancer, one for bladder cancer and one for Paget disease. The patterns of oedema varied widely with respect to the affected area, the onset of symptoms and progression. Eighteen of the 29 patients first noticed swelling of the entire leg, while eight noticed swelling of the entire thigh, and three noticed swelling of the lower thigh. In addition, eight of the patients suffered from bilateral leg lymphoedema. In two of these patients, the lymphoedema appeared on both sides at the same time; in the others the lymphoedema broadened to affect both sides over periods ranging from 2 months to 6 years (mean 277 months) after the appearance of lymphoedema on one side. The mean latent duration (from surgical treatment to the onset of oedema) was 415 months. The mean duration of oedema (from onset to start of our treatment) was 440 months. Prior to surgery, lymphangiography was performed to identify lymphatic vessels and to determine the sites for skin incision. After skin incision, lymphatic vessels were identified using a surgical microscope. The lymphatic vessels examined for this study were collected from the foot or ankle. Three lymphatic vessels were obtained from normal legs (an ankle and dorsal side of the foot in two persons) as normal controls. These patients had no symptoms of lymphoedema and were operated on for cervical cancer, after which they underwent prophylactic lymphaticovenous anastomosis in our hospital. British Journal of Dermatology (2015) 172, pp1286–1293
1288 Phenotypic modulation of SMCs in lymphoedema, F. Ogata et al.
Written informed consent was obtained from each subject after a full explanation of the study, which was approved by the Ethics Committee of the University of Tokyo Hospital. Clinical and pathological grading of lymphoedema The clinical severity of lymphoedema was graded according to International Society of Lymphology (ISL) staging (Table S1; see Supporting Information).22 Because the clinical manifestations of lymphoedema (e.g. skin changes and oedema) are often milder in the feet and ankles than in the thighs (Supplementary Fig. S1), we also used ISL staging to grade the severity of local clinical manifestations at the sites of sample collection, based on the skin changes and the level of oedema. We graded the macroscopic pathological changes at the sites of sample collection into three groups. The skin of grade 1 lesions was soft, and subcutaneous tissue was not fibrotic. The subcutaneous collector lymphatic vessel was transparent, the vessel wall was pliant, and lymph within the vessel was easily detectable when the proximal portion of the vessel was closed using forceps. The skin of grade 2 lesions was harder than unaffected skin. While the subcutaneous tissue was apparently fibrotic, the subcutaneous fibrotic layers could be easily cut with a forceps, and the subcutaneous lymphatic vessel could be separated from the fibrotic subcutaneous tissue. The lymphatic vessel was less translucent than grade 1 vessels, but the lumen was more visible than in grade 1 vessels because the vessel wall was stiff and not elastic. Lymph could be identified. The skin of grade 3 lesions was thickened and verrucous with lymphorrhea and/or ulcer formation. The subcutaneous tissue was markedly fibrotic, and firm white fibrotic septa had formed. The lymphatic vessel was opaque and white, and the lumen was invisible in most cases. Although these severely affected lymphatic vessels may resemble peripheral nerves, they lack the transverse stripe pattern of myelin sheaths (Fig. 1g–i). Histological and immunohistochemical analysis Paraffin-embedded sections (5-lm thick) were deparaffinized and blocked with 2% bovine serum albumin. To evaluate histopathological changes and tissue fibrosis, elastica van Gieson and Masson’s trichrome staining were performed. For immunohistochemical staining, anti-SM1 antibody (Kyowa Medex, Shizuoka, Japan), anti-SM2 antibody (Yamasa, Tokyo, Japan), anti-SMaA antibody (Sigma-Aldrich, St Louis, MO, U.S.A.), anti-CD31 antibody (BD Biosciences, San Diego, CA, U.S.A.) and anticollagen type 1 antibody (Abcam, Cambridge, U.K.) were used with the avidin–biotin complex technique and Vector Red substrate (Vector Laboratories, Burlingame, CA, U.S.A.). Sections were counterstained with haematoxylin. For each section, the area of immunoreactive lymphatic SMCs and collagen type I in the lymphatic vessels was quantified by imaging software (WinROOF, Mitani, Tokyo, Japan). British Journal of Dermatology (2015) 172, pp1286–1293
Transmission electron microscopy Samples were fixed with 25% glutaraldehyde in 01 mol L 1 cacodylate buffer (pH 74) for 1 h at 4 °C and then postfixed with 1% osmium tetroxide in cacodylate buffer (pH 74) for 1 h at 4 °C. After staining with 1% aqueous uranyl acetate solution for 10 min at room temperature, the samples were dehydrated in a graded series of ethanol solutions. For transmission electron microscopy (TEM), the blocks were embedded in Epon after dehydration. Ultrathin sections were cut at 50 nm with a diamond knife, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (H-7000; Hitachi, Tokyo, Japan) at an acceleration voltage of 75 kV. Statistical analysis Data are shown as means SEM. Differences among multiple groups were analysed using one-way ANOVA followed by posthoc Tukey–Kramer tests. Values of P < 005 were considered significant.
Results Pathological grading of lymphoedema tissues Lymphatic samples were collected from the dorsal area of the foot and the ankle of patients who underwent lymphaticovenous anastomosis. We graded the macroscopic pathology of the sites of sample collection into three groups based on changes in the skin, subcutaneous tissue and lymphatic vessels (Fig. 1). The collector lymphatic vessels in grade 1 samples were translucent and pliant. The skin in the region from which the tissue was collected was soft, and the subcutaneous tissue was not fibrotic. The lymphatic vessels in grade 2 samples were less translucent than those in grade 1. The skin was clearly harder than in unaffected regions, and the subcutaneous tissue was moderately fibrotic but was soft and easily cut with a forceps. The lymphatic vessels in grade 3 samples were white and opaque. The vessels were stiff, and the lumens were mostly invisible. The skin was clearly thickened and the subcutaneous tissues were markedly fibrotic. For ISL staging, the clinical severity of lymphoedema is graded based on oedematous and trophic changes in the affected limb (Supplementary Table S1).22 However, the severity of skin changes and oedema at the site of sample collection may differ from the clinical staging of the entire leg. In fact, oedema and skin changes were often milder in the feet and ankles than in the thighs. For that reason, we graded the local manifestation of lymphoedema at the sites of sample collection based on the state of the skin and oedema, as defined in the ISL staging. We found that the macroscopic pathological grades were well correlated with the stages of local clinical manifestation of lymphoedema (Fig. S1; see Supporting Information), which suggests the progression of pathology from grade 1 to grade 3. © 2014 British Association of Dermatologists
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Lymphatic smooth muscle cells in lymphoedema Like the walls of blood vessels, the walls of normal collector lymphatic vessels in the legs consist of three layers. The tunica intima is comprised of CD31+ endothelial cells (Fig. 2). Unlike arterial walls, normal collector lymphatic vessels do not have a well-defined internal elastic lamina. The tunica media contains one to three rows of SMCs that are positive for SMaA and SM1 and SM2 SM-MHC (Fig. 2). Compared with normal lymphatic vessels, all lymphatic vessels from patients with chronic lymphoedema showed marked wall thickening (Fig. 3a). Most of the medial cells were positive for both SMaA and SM1, indicating these cells were SMCs (Fig. 2). The medial layer was thickened and consisted of 3– 10 or more rows of SMCs. More strikingly, SMaA+ SM1+ SMCs were also observed in the subendothelial region. The subendothelial SM layer was particularly thick in greatly obstructed lymphatic vessels, and between the medial and subendothelial SM layers there was a fibrous intermediate region that contained far fewer cells than the adjacent SMC-rich regions. Within this region, most of the cells were negative for SMC markers, suggesting they were either not © 2014 British Association of Dermatologists
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Fig 1. Gross pathological changes in lymphoedema. Lymphoedema lesions were graded into three groups based on the macroscopic pathological changes. Shown are representative lesions. The sites of surgery and sample collection are marked with bars in panels (a), (d) and (g). The photographs shown in (b), (e) and (h) were taken during lymphaticovenous anastomosis surgery, when the affected collector lymphatic vessels were identified. Panels (c), (f) and (i) provide schematic representations of the gross appearance of the images in (b), (e) and (h). (a–c) A representative grade 1 lesion. Although the patient suffered from clinical stage 2 lymphoedema in the affected (left) thigh, the tissue sample was collected from the foot, which showed only mild oedema and minimal skin changes that were considered to indicate local stage 1 lymphoedema. Note that the blue nylon suture is clearly visible behind the lymphatic vessel (b). (d–f) A representative grade 2 lesion. The patient suffered from stage 2 lymphoedema in the entire affected (left) leg, including the foot. Note that the nylon suture behind the lymphatic vessel is barely visible (e). (g–i) A representative grade 3 lesion. The affected leg exhibited lymphostatic elephantiasis in which pitting was absent. The lumen of the affected lymphatic vessel (arrow heads) was obstructed. The areas shown in grey in panel (i) indicate firm white septa. Scale bars, 1 mm (b, e, h).
(b)
SMCs or they were highly modulated SMCs. As mentioned, the normal lymphatic wall does not have a well-defined internal elastic lamina but, based on the clear distinction between the two SM layers, we will hereafter refer to the subendothelial SMCs as intimal SMCs. Phenotypic modulation of lymphatic SMCs appeared to be more pronounced in advanced lymphoedema. In grade 1 lesions, SMC layers were clearly thickened, but most SMCs stained intensely for SMaA, SM1 and SM2 (Fig. 2). In grade 2 and 3 lymphatic vessels, levels of SM1 were clearly reduced in intimal and medial SMCs, although most mural cells still weakly stained for SM1 (Figs 2, 3b). While SM1 is widely expressed in SMCs, expression of SM2 is known to be more restricted to differentiated SMCs.18,21 SM2 levels were markedly reduced in grade 2 and 3 lymphatic vessels, and the mural cells were nearly negative for SM2 in grade 3 (Figs 2, 3c). By contrast, the cells were clearly positive for SMaA, even in grade 3 lymphatic vessels. Smooth muscle cells in affected lymphatic vessels were smaller than those in normal vessels, and they appeared to be misaligned. On electron microscopy, SMCs in normal collector lymphatic vessels were long and fusiform and were rich in British Journal of Dermatology (2015) 172, pp1286–1293
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Grade 1
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Fig 2. Histopathology of lymphatic vessels in lymphoedema. Histopathological features of representative lymphatic vessels at each pathological group. Serial sections were immunostained for CD31, SMaA, SM1, SM2 and collagen type 1. The sections were also stained with Masson’s trichrome (MTC). Scale bars, 100 lm. SMaA, smooth muscle a actin.
myofilaments and dense bodies, but contained few organelles such as Golgi complexes, rough endoplasmic reticulum or mitochondria (Fig. 4a). In sharp contrast, the SMCs in lymphatic vessels from a lymphoedematous leg had lost their normal fusiform shape, and the cell membranes appeared to be more convoluted. The cells also contained more intracellular vesicles and mitochondria than SMCs in normal lymphatic vessels (Fig. 4b,c). These features indicate that lymphatic SMCs are phenotypically modulated in lymphoedema. There were also British Journal of Dermatology (2015) 172, pp1286–1293
large amounts of irregularly disposed, extracellular collagen fibres between the SMCs (Fig. 4b,c). In addition, some of the SMCs were apoptotic, and apoptotic bodies were observed (Fig. 4d). Vessel wall fibrosis in lymphoedema The walls of lymphatic vessels from lymphoedematous legs showed clear fibrosis spreading between the SMCs in the intima, media and adventitia, particularly in grade 2 and grade © 2014 British Association of Dermatologists
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Fig 3. Phenotypic modulation of SMCs and collagen deposition in lymphatic vessels. (a) Total area of the intimal + medial layers. Normal, n = 3; grade 1, n = 12; grade 2, n = 16; grade 3, n = 17. (b, c) Fractional SM1+ (b) and SM2+ (c) areas within SMaA+ areas. Normal, n = 3; grade 1, n = 11 (b), 4 (c); grade 2, n = 14 (b), 6 (c); grade 3, n = 16 (b), 5 (c). (d) Fractional collagen type I+ area within the intimal + medial area; n = 3, each. *P < 005 vs. normal. #P < 005. SMCs, smooth muscle cells; SMaA, smooth muscle a actin.
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Fig 4. Transmission electron micrographs of lymphatic SMCs. (a) SMCs in a normal lymphatic vessel. The cytoplasm is predominantly occupied by myofilaments (m) and a few synthetic organelles can be seen. (b) SMCs in a grade 1 lesion. Although the cells contain abundant myofilaments, Golgi vesicles (G), rough endoplasmic reticulum (rER) and mitochondria (mt) are also prominent. (c) SMCs in a grade 2 lymphatic vessel. The cells show a characteristic synthetic phenotype, with numerous synthetic organelles and few myofilaments. (d) An apoptotic SMC in a grade 3 lymphatic vessel. The cell contains residual myofilaments, and condensed chromatin. Scale bars, 5 lm. SMCs, smooth muscle cells.
mt G
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3 lesions (Figs 2, 5). The area of collagen type I+ grew significantly with the progression of lymphoedema (Fig. 3d).
Discussion In the present study, we showed that within lymphatic oedematous lesions the walls of collecting lymphatic vessels were © 2014 British Association of Dermatologists
G mt
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thickened, mainly due to hyperplasia of SMCs exhibiting modulated phenotypes. Expression of two markers of differentiated SMCs, SM1 and SM2, was downregulated, and TEM revealed features of the SMC synthetic phenotype, including increased numbers of synthetic organelles, such as endoplasmic reticulum and Golgi complex, and decreased numbers of contractile elements.23 Lymphatic vessel wall fibrosis was also prominent British Journal of Dermatology (2015) 172, pp1286–1293
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Normal
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Fig 5. Collagen deposition in lymphatic vessel walls. Transmission electron micrograph of normal and grade 2 lymphatic vessels. Scale bars, 5 lm.
in advanced cases. And in highly advanced cases, the vessel lumen was severely obstructed. These results strongly suggest that lymphatic contractility is greatly diminished in patients with grade 2 or 3 pathology. Even in grade 1 lesions, the vessel walls were thickened with misaligned SMCs. Although oedema may not be apparent with grade 1 lesions, these observations suggest that lymphatic function is already impaired. Indeed, even in cases of latent lymphoedema, lymphoscintigraphy showed clear evidence of deficient lymphatic flow.9,24,25 Lymphography using indocyanine green dye to detect subcutaneous lymphatic vessels also showed retarded lymphatic flow at stage 0 and dermal backflow at stage 1.25,26 These results strongly suggest that SMC proliferation and phenotypic modulation make a critical contribution to lymphatic dysfunction. The results of the present study demonstrate that SMCs undergo phenotypic modulation in lymphoedema. Similar changes are commonly observed in arterial disease, such as atherosclerosis, but we noted several differences between SMCs in lymphoedema and arterial disease. For instance, the SMCs in lymphatic vessels did not show foam cell-like phenotypes in lymphoedema, and we found only a few CD68+ macrophages in our lymphatic samples (data not shown). Given that macrophages are integral to atherogenesis, this suggests the processes underlying the lymphatic vessel pathology seen in lymphoedema differ somewhat from those involved in atherogenesis. In addition to macrophages, endothelial cells (ECs) as well as other immune cells, including lymphocytes, have been shown to affect SMC proliferation, migration and death.27 It will be important to assess the changes in those cells in future studies. A related question that remains to be addressed is how SMC phenotypic modulation and proliferation are induced during the development of lymphoedema. An attractive hypothesis is that disturbance of the lymphatic flow modulates lymphatic EC function, which in turn promotes lymphatic SMC growth. Within arteries, laminar flow shear stress is known to increase nitric oxide (NO) production by ECs and maintain healthy EC function. Disruption of the laminar flow reduces NO production and induces expression of adhesion molecules and proinflammatory cytokines in ECs, promoting vascular inflammation and SMC proliferation.27 Recent studies have shown that flow-mediated signalling also British Journal of Dermatology (2015) 172, pp1286–1293
controls lymphatic EC function,28 and that lymphatic flow plays a key role in lymphatic vessel development and regeneration. It is tempting to speculate that disturbance of the normal lymphatic flow contributes to lymphatic EC dysfunction and SMC phenotypic modulation. Consistent with that idea, lymphatic flow is disturbed in lymphoedema, even at very early stages of the condition.25,26 Although our results strongly suggest proliferation of lymphatic SMCs during development of lymphoedema, we observed very few SMCs that stained positively for Ki-67 in our lymphoedema samples (data not shown). One possible explanation for the small Ki-67+ SMC fraction is that SMCs in lymphatic vessels were replicating at very low rates when the samples were collected. In fact, a previous study of human coronary artery plaques showed that although human coronary plaques clearly have more layers of intimal and medial SMCs than healthy arteries, proliferating cell nuclear antigen positive and Ki-67+ cells were very rare (< 1%).29 Given the long period of time needed for development of atherosclerotic plaques, this low proliferation rate is not surprising. Development of lymphoedema also takes a long period of time. In fact, the mean time from surgical treatment to lymphaticovenous anastomosis surgery, during which the samples were collected, was 887 months (range 10–264 months). We suggest this reflects the very low rates of SMC proliferation at the time the samples were collected. We found apoptotic SMCs in samples of advanced lymphoedema (Fig. 4d). SMC apoptosis is reportedly induced during both physiological and pathological vessel remodelling. For instance, forced induction of low-level SMC apoptosis promotes neointima formation and medial repopulation by increasing SMC proliferation, migration and ECM production in a mouse carotid artery ligation model.30 Chronic low-level SMC apoptosis has also been shown to promote medial expansion and degeneration, plaque calcification and fibrous cap thinning.31 These findings suggest that SMC apoptosis actively contributes to vessel wall remodelling. It is currently unknown whether SMC apoptosis is also involved in lymphatic vessel remodelling in lymphoedema. In addition, cellular senescence has been noted among SMCs in several arterial disease models.32 Given that cell senescence promotes tissue remodelling by activating inflammatory processes and clearance of the senescent © 2014 British Association of Dermatologists
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cells, it would be interesting to assess the involvement of SMC senescence in lymphatic vessel remodelling. It is presently difficult to predict a patient’s risk of developing lymphoedema after a surgical procedure, and the therapeutic options are limited for those who do develop the condition. To stratify high-risk patients and provide them with effective prevention and therapy, it will be essential to further clarify the molecular mechanisms underlying lymphoedema. The results of the present study demonstrate that phenotypic modulation and proliferation of SMCs are crucially involved in the development of lymphoedema. However, a limitation of this study is the small number of samples, particularly from early-stage lymphoedema. To better understand the mechanisms leading to SMC phenotypic modulation and proliferation, it will be important to analyse pathological processes going on earlier during lymphoedema development. It is anticipated that further analysis of the mechanisms controlling lymphatic SMCs (e.g. identification of the stimuli and signals inducing phenotypic modulation of lymphatic SMCs) will not only provide a better understanding of the pathogenesis lymphoedema, but also enable identification of therapeutic targets.
Acknowledgments We thank N. Yamanaka, M. Hayashi, Y. Xiao and A. Ono for their excellent technical assistance and S. Fukuda at Electron Microscopic Research Room, Graduated School of Medicine, University of Tokyo for assistance with electron microscopic techniques. We also thank the patients who agreed to take part in this study.
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Fig S1. Relationship between clinical stage, local manifestation and pathological grade. Table S1. Staging of lymphoedema.
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