Inhibition of the mammalian target of rapamycin impedes lymphangiogenesis
2007; Elsevier BV; Volume: 71; Issue: 8 Linguagem: Inglês
10.1038/sj.ki.5002112
ISSN1523-1755
AutoresStephan Huber, Christiane J. Bruns, G. Schmid, Patrick Hermann, Claudius Conrad, Hanno Nieß, Ralf Huss, Christian Graeb, K.‐W. Jauch, Christopher Heeschen, Markus Guba,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoLymphatic complications are common side effects of mammalian target of rapamycin (mTOR) inhibitor-based immunosuppression in kidney transplantation. Therefore, we investigated whether the mTOR inhibitor rapamycin, besides its known antihemangiogenic effect, also impedes regenerative lymphangiogenesis. In a murine skin flap model, rapamycin impaired recovery of lymphatic flow across surgical incisions resulting in prolonged wound edema in these animals. Importantly, the antilymphangiogenic effect of rapamycin was not related to a general inhibition of wound healing as demonstrated an in vivo Matrigel™ lymphangiogenesis assay and a model of lymphangioma. Rapamycin concentrations as low as 1 ng/ml potently inhibited vascular endothelial growth factor (VEGF)-C driven proliferation and migration, respectively, of isolated human lymphatic endothelial cells (LECs) in vitro. Mechanistically, mTOR inhibition impairs downstream signaling of VEGF-A as well as VEGF-C via mTOR to the p70S6 kinase in LECs. In conclusion, we provide extensive experimental evidence for an antilymphangiogenic activity of mTOR inhibition suggesting that the early use of mTOR inhibitor following tissue injury should be avoided. Conversely, the antilymphangiogenic properties of rapamycin and its derivates may provide therapeutic value for the prevention and treatment of malignancies, respectively. Lymphatic complications are common side effects of mammalian target of rapamycin (mTOR) inhibitor-based immunosuppression in kidney transplantation. Therefore, we investigated whether the mTOR inhibitor rapamycin, besides its known antihemangiogenic effect, also impedes regenerative lymphangiogenesis. In a murine skin flap model, rapamycin impaired recovery of lymphatic flow across surgical incisions resulting in prolonged wound edema in these animals. Importantly, the antilymphangiogenic effect of rapamycin was not related to a general inhibition of wound healing as demonstrated an in vivo Matrigel™ lymphangiogenesis assay and a model of lymphangioma. Rapamycin concentrations as low as 1 ng/ml potently inhibited vascular endothelial growth factor (VEGF)-C driven proliferation and migration, respectively, of isolated human lymphatic endothelial cells (LECs) in vitro. Mechanistically, mTOR inhibition impairs downstream signaling of VEGF-A as well as VEGF-C via mTOR to the p70S6 kinase in LECs. In conclusion, we provide extensive experimental evidence for an antilymphangiogenic activity of mTOR inhibition suggesting that the early use of mTOR inhibitor following tissue injury should be avoided. Conversely, the antilymphangiogenic properties of rapamycin and its derivates may provide therapeutic value for the prevention and treatment of malignancies, respectively. In the vast majority of tissues and organs, the lymphatic network is responsible for the removal of interstitial protein and fluid, thereby maintaining the interstitial fluid balance and providing lymphatic clearance of macromolecules.1.Saharinen P. Tammela T. Karkkainen M.J. Alitalo K. Lymphatic vasculature: development molecular regulation and role in tumor metastasis and inflammation.Trends Immunol. 2004; 25: 387-395Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar Disintegration of the lymphatic drainage by chronic inflammation, infection, trauma, or surgical procedures can give rise to lymphedema and lymphatic malformations which may result in large cystic structures accumulating lymphatic fluid (lymphoceles).1.Saharinen P. Tammela T. Karkkainen M.J. Alitalo K. Lymphatic vasculature: development molecular regulation and role in tumor metastasis and inflammation.Trends Immunol. 2004; 25: 387-395Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar In addition to its role in fluid drainage, the lymphatic system also represents an important route for circulating immune cells that function in immune surveillance and for metastatic dissemination of tumor cells via pre-existing and possibly also via newly formed lymphatic vessels.2.Skobe M. Hamberg L.M. Hawighorst T. et al.Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.Am J Pathol. 2001; 159: 893-903Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar Under physiological conditions, the lymphatic vasculature is a rather quiescent system, but several pathological conditions such as lymphatic vessel injury, inflammation, and cancer are characterized by lymphatic vessel growth through stimulation by an orchestra of growth factors.1.Saharinen P. Tammela T. Karkkainen M.J. Alitalo K. Lymphatic vasculature: development molecular regulation and role in tumor metastasis and inflammation.Trends Immunol. 2004; 25: 387-395Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar,3.Karkkainen M.J. Petrova T.V. Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis.Oncogene. 2000; 19: 5598-5605Crossref PubMed Scopus (351) Google Scholar The recent introduction of rapamycin into clinical kidney transplantation allowed transplant physicians to develop novel immunosuppressive strategies providing adequate immunosuppression for the transplant recipient while avoiding the nephrotoxic side effects associated with calcineurin inhibitor (CNI) therapy. Rapamycin forms a complex with the FK506-binding protein complex that binds with high affinity to the mammalian target of rapamycin (mTOR). This interaction causes dephosphorylation and inactivation of the p70S6 kinase, which, when activated, stimulates the production of ribosomal components necessary for protein synthesis and cell cycle progression. This activity, which effectively blocks interleukin-2 stimulation of lymphocyte division, is the basis for the successful clinical use of rapamycin to prevent allograft rejection in organ transplantation. However, a number of side effects may limit the use of rapamycin in kidney transplantation. In addition to general complications such as deregulation of the lipid metabolism or the risk of thrombo- and leucopenia, a number of complications have been reported that could be well rationalized by a potential antilymphangiogenic activity of mTOR inhibitors. Specifically, impaired wound healing and formation of lymphoceles have been associated with rapamycin-based immunosuppression in kidney transplant patients.4.Giessing M. Budde K. Sirolimus and lymphocele formation after kidney transplantation: an immunosuppressive medication as co-factor for a surgical problem?.Nephrol Dial Transplant. 2003; 18: 448-449Crossref PubMed Scopus (20) Google Scholar,5.Aboujaoude W. Milgrom M.L. Govani M.V. Lymphedema associated with sirolimus in renal transplant recipients.Transplantation. 2004; 77: 1094-1096Crossref PubMed Scopus (52) Google Scholar Spontaneous formation of severe lymphedema has also been observed in a number of patients,6.Chhajed P.N. Dickenmann M. Bubendorf L. et al.Patterns of pulmonary complications associated with sirolimus.Respiration. 2006; 73: 367-374Crossref PubMed Scopus (72) Google Scholar, 7.Fuchs U. Zittermann A. Berthold H.K. et al.Immunosuppressive therapy with everolimus can be associated with potentially life-threatening lingual angioedema.Transplantation. 2005; 79: 981-983Crossref PubMed Scopus (47) Google Scholar, 8.Mahe E. Morelon E. Lechaton S. et al.Cutaneous adverse events in renal transplant recipients receiving sirolimus-based therapy.Transplantation. 2005; 79: 476-482Crossref PubMed Scopus (175) Google Scholar, 9.Wadei H. Gruber S.A. El-Amm J.M. et al.Sirolimus-induced angioedema.Am J Transplant. 2004; 4: 1002-1005Crossref PubMed Scopus (38) Google Scholar which regressed after conversion to mTOR inhibitor-free immunosuppressant. Indeed, these effects may be explained by the fact that not only lymphocytes but also other cell types such as smooth muscle cells and vascular endothelial cells depend on mTOR signaling as a major downstream pathway of various growth factors, energy sensors, hypoxia sensors as well as cell components that are regulating cell growth and cell division.10.Rowinsky E.K. Targeting the molecular target of rapamycin (mTOR).Curr Opin Oncol. 2004; 16: 564-575Crossref PubMed Scopus (145) Google Scholar Therefore, blockade of mTOR function mimics deprivation of amino acids and, to a limited extent, lack of growth factors. With respect to endothelial cells, mTOR inhibition translates into a powerful antihemangiogenic effect.11.Guba M. von Breitenbuch P. Steinbauer M. et al.Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor.Nat Med. 2002; 8: 128-135Crossref PubMed Scopus (1506) Google Scholar We have previously shown that rapamycin blocks blood vessels growth via inhibition of vascular endothelial growth factor (VEGF)-A production and reduced responsiveness of vascular endothelial cells to VEGF-A, respectively.11.Guba M. von Breitenbuch P. Steinbauer M. et al.Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor.Nat Med. 2002; 8: 128-135Crossref PubMed Scopus (1506) Google Scholar,12.Guba M. Yezhelyev M. Eichhorn M.E. et al.Rapamycin induces tumor-specific thrombosis via tissue factor in the presence of VEGF.Blood. 2005; 105: 4463-4469Crossref PubMed Scopus (112) Google Scholar Considering the lymphatic complications observed in kidney transplant patients, we hypothesized that rapamycin may also inhibit lymphatic endothelial cells (LECs) and, thus, may act as an antilymphangiogenic agent. The potential of rapamycin to delay regeneration of lymphatic vessels after surgical incision was determined using an epigastric skin flap model. Rapamycin markedly inhibited the lymphatic drainage of skin flaps during 14 days of treatment compared with control mice (Figure 1a and b; Table 1). Specifically, the number of Patent Blue marked lymphatic vessels crossing the flap border, which was used as an indicator of reconstitution of lymph drainage, was significantly reduced in rapamycin-treated mice compared with controls. Drainage of Patent Blue to the regional, sentinel lymph nodes was observed in all control mice but only in 25% of the rapamycin-treated mice (P<0.01). Consequently, this impaired lymphatic drainage was associated with macroscopic lymphedema in all rapamycin-treated animals whereas in control animals, lymphedema was present in only 33% of the mice. Moreover, the ability to reconstitute lymphatic drainage by recruiting collateral vessels in the preserved caudal skin bridge was reduced in rapamycin-treated animals, although this difference did not reach a significant level. Consistently, the histological analysis of the regenerative areas of the skin flaps (Figure 1c) revealed a significantly lower number of lymphatic vessel endothelial receptor (LYVE)-positive lymphatic vessels in rapamycin-treated mice compared with control mice (Figure 1d and e).Table 1Skin flap model – number of positive sentinel lymph nodes, collateral vessels, and presence of lymphedema in individual animals treated with vehicle or rapamycin (n≥5 per group)Control (C1, C2, C3, C4, C5, C6)Rapamycin (5 mg/kg/day) (R1, R2, R3, R4, R5)Positive sentinel lymph nodesaPatent Blue-positive right and/or left axillary lymph nodes. (n)2, 2, 2, 1, 1, 21, 0, 0, 1, 0P<0.01Collateral vessels (n)1, 1, 3, 1, 2, 20, 0, 2, 1, 2NSLymphedemab+, macroscopic lymphedema.−, −, +, −, +, −+, +, +, +, +P<0.05NS, not significant.a Patent Blue-positive right and/or left axillary lymph nodes.b +, macroscopic lymphedema. Open table in a new tab NS, not significant. To provide further evidence that rapamycin inhibits lymphatic vessel regeneration independent of the well-known effects on general wound healing, we used the Matrigel™ plug in vivo assay. Two weeks after implantation of the Matrigel™ plugs, the ingrowth of lymphatic vessels was visualized by LYVE-1 immunostaining. In control animals, Matrigel™ plugs were strongly invaded by LYVE-1-positive lymphatic vessels (Figure 2). In contrast, in rapamycin-treated animals, lymphatic neovascularization was markedly inhibited. The ability of rapamycin to delay lymphatic proliferation in vivo was further investigated in a distinct murine model of lymphangioma (benign tumor of LECs). In this model, two consecutive intraperitoneal injections of incomplete Freund's adjuvant on days 0 and 1513.Mancardi S. Stanta G. Dusetti N. Bestagno M. et al.Lymphatic endothelial tumors induced by intraperitoneal injection of incomplete Freund's adjuvant.Exp Cell Res. 1999; 246: 368-375Crossref PubMed Scopus (67) Google Scholar result in the development of lymphangiomas in the peritoneal cavity (Figure 3a and b). The lymphatic phenotype of the formed structures was confirmed by staining for LYVE-1 (Figure 3c). Compared with the control group, rapamycin-treated animals showed a significantly reduced formation of lymphangiomas on day 28 (Figure 3d). This inhibition of lymphangioma growth was confirmed in NMRI nu/nu mice, lacking T cells, indicating that the antiproliferative effect of rapamycin is independent of its immunosuppressive mode of action. In contrast, the CNI cyclosporine had no significant inhibitory effect on the de novo generation of lymphatic vessels (Figure 3e). Moreover, rapamycin was also capable of significantly decreasing the tumor volumes of pre-existing lymphangioma formations (Figure 3f). The effect of rapamycin on VEGF-C driven proliferation of the LYVE+ LEC fraction of human dermal microvascular endothelial cell (HDMEC), identified by standard flow cytometry, was tested in a bromodeoxyuridine (BrdU) incorporation assay. Rapamycin inhibited proliferation in a concentration-dependent manner (Figure 4a). A rapamycin concentration as low as 1 ng/ml resulted in an ~50% inhibition of cell proliferation. Concentrations of 10 ng/ml or above virtually abrogated cell proliferation. As migration is an integral part of the lymphangiogenic process, we also investigated the migratory activity of isolated LECs following treatment with rapamycin. Indeed, rapamycin also resulted in a significant reduction in the migratory activity of the isolated cells (Figure 4b). These data demonstrate that rapamycin significantly inhibits both LEC proliferation and migration in a concentration range that is highly clinically relevant for transplant patients (5–20 ng/ml). Mechanistically, we investigated whether the antiproliferative effect of rapamycin on LECs is generated through inhibition of p70S6 kinase phosphorylation. Western blot analysis revealed that mTOR inhibition virtually abolished VEGF-C-stimulated phosphorylation of the p70S6 kinase in isolated LECs (Figure 5a and b). We also investigated the ability of rapamycin to block p70S6 kinase phosphorylation in the LECs stimulated with different VEGF isoforms known to primarily activate either the vascular endothelial growth factor receptor (VEGFR)-2 (VEGF-A) or the VEGFR-3 (VEGF-C*). Both VEGF-A and VEGF-C exhibited a solid phosphorylation of the p70S6 kinase in LECs. In contrast, treatment with VEGF-C*, which selectively activates VEGFR-3, resulted in a more modest phosphorylation of the p70S6 kinase. In either case, however, the phosphorylation was abrogated by treatment with rapamycin indicating that mTOR-mediated phosphorylation of the p70S6 kinase is crucial for both the VEGF-A- and VEGF-C-mediated proliferation of LECs. The salient findings of this study are that the mTOR inhibition potently decreases regenerative and neoplastic lymphangiogenesis. The antilymphangiogenic effect during tissue regeneration resulted in a prolonged occurrence of lymphedema in rapamycin-treated animals emphasizing the clinical relevance of the antilymphangiogenic effect of mTOR inhibition in a transplant setting. Of note, the observed in vivo effects were not secondary to impaired wound healing but rather due to direct antilymphangiogenic effects. Indeed, the potent antilymphangiogenic effect of rapamycin was confirmed in a wound-healing independent Matrigel™ plug assay as well as a reduced LEC proliferation and migration in vitro using clinically relevant concentrations. Mechanistically, rapamycin administration, in analogy to our previous studies in hemangiogenesis, impairs downstream signaling of VEGF-A through inhibition of the mTOR/p70S6K pathway in LECs. Even more important, however, we show here for the first time that rapamycin also interferes with the intracellular pathway activation of LEC by VEGF-C, the main initiator of lymphangiogenesis. Finally, our data indicate that the antilymphangiogenic activity is not restricted to a specific mTOR inhibitor rather than is a general phenomenon of mTOR inhibition. All effects could be elicited with both clinically approved mTOR inhibitors rapamycin (sirolimus, Rapamune™) and RAD-001 (everolimus, Certican™). Our findings bear important clinical relevance as mTOR inhibitors are increasingly used in immunosuppressive regimens following organ transplantation to prevent acute and chronic CNI nephrotoxicity.14.Fritsche L. Dragun D. Neumayer H.H. Budde K. Impact of cyclosporine on the development of immunosuppressive therapy.Transplant Proc. 2004; 36: 130S-134SAbstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar However, while providing similar immunosuppressive efficacy, their use as a first-line agents has been challenged by the observed negative effects on postoperative wound healing. Wound edema, wound dehiscence, anastomosis insufficiency and the development of lymphoceles are more frequently observed in mTOR inhibitor-based immunosuppressive regimes compared with conventional CNI treatment. This clinical experience with the use of mTOR inhibitors during the early post-transplant phase is suggestive for a potential antilymphangiogenic effect of mTOR inhibitors. Moreover, even during the later phase following transplantation well beyond the completion of wound healing, lymph edemas at remote locations can complicate mTOR inhibitor-based immunosuppression.6.Chhajed P.N. Dickenmann M. Bubendorf L. et al.Patterns of pulmonary complications associated with sirolimus.Respiration. 2006; 73: 367-374Crossref PubMed Scopus (72) Google Scholar, 7.Fuchs U. Zittermann A. Berthold H.K. et al.Immunosuppressive therapy with everolimus can be associated with potentially life-threatening lingual angioedema.Transplantation. 2005; 79: 981-983Crossref PubMed Scopus (47) Google Scholar, 8.Mahe E. Morelon E. Lechaton S. et al.Cutaneous adverse events in renal transplant recipients receiving sirolimus-based therapy.Transplantation. 2005; 79: 476-482Crossref PubMed Scopus (175) Google Scholar, 9.Wadei H. Gruber S.A. El-Amm J.M. et al.Sirolimus-induced angioedema.Am J Transplant. 2004; 4: 1002-1005Crossref PubMed Scopus (38) Google Scholar Of note, in patients with this adverse effect elicited by a mTOR treatment, withdrawal of the drug regularly leads to a regression of these edemas. Our models of regenerative lymphangiogenesis recapitulate some of the above-mentioned problems and show that the postoperative wound healing problems can at least in part attributed to antilymphangiogenic effects. The skin flap model as well as wound healing assays after skin biopsy (data not shown) suggest that inhibition of lymphangiogenesis is largely independent of the primary wound closure by granulation/scar tissue. These data are further corroborated by the clear inhibition of lymphangiogenesis into Matrigel™ plugs. Furthermore, our in vitro data show an extremely high sensitivity of LECs for mTOR inhibitors. Even at very low concentrations (5–10 ng/ml), far below fibroblast and most tumor cells are affected rapamycin led to an almost complete inhibition of cell proliferation. Taken together, the clinical experience with this new class of immunosuppressive agents in association with our experimental findings strongly support the idea of antilymphangiogenesis in the pathogenesis of some of the mTOR inhibitor induced adverse effects. On the other hand, lymphatic vessels play a major role in cancer biology, as the spread of tumor cells to lymph nodes implicates the lymphatic system as an important route of metastasis and is often an early event in metastatic disease. Therefore, the presence of tumor cells in local lymph nodes is significant for the staging of cancer and largely determines the outcome of tumor patients. It was long supposed that lymphatic metastasis was a passive process whereby detached tumor cells reached lymph nodes via drainage through pre-existing local lymphatic vessels. More recently, it is becoming more apparent that lymphangiogenesis contributes actively to metastasis. Importantly, antilymphangiogenic strategies improved survival in animal tumor models by reducing tumor metastasis.15.Pepper M.S. Lymphangiogenesis and tumor metastasis: myth or reality?.Clin Cancer Res. 2001; 7: 462-468PubMed Google Scholar First clinical evidence for an antilymphatic effect of rapamycin relates to the therapeutic experience in Kaposi sarcomas.16.Stallone G. Schena A. Infante B. et al.Sirolimus for Kaposi's sarcoma in renal-transplant recipients.N Engl J Med. 2005; 352: 1317-1323Crossref PubMed Scopus (778) Google Scholar Kaposi sarcomas are characterized by a high expression of VEGFR-3 suggesting a lymphatic origin of these tumors.17.Weninger W. Partanen T.A. Breiteneder-Geleff S. et al.Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi's sarcoma tumor cells.Lab Invest. 1999; 79: 243-251PubMed Google Scholar Interestingly, in the instance of developing Kaposi sarcomas, conversion to an mTOR inhibitor-based immunosuppression commonly results in regression of these Kaposi sarcomas further corroborating our principle idea that rapamycin may inhibit lymphangiogenesis. Therefore, the antilymphangiogenic as well as antiangiogenic effects of mTOR inhibition may well translate into a reduced incidence of clinically apparent malignancies through reduced tumor growth and lymphatic metastasis, respectively. Indeed, we already demonstrated in our previous experimental studies that rapamycin reduces lymphatic metastasis in a CT26 colon cancer and a L3.6pl pancreatic cancer model.11.Guba M. von Breitenbuch P. Steinbauer M. et al.Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor.Nat Med. 2002; 8: 128-135Crossref PubMed Scopus (1506) Google Scholar Here, we show in a murine lymphangioma model that rapamycin significantly inhibits the development and progression of these benign tumors, suggesting a therapeutic potential of mTOR inhibitors in benign and malignant lymphoproliferative diseases. Thus, the antilymphangiogenic effects of mTOR inhibition complicating the early phase after transplantation may actually be advantageous during later stages to prevent the development, progression, and metastasis of certain tumor entities. Indeed, increasing evidence suggests that mTOR inhibitors reduce the high rate of de novo malignancies after organ transplantation. In conclusion, here we demonstrate that mTOR inhibitors act as antilymphangiogenic agents resulting in adverse effects with respect to postoperative wound healing and edema formation. Therefore, early mTOR inhibition following tissue injury should be avoided. Our data provide the rationale for a delayed used of mTOR inhibitors following surgical interventions such as organ transplantation. Currently, several transplantation centers, including our own, are investigating newly developed early conversion protocols. These protocols are designed to avoid the early side effects of mTOR inhibitors which are, as shown here, very likely to be at least in part related to their antilymphangiogenic properties while maintaining the beneficial effects related to the long-term use of mTOR inhibitors. Moreover, the antilymphangiogenic properties of rapamycin and its derivates may actually provide additional therapeutic value for the prevention and treatment of malignancies, respectively. Currently, mTOR inhibitors are tested in phase I and II trials for their antitumoral effects in a variety of cancer entities. As no pharmacological modulator of lymphangiogenesis has been established to date,18.Saaristo A. Karkkainen M.J. Alitalo K. Insights into the molecular pathogenesis and targeted treatment of lymphedema.Ann N Y Acad Sci. 2002; 979: 94-110Crossref PubMed Scopus (39) Google Scholar mTOR inhibitors may indeed serve as a valuable tool to modulate lymphangiogenesis in different pathological conditions. The effect of rapamycin on lymphatic vessel regeneration was examined in a murine skin flap assay as described elsewhere.19.Saaristo A. Tammela T. Timonen J. et al.Vascular endothelial growth factor-C gene therapy restores lymphatic flow across incision wounds.FASEB J. 2004; 18: 1707-1709PubMed Google Scholar Briefly, under anesthesia (ketamine, xylazine) nude NMRI-mice (Harlan Winkelmann, Borchen, Germany) received an approximately 1 cm2 3/2 oval skin incision to create an epigastric flap. Incisions were closed with single stitched maxon sutures. Loss of lymphatic drainage across the flap was confirmed by intradermal injection of 50 μl of Patent Blue V (2.5%; Guerbet, Roissy, France) into the center of the flap. After surgery, animals were then randomized for treatment with vehicle or rapamycin. Rapamycin (Rapamune™ oral solution dissolved in tap water; 5 mg/kg/day) was orally administered via the drinking water. Control mice received tap water instead. Previous experiments indicated that this rapamycin concentration results in clinically relevant rapamycin blood levels (5–20 ng/ml) (Table 2; high-performance liquid chromatography measurements; courtesy of Dr Vogeser, Central Laboratory, Klinikum Großhadern, Munich, Germany). After 4 weeks, 50 μl of Patent Blue was intradermally injected into the flap. The blue lymphatic vessels were counted after 15 min. All lymphatic vessels that crossed the scar area of the flap incision were considered as crossing vessels. Lymphatic vessels draining Patent Blue caudal of the flap were considered as collateral vessels. After 20 min, lymphatic drainage of Patent Blue was assessed by exposing the axillary lymph nodes. If blue color was detectable in the node, it was considered as a positive lymph node.Table 2Rapamycin blood levels in mice following oral treatment via the drinking water (n≥4 per group)Rapamycin oral dose (mg/kg/day)Rapamycin blood levels (ng/ml)1.01.7±0.32.02.7±0.45.014.4±3.7 Open table in a new tab To determine the effect of rapamycin on lymphatic sprouting in vivo, a Matrigel™ plug assay was carried out as described previously.20.Passaniti A. Taylor R.M. Pili R. et al.A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor.Lab Invest. 1992; 67: 519-528PubMed Google Scholar Balb/c mice were anesthetized as described above. An area around the left hip was shaved and an amount of 500 μl of Matrigel™ (Becton Dickinson, Two Oak Park, Bedford, MA, USA) supplied with VEGF-C (R&D Systems, Minneapolis, MN, USA) at a concentration of 100 ng/ml was injected subcutaneously in the left inguinal area. After 21 days, mice were killed for harvesting of the plug followed by embedding in paraffin and preparation of 5 μm sections. Lymphatic vessels were identified by immunohistochemistry using rabbit primary antibodies against murine LYVE-1 (Acris Antibodies) and a biotinylated goat secondary antibody to rabbit IgG (DakoCytomation, Glostrup, Denmark). Fluorescence labeling was performed with Texas Red Avidin D (Vector, Burlingame, CA, USA) and nuclei were stained with Vectashield-DAPI (Vector). Sections were analyzed using an Axiovert 40 fluorescence microscope (Zeiss, Oberkochen, Germany), the AxioCam MRm camera (Zeiss), and AxioVision 4.4 Software (Zeiss). Ten randomly selected fields from each section were counted to assess the number of lymphatic vessels. C57/BL6 mice were intraperitoneally injected twice on days 0 and 14 with 200 ml of emulsified (1:1 with phosphate-buffered saline) incomplete Freund's adjuvant and simultaneously treated with oral rapamycin or control. After 4 weeks, intra-abdominal angiomas were assessed and then surgically removed for histological analysis. The tissue was fixed in 10% formalin, embedded in paraffin, and cut into sections of 5 μm thickness. Lymphatic vessels were identified by immunostaining for LYVE-1. To further evaluate the effect of rapamycin and the widely used immunosuppressive agent cyclosporine on lymphangioma growth, independent of the immunosuppressive effects on T cells, we performed experiments in athymic NMRI nu/nu mice (8–10 weeks). The therapeutic effect of rapamycin on established lymphangiomas that were generated by injection of Freund's adjuvant on days 0 and 14 followed by randomization to treatment with rapamycin or control on day 16. HDMECs were purchased from PromoCell (Heidelberg, Germany) and cultivated in NUNClonCell T-75 or T-175 flasks (NUNC, Roskilde, Denmark) for up to four passages using Endothelial Cell Growth Medium MV (PromoCell). LECs were isolated by magnetic bead sorting. For this purpose, HDMECs were trypsinized, washed, and resuspended in phosphate-buffered saline. Then, cells were incubated with a biotinylated VEGFR-3 antibody (R&D Systems) for 30 min at 4°C. After another washing step, the cells were incubated with streptavidin-MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) for 20 min at room temperature. Finally, VEGFR-3+ cells were enriched using a MidiMACS magnet and MS columns (Miltenyi Biotech). All magnetic activated cell-sorting procedures were performed according to the manufacturer's instructions. The purity of isolated cells was determined by standard flow cytometry analysis using a primary antibody against human podoplanin (Acris, Hiddenhausen, Germany) and a secondary fluorescein isothiocyanate-labeled antibody against mouse IgG1 (BD Pharmingen, San Diego, CA, USA). The purity of isolated LECs exceeded 95%. HDMECs were incubated with BrdU (10 μM) for 2 h. Then, cells were washed in phosphate-buffered saline and incubated with anti-BrdU-fluorescein isothiocyanate (1:50) for 20 min according to the manufacturer's instructions (BD Pharmingen). LECs were identified by simultaneous staining for the lymphatic marker LYVE-1. For this purpose, HDMECs were incubated with biotinylated antibodies for LYVE-1 (R&D Systems) and PerCP-labeled streptavidin (BD Pharmingen). A total of 2.5 × 105 pretreated LECs were resuspended in 250 μl of MV Basal Medium (PromoCell) containing 2% fetal calf serum and placed in the upper chamber of a modified Boyden chamber filled with Matrigel™ (BioCoat® invasion assay, 8 μm pore size, Becton Dickinson Labware). The upper chamber was placed in a 24–well culture dish containing 500 μl of the medium described above supplemented with 100 ng/ml VEGF-C and rapamycin at a concentration of 20 ng/ml. After 24 h of incubation at 37°C, transmigrated cells were pelleted and scored using a Neubauer chamber. Phosphorylation of the p70S6 kinase at the Thr389 site, the specific phosphorylation site of mTOR,21.Aoki M. Blazek E. Vogt P.K. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt.Proc Natl Acad Sci USA. 2001; 98: 136-141Crossref PubMed Scopus (279) Google Scholar was assessed by Western blotting. Isolated LECs were cultured in Endothelial Cell Growth Medium MV containing all supplements from the original kit except for growth factors and added VEGF-C (R&D Systems) at a concentration of 100 ng/ml, starved (‘diet’ medium: serum and supplement depleted) for 16 h, and subsequently cultivated for 1 h with diet medium containing rapamycin (20 ng/ml) or control. Following this conditioning period, LECs were stimulated with recombinant VEGF-A165 (50 ng/ml; PromoCell), recombinant VEGF-C (200 ng/ml; R&D Systems; binding to both VEGFR-2 and VEGFR-3), or recombinant VEGF-C in which Cys156 is replaced by a Ser residue (VEGF-C*; R&D Systems) as a selective agonist of VEGFR-3.22.Joukov V. Kumar V. Sorsa T. et al.A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities.J Biol Chem. 1998; 273: 6599-6602Crossref PubMed Scopus (190) Google Scholar,23.Veikkola T. Jussila L. Makinen T. et al.Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.EMBO J. 2001; 20: 1223-1231Crossref PubMed Scopus (544) Google Scholar After a 30-min incubation period, cells were lyzed (Tris (tris(hydroxymethyl) aminomethane)–HCl 20 mM, pH 7.5, NaCl 150 mM, Na2EDTA (ethylenediaminetetraacetic acid) 1 mM, EGTA (ethylene glycol tetraacetic acid) 1 mM, 1% Triton-100, sodium pyrophosphate 2.5 mM, β-glycerophosphate 1 mM, leupeptin 1 μg/ml, Na3VO4 1 mM, phenylmethylsulfonyl fluoride 1 mM) and protein degradation was inhibited by protease inhibitor tablets (Cell Signaling Technology, Beverly, MA, USA). Equal amounts of protein extract were separated on polyacrylamide-sodium dodecyl sulfate gels (8%), transferred onto a polyvinylidene difluoride membrane, and probed with rabbit-phospho (Thr389) p70S6 kinase antibody (Cell Signaling Technology). Primary antibody binding was detected with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (sc-2004; Santa Cruz Biotechnology, St Cruz, CA, USA) in an enhanced chemiluminescence Western blotting system (Amersham, GE Healthcare Europe, Munich, Germany). To confirm total protein loading, total actin was detected using a mouse antibody against all six isoforms of actin (ICN Pharmaceuticals Inc., Cappel Product, Aurora, OH, USA) as the primary antibody, followed by a sheep anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Amersham). Results for continuous variables are expressed as means±s.d. Treatment groups were compared with the independent samples t-test. In the case of non-normal distribution, the Mann–Whitney U-test was used. Pair-wise multiple comparisons were performed with the t-test (two-sided) with LSD adjustment. P<0.05 were considered statistically significant. All analyses were performed with SPSS 11.5 (SPSS Inc., Chicago, IL, USA). We thank Sabine Schrepfer, Michael Brueckel, and Christoph von Hesler for their excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SPP 1190: GU 489/3-1 to MG and CJB; FOR 501: HE 3044/2-2 to CH), the Wilhelm Sander Stiftung (no. 2003.133.1 to MG and CJB), Deutsche Krebshilfe/Dr Miltred Scheel Stiftung für Krebsforschung; I0-2029-Br I to CJB) and Baxter BioSurgery, Vienna, Austria (to CC and RH). This report includes data that were generated during the doctoral thesis of Stephan Huber at the medical school of the Ludwig-Maximilians-University Munich, in preparation.
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