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Endogenous Calcitonin Gene–Related Peptide Deficiency Exacerbates Postoperative Lymphedema by Suppressing Lymphatic Capillary Formation and M2 Macrophage Accumulation

2019; Elsevier BV; Volume: 189; Issue: 12 Linguagem: Inglês

10.1016/j.ajpath.2019.08.011

1525-2191

Shuhei Matsui, Megumu Tanaka, Akiko Kamiyoshi, Takayuki Sakurai, Yuka Ichikawa‐Shindo, Hisaka Kawate, Kun Dai, Nanqi Cui, Yangxuan Wei, Masaaki Tanaka, Shinji Kakihara, Keisei Nakamura, Akihiro Yamauchi, Kumiko Ishida, Satoshi Tanaka, Mikito Kawamata, Takayuki Shindo,

Biomarkers in Disease Mechanisms

Lymphedema is a chronic condition caused by disruption of lymphatic vessels, which often occurs after invasive surgery. Calcitonin gene–related peptide (CGRP) is a 37-amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene (Calca). CGRP was initially identified as a neuropeptide released primarily from sensory nerves and involved in regulating pathophysiological nociceptive pain. However, recent studies have shown CGRP is also released from a variety of other cells and possesses multiple functions. In this study, CGRP knockout (−/−) mice were used to show the actions of endogenous CGRP in postoperative lymphedema. After generating a mouse postoperative tail lymphedema model, the edema was observed to be more severe in CGRP−/− mice than in wild-type mice. Numbers of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)–positive lymphatic capillaries were decreased and lymphatic capillary formation–related factors were down-regulated in CGRP−/− mice. In addition, accumulation of M2 but not M1 macrophages was selectively reduced in the edematous tissue of CGRP−/− mice. Selective depletion of M2 macrophages decreased lymphatic capillary formation and worsened lymphedema in wild-type mice but not CGRP−/− mice, where numbers of M2 macrophages were already diminished. These findings suggest that endogenous CGRP acts to ameliorate postoperative lymphedema by enhancing lymphatic capillary formation and that M2 macrophages play critical roles. CGRP may be a useful therapeutic target for the treatment of postoperative lymphedema. Lymphedema is a chronic condition caused by disruption of lymphatic vessels, which often occurs after invasive surgery. Calcitonin gene–related peptide (CGRP) is a 37-amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene (Calca). CGRP was initially identified as a neuropeptide released primarily from sensory nerves and involved in regulating pathophysiological nociceptive pain. However, recent studies have shown CGRP is also released from a variety of other cells and possesses multiple functions. In this study, CGRP knockout (−/−) mice were used to show the actions of endogenous CGRP in postoperative lymphedema. After generating a mouse postoperative tail lymphedema model, the edema was observed to be more severe in CGRP−/− mice than in wild-type mice. Numbers of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)–positive lymphatic capillaries were decreased and lymphatic capillary formation–related factors were down-regulated in CGRP−/− mice. In addition, accumulation of M2 but not M1 macrophages was selectively reduced in the edematous tissue of CGRP−/− mice. Selective depletion of M2 macrophages decreased lymphatic capillary formation and worsened lymphedema in wild-type mice but not CGRP−/− mice, where numbers of M2 macrophages were already diminished. These findings suggest that endogenous CGRP acts to ameliorate postoperative lymphedema by enhancing lymphatic capillary formation and that M2 macrophages play critical roles. CGRP may be a useful therapeutic target for the treatment of postoperative lymphedema. Lymphedema is a chronic condition caused by disruption of lymphatic vessels, often after lymph node resection and radiation therapy. In extreme cases, supermicrosurgical lymphaticovenular anastomosis is applied,1Maegawa J. Yabuki Y. Tomoeda H. Hosono M. Yasumura K. Outcomes of lymphaticovenous side-to-end anastomosis in peripheral lymphedema.J Vasc Surg. 2012; 55: 753-760Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar,2Demirtas Y. Ozturk N. Yapici O. Topalan M. Supermicrosurgical lymphaticovenular anastomosis and lymphaticovenous implantation for treatment of unilateral lower extremity lymphedema.Microsurgery. 2009; 29: 609-618Crossref PubMed Scopus (49) Google Scholar but the operation is not curative, and continued therapy remains necessary. A better understanding of the molecular and cellular mechanisms involved in the regulation of lymphatic vessels will bring new insight to lymphedema treatment. Calcitonin gene–related peptide (CGRP) is a 37-amino acid peptide produced through alternative splicing of the primary transcript of the calcitonin/CGRP gene (Calca).3Rosenfeld M.G. Mermod J.J. Amara S.G. Swanson L.W. Sawchenko P.E. Rivier J. Vale W.W. Evans R.M. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing.Nature. 1983; 304: 129-135Crossref PubMed Scopus (1957) Google Scholar Originally identified as a strong vasodilatory neuropeptide, CGRP is released primarily from sensory nerves and is widely distributed in the central and peripheral nervous systems.3Rosenfeld M.G. Mermod J.J. Amara S.G. Swanson L.W. Sawchenko P.E. Rivier J. Vale W.W. Evans R.M. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing.Nature. 1983; 304: 129-135Crossref PubMed Scopus (1957) Google Scholar, 4Eftekhari S. Salvatore C.A. Johansson S. Chen T.B. Zeng Z. Edvinsson L. Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.Brain Res. 2015; 1600: 93-109Crossref PubMed Scopus (165) Google Scholar, 5Kruger L. Mantyh P.W. Sternini C. Brecha N.C. Mantyh C.R. Calcitonin gene-related peptide (CGRP) in the rat central nervous system: patterns of immunoreactivity and receptor binding sites.Brain Res. 1988; 463: 223-244Crossref PubMed Scopus (190) Google Scholar In addition, CGRP is also expressed in the heart, lungs, digestive system, kidney, and blood vessels.6Kawasaki H. Takasaki K. Saito A. Goto K. Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat.Nature. 1988; 335: 164-167Crossref PubMed Scopus (417) Google Scholar, 7Uddman R. Edvinsson L. Ekblad E. Hakanson R. Sundler F. Calcitonin gene-related peptide (CGRP): perivascular distribution and vasodilatory effects.Regul Pept. 1986; 15: 1-23Crossref PubMed Scopus (285) Google Scholar, 8Villarreal D. Reams G. Freeman R. Calcitonin gene-related peptide and the kidney.Curr Opin Nephrol Hypertens. 1994; 3: 453-458Crossref PubMed Scopus (4) Google Scholar Of the two major forms of CGRP, α and β, α-CGRP is predominantly expressed throughout the central and peripheral nervous systems, whereas β-CGRP is found mainly in the enteric nervous system.9Matteoli M. Haimann C. Torri-Tarelli F. Polak J.M. Ceccarelli B. De Camilli P. Differential effect of alpha-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction.Proc Natl Acad Sci U S A. 1988; 85: 7366-7370Crossref PubMed Scopus (192) Google Scholar,10Rezaeian A.H. Isokane T. Nishibori M. Chiba M. Hiraiwa N. Yoshizawa M. Yasue H. AlphaCGRP and betaCGRP transcript amount in mouse tissues of various developmental stages and their tissue expression sites.Brain Dev. 2009; 31: 682-693Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar CGRP exhibits a wide variety of biological effects in addition to its vasodilatory activity. For example, it now appears to be involved in the pathophysiology of migraine,11Ramos-Romero M.L. Sobrino-Mejia F.E. Rev Neurol. 2016; 63 (Spanish): 460-468PubMed Google Scholar and in the heart it may play a protective role against myocardial ischemia and heart failure.12Chai W. Mehrotra S. Jan Danser A.H. Schoemaker R.G. The role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning in isolated rat hearts.Eur J Pharmacol. 2006; 531: 246-253Crossref PubMed Scopus (84) Google Scholar,13Li J. Levick S.P. DiPette D.J. Janicki J.S. Supowit S.C. Alpha-calcitonin gene-related peptide is protective against pressure overload-induced heart failure.Regul Pept. 2013; 185: 20-28Crossref PubMed Scopus (39) Google Scholar CGRP also reportedly facilitates angiogenesis in response to ischemia,14Mishima T. Ito Y. Hosono K. Tamura Y. Uchida Y. Hirata M. Suzsuki T. Amano H. Kato S. Kurihara Y. Kurihara H. Hayashi I. Watanabe M. Majima M. Calcitonin gene-related peptide facilitates revascularization during hindlimb ischemia in mice.Am J Physiol Heart Circ Physiol. 2011; 300: H431-H439Crossref PubMed Scopus (57) Google Scholar,15Zheng S. Li W. Xu M. Bai X. Zhou Z. Han J. Shyy J.Y. Wang X. Calcitonin gene-related peptide promotes angiogenesis via AMP-activated protein kinase.Am J Physiol Cell Physiol. 2010; 299: C1485-C1492Crossref PubMed Scopus (57) Google Scholar improves blood flow and vascular endothelial function,16Just S. Arndt K. Doods H. The role of CGRP and nicotinic receptors in centrally evoked facial blood flow changes.Neurosci Lett. 2005; 381: 120-124Crossref PubMed Scopus (23) Google Scholar,17Wang Z. Martorell B.C. Walchli T. Vogel O. Fischer J. Born W. Vogel J. Calcitonin gene-related peptide (CGRP) receptors are important to maintain cerebrovascular reactivity in chronic hypertension.PLoS One. 2015; 10: e0123697Crossref PubMed Scopus (21) Google Scholar and plays an important role in inflammation.18Fernandes E.S. Schmidhuber S.M. Brain S.D. Sensory-nerve-derived neuropeptides: possible therapeutic targets.Handb Exp Pharmacol. 2009; : 393-416Crossref PubMed Scopus (40) Google Scholar,19Permpoonputtana K. Porter J.E. Govitrapong P. Calcitonin gene-related peptide mediates an inflammatory response in Schwann cells via cAMP-dependent ERK signaling cascade.Life Sci. 2016; 144: 19-25Crossref PubMed Scopus (18) Google Scholar From its structural homology and similar vasodilatory effects, CGRP is thought to be part of the peptide family that also includes adrenomedullin (ADM). Like CGRP, ADM is secreted by numerous tissues and organs and exerts a variety of effects. Moreover, CGRP, ADM, and other peptides in this family share a common seven transmembrane G protein–coupled receptor, calcitonin receptor–like receptor (CLR).20McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor.Nature. 1998; 393: 333-339Crossref PubMed Scopus (1854) Google Scholar The specific affinities of CLR for its various ligands are determined by three accessory proteins called receptor activity–modifying proteins (RAMP1-3).20McLatchie L.M. Fraser N.J. Main M.J. Wise A. Brown J. Thompson N. Solari R. Lee M.G. Foord S.M. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor.Nature. 1998; 393: 333-339Crossref PubMed Scopus (1854) Google Scholar When associated with RAMP1, CLR has higher affinity for CGRP; association with RAMP2 or RAMP3 gives CLR a higher affinity for ADM. With the use of RAMP1 knockout (−/−) mice, Kurashige et al21Kurashige C. Hosono K. Matsuda H. Tsujikawa K. Okamoto H. Majima M. Roles of receptor activity-modifying protein 1 in angiogenesis and lymphangiogenesis during skin wound healing in mice.FASEB J. 2014; 28: 1237-1247Crossref PubMed Scopus (39) Google Scholar provided evidence that RAMP1 expressed by macrophages is an important mediator of lymphatic capillary formation during skin wound healing. However, the RAMP2/CLR and RAMP3/CLR complexes also have affinity for CGRP, although lower than that of RAMP1/CLR complex. In addition, the RAMP1/CLR complex also functions as a receptor for other members of the calcitonin superfamily, including calcitonin, intermedin, and amylin. Consequently, it remains unclear whether CGRP directly regulates lymphatic capillary formation. Clarification of the precise function of CGRP in lymphatic capillary formation is necessary for the potential therapeutic application of CGRP to the treatment of lymphedema. In this study, therefore, CGRP−/− mice were used to analyze the actions of endogenous CGRP in postoperative lymphedema. CGRP is encoded by the same gene as calcitonin. α-CGRP−/− mice were generated with the use of a targeting DNA construct that replaced exon 5, encoding a CGRP-specific region, to prevent the effects of calcitonin deficiency.22Oh-hashi Y. Shindo T. Kurihara Y. Imai T. Wang Y. Morita H. Imai Y. Kayaba Y. Nishimatsu H. Suematsu Y. Hirata Y. Yazaki Y. Nagai R. Kuwaki T. Kurihara H. Elevated sympathetic nervous activity in mice deficient in alphaCGRP.Circ Res. 2001; 89: 983-990Crossref PubMed Scopus (135) Google Scholar C57BL/6J pure-background male mice were used. Male 8- to 10-week–old CGRP−/− mice and their wild-type (WT) littermates were used in this study. All animal experiments were performed in accordance with the ethical guidelines of Shinshu University. Before all invasive procedures, mice were anesthetized through intraperitoneal injection of 2,2,2-tribromoethanol (240 mg/kg; Wako, Osaka, Japan). Tail lymphedema was induced as described previously with modification.23Jin D. Harada K. Ohnishi S. Yamahara K. Kangawa K. Nagaya N. Adrenomedullin induces lymphangiogenesis and ameliorates secondary lymphoedema.Cardiovasc Res. 2008; 80: 339-345Crossref PubMed Scopus (50) Google Scholar, 24Mishima T. Ito Y. Nishizawa N. Amano H. Tsujikawa K. Miyaji K. Watanabe M. Majima M. RAMP1 signaling improves lymphedema and promotes lymphangiogenesis in mice.J Surg Res. 2017; 219: 50-60Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 25Kashiwagi S. Hosono K. Suzuki T. Takeda A. Uchinuma E. Majima M. Role of COX-2 in lymphangiogenesis and restoration of lymphatic flow in secondary lymphedema.Lab Invest. 2011; 91: 1314-1325Crossref PubMed Scopus (34) Google Scholar In brief, a 3-mm–wide ring of skin was removed 1 cm distal to the base of the tail. Mouse tail skin consists of a regular and hexagonal network of dermal lymphatic capillaries.26Hagendoorn J. Padera T.P. Kashiwagi S. Isaka N. Noda F. Lin M.I. Huang P.L. Sessa W.C. Fukumura D. Jain R.K. Endothelial nitric oxide synthase regulates microlymphatic flow via collecting lymphatics.Circ Res. 2004; 95: 204-209Crossref PubMed Scopus (82) Google Scholar, 27Pytowski B. Goldman J. Persaud K. Wu Y. Witte L. Hicklin D.J. Skobe M. Boardman K.C. Swartz M.A. Complete and specific inhibition of adult lymphatic regeneration by a novel VEGFR-3 neutralizing antibody.J Natl Cancer Inst. 2005; 97: 14-21Crossref PubMed Scopus (214) Google Scholar, 28Swartz M.A. Berk D.A. Jain R.K. Transport in lymphatic capillaries. I. Macroscopic measurements using residence time distribution theory.Am J Physiol. 1996; 270: H324-H329Crossref PubMed Google Scholar A circumferential incision was made through the dermis to sever the dermal lymphatic capillaries without damaging the major blood or lymphatic vessels, which are located deeper within the tail, close to the bony/cartilaginous portion. Care was taken to maintain the integrity of the major underlying vessels and tendons so that the tail distal to the incision did not become necrotic. The injured region of the tail was then wrapped with adhesive tape to protect the surgical site from infection. The horizontal tail diameter was measured with a Vernier caliper 1 cm from the distal edge of the injury, where a circumferential mark was made on the tail. For pathologic examination, sections of the complete tail were collected 0.5 to 1 cm distal to the wounds. For gene expression analysis, tail skin and subcutaneous tissues (without muscle, bone, or cartilage) were collected 1 to 2 cm distal to the wounds. Mannosylated clodronate liposomes (MCLs) selectively deplete M2 macrophages.29Zhou Y. Yoshida S. Nakao S. Yoshimura T. Kobayashi Y. Nakama T. Kubo Y. Miyawaki K. Yamaguchi M. Ishikawa K. Oshima Y. Akashi K. Ishibashi T. M2 macrophages enhance pathological neovascularization in the mouse model of oxygen-induced retinopathy.Invest Ophthalmol Vis Sci. 2015; 56: 4767-4777Crossref PubMed Scopus (52) Google Scholar MCLs or control liposomes (Encapsular Nano Sciences, Brentwood, TN) were injected into the tail vein. In each mouse, 0.01 mg/g MCLs were injected 3 days before (day −3) and on days 0, 4, and 7 after the operation. Tissues were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm–thick sections for histologic examination. The specimens were then deparaffinized for hematoxylin/eosin staining or immunohistochemistry with anti-CD31 (BD Biosciences, San Jose, CA), anti–LYVE-1 (Relia Tech GmbH, Wolfenbüttel Germany), anti–α-CGRP (Sigma-Aldrich, St. Louis, MO), anti-CD68 antibody (Abcam, Cambridge, UK), anti-F4/80 (Bio-Rad, Hercules, CA), anti-CD11c (Abcam), anti-Arginase1 (Genetex, Irvine, CA), or anti-CD206 antibodies (Abcam). Nuclei were stained with DAPI (Life Technologies, Carlsbad, CA). Fluorescence was observed with a KEYENCE model BZ-X710 fluorescence microscope (Osaka, Japan) equipped with the appropriate filter sets. Measurements of the luminal areas of capillaries and numbers of macrophages were made with Image J software version 1.52 (https://imagej.nih.gov/ij/download.html). To examine the morphologic structure of tail lymphatic capillaries, the tails of anesthetized mice were injected intradermally with 10 μL of indocyanine green (Daiichi-Sankyo, Tokyo, Japan) at a site 3 cm from the base of the tail. Images were then taken with a Heidelberg Retina Angiograph 2 (Heidelberg Engineering GmbH, Heidelberg, Germany) 30 minutes after the tail injection. Total RNA was extracted from tissues with the use of Trireagent (Molecular Research Center, Inc., Cincinnati, OH), after which time the RNA was treated with DNA-Free (Ambion, Naugatuck, CT) to remove contaminating DNA and was reverse transcribed with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time quantitative RT-PCR was performed with an Applied Biosystems 7300 real-time PCR System with SYBR green (Toyobo, Osaka, Japan) or Realtime PCR Master Mix (Toyobo) and TaqMan probes (MBL, Aichi, Japan). The primers and probes used are listed in Table 1. Values were normalized to mouse glyceraldehyde-3-phosphate dehydrogenase (Pre-Developed TaqMan assay reagents; Applied Biosystems).Table 1Primers and Probes Used for Real-Time Quantitative RT-PCRGenePrimerCalca (CGRP)Forward5′-TCCTGCAACACTGCCACCT-3′Reverse5′-ACACCTCCTGATCTGCTCAGC-3′Probe5′-TGTGACCCATCGGCTGGCAGG-3′Adm (Adrenomedullin)Forward5′-CTACCGCCAGAGCATGAACC-3′Reverse5′-GAAATGTGCAGGTCCCGAA-3′Probe5′-CCCGCAGCAATGGATGCCG-3′Calcr (CLR)Forward5′-AGGCGTTTACCTGCACACACT-3′Reverse5′-CAGGAAGCAGAGGAAACCCC-3′Probe5′-ATCGTGGTGGCTGTGTTTGCGGAG-3′Ramp1 (RAMP1)Forward5′-GCACTGGTGGTCTGGAGGA-3′Reverse5′-CCCTCATCACCTGGGATACCT-3′Probe5′-CAAGCGCACAGAGGGCATCGTG-3′Ramp2 (RAMP2)Forward5′-GCAGCCCACCTTCTCTGATC-3′Reverse5′-AACGGGATGAGGCAGATGG-3′Probe5′-CCCAGAGGATGTGCTCCTGGCCAT-3′Ramp3 (RAMP3)Forward5′-TGCAACGAGACAGGGATGC-3′Reverse5′-GCATCATGTCAGCGAAGGC-3′Probe5′-AGAGGCTGCCTCGCTGTGGGAA-3′Pecam1 (CD31)Forward5′-CTGCAGGCATCGGCAAA-3′Reverse5′-GCATTTCGCACACCTGGAT-3′Vegfa (VEGF-A)Forward5′-GCAGGCTGCTGTAACGATGA-3′Reverse5′-CGCATGATCTGCATGGTGAT-3′Flt1 (VEGFR-1)Forward5′-CAAGGACGGCTTTGCAGATC-3′Reverse5′-GCTCATGAATTTGAAAGCGTTTAC-3′Lyve1 (LYVE-1)Forward5′-AAGCAGCTGGGTTTGGAGGT-3′Reverse5′-CACCAAAGAAGAGGAGAGCCA-3′Prox1 (PROX1)Forward5′-CGGGTTGAGAATATCATTC-3′Reverse5′-TCTTTCGTTTTCATTGCCCC-3′Pdpn (Podoplanin)Forward5′-TGGCAAGGCACCTCTGGTA-3′Reverse5′-TGAGGTGGACAGTTCCTCTAAGG-3′Vegfc (VEGF-C)Forward5′-CCTCTCTCACAAGGCCCCA-3′Reverse5′-TAGACATGCACCGGCAGGA-3′Flt4 (VEGFR-3)Forward5′-AAGGCCTGCCCATGCA-3′Reverse5′-TCGCCAGGGTCCATGATG-3′Il1b (IL-1β)Forward5′-CTACAGGCTCCGAGATGAACAAC-3′Reverse5′-TCCATTGAGGTGGAGAGCTTTC-3′Il6 (IL-6)Forward5′-CCCAATTTCCAATGCTCTCC-3′Reverse5′-TGAATTGGATGGTCTTGGTCC-3′Tnf (TNFα)Forward5′-ACGGCATGGATCTCAAAGAC-3′Reverse5′-AGATAGCAAATCGGCTGACG-3′Il10 (IL-10)Forward5′-CAGCCGGGAAGACAATAACTG-3′Reverse5′-CCGCAGCTCTAGGAGCATGT-3′Adgre1 (F4/80)Forward5′-GATGAATTCCCGTGTTGTTGGT-3′Reverse5′-ACATCAGTGTTCCAGGAGACACA-3′Cd68 (CD68)Forward5′-TGGCGGTGGAATACAATGTG-3′Reverse5′-GAGATGAATTCTGCGCCATGA-3′Nos2 (iNOS)Forward5′-ATGTGGCTACCACATTGAAGAAGC-3′Reverse5′-AAGACTGCACCGAAGATATCTTCATG-3′Itgax (CD11c)Forward5′-CTGGATAGCCTTTCTTCTGCTG-3′Reverse5′-GCACACTGTGTCCGAACTC-3′Arg1 (Arginase1)Forward5′-TTAGGCCAAGGTGCTTGCTGCC-3′Reverse5′-TACCATGGCCCTGAGGAGGTTC-3′Mrc1 (CD206)Forward5′-TCAGCTATTGGACGCGAGGCA-3′Reverse5′-TCCGGGTTGCAAGTTGCCGT-3′Retnla (Fizz1)Forward5′-TCCCAGTGAATACTGATGAGA-3′Reverse5′-CCACTCTGGATCTCCCAAGA-3′CGRP, calcitonin gene–related peptide; CLR, calcitonin receptor–like receptor; iNOS, inducible nitric oxide synthase; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; RAMP, receptor activity–modifying protein; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Open table in a new tab CGRP, calcitonin gene–related peptide; CLR, calcitonin receptor–like receptor; iNOS, inducible nitric oxide synthase; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; RAMP, receptor activity–modifying protein; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Statistical analysis was performed with GraphPad Prism software version 7.03. (GraphPad Software Inc., San Diego, CA). Values are expressed as means ± SEM. Comparisons among multiple groups were made with analysis of variance, followed by Sidak's test. Comparisons between two groups were made with t-test. P < 0.05 was considered statistically significant. The tails of mice contain a highly regular network of lymphatic capillaries in the skin. Consequently, circumferential removal of the skin from the tail obstructs lymphatic flow, resulting in acute lymphedema and enabling this system to serve as a model of secondary lymphedema.23Jin D. Harada K. Ohnishi S. Yamahara K. Kangawa K. Nagaya N. Adrenomedullin induces lymphangiogenesis and ameliorates secondary lymphoedema.Cardiovasc Res. 2008; 80: 339-345Crossref PubMed Scopus (50) Google Scholar The time course of postoperative lymphedema was evaluated by measuring tail diameters in WT and CGRP−/− mice. After the surgical procedure, the tails of all treated mice became edematous, with the edema reaching a peak on postoperative days 10 to 12 in both WT and CGRP−/− mice. Thereafter, the tail edema gradually resolved. Notably, from postoperative day 6 to day 16, tail thickness was significantly greater in CGRP−/− mice than in WT mice (Figure 1, A and B ). Cross sections of the tails also revealed enhanced postoperative edema in CGRP−/− mice (Figure 1C). Although no difference between WT and CGRP−/− mice was detected before surgery (day 0), the tails of CGRP−/− mice showed significantly greater swelling on postoperative day 10. With the use of this tail lymphedema model, the lymphatic capillaries within lymphedematous sites were visualized by immunostaining sections for the lymphatic marker LYVE-1 (Figure 2A). Although the numbers of LYVE-1–positive lymphatic capillaries (Figure 2B) and the sums of their luminal areas (Figure 2C) were increased after the surgery in both WT and CGRP−/− mice, they were significantly smaller in CGRP−/− mice than in WT mice on day 10. Immunostaining was also performed for CD31 to assess changes in blood vessels within the injury site, but neither the numbers nor the luminal areas differed between WT and CGRP−/− mice (Supplemental Figure S1, A and B). These results clearly indicated specific impairment of lymphatic capillary formation with no effect on angiogenesis in CGRP−/− mice. To analyze the morphologic structure of lymphatic capillaries, indocyanine green was subcutaneously injected into the tail tip and then observed with fluorescence microscopy 30 minutes after the injection. WT mice showed a honeycomb-like pattern of lymphatic vessels, whereas this pattern was disrupted in CGRP−/− mice (Figure 2D). The gene expression levels of Calca (CGRP), Adm (Adrenomedullin), Calcr (CLR), Ramp1, Ramp2, and Ramp3 were analyzed in the lymphedematous tails of WT and CGRP−/− mice (Figure 3). Calca (CGRP) expression was significantly lower in CGRP−/− than in WT mice, although a slight and nonspecific signal was detected, which is consistent with earlier observations.30Zhai L. Sakurai T. Kamiyoshi A. Ichikawa-Shindo Y. Kawate H. Tanaka M. Xian X. Hirabayashi K. Dai K. Cui N. Tanimura K. Liu T. Wei Y. Tanaka M. Tomiyama H. Yamauchi A. Igarashi K. Shindo T. Endogenous calcitonin gene-related peptide suppresses ischemic brain injuries and progression of cognitive decline.J Hypertens. 2018; 36: 876-891Crossref PubMed Scopus (26) Google Scholar Expression of Adm (Adrenomedullin), whose functions somewhat overlap those of CGRP, was significantly elevated in CGRP−/− mice on postoperative day 10, possibly as a compensatory response to the CGRP deficiency. However, components of the CGRP and ADM receptors [Calcr (CLR) and Ramp1-3] were all down-regulated in CGRP−/− mice compared with WT mice. Immunostaining was also performed for CGRP to compare its distribution in WT and CGRP−/− mice (Supplemental Figure S2, A and B). CGRP was significantly but transiently up-regulated in the postoperative lymphedematous tails of WT mice, with levels peaking on postoperative day 10. By contrast, no CGRP was detected in CGRP−/− mice. With the use of real-time RT-PCR, it was observed that in both WT and CGRP−/− mice, expression of the angiogenesis-related factors Pecam1 (CD31), Vegfa, and Flt1 [vascular endothelial growth factor receptor (VEGFR)-1] was unaffected by the operative procedure (Figure 4A). By contrast, the lymphatic capillary formation–related factors Lyve1, Prox1, Vegfc, and Flt4 (VEGFR-3) were transiently up-regulated on postoperative day 10. However, expression of these factors was significantly lower in CGRP−/− than in WT mice on postoperative day 10 (Figure 4B). Analysis of cytokine expression revealed that, although the expression of the inflammatory cytokine Il1b was slightly up-regulated on postoperative day 10 in WT mice, the up-regulation was significantly more prominent in CGRP−/− mice (Figure 5A). However, expression of anti-inflammatory cytokine Il10 was significantly lower in CGRP−/− than in WT mice before surgery (day 0). These results suggested there was greater postoperative inflammation in CGRP−/− mice than in WT mice. The expression of macrophage markers in the postoperative lymphedema model was also analyzed (Figure 5B). The pan-macrophage markers Adgre1 (F4/80) and Cd68 were significantly lower in CGRP−/− mice than in WT mice before surgery (day 0) and on postoperative day 10. Although expression of Itgax (CD11c) and Nos2 (inducible nitric oxide synthase), two M1 macrophage markers, were reduced before the surgery in CGRP−/− mice, the difference became undetectable after the surgery. By contrast, expression of three M2 macrophage markers, Mrc1 (CD206), Arg1 (Arginase1), and Retnla (Fizz1), were significantly lower in CGRP−/− mice than in WT mice on postoperative day 10. These observations suggested that the reduction in F4/80 and CD68+ macrophages in CGRP−/− mice on postoperative day 10 mainly reflected a reduction of M2 macrophages. To confirm this finding, double immunostaining was performed for the pan-macrophage marker CD68 and the M1 macrophage marker CD11c. The number of M1 macrophages positive for both CD68 and CD11c did not differ between WT and CGRP−/− mice (Figure 6). Double-immunostaining was performed for the pan-macrophage marker CD68 and the M2 macrophage marker CD206, or the pan-macrophage marker F4/80 and the M2 macrophage marker Arginase1. In contrast to M1 macrophages, numbers of M2 macrophages, positive for both CD68 and CD206 (Figure 7, A–C ) or F4/80 and Arginase1 (Figure 7, D–F), were significantly lower in CGRP−/− mice than in WT mice.Figure 7M2 macrophage numbers are decreased in calcitonin gene–related peptide knockout (CGRP−/−) mice. A: Fluorescently immunostained cross sections from the tails of wild-type (WT) and CGRP−/− mice in the postoperative lymphedema model on day 10 after surgery: red indicates CD68 (pan-macrophage marker); green, CD206 (M2 macrophage marker); blue, DAPI. Sections were collected 0.5 to 1 cm distal to the wounds. B: Numbers of CD68+ cells in cross sections from the tails of WT and CGRP−/− mice on day 10 after surgery. C: Numbers of CD68+ and CD206+ cells in cross sections from the tails of WT and CGRP−/− mice on postoperative day 10. D: Fluorescently immunostained cross sections from the tails of WT and CGRP−/− mice in the postoperative lymphedema model on day 10 after surgery: red indicates F4/80 (pan-macrophage marker); green, Arginase1 (M2 macrophage marker); blue, DAPI. Sections were collected 0.5 to 1 cm distal to the wounds. E: Numbers of F4/80-positive cells in cross sections from the tails of WT and CGRP−/− mice on day 10 after surgery. F: Numbers of F4/80 and Arginase1–double-positive (F4/80+ and Arginase1+) cells in cross sections from the tails of WT and CGRP−/− mice on postoperative day 10. Data are expressed as means ± SEM. n = 5 in each group (B, C, E, and F). Scale bars: 100 μm (A and D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To clarify the relationship between the reduction in M2 macrophages and edema in CGRP−/− mice, the effect of M2 macrophage depletion was assessed with MCLs in the postoperative lymphedema model. MCLs are selectively phagocytosed by M2 macrophages expressing mannose receptors, which causes cell death.29Zhou Y. Yoshida S. Nakao S. Yoshimura T. Kobayashi Y. Nakama T. Kubo Y. Miyawaki K. Yamaguchi M. Ishikawa K. Oshima Y. Akashi K. Ishibashi T. M2 macrophages enhance pathological neovascularization in the mouse model of oxygen-induced retinopathy.Invest Ophthalmol Vis Sci. 2015; 56: 4767-4777Crossref PubMed Scopus (52) Goo