The Vascular Endothelial Growth Factor Inhibitor Soluble FLT-1 Ameliorates Atopic Dermatitis in APOC1 Transgenic Mice
2019; Elsevier BV; Volume: 140; Issue: 2 Linguagem: Inglês
10.1016/j.jid.2019.07.700
ISSN1523-1747
AutoresCleo C. L. van Aanhold, Pascal Bus, Malu Zandbergen, Manon Bos, Jimmy F.P. Berbée, Koen D. Quint, Jan A. Bruijn, Hans J. Baelde,
Tópico(s)Circadian rhythm and melatonin
ResumoAtopic dermatitis (AD) is an inflammatory skin condition characterized by scaling, lichenification, excoriations, and pruritus. Vascular endothelial growth factor (VEGF) has previously been underscored as a contributor to skin inflammation in AD (Varricchi et al., 2015Varricchi G. Granata F. Loffredo S. Genovese A. Marone G. Angiogenesis and lymphangiogenesis in inflammatory skin disorders.J Am Acad Dermatol. 2015; 73: 144-153Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This is supported by the evidence that (1) VEGF causes hyperpermeability of vessels (Ferrara et al., 2003Ferrara N. Gerber H.P. LeCouter J. The biology of VEGF and its receptors.Nat Med. 2003; 9: 669-676Crossref PubMed Scopus (7333) Google Scholar) leading to edema and spongiosis, (2) VEGF induces chemotaxis of myeloid cells expressing VEGF receptor 1 (e.g., macrophages, mast cells) (Sawano et al., 2001Sawano A. Iwai S. Sakurai Y. Ito M. Shitara K. Nakahata T. et al.Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans.Blood. 2001; 97: 785-791Crossref PubMed Scopus (397) Google Scholar), and (3) VEGF activates endothelial cells, leading to increased expression of adhesion molecules in these cells (Kim et al., 2001Kim I. Moon S.O. Kim S.H. Kim H.J. Koh Y.S. Koh G.Y. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells.J Biol Chem. 2001; 276: 7614-7620Crossref PubMed Scopus (598) Google Scholar), thereby promoting tissue infiltration of leukocytes. Moreover, VEGF levels are increased in skin lesions and plasma of patients with AD (Varricchi et al., 2015Varricchi G. Granata F. Loffredo S. Genovese A. Marone G. Angiogenesis and lymphangiogenesis in inflammatory skin disorders.J Am Acad Dermatol. 2015; 73: 144-153Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Thus, VEGF-inhibiting therapy may be a promising treatment for AD. Soluble VEGF receptor 1 or soluble fms-like tyrosine kinase-1 (sFLT-1) is a natural inhibitor of VEGF-A by acting as a decoy receptor (Shibuya, 2015Shibuya M. VEGF-VEGFR system as a target for suppressing inflammation and other diseases.Endocr Metab Immune Disord Drug Targets. 2015; 15: 135-144Crossref PubMed Scopus (60) Google Scholar). Treatment with low doses of sFLT-1 reduces inflammation and disease severity in preclinical models of several inflammatory diseases. In this study, we investigated the anti-inflammatory effects of sFLT-1 on AD in a mouse model. APOC1 transgenic (APOC1-tg) mice develop AD because of a disruption of the skin lipid barrier (Nagelkerken et al., 2008Nagelkerken L. Verzaal P. Lagerweij T. Persoon-Deen C. Berbee J.F. Prens E.P. et al.Development of atopic dermatitis in mice transgenic for human apolipoprotein C1.J Invest Dermatol. 2008; 128: 1165-1172Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and a triggered immune system (Bus et al., 2017aBus P. Pierneef L. Bor R. Wolterbeek R. van Es L.A. Rensen P.C. et al.Apolipoprotein C-I plays a role in the pathogenesis of glomerulosclerosis.J Pathol. 2017; 241: 589-599Crossref PubMed Scopus (5) Google Scholar). While manifesting many AD hallmarks, the APOC1-tg model does not fully replicate human disease. In this study, we transfected APOC1-tg mice with sFlt-1 (Supplementary Figure S1). Detailed information on materials and methods used is described in Supplementary Materials. All work was approved by the Animal Experiments Committee (DEC license 13163). Consistent with a previous report (Nagelkerken et al., 2008Nagelkerken L. Verzaal P. Lagerweij T. Persoon-Deen C. Berbee J.F. Prens E.P. et al.Development of atopic dermatitis in mice transgenic for human apolipoprotein C1.J Invest Dermatol. 2008; 128: 1165-1172Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), APOC1-tg mice, and not age-matched wild-type (WT) mice, spontaneously developed AD from 6 weeks of age onward (Figure 1). Transfection of 8-week-old APOC1-tg mice with sFlt-1 completely resolved skin lesions, and hair growth recurred (Figure 1c and f). Quantification of the thickness of the epidermal layer at 15 weeks after transfection showed that sFLT-1 normalized epidermal thickening in APOC1-tg mice (Figure 1g). Furthermore, F4/80-positive areas and numbers of CD3+ cells were increased in the skin of nontransfected APOC1-tg mice and were reduced to levels observed in WT in sFlt-1–transfected APOC1-tg mice (Figure 1h and i, and Supplementary Figure S2). Neutrophil infiltrates were also present in the skin of APOC1-tg mice (Supplementary Figure S2g) but were not observed in transfected mice (Supplementary Figure S2h). We next determined whether sFLT-1 reduces serum levels of proinflammatory cytokines in APOC1-tg mice. No differences were observed in tumor necrosis factor–α levels among groups in sera collected at 15 weeks after transfection. Nonetheless, APOC1-tg mice had markedly increased serum IL-6 levels compared with those of WT mice, which were normalized after transfection with sFlt-1 (Figure 1j). Thus, sFLT-1 reduces skin lesions and inflammation in APOC1-tg mice. Because we previously demonstrated that sFLT-1 decreases VEGF-induced endothelial cell activation (Bus et al., 2017bBus P. Scharpfenecker M. Van Der Wilk P. Wolterbeek R. Bruijn J.A. Baelde H.J. The VEGF-A inhibitor sFLT-1 improves renal function by reducing endothelial activation and inflammation in a mouse model of type 1 diabetes.Diabetologia. 2017; 60: 1813-1821Crossref PubMed Scopus (0) Google Scholar), we next determined whether sFLT-1 reduces skin inflammation in APOC1-tg mice by decreasing expression of the adhesion molecule, VCAM-1. Compared with WT mice, VCAM-1 was markedly increased in endothelial cells in the skin of APOC1-tg mice (Figure 2a and Supplementary Figure S3). Transfection with sFlt-1 reduced VCAM-1 expression to WT (Figure 2c and d). In line with this, VEGF expression was increased in the skin of APOC1-tg mice compared with that of WT mice (Figure 2e). This suggests that sFLT-1 reduces VEGF-increased endothelial activation in the skin of APOC1-tg mice by sequestering increased VEGF levels. Other functions of sFLT-1 besides sequestering VEGF have been described; sFLT-1 plays an essential role in podocyte cell morphology and glomerular barrier function—independent of VEGF—by binding directly to the glycosphingolipid monosialodihexosylganglioside (GM3) in lipid rafts on the surface of the podocytes (Jin et al., 2012Jin J. Sison K. Li C. Tian R. Wnuk M. Sung H.K. et al.Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function.Cell. 2012; 151: 384-399Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Monocytes also express GM3, and this expression is increased upon differentiation of monocytes into macrophages and during inflammatory responses (Gracheva et al., 2007Gracheva E.V. Samovilova N.N. Golovanova N.K. Andreeva E.R. Andrianova I.V. Tararak E.M. et al.Activation of ganglioside GM3 biosynthesis in human monocyte/macrophages during culturing in vitro.Biochemistry (Mosc). 2007; 72: 772-777Crossref PubMed Scopus (15) Google Scholar, Puryear et al., 2012Puryear W.B. Yu X. Ramirez N.P. Reinhard B.M. Gummuluru S. HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells.Proc Natl Acad Sci USA. 2012; 109: 7475-7480Crossref PubMed Scopus (78) Google Scholar). Therefore, sFLT-1 may bind to monocytes or macrophages via glycosphingolipid monosialodihexosylganglioside, subsequently altering cellular function. This notion is supported by the finding that sFLT-1 downregulates the FLT-1 receptor in leukocytes by decreasing the activity of the FLT-1 promoter, thereby preventing subsequent migration of these cells upon stimulation with VEGF (Krysiak et al., 2005Krysiak O. Bretschneider A. Zhong E. Webb J. Hopp H. Verlohren S. et al.Soluble vascular endothelial growth factor receptor-1 (sFLT-1) mediates downregulation of FLT-1 and prevents activated neutrophils from women with preeclampsia from additional migration by VEGF.Circ Res. 2005; 97: 1253-1261Crossref PubMed Scopus (34) Google Scholar). Thus, the anti-inflammatory effects of sFLT-1 on AD in APOC1-tg mice may be explained by VEGF sequestration and by a VEGF-independent mechanism. Treatment with VEGF inhibitors has been widely associated with nephrotoxicity (Estrada et al., 2019Estrada C.C. Maldonado A. Mallipattu S.K. Therapeutic inhibition of VEGF signaling and associated nephrotoxicities.J Am Soc Nephrol. 2019; 30: 187-200Crossref PubMed Scopus (37) Google Scholar). We previously found that sFlt-1 transfection reversed kidney inflammation and damage in diabetic mice (Bus et al., 2017bBus P. Scharpfenecker M. Van Der Wilk P. Wolterbeek R. Bruijn J.A. Baelde H.J. The VEGF-A inhibitor sFLT-1 improves renal function by reducing endothelial activation and inflammation in a mouse model of type 1 diabetes.Diabetologia. 2017; 60: 1813-1821Crossref PubMed Scopus (0) Google Scholar). In line with this, this study found that APOC1-tg mice transfected with sFlt-1 do not develop renal dysfunction or histological changes in the kidney (Supplementary Figure S4), although APOC1-tg mice are prone to develop kidney disease at a later age (Bus et al., 2017aBus P. Pierneef L. Bor R. Wolterbeek R. van Es L.A. Rensen P.C. et al.Apolipoprotein C-I plays a role in the pathogenesis of glomerulosclerosis.J Pathol. 2017; 241: 589-599Crossref PubMed Scopus (5) Google Scholar). In contrast, sFLT-1 reduced glomerular inflammation in APOC1-tg mice; however, this did not reach significance within the studied time frame (Supplementary Figure S4). This suggests that sFLT-1 does not lead to kidney dysfunction and exerts its anti-inflammatory effects systemically. sFLT-1 may be a safe and promising treatment for several inflammatory diseases. In summary, we show that sFLT-1 ameliorates skin lesions and inflammation in an AD mouse model. In APOC1-tg mice transfected with sFlt-1, thickening of the epidermis and inflammatory infiltrates in the skin remitted, along with normalization of serum levels of IL-6 and skin expression of VCAM-1, without developing renal problems during the studied period. Thus, this study shows that sFLT-1 may be a valuable treatment for patients with AD. Datasets related to this article can be found at https://doi.org/10.17632/3rrcdhcmgr.2, hosted at Mendeley (Van Aanhold, 2019Van Aanhold C.C.L. Dataset sFLT-1 ameliorates AD in APOC1-tg mice.Mendeley. 2019; Google Scholar). Cleo C.L. van Aanhold: http://orcid.org/0000-0001-7760-7781 Pascal Bus: http://orcid.org/0000-0001-9833-2887 Malu Zandbergen: http://orcid.org/0000-0003-3671-4059 Manon Bos: http://orcid.org/0000-0002-8636-888X Jimmy F.P. Berbée: http://orcid.org/0000-0001-9133-3297 Koen D. Quint: http://orcid.org/0000-0002-3267-5630 Jan A. Bruijn: http://orcid.org/0000-0001-7592-250X Hans J. Baelde: http://orcid.org/0000-0002-1214-500X The authors state no conflicts of interest. Conceptualization: PB, HJB; Investigation: CCLvA, PB, MZ; Resources: JFPB; Supervision: HJB; Visualization: CCLvA; Writing - Original Draft Preparation: CCLvA; Writing - Review and Editing: CCLvA, PB, MZ, JFPB, MB, KDQ, JAB, HJB. Two pcDNA3.1 vectors (Invitrogen, Breda, The Netherlands) containing either sFlt-1-VSV or the luciferase gene were generated as described previously (Bus et al., 2017bBus P. Scharpfenecker M. Van Der Wilk P. Wolterbeek R. Bruijn J.A. Baelde H.J. The VEGF-A inhibitor sFLT-1 improves renal function by reducing endothelial activation and inflammation in a mouse model of type 1 diabetes.Diabetologia. 2017; 60: 1813-1821Crossref PubMed Scopus (0) Google Scholar, Eefting et al., 2007Eefting D. Grimbergen J.M. de Vries M.R. van Weel V. Kaijzel E.L. Que I. et al.Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA.Hum Gene Ther. 2007; 18: 861-869Crossref PubMed Scopus (16) Google Scholar). The resulting plasmids were amplified in DH5α E. coli (Invitrogen), purified using the QIAfilter Plasmid Maxi-prep kit (Qiagen, Venlo, the Netherlands) and dissolved in Endo-Free Tris–EDTA buffer (Qiagen). Mice were transfected by electroporation of the sFlt-1-VSV and luciferase constructs into both gastrocnemius muscles (20 μg each) as described previously (Eefting et al., 2007Eefting D. Grimbergen J.M. de Vries M.R. van Weel V. Kaijzel E.L. Que I. et al.Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA.Hum Gene Ther. 2007; 18: 861-869Crossref PubMed Scopus (16) Google Scholar). To monitor transfection efficiency, the mice were injected intraperitoneally with luciferin at 2-week intervals. Five minutes after each luciferin injection, luciferase activity was visualized at the transfection sites using a Night-OWL bioluminescence camera (Xenogen Ivis Spectrum, Alameda, CA) as described previously (Eefting et al., 2007Eefting D. Grimbergen J.M. de Vries M.R. van Weel V. Kaijzel E.L. Que I. et al.Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA.Hum Gene Ther. 2007; 18: 861-869Crossref PubMed Scopus (16) Google Scholar). A radiance above 1×106 p/sec/cm2/sr was considered as a successful transfection. For this study, we used 8-week-old C57BL/6J mice (n = 9 males, n = 11 females; Harlan Laboratories, Indianapolis, IN) and age-matched APOC1-tg mice (n = 9 males, n = 11 females). All experiments were conducted in accordance with national guidelines for the care and use of experimental animals (DEC license 13163). Mice were housed in individually ventilated cages in groups of up to five mice per cage, with food and water provided ad libitum. For the experiments, mice were randomly assigned to groups: sFlt-1-transfected and nontransfected APOC1-tg mice (n = 10 per group) and sFlt-1–transfected and nontransfected C57BL/6J (WT) mice (n = 10 per group). At 8 weeks of age, the mice were transfected with sFlt-1. Fifteen weeks after transfection, the mice were killed, skin and renal tissue were collected for histological analysis, and sera were collected for cytokine quantification; this time point was chosen because we previously found that treatment with sFLT-1 for 15 weeks resulted in marked changes in kidney inflammation and histology in diabetic mice (Bus et al., 2017bBus P. Scharpfenecker M. Van Der Wilk P. Wolterbeek R. Bruijn J.A. Baelde H.J. The VEGF-A inhibitor sFLT-1 improves renal function by reducing endothelial activation and inflammation in a mouse model of type 1 diabetes.Diabetologia. 2017; 60: 1813-1821Crossref PubMed Scopus (0) Google Scholar). One mouse in the nontransfected APOC1-tg group was removed from the study because the development of severe AD complicated by open wounds. Missing samples or samples with poor tissue quality for immunohistochemistry were excluded from analysis. The skin and kidney tissues were cut at 4-μm thickness, using a microtome (RM2255, Leica, Wetzlar, Germany) for paraffin-embedded tissues and a Reichert cryostat (CM3050S, Leica) for frozen tissues, and were stained with hematoxylin and eosin and periodic acid-Schiff using a standard protocol. The frozen skin tissues were immunostained with rat anti-mouse F4/80 (1:100; kindly provided by the Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands), rat anti-mouse CD3 (1:20; Abcam, Cambridge, MA), rat anti-mouse VCAM-1 (1:400; BD Pharmingen, San Diego, CA), rabbit anti-mouse VEGF (1:100; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-VSV (1:2400; Abcam) primary antibodies, followed by anti-rat–IgG-Impress (Vector Laboratories, Burlingame, CA) and anti-rabbit-Envision (Dako, Glostrup, Denmark) horseradish peroxidase-conjugated secondary antibodies with DAB+ as the chromogen. Paraffin-embedded kidney tissues were immunostained with a rabbit anti-human Wilms tumor 1 (1:500; Santa Cruz Biotechnology), followed by anti-rabbit–Envision horseradish peroxidase-conjugated secondary antibody with DAB+ as the chromogen. The frozen kidney tissues were immunostained using rat anti-mouse CD68 (1:15; Abcam), followed by anti-rat–IgG-Impress horseradish peroxidase-conjugated secondary antibody with DAB+ as the chromogen. Double-label immunofluorescence was performed with rat anti–VCAM-1 and goat anti–von Willebrand factor (Affinity Biologicals Inc, Ancaster, Canada), followed by the appropriate secondary antibodies, after which the slides were mounted using Vectashield plus DAPI (Vector Laboratories). For each immunostaining experiment, a nonspecific isotype-matched antibody was used as a negative control. Images of tissue sections were digitized using a Philips Ultra-Fast Scanner 1.6 RA (Philips Digital Pathology, Best, The Netherlands). For the measurement of epidermal thickness, 10 measurements per field in five randomly selected fields per sample were determined at ×20 magnification using ImageJ software (National Institutes of Health, Bethesda, MD). To determine skin expression of F4/80, VCAM-1, and VEGF, the immunostained surface was measured relative to the total surface area of the skin, in five randomly selected fields per sample at ×20 magnification using ImageJ. Both dermal and epidermal staining of VEGF were measured. CD3-positive cells were counted in five randomly selected fields per sample at ×20 magnification. The surface area of the glomerular tuft, the number of podocytes, and the number of macrophages were quantified as described previously (Bus et al., 2017aBus P. Pierneef L. Bor R. Wolterbeek R. van Es L.A. Rensen P.C. et al.Apolipoprotein C-I plays a role in the pathogenesis of glomerulosclerosis.J Pathol. 2017; 241: 589-599Crossref PubMed Scopus (5) Google Scholar). Sera collected at week 15 posttransfection were quantified for tumor necrosis factor-α and IL-6 by Luminex according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). To measure the urine albumin-to-creatinine ratio, spot urine was collected at 5 and 15 weeks after transfection. Urine albumin levels were measured using rocket immunoelectrophoresis with a rabbit anti-mouse albumin; purified mouse serum albumin (Sigma-Aldrich, Saint Louis, MO) was used as a standard. Urine creatinine was measured using a creatinine assay, with picric acid, sodium hydroxide, and creatinine standards (Sigma-Aldrich); the albumin-to-creatinine ratio was then calculated. For comparison of groups, we used one-way analysis of variance, thereby taking into account possible differences in variances among groups, based on the Levene's test. Where appropriate, one-way analysis of variance was tested using a log scale. Differences were considered significant at P < 0.05.
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