{"paper_id":"0752df60-aee8-4107-83eb-0e93f48bbdf1","body_text":"Tolerability and effect of inhibiting microfibrillar-associated protein 4 in small intestinal anastomotic healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tolerability and effect of inhibiting microfibrillar-associated protein 4 in small intestinal anastomotic healing Rasmus Refshauge Andresen, Jesper Brandt Pedersen, Paula Frederikke Hellsegg Grünfeld, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7132995/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Background Crohn’s disease often leads to strictures due to excessive extracellular matrix deposition and smooth muscle cell hyperplasia. Current stricture treatments include surgical and endoscopic interventions, but high recurrence rates remain a challenge. Microfibrillar-associated protein 4 (MFAP4) contributes to fibrosis in various tissues. The anti-MFAP4 antibody was evaluated for its tolerability and anti-fibrotic efficacy on small intestinal anastomotic healing in pig models. Methods Two small intestinal anastomoses were made in 45 pigs. Fibrosis was induced using aethoxysklerol injections. Each anastomosis was locally injected with either anti-MFAP4, positive control anakinra (interleukin-1 receptor antagonist) or negative vehicle control. Tolerability of anti-MFAP4 was observed across three observation durations (5, 10, and 28 days) and assessed by weight gain, anastomotic tissue strength, and histological evaluation. Anti-fibrotic efficacy was tested using semi-quantitative collagen scoring from the 28-days study. Proteome analysis of tissue sections was applied for mechanistic analyses. Results Local anastomotic injections of 16 mg and 32 mg anti-MFAP4 were well tolerated. Anastomotic fibrosis was significantly reduced both by positive control anakinra-treatment and anti-MFAP4-treatment. Anti-MFAP4 reduced fibrosis by 23% relative to the negative control. Gene ontology term analysis showed up-regulation of muscle cell contractile apparatus and down-regulation of transcription and translation in the anti-MFAP4 group. Conclusions The data supported that anti-MFAP4-treatment induced smooth muscle cell switching from the synthetic phenotype involved in fibrosis to the contractile phenotype essential for homeostatic gut motor activity. Anti-MFAP4 has potential as treatment of Crohn’s patients at risk of developing fibrostenosis and as a prophylactic treatment to support post-surgical anastomotic healing. Health sciences/Diseases Health sciences/Gastroenterology Health sciences/Medical research Intestinal anastomoses Microfibrillar-associated protein 4 fibrosis smooth muscle cells phenotype switch Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Crohn's disease (CD) is a type of inflammatory bowel disease characterized by chronic transmural inflammation, most commonly affecting the terminal ileum [ 1 ]. The inflammation may lead to intestinal wall fibrostenosis, and up to 70% of the Crohn’s disease patients will develop fibrotic stricture at 10 years after diagnosis [ 2 ]. Fibrostenotic complications are the most common driver for intestinal resection. After introduction of biologics then cumulative 5-year resection rate decreased from 12,5% to 9,3% after diagnosis [ 3 ]. A similar trend in the re-resection rate has been demonstrated. The disease-driven re-resection rates after 1, 5 and 10-years was 3,6%, 10,1% and 14,1% respectively in a population study [ 4 ]. The development of intestinal strictures in CD is multifactorial. Aberrant inflammatory wound healing characterized by excessive collagen-rich extracellular matrix (ECM) deposition by increased numbers of mesenchymal cells is one factor and smooth muscles cell (SMC) hyperplasia/hypertrophy is another important factor [ 5 , 6 ]. SMC are recognized with the capacity to dedifferentiate from a normal contractile, quiescent phenotype to a synthetic phenotype characterized with increased ECM synthesis and proliferation during wound healing and pathophysiological conditions [ 7 ]. Excessive SMC proliferation has been demonstrated in both Crohn’s strictures [ 8 ] as well as in animal models of induced intestinal inflammation [ 9 ] and is recognized as the most prominent histological change in crohn's fibrostenosing strictures[ 5 ]. The pathophysiological process includes increasing expression of integrin α V β 3 , which has been shown to increase SMC proliferation and hyperplasia in stricturing CD [ 8 ]. In the earliest phase of wound healing (day 1–4) hemostasis results in clot-formation and the release of pro-inflammatory cytokines. Inflammation is induced by the cytokines and the accumulation of apoptotic and necrotic cellular material followed by migration of neutrophiles and macrophages. This is followed by a proliferative phase (day 4–14) where mesenchymal cells migrate into the healing tissue and start to proliferate. These cells; including fibroblasts, myofibroblasts and SMCs, are responsible for the formation of collagen. The resulting collagen deposition provides anastomotic strength and stability and the tissue continuity is dynamically restored during this phase [ 10 ]. Also, a shift from pro- to anti-inflammatory signaling restricts the inflammatory response in this phase. In the final remodeling phase (day 14 ◊), deposited collagen is remodeled by fibrolytic activity. Collagen type III is partly replaced with the more durable collagen type I and tissue strength is plateaued [ 11 ]. A disturbed wound healing from an underlying disease such as CD may result in the formation of excessive extracellular matrix resulting in a fibrotic stenosis in the anastomotic area and recurring after surgical resection [ 6 ]. The ECM protein microfibrillar-associated protein 4 (MFAP4) co-localizes with elastin and collagen fibers, especially in small intestine, the heart, and lungs [ 12 – 14 ]. MFAP4-deficiency is demonstrated to be protective of development of fibrosis in heart and kidney [ 15 – 17 ]. Recently, an animal model of inflammatory bowel disease showed that MFAP4 colocalized with fibrotic markers suggesting it to also be a profibrotic driver in the intestines [ 18 ]. MFAP4 is found significantly increased in response to TGF-β treatment in human mesenchymal cells [ 19 ], and in line with this, transcriptomic analysis has demonstrated that MFAP4 is expressed in SMC, fibroblasts and activated fibroblasts in ileal resections from CD patients [ 18 ]. The above observations provides the rationale for testing the role of MFAP4 in intestinal fibrostenotic processes.We hypothesized that MFAP4 could serve as therapeutic target in anastomotic fibrostenotic processes. We have previously developed an anti-MFAP4 antibody with the capacity to block MFAP4 binding to integrin α V β 3 and the consective activation of integrin expressing cells [ 20 ]. We tested if local pharmacological treatment with anti-MFAP4 was tolerated, tested therapeutic efficacy of reducing fibrosis in pig intestinal anastomotic healing, and investigated the therapeutic effects by proteome analysis. Materials and methods Ethical approval All experiments were performed in accordance with relevant guidelines and regulations, and all live animal experimental protocols were conducted according to ARRIVE 2.0 guidelines [21] and were approved by the Danish Animal Experiments Inspectorate (j. nr. 2018-15-0201-01583). Study design Three separate interventional randomized and investigator blinded studies were performed using Danish Landrace pigs. In each study two hand sewn anastomoses were created in the small intestines. The different lengths of the studies, 5 days, 10 days and 28 days, were chosen for the observation of tolerability of injecting anti-MFAP4 in the different phases of wound healing [10]. Tolerability outcomes were anastomotic healing following initial inflammatory healing phase with provisional wound closure (Day 5), during the proliferative phase where the tissue strength is dynamically restored (Day 10) and during the remodeling phase (Day 28). The tolerability was evaluated through patological scoring, macroscopical findings, measurements of tissue strength and formation of adherences. The primary efficacy outcome was collagen-deposition following anti-MFAP4 treatment observed in the 28-days study. The secondary outcome was therapeutic mechanisms evoked by anti-MFAP4 in the 28-days study and observed by proteome analysis . 5-days study: Each pig received either vehicle control treatment (n=6) or anti-MFAP4 treatment (20 mg/ml) (n=7). A total of 0.8 ml of vehicle or anti-MFAP4 was injected in 8 predefined subserosal depots on each side of the anastomosis at a distance of 2 mm from the anastomotic line. Pigs were euthanized at postoperative day 5 and the anastomotic site was resected. After a tensile strength test the anastomotic tissues was fixed in formalin for later histological examination. 10-days study: All pigs received aethoxysklerol (5 mg/ml) injections with a total of 0.8 ml distributed on 8 predefined subserosal depots on both sides of the anastomotic side together with either vehicle control treatment (n=7) or anti-MFAP4 treatment (20 mg/ml) (n=7) as described in the 5-days study. Pigs were euthanized on postoperative day 10. The anastomotic area was resected after in situ contrast study (X-ray). After a tensile strength test the anastomotic tissue was fixed in formalin for later histological examination. 28-days study: In all pigs, aethoxysklerol injections was performed as described above. On postoperative day 14, a re-laparotomy was performed, and the pigs were randomized to receive either positive control anakinra (Interleukin-1 receptor antagonist (IL-1Ra)) treatment (40 mg/ml) (n=9), vehicle control treatment (n=9), or anti-MFAP4 treatment (40 mg/ml). In total, a volume of 0.8 ml of each was injected around the anastomotic area as described above. On postoperative day 28, in situ contrast study (X-ray) of the anastomotic area was performed, and the anastomotic area was resected. After a tensile strength test the anastomotic tissue was fixed in formalin for later histological examination. Figure 1 summarizes the study outline. Animals Weaned female Danish Landrace & Yorkshire pigs (obtained from the breader Kokkenborg, Stenstrup, Denmark) of approximately 20 kg were included in the study. The pigs were acclimatized to their new environment for at least one week prior to surgery. They were housed at a conventional large animal housing facility with light/dark cycles (twelve hours with gradually dimmed light and natural light from windows) at a constant temperature of 20-21°C. The pigs had access to food twice daily (0.9 kg/20 kg body weight) and free access to water. Before surgical procedures the pigs were weighed. To ensure animal welfare, the pigs were intensively inspected daily, and during the first 24 hours following surgery. Anesthesia Pigs were subjected to a fasting period of 12 hours before surgery with free access to drinking water. Pre-anesthetic sedation was a combination of 0.25 mg/kg midazolam (Midazolam Hameln®, Hameln Pharmaceuticals GmbH, Hameln, Germany), 0.03 mg/kg medetomidine (Cepetor, Scanvet), 0.2 mg/kg butorphanol (Butomidor, Salfarm Danmark A/S, Kolding, Denmark) and 5 mg/kg ketamine (Ketaminol Vet.,100 mg/ml, MSD Animal Health) administered intramuscularly (im.). Anaesthesia was induced with 5 mg/kg propofol (B. Braun Medical A/S, Copenhagen, Denmark) intravenously (iv.) through an ear vein and the pigs were intubated with a cuffed tube size 4.0 and connected to a Siesta i TS Anaesthesia Machine (Dameca, Rødovre, Denmark). Anaesthesia was maintained with a continuously infusion of 15 mg/kg/hour propofol (B. Braun Melsungen AG, Melsungen, Germany) and 25-50 µg/kg/hour fentanyl (B. Braun Melsungen AG, Melsungen, Germany). Perioperative analgesia consisted of fentanyl (B. Braun Melsungen AG, Melsungen, Germany) iv. administered, in a dose of 50 μg/kg/hour. The pigs were mechanically ventilated with a tidal volume of 7-10 ml/kg. The respiratory frequency was adapted to the ETCO 2 level. Blood pressure, electrocardiogram, heart rate and oxygen saturation were monitored continuously. The pigs received prophylactic antibiotics preoperatively with 15 mg/kg amoxicillin (Curamox ® Prolongatum Vet. Boehringer Ingelheim Danmark A/S, København Ø, Denmark), perioperatively 20 mg/kg metronidazole (B. Braun, Melsungen AG, Germany), and 48 and 96 hours postoperatively 15 mg/kg amoxicillin (Curamox ® Prolongatum Vet. Boehringer Ingelheim Danmark A/S, København Ø, Denmark). Postoperative analgesia consisted of fentanyl transdermal patch 2 µg/kg/hour (Matrifen® Takeda Pharma A/S, Taastrup, Denmark) rounded down to the nearest patch dose in increments of 12 µg/hour. The fentanyl patch was removed after 3 days. Additionally, some pigs received buprenorphine 0.03 mg/kg (Bupaq ® Multidose, Salfarm Danmark A/S, Kolding, Denmark) i.m. every fourth to six hours for the first 24 hours after surgery, while pigs in the 28-days study received a subcutaneous injection with a weight-adjusted dose of sustained-release formulation of buprenorphine preoperatively, ensuring analgesia for at least 48 hours postoperatively. Euthanization Prior to euthanization the pigs underwent general anesthesia as described above. The pigs were euthanized using 140 mg/kg pentobarbital (400 mg/ml) (Euthanimal, ScanVet Animal Health A/S, Fredensborg, Danmark) administered via the ear vein. Surgery During surgery, the pigs were placed on a heating pad set to 32°C, to avoid hypothermia. They received Ringer's acetate i.v. at a rate of 4-10 ml/kg/h. The small intestines were exposed through a 10 cm lower midline laparotomy. Two end-to-end anastomoses were created 50 cm and 150 cm orally to the ileoceacal junction with a seromuscular running suture using monocryl 4-0 (Ethicon, Johnson & Johnson, Diegem, Belgium). After interventional procedures the abdominal fascia was closed with running PDS*II® 0 (Ethicon, Johnson & Johnson, Somerville, New Jersey, USA). The skin was closed intracutaneously with a running monocryl® 3-0 (Ethicon, Johnson & Johnson, Diegem, Belgium). The skin incision was sealed with a liquid bandage (KRUUSE Wound Plast, Cat. no: 161020). At study completion, a re-laparotomy was performed, and all the anastomoses were identified and freed from adhesions, which were graded in using a modified Leach grading of adhesion (Supplemental Table S2). All anastomoses were examined for macroscopical findings such as pseudo-diverticulosis, abscesses, visible leakage, fistulas, or signs of ileus. In 5 pigs, anastomotic tissue, lung, and artery biopsies from the 28-days study were collected at end-study for analysis of anti-MFAP4 deposition. An incision in the right diaphragmatic dome allowed harvesting of the biopsy from the basis of the middle lobe of the right lung. The arterial biopsy was harvested from the left common iliac artery. Biopsies were snap frozen with liquid nitrogen, and stored at -80 °C. Interventional agents and controls Anti-MFAP4 is a humanized, monoclonal IgG1 antibody, that binds to MFAP4 with high affinity and blocks its interaction with integrins [20]. Wild-type anti-MFAP4 was used for the 5-days and the 10-days study. For the 28-days study, we applied a point-mutated variant with minimized Fc-mediated effects the so-called hIgG1-P329G LALAvariant [20]. Aethoxysclerol ® (Kreussler Pharma) causes submucosal fibrosis by local injection [22]. The positive control was anakinra (40 mg/mL, Kineret ® . Swedish Orphan Biovitrum. Batch 3798201c), vehicle control (10 mM histidine, 10% w/w trehalose pH 6.0), or anti-MFAP4 (20 or 40 mg/mL). For both anti-MFAP4- and aethoxysklerol-treatment and for control-treatments (vehicle and anakinra) a volume of 0.8 ml was injected into the anastomotic area. Radiography The wound contraction at the anastomotic site was assessed in situ by a contrast study. The intestinal loop was clamped approximately 10 cm proximally and distally to each anastomosis. Water-soluble contrast (Omnipaque®, GE HealthCare) was infused to a pressure of 20 mmHg and an image in one plane was obtained. The intestinal diameters were measured orally and aborally to the anastomotic site using an image analysis software (Image J, NIH, Bethesda, USA). An anastomotic index (AI) was calculated for each anastomosis by dividing the diameter of the anastomosis sites by a mean pre- and postanastomotic diameter based on five measure points at 1 cm intervals from the anastomotic line (Supplemental Figure S2). Maximal anastomotic tensile strength (MATS) Following radiography, the anastomosis was resected with approximately 5 cm margin orally and aborally and cleaned of fecal contents with water prior to maximal anastomotic tensile strength (MATS) test. The resected intestinal segment was mounted by two clamps in the tensile testing machine (LF Plus®; Lloyds Instruments, Fareham, UK) equipped with an XCL 100 N loadcell (Lloyds Instruments, Fareham, UK) 5 minutes after resection to prevent the influence of cold ischemia. The position of the clamps was in a distance of 6 cm with the anastomosis placed in the center (Supplemental Figure S3). The segment was gradually stretched at a constant deformation rate of 15 mm/min until rupture occurred. The applied force in Newton was measured at three points: When a tear became visible in the serosa (MATS 1). When a transmural lesion was visible (MATS 2). The rupture was confirmed by a simultaneous drop in the load-strain curve calculated by the software NEXYGEN Plus ® (version 3.0; Lillerød, Denmark). The maximal force applied during the tensile strength test (MATS 3) was calculated by the software. Peparation for histology A ten mm long intestinal tissue with central intact anastomotic line was prepared and fixed in a 4% formaldehyde solution for 48 hours and subsequently embedded in paraffin. Three micrometer thick slices were stained with hematoxylin and eosin or picrosirius red for histological evaluation. Histological evaluation The anastomotic healing of each anastomosis was visually assessed according to the Verhofstad scale [23] (Supplemental Table S3), evaluating necrosis, polymorph nucleated cells, lymphocyte infiltration and macrophages, degree of edema, state of epithelial layer and bridging of the submucosal layer. Collagen deposition at the anastomotic line was scored in percentage of the anastomotic area. Each anastomosis was assessed independently using a mean score from two sections from the same anastomosis. Treatment group was blinded to the assessor. Proteome analysis Three µm sections of formalin-fixed, paraffin-embedded tissue from the distal anastomosis of each animal (n=9/group) were deparaffinized by using xylene followed by extraction of proteins by dissolving the deparaffinized tissue sections in extraction buffer (1M dithiothreitol (DTT), 0.2 M tetraethylammonium bicarbonate and 10% sodium dodecyl sulfate, then subjected to two rounds of ultra-sonification (15 minutes of ultra-sonification/15 minutes of cooling on ice), and incubated at 99 ᵒC for 20 minutes then at 80 ᵒC for 2 hours. Protein alkylation was done by adding a 200 mM iodoacetamide (IAA) solution to a final DTT/IAA concentration ratio of 1:3. The acetone-precipitated proteins were re-dissolved in 5 μL 8 M urea with 1 μg LysC and incubated at 30 ᵒC for 4 hours, followed by a further dilution to 1 M urea, the addition of 2 μg trypsin, and an overnight incubation at 30 ᵒC. The resulting tryptic peptide samples were isotopically labeled using the 16-plex tandem mass tag (TMTpro) (Thermo Fisher, USA) where a pool of all samples was labeled with the mass tag 126 that served as an internal standard. Tagged peptides were mixed into three mixed peptide samples that were fractionated into 7 fractions using high pH chromatography and analyzed by nano-HPLC-MSMS also as previously described [24] with the following modifications: LC gradient length was 100 minutes, higher-energy collisional dissociation was set to 36. All Thermo Scientific Exploris 480 raw data files were processed and quantified using Proteome Discoverer version 3.0 (Thermo Scientific, Waltham, MA, UnitedStates) using a combined Chimerys, Sequest and MS Amanda search. For Sequest and MS Amanda searches TMTpro (N-terminal and lysine) and cysteine carbamidomethylation set as fixed modifications, and methionine oxidation and asparagine and glutamine deamidation as variable modifications whereas for Chimerys TMTpro (N-terminal and lysine) and cysteine carbamidomethylation were set as fixed. Data were searched against the Uniprot pig proteome (Sus scrofa, UP000008227, 46225 entries, downloaded 12 th June 2024). Pathway enrichment analyses were performed using clusterProfiler 4.0 package [25] and org.Hs.eg.db in R. Immunohistochemistry Three µm thick formalin-fixed, paraffin-embedded sections were mounted on FLEX IHC Microscope slides (Dako/Agilent, Glostrup, Denmark). Sections were dried at room temperature and baked at 60°C for 60 minutes. Following, epitope retrieval was performed by processing samples in DISCOVERY Cell Conditioning solution (Ventana) for 32 min at 100°C. Humanized anti-MFAP4 (human IgG) and Forkhead box P3 (FOXP3) were detected by immunohistochemistry using Goat anti-Human IgG Fc (Abcam, Cambridge, UK, cat# ab ab97221) diluted 1:2000 or rat anti-FOXP3 (Thermo Fischer Scientific, cat# ab ab97221) diluted 1:100 and incubated for 32 mins at 36°C. Staining was automated using the Discovery Ultra immunostainer (Ventana Medical Systems, Tucson, AZ) and using the OmniMap anti-Goat-HRP detection system (Roche cat# 760-4647) for anti-MFAP4 or OmniMap anti-Rat-HRP (Roche cat#760-4457) for anti-FOXP3 followed by Discovery ChromoMap DAB (Ventana Medical Systems, Tucson, AZ). Nuclear counter staining was performed using Hematoxylin II (Ventana Medical Systems, Tucson, AZ). Finally, slides were washed, dehydrated, and coverslipped using an automated Dako coverslipper (Dako/Agilent, Glostrup, Denmark). For a-smooth muscle actin (a-SMA) staining the tissues were processed as described above. Epitope retrieval was performed in Target Retrieval Solution-H (Agilent) for 30 min at 97°C. Staining was performed using the OMNIS automated immunohistochemistry platform (Agilent) using mouse anti-a-SMA (BS66, Nordic Biosite, cat#BSH-7459) diluted 1:1,000 and detected using EnVision FLEX/HRP+ Mouse LINK and EnVision FLEX DAB+ Substrate Chromogen System (Agilent). Nuclear counter staining was performed using Hematoxylin, Mayers ready to use solution (Agilent). Finally, slides were washed, dehydrated, and cover slipped using an automated Dako cover slipper (Dako/Agilent, Glostrup, Denmark). ELISA detection of anti-MFAP4 (human IgG) in tissue samples Buffers: Tris-buffered saline (TBS): 140 mM NaCl (Merck Millipore), 10 mM Tris-HCl (Sigma-Aldrich), 0.02% (w/v) NaN 3 (Sigma-Aldrich), pH 7.4; TBS/Tw: TBS containing 0.05% (v/v) Tween 20 (polyoxyethylene sorbitan monolaurate, Merck Millipore); phosphate-buffered saline (PBS): 137 mM NaCl (Merck Millipore), 3 mM KCl (Merck Millipore), 8 mM Na 2 HPO 4 (VWR) , 1.5 mM KH 2 PO 4 (Merck Millipore), pH 7.4; PBS/Tw: TBS containing 0.05% (v/v) Tween 20 (polyoxyethylene sorbitan monolaurate, Merck Millipore); PBS/Tw/Bovine serum albumin (BSA): PBS/Tw, 0.1% BSA; substrate buffer: 35 mM citric acid (Sigma-Aldrich), 67 mM Na 2 HPO 4 (VWR), pH 5.0. Snap-frozen tissues from each of two anastomoses/pig, one pulmonary or arterial sample were included in ELISA analysis. 96-well MaxiSorp plates (NUNC MaxiSorp) were coated with in house produced recombinant MFAP4 (0.25 μg/ml in PBS) overnight at 4°C, washed with PBS/Tw/BSA, and then blocked with PBS/Tw/BSA for 1 hour at room temperature. To create a standard curve, Anti-MFAP4 was diluted to 10 ng/ml in PBS/Tw/BSA, followed by 2-fold dilutions in PBS/Tw/BSA. Intestinal, pulmonary and arterial tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich cat#R0278) using a Precellys 24 tissue homogenizer (Bertin Instruments) and protein concentrations of the lysates determined using the Biorad DC protein assay (Biorad cat nr 500112). The lysates were diluted in PBS/Tw/BSA, to be within the linear range of the assay and aliquoted onto the MFAP4-coated plates (100 μl per well). All samples were incubated 1 hour at room temperature, washed with PBS/Tw/BSA and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (ab97225, Abcam) at 1:10,000 dilution in PBS/Tw/BSA for 30 min at room temperature. The plates were then washed and incubated with streptavidin-HRP conjugate (Invitrogen) diluted 1:2,000 in TBS/Tw for 15 min, washed again and incubated with o-phenylenediamine dihydrochloride ( OPD) substrate (0.8 mg/ml, Kementec) dissolved in substrate buffer (0.03% freshly prepared H 2 O 2 (Sigma-Aldrich) in the dark at room temperature. Color development was stopped by the addition of 100 µl 1 M H 2 SO 4 , (Sigma-Aldrich) and the plates were read at OD 492 nm with OD 600 nm as reference. Statistical methods For each continuous outcome variable student’s t-test was performed for the comparison between anti-MFAP4 or positive control anakinra-treatment relative to the negative control vehicle-treatment. Mann-Whitney test was performed for comparisons of leach-scoring and Verhofstad scale scoring. Results were considered statistically significant if p<0.05. Statistical analyses were performed using GraphPad Prism 10 (https://www.graphpad.com). Pathway enrichment analyses were based on ranked log2-transformed fold changes (gse function) and unadjusted p < 0.05 (unpaired, two-tailed student’s t-test for each protein per two-group comparison). The Benjamini-Hochberg procedure was applied to correct for multiple testing. Results Tolerability of local injection of anti-MFAP4 in the anastomosis in early healing phases In the 5-days study, the mean body weight increase as well as the anastomotic tensile strength as assessed by MATS by day-5 was similar between vehicle and anti-MFAP4 treated groups (Table 1). The histological Verhofstad scoring was similar between the two groups. No macroscopical findings such as pseudo-diverticulosis, abscesses, leakage, fistulas, or signs of ileus was observed in any of the anastomoses (Table 1). The addition of aethoxysklerol injections and 5-days longer post-surgery period (10-days study) had no effect on the results (Table 1). The anastomotic index in the 10-days study was similar in the vehicle group and the anti-MFAP4 group with a median of 0.56 in both groups. The scorings of sirius red-determined collagen percentage was similar between the 5-days study, which represents the inflammatory stage where collagen deposition is expected to be limited, and the 10 days-study. Tolerability of local injection of anti-MFAP4 in the anastomosis in the late healing phase In the 28-days study we increased the anti-MFAP4 dose to 40 mg/ml to test an escalated dose and we introduced anakinra. We used a variant form of anti-MFAP4, that abolish potential Fc-mediated undesirable functions, which are immune-activating effects of native antibodies potentially casing cytotoxicity. Initially, we tested for potential systemic deposition of the locally administered anti-MFAP4 through investigations of samples obtained from lung and artery. We did not observe any detectable anti-MFAP4 (human IgG) in lung and artery, while anti-MFAP4 was detected in the anastomosis 28 days post-surgery (14 days post-injection) (Figure 2A). Immunostaining of anti-MFAP4 in anastomotic sections from the 28-days study showed no specific staining in vehicle treated sample, and specific staining of elastic fibers (the main location of MFAP4 [26, 27]) and additional perivascular staining in the anastomotic tissue of anti-MFAP4 treated samples (Figure 2B-C). The anastomotic index was significantly lower both in the anti-MFAP4 treatment group and the anakinra treatment group relative to vehicle control treatment, suggesting that wound contraction was sustained by both treatments (Table 2). Adhesion score was not affected by anti-MFAP4 but was significantly reduced by anakinra. The histological examination showed a significant increase in lymphocytes in both the anakinra- and anti-MFAP4 treatment groups relative to vehicle treatment (Table 2). There were no macroscopical findings of pseudo-diverticulosis, abscesses, visible leakage, fistulas, or signs of ileus in any of the anastomoses. Anti-MFAP4 and total collagen deposition in late anastomotic healing phase The mean anastomotic collagen scoring was approximately doubled in the vehicle-treated 28-days study (Table 2) relative to the earlier healing phase (10-days model, Table 1). Both the positive control anakinra- and anti-MFAP4-treatment resulted in significant reduction of the anastomotic collagen deposition relative to vehicle treatment (Table 2). The anti-MFAP4-treated group showed a significant 23% mean score reduction relative to vehicle treatment. Representative images of collagen deposition in anakinra-, vehicle- and anti-MFAP4-treated groups are shown in Figure 3. Proteomic changes inferred by anti-MFAP4 treatment in the anastomosis in late healing phase A total of 3075 unique proteins were identified by proteomic analysis in all samples (Supplemental Table S1). Nominally regulated proteins from the positive control anakinra- and anti-MFAP4-treated anastomoses are displayed on volcano plots in Figure 4A-B. Remarkably, the most significantly down-regulated proteins by the positive anti-fibrotic control treatment anakinra included Cartilage Associated Protein (CRTAP), Collagen type II alpha 1 chain (COL2A1) and Collagen type III alpha 1 chain (COL3A1), which are all related with fibrosis (Figure 4A). This supported the observation of anakinra-mediated reduction of Sirius red-determined fibrosis. Amongst the most significantly up-regulated proteins in the anti-MFAP4 treated groups were markers of epithelial healing; Olfactomedin 4 (OLMF4), which is a marker of intestinal epithelial stem cells [28], and Pytuvate Kinase M (PKM), which may repress apoptosis and facilitate survival of intestinal epithelial cells [29]. We analyzed the proteome data using an unassisted Gene Ontology (GO) enrichment analysis (Supplemental Tables S4 and S5). Several GO terms were nominally regulated in the treatment-groups. The seven most significantly anakinra down-regulated GO terms were related to extracellular matrix- and collagen-formation (Figure 4C). The GO-term “collagen fibril organization” remained significantly downregulated after controlling for multiple testing. The ten most significantly anakinra up-regulated GO terms included chaperone-related effects, nucleosome related effects and heat-shock protein related effects (response to temperature and heat (Supplemental Table S4)) (Figure 4D), which are important for proteostasis during mechanical stress [30]. All top-ten anti-MFAP4 up- and down-regulated GO-terms remained significant after correcting for multiple testing. Eight out of the ten most significantly down-regulated GO terms were associated with transcriptional or translational activity (Figure 4E). The top-ten most significantly up-regulated GO terms were all associated with muscle contractility or cytoskeletal organization (Figure 4F). Surprisingly, some GO terms were associated with skeletal muscle, which is not present in the small intestine (I-band, sarcomere, myofibril, Z-disc). However, all the enriched proteins underlying these terms (Supplemental Table S5) are also expressed by smooth muscle cells, albeit with lower transcripts per million relative to skeletal muscle [31]. Therefore, the observed regulations reflect the effects of the contractile apparatus of smooth muscle cells. The data supported that anti-MFAP4-treatment induced SMC-switching from the synthetic phenotype involved in development of fibrostenosis to the contractile phenotype essential for homeostatic gut motor activity. We analyzed a-SMA immuno-staining in the anastomotic sections and observed a low muscularis propria staining-intensity in the vehicle treated group. The a-SMA immuno-staining was variable between analyzed anastomoses for both the anakinra- and anti-MFAP4-treated groups. Yet, higher intensity of a-SMA immuno-staining was observed in the anakinra-treated group, and the highest a-SMA intensity was observed in the anti-MFAP4-treated group. Representative images are shown in Figure 5. Anti-MFAP4 and regulation of lymphocytes Histochemical evaluation showed that lymphocytes were upregulated by anti-MFAP4 (Table 2). Yet, GO terms related with lymphocytes included downregulation of “gamma-delta T cell activation” (Figure 4E), and down-regulation of “T-cell proliferation”, and “T cell activation” (Supplemental Table S5). This suggested that a general negative regulation of T-cells was present. On this basis, we specifically investigated the presence of regulatory T cells (FOXP3-positive cells), and representative sections are shown in Supplemental Figure S4. Inspection of the stained sections supported that FOXP3-positive cells were present in all three treatment groups and that areas with high density of regulatory T-cells were present in the lamina propria of anakinra- and anti-MFAP4-treated samples relative to the control-treated samples. Discussion Observations from the present study support that pharmacological blocking of MFAP4, using anti-MFAP4, has potential as therapeutic intervention to prevent or treat fibrostenotic processes in intestinal anastomotic healing. One local dose of 16 mg or 32 mg anti-MFAP4 did not affect weight gain, the anastomotic strength or histochemical evaluation of healing in early and late healing phases, respectively. Local injection with anti-MFAP4 showed reduction of collagen deposition and the anastomotic SMC phenotype was switched from an ECM synthetic phenotype towards a contractile homeostatic phenotype in the late healing phase. Anti-MFAP4 is raised against human MFAP4. Pigs may be an ideal species for the creation of a large animal model given that their gastrointestinal anatomy closely resembles that of humans [32]. The homology between pig and human MFAP4 is 94% supporting the use of anti-MFAP4 in pigs. Anti-MFAP4 binds with high affinity to MFAP4, which is embedded in the ECM and therefore it may be retained to a high degree at the injections site. To test if locally injected anti-MFAP4 could reach the circulation and other organs and thereby exert unknown effects tat these sites, we harvested lung and arterial tissue. These tissues represents MFAP4-rich organs. However, we did not observe any anti-MFAP4 deposition in these organs after local injection, whereasapproximately 1 ng/ml tissue was retained in the anastomoses 14 days post-injection. In a prior study we observed a duration of efficacy of one dose anti-MFAP4 in reduction of vascular leakage for at least three months [20]. Together, these observations suggest that one dosing of anti-MFAP4 might be retained locally and be sufficient for obtaining a treatment effect through out a months-long period. We applied local injection of anti-MFAP4 because other tissues might absorb systemically injected anti-MFAP4. For systemic injection, it would be required to test systemic tolerability, and a scale-up of our recombinant antibody production would be needed. Therefore, it is unknown if systemic administration of anti-MFAP4 is possible. However, endoscopic submucosal injection is demonstrated as a feasible approach for local delivery of small interfering RNA for other target [33]. Due to previously observed long duration of efficacy of anti-MFAP4 [20], anti-MFAP4 could potentially also be delivered locally through endoscopic injection. The effective therapeutic dose of anti-MFAP4 for local injection into the pig intestinal anastomosis was unknown when this study was initiated. In a prior study we have observed retinal anti-angiogenic therapeutic efficacy of 2 mg anti-MFAP4 intravitreal injection in non-human primates [20]. This species has a vitreous volume of approximately 2.4 ml [34], and thereby the effective starting dose in that prior study was 0.8 mg/ml. In the present study, we aimed to reach far higher doses and locally injected 20 mg/ml (16 mg total) or 40 mg/ml doses (32 mg total) to accommodate for a higher, yet unknown, distribution volume. Pro-inflammatory effects of MFAP4 have previously been described [17, 26], and consequently anti-MFAP4 might potentially compromise the early inflammatory phase of wound healing. In addition, the anti-angiogenic effects of anti-MFAP4 [20] might potentially compromise angiogenesis during the proliferative phase of wound healing. However, we did not see any significant changes in anastomotic tensile strength, or the histological scores following dosing of 20 mg/ml anti-MFAP4 in the early healing phases (5-days and 10-days studies). The tensile strength was measured using MATS test, which is considered appropriate for evaluation of the healing tissue strength [35]. On this basis, we do not expect that anti-MFAP4 compromised early inflammation or angiogenesis to a significant degree. For testing therapeutic efficacy, we increased the anti-MFAP4 dose to 40 mg/ml in the 28-days study. This higher dose did not weaken the anastomotic strength but appeared to induce lymphocytic infiltration into the tissue. For the 28-days study, we applied a Fc-neutralized variant of anti-MFAP4 to dampen potential antibody-dependent or complement-dependent cytotoxicity. Moreover, antibodies are expected to arise 9-35 days after antigen exposure in pigs [36]. Therefore, we only applied 14 days of anti-MFAP4-administration to reduce the potential development of anti-antibodies and the associated risk of drug-induced inflammation in the pigs. Proteome analysis supported an immune-suppressive state of the anastomosis in the anakinra- and anti-MFAP4-treated samples. In line with this, specific analysis of immuno-stained anastomotic sections supported that FOXP3-positive regulatory lymphocytes involved in dampening of local intestinal inflammation [37] were enriched in the anakinra- and anti-MFAP4-treated samples. However, detailed analysis of lymphocyte populations requires flow- or tissue-cytometry and was out of scope for the present study. The normal pig small intestinal anastomosis has non-significantly different collagen volume in the muscularis and subserosa than outside of the anastomosis region when observed in the late healing phase [38]. We injected aethoxysklerol locally in the anastomotic line at the time of surgery in order to enhance the fibrotic process. The clinical fibrotic effect of aethoxysklerol is expected to occur week-to-months after sclerotherapy injection. The anti-fibrotic treatment effects were therefore analyzed in the 28-days study. Dampening of inflammation is expected to reduce fibrogenesis. In line with this, the use of anti-TNF-α antibodies is safe in patients with inflammatory stricturing CD and could decrease the need for surgery over time [39-41]. However, the surgery-free interval is still short and data on anti-fibrotic effect of anti-TNF are lacking [42, 43]. In contrast, an anti-fibrotic effect of IL-1-inhibition in the gastrointestinal system and skin is well recognized [44-46]. Anakinra is a recombinantly expressed IL-1 receptor antagonist molecule and was applied as locally injected therapy in the same dose as anti-MFAP4. For clinical use, anakinra is applied by daily subcutaneous injections and such alternative dosing regimen might have provided a higher anti-fibrotic effect than the dosing used in our study. Dysregulation of interleukins decreases the fibrinolytic capacity of the peritoneum, increases adhesion formation, and is initiated in early hemostasis/inflammatory phase [47]. In line with this, anakinra treatment had capacity to reduce adherence formation in our study supported by observations from previous publications applying IL-1R inhibition [44, 45]. Human IL-1Ra is previously shown to be well tolerated and to show therapeutic efficacy in pigs [48, 49]. Semiquantitative collagen scoring, using transmural assessment of Sirius red-determined collagen deposition, is reported to be fast, reliable and reproducible [38] and was applied as a main outcome in our study. However, it considers the entire intestinal wall as a single region of interest and is not as sensitive as stereological assessment of the intestinal anastomotic collagen volume [38]. Nevertheless, semiquantitative collagen scoring showed a significantly reduced anastomotic collagen deposition with both the positive control anakinra-treatment and anti-MFAP4-treatment in the present study. The observed anti-fibrotic activity of anti-MFAP4 is supported by observations of reduced fibrogenesis in gene-deficient mouse models of organ fibrosis [15, 17]. Wound contraction refers to the process during initial wound closure and healing, where fibroblasts generate forces that pull the edges of the open wound together to accelerate closure and reduce the resulting scar size. Scar contraction occurs later, involving the differentiated myofibroblasts that cause shrinkage in a fully healed scar [50].We assessed wound/scar contraction using x-ray analysis and observed a reduced anastomotic index in the 10-days study. Moreover, both anti-MFAP4- and anakinra-treatment reduced the anastomotic index relative to vehicle control in the 28-days study. Myo(fibroblast)-mediated contraction is dependent upon integrin a V b 3 -mediated focal adhesion formation [51, 52], but antibody-mediated blocking of MFAP4 is previously shown to reduce focal adhesion formation [26] and could therefore be expected to reduce wound contraction. On the other hand, relaxation of the contraction occurs due to myofibroblast senescence or apoptosis, which is also dependent on integrin a V b 3 [52]. Our observations suggested that the anti-MFAP4- and anakinra-treatments prolonged the myofibroblast-mediated contraction, while at the same time reducing the collagen-accumulation. Importantly, while myofibroblast contraction is reversible, the deposition and quality of the collagen can make the contraction irreversible [52]. Detailed analysis of the role of anti-MFAP4-treatment in wound/scar contraction would require dynamic, cellular, and ECM constitutional analysis and was not further explored in the present study. We applied proteome analysis using fixed tissue sections. Formaldehyde fixation limits the extraction efficiency of proteins and thereby the sensitivity. Moreover, proteome analysis is likely to underrepresent ECM proteins, which are often crosslinked and hard to extract even without the fixation. Nevertheless, our analysis showed regulation of collagens in the anakinra treatment group. Each tissue section represented tissue areas being transmural and containing the anastomotic line and additional tissue on each side. The most significantly anti-MFAP4-upregulated proteins were reflecting growth and survival of epithelial cells, and the most significantly anti-MFAP4-regulated GO terms were related to upregulation of the SMC contractile apparatus and down-regulation of transcriptional and translational activity. The main observations from the proteomic analysis supported that anti-MFAP4 induced a phenotypical shift from the synthetic/proliferative to the contractile, homeostatic phenotype and a-SMA immuno-staining of the anastomoses supported this observation. This further suggests that the anti-fibrotic effect observed with anti-MFAP4-treatment was secondary to the SMC switch. Integrin a V b 3 ligands are previously shown to regulate increased SMC hyperplasia in stricturing Crohn's disease [8]. In parallel, we have previously observed that MFAP4 induced vascular SMC proliferation through integrin a V b 3 -engagement [26]. Hyperplasia/hypertrophy of the SMC layer contributing to stricture formation is not usually recognized in intestinal anastomoses but often in patients with chronic inflammatory bowel disease. It is responsible for intestinal wall thickening and formation of strictures in disease, where chronic inflammation causes a change of metabolism of SMCs that start producing ECM [53]. The observation that anti-MFAP4’s major therapeutic effect is related to a beneficial phenotypical switch of SMC may therefore be especially relevant for Chron’s patients. However, the anti-fibrotic effect of anti-MFAP4 may potentially be relevant for various types of patients with increased risk of developing gastrointestinal fibrosis or fibrostenosis. Limitations of the study included that therapeutic effects were only tested in female animals, we did not test increasing or repeated dosing of anti-MFAP4, and our outcome analysis was based on fixed tissue sections rather than frozen tissue. One general limitation to advancing anti-fibrotic therapies is the lack of a standardized large animal model of fibrosis-driven luminal stricture. Our study was designed to investigate fibrosis deposition in the anastomosis. Others have developed several months long pig-model receiving 5% phenol and 0.2% 2,4,6-trinitrobenzenesulfonic acid solution for repeated submucosal injection to develop true anastomotic fibrostenosis [54] of relevance for potential additional efficacy-testing. Conclusion To our knowledge, this is the first study of tolerability and anti-MFAP4-mediated anti-fibrotic effect in post-surgery anastomotic healing. We identified MFAP4 as a new target and pathophysiological factor. Therapeutical blocking of MFAP4 maintained the contractile SMC phenotype and reduced fibrosis. The study further supported previous studies of anakinra-mediated reduction of fibrosis. Our observations warrant further testing of anti-MFAP4 as candidate therapy in Crohn’s patients in risk of developing fibrostenosis or as prophylactic treatment in post-surgical anastomotic healing. Abbreviations Crohn’s disease CD Extracellular matrix ECM Smooth muscles cells SMC Microfibrillar associated protein 4–MFAP4 Single cell RNA sequencing–scRNA–seq Interleukin 1 receptor antagonist–IL–1Ra Anastomotic index AI Maximal anastomotic tensile strength MATS Forkhead box P3 FOXP3 α smooth muscle actin–α–SMA Tris buffered saline–TBS Phosphate buffered saline–PBS Bovine serum albumin BSA Radioimmunoprecipitation assay RIPA Horseradish peroxidase HRP o phenylenediamine dihydrochloride–OPD Cartilage Associated Protein CRTAP Collagen type II alpha 1 chain COL2A1 Collagen type III alpha 1 chain COL3A1 Olfactomedin 4 OLMF4 Pyruvate Kinase M PKM Gene Ontology GO Gastrointestinal Diseases and Malformations in Infancy and Childhood GAIN Declarations Consent for publication Not applicable Data availability All data underlying this study are available from the corresponding author upon reasonable request. Competing interests A.S and G.L.S are inventors on U.S. Patent No. 9,988,442 and EP17199552.5 owned by University of Southern Denmark. Funding This work was supported by Novo Nordisk Foundation – Pioneer Innovator grant [NNF22OC0076385]; Clinical center of excellence ‘Gastrointestinal Diseases and Malformations in Infancy and Childhood (GAIN) [20/62844], Odense University Hospital; Overlægerådets forskningsfond [A6053], Odense University Hospital; Odense University Hospital Free Funding [A4431]; A.P. Møller Fonden [2024-00886]; A.J. Andersen og Hustrus Fond [01737-0005 FHP] Author contribution Conceptualization: MBE, NQ and GLS Contributed to study design: MBE, NQ and GLS Methodology and investigations: RRA, JBP, PFHG, CSN, ALK, AFHK, NG, KER, HKH, JK, MD, GIM, LBS and HCB Original draft: RRA and JBP Funding acquisition: MBE and GLS Writing review and editing: All authors. All authors approved the manuscript for publication Acknowledgements The authors wish to thank veterinarians Charlotte Laurfelt Munch Rasmussen and Louise Langhorn, Diana Bianca Hansen, Pernille Simonsen, Kristoffer Augustesen and laboratory technician Vivi Monrad for their assistance with animal care. Moreover, we wish to thank technicians Lone Christiansen, Department of Pathology at Odense University Hospital and Tine Rasmussen, Institute of Molecular Medicine, University of Southern Denmark, for help and assistance. Generative AI was used to correct syntax of long sentences. References Torres J, Mehandru S, Colombel JF, Peyrin-Biroulet L. Crohn's disease. Lancet. 2017;389(10080):1741-55. Lin X, Wang Y, Liu Z, Lin S, Tan J, He J, et al. Intestinal strictures in Crohn's disease: a 2021 update. Therap Adv Gastroenterol. 2022;15:17562848221104951. Khoudari G, Mansoor E, Click B, Alkhayyat M, Saleh MA, Sinh P, et al. 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Body weight, anastomotic index, adhesion formation and histochemical scoring in 5- and 10-days studies Vehicle treatment Anti-MFAP4 treatment 5-days study n=6 n=7 Baseline body weight 19.1(1.2) kg 20.0(1.9) kg Body weight increase 2.6(1.2) kg 2.1(0.4) kg MATS 1 6.3(2.6) N 6.2(2.6) N MATS 2 10.4(1.6) N 9.4(2.7) N MATS 3 10.7(1.6) 10.0(3.1) N Leach score 1.1(1.0) 1.2(0.4) Necrosis 1.1(0.8) 1.3(0.6) Polymorphonuclear cells 1.3(0.6) 1.6(0.6) Lymphocytes 1.3(0.8) 1.3(0.9) Macrophages 0.2(0.4) 0.1(0.3) Oedema 0.8(0.8) 1.1(0.8) Mucosal epithelium 2.3(1.0) 2.4(0.6) Submucosal muscular layer 1.6(0.7) 1.2(0.6) Collagen 25.4(8.9)% 23.2(7.5)% 10-days study + aethoxysklerol n=7 +aethoxysklerol n=7 Baseline body weight 20.1(1.8) 19.0(1.1) Body weight increase 6.9(0.9) 7.1(1.1) Anastomotic index 0.56(0.08) 0.56(0.09) MATS 1 8.9(3.2) 7.5(4.1) MATS 2 9.1(3.1) 9.5(3.3) MATS 3 9.6(3.7) 9.7(3.4) Leach score 1.0(0.6) 0.8(0.4) Necrosis 1.4(1.0) 1.6(0.5) Polymorphonuclear cells 1.4(1.0) 1.6(0.7) Lymphocytes 1.1(0.7) 1.4(0.7) Macrophages 0.2(0.4) 0.1(0.3) Oedema 0.8(0.9) 0.7(0.6) Mucosal epithelium 2.2(1.3) 1.8(1.3) Submucosal muscular layer 1.7(0.7) 1.6(0.9) Collagen 24.3(7.0)% 27.3(10.7)% Anti-MFAP4 treatment was compared with the negative control vehicle-treatment. Leach scoring and Verhofstad scale scorings were analyzed by Mann-Whitney test. Additional variables were analyzed by t-test. No significant changes were found. Data are mean(SD). Table 2. Body weight, anastomotic index, adhesion formation and histochemical scoring in 28-days study Anakinra treatment Vehicle treatment Anti-MFAP4 treatment p -value 28-days study + aethoxysklerol N=9 + aethoxysklerol N=9 + aethoxysklerol N=9 Baseline body weight 29.0(3.6) 28.2(4.4) 29.3(3.2) Body weight increase 22.4(5.1) 24.4(3.9) 25.0(4.5) Anastomotic index 0.62(0.07) a 0.72(0.06) 0.63(0.16) b a p =4.9*10 -5 , b p =0.04 MATS 1 8.3(3.9) 8.5(4.7) 8.8(5.5) MATS 2 11.4(3.6) 10.4(3.3) 11.7(4.8) MATS 3 13.5(4.1) 12.3(3.7) 13.9(4.7) Leach score 0.1(0.3) c 0.8(1.0) 0.8(1.3) c p =0.01 Necrosis 1.0(0.9) 0.9(0.7) 1.1(1.0) Polymorphonuclear cells 0.9(0.6) 0.8(0.7) 1.2(0.9) Lymphocytes 0.9(0.6) d 0.4(0.6) 1.2(0.7) e d p =0.03, e p =0.003 Macrophages 0.4(0.5) 0.3(0.5) 0.7(0.8) Oedema 0.9(0.6) 0.8(0.6) 0.6(0.5) Mucosal epithelium 0.3(0.8) 0.5(1.0) 0.3(0.5) Submucosal muscular layer 1.4(1.0) 1.2(0.7) 1.1(1.0) Collagen 36.1(14.2) f 52.7(17.8) 40.6(18.0) g f p =0.003, g p =0.048 Anakinra- or anti-MFAP4 treatments were compared with the negative control vehicle-treatment. Leach scoring and Verhofstad scale scorings were analyzed by Mann-Whitney test. Additional variables were analyzed by t-test. Data are mean(SD). Additional Declarations Competing interest reported. Anders Schlosser and Grith Lykke Sorensen are inventors on U.S. Patent No. 9,988,442 and EP17199552.5 owned by the University of Southern Denmark. Rasmus Refshauge Andresen, Jesper Brandt Pedersen, Paula Frederikke Hellsegg Grünfeld, Charlotte Skoie Nielsen, Anna Lings Kjelgaard, Anders Frederik Højer Kolind, Nils Grimm, Kasper Emil Rosenbech, Henriette Kirkeby Høiberg, Johanne Kalland, Mie Dilling, Gunvor Iben Madsen, Lasse Bach Steffensen, Hans Christian Beck, Sören Möller, Niels Qvist, and Mark Ellebæk have no competing interests. Supplementary Files TableS1.xlsx TableS4.xlsx TableS5.xlsx SupplementalTablesandFigures.docx Cite Share Download PDF Status: Published Journal Publication published 29 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 06 Oct, 2025 Reviews received at journal 27 Sep, 2025 Reviews received at journal 19 Sep, 2025 Reviews received at journal 09 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 05 Sep, 2025 Reviewers agreed at journal 04 Sep, 2025 Reviewers invited by journal 04 Sep, 2025 Editor assigned by journal 01 Sep, 2025 Editor invited by journal 31 Jul, 2025 Submission checks completed at journal 29 Jul, 2025 First submitted to journal 29 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7132995\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":513280318,\"identity\":\"5eb02981-7bb8-4052-9dc4-ed846514fe5b\",\"order_by\":0,\"name\":\"Rasmus Refshauge Andresen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Rasmus\",\"middleName\":\"Refshauge\",\"lastName\":\"Andresen\",\"suffix\":\"\"},{\"id\":513280319,\"identity\":\"2683560e-1768-42e2-8c67-dcc965d04674\",\"order_by\":1,\"name\":\"Jesper Brandt 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Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Charlotte\",\"middleName\":\"Skoie\",\"lastName\":\"Nielsen\",\"suffix\":\"\"},{\"id\":513280322,\"identity\":\"1ab42e63-e7b4-41ed-81fb-a1d2ae9dd025\",\"order_by\":4,\"name\":\"Anna Lings Kjelgaard\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Anna\",\"middleName\":\"Lings\",\"lastName\":\"Kjelgaard\",\"suffix\":\"\"},{\"id\":513280323,\"identity\":\"5d277213-99f8-41a9-a111-b908369f7ec4\",\"order_by\":5,\"name\":\"Anders Frederik Højer Kolind\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Anders\",\"middleName\":\"Frederik Højer\",\"lastName\":\"Kolind\",\"suffix\":\"\"},{\"id\":513280326,\"identity\":\"6e9b4568-aab9-4d1e-b968-2ce69e881dbb\",\"order_by\":6,\"name\":\"Nils Grimm\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nils\",\"middleName\":\"\",\"lastName\":\"Grimm\",\"suffix\":\"\"},{\"id\":513280334,\"identity\":\"107e8ea0-3f0c-4cf2-b661-0c8f4a3b1348\",\"order_by\":7,\"name\":\"Kasper Emil Rosenbech\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kasper\",\"middleName\":\"Emil\",\"lastName\":\"Rosenbech\",\"suffix\":\"\"},{\"id\":513280335,\"identity\":\"aa6c8231-33c6-4dcd-a664-56d41a20ef67\",\"order_by\":8,\"name\":\"Henriette Kirkeby Høiberg\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Henriette\",\"middleName\":\"Kirkeby\",\"lastName\":\"Høiberg\",\"suffix\":\"\"},{\"id\":513280336,\"identity\":\"fa98cf28-1809-4688-9a14-89c9d91d89d7\",\"order_by\":9,\"name\":\"Johanne Kalland\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Johanne\",\"middleName\":\"\",\"lastName\":\"Kalland\",\"suffix\":\"\"},{\"id\":513280339,\"identity\":\"d1a40a91-6c3b-4bc4-86b1-f8768c3499b7\",\"order_by\":10,\"name\":\"Mie Dilling\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mie\",\"middleName\":\"\",\"lastName\":\"Dilling\",\"suffix\":\"\"},{\"id\":513280340,\"identity\":\"57e6ac71-aa1b-48b9-a5b4-7a504f8ac425\",\"order_by\":11,\"name\":\"Gunvor Iben Madsen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Gunvor\",\"middleName\":\"Iben\",\"lastName\":\"Madsen\",\"suffix\":\"\"},{\"id\":513280341,\"identity\":\"f8766ff9-8761-4aab-acd4-a083acc412ca\",\"order_by\":12,\"name\":\"Anders Schlosser\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Southern Denmark\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Anders\",\"middleName\":\"\",\"lastName\":\"Schlosser\",\"suffix\":\"\"},{\"id\":513280342,\"identity\":\"cb0e651f-3305-4738-8651-02260d3d814f\",\"order_by\":13,\"name\":\"Lasse Bach Steffensen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Southern Denmark\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lasse\",\"middleName\":\"Bach\",\"lastName\":\"Steffensen\",\"suffix\":\"\"},{\"id\":513280343,\"identity\":\"125632fb-19fe-4ef3-8eab-a44e09b03b8d\",\"order_by\":14,\"name\":\"Hans Christian Beck\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hans\",\"middleName\":\"Christian\",\"lastName\":\"Beck\",\"suffix\":\"\"},{\"id\":513280344,\"identity\":\"8969b99b-6cc2-4fe3-a910-dbfc3fd109bf\",\"order_by\":15,\"name\":\"Sören Möller\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sören\",\"middleName\":\"\",\"lastName\":\"Möller\",\"suffix\":\"\"},{\"id\":513280346,\"identity\":\"e3cd5674-e45e-41ac-8d03-63fe5baf58f9\",\"order_by\":16,\"name\":\"Niels Qvist\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Niels\",\"middleName\":\"\",\"lastName\":\"Qvist\",\"suffix\":\"\"},{\"id\":513280347,\"identity\":\"d17a605b-f6ef-4017-81bc-5892c335544f\",\"order_by\":17,\"name\":\"Mark Bremholm Ellebaek\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIie3PsUrDQBzH8f9xayCOFxTzCj8pBAvFvspJ4LoUDPgCBYcuPkDAPET6BidCXaJZD+qQImTq0E4qZLBJpwpJcBO57/Y/+PD/H5HN9geDJk6sujwjQcRmGDWP5Oh2MmzITDgHEql+Mj4m26d+Ejy/vKMm7undeh0jv/HjMKVN1kGyyUCyT+F4yXJwkWI1TI2KWGI6iFZc11tgZOAVWAFiCu5s2wny8kDGZvLxVeAVftxHjOKy2SKmAUuhQaYmHYfBlBzXeyLeslsvRghkZfSYdHwfueJiV43O3Yf5YndfXcGfh4tis2wnTfJoOpGke8DP3N8Cm81m++99A37eWRzbwCcVAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Odense University Hospital\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Mark\",\"middleName\":\"Bremholm\",\"lastName\":\"Ellebaek\",\"suffix\":\"\"},{\"id\":513280348,\"identity\":\"adfad2d2-e3f0-4107-aaca-baf6ec74bf70\",\"order_by\":18,\"name\":\"Grith Lykke Sorensen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Southern Denmark\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Grith\",\"middleName\":\"Lykke\",\"lastName\":\"Sorensen\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-07-15 17:23:19\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7132995/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7132995/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41598-025-30123-0\",\"type\":\"published\",\"date\":\"2025-11-29T15:57:38+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":91086692,\"identity\":\"0d2b3d8d-282c-4f99-881f-e45218130e3f\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:29:57\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":74311,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStudy outline.\\u003c/strong\\u003e Three interventional studies of pig anastomosis healing were performed. In each study two hand sewn anastomoses were created in the small intestines on day 0. The length of the studies was varied to allow observation of tolerability of intervention (\\u003cstrong\\u003eA\\u003c/strong\\u003e, 5-days study) following the initial inflammatory healing phase with provisional wound closure, (\\u003cstrong\\u003eB\\u003c/strong\\u003e, 10-days study) during the proliferative phase where the tissue strength is dynamically restored, and (\\u003cstrong\\u003eC\\u003c/strong\\u003e, 28-days study) following the proliferative phase of healing. The sclerotic agent aethoxyklerol and anakinra was used to induce fibrosis and as positive anti-fibrotic treatment control, respectively. Image is created using BioRender.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/3f47dcafc6ab70c319f2cc02.jpg\"},{\"id\":91085535,\"identity\":\"b4808aa5-5a65-46e9-b5d6-3c08dac331e7\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:21:57\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":77071,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnti-MFAP4 detection in intestinal anastomoses, lung tissue and common iliac artery 14 days post-injection of Anakinra or anti-MFAP4 in the anastomotic area.\\u003c/strong\\u003e Anti-MFAP4 was locally injected 14-days after surgical formation of intestinal anastomosis in pigs and samples are obtained 28 days post-surgery. (\\u003cstrong\\u003eA\\u003c/strong\\u003e) Anti-MFAP4 was measured by ELISA in protein lysates from two intestinal anastomosis, lung and artery samples treated with either anakinra or anti-MFAP4, (n =2-3). Data are individual measurement points with mean (SD). Unspecific staining of anti-MFAP4 is seen in blood (blue arrow, arterial blood) in (\\u003cstrong\\u003eB\\u003c/strong\\u003e) vehicle- or (\\u003cstrong\\u003eC\\u003c/strong\\u003e) anti-MFAP4-treated sample. Anti-MFAP4 was specifically immunodetected (red arrows) in (C). Bar = 100 µm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/d9bd2ea1020c97daeb90f44d.jpg\"},{\"id\":91085534,\"identity\":\"c0a042db-71a3-455a-8602-e463299aa078\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:21:57\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":130671,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCollagen deposition in the late anastomotic healing phase. \\u003c/strong\\u003eRepresentative images of collagen deposition (Sirius Red staining) in anastomotic areas in the 28-days study with (\\u003cstrong\\u003eA\\u003c/strong\\u003e) anakinra-treatment, (\\u003cstrong\\u003eB\\u003c/strong\\u003e) vehicle-treatment or (\\u003cstrong\\u003eC\\u003c/strong\\u003e) anti-MFAP4-treatment, respectively. Anakinra, vehicle and anti-MFAP4 were locally injected 14-days post-surgery in the 28-days study. Black lines frame the anastomotic area. Bar = 1000 µm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/929327f6fea7363591ecb368.jpg\"},{\"id\":91085538,\"identity\":\"e40422b1-bd65-42f9-b9dc-19c083e0e322\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:21:57\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":122587,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eProteomic analysis of intestinal anastomosis. \\u003c/strong\\u003eTissue sections from anastomoses obtained from the 28-days study were subjected to proteome analysis. Volcano plots are showing regulation of identified proteins in (\\u003cstrong\\u003eA\\u003c/strong\\u003e) anakinra- (n=9) or (\\u003cstrong\\u003eB\\u003c/strong\\u003e) anti-MFAP4-treated (n=9) anastomoses relative to negative control vehicle treated anastomoses (n=9). The five most significantly down-regulated proteins in volcano plots are shown in red and most significantly up-regulated proteins are shown in green. The ten most significantly up- and down-regulated GO terms are shown for each treatment in (\\u003cstrong\\u003eC-D\\u003c/strong\\u003e) anakinra-treated or (\\u003cstrong\\u003eE-F\\u003c/strong\\u003e) anti-MFAP4-treated anastomoses. Nominal \\u003cem\\u003ep\\u003c/em\\u003e-values are shown.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/c1fe3a4fb88cba1e27e53ca4.jpg\"},{\"id\":91088629,\"identity\":\"5b7b54a3-d5db-4b9f-ab30-f6efa470ee08\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:45:57\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":155465,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnti-MFAP4 induces a-SMA in the muscular layers in the late anastomotic healing phase. \\u003c/strong\\u003eRepresentative images of a-SMA\\u003cstrong\\u003e \\u003c/strong\\u003estaining (brown staining) of muscular layers in\\u003cstrong\\u003e \\u003c/strong\\u003eanastomotic and peri-anastomotic areas in 28-days study with (\\u003cstrong\\u003eA\\u003c/strong\\u003e) anakinra treatment, (\\u003cstrong\\u003eB\\u003c/strong\\u003e) vehicle treatment or (\\u003cstrong\\u003eC\\u003c/strong\\u003e) anti-MFAP4 treatment, respectively. Red arrows indicate areas of intact circular and longitudinal smooth muscle layers of muscularis propria. Anakinra, vehicle and anti-MFAP4 were locally injected 14-days post-surgery in the 28-days study. Bar = 1000 µm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/b9570f35431b6395f85a75b3.jpg\"},{\"id\":97178381,\"identity\":\"ee27e8c6-e3bd-402e-ad37-dc3e79b83ca9\",\"added_by\":\"auto\",\"created_at\":\"2025-12-01 16:09:24\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1994613,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/8207d7e7-e379-48ae-b921-7a32b68129f1.pdf\"},{\"id\":91085545,\"identity\":\"c24317b8-cd2a-4633-98ff-b20874e7178d\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:21:57\",\"extension\":\"xlsx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1172644,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"TableS1.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/a0476156ac084d9ed745b8b0.xlsx\"},{\"id\":91087474,\"identity\":\"1349f078-bc0b-4946-95ed-ab9d5602ef9a\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:37:57\",\"extension\":\"xlsx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1011599,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"TableS4.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/685532d730c2a746aea581b3.xlsx\"},{\"id\":91085544,\"identity\":\"035a9285-5839-4bf5-8ed5-6b1ff2cd005c\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:21:57\",\"extension\":\"xlsx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1195989,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"TableS5.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/03cfa7ce4bf42ccc66f4db09.xlsx\"},{\"id\":91086699,\"identity\":\"e3dd6db6-9c2f-4e5d-8ae5-9bfa092abb12\",\"added_by\":\"auto\",\"created_at\":\"2025-09-11 12:29:57\",\"extension\":\"docx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":8560221,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementalTablesandFigures.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7132995/v1/8c44e337f40101fefbe83dd0.docx\"}],\"financialInterests\":\"Competing interest reported. Anders Schlosser and Grith Lykke Sorensen are inventors on U.S. Patent No. 9,988,442 and EP17199552.5 owned by the University of Southern Denmark.\\nRasmus Refshauge Andresen, Jesper Brandt Pedersen, Paula Frederikke Hellsegg Grünfeld, Charlotte Skoie Nielsen, Anna Lings Kjelgaard, Anders Frederik Højer Kolind, Nils Grimm, Kasper Emil Rosenbech, Henriette Kirkeby Høiberg, Johanne Kalland, Mie Dilling, Gunvor Iben Madsen, Lasse Bach Steffensen, Hans Christian Beck, Sören Möller, Niels Qvist, and Mark Ellebæk have no competing interests.\",\"formattedTitle\":\"Tolerability and effect of inhibiting microfibrillar-associated protein 4 in small intestinal anastomotic healing\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eCrohn's disease (CD) is a type of inflammatory bowel disease characterized by chronic transmural inflammation, most commonly affecting the terminal ileum [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. The inflammation may lead to intestinal wall fibrostenosis, and up to 70% of the Crohn\\u0026rsquo;s disease patients will develop fibrotic stricture at 10 years after diagnosis [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Fibrostenotic complications are the most common driver for intestinal resection. After introduction of biologics then cumulative 5-year resection rate decreased from 12,5% to 9,3% after diagnosis [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. A similar trend in the re-resection rate has been demonstrated. The disease-driven re-resection rates after 1, 5 and 10-years was 3,6%, 10,1% and 14,1% respectively in a population study [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eThe development of intestinal strictures in CD is multifactorial. Aberrant inflammatory wound healing characterized by excessive collagen-rich extracellular matrix (ECM) deposition by increased numbers of mesenchymal cells is one factor and smooth muscles cell (SMC) hyperplasia/hypertrophy is another important factor [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. SMC are recognized with the capacity to dedifferentiate from a normal contractile, quiescent phenotype to a synthetic phenotype characterized with increased ECM synthesis and proliferation during wound healing and pathophysiological conditions [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Excessive SMC proliferation has been demonstrated in both Crohn\\u0026rsquo;s strictures [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e] as well as in animal models of induced intestinal inflammation [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] and is recognized as the most prominent histological change in crohn's fibrostenosing strictures[\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. The pathophysiological process includes increasing expression of integrin α\\u003csub\\u003eV\\u003c/sub\\u003eβ\\u003csub\\u003e3\\u003c/sub\\u003e, which has been shown to increase SMC proliferation and hyperplasia in stricturing CD [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eIn the earliest phase of wound healing (day 1\\u0026ndash;4) hemostasis results in clot-formation and the release of pro-inflammatory cytokines. Inflammation is induced by the cytokines and the accumulation of apoptotic and necrotic cellular material followed by migration of neutrophiles and macrophages. This is followed by a proliferative phase (day 4\\u0026ndash;14) where mesenchymal cells migrate into the healing tissue and start to proliferate. These cells; including fibroblasts, myofibroblasts and SMCs, are responsible for the formation of collagen. The resulting collagen deposition provides anastomotic strength and stability and the tissue continuity is dynamically restored during this phase [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Also, a shift from pro- to anti-inflammatory signaling restricts the inflammatory response in this phase. In the final remodeling phase (day 14 \\u0026loz;), deposited collagen is remodeled by fibrolytic activity. Collagen type III is partly replaced with the more durable collagen type I and tissue strength is plateaued [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. A disturbed wound healing from an underlying disease such as CD may result in the formation of excessive extracellular matrix resulting in a fibrotic stenosis in the anastomotic area and recurring after surgical resection [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eThe ECM protein microfibrillar-associated protein 4 (MFAP4) co-localizes with elastin and collagen fibers, especially in small intestine, the heart, and lungs [\\u003cspan additionalcitationids=\\\"CR13\\\" citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. MFAP4-deficiency is demonstrated to be protective of development of fibrosis in heart and kidney [\\u003cspan additionalcitationids=\\\"CR16\\\" citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Recently, an animal model of inflammatory bowel disease showed that MFAP4 colocalized with fibrotic markers suggesting it to also be a profibrotic driver in the intestines [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. MFAP4 is found significantly increased in response to TGF-β treatment in human mesenchymal cells [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e], and in line with this, transcriptomic analysis has demonstrated that MFAP4 is expressed in SMC, fibroblasts and activated fibroblasts in ileal resections from CD patients [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eThe above observations provides the rationale for testing the role of MFAP4 in intestinal fibrostenotic processes.We hypothesized that MFAP4 could serve as therapeutic target in anastomotic fibrostenotic processes.\\u003c/p\\u003e\\u003cp\\u003eWe have previously developed an anti-MFAP4 antibody with the capacity to block MFAP4 binding to integrin α\\u003csub\\u003eV\\u003c/sub\\u003eβ\\u003csub\\u003e3\\u003c/sub\\u003e and the consective activation of integrin expressing cells [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. We tested if local pharmacological treatment with anti-MFAP4 was tolerated, tested therapeutic efficacy of reducing fibrosis in pig intestinal anastomotic healing, and investigated the therapeutic effects by proteome analysis.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthical approval\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll experiments were performed in accordance with relevant guidelines and regulations, and all live animal experimental protocols were conducted according to ARRIVE 2.0 guidelines [21] and were approved by the Danish Animal Experiments Inspectorate (j. nr. 2018-15-0201-01583).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStudy design\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThree separate interventional randomized and investigator blinded studies were performed using Danish Landrace pigs. In each study two hand sewn anastomoses were created in the small intestines.\\u0026nbsp;The different lengths of the studies, 5 days, 10 days and 28 days, were chosen for the observation of tolerability of injecting anti-MFAP4 in the different phases of wound healing [10]. Tolerability outcomes were anastomotic healing following initial inflammatory healing phase with provisional wound closure (Day 5), during the proliferative phase where the tissue strength is dynamically restored (Day 10) and during the remodeling phase (Day 28). The tolerability was evaluated through patological scoring, macroscopical findings, measurements of tissue strength and formation of adherences. The primary efficacy outcome was collagen-deposition following anti-MFAP4 treatment observed in the 28-days study. The secondary outcome was therapeutic mechanisms evoked by anti-MFAP4 in the 28-days study and observed by proteome analysis .\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e5-days study:\\u0026nbsp;\\u003c/strong\\u003eEach pig received either vehicle control treatment (n=6) or anti-MFAP4 treatment (20 mg/ml) (n=7). A total of 0.8 ml of vehicle or anti-MFAP4 was injected in\\u0026nbsp;8 predefined subserosal depots on each side of the anastomosis at a distance of 2 mm from the anastomotic line. Pigs were euthanized at postoperative day 5 and the anastomotic site was resected. After a tensile strength test the anastomotic tissues was fixed in formalin for later histological examination.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e10-days study:\\u003c/strong\\u003e All pigs received aethoxysklerol (5 mg/ml) \\u0026nbsp;injections with a total of 0.8 ml distributed on 8 predefined subserosal depots on both sides of the anastomotic side together with either vehicle control treatment (n=7) or anti-MFAP4 treatment (20 mg/ml) (n=7) as described in the 5-days study. Pigs were euthanized on postoperative day 10. The anastomotic area was resected after in situ contrast study (X-ray). After a tensile strength test the anastomotic tissue was fixed in formalin for later histological examination.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e28-days study:\\u003c/strong\\u003e In all pigs, aethoxysklerol injections was performed as described above. On postoperative day 14, a re-laparotomy was performed, and the pigs were randomized to receive either positive control anakinra (Interleukin-1 receptor antagonist (IL-1Ra)) treatment (40 mg/ml) (n=9), vehicle control treatment (n=9), or anti-MFAP4 treatment (40 mg/ml). In total, a volume of 0.8 ml of each was injected around the anastomotic area as described above. On postoperative day 28, in situ contrast study (X-ray) of the anastomotic area was performed, and the anastomotic area was resected. After a tensile strength test the anastomotic tissue was fixed in formalin for later histological examination.\\u003c/p\\u003e\\n\\u003cp\\u003eFigure 1 summarizes the study outline.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnimals\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWeaned female Danish Landrace \\u0026amp; Yorkshire pigs (obtained from the breader Kokkenborg, Stenstrup, Denmark) of approximately 20 kg were included in the study. The pigs were acclimatized to their new environment for at least one week prior to surgery. They were housed at a conventional large animal housing facility with light/dark cycles (twelve hours with gradually dimmed light and natural light from windows) at a constant temperature of 20-21\\u0026deg;C. The pigs had access to food twice daily (0.9 kg/20 kg body weight) and free access to water. Before surgical procedures the pigs were weighed. To ensure animal welfare, the pigs were intensively inspected daily, and during the first 24 hours following surgery.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnesthesia\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePigs were subjected to a fasting period of 12 hours before surgery with free access to drinking water. Pre-anesthetic sedation was a combination of 0.25 mg/kg midazolam (Midazolam Hameln\\u0026reg;, Hameln Pharmaceuticals GmbH, Hameln, Germany), 0.03 mg/kg medetomidine (Cepetor, Scanvet), 0.2 mg/kg butorphanol (Butomidor, Salfarm Danmark A/S, Kolding, Denmark) and 5 mg/kg ketamine (Ketaminol Vet.,100 mg/ml, MSD Animal Health) administered intramuscularly (im.). Anaesthesia was induced with 5 mg/kg propofol (B. Braun Medical A/S, Copenhagen, Denmark) intravenously (iv.) through an ear vein and the pigs were intubated with a cuffed tube size 4.0 and connected to a Siesta i TS Anaesthesia Machine (Dameca, R\\u0026oslash;dovre, Denmark). Anaesthesia was maintained with a continuously infusion of 15 mg/kg/hour propofol (B. Braun Melsungen AG, Melsungen, Germany) and 25-50 \\u0026micro;g/kg/hour fentanyl (B. Braun Melsungen AG, Melsungen, Germany). Perioperative analgesia consisted of fentanyl (B. Braun Melsungen AG, Melsungen, Germany) iv. administered, in a dose of 50 \\u0026mu;g/kg/hour.\\u0026nbsp;The pigs were mechanically ventilated with a tidal volume of 7-10 ml/kg. The respiratory frequency was adapted to the ETCO\\u003csub\\u003e2\\u003c/sub\\u003e level. Blood pressure, electrocardiogram, heart rate and oxygen saturation were monitored continuously.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe pigs received prophylactic antibiotics preoperatively with 15 mg/kg amoxicillin (Curamox \\u0026reg; Prolongatum Vet. Boehringer Ingelheim Danmark A/S, K\\u0026oslash;benhavn \\u0026Oslash;, Denmark), perioperatively 20 mg/kg metronidazole (B. Braun, Melsungen AG, Germany), and 48 and 96 hours postoperatively 15 mg/kg amoxicillin (Curamox \\u0026reg; Prolongatum Vet. Boehringer Ingelheim Danmark A/S, K\\u0026oslash;benhavn \\u0026Oslash;, Denmark).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePostoperative analgesia consisted of fentanyl transdermal patch 2 \\u0026micro;g/kg/hour (Matrifen\\u0026reg; Takeda Pharma A/S, Taastrup, Denmark)\\u0026nbsp;rounded down to the nearest patch dose in increments of 12 \\u0026micro;g/hour. The fentanyl patch was removed after 3 days. Additionally, some pigs received buprenorphine 0.03 mg/kg (Bupaq \\u0026reg; Multidose, Salfarm Danmark A/S, Kolding, Denmark) i.m. every fourth to six hours for the first 24 hours after surgery, while pigs in the 28-days study received a subcutaneous injection with a weight-adjusted dose of sustained-release formulation of buprenorphine preoperatively, ensuring analgesia for at least 48 hours postoperatively.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEuthanization\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePrior to euthanization the pigs underwent general anesthesia as described above. The pigs were euthanized using 140 mg/kg pentobarbital (400 mg/ml) (Euthanimal, ScanVet Animal Health A/S, Fredensborg, Danmark) administered via the ear vein.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSurgery\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDuring surgery, the pigs were placed on a heating pad set to 32\\u0026deg;C, to avoid hypothermia. They received Ringer\\u0026apos;s acetate i.v. at a rate of 4-10 ml/kg/h. The small intestines were exposed through a 10 cm lower midline laparotomy. Two end-to-end anastomoses were created 50 cm and 150 cm orally to the ileoceacal junction with a seromuscular running suture using monocryl 4-0 (Ethicon, Johnson \\u0026amp; Johnson, Diegem, Belgium). After interventional procedures the abdominal fascia was closed with running PDS*II\\u0026reg; 0 (Ethicon, Johnson \\u0026amp; Johnson, Somerville, New Jersey, USA). The skin was closed intracutaneously with a running monocryl\\u0026reg; 3-0 (Ethicon, Johnson \\u0026amp; Johnson, Diegem, Belgium). The skin incision was sealed with a liquid bandage (KRUUSE Wound Plast, Cat. no: 161020).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAt study completion, a\\u0026nbsp;re-laparotomy was performed, and all the anastomoses were identified and freed from adhesions, which were graded in using a modified Leach grading of adhesion (Supplemental Table S2). All anastomoses were examined for macroscopical findings such as pseudo-diverticulosis, abscesses, visible leakage, fistulas, or signs of ileus.\\u003c/p\\u003e\\n\\u003cp\\u003eIn 5 pigs, anastomotic tissue, lung, and artery biopsies from the 28-days study were collected at end-study for analysis of anti-MFAP4 deposition.\\u0026nbsp;An incision in the right diaphragmatic dome allowed harvesting of the biopsy from the basis of the middle lobe of the right lung. The arterial biopsy was harvested from the left common iliac artery. Biopsies were snap frozen with liquid nitrogen, and stored at -80 \\u0026deg;C. \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInterventional agents and controls\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAnti-MFAP4 is a humanized, monoclonal IgG1 antibody, that binds to MFAP4 with high affinity and blocks its interaction with integrins [20]. Wild-type anti-MFAP4 was used for the 5-days and the 10-days study. For the 28-days study, we applied a point-mutated variant with minimized Fc-mediated effects the so-called hIgG1-P329G\\u0026nbsp;LALAvariant [20]. Aethoxysclerol\\u003csup\\u003e\\u0026reg;\\u0026nbsp;\\u003c/sup\\u003e(Kreussler Pharma) causes submucosal fibrosis by local injection\\u0026nbsp;[22]. The positive control was anakinra (40 mg/mL, Kineret\\u003csup\\u003e\\u0026reg;\\u003c/sup\\u003e. Swedish Orphan Biovitrum.\\u0026nbsp;Batch\\u0026nbsp;3798201c), vehicle control (10 mM histidine, 10% w/w trehalose pH 6.0), or anti-MFAP4 (20 or 40 mg/mL). For both anti-MFAP4- and aethoxysklerol-treatment and for control-treatments (vehicle and anakinra) a volume of 0.8 ml was injected into the anastomotic area.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRadiography\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe wound contraction at the anastomotic site was assessed in situ by a contrast study. The intestinal loop was clamped\\u0026nbsp;approximately 10 cm proximally and distally to each anastomosis. Water-soluble contrast (Omnipaque\\u0026reg;, GE HealthCare) was infused to a pressure of 20 mmHg and an image in one plane was obtained. The intestinal diameters were measured orally and aborally to the anastomotic site using an image analysis software (Image J, NIH, Bethesda, USA). An anastomotic index (AI) was calculated for each anastomosis by dividing the diameter of the anastomosis sites by a mean pre- and postanastomotic diameter based on five measure points at 1 cm intervals from the anastomotic line (Supplemental Figure S2).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMaximal anastomotic tensile strength (MATS)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFollowing radiography, the anastomosis was resected with approximately 5 cm margin orally and aborally and cleaned of fecal contents with water prior to maximal anastomotic tensile strength (MATS) test. The resected intestinal segment was mounted by two clamps in the tensile testing machine (LF Plus\\u0026reg;; Lloyds Instruments, Fareham, UK) equipped with an XCL 100 N loadcell (Lloyds Instruments, Fareham, UK) 5 minutes after resection to prevent the influence of cold ischemia. The position of the clamps was in a distance of 6 cm with the anastomosis placed in the center (Supplemental Figure S3). The segment was gradually stretched at a constant deformation rate of 15 mm/min until rupture occurred.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe applied force in Newton was measured at three points:\\u0026nbsp;\\u003c/p\\u003e\\n\\u003col start=\\\"1\\\" type=\\\"1\\\"\\u003e\\n \\u003cli\\u003eWhen a tear became visible in the serosa (MATS 1).\\u0026nbsp;\\u003c/li\\u003e\\n \\u003cli\\u003eWhen a transmural lesion was visible (MATS 2). The rupture was confirmed by a simultaneous drop in the load-strain curve calculated by the software NEXYGEN Plus \\u0026reg; (version 3.0; Liller\\u0026oslash;d, Denmark).\\u0026nbsp;\\u003c/li\\u003e\\n \\u003cli\\u003eThe maximal force applied during the tensile strength test (MATS 3) was calculated by the software.\\u0026nbsp;\\u003c/li\\u003e\\n\\u003c/ol\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePeparation for histology\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA ten mm long intestinal tissue with central intact anastomotic line was prepared and fixed in a 4% formaldehyde solution for 48 hours and subsequently embedded in paraffin. Three micrometer thick slices were stained with hematoxylin and eosin or picrosirius red for histological evaluation.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eHistological evaluation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe anastomotic healing of each anastomosis was visually assessed according to the Verhofstad scale [23] (Supplemental Table S3), evaluating necrosis, polymorph nucleated cells, lymphocyte infiltration and macrophages, degree of edema, state of epithelial layer and bridging of the submucosal layer. Collagen deposition at the anastomotic line was scored in percentage of the anastomotic area. Each anastomosis was assessed independently using a mean score from two sections from the same anastomosis. Treatment group was blinded to the assessor.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eProteome analysis\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThree \\u0026micro;m sections of formalin-fixed, paraffin-embedded tissue from the distal anastomosis of each animal (n=9/group) were deparaffinized by using xylene followed by extraction of proteins by dissolving the deparaffinized tissue sections in extraction buffer (1M dithiothreitol (DTT), 0.2 M tetraethylammonium bicarbonate and 10% sodium dodecyl sulfate, then subjected to two rounds of ultra-sonification (15 minutes of ultra-sonification/15 minutes of cooling on ice), and incubated at 99 ᵒC for 20 minutes then at 80 ᵒC for 2 hours. Protein alkylation was done by adding a 200 mM iodoacetamide (IAA) solution to a final DTT/IAA concentration ratio of 1:3. The acetone-precipitated proteins were re-dissolved in 5 \\u0026mu;L 8 M urea with 1 \\u0026mu;g LysC and incubated at 30 ᵒC for 4 hours, followed by a further dilution to 1 M urea, the addition of 2 \\u0026mu;g trypsin, and an overnight incubation at 30 ᵒC. The resulting tryptic peptide samples were isotopically labeled using the 16-plex tandem mass tag (TMTpro) (Thermo Fisher, USA) where a pool of all samples was labeled with the mass tag 126 that served as an internal standard. Tagged peptides were mixed into three mixed peptide samples that were fractionated into 7 fractions using high pH chromatography and analyzed by nano-HPLC-MSMS also as previously described [24] with the following modifications: LC gradient length was 100 minutes, higher-energy collisional dissociation was set to 36. All Thermo Scientific Exploris 480 raw data files were processed and quantified using Proteome Discoverer version 3.0 (Thermo Scientific, Waltham, MA, UnitedStates) using a combined Chimerys, Sequest and MS Amanda search. For Sequest and MS Amanda searches TMTpro (N-terminal and lysine) and cysteine carbamidomethylation set as fixed modifications, and methionine oxidation and asparagine and glutamine deamidation as variable modifications whereas for Chimerys TMTpro (N-terminal and lysine) and cysteine carbamidomethylation were set as fixed. Data were searched against the Uniprot pig proteome (Sus scrofa, UP000008227, 46225 entries, downloaded 12\\u003csup\\u003eth\\u003c/sup\\u003e June 2024).\\u003c/p\\u003e\\n\\u003cp\\u003ePathway enrichment analyses were performed using clusterProfiler 4.0 package [25] and org.Hs.eg.db in R.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemistry\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThree \\u0026micro;m thick\\u0026nbsp;formalin-fixed, paraffin-embedded sections were mounted on FLEX IHC Microscope slides (Dako/Agilent, Glostrup, Denmark). Sections were dried at room temperature and baked at 60\\u0026deg;C for 60 minutes. Following, epitope retrieval was performed by processing samples in DISCOVERY Cell Conditioning solution (Ventana) for 32 min at 100\\u0026deg;C. Humanized anti-MFAP4 (human IgG) and Forkhead box P3 (FOXP3) were detected by immunohistochemistry using Goat anti-Human IgG Fc (Abcam, Cambridge, UK, cat# ab ab97221) diluted 1:2000 or rat anti-FOXP3 (Thermo Fischer Scientific, cat# ab ab97221) diluted 1:100 and incubated for 32 mins at 36\\u0026deg;C. Staining was automated using the Discovery Ultra immunostainer (Ventana Medical Systems, Tucson, AZ) and using the OmniMap anti-Goat-HRP detection system (Roche cat#\\u0026nbsp;760-4647) for anti-MFAP4 or OmniMap anti-Rat-HRP (Roche cat#760-4457) for anti-FOXP3 followed by Discovery\\u0026nbsp;ChromoMap DAB\\u0026nbsp;(Ventana Medical Systems, Tucson, AZ). Nuclear counter staining was performed using\\u0026nbsp;Hematoxylin\\u0026nbsp;II (Ventana Medical Systems, Tucson,\\u0026nbsp;AZ). Finally, slides were washed, dehydrated, and\\u0026nbsp;coverslipped\\u0026nbsp;using an automated\\u0026nbsp;Dako\\u0026nbsp;coverslipper (Dako/Agilent,\\u0026nbsp;Glostrup, Denmark).\\u003c/p\\u003e\\n\\u003cp\\u003eFor\\u0026nbsp;a-smooth muscle actin (a-SMA) staining the tissues were processed as described above.\\u0026nbsp;Epitope retrieval was performed in Target Retrieval Solution-H (Agilent) for 30 min at 97\\u0026deg;C. Staining was performed using\\u0026nbsp;the\\u0026nbsp;OMNIS\\u0026nbsp;automated immunohistochemistry\\u0026nbsp;platform (Agilent) using mouse anti-a-SMA (BS66, Nordic Biosite, cat#BSH-7459) diluted 1:1,000 and detected using\\u0026nbsp;EnVision FLEX/HRP+ Mouse LINK and\\u0026nbsp;EnVision FLEX DAB+ Substrate Chromogen System (Agilent).\\u0026nbsp;Nuclear counter staining was performed using\\u0026nbsp;Hematoxylin, Mayers ready to use solution (Agilent). Finally, slides were washed, dehydrated, and\\u0026nbsp;cover slipped\\u0026nbsp;using an\\u0026nbsp;automated Dako cover slipper (Dako/Agilent, Glostrup, Denmark).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eELISA detection of anti-MFAP4 (human IgG) in tissue samples\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBuffers: Tris-buffered saline (TBS): 140 mM NaCl (Merck Millipore), 10 mM Tris-HCl (Sigma-Aldrich), 0.02% (w/v) NaN\\u003csub\\u003e3\\u003c/sub\\u003e (Sigma-Aldrich), pH 7.4; TBS/Tw: TBS containing 0.05% (v/v) Tween 20 (polyoxyethylene sorbitan monolaurate, Merck Millipore); phosphate-buffered saline (PBS): 137 mM NaCl (Merck Millipore), 3 mM KCl (Merck Millipore), 8 mM Na\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e (VWR) , 1.5 mM KH\\u003csub\\u003e2\\u003c/sub\\u003ePO\\u003csub\\u003e4\\u003c/sub\\u003e (Merck Millipore), pH 7.4; PBS/Tw: TBS containing 0.05% (v/v) Tween 20 (polyoxyethylene sorbitan monolaurate, Merck Millipore);\\u0026nbsp;PBS/Tw/Bovine serum albumin (BSA): PBS/Tw, 0.1% BSA; substrate buffer: 35 mM citric acid (Sigma-Aldrich), 67 mM Na\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e (VWR), pH 5.0.\\u003c/p\\u003e\\n\\u003cp\\u003eSnap-frozen tissues from each of two anastomoses/pig, one pulmonary or arterial sample were included in ELISA analysis. 96-well MaxiSorp plates (NUNC MaxiSorp) were coated with in house produced recombinant MFAP4 (0.25 \\u0026mu;g/ml in PBS) overnight at 4\\u0026deg;C, washed with PBS/Tw/BSA, and then blocked with PBS/Tw/BSA for 1 hour at room temperature. To create a standard curve, Anti-MFAP4 was diluted to 10 ng/ml in PBS/Tw/BSA, followed by 2-fold dilutions in PBS/Tw/BSA. Intestinal, pulmonary and arterial tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich cat#R0278) using a Precellys 24 tissue homogenizer (Bertin Instruments) and protein concentrations of the lysates determined using the Biorad DC protein assay (Biorad cat nr 500112). \\u0026nbsp;The lysates were diluted in PBS/Tw/BSA, to be within the linear range of the assay and aliquoted onto the MFAP4-coated plates (100 \\u0026mu;l per well). All samples were incubated 1 hour at room temperature, washed with PBS/Tw/BSA and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (ab97225, Abcam) at 1:10,000 dilution in PBS/Tw/BSA for 30 min at room temperature. The plates were then washed and incubated with streptavidin-HRP conjugate (Invitrogen) diluted 1:2,000 in TBS/Tw for 15 min, washed again and incubated with o-phenylenediamine dihydrochloride \\u003cstrong\\u003e(\\u003c/strong\\u003eOPD) substrate (0.8\\u0026nbsp;mg/ml, Kementec) dissolved in substrate buffer (0.03% freshly prepared H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u0026nbsp;\\u003c/sub\\u003e(Sigma-Aldrich) in the dark at room temperature. Color development was stopped by the addition of 100 \\u0026micro;l 1 M H\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e, (Sigma-Aldrich) and the plates were read at OD\\u003csub\\u003e492\\u003c/sub\\u003e nm with OD\\u003csub\\u003e600\\u003c/sub\\u003e nm as reference.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStatistical methods\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFor each continuous outcome variable student\\u0026rsquo;s t-test was performed for the comparison between anti-MFAP4 or positive control anakinra-treatment relative to the negative control vehicle-treatment. Mann-Whitney test was performed for comparisons of leach-scoring and Verhofstad scale scoring. Results were considered statistically significant if p\\u0026lt;0.05. Statistical analyses were performed using GraphPad Prism 10 (https://www.graphpad.com).\\u003c/p\\u003e\\n\\u003cp\\u003ePathway enrichment analyses were based on ranked log2-transformed fold changes (gse function) and unadjusted \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 (unpaired, two-tailed student\\u0026rsquo;s t-test for each protein per two-group comparison). The \\u003cem\\u003eBenjamini-Hochberg\\u0026nbsp;\\u003c/em\\u003eprocedure was applied to correct for multiple testing.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eTolerability of local injection of anti-MFAP4 in the anastomosis in early healing phases\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn the 5-days study, the mean body weight increase \\u0026nbsp;as well as the anastomotic tensile strength as assessed by MATS by day-5 was similar between vehicle and anti-MFAP4 treated groups (Table 1).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe histological Verhofstad scoring was similar between the two groups. No macroscopical findings such as \\u0026nbsp;pseudo-diverticulosis, abscesses, leakage, fistulas, or signs of ileus was observed in any of the anastomoses (Table 1).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe addition of aethoxysklerol injections and 5-days longer post-surgery period (10-days study) had no effect on the results (Table 1). The anastomotic index in the 10-days study was similar in the vehicle group and the anti-MFAP4 group with a median of 0.56 in both groups. The scorings of sirius red-determined collagen percentage was similar between the 5-days study, which represents the inflammatory stage where collagen deposition is expected to be limited, and the 10 days-study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTolerability of local injection of anti-MFAP4 in the anastomosis in the late healing phase\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn the 28-days study we increased the anti-MFAP4 dose to 40 mg/ml to test an escalated dose and we introduced anakinra. We used a variant form of anti-MFAP4, that abolish potential Fc-mediated undesirable functions, which are immune-activating effects of native antibodies potentially casing cytotoxicity. \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eInitially, we tested for potential systemic deposition of the locally administered anti-MFAP4 through investigations of samples obtained from lung and artery. We did not observe any detectable anti-MFAP4 (human IgG) in lung and artery, while anti-MFAP4 was detected in the anastomosis 28 days post-surgery (14 days post-injection) (Figure 2A). Immunostaining of anti-MFAP4 in anastomotic sections from the 28-days study showed no specific staining in vehicle treated sample, and specific staining of elastic fibers (the main location of MFAP4\\u0026nbsp;[26, 27]) and additional perivascular staining in the anastomotic tissue of anti-MFAP4 treated samples (Figure 2B-C).\\u003c/p\\u003e\\n\\u003cp\\u003eThe anastomotic index was significantly lower both in the anti-MFAP4 treatment group and the anakinra treatment group relative to vehicle control treatment, suggesting that wound contraction was sustained by both treatments (Table 2). Adhesion score was not affected by anti-MFAP4 but was significantly reduced by anakinra. The histological examination showed a significant increase in lymphocytes in both the anakinra- and anti-MFAP4 treatment groups relative to vehicle treatment (Table 2). There were no macroscopical findings of pseudo-diverticulosis, abscesses, visible leakage, fistulas, or signs of ileus in any of the anastomoses.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnti-MFAP4 and total collagen deposition in late anastomotic healing phase\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe mean anastomotic collagen scoring was approximately doubled in the vehicle-treated 28-days study (Table 2) relative to the earlier healing phase (10-days model, Table 1). Both the positive control anakinra- and anti-MFAP4-treatment resulted in significant reduction of the anastomotic collagen deposition relative to vehicle treatment (Table 2). The anti-MFAP4-treated group showed a significant 23% mean score reduction relative to vehicle treatment. Representative images of collagen deposition in anakinra-, vehicle- and anti-MFAP4-treated groups are shown in Figure 3.\\u003cbr\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eProteomic changes inferred by anti-MFAP4 treatment in the anastomosis in late healing phase\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA total of 3075 unique proteins were identified by proteomic analysis in all samples (Supplemental Table S1). Nominally regulated proteins from the positive control anakinra- and anti-MFAP4-treated anastomoses are displayed on volcano plots in Figure 4A-B. Remarkably, the most significantly down-regulated proteins by the positive anti-fibrotic control treatment anakinra included Cartilage Associated Protein (CRTAP), Collagen type II alpha 1 chain (COL2A1) and Collagen type III alpha 1 chain (COL3A1), which are all related with fibrosis (Figure 4A). This supported the observation of anakinra-mediated reduction of Sirius red-determined fibrosis. Amongst the most significantly up-regulated proteins in the anti-MFAP4 treated groups were markers of epithelial healing; Olfactomedin 4 (OLMF4), which is a marker of intestinal epithelial stem cells [28], and Pytuvate Kinase M (PKM), which may repress\\u0026nbsp;\\u003ca href=\\\"https://www.sciencedirect.com/topics/medicine-and-dentistry/programmed-cell-death\\\" title=\\\"Learn more about apoptosis from ScienceDirect's AI-generated Topic Pages\\\"\\u003eapoptosis\\u003c/a\\u003e and facilitate survival of intestinal epithelial cells\\u0026nbsp;[29].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eWe analyzed the proteome data using an unassisted Gene Ontology (GO) enrichment analysis (Supplemental Tables S4 and S5). Several GO terms were nominally regulated in the treatment-groups. The seven most significantly anakinra down-regulated GO terms were related to extracellular matrix- and collagen-formation (Figure 4C). The GO-term “collagen fibril organization” remained significantly downregulated after controlling for multiple testing. The ten most significantly anakinra up-regulated GO terms included chaperone-related effects, nucleosome related effects and heat-shock protein related effects (response to temperature and heat (Supplemental Table S4)) (Figure 4D), which are important for proteostasis during mechanical stress [30].\\u003c/p\\u003e\\n\\u003cp\\u003eAll top-ten anti-MFAP4 up- and down-regulated GO-terms remained significant after correcting for multiple testing. Eight out of the ten most significantly down-regulated GO terms were associated with transcriptional or translational activity (Figure 4E). The top-ten most significantly up-regulated GO terms were all associated with muscle contractility or cytoskeletal organization (Figure 4F). Surprisingly, some GO terms were associated with skeletal muscle, which is not present in the small intestine (I-band, sarcomere, myofibril, Z-disc). However, all the enriched proteins underlying these terms (Supplemental Table S5) are also expressed by smooth muscle cells, albeit with lower transcripts per million relative to skeletal muscle [31]. \\u0026nbsp;Therefore, the observed regulations reflect the effects of the contractile apparatus of smooth muscle cells. The data supported that anti-MFAP4-treatment induced SMC-switching from the synthetic phenotype involved in development of fibrostenosis to the contractile phenotype essential for homeostatic gut motor activity.\\u003c/p\\u003e\\n\\u003cp\\u003eWe analyzed\\u0026nbsp;a-SMA immuno-staining in the anastomotic sections and observed a low muscularis propria staining-intensity in the vehicle treated group. The\\u0026nbsp;a-SMA immuno-staining was variable between analyzed anastomoses for both the anakinra- and anti-MFAP4-treated groups. Yet, higher intensity of\\u0026nbsp;a-SMA immuno-staining was observed in the anakinra-treated group, and the highest\\u0026nbsp;a-SMA intensity was observed in the anti-MFAP4-treated group. Representative images are shown in Figure 5.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnti-MFAP4 and regulation of lymphocytes\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHistochemical evaluation showed that lymphocytes were upregulated by anti-MFAP4 (Table 2). Yet, GO terms related with lymphocytes included downregulation of “gamma-delta T cell activation” (Figure 4E), and down-regulation of “T-cell proliferation”, and “T cell activation” (Supplemental Table S5). This suggested that a general negative regulation of T-cells was present. On this basis, we specifically investigated the presence of regulatory T cells (FOXP3-positive cells), and representative sections are shown in Supplemental Figure S4. Inspection of the stained sections supported that FOXP3-positive cells were present in all three treatment groups and that areas with high density of regulatory T-cells were present in the lamina propria of anakinra- and anti-MFAP4-treated samples relative to the control-treated samples.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eObservations from the present study support that pharmacological blocking of MFAP4, using anti-MFAP4, has potential as therapeutic intervention to prevent or treat fibrostenotic processes in intestinal anastomotic healing. One local dose of 16 mg or 32 mg anti-MFAP4 did not affect weight gain, the anastomotic strength or histochemical evaluation of healing in early and late healing phases, respectively. Local injection with anti-MFAP4 showed reduction of collagen deposition and the anastomotic SMC phenotype was switched from an ECM synthetic phenotype towards a contractile homeostatic phenotype in the late healing phase.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAnti-MFAP4 is raised against human MFAP4. Pigs may be an ideal species for the creation of a large animal model given that their gastrointestinal anatomy closely resembles that of humans [32]. The homology between pig and human MFAP4 is 94% supporting the use of anti-MFAP4 in pigs.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAnti-MFAP4 binds with high affinity to MFAP4, which is embedded in the ECM and therefore it may be retained to a high degree at the injections site. To test if locally injected anti-MFAP4 could reach the circulation and other organs and thereby exert unknown effects tat these sites, we harvested lung and arterial tissue. These tissues represents MFAP4-rich organs. However, we did not observe any anti-MFAP4 deposition in these organs after local injection, whereasapproximately 1 ng/ml tissue was retained in the anastomoses 14 days post-injection. In a prior study we observed a duration of efficacy of one dose anti-MFAP4 in reduction of vascular leakage for at least three months [20]. Together, these observations suggest that one dosing of anti-MFAP4 might be retained locally and be sufficient for obtaining a treatment effect through out a months-long period.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eWe applied local injection of anti-MFAP4 because other tissues might absorb systemically injected anti-MFAP4. For systemic injection, it would be required to test systemic tolerability, and a scale-up of our recombinant antibody production would be needed. Therefore, it is unknown if systemic administration of anti-MFAP4 is possible. However, endoscopic submucosal injection is demonstrated as a feasible approach for local delivery of small interfering RNA for other target [33]. Due to previously observed long duration of efficacy of anti-MFAP4 [20], anti-MFAP4 could potentially also be delivered locally through endoscopic injection. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe effective therapeutic dose of anti-MFAP4 for local injection into the pig intestinal anastomosis was unknown when this study was initiated. In a prior study we have observed retinal anti-angiogenic therapeutic efficacy of 2 mg anti-MFAP4 intravitreal injection in non-human primates [20]. This species has a vitreous volume of approximately 2.4 ml [34], and thereby the effective starting dose in that prior study was 0.8 mg/ml. In the present study, we aimed to reach far higher doses and locally injected 20 mg/ml (16 mg total) or 40 mg/ml doses (32 mg total) to accommodate for a higher, yet unknown, distribution volume.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePro-inflammatory effects of MFAP4 have previously been described [17, 26], and consequently anti-MFAP4 might potentially compromise the early inflammatory phase of wound healing. In addition, the anti-angiogenic effects of anti-MFAP4 [20] might potentially compromise angiogenesis during the proliferative phase of wound healing. However, we did not see any significant changes in anastomotic tensile strength, or the histological scores following dosing of 20 mg/ml anti-MFAP4 in the early healing phases (5-days and 10-days studies). The tensile strength was measured using MATS test, which is considered appropriate for evaluation of the healing tissue strength [35].\\u0026nbsp;On this basis, we do not expect that anti-MFAP4 compromised early inflammation or angiogenesis to a significant degree.\\u003c/p\\u003e\\n\\u003cp\\u003eFor testing therapeutic efficacy, we increased the anti-MFAP4 dose to 40 mg/ml in the 28-days study. This higher dose did not weaken the anastomotic strength but appeared to induce lymphocytic infiltration into the tissue. For the 28-days study, we applied a Fc-neutralized variant of anti-MFAP4 to dampen potential antibody-dependent or complement-dependent cytotoxicity. Moreover, antibodies are expected to arise 9-35 days after antigen exposure in pigs [36]. Therefore, we only applied 14 days of anti-MFAP4-administration to reduce the potential development of anti-antibodies and the associated risk of drug-induced inflammation in the pigs.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eProteome analysis supported an immune-suppressive state of the anastomosis in the anakinra- and anti-MFAP4-treated samples. In line with this, specific analysis of immuno-stained anastomotic sections supported that FOXP3-positive regulatory lymphocytes involved in dampening of local intestinal inflammation [37] were enriched in the anakinra- and anti-MFAP4-treated samples. However, detailed analysis of lymphocyte populations requires flow- or tissue-cytometry and was out of scope for the present study.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe normal pig small intestinal anastomosis has non-significantly different collagen volume in the muscularis and subserosa than outside of the anastomosis region when observed in the late healing phase [38]. We injected aethoxysklerol locally in the anastomotic line at the time of surgery in order to enhance the fibrotic process. The clinical fibrotic effect of aethoxysklerol is expected to occur week-to-months after sclerotherapy injection. The anti-fibrotic treatment effects were therefore analyzed in the 28-days study.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eDampening of inflammation is expected to reduce fibrogenesis. In line with this, the use of anti-TNF-α antibodies is safe in patients with inflammatory stricturing CD and could decrease the need for surgery over time [39-41]. However, the surgery-free interval is still short and data on anti-fibrotic effect of anti-TNF are lacking [42, 43]. In contrast, an anti-fibrotic effect of IL-1-inhibition in the gastrointestinal system and skin is well recognized [44-46]. Anakinra is a recombinantly expressed IL-1 receptor antagonist molecule and was applied as locally injected therapy in the same dose as anti-MFAP4. For clinical use, anakinra is applied by daily subcutaneous injections and such alternative dosing regimen might have provided a higher anti-fibrotic effect than the dosing used in our study. Dysregulation of interleukins decreases the fibrinolytic capacity of the peritoneum, increases adhesion formation, and is initiated in early hemostasis/inflammatory phase [47]. In line with this, anakinra treatment had capacity to reduce adherence formation in our study supported by observations from previous publications applying IL-1R inhibition [44, 45].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHuman IL-1Ra is previously shown to be well tolerated and to show therapeutic efficacy in pigs [48, 49].\\u003c/p\\u003e\\n\\u003cp\\u003eSemiquantitative collagen scoring, using transmural assessment of Sirius red-determined collagen deposition, is reported to be fast, reliable and reproducible [38] and was applied as a main outcome in our study. However, it considers the entire intestinal wall as a single region of interest and is not as sensitive as stereological assessment of the intestinal anastomotic collagen volume [38]. Nevertheless, semiquantitative collagen scoring showed a significantly reduced anastomotic collagen deposition with both the positive control anakinra-treatment and anti-MFAP4-treatment in the present study. The observed anti-fibrotic activity of anti-MFAP4 is supported by observations of reduced fibrogenesis in gene-deficient mouse models of organ fibrosis [15, 17].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eWound contraction refers to the process during initial wound closure and healing, where fibroblasts generate forces that pull the edges of the open wound together to accelerate closure and reduce the resulting scar size. Scar contraction occurs later, involving the differentiated myofibroblasts that cause shrinkage in a fully healed scar [50].We assessed wound/scar contraction using x-ray analysis and observed a reduced anastomotic index in the 10-days study. Moreover, both anti-MFAP4- and anakinra-treatment reduced the anastomotic index relative to vehicle control in the 28-days study. Myo(fibroblast)-mediated contraction is dependent upon integrin\\u0026nbsp;a\\u003csub\\u003eV\\u003c/sub\\u003eb\\u003csub\\u003e3\\u003c/sub\\u003e-mediated focal adhesion formation\\u0026nbsp;[51, 52], but antibody-mediated blocking of MFAP4 is previously shown to reduce focal adhesion formation\\u0026nbsp;[26]\\u0026nbsp;and could therefore be expected to reduce wound contraction. On the other hand, relaxation of the contraction occurs due to myofibroblast senescence or apoptosis, which is also dependent on integrin\\u0026nbsp;a\\u003csub\\u003eV\\u003c/sub\\u003eb\\u003csub\\u003e3\\u003c/sub\\u003e [52]. Our observations suggested that the anti-MFAP4- and anakinra-treatments prolonged the myofibroblast-mediated contraction, while at the same time reducing the collagen-accumulation. Importantly, while myofibroblast contraction is reversible, the deposition and quality of the collagen can make the contraction irreversible\\u0026nbsp;[52]. Detailed analysis of the role of anti-MFAP4-treatment in wound/scar contraction would require dynamic, cellular, and ECM constitutional analysis and was not further explored in the present study.\\u003c/p\\u003e\\n\\u003cp\\u003eWe applied proteome analysis using fixed tissue sections. Formaldehyde fixation limits the extraction efficiency of proteins and thereby the sensitivity. Moreover, proteome analysis is likely to underrepresent ECM proteins, which are often crosslinked and hard to extract even without the fixation. Nevertheless, our analysis showed regulation of collagens in the anakinra treatment group. Each tissue section represented tissue areas being transmural and containing the anastomotic line and additional tissue on each side. The most significantly anti-MFAP4-upregulated proteins were reflecting growth and survival of epithelial cells, and the most significantly anti-MFAP4-regulated GO terms were related to upregulation of the SMC contractile apparatus and down-regulation of transcriptional and translational activity. The main observations from the proteomic analysis supported that anti-MFAP4 induced a phenotypical shift from the synthetic/proliferative to the contractile, homeostatic phenotype and\\u0026nbsp;a-SMA immuno-staining of the anastomoses supported this observation. This further suggests that the anti-fibrotic effect observed with anti-MFAP4-treatment was secondary to the SMC switch. Integrin a\\u003csub\\u003eV\\u003c/sub\\u003eb\\u003csub\\u003e3\\u0026nbsp;\\u003c/sub\\u003eligands are previously shown to regulate increased SMC hyperplasia in stricturing Crohn's disease [8]. In parallel, we have previously observed that MFAP4 induced vascular SMC proliferation through integrin\\u0026nbsp;a\\u003csub\\u003eV\\u003c/sub\\u003eb\\u003csub\\u003e3\\u003c/sub\\u003e-engagement\\u0026nbsp;[26].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHyperplasia/hypertrophy of the SMC layer contributing to stricture formation is not usually recognized in intestinal anastomoses but often in patients with chronic inflammatory bowel disease. It is responsible for intestinal wall thickening and formation of strictures in disease, where chronic inflammation causes a change of metabolism of SMCs that start producing ECM [53]. The observation that anti-MFAP4’s major therapeutic effect is related to a beneficial phenotypical switch of SMC may therefore be especially relevant for Chron’s patients. However, the anti-fibrotic effect of anti-MFAP4 may potentially be relevant for various types of patients with increased risk of developing gastrointestinal fibrosis or fibrostenosis.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eLimitations of the study included that therapeutic effects were only tested in female animals, we did not test increasing or repeated dosing of anti-MFAP4, and our outcome analysis was based on fixed tissue sections rather than frozen tissue. One general limitation to advancing anti-fibrotic therapies is the lack of a standardized large animal model of fibrosis-driven luminal stricture.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eOur study was designed to investigate fibrosis deposition in the anastomosis. Others have developed several months long pig-model receiving 5% phenol and 0.2% 2,4,6-trinitrobenzenesulfonic acid solution for repeated submucosal injection to develop true anastomotic fibrostenosis [54] of relevance for potential additional efficacy-testing.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eTo our knowledge, this is the first study of tolerability and anti-MFAP4-mediated anti-fibrotic effect in post-surgery anastomotic healing. We identified MFAP4 as a new target and pathophysiological factor. Therapeutical blocking of MFAP4 maintained the contractile SMC phenotype and reduced fibrosis. The study further supported previous studies of anakinra-mediated reduction of fibrosis. Our observations warrant further testing of anti-MFAP4 as candidate therapy in Crohn\\u0026rsquo;s patients in risk of developing fibrostenosis or as prophylactic treatment in post-surgical anastomotic healing.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cdiv class=\\\"DefinitionList\\\"\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eCrohn\\u0026rsquo;s disease\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eCD\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eExtracellular matrix\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eECM\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eSmooth muscles cells\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eSMC\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eMicrofibrillar\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eassociated protein 4\\u0026ndash;MFAP4\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eSingle\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003ecell RNA sequencing\\u0026ndash;scRNA\\u0026ndash;seq\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eInterleukin\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003e1 receptor antagonist\\u0026ndash;IL\\u0026ndash;1Ra\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eAnastomotic index\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eAI\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eMaximal anastomotic tensile strength\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eMATS\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eForkhead box P3\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eFOXP3\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eα\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003esmooth muscle actin\\u0026ndash;α\\u0026ndash;SMA\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eTris\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003ebuffered saline\\u0026ndash;TBS\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003ePhosphate\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003ebuffered saline\\u0026ndash;PBS\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eBovine serum albumin\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eBSA\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eRadioimmunoprecipitation assay\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eRIPA\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eHorseradish peroxidase\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eHRP\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eo\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003ephenylenediamine dihydrochloride\\u0026ndash;OPD\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eCartilage Associated Protein\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eCRTAP\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eCollagen type II alpha 1 chain\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eCOL2A1\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eCollagen type III alpha 1 chain\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eCOL3A1\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eOlfactomedin 4\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eOLMF4\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003ePyruvate Kinase M\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003ePKM\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eGene Ontology\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eGO\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e\\u003cdiv class=\\\"Term\\\"\\u003eGastrointestinal Diseases and Malformations in Infancy and Childhood\\u003c/div\\u003e\\u003cdiv class=\\\"Description\\\"\\u003e\\u003cp\\u003eGAIN\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll data underlying this study are available from the corresponding author upon reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA.S and G.L.S are inventors on U.S.\\u0026nbsp;Patent\\u0026nbsp;No. 9,988,442 and EP17199552.5 owned by University of Southern Denmark.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by Novo Nordisk Foundation – Pioneer Innovator grant [NNF22OC0076385]; Clinical\\u0026nbsp;center of excellence ‘Gastrointestinal Diseases and Malformations in Infancy and Childhood (GAIN) [20/62844], Odense University Hospital;\\u0026nbsp;Overlægerådets forskningsfond [A6053], Odense University Hospital; Odense University Hospital Free Funding [A4431]; A.P. Møller Fonden [2024-00886]; A.J. Andersen og Hustrus Fond [01737-0005 FHP]\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization: MBE, NQ and GLS\\u003c/p\\u003e\\n\\u003cp\\u003eContributed to study design: MBE, NQ and GLS\\u003c/p\\u003e\\n\\u003cp\\u003eMethodology and investigations: RRA, JBP, PFHG, CSN, ALK, AFHK, NG, KER, HKH, JK, MD, GIM, LBS and HCB\\u003c/p\\u003e\\n\\u003cp\\u003eOriginal draft: RRA and JBP\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFunding acquisition: MBE and GLS\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eWriting review and editing: \\u0026nbsp;All authors. All authors approved the manuscript for publication\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors wish to thank veterinarians Charlotte Laurfelt Munch Rasmussen and Louise Langhorn,\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eDiana Bianca Hansen, Pernille Simonsen, Kristoffer Augustesen and laboratory technician Vivi Monrad for their assistance with animal care. Moreover, we wish to thank technicians Lone Christiansen, Department of Pathology at Odense University Hospital and Tine Rasmussen, Institute of Molecular Medicine, University of Southern Denmark, for help and assistance. Generative AI was used to correct syntax of long sentences.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eTorres J, Mehandru S, Colombel JF, Peyrin-Biroulet L. Crohn\\u0026apos;s disease. 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Histological mapping of healing of the small and large intestine - A quantitative study in a porcine model. Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft. 2023;249:152095.\\u003c/li\\u003e\\n\\u003cli\\u003eBouhnik Y, Carbonnel F, Laharie D, Stefanescu C, Hebuterne X, Abitbol V, et al. Efficacy of adalimumab in patients with Crohn\\u0026apos;s disease and symptomatic small bowel stricture: a multicentre, prospective, observational cohort (CREOLE) study. Gut. 2018;67(1):53-60.\\u003c/li\\u003e\\n\\u003cli\\u003eEl-Hussuna A, Qvist N, Zangenberg MS, Langkilde A, Siersma V, Hjort S, et al. No effect of anti-TNF-alpha agents on the surgical stress response in patients with inflammatory bowel disease undergoing bowel resections: a prospective multi-center pilot study. BMC Surg. 2018;18(1):91.\\u003c/li\\u003e\\n\\u003cli\\u003eLenti MV, Di Sabatino A. Intestinal fibrosis. Mol Aspects Med. 2019;65:100-9.\\u003c/li\\u003e\\n\\u003cli\\u003ePapaconstantinou I, Zeglinas C, Gazouli M, Nastos K, Yiallourou A, Lykoudis P, et al. Effect of infliximab on the healing of intestinal anastomosis. An experimental study in rats. Int J Surg. 2014;12(9):969-75.\\u003c/li\\u003e\\n\\u003cli\\u003eKarakose O, Eken H, Ulusoy AN, Topgul HK, Bilgin M, Yuruker SS, et al. The Effect of Infliximab on Intestinal Anastomosis Healing in Rats. Prague Med Rep. 2016;117(2-3):108-16.\\u003c/li\\u003e\\n\\u003cli\\u003eHershlag A, Otterness IG, Bliven ML, Diamond MP, Polan ML. The effect of interleukin-1 on adhesion formation in the rat. American journal of obstetrics and gynecology. 1991;165(3):771-4.\\u003c/li\\u003e\\n\\u003cli\\u003eKaidi AA, Nazzal M, Gurchumelidze T, Ali MA, Dawe EJ, Silva YJ. Preoperative administration of antibodies against tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) and their impact on peritoneal adhesion formation. Am Surg. 1995;61(7):569-72.\\u003c/li\\u003e\\n\\u003cli\\u003eThomay AA, Daley JM, Sabo E, Worth PJ, Shelton LJ, Harty MW, et al. Disruption of interleukin-1 signaling improves the quality of wound healing. The American journal of pathology. 2009;174(6):2129-36.\\u003c/li\\u003e\\n\\u003cli\\u003eCorrea-Rovelo JM, Villanueva-Lopez GC, Medina-Santillan R, Carrillo-Esper R, Diaz-Giron-Gidi A. [Intestinal obstruction secondary to postoperative adhesion formation in abdominal surgery. Review]. Cir Cir. 2015;83(4):345-51.\\u003c/li\\u003e\\n\\u003cli\\u003eChada M, Nogel S, Schmidt AM, Ruckel A, Bosselmann S, Walther J, et al. Anakinra (IL-1R antagonist) lowers pulmonary artery pressure in a neonatal surfactant depleted piglet model. Pediatr Pulmonol. 2008;43(9):851-7.\\u003c/li\\u003e\\n\\u003cli\\u003eMorton AC, Arnold ND, Gunn J, Varcoe R, Francis SE, Dower SK, et al. Interleukin-1 receptor antagonist alters the response to vessel wall injury in a porcine coronary artery model. Cardiovascular Research. 2005;68(3):493-501.\\u003c/li\\u003e\\n\\u003cli\\u003eKwan P, Hori K, Ding J, Tredget EE. Scar and contracture: biological principles. Hand Clin. 2009;25(4):511-28.\\u003c/li\\u003e\\n\\u003cli\\u003eFiore VF, Wong SS, Tran C, Tan C, Xu W, Sulchek T, et al. alphavbeta3 Integrin drives fibroblast contraction and strain stiffening of soft provisional matrix during progressive fibrosis. JCI Insight. 2018;3(20).\\u003c/li\\u003e\\n\\u003cli\\u003eVan De Water L, Varney S, Tomasek JJ. Mechanoregulation of the Myofibroblast in Wound Contraction, Scarring, and Fibrosis: Opportunities for New Therapeutic Intervention. Adv Wound Care (New Rochelle). 2013;2(4):122-41.\\u003c/li\\u003e\\n\\u003cli\\u003eRosendorf J, Klicova M, Herrmann I, Anthis A, Cervenkova L, Palek R, et al. Intestinal Anastomotic Healing: What do We Know About Processes Behind Anastomotic Complications. Front Surg. 2022;9:904810.\\u003c/li\\u003e\\n\\u003cli\\u003eLukas M, Kolar M, Ryska O, Juhas S, Juhasova J, Kalvach J, et al. Novel porcine model of Crohn\\u0026apos;s disease anastomotic stricture suitable for evaluation and training of advanced endoscopic techniques. Gastrointest Endosc. 2021;93(1):250-6.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eTable 1. Body weight, anastomotic index, adhesion formation and histochemical scoring in 5- and 10-days studies\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eVehicle\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etreatment\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAnti-MFAP4\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etreatment\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e5-days study\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003en=6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003en=7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBaseline body weight\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e19.1(1.2) kg\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e20.0(1.9) kg\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBody weight increase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e2.6(1.2) kg\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e2.1(0.4) kg\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e6.3(2.6) N\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e6.2(2.6) N\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e10.4(1.6) N\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e9.4(2.7) N\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e10.7(1.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e10.0(3.1) N\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLeach score\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.1(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.2(0.4)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNecrosis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.1(0.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.3(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePolymorphonuclear cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.3(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.6(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLymphocytes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.3(0.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.3(0.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMacrophages\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.2(0.4)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.1(0.3)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOedema\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.8(0.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.1(0.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMucosal epithelium\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e2.3(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e2.4(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSubmucosal muscular layer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.6(0.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.2(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCollagen\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e25.4(8.9)%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e23.2(7.5)%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e10-days study\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e+ aethoxysklerol\\u003c/p\\u003e\\n \\u003cp\\u003en=7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e+aethoxysklerol\\u003c/p\\u003e\\n \\u003cp\\u003en=7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBaseline body weight\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e20.1(1.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e19.0(1.1)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBody weight increase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e6.9(0.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e7.1(1.1)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAnastomotic index\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.56(0.08)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.56(0.09)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e8.9(3.2)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e7.5(4.1)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e9.1(3.1)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e9.5(3.3)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e9.6(3.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e9.7(3.4)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLeach score\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.0(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.8(0.4)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNecrosis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.4(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.6(0.5)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePolymorphonuclear cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.4(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.6(0.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLymphocytes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.1(0.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.4(0.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMacrophages\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.2(0.4)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.1(0.3)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOedema\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.8(0.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.7(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMucosal epithelium\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e2.2(1.3)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.8(1.3)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSubmucosal muscular layer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.7(0.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.6(0.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCollagen\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e24.3(7.0)%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e27.3(10.7)%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eAnti-MFAP4 treatment was compared with the negative control vehicle-treatment. Leach scoring and Verhofstad scale scorings were analyzed by Mann-Whitney test. Additional variables were analyzed by t-test. No significant changes were found. Data are mean(SD).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 2. Body weight, anastomotic index, adhesion formation and histochemical scoring in 28-days study\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAnakinra\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etreatment\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eVehicle\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etreatment\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eAnti-MFAP4\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003etreatment\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003ep\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e-value\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e28-days study\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e+ aethoxysklerol\\u003c/p\\u003e\\n \\u003cp\\u003eN=9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e+ aethoxysklerol\\u003c/p\\u003e\\n \\u003cp\\u003eN=9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e+ aethoxysklerol\\u003c/p\\u003e\\n \\u003cp\\u003eN=9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBaseline body weight\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e29.0(3.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e28.2(4.4)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e29.3(3.2)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBody weight increase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e22.4(5.1)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e24.4(3.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e25.0(4.5)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAnastomotic index\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.62(0.07)\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.72(0.06)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.63(0.16)\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003csup\\u003ea\\u0026nbsp;\\u003c/sup\\u003e\\u003cem\\u003ep\\u003c/em\\u003e=4.9*10\\u003csup\\u003e-5\\u003c/sup\\u003e,\\u003csup\\u003e\\u0026nbsp;b\\u003c/sup\\u003e \\u003cem\\u003ep\\u003c/em\\u003e=0.04\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e8.3(3.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e8.5(4.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e8.8(5.5)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e11.4(3.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e10.4(3.3)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e11.7(4.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMATS 3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e13.5(4.1)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e12.3(3.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e13.9(4.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLeach score\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.1(0.3)\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.8(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.8(1.3)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003csup\\u003ec\\u0026nbsp;\\u003c/sup\\u003e\\u003cem\\u003ep\\u003c/em\\u003e=0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNecrosis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.0(0.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.9(0.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.1(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePolymorphonuclear cells\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.9(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.8(0.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.2(0.9)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eLymphocytes\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.9(0.6)\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.4(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.2(0.7)\\u003csup\\u003ee\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003csup\\u003ed\\u003c/sup\\u003e \\u003cem\\u003ep\\u003c/em\\u003e=0.03, \\u003csup\\u003ee\\u003c/sup\\u003e \\u003cem\\u003ep\\u003c/em\\u003e=0.003\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMacrophages\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.4(0.5)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.3(0.5)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.7(0.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOedema\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.9(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.8(0.6)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.6(0.5)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMucosal epithelium\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.3(0.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.5(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0.3(0.5)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSubmucosal muscular layer\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.4(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.2(0.7)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e1.1(1.0)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCollagen\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e36.1(14.2)\\u003csup\\u003ef\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e52.7(17.8)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e40.6(18.0)\\u003csup\\u003eg\\u003c/sup\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003e\\u003csup\\u003ef\\u003c/sup\\u003e\\u003c/em\\u003e\\u003cem\\u003ep\\u003c/em\\u003e=0.003, \\u003csup\\u003eg\\u003c/sup\\u003e\\u003cem\\u003ep\\u003c/em\\u003e=0.048\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eAnakinra- or anti-MFAP4 treatments were compared with the negative control vehicle-treatment. Leach scoring and Verhofstad scale scorings were analyzed by Mann-Whitney test. Additional variables were analyzed by t-test. \\u0026nbsp;Data are mean(SD).\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Intestinal anastomoses, Microfibrillar-associated protein 4, fibrosis, smooth muscle cells, phenotype switch\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7132995/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7132995/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e\\u003cp\\u003eCrohn\\u0026rsquo;s disease often leads to strictures due to excessive extracellular matrix deposition and smooth muscle cell hyperplasia. Current stricture treatments include surgical and endoscopic interventions, but high recurrence rates remain a challenge. Microfibrillar-associated protein 4 (MFAP4) contributes to fibrosis in various tissues. The anti-MFAP4 antibody was evaluated for its tolerability and anti-fibrotic efficacy on small intestinal anastomotic healing in pig models.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e\\u003cp\\u003eTwo small intestinal anastomoses were made in 45 pigs. Fibrosis was induced using aethoxysklerol injections. Each anastomosis was locally injected with either anti-MFAP4, positive control anakinra (interleukin-1 receptor antagonist) or negative vehicle control. Tolerability of anti-MFAP4 was observed across three observation durations (5, 10, and 28 days) and assessed by weight gain, anastomotic tissue strength, and histological evaluation. Anti-fibrotic efficacy was tested using semi-quantitative collagen scoring from the 28-days study. Proteome analysis of tissue sections was applied for mechanistic analyses.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e\\u003cp\\u003eLocal anastomotic injections of 16 mg and 32 mg anti-MFAP4 were well tolerated. Anastomotic fibrosis was significantly reduced both by positive control anakinra-treatment and anti-MFAP4-treatment. Anti-MFAP4 reduced fibrosis by 23% relative to the negative control. Gene ontology term analysis showed up-regulation of muscle cell contractile apparatus and down-regulation of transcription and translation in the anti-MFAP4 group.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e\\u003cp\\u003eThe data supported that anti-MFAP4-treatment induced smooth muscle cell switching from the synthetic phenotype involved in fibrosis to the contractile phenotype essential for homeostatic gut motor activity. Anti-MFAP4 has potential as treatment of Crohn\\u0026rsquo;s patients at risk of developing fibrostenosis and as a prophylactic treatment to support post-surgical anastomotic healing.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Tolerability and effect of inhibiting microfibrillar-associated protein 4 in small intestinal anastomotic healing\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-09-11 12:21:52\",\"doi\":\"10.21203/rs.3.rs-7132995/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-10-06T11:16:41+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-09-28T00:39:11+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-09-19T13:45:02+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-09-09T08:26:35+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"34841505223048848503010884468505701827\",\"date\":\"2025-09-08T11:06:58+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"284525378453701096791245262001481460682\",\"date\":\"2025-09-08T10:45:41+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"110154082173862373328104652358177637003\",\"date\":\"2025-09-05T07:38:22+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"279034921584075676153122560920584942247\",\"date\":\"2025-09-05T00:30:18+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-09-04T20:16:24+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-09-01T05:22:16+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2025-07-31T08:41:05+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-07-29T10:53:04+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2025-07-29T10:48:30+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"74c0024e-c680-4893-a36d-9a2b56b5c6b6\",\"owner\":[],\"postedDate\":\"September 11th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":54529724,\"name\":\"Health sciences/Diseases\"},{\"id\":54529725,\"name\":\"Health sciences/Gastroenterology\"},{\"id\":54529726,\"name\":\"Health sciences/Medical research\"}],\"tags\":[],\"updatedAt\":\"2025-12-01T16:01:34+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7132995\",\"link\":\"https://doi.org/10.1038/s41598-025-30123-0\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2025-11-29 15:57:38\",\"publishedOnDateReadable\":\"November 29th, 2025\"},\"versionCreatedAt\":\"2025-09-11 12:21:52\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-025-30123-0\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-025-30123-0\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7132995\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7132995\",\"identity\":\"rs-7132995\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}