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. 2019 Aug 1;2(5):342–352. doi: 10.1021/acsptsci.9b00037

Antibody-Mediated Delivery of VEGFC Ameliorates Experimental Chronic Colitis

Carlotta Tacconi †, Simon Schwager †, Nikola Cousin †, Davor Bajic †, Marko Sesartic †, John P Sundberg ‡, Dario Neri †, Michael Detmar †,*
PMCID: PMC7088901  PMID: 32259068

Abstract

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Crohn’s disease (CD) and ulcerative colitis (UC) are two distinct forms of inflammatory bowel disease (IBD) characterized by an expanded lymphatic network with impaired functionality both in mouse models and in human patients. In this study, we investigated whether targeted delivery of the pro-lymphangiogenic vascular endothelial growth factor C (VEGFC) to the site of inflammation may represent a new, clinically feasible strategy for treating IBD. To achieve targeting of inflamed tissue, we developed a fusion protein consisting of human VEGFC fused to the F8 antibody (F8-VEGFC), which specifically binds to the extradomain A (EDA) of fibronectin, a spliced isoform almost exclusively expressed in inflamed tissues. The therapeutic activity of intravenously administered F8-VEGFC, compared to a targeted construct lacking VEGFC (F8-SIP), was investigated in a mouse model of dextran sodium sulfate (DSS)-induced colitis. The presence of EDA fibronectin was detected in both human and mouse inflamed colon tissue. Biodistribution studies of radiolabeled F8-VEGFC revealed a specific accumulation of the antibody in the colon of DSS-administered mice, as compared to an untargeted VEGFC fusion protein (KSF-VEGFC) (binding the irrelevant hen egg lysozyme antigen). Systemic treatment with F8-VEGFC significantly reduced the clinical and histological signs of inflammation, expanded the lymphatic vascular network, reduced the density of immune cells, and also decreased the expression of inflammatory cytokines in the inflamed colon. Overall, these results reveal that administration of F8-VEGFC represents a novel and promising approach for the treatment of IBD.

Keywords: lymphatic vessels, lymphangiogenesis, antibody therapy, inflammatory bowel disease, VEGFC

The lymphatic vasculature is of critical importance for the maintenance of tissue fluid homeostasis, absorption of dietary lipids in the intestine and immune surveillance. Lymphatic vessels (LVs) serve as transport routes for antigens, inflammatory mediators, and cells from peripheral tissues to lymph nodes, rendering them critical for the efficient initiation of immune responses.1

Inflammatory bowel disease (IBD) comprises Crohn’s disease (CD) and ulcerative colitis (UC), chronic inflammatory diseases of the gastrointestinal tract whose pathogenesis remains incompletely understood. While past research has mainly focused on immune cells and their role in IBD, dynamic changes in the lymphatic vasculature (LV) have also been reported, including dilation of existing LVs as well as the formation of new LVs (lymphangiogenesis).2,3 LVs were often found to be obstructed4 and a reduced LV function has been reported in human patients suffering from Crohn’s disease.5 A lower density of LVs has been associated with an increased risk of Crohn’s disease recurrence in human patients,6 suggesting a protective role of the lymphatic vasculature in IBD pathogenesis.

One of the majors signaling axes implicated in the growth and function of the lymphatic vasculature consists of the vascular endothelial growth factor (VEGF)C and the cognate vascular endothelial growth factor receptor (VEGFR)-3, which is almost exclusively expressed by lymphatic endothelial cells (LECs).7,8

Strikingly, stimulation of the lymphatic vasculature by viral or transgenic expression of VEGFC reduced inflammation in mouse models of skin inflammation,9,10 rheumatoid arthritis,11 and IBD.12 In line with these findings, a blockade of VEGFC/VEGFR-3 signaling resulted in increased severity of inflammation in experimental models of psoriasis,9 arthritis,13 airway inflammation,14 and IBD.12,15 Unfortunately, despite these beneficial effects of LV stimulation in chronic inflammatory diseases, no drugs are currently available that specifically activate the lymphatic vasculature. While the VEGFC protein might be a strong candidate to fill this therapeutic gap, clinically viable delivery strategies are currently lacking. Indeed, previous experimental studies have mainly used transgenic or viral overexpression, or topical application of VEGFC protein. While adequate for experimental animal studies, these approaches are not practical in a clinical setting. Local administration, for example, is challenging if the inflammation site is not easily accessible, as in the case of IBD, or covers a larger area, as in the case of psoriasis. Systemic injections of recombinant growth factors, while intuitive, have been unsuccessful in multiple cases,16,17 most likely due to their short circulation half-life which necessitates higher doses and results in small therapeutic indices. In addition, their cognate receptors are often widely expressed, thus increasing the risk of toxicities.

Targeted, antibody-mediated delivery of VEGFC may overcome these limitations. Thus, we developed a fusion protein that consists of VEGFC linked to an antibody-fragment specific for the extradomain A (EDA) of fibronectin (FN).

Fibronectin is subject to alternative splicing and EDA is only expressed during tissue remodeling and angiogenesis, two processes commonly occurring during inflammation.17,18 Systemic treatment with this fusion protein potently alleviated inflammation in two mouse models of skin inflammation.10 We hypothesized that treatment with F8-VEGFC might also exert beneficial effects in IBD.

In the present study, we investigated the biodistribution, biological activity, and therapeutic effectiveness of the fully human fusion protein F8-VEGFC in a chronic mouse model of dextran sodium sulfate (DSS)-induced colitis. We found that F8-VEGFC specifically accumulated in the inflamed colon where it induced an expansion of the lymphatic vasculature. Systemic treatment with F8-VEGFC also significantly reduced disease severity, levels of inflammatory mediators, and the number of inflammatory cells in the inflamed tissue. This study revealed that targeted stimulation of the lymphatic vasculature by the fusion protein F8-VEGFC is a new, promising therapeutic approach for the treatment of IBD, suitable for clinical development.

Results and Discussion

EDA-Fibronectin Is Upregulated in Inflamed Human and Mouse Colon

EDA-FN was recently shown to be upregulated in human inflamed psoriatic skin.10 However, it has remained unclear whether EDA-FN is also specifically upregulated in inflamed versus uninflamed colon. To answer this question, publicly available microarray data of human UC-, CD-, and healthy colon-derived biopsies were investigated. By selection of a probe specific for the EDA-FN region, a significant increase of EDA-FN in both UC (log2 fold change of 1.5 and p value of 0.004) and CD (log2 fold change of 1.2 and p value of 0.042) inflamed tissue was observed compared to healthy controls (Figure 1A). The DSS chronic mouse model of colitis was used to determine if it could replicate this observation in human IBD. Mice were exposed to two cycles of DSS in drinking water for 1 week separated by 2 weeks of normal drinking water. On day 3 of the second DSS cycle, colons were processed and immunofluorescently labeled for EDA-FN. In line with the findings in human tissue, EDA-FN was expressed in inflamed colons, while EDA-FN was not detectable in naïve control colons (Figure 1B). Notably, EDA-FN was localized around blood vessels as previously seen in skin inflammation.10

Figure 1.

Figure 1

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EDA-FN is increased in inflamed colon tissue. (A) EDA-FN expression levels were assessed in healthy (n = 6), ulcerative colitis (UC, n = 24), and Crohn’s disease (CD, n = 19) endoscopic colonic biopsies, and absolute values for the probe 212464_s_at, recognizing EDA-FN, are shown. (B) Representative immunofluorescence pictures of naïve (CTRL, n = 3) and inflamed colons at day 24 post DSS administration (DSS, n = 3) stained for CD31 (red), EDA-FN (green), and Hoechst (blue). Scale bar: 100 μm. All data are presented as mean ± SD. Adjusted p value *p < 0.05, **p < 0.01.

To determine if EDA-targeting F8-VEGFC fusion protein accumulated in inflamed colons, radioiodinated preparations of F8-VEGFC and KSF-VEGFC (an untargeted control specific to hen egg lysozyme) were intravenously injected in naïve or DSS-induced colitic mice. Colons were removed 24 h later for autoradiography evaluation, which indicated specific targeting of F8-VEGFC to the inflamed colons (Figure 2A,B). Importantly, the radioactive signal was mainly localized in the more severely inflamed distal colon and rectum while only a lower signal was detectable in the proximal colon and the cecum (Figure 2A), locations which are known to be less affected by DSS induced colitis. Of note, the untargeted control KSF-VEGFC fusion protein did not preferentially accumulate in more inflamed regions and no differences were observed between naïve and DSS-inflamed colons (Figure 2A,B).

Figure 2.

Figure 2

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F8-VEGFC preferentially localizes to the inflamed colon tissue. (A) Autoradiography of radioiodinated KSF-VEGFC and F8-VEGFC in inflamed (right, DSS) compared to uninflamed (left, CTRL) colon. (B) Quantification of hot-averaged values in C expressed as a fold change (FC) relative to control KSF-VEGFC treated mice (n = 5 mice per group). (C) Quantitative biodistribution analysis of radioiodinated F8-VEGFC and KSF-VEGFC of colon, liver, lung, lung, spleen, heart, kidney, and small intestine in mice with uninflamed or inflamed colons, expressed as percent of injected dose per gram of tissue normalized over control KSF-VEGFC-injected mice (n = 5 mice per group). All data are presented as mean ± SD. Statistical significance was determined by two-way ANOVA with Sidak’s correction for multiple comparisons. Asterisks indicate statistical significance with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

The quantitative biodistribution analysis confirmed the specific accumulation of F8-VEGFC in inflamed compared to uninflamed colons (Figure 2C). In mice with colon inflammation, the F8-VEGFC exclusively targeted the inflamed colon, but not the uninflamed small intestine or any other organs analyzed. Untargeted KSF-VEGFC did not accumulate in any of the assessed organs, including the colon, indicating that the accumulation of F8-VEGFC in the colon was not merely due to increased vascular leakiness. These results are in accordance with a recent study in which a favorable biodistribution of F8-VEGFC in mouse models of chronic skin inflammation was observed.10 Overall, these data reveal that using the F8 diabody as targeting moiety is a suitable and functional strategy for the specific delivery of VEGFC to the inflamed colon.

F8-VEGFC Delivery Reduces the Severity of Chronic Colitis

LVs play an important role in IBD, and dysfunctional lymphatic vessels have been reported in both human and mouse IBD.5,12 To assess the therapeutic potential of F8-VEGFC, the DSS chronic model of colitis was used. The DSS-induced colitis model is considered a highly relevant model for IBD since it is sensitive to drugs commonly used for the treatment of this disease, has a similar pattern of deregulated genes, is characterized by T cell accumulation in the inflamed colon and, most importantly, has aberrant LVs. Mice received DSS in drinking water for 1 week, followed by 2 weeks of normal drinking water and a final week of DSS. On day 3 of the second DSS cycle, mice were randomized according to the disease activity index (DAI),19 a combinatorial clinical score of colitis considering stool consistency, fecal blood, and body weight loss. Animals were injected intravenously every second day with lymphangiogenic F8-VEGFC or F8-SIP, which lacks lymphangiogenic activity. Mice treated with F8-VEGFC had a significant improvement of the DAI score (Figure 3A) and experienced a smaller bodyweight loss than the F8-SIP controls (Supporting Information, Figure S1A ), indicating reduced inflammation severity. Colitis alleviation was confirmed by increased colon length (Figure 3B) and a reduction of the severity of histological inflammation (Figure 3C,D) and of the ulceration score (Figure S1B). These findings are in line with previous studies in which therapeutic properties of VEGFC in IBD have been reported. In two studies, adenoviral delivery of human VEGFC ameliorated inflammation in both the IL10 knockout mouse model and the chronic DSS model of colitis.12,20 The pathogenic importance of the lymphatic vasculature was confirmed since colon inflammation was increased in two different mouse models of IBD after antibody blockade of VEGFR3.12,15,21 Overall, these data indicate that targeted delivery of VEGFC can improve established chronic colitis.

Figure 3.

Figure 3

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F8-VEGFC ameliorates chronic DSS-induced colitis in mice. (A) Disease activity index (DAI) score of mice treated at day 24 from the beginning of the study by intravenous injections (i.v.) with F8-SIP or F8-VEGFC every other day (n = 8 per group), as indicated by the black arrows. (B) Total colon length was measured with a ruler at the end point on day 32 (n ≥ 8 per group). (C) Representative H&E stained sections of F8-SIP- (left) and F8-VEGFC- (right) treated mice. Scale bars: 100 μm. (D) Histopathological colitis scoring system: evaluation of inflammation severity, epithelial hyperplasia, mucosal ulceration, and extent of area involved along the distal and rectal part of the colon (n = 8 per group). All data are presented as mean ± SD. Statistical significance was determined by two-way ANOVA with Sidak’s correction for multiple comparisons or by two-tailed Student’s t test. Asterisks indicate statistical significance with p < 0.05 (*) and p < 0.01 (**).

F8-VEGFC Administration Induces Lymphatic Vessel Expansion in DSS-Induced Chronic Colitis

VEGFC is one of the most important lymphangiogenic growth factors, and LV stimulation by VEGFC has been associated with inflammation resolution in various disease models.10,12 This study investigated whether systemic application of F8-VEGFC induced LV expansion in the inflamed colon. Colon tissue samples were stained for the LV marker LYVE-1 and the pan-endothelial marker CD31. F8-VEGFC application induced a significant expansion of LVs in terms of number (Figure 4A,B) and total area covered by LVs, compared with F8-SIP (Figure 4A,C), similar to previous findings in skin inflammation models.10 LV size was not affected (Figure S2A). Induced lymphatic vessels (LVs) have been reported to regress after inflammation resolution, notably in the cornea and in the lymph nodes,22,23 while they persisted in the airways and the skin.14,24 Similarly, it has been reported that increased LVs were also found in noninflamed intestinal tissue from IBD patients, indicating persistence of LVs after disease resolution.25 Future studies are needed to investigate the long-term persistence of induced LVs after cessation of the F8-VEGFC treatment, which might potentially prevent disease recurrence.

Figure 4.

Figure 4

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F8-VEGFC induces expansion of the lymphatic vasculature in chronic colitis. (A) Representative pictures of immunofluorescence labeling of the inflamed colon after F8-SIP (upper panels) or F8-VEGFC (lower panels) treatment for LYVE-1 (green), CD31 (red), and Hoechst (blue). Dotted white lines mark the separation between the mucosa (M) and the submucosa (SM). Scale bars: 100 μm. LYVE-1 + LVs were quantified in terms of number (B) and percentage of stained area (C) in 5–8 fields per colon per mouse (n = 5 per group). CD31+LYVE-1- blood vessels were quantified in terms of number (D) and percentage of stained area (E) in 5–8 fields per colon per mouse (n = 5 per group). All data are presented as mean ± SD. Statistical significance was determined by two-tailed Student’s t test. Asterisks indicate statistical significance with p < 0.01 (**).

Besides VEGFR-3, the fully processed VEGFC form present in the fusion protein could potentially also activate VEGFR-2 that is expressed on blood vascular endothelial cells, eventually resulting in angiogenesis. Moreover, besides LECs, VEGFR-3 expression has also been reported on some blood vascular endothelial cells under pathological conditions.26 Since angiogenesis plays a pathogenesis-promoting role in IBD,27−29 the potential effects of F8-VEGFC was evaluated on blood vessels. Administration of F8-VEGFC did not affect the blood vessel number (Figure 4A,D), the area covered by blood vessels (Figure 4A,E) or blood vessel size (Figure S2B). Thus, the reduction in disease severity upon treatment with F8-VEGFC was mediated specifically by its effects on lymphatic rather than blood vessels. These findings are compatible with previous reports using VEGFR-3-blocking antibodies or adenoviral delivery of VEGFC in mouse models of IBD in which blood vessel numbers and area covered by blood vessels were not affected by modulation of the VEGFC/VEGFR-3 pathway.12,15

F8-VEGFC Treatment Reduces the Number of Macrophages and T Regulatory Cells in Colitis

Since F8-VEGFC promoted lymphangiogenesis and one of the main functions of the lymphatic vasculature is to drain immune cells from the inflamed tissue,10,12 the effects of F8-VEGFC treatment on leukocyte infiltration in the colon was investigated. Despite reduced hallmarks of inflammation (Figure 3), a significant difference in the total leukocyte infiltrate (Figure 5A), defined as the percentage of viable CD45+ cells (Figure S3A), was not observed. However, there were significant differences in specific immune cell populations. Specifically, the number of F4/80+ macrophages was significantly reduced in F8-VEGFC administered mice compared with F8-SIP controls (Figure 5B,C), while there were no major differences in the number of CD11c+/F4/80- dendritic cells (Figure 5B,D) and CD11b+/CD11c-/F4/80- monocyte-like and CD11b+/CD11c-/F4/80- myeloid cells (Figure 5 E and Figure S3B). A reduced number of F4/80+ macrophages in F8-VEGFC-treated mice was further confirmed by immunofluorescent labeling of tissue sections (Figure 5F,G). Similar effects were shown by Wang and colleagues, who reported reduced macrophage numbers in mice injected with an adenovirus overexpressing VEGFC in the DSS model of colitis,20 indicating enhanced lymphatic drainage of macrophages to draining lymph nodes.12,20,30

Figure 5.

Figure 5

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F8-VEGFC reduces macrophage infiltration in chronically inflamed colons. Flow cytometry quantification of total leukocytes pregated on single living cells. (A) Percentage of total CD45+ cells. (B) Representative flow cytometry plots of live cells pregated for CD45 positivity of dendritic cells (DC) and macrophages in inflamed colons of F8-SIP (left panel) and F8-VEGFC (right panel) treated mice. (C) Percentage of F4/80+ macrophages of total CD45+ cells (n = 8 per group). (D) Percentage of CD11c+F4/80- dendritic cells of total CD45+ (n = 8 per group). (E) Percentage of CD11c-F480-CD11b+ myeloid cells of total CD45+ cells (n = 8 per group). (F) Representative pictures of immunofluorescence staining of the inflamed colon after F8-SIP (left panel) or F8-VEGFC (right panel) treatment for F4/80 (green) and Hoechst (blue). Scale bars: 100 μm. (G) Percentage of F4/80-positive staining area in 5–8 fields per colon per mouse (n = 5 per group).

In line with decreased inflammation, flow cytometry analysis revealed a trend toward a reduction of γδ T cells (Figure 6 A and Figure S3C) and CD8 T cells (Figure 6B and Figure S3D) in F8-VEGFC-treated mice. Notably, we did not observe significant differences in the percentage of total CD4 T cells (Figure 6C and Figure S3D), while CD4 T regulatory cells were significantly diminished in animals injected with F8-VEGFC (Figure 6D,E), indicating that the less inflamed tissue of F8-VEGFC-treated mice might recruit fewer T regulatory cells. In line with this concept, it was recently reported that infliximab-treated IBD patients in remission displayed increased levels of circulating T regulatory cells in the blood and significantly reduced levels in colonic biopsies.31

Figure 6.

Figure 6

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F8-VEGFC reduces lymphocyte infiltration in chronically inflamed colons. FACS quantification of respective cells pregated on single living CD45 positive cells. (A) Percentage of γδ T cells of total CD45+ cells (n = 8 per group). (B) Percentage of CD8 T cells of total CD45+ cells (n = 8 per group). (C) Percentage of CD4 T cells of total CD45+ cells (n = 8 per group). (D) Representative flow cytometry plots of live cells pregated for CD45 positivity of T regulatory cells (Treg) in inflamed colons of F8-SIP- (left panel) or F8-VEGFC- (right panel) treated mice. (E) Percentage of T regulatory cells of total CD4 T cells (n = 8 per group). All data are presented as mean ± SD. Statistical significance was determined by two-tailed Student’s t test. Asterisks indicate statistical significance with p < 0.05 (*) and p < 0.01 (**).

Overall, these data indicate that F8-VEGFC influences the composition of the inflammatory infiltrate by reducing macrophages, CD4 T regulatory cells, and γδ T cells as well as CD8 T cells.

Treatment with F8-VEGFC Reduces Expression of Pro-inflammatory Cytokines in Colitis

To investigate whether, in addition to inflammatory cells, targeted delivery of VEGFC might also reduce the tissue levels of inflammatory cytokines, RNA was isolated from inflamed colons of F8-SIP- and F8-VEGFC-treated mice and qPCR analyses were performed. F8-VEGFC treatment significantly downregulated the expression of tumor necrosis factor alfa (Tnf) (Figure 7A) and interferon gamma (Ifng) (Figure 7B), and a strong trend was observed for downregulation of other pro-inflammatory genes, namely chemokine (C–X–C motif) ligand10 (Cxcl10) (Figure 7C), interleukin 1 beta (Il1b) (Figure 7D), and inducible nitric oxide synthase (iNOS) (Nos2) (Figure 7E). Interleukin-10 (Il10) was slightly downregulated in F8-VEGFC-treated mice (Figure 7F), a finding compatible with reduced numbers of T regulatory cells in the colon.

Figure 7.

Figure 7

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Targeted delivery of VEGFC reduces inflammatory cytokine expression. Colonic expression of Tnfa (A), Ifng (B), Cxcl10 (C), Il1b (D), Nos2 (E), and Il10 (F) mRNA as examined by qRT-PCR in F8-SIP- and F8-VEGFC-injected mice (n ≥ 8 per group). Rplp0 was used as an endogenous reference gene. Comparison between experimental groups was done by the 2–ΔCT method. All data are presented as mean ± SEM. Statistical significance was determined by two-tailed Student’s t test. Asterisks indicate statistical significance with p < 0.05 (*).

Conclusions

Overall, our results, together with previous studies that reported beneficial effects of adenoviral overexpression of VEGFC in intestinal inflammation, strongly support the use of F8-VEGFC for the treatment of IBD. Importantly, our study reveals a therapeutic activity of VEGFC which goes beyond the prevention potential that was shown in previous studies. From a clinical point of view, targeted delivery of VEGFC via the F8-VEGFC fusion protein may reduce the dose of VEGFC needed to achieve therapeutic efficacy and may represent a safer approach compared to the adenoviral systemic delivery of this pro-lymphangiogenic factor. Thus, F8-VEGFC represents a clinically relevant fusion protein for IBD treatment, which possesses the anti-inflammatory properties of VEGFC while lacking potential undesired off-target effects.

Methods

Meta-Analysis of EDA-Fibronectin Expression in Human IBD Tissue

The data set GSE16879 was selected in the data repository NCBI GEO, which contains biopsies of actively inflamed colon mucosa obtained at endoscopy from IBD patients (24 ulcerative colitis (UC) and 19 Crohn’s disease (CD) patients) and from normal colon mucosa (N = 6). Total RNA was isolated from intestinal mucosal biopsies, labeled and hybridized to Affymetrix Human Genome U133 Plus 2.0 Arrays.32 Differential expression analysis was performed using the GEO2R tool available at www.ncbi.nlm.nih.gov/geo, which uses the GEOquery and limma R packages from the Bioconductor project. We compared UC samples and colonic CD samples with normal reference controls and used the values for the probe 212464_s_at covering the EDA-FN region.33

Animals

Female C57BL/6JRj (referred to as C57BL/6) mice were purchased from Janvier (Laval, France). All mice were housed in a specific opportunistic pathogen-free (SOPF) facility. Housing was temperature controlled, with a 12 h light/12 h dark cycle. Procedures involving mice conformed to institutional guidelines in agreement with national and international law and were approved by the Kantonales Veterinäramt Zurich (license number 265/16).

DSS-Induced Chronic Colitis

Chronic colitis was induced in 8-week-old C57BL/6 female mice subjected to two 7-day cycles of 1.5% DSS (molecular mass, 40 kDa, Alfa Aesar) in drinking water ad libitum. The DSS cycles were interspersed with 2 weeks of providing normal drinking water. The severity of colitis was quantified using a disease activity index (DAI) score based on the evaluation of body weight, diarrhea, and presence of blood in stools.19 The DAI was determined by scoring changes in weight loss (0 = none; 1 = 1 to 5%; 2 = 5 to 10%; 3 = 10 to 20%; 4 = >20%); stool consistency (0 = normal; 1 = moist/sticky stool; 2 = soft stool; 3 = very loose and wet stools; 4 = diarrhea) and presence of blood in the feces (0 = no blood; 1 = moderate occult bleeding; 2 = strong occult bleeding; 3 = visible blood in the feces; 4 = gross rectal bleeding) by Hemoccult SENSA (HemoCue). A three grade (0–4) DAI was thus obtained.19 Surviving mice were euthanized by overdose of anesthetics (1000 mg/kg ketamine–3.5 mg/kg medetomidine) injected intraperitoneally at the indicated time by after the first DSS exposure. Colon length was measured and the tissue processed as described below.

Immunofluorescent EDA-Fibronectin Labeling

Optimum cutting temperature (OCT) solution (Tissue-Tek)-embedded colon samples of naïve and colitic mice after 3 days of the second DSS cycle were frozen on liquid nitrogen, and 7 μm cryostat sections were prepared. After fixation in acetone and rehydration in 80% methanol, the sections were incubated with blocking solution (5% donkey serum, 1% bovine serum albumin (BSA), 0.1% Triton-X in PBS) for 1 h. EDA-FN staining was performed using a biotinylated F8 antibody in the small immunoprotein (SIP) format which was self-produced (400 μg/mL)34 and a rat anti-CD31 (Dianova DIA-310, 1:20) primary antibody in blocking solution at 4 °C overnight. After washing, sections were incubated with streptavidin Alexa 488, donkey antirat Alexa 594 (both Invitrogen, 1:200), and Hoechst (1:1000) for 30 min at room temperature and washed. Images were acquired on an Axioskop2 mot plus microscope (Carl Zeiss) with an AxioCam MRc camera (Carl Zeiss) and a Plan-APOCHROMAT ×10 or ×20 objective (Carl Zeiss) using the AxioVision software 4.8 (Carl Zeiss).

Production of Fusion Proteins

Fusion proteins were produced as described previously.10 Briefly, FreeStyle CHO-S cells (Thermo Fisher) were cultured in PowerCHO-2CD medium (Lonza), supplemented with 2 mM l-glutamine (Life Technologies) and HT supplement (Lonza, 100 μM hypoxanthine, 16 μM thymidine). For transfection, the medium was replaced with identically supplemented ProCHO-4. Plasmids encoding F8-VEGFC or KSF-VEGFC were mixed with polethylenimine (PEI) in a DNA/PEI ratio of 1:4 and subsequently added to the cells. Cells were incubated at 37 °C for 3–4 h before PowerCHO-2CD was added. After 24 h, the incubation temperature was changed to 32 °C and transfected cells were incubated for 5–7 days.

For F8-VEGFC, stably transfected monoclonal cells were generated by serial dilution of transiently transfected cells and selection with G-418 (Life Technologies) for >28 days. For KSF-VEGFC, cells were transfected using electroporation (Nucleofector V kit, Lonza, employed according to the manufacturer’s instructions). After selection with G-418 (Roche) for >28 days, cells were stained with rabbit antihuman IgG (A0423, Dako) and Alexa 488-conjugated goat antirabbit secondary antibody (Invitrogen). Single cells were sorted on a FACSAria IIu (BD Biosciences). Fusion proteins were purified by protein A affinity chromatography (HiTrap Protein A HP, GE Healthcare Life Sciences) as described.35

F8-VEGFC Biodistribution Assay and Autoradiography

To investigate the targeting performance of the F8 fusion protein, 8-week-old female C57BL/6 mice were subjected to two 7-day cycles of 1.5% DSS (molecular mass 40 kDa, Alfa Aesar) in autoclaved sterile filtered drinking water ad libitum. The DSS cycles were interspersed with 2 weeks of normal drinking water. A 10 μg aliquot of radioiodinated fusion protein F8-VEGFC or control nontargeted KSF-VEGFC was injected intravenously into the lateral tail vein of inflamed and uninflamed mice (for details of the labeling protocol see ref (36)). At 24 h after injection, colon, liver, lungs, spleen, heart, kidney, and small intestine were collected, weighed, and their radioactivity was measured by a Packard Cobra γ counter. The radioactivity of individual organs was expressed as percentage of injected dose per gram of tissue (%ID/g), normalized over uninflamed KSF-VEGFC controls. Colons were further exposed to a phosphorimaging plate (Fujifilm Holdings Corporation, Tokyo, Japan) for 16 h. All autoradiographic figures were acquired on the same day with BASReader 3.14 software. Images were processed and quantified using identical parameters with the Software Pmod 3.9.

Histological Evaluation of Colonic Inflammation

At 32 days after the first DSS exposure, mice were euthanized. The colon was removed and fixed in 4% paraformaldehyde. Tissues were then embedded in paraffin and cut into 5 μm sections (longitudinal sections of the rolled colon, so-called “Swiss Rolls”). Routine hematoxylin and eosin staining were performed on one section per case. The scoring of colon inflammation was done by an experienced pathologist (JPS) in a blinded fashion. The histopathological colitis score was obtained according to a system previously shown to be sensitive and robust.37,38 In brief, four general criteria were evaluated in sections, separately for the mid and distal colon: severity of inflammation, degree of epithelial hyperplasia, degree of ulceration (if present), and percentage of area involved. Each of the criteria was graded on a 0–3 scale (0 = absent; 1 = mild; 2 = moderate; 3 = severe). The cumulative colitis score was obtained by summing up the scores for the mid and distal colon (0–24).

Histological Evaluation of the Colon Vasculature and Macrophages

Sections of formalin-fixed, paraffin-embedded tissues, thickness 5 μm, were deparaffinized in xylene and rehydrated in a descending ethanol series. Following antigen retrieval by heating 20 min in citrate buffer, slides were incubated with blocking solution (5% donkey serum, 1% BSA, 0.1% Triton-X in PBS) for 1 h. Tissue sections were then incubated with biotinylated goat anti-LYVE-1 (R&D BAF2125, 1:50) and rat anti-CD31 (Dianova DIA-310, 1:20) or with rat anti-F4/80 (Abcam ab6640, 1:50) and goat anti-CD206 (R&D AF2535, 1:50) primary antibodies in blocking solution at 4 °C overnight. After being washed, sections were incubated with streptavidin Alexa 488 (Invitrogen, 1:200) and appropriate secondary antibodies (donkey antirat Alexa 594, donkey antirat Alexa 488, and donkey antigoat Alexa 594; all from Invitrogen, 1:200) and Hoechst (1:1000) for 30 min at room temperature and washed. Images were acquired on a Pannoramic Digital Slide Scanner (3DHISTECH, Pannoramic 250 Flash III). The acquired fields of interest were then analyzed using the ImageJ software version 1.52i as follows: Excluding signal particles less than 25 μm2, blood vessels (CD31+LYVE-1-) and lymphatic vessels (LYVE-1+) were counted. Blood and lymphatic vessel densities were calculated as the area (in %) covered by CD31+LYVE-1- or LYVE-1+ vessels. To calculate blood and LV size, the total positive area was divided by the number of vessels. The density of macrophages was analyzed in a similar manner by evaluating the F4/80 positive area after exclusion of particles less than 15 μm2. Results were expressed as percentage of area covered by macrophages. Data analysis was performed in a blinded fashion.

FACS Analysis of Colon Tissues

Colons were cleaned with Hanks’ Balanced Salt solution (HBSS) containing antibiotics and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) and cut in small pieces. Colon fragments were incubated for 30 min at room temperature in HBSS and 1 mM of DTT under agitation. Tissue was minced and digested with HBSS containing 5% fetal calf serum, Hepes, 5 mM CaCl2, 10 mg/mL Dispase and collagenase (Roche), and 40 μg/mL DNaseI (Roche) at 37 °C under agitation. Digested fragments were passed through a 100 μm filter (BD Falcon) with a 1 mL syringe plunger and washed with 10 mL of RPMI medium, supplemented with 10% fetal calf serum and 20 mM Hepes. Single cell suspensions were further passed through a 70 μm filter (BD Falcon). Cells were incubated with Fc block (BioLegend 101302, 1:50) prior to staining with fluorescent antibodies for 20 min on ice. Single cell suspensions were incubated for 30 min at 4 °C with CD11b-BV605 (BioLegend 101237, 1:100), F4/80-AF647 (AbD MCA497A647, 1:100), CD45-PB (BioLegend 103126, 1:200), CD11c-PE-Cy7 (BioLegend 117318, 1:400), CD4-FITC (BioLegend 100405, 1:100), CD8-APC-Cy7 (BioLegend 100714, 1:100), GDTCR-Percp710 (eBioscience 46-5711-82, 1:400) diluted in PBS. Live/dead cell labeling was performed together with the antibody incubation (Zombie Aqua, BioLegend). To identify CD4-positive T regulatory cells, intracellular labeling with Foxp3-PE (eBioscience 12-5773-80, 1:100) was performed using an intracellular labeling kit (eBioscience Affymetrix 72-5775) according to the manufacturer’s instructions. After being washed, cells were resuspended in FACS buffer for acquisition. Samples were acquired on a LSRFortessa cell analyzer (BD). FACS data were analyzed using FlowJo v10.2 software (Tree Star).

RNA Extraction from Formalin-Fixed Paraffin-Embedded Colon Tissue and qPCR

Total RNA was isolated from two 10-μm-thick paraffin-embedded tissue slides per sample using the RNeasy FFPE kit (Qiagen) according to the manufacturer’s instructions. RNA concentration was measured using a ND-1000 NanoDrop. Equal amounts of RNA were reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The expression of selected genes was quantified by qPCR using the PowerUp SYBR green master mix (Applied Biosystems) on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Primers were designed according to RefSeq published sequences and are listed in Table 1.

Table 1. qPCR Primer Sequences.

graphic file with name pt9b00037_0009.jpg

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Statistical Analyses

Statistical analyses were performed using Prism version 7.0a (GraphPad Software, Inc.). Data are shown as mean ± SD or SEM as described in the figure legends. To determine statistical significance, either a two-tailed, unpaired Student’s t test (for the comparison of two groups) or a two-way ANOVA (for repeated measurements) with Sidak’s post-test was performed. Samples failing a Grubbs’ outlier test were excluded. Differences were considered statistically significant if p < 0.05.

Ethical Consideration

All animal experiments were performed in accordance with license ZH265/16 approved by the local veterinary authorities (Kantonales Veterinäramt Zürich). Special care was taken to limit animal discomfort throughout the whole study.

Acknowledgments

This study was supported by Swiss National Science Foundation Grants 310030_166490 and 310030B_185392, European Research Council Grant LYVICAM, Oncosuisse, and Leducq Foundation Transatlantic Network of Excellence Grant Lymph Vessels in Obesity and Cardiovascular Disease (11CVD03) (to M.D.). The authors thank Dr. Samia Bachmann and Jeanette Scholl for excellent technical assistance and Dr. Marco Taddio for helping with the autoradiography acquisition and quantification.

Glossary

Abbreviations

BSA

bovine serum albumin

CXCL10

chemokine (C-X-C motif) ligand10

CD

Crohn’s disease

DSS

dextran sodium sulfate

DAI

disease activity index

EDA

extradomain A

FN

fibronectin

iNOS

inducible nitric oxide synthase

IBD

inflammatory bowel disease

IFNG

interferon gamma

IL1b

interleukin 1 beta

Interleukin-10

I.V., intravenous injections

LEC

lymphatic endothelial cells

LVs

lymphatic vessels

OCT

optimum cutting temperature

PEI

polethylenimine

SIP

small immunoprotein

TNF

tumor necrosis factor alfa

UC

ulcerative colitis

VEGFC

vascular endothelial growth factor C

VEGFR-3

vascular endothelial growth factor receptor 3.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsptsci.9b00037.

  • F8-VEGFC delivery reduces body weight loss and histological ulceration score; F8-VEGFC does not change lymphatic and blood vessel size in colitis; FACS gating strategy for immune cells (PDF)

Author Contributions

# These authors contributed equally. C.T. and S.S. designed the research, performed experiments, analyzed results, and wrote the manuscript; N.C., D.B., M.S., and J.P.S. performed experiments and analyzed results, D.N. designed the research and analyzed results, M.D. designed the research, analyzed results, and revised the manuscript. All authors have given approval to the final version of the manuscript.

The authors declare the following competing financial interest(s): D.N. is a shareholder and board member of Philogen, a biotech company which shares with ETH Zurich commercialization rights for F8-VEGFC. M.D., D.N., and S.S. are inventors on a pending patent for F8-VEGF-C. The other authors declare no competing financial interest.

Supplementary Material

pt9b00037_si_001.pdf (487.2KB, pdf)

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Supplementary Materials

pt9b00037_si_001.pdf (487.2KB, pdf)

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