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From the Departments of Pathology,* Molecular and Cellular Physiology, and Cardiology, Louisiana State University Health Sciences Center–Shreveport, Shreveport, Louisiana; and Attenuon, San Diego, California
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Abstract |
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Angiogenic cytokine production has been reported to be elevated during IBD, possibly governing increased neovascularization during disease. Serum from IBD patients was reported to contain elevated concentrations of vascular endothelial growth factor (VEGF)-A during active disease versus remission.9-11 A recent report by Konno and colleagues12 has also shown elevated local tissue concentrations of VEGF-A in IBD specimens that may be more relevant in relation to IBD pathology, as it has been shown that local microenvironment tissue levels of VEGF-A are key in determining normal versus pathological angiogenesis.12,13 Moreover, studies have also shown increases in serum levels of basic fibroblast growth factor (b-FGF) and transforming growth factor (TGF)-ß in patients with active ulcerative colitis (UC) or Crohn’s disease (CD).10,14 Together, these reports clearly demonstrate that angiogenic cytokines are increased in patients with active IBD. However, no information is available regarding distinct expression profiles of pro- versus anti-angiogenic molecules during colitis.
Increased leukocyte infiltration is a hallmark feature of IBD and experimental colitis, with T cells, monocytes, and neutrophils contributing to disease initiation and subsequent tissue damage.15-18
In addition to their role in the inflammatory response, leukocytes may also regulate angiogenic activity. Several studies have shown that various leukocyte types produce diverse angiogenic factors and can also modify extracellular matrix thereby promoting neovascularization.19-21
Neutrophils have been reported to be significant sources of cytokines, such as interleukin (IL)-8 and VEGF, as well as matrix metalloproteinases (MMP-2 and -9) that are released at the site of inflammation to regulate angiogenesis.22,23
Monocytes and macrophages also have corresponding roles in angiogenesis as sources of cytokines such as VEGF, FGFs, interferon-
, tumor necrosis factor (TNF)-
, and many others.24-26
It is widely accepted that T cells regulate IBD pathogenesis through the production of proinflammatory cytokines, such as Th1 cytokines, that are primarily proangiogenic concomitant with reduced production of regulatory cytokines, such as Th2 cytokines, that may have angiostatic or anti-angiogenic effects.27-29
Therefore, infiltrated T cells could elicit diverse angiogenic regulation through the production of a wide array of angiogenic molecules including Th1 and Th2 cytokines and chemokines. Thus it is very likely that leukocyte infiltration with concomitant production of angiogenic mediators facilitates neovascularization and chronic inflammation observed in IBD.
Here we examine the hypothesis that increased angiogenic activity plays an important pathological role in two different models of experimental colitis. Data contained herein demonstrate that blood vessel density increases during colitis and strongly correlates with pathological scores of affected tissue. Moreover, we report that angiogenic stimulation in both models shows some similarities, as well as distinct differences, in angiogenic gene and protein profile expression, demonstrating a diverse angiogenic response. Data are also presented that demonstrate an essential requirement of leukocyte infiltration for angiogenic stimulation during colitis. Last, we report that anti-angiogenic intervention significantly attenuates leukocyte infiltration and tissue damage associated with experimental colitis, indicating that increased angiogenesis during colitis plays an important pathological role during disease. Together, these data provide compelling evidence for an important pathophysiological role of increased angiogenic activity during experimental colitis and highlight the potential for anti-angiogenic intervention as a novel therapeutic target for IBD.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Mice used for this study were bred and housed at the Association for Assessment and Accreditation of Laboratory Animal Care, international-accredited Louisiana State University Health Sciences Center–Shreveport animal resource facility and maintained according to the National Research Council’s Guide for Care and Use of Laboratory Animals. CD18-null–/– (Itgb2tm2Bay) C57BL/6J and wild-type C57BL/6J mice were bred in-house. Male Rag-1–/–-null (Rag1tm1Mom) C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME).
3% Dextran Sodium Sulfate (DSS) and CD4+CD45RBhigh T-Cell Transfer Models of Experimental Colitis
The 3% DSS and CD4+CD45RBhigh T-cell transfer models of colitis were performed as previously reported.16,30 Ten- to 12-week-old wild-type or CD18–/–-null male C57BL/6J mice were administered 3% DSS (TDB Consultancy AB, Uppsala, Sweden) in their drinking water for 6 days. Control cohorts were given regular drinking water. Animals were monitored for symptoms of disease progression as previously reported and sacrificed on the 7th day for analysis of various parameters described below. The 3% DSS cyclic colitis model was performed with slight modifications as previously reported.31 In brief, 3% DSS was administered for 5 days (on) and then switched to regular water for another 5 days (off). This cycle was repeated three times, and mice were sacrificed at the end of the third off cycle.
CD4+CD45RBhigh T-cell-dependent colitis was induced in male Rag-1-null mice between 10 and 12 weeks of age using CD4+ T cells from the spleens of donor female C57/B6J mice as previously reported.16,32 In brief, CD4+ T cells were enriched using the MACS system from Miltenyi Biotech for negative selection by magnetic cell sorting. Cells were then labeled with anti-fluorescein isothiocyanate (FITC) microbeads, and unlabeled cells were separated on a depletion column (column type CS; Miltenyi Biotech, Auburn, CA). Enriched CD4+ T cells were labeled with biotin-conjugated anti-CD4 monoclonal antibody followed with streptavidin-670 and phycoerythrin-conjugated anti-CD45RB monoclonal antibody (PharMingen, La Jolla, CA) and fractioned into CD4+CD45RBhigh and CD4+CD45RBlow cell populations by two-color sorting on a FACS Vantage (Becton, Dickinson and Company, Mountain View, CA). The CD45RBhigh population was defined as the brightest 40% and that of the CD45RBlow were the dimmest 15%. Next, 5 x 105 CD4+CD45RBhigh or CD4+CD45RBlow cells were injected intraperitoneally in 500 µl of phosphate-buffered saline (PBS). Animals were monitored throughout 8 weeks for the development of experimental colitis and sacrificed at the end of week 8 for analysis of the various parameters described below.
Histopathological Scoring of Experimental Colitis
Pathological scoring of paraffin-embedded hematoxylin and eosin (H&E)-stained sections of the distal colons from DSS and CD4+CD45RBhigh T-cell transfer experimental colitis mice was performed in a double-blinded manner. Histopathological scoring of DSS-treated colons was performed as previously reported.30 In brief, these are severity of inflammation, scored 0 to 3 for none, slight, moderate, and severe; depth of injury, scored 0 to 3 for none, mucosal, mucosal, and submucosal, transmural; and crypt damage, scored 0 to 4 for none, basal one-third damaged, basal two-thirds damaged, only surface epithelium intact, entire crypt epithelium lost. Each sample received a score of 0 to 40 based on multiplying the score of each parameter by the percentage of tissue involvement (x1, 0 to 25%; x2, 25 to 50%; x3, 50 to 75%; x4, 75 to 100%) and summing these values. Histopathological analysis of CD4+CD45RBhigh-induced colitis was performed as previously reported.16 Eight parameters were used and included 1) the degree of inflammation in the lamina propria (range, 1 to 3); 2) goblet cell loss indicative of mucin depletion (range, 0 to 2); 3) reactive epithelial hyperplasia/atypia with nuclear changes (range, 0 to 3); 4) number of intraepithelial lymphocytes in the epithelial crypt (range, 0 to 3); 5) abnormal crypt architecture (range, 0 to 3); 6) number of crypt abscesses (range, 0 to 2); 7) mucosal erosion to frank ulceration (range, 0 to 2); and 8) submucosal spread to transmural involvement (range, 0 to 2). The severity of inflammatory changes is based on the sum of the scores in each parameter with a maximal score of 20.
Determination of Tissue Angiogenic Index
Distal colon segments from both models were frozen in OCT compound (Sakura Finetek USA, Inc., Torrance, CA). Ten-µm frozen cross-sections were fixed with 95% ethanol/5% glacial acetic acid for 20 minutes at –20°C, washed three times with PBS, blocked with 5% horse serum/PBS, and then stained with anti-mouse PECAM-1 (CD31) antibody (BD PharMingen) diluted 1:200 in PBS/0.05% horse serum followed by Cy3-conjugated anti-rat antibody diluted 1:250 in PBS with 0.05% horse serum. Slides were then mounted with Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (Vector Laboratories, Inc., Burlingame, CA). Digital images were taken at a magnification of x200 on a Nikon Eclipse TE2000-S epifluorescent scope (Melville, NY) using Texas Red and DAPI excitation/emission filters in conjunction with a Hamamatsu Orca-ER digital camera (Bridgewater, NJ). The total pixel density of PECAM-1 and DAPI staining was quantified for each image using Simple PCI software (C-Imaging Systems; Compix Inc., Sewickley, PA). All imaging settings for each respective fluorochrome were kept constant between specimens and the amount of PECAM-1 and DAPI pixel density measured using the image processing and analysis feature of Simple PCI. Total PECAM-1 pixel density was divided by the total DAPI pixel density for each image to calculate a vascular density ratio referred to as the angiogenic index.
Dual Radiolabel Antibody Measurement of Vascular Surface Area
A PECAM-1 dual radiolabel assay was used to quantify vascular surface area in the colons of DSS and CD4+CD45RBhigh T-cell transfer experimental colitis mice. On completion of the experimental protocols, the animals were volume exchanged with radiolabeled PECAM-1 and control antibodies as previously reported.33 Binding antibodies against PECAM-1 were labeled with 125I (DuPont NEN, Boston, MA), and nonbinding control antibodies were labeled with 131I (DuPont NEN) using the chloramine T method with resulting specific activities of 0.5 µCi/µg. Animals were anesthetized with xylazine/ketamine, and the carotid artery and jugular vein were cannulated and injected with the radiolabeled antibodies through the jugular vein with 0.2 ml of heparinized saline. After 4 minutes and 45 seconds, 250 µl of blood was obtained from the carotid line to determine end-point binding. The animal was then flushed with 6 ml of bicarbonate-buffer salt solution at pH 7.4 through the jugular to clear the thoracic organs. The inferior vena cava was then cut, and the animal was flushed with 15 ml of bicarbonate-buffer salt solution through the carotid to clear the abdominal organs. Organs were then harvested and weighed, and the activity of 125I and 131I in the colons was measured using a 14800 Wizard 3 gamma counter (Wallac Instruments, Wellesley, MA) with automatic correction for background activity and spillover. The total injected activity in each experiment was calculated by counting a 2-µl sample of the radiolabeled monoclonal antibody (mAb) mixture. The radioactivities remaining in the tube used to mix the mAbs and the syringe used to inject the mixture were subtracted from the total injected activity. PECAM-1 antibody binding was calculated by subtracting the accumulated activity per gram of tissue of the nonbinding mAb from the activity of the binding mAb and expressed as micrograms of mAb per gram of tissue using the following equation: (µg mAb/g) = {[%ID[125I/g tissue] – %ID[131I/g tissue] x %ID(125I)plasma/%ID (131I)plasma} x total binding mAb (µg)/100, where ID represents injected dose.
FITC-Labeled Lycopersicon esculentum Lectin Labeling of Colon Vasculature
FITC-labeled L. esculentum lectin (Vector Laboratories) labeling was used to independently validate the PECAM-1 staining technique and to evaluate if the neovasculature was perfused. Lectin labeling of the vasculature in distal colons of DSS and CD4+CD45RBhigh T-cell transfer colitis models was performed as previously reported.34 On completion of the colitis protocols, mice were anesthetized, and the inferior vena cava was cannulated. One hundred µg of the lectin in 100 µl of 0.9% NaCl was perfused into the tail vein. The lectin was allowed to circulate for 2 minutes before fixative, 0.5% glutaraldehyde and 1% paraformaldehyde in PBS, was perfused through the vena cava. Eight-µm frozen cross-sections were made using the same tissue processing method described for PECAM-1 staining. The slides were then mounted in DAPI Vectashield mounting medium (Vector Laboratories) and analyzed for FITC-lectin pixel density to determine a lectin angiogenic index. Additional sections were also stained for PECAM-1 and three-color images acquired (PECAM-1, lectin, and DAPI).
Angiogenesis Gene Array
Mouse Angiogenesis Gene Array kits from SuperArray Biosciences Corp., Frederick, MD, were used to assay levels of angiogenic gene expression in experimental colitis. Total RNA was isolated from colon tissue of DSS and CD4+CD45RBhigh colitic and control mice as previously reported.16,35 Colon samples were snap-frozen in liquid nitrogen and then ground with a mortar and pestle kept cold on liquid nitrogen. Samples were then placed in Dounce homogenizers containing 600 µl of buffer RLT as specified by Qiagen’s RNeasy protocol (Qiagen, Valencia, CA). RNA isolation was performed according to the manufacturer’s protocol. RNA concentration and purity were measured by UV absorbance at 260 nm and 280 nm.
Gene array membranes were prehybridized using the GEAhyb hybridization solution containing sheared salmon sperm DNA and incubated at 60°C for 2 hours using a VWR Scientific Products (West Chester, PA) hybridization oven. Probe synthesis was done according to the GE Array Ampo Labeling-LPR kit protocol using total RNA from colon tissue. After prehybridization, the labeled cDNA probes were denatured and added to the arrays. Gene arrays were hybridized overnight at 60°C at 10 rotations per minute. Arrays were then washed three times in 1x standard saline citrate and washed again three times in 0.1x standard saline citrate. The arrays were then blocked for 1 hour in blocking buffer and incubated with alkaline phosphatase for 1 hour in 1x buffer F (SuperArray). The arrays were then washed and incubated in CPD star for 5 minutes and exposed to Blue Bio film (Denville Scientific, Inc., Metuchen, NJ) and developed using a Konica SRX-101 film developer (Konica Inc., Ramsey, NJ).
VEGF-A Western Blotting and Serum and Tissue Enzyme-Linked Immunosorbent Assays (ELISAs)
VEGF-A Western blot and ELISA were performed as previously reported.36,37
Serum was obtained from controls, DSS-treated, and CD4+CD45RBhigh-treated mice as follows. A retro-orbital bleed was used to collect 
1 ml of blood from all mice at the time of death. These samples were kept on ice for 30 minutes and then spun at 3000 rpm for 10 minutes to obtain serum. Serum was collected in 50-µl aliquots and stored at –80°C. Tissue lysates were made from 15- to 20-mm segments of distal colon tissue from controls, DSS, and CD4+CD45RBhigh colitic mice as follows. Samples were snap-frozen in liquid nitrogen and later homogenized in 500 µl of radioimmunoprecipitation assay lysis buffer containing protease inhibitors. The homogenized tissue was placed in a 1.5-ml microcentrifuge tube and spun for 30 minutes at 12,000 rpm at 4°C. The supernatant was removed and placed in a clean microcentrifuge tube and spun down two more times to obtain a clear lysate. The samples were aliquoted, and the protein concentration determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA).
VEGF-A Western blot analysis was performed as previously reported.36,37 In brief, 50 µg of total protein from each sample was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 150 V. Proteins were transferred to Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad) and subsequently blocked for 6 hours at 4°C in 5% milk TBS-T. Polyclonal anti-murine VEGF-A164 antibody (Peprotech, Inc., Rocky Hill, NJ) was applied at a concentration of 2 µg/ml and incubated overnight at 4°C. Anti-rabbit IgG-horseradish peroxidase-linked secondary antibody (Amersham Biosciences, Arlington Heights, IL) was applied at a 1:2000 dilution and incubated for 3 hours at room temperature in 5% milk TBS-T. The membrane was then incubated in ECL Western blotting detection reagents (Amersham Biosciences) for 10 minutes, exposed to Blue Bio film (Denville Scientific, Inc.), and developed using a Konica SRX-101 film developer (Konica, Inc.).
Quantitative measurement of VEGF-A levels was performed using a mouse VEGF-A ELISA kit according to the manufacturer’s protocol (Oncogene, Boston, MA). In brief, 50 µl of assay diluent RD1N was pipetted into each well along with 50 µl of the samples and mixed by shaking the plate for 1 minute. The plate was then sealed and incubated at room temperature for 2 hours. Wells were washed five times with 1x wash buffer, and 100 µl of 1x mouse VEGF conjugate concentrate was then added to each well. The plate was again sealed and incubated for 2 hours. Wells were washed five times, and 100 µl of substrate solution was then added and incubated for 30 minutes. The reaction was terminated using 100 µl of stop solution and then read on a TECAN GENios Plus multiscan plate reader (Phenix Research Products, Candler, NC) at dual wavelengths of 450/540 nm.
Use of Anti-Angiogenic Agents during Experimental Colitis
Ten- to 12-week-old wild-type C57BL/6J mice undergoing DSS-induced colitis were treated with the anti-angiogenic agent thalidomide (Calbiochem, La Jolla, CA). Thalidomide (200 mg/kg) in dimethyl sulfoxide was administered intraperitoneally on days 0, 2, 4, and 6 of the model. Control animals were injected with an equal amount of dimethyl sulfoxide alone at the same time points. Colon tissue was harvested and processed for H&E pathological scoring and angiogenic index determination as described above.
ATN-161 (Ac-PHSCN-NH2), a 5-mer peptide that binds to 5ß1 and Vß3, was used in the CD4+CD45RBhigh colitis model to evaluate the effects of target-specific anti-angiogenic intervention.38
Two weeks after CD4+CD45RBhigh reconstitution of Rag-1-deficient mice, 1 mg/kg ATN-161 (active peptide) or ATN-163 (scrambled peptide) in PBS was administered by intraperitoneal injection three times a week for the remaining 6 weeks of the study protocol. On completion of week 8, animals were sacrificed, and tissue was processed for various parameters described above.
Leukocyte Adhesion Under Hydrodynamic Flow Conditions
Hydrodynamic parallel plate flow chamber studies were performed as we have previously reported.39
In brief, freshly isolated mouse splenocytes were labeled with a fluorescent dye by 30-minute incubation at 37°C with 200 nmol/L Cell Tracker Green purchased from Molecular Probes, Eugene, OR. The labeled cells were resuspended in Hanks’ balanced salt solution at 2 x 105 cells/ml in a 200-ml beaker kept at 37°C and stirred at 60 revolutions per minute. A Glycotech flow chamber insert and gasket were used to form a laminar plate flow chamber that could be viewed on a microscope. Labeled splenocytes mixed with or without ATN-161 (10 µg/ml) were drawn from the beaker into the flow chamber across a monolayer of either unstimulated or TNF- (10 ng/ml)-stimulated murine endothelial cells at a physiological shear rate of 1.5 dynes/cm2 using a programmable digital syringe pump. The fluorescent cells were visualized using a Nikon Eclipse TE-2000 epifluorescent microscope equipped with a Hamamatsu digital camera, and digital video was captured at 29 images/second using SIMPLE PCI software from Compix. Firmly adherent cells were counted and defined as cells that did not move one cell diameter throughout a 5-second period as determined by automated tracking and manual review of individual cells in each experimental field of view.
Statistical Analysis
Control versus colitis data were compared against one another for each model of colitis. Statistical analysis of the data were determined using a Student’s t-test with a value of P < 0.05 required to achieve significance. Temporal comparisons were performed by one-way analysis of variance with Dunnett’s posttest. A value of P < 0.05 was required to achieve significance. n values for each experiment are reported in the figures. Data are reported as mean ± SE.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Several different models of experimental colitis have been reported that display various pathophysiological aspects of IBD disorders including CD and UC.15,40 Although no model serves as a complete surrogate for human disease, many salient clinical features can be examined depending on the agent or method used to induce experimental colitis. In this study, we used the DSS and CD4+CD45RBhigh T-cell transfer models of colitis, which manifest similar disease characteristics of UC and CD. DSS induces colitis primarily by irritation and denudation of surface epithelium that quickly results in weight loss, loose stool/diarrhea, and occult and gross rectal bleeding.41,42 We analyzed vehicle control and DSS-treated distal colons of C57/B6J mice. Prominent mucosal and submucosal leukocyte infiltrates were observed in 3% DSS-treated colons along with severe erosion of the epithelial cell layer often resulting in ulceration, significant mucosal/crypt damage, and submucosal edema. Lesions were intermittently located within the colon with the majority of histopathological changes observed in the distal segment (data not shown). CD4+CD45RBhigh T-cell-induced colitis results from differential expansion of Th1 cells in Rag-1–/– mice, driven by enteric bacteria.43,44 Similar, but less severe, leukocyte infiltration was observed in the CD4+CD45RBhigh T-cell transfer model along with moderate erosion of the epithelial cell layer and mucosal/crypt damage compared with CD4+CD45RBlow control colons that do not develop disease (data not shown). Blinded analysis of the histopathology was done, and the scoring of pathological changes showed a significant increase in histopathological scores in both models, these being 23- and 17-fold in the DSS and CD4+CD45RBhigh models, respectively, with the DSS model displaying greater disease severity. Together, these models display several important inflammatory pathophysiological features of human IBD.
Measurement of Blood Vessel Density in Experimental Colitis
Several clinical reports of IBD have observed an increase in colon vascular density during active disease.6,7
However, no studies have examined the vascular density of actively inflamed colons from experimental models of colitis. Therefore, we used the well-established technique of anti-PECAM-1 (CD31) immunohistochemistry to measure changes in vascular density between the two colitis models.45-48
Figure 1, A and B
, illustrates the amount of PECAM-1 staining (red) in control and DSS-treated distal colons, respectively. Figure 1, C and D
, shows secondary antibody control and DAPI alone staining of DSS-treated colons, respectively. Likewise, Figure 1, E and F
, illustrates the amount of PECAM-1 staining in control CD4+CD45RBlow colons and CD4+CD45RBhigh colons, respectively. Figure 1, G and H
, demonstrates secondary antibody control and DAPI alone staining of CD4+CD45RBhigh distal colons, respectively. To determine differences in vessel density, quantitative measurements of total PECAM-1 pixel surface area were made in conjunction with total surface area measurement of DAPI nuclear staining (blue) to calculate an angiogenic index. Figure 1I
illustrates that both the DSS and CD4+CD45RBhigh colitis models show a significant increase in the angiogenic index compared with respective controls. The PECAM-1 angiogenic index showed significant increases in vascular density in both models with the CD4+CD45RBhigh model displaying a 188 ± 2.1% increase in vascular density and the DSS model an 89.7 ± 1.9% increase in vascular density. We further validated these observations by performing dual radiolabel experiments using 125I-labeled anti-PECAM-1 to quantitatively measure the colon vascular surface area.33,49
Figure 1J
demonstrates increased anti-PECAM-1 antibody binding in both models of experimental colitis, confirming an increase in vascular density. Moreover, anti-PECAM-1 radiolabel data show a similar increase in blood vessel surface area compared with PECAM-1 immunofluorescent measurement of the angiogenic index. These data clearly demonstrate that experimental colitis results in a significant increase in colon vascular density.
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shows PECAM-1 staining of DSS distal colon, whereas Figure 2B
illustrates the FITC lectin binding of the same section. Figure 2C
shows the three-color merged image of lectin (green), PECAM-1 (red), and DAPI nuclear stain (blue). The majority of lectin and PECAM-1 staining co-localized throughout the tissue, with few areas showing distinct PECAM-1 staining alone. Figure 2D
reports the FITC-lectin angiogenic index in both the DSS and CD4+CD45RBhigh models compared with controls. The FITC-lectin angiogenic index corroborates the PECAM-1 angiogenic index for determining vascular density, with the lectin angiogenic index showing a 153 ± 1.4% increase in blood vessel density for the CD4+CD45RBhigh model and 95 ± 1.3% in the DSS model. Moreover, the high degree of co-localization between lectin and PECAM-1 indicates perfusion of these vessels.
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Several chronic disease conditions have been associated with increased angiogenic activity.55-57
Given the cyclical nature of IBD activity and remission, increased angiogenic activity may serve to facilitate tissue repair. However, no clear information exists regarding the relationship between angiogenic activity and disease severity in IBD or other chronic inflammatory disorders. Therefore, we examined the association of increased angiogenic activity with disease severity in specimens from both experimental colitis models. Ten-µm serial sections of distal colons were processed for H&E histopathology or anti-PECAM-1 angiogenic index to compare these parameters within the same tissue specimen. Figure 3, A and B
, shows the correlation between angiogenic index and histopathology score for the DSS and CD4+CD45RBhigh colitis models, respectively. Measurements made from identical specimens from either model show a strong and significant correlation between the angiogenic index and tissue histopathology score with the DSS model having an r2 of 0.90 and the CD4+CD45RBhigh model an r2 of 0.91. Moreover, we also examined the temporal relationship between increased angiogenic activity and tissue histopathology in the DSS colitis model. Figure 3C
illustrates that DSS-mediated angiogenic activity becomes significantly elevated at 4 days, which is precisely when significant tissue histopathology is observed (Figure 3D)
. We also examined angiogenic activity and tissue pathology on completion of a cyclical DSS regimen, in which 3% DSS is administered for 5 days and then withdrawn for 5 days (and switched to regular water), with this cycle being repeated three times. Cycling of the DSS treatment results in acute inflammatory periods followed by tissue restitution and repair, which may emulate cyclical aspects of human IBD.31
Tissues were harvested at the end of the last off cycle that revealed histopathological changes, albeit greatly reduced, but that angiogenic activity was not significantly different from control (Figure 3D)
. These results combined with data from the acute DSS model indicate that increased angiogenic activity is associated with active inflammation and diminished during restitution. Together, these data strongly suggest that increased angiogenic activity contributes to tissue histopathology during active experimental colitis.
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It is now well accepted that the presence of high levels of proangiogenic factors play key roles in the process of pathological neovascularization as shown by studies that inhibit them.58-60
Therefore, we examined expression profiles of angiogenic regulatory genes to gain a better understanding of the factors that may be involved in controlling angiogenesis during experimental colitis. Angiogenesis gene arrays were performed using total RNA isolated from four different control and DSS-treated colons on completion of a 6-day study and from four different CD4+CD45RBlow control or CD4+CD45RBhigh experimental colons on completion of the 8-week study. Differentially regulated genes were identified based on a twofold change in signal intensity (up or down) compared with control signals. A complete list of the genes that were similarly or differentially regulated in the DSS and CD4+CD45RBhigh colons as compared with their respective controls is shown in Table 1
.
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, chemokine (CXC) ligand 1 (Gro1), and hepatocyte growth factor (HGF). Additional mediators that can exert either pro- or anti-angiogenic effects, depending on stimulation, were also similarly up-regulated in both models including TGF-ßR2 and TGF-ßR3, macrophage scavenger receptor 1 (Msr1), and thrombospondin-1 (Thbs 1). Three genes were also similarly down-regulated including CD36 antigen and chromogranin A, which are anti-angiogenic, and TGF-ß2, which may act in either capacity. Of the similarly regulated genes, most up-regulated genes exhibit proangiogenic effects and most down-regulated ones exhibit anti-angiogenic effects. This suggests that the inflammatory stimulus involved with either model of experimental colitis elicits a proangiogenic response consistent with underlying pathological features between the two models.
Differential regulation of numerous angiogenic mediators also occurred, revealing specific angiogenic differences between the two models. In the DSS model, several pro- and anti-angiogenic genes were up-regulated, but the same genes were unchanged in the CD4+CD45RBhigh model. The proangiogenic factors include fibroblast growth factor receptors 1 and 3 (FGFR1, 3), serine protease inhibitor 1 (PAI-1), and colony stimulating factor 3 (CSF3). Those with anti-angiogenic effects are IL-10, thrombospondin 2 (Thbs2), and a disintegrin metalloproteinase thrombospondin type 1 motif (Adamts 1). However, many more anti-angiogenic genes (eg, Col18, TIMP-2, and so forth) were significantly down-regulated in DSS colitis, suggesting that angiogenic stimulation in this model is likely attributable to loss of negative regulators. Conversely, proangiogenic me-diators were more substantially up-regulated in the CD4+CD45RBhigh colitis model. Moreover, several molecules were distinctly up-regulated in CD4+CD45RBhigh colitis yet down-regulated in DSS colitis, including connective tissue growth factor (CTGF), fibroblast growth factor-1 (FGF1), inhibitor of DNA binding 3 (ID3), and interferon-. Most interestingly, VEGF-A, -B, -C, and -D were all up-regulated in CD4+CD45RBhigh colitis yet down-regulated in the DSS model, illustrating the unique nature of angiogenic gene expression between the two models. Together, these data suggest that neovascularization in the CD4+CD45RBhigh model primarily involves up-regulation of proangiogenic factors, whereas neovascularization in the DSS model is associated with an abundant decrease in anti-angiogenic gene expression.
Differential expression of angiogenic factors, such as VEGF-A, have recently been reported in UC and CD.9,11,12
Having observed distinct differences in VEGF-A gene expression between the two models, we examined VEGF-A protein levels present in serum and colon tissue from the DSS and CD4+CD45RBhigh models by Western blot and ELISA. Western blots for VEGF-A did not reveal any sig-nificant difference in VEGF-A levels during DSS colitis (Figure 4A)
; however, densitometric analysis of the CD4+CD45RBhigh VEGF-A Westerns showed a significant 9.5-fold increase in tissue VEGF levels (Figure 4B)
. VEGF-A ELISA was performed with tissue lysates to quantitate precisely the amount of VEGF-A produced. Having observed no change in VEGF-A protein levels on completion of the DSS model, we examined VEGF-A tissue levels at various time points in the DSS model to address the possibility of early changes in expression that could be lost because of tissue damage. Consistent with the angiogenesis gene array results, VEGF-A expression was not significantly different throughout DSS colitis. However, we observed a trend of decreased expression early during the colitis model that resolved by day 6 of the DSS model (Figure 4C)
. Thus, alteration of VEGF-A expression in the DSS model is not attributable to excessive tissue damage and/or protein loss. Moreover, VEGF-A expression was not increased on completion of the chronic DSS model. Conversely, a sevenfold increase in VEGF-A was observed in the colon tissue of CD4+CD45RBhigh mice with a control concentration of 450 pg/µg of total protein versus 3300 pg/µg of total protein for colitic specimens (Figure 4D)
. Last, serum VEGF-A levels from both models did not reveal any significant differences between experimental and control groups (data not shown). Together, these data confirm results from the angiogenesis gene array experiments and provide clear evidence demonstrating different mechanisms of angiogenic stimulation between the two models.
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Infiltration of leukocytes into tissue has been shown to be associated with increased angiogenesis in various chronic inflammatory disorders and is believed to be an important mediator of pathological angiogenesis.61-64
Therefore, we examined whether increased leukocyte infiltration is necessary for increased angiogenic activity observed during experimental colitis. The CD18-null mutant mouse, with its complete loss of leukocyte ß2 integrins, has a severely blunted ability to recruit leukocytes under inflammatory conditions and its loss attenuates chronic inflammatory disease.65-67
We have recently shown that genetic ablation of CD18 confers protection against DSS colitis attributable to defective leukocyte infiltration.30
Therefore, we determined the importance of leukocyte recruitment on angiogenic stimulation during experimental colitis using CD18-null mice treated with DSS. Histopathological analysis of wild-type mice revealed a typical increase in leukocyte infiltrates in the mucosa and submucosa as well as extensive mucosal/crypt and epithelial cell layer damage, which was essentially absent in CD18-null colons treated with DSS (Figure 5, A and B
, respectively). Histopathological scoring revealed an absence of DSS-mediated tissue damage as we have recently reported (Figure 5C)
.30
Importantly, the angiogenic index of DSS CD18-null colons was found to be significantly reduced compared with DSS wild-type colons, 0.096 ± 0.003 versus 0.254 ± 0.034, respectively (Figure 5D)
. Together, these data demonstrate that leukocyte infiltration is an obligatory requirement for angiogenic stimulation during experimental colitis.
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Data presented thus far demonstrate increased angiogenic activity during experimental colitis that is primarily dependent on inflammatory cells and mediators. However, no experimental evidence exists demonstrating that increased angiogenic activity plays a pathological role during colitis. Recent clinical reports suggest that the anti-angiogenic agent thalidomide is capable of inducing remission of active CD.8
Given the unique nature of thalidomide as an anti-angiogenic and immunomodulatory agent, we determined whether thalidomide treatment affects DSS-induced colitis and neovascularization.68,69
Figure 6, A and B
, illustrates histopathological features of DSS plus dimethyl sulfoxide vehicle or DSS plus 200 mg/kg thalidomide, respectively. DSS-mediated histopathology showed that vehicle control mice develop significant tissue damage with perfuse immune cell infiltration associated with prominent mucosal/crypt and epithelial cell damage (Figure 6A)
. Conversely, thalidomide significantly blunted mucosal injury and leukocyte recruitment and preserved the mucosal epithelium compared with vehicle control colons (Figure 6B)
. DSS cumulative histopathology scores are shown in Figure 6C
demonstrating a significant decrease in tissue damage in colons from thalidomide-treated mice. Moreover, the angiogenic index was also significantly decreased in thalidomide-treated mice compared with control mice (Figure 6D)
. These data demonstrate that angiogenic and inflammatory activity play key pathophysiological roles in the development of DSS-induced colitis.
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To substantiate that the therapeutic effects observed with thalidomide were attributable to the inhibition of angiogenesis, we used a selective anti-angiogenic peptide ATN-161 in the CD4+CD45RBhigh colitis model. Angiogenesis gene array analysis of the CD4+CD45RBhigh model revealed distinct up-regulation of 5, V, and ß3 integrins and matrix ligands associated with neovascularization. The anti-angiogenic peptide ATN-161 attenuates neovascularization by binding 5ß1 and Vß3 and has been reported to significantly attenuate tumor-mediated angiogenesis.38
Figure 7
shows that 1 mg/kg ATN-163 (scrambled peptide) treatment did not alter histopathology of CD4+CD45RBhigh colitis, whereas 1 mg/kg ATN-161 (active peptide) significantly decreased immune cell infiltration and edema and attenuated crypt destruction compared with ATN-163 control drug (Figure 7, A and B
, respectively). Moreover, Figure 7, C and D
, demonstrates that ATN161 significantly decreases both the tissue histopathology score and the angiogenic index, respectively. Studies have verified this compound as a potent and selective inhibitor of angiogenesis with no indication of a role in modulating inflammatory responses. ATN-161 was shown to be highly active in Matrigel plugs where angiogenesis is driven by VEGF or FGF-2, where the compound bound exclusively to neovessels.38
In addition, ATN-161 selectively decreases viable EPCs, which are known to be involved in angiogenesis, while having no effect on CECs, which are associated with inflammation (A.P.M., personal communication). Nonetheless, given the possibility that ATN-161 might affect leukocyte function, we examined whether ATN-161 altered leukocyte recruitment and adhesion to activated endothelial monolayers. MS-1 mouse endothelial cells were stimulated with 10 ng/ml TNF- and assembled in a parallel plate flow chamber system to evaluate mouse splenocyte adhesion under hydrodynamic flow conditions as we have previously reported.39
Figure 7E
clearly illustrates that 10 µg/ml ATN-161, the therapeutic dose used for in vivo experiments based on a total blood volume of 2.5 ml, did not alter TNF--mediated leukocyte adhesion. This finding corroborates the selectivity of ATN-161 and demonstrates that the agent does not interfere with inflammatory responses. Together, these data using a select agent against angiogenic integrins demonstrate that increased neovascularization plays an important role in facilitating tissue damage and dysfunction during CD4+CD45RBhigh colitis.
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Increased angiogenic activity has been observed in several chronic inflammatory diseases, yet the importance of this association and the manner in which angiogenesis is stimulated in IBD or experimental colitis remains unknown.29,73,74 We found that blood vessel densities were significantly increased in both the DSS and CD4+CD45RBhigh models of colitis. Moreover, the majority of the vasculature was perfused as determined by intravascular FITC-lectin binding to endothelium.54 A strong correlation between the overall angiogenic index and histopathological score was also found in tissue sections from both models. In addition, the increase in neovascularization temporally coincided with increased tissue histopathology. Together, these data strongly suggest that stimulation of angiogenic activity is an active participant in the pathological process of experimental colitis.
Stimulation of angiogenesis is complex and dynamic with different mediators influencing neovascularization in a tissue- and disease-specific manner.56,75 It is increasingly accepted that angiogenic activity occurs in both physiological and pathological settings resulting in clear differences in vascular phenotypes.56 As such, differential regulation of the angiogenic response is influenced by the type of mediator, either positive or negative, and the degree of its expression, either up or down.76-78 Here we show that there are a small group of angiogenic mediators that are similarly controlled between the two models of experimental colitis. However, we also found differential regulation of numerous angiogenic genes between the two models, with the majority being down-regulated in DSS colitis and up-regulated in CD4+CD45RBhigh colitis. Importantly, several potent anti-angiogenic or angiostatic genes were significantly down-regulated in the DSS model, including CD36, chromogranin A, thrombospondin 4, collagen type 18 (endostatin precursor), and TIMP-2 versus the CD4+CD45RBhigh model that only included CD36 and chromogranin A. These data suggest that angiogenesis may primarily occur in the DSS model through loss of angiogenic inhibition. Conversely, angiogenesis in the CD4+CD45RBhigh model likely occurs because of dramatic differential up-regulation of proangiogenic mediators (34 genes) compared with anti-angiogenic mediators (three genes). Several explanations could account for the difference in angiogenic gene expression between the two models, with the manner of colitis induction and the type of immune cell involved being obvious possibilities. Together, our findings suggest that interventions aimed at increasing and restoring anti-angiogenic activity versus blockade of proangiogenic mediators may provide additional selective means for treating various forms of IBD.
Protein expression analysis of VEGF-A between the two models confirmed the differential gene array data reinforcing the notion that angiogenic stimulation in the two models is clearly different. VEGF-A is widely regarded as a classic angiogenic cytokine because of its plethoric role in stimulating various endothelial cell responses necessary for angiogenesis.79-84 Moreover, VEGF-A has been reported to be important for both physiological and pathological angiogenesis, indicating that this molecule may be central for multiple angiogenic responses.85 We were surprised to find that VEGF-A expression was significantly enhanced in the CD4+CD45RBhigh T-cell transfer model but not in the DSS model. However, a recent study by Konno and colleagues12 has reported differential VEGF expression in UC versus CD tissue, corroborating our findings. These data reinforce the idea that targeted anti-angiogenic therapy in IBD may be most useful in treating specific classifications of colitis. Additional clinical and experimental investigation is required to better understand such differences and whether they may be exploited for intervention.
Palliative treatment of IBD has historically been accomplished through the use of aminosalicylates, steroids, or antibiotics along with the more recent use of immunomodulators including azathioprine, mercaptopurine, methotrexate, cyclosporine, and TNF- blockade (Remicade). Unfortunately, all of these interventions may be limited in their efficacy and are associated with substantial side effects.5,86,87
Thus, there have been minimal advances in the ability to prevent or cure IBD. Therefore, we examined the effect of anti-angiogenic intervention on experimental colitis. Thalidomide is an anti-angiogenic and immunomodulatory agent that has successfully induced remission of active IBD, but its ability to attenuate experimental colitis and its associated angiogenic activity is not known.8,88,89
The molecular effects of thalidomide are many and include increased endothelial ceramide production that could facilitate apoptosis, decreased endothelial cell Akt activation that diminishes cell motility, differential gene regulation of endothelial cell growth factors and their receptors, and attenuation of TNF- and leukocyte responses, all of which are likely to be involved in the angiogenic response during experimental colitis.68,69,90-92
Thalidomide treatment in the DSS model significantly attenuated disease as observed by a dramatic decrease in pathology score and angiogenic index compared with vehicle control mice. Given the biological mechanisms of thalidomide, these findings further demonstrate an important link between angiogenesis and inflammation during experimental colitis.
We also showed that selective anti-angiogenic inhibition of 5ß1 and Vß3, which were selectively up-regulated in the CD4+CD45RBhigh model, by ATN-161 significantly attenuated experimental colitis, providing evidence that targeting the neovasculature alone clearly protects against tissue damage. Thus, our results provide strong evidence for a pathological angiogenesis paradigm and that therapeutic targeting of angiogenesis may be clinically useful for IBD. However, future studies directly targeting additional angiogenic factors are needed to understand better the key mediators of angiogenesis during experimental colitis.
In summary, these data demonstrate that experimental colitis is characterized by angiogenic inflammation that contributes to the development and sustenance of experimental colitis. Our data provide a compelling argument that increased leukocyte recruitment is required for angiogenic stimulation during colitis. The ability to decrease angiogenic activity by preventing the inflammatory response with the CD18-null mouse model further solidifies the link between the two. Moreover, the angiogenic responses seen in experimental colitis may contribute to chronic inflammation, which likely enables a vicious cycle of disease activity. Indeed, we have recently reported that high concentrations of angiogenic cytokines, such as VEGF-A, increase leukocyte interactions with colon microvascular endothelial cells similar to proinflammatory agents such as TNF-.39
The ability of the anti-angiogenic agents thalidomide and ATN-161 to significantly attenuate experimental colitis demonstrates that anti-angiogenic therapies could provide novel treatments for IBD.
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Acknowledgements |
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Supported by the National Institutes of Health (grant DK 65649) and the Center for Excellence in Arthritis and Rheumatology at Louisiana State University Health Sciences Center–Shreveport.
Accepted for publication August 15, 2006.
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