Introduction
Ischemia and dysfunctional endothelium are common components of many clinically relevant disease processes in horses, including intestinal disease, laminitis, and chronic wounds in the distal portion of the limbs (ie, distal limb wounds). Endothelial colony-forming cells are a subtype of endothelial progenitor cells that can be isolated from umbilical cord blood or peripheral blood samples.1 These cells have an endothelial phenotype, a high proliferative potential, and the capacity to form vascular networks in vitro and in vivo.1,2 The ability of ECFCs to undergo vasculogenesis makes them attractive for the treatment of horses with ischemic diseases such as laminitis, intestinal disease, and chronic wounds.1–3
Treatment of ischemic disease with ECFCs has yielded promising results in humans and other animals. For example, exogenously administered endothelial progenitor cells improved neovascularization and tissue perfusion in mice with experimentally induced hind limb ischemia.4,5 In humans with diabetic foot disease, treatment with endothelial progenitor cells resulted in greater oxygenation and improved healing relative to that achieved with a control treatment.6
An inflammatory environment, such as that present during ischemic disease, likely influences function of circulating or implanted ECFCs. In humans, conditions characterized by inflammation, including autoimmune diseases, rheumatoid arthritis, and type 2 diabetes, are associated with a high risk of endothelial dysfunction.7 Decreases in functional abilities of circulating endothelial progenitor cells have also been demonstrated in diabetic humans. Endothelial progenitor cells from diabetic humans have impaired proliferation, adhesion, and incorporation into blood vessels.8
Determination of the ways in which ECFCs respond to an inflammatory microenvironment would be key to both understanding the function of ECFCs and developing techniques to enhance their therapeutic potential. An inflammatory microenvironment has been shown to influence stem cell function by modulation of inflammation through interactions with innate and adaptive immune cells.9 These immunomodulatory effects are not constitutive but are induced by inflammatory cytokines within an inflammatory microenvironment.10 Research findings suggest that inflammation is required to induce immunomodulatory effects of mesenchymal stem cells, but inflammation can also have a negative impact on mesenchymal stem cell function.11 An inflammatory microenvironment likely also influences other types of stem cells, such as ECFCs.
The ability of ECFCs to successfully promote neovascularization requires a coordinated sequence of events that includes cell migration, adhesion, and proliferation; vessel lumen formation; and ultimately differentiation into endothelial cells. Failure of any of these steps will result in altered healing.
Limited information is available about the effects of inflammatory cytokines on ECFC function. In vitro treatment of healthy human ECFCs with the inflammatory cytokine tumor necrosis factor-α results in impaired proliferation, migration, and tube formation.12 In contrast, in vitro stimulation of healthy human endothelial progenitor cells with IL-1β causes increased proliferation, migration, and adhesion.13
To the authors’ knowledge, no information is available on the effects of proinflammatory cytokines on ECFC function in horses. Therefore, the objective of the study reported here was to investigate the effects of recombinant equine IL-1β on function of equine ECFCs in vitro. We hypothesized that challenge of equine ECFCs with recombinant equine IL-1β would increase cell migration, tubule formation, adhesion, proliferation, and DiI-Ac-LDL uptake.
Materials and Methods
Animals
Protocols involving animals in this study were approved by the Auburn University Animal Care and Use Committee (protocol No. 2014-2408). An a priori sample size calculation based on the effects of IL-1β on human ECFCs13 was performed, indicating that 3 lines of ECFCs would be sufficient for all experiments. Data suggest that ECFC function and number vary with age, gender, and health status in humans.14–16 Therefore, to reduce variability among horses, 3 healthy geldings of similar ages (14 to 17 years) were selected from the population of horses owned by Auburn University for blood sample collection. Geldings included 2 American Quarter Horses and 1 warmblood horse. Health status was determined by physical examination, CBC, and serum biochemical analysis. Body condition score was 5/9 for all 3 horses.
Isolation, storage, and culture of ECFCs
A jugular or cephalic venous blood sample (60 mL) was obtained from each horse with a 1.5-inch, 16-gauge needle into two 60 mL sterile syringes containing 600 U of heparin each. Samples were placed on ice and immediately transported to the laboratory for processing. The ECFCs were isolated, cultured, and characterized as described elsewhere.17,18 Cells formed tubules in vitro, demonstrated uptake of Di-Ac-LDL, and had positive results of indirect immunofluorescence testing for CD105, von Willebrand factor, and vascular endothelial growth factor receptor-2.17 Cells were cryopreserved in freezing medium consisting of 95% equine serum and 5% dimethyl sulfoxide.
Thawed ECFCs were cultured in collagen-coated tissue culture polystyrene flasks in endothelial cell growth medium containing 10% equine serum and manufacturer-supplied growth factors and antimicrobials.a Standard culture conditions (37°C, 5% CO2, and 95% humidity) were used for all experiments. Unless otherwise stated, cells were detached with trypsin-like dissociation enzyme.b Experiments were performed on ECFCs between passages 2 and 7.
Recombinant equine IL-1β
Recombinant equine IL-1βc was purchased as a 10-μg powder and reconstituted in PBS solution containing 0.1% bovine serum albumin to generate a 1-ng/μL working solution. For the experiments, IL-1β was directly diluted in culture medium.
In vitro experiments
Wound healing—To assess in vitro wound healing, ECFC lines from the 3 horses were individually evaluated for changes in wound healing and migration after exposure to recombinant equine IL-1β at various concentrations for 6 and 24 hours and continuously from seeding throughout the assay (54 hours). Three replicates were performed for each condition. To perform the assay, equine ECFCs were seeded into a collagen-coated 12-well plate at a cell density of 12,500 cells/cm2. The ECFCs from all lines and under all conditions grew consistently to 80% to 100% confluency by 48 hours after seeding at this cell density. Medium containing IL-1β (0, 0.1, 1, and 10 ng/mL) was added at the time of seeding, at 6 hours before confluency, or at 24 hours before confluency. Once cells were confluent at 48 hours, a scratch was created by use of a 200-μL pipette tip in accordance with standard scratch assay protocols.19–21 At the time of the scratch, medium was replaced with regular endothelial cell growth medium without IL-1β for the 6- and 24-hour exposure times and left unchanged for the continuous (54-hour) exposure. For all wound healing assays, images were obtained with an inverted light microscope (10× magnification) at the time of wound creation (0 hours) and 6 hours after wound creation. The underside of the plate was marked to ensure that images were consistently obtained at the same location. The wound area was measured in each image by use of a software program.d Gap closure was calculated as the wound area at 6 hours minus the wound area at 0 hours in accordance with standard scratch assay protocols.20,21 Assay results were reported as a percentage change in gap closure from untreated control cell values and compared among different IL-1β concentrations and exposure times.
Tubule formation—In vitro tubule formation by the 3 lines of ECFCs was evaluated following 6 and 48 hours of cell incubation with recombinant equine IL-1β at various concentrations. To perform this assay, ECFCs were seeded into collagen-coated 25-cm2 tissue culture polystyrene plates at a cell density of 6,000 to 8,000 cells/cm2 and then seeded onto the basement membrane matrixe when they reached 75% to 90% confluency, which was predictably at 48 hours as shown by daily observations of the cells. For the IL-1β challenge, medium containing IL-1β (0, 0.1, 1, and 10 ng/mL) was added 6 or 48 hours prior to seeding onto the basement membrane. Cells from all conditions were seeded at cell densities of 37,500 cells/cm2 into 2 wells of a 96-well tissue culture polystyrene plate containing 75 μL of solubilized basement membrane and regular endothelial growth medium without IL-1β.
Vascular tubule formation was assessed by examination of light microscopy images obtained at 4× magnification at 0, 5, and 24 hours after cells had been seeded onto the basement membrane. Images were evaluated for number of segments, number of branches, number of meshes, number of junctions, tube length, and total mesh area with the aid of a software program.f Two replicates were performed for each condition. The IL-1β challenge results were reported as the percentage change in each parameter from untreated control cell values and compared among different IL-1β concentrations and exposure times.
ECFC adhesion—In vitro adhesion of the 3 lines of ECFCs was evaluated following 6 and 48 hours of cell incubation with recombinant equine IL-1β at various concentrations. To perform the assay, ECFCs were seeded into collagen-coated 25-cm2 plates at a cell density of 6,000 of 8,000 cells/cm2 and were passaged for the adhesion assay when they reached 75% to 90% confluency, which was predictable at 48 hours as shown by daily observations of the cells. For the IL-1β challenge, medium containing IL-1β (0, 0.1, 1, and 10 ng/mL) was added 6 hours prior to passage or 48 hours prior to passage into the new plate for the adhesion assay.
To evaluate adhesion after IL-1β exposure, cells from all conditions were seeded at a cell density of 7,500 cells/cm2 into 3 wells of a new collagen-coated 12-well plate with regular endothelial growth medium and incubated at 37°C, 5% CO2, and 95% humidity. After 60 minutes of incubation, adherent cells were counted with the aid of an inverted microscope in 5 fields in each well at 20× magnification. Three replicates were performed for each condition. Numbers of adherent cells per field of view were averaged for each condition and compared with untreated control cell values.
Cell proliferation and viability—For in vitro assessment of cell proliferation and viability, the effect of recombinant equine IL-1β on proliferation of the 3 lines of ECFCs was examined with an XTT assay.g The XTT is a tetrazolium salt that is reduced by mitochondrial enzymes in live cells to a water-soluble orange formazan product. This product can be measured photometrically to quantify cell viability and proliferation. The ECFCs were seeded at cell densities of 15,625 cells/cm2, 37,500 cells/cm2, and 156,250 cells/cm2 into wells of a collagen-coated 96-well plate with medium containing IL-1β (0, 0.1, 1, and 10 ng/mL). Three replicates were performed for each condition. After 12 hours of incubation of ECFCs with IL-1β, 50 μL of activated XTT solution was added to each well without changing the medium, and cells were incubated for an additional 4 hours.
Formazan production was quantified by spectrophotometry at a wavelength of 455 nm by use of a microplate reader. Background absorption was measured at 650 nm and was subtracted from the absorbance signal to obtain normalized absorbance values, which were compared between concentrations of IL-1β for proliferation or via linear regression for viability.
DiI-Ac-LDL uptake—To evaluate whether IL-1β altered the phenotype of equine ECFCs in vitro, uptake of DiI-Ac-LDL was evaluated in the 3 ECFC lines following cell exposure to recombinant equine IL-1β at various concentrations for 24 hours. A phenotypic property of endothelial progenitor cells is receptor-mediated uptake of acetylated low-density lipoprotein. The DiI-Ac-LDL is taken up by ECFCs, and the probe is detected by flow cytometry. Two replicates were performed for each condition.
To perform the assay, ECFCs were seeded into a collagen coated 12-well plate with IL-1β–containing endothelial cell growth medium at concentrations of 0, 0.1, 1, and 10 ng/mL and at a cell density of 12,500 cells/cm2. Cells were incubated under standard culture conditions for 24 hours to approximately 50% confluence. Cells were then washed with PBS solution and incubated for an additional 4 hours with DiI-Ac-LDLh diluted in regular endothelial growth medium to a concentration of 10 μg/mL. After incubation, cells were washed with PBS solution, enzymatically detachedi from the plate, rinsed twice with HEPES-buffered PBS solution, rinsed with HEPES-buffered 1% bovine serum albumin, and then resuspended in sorting medium. Cells were filtered through a 35-μm mesh filter, placed on ice, and analyzed by flow cytometry.j A total of 15,000 events were collected for each duplicate sample and unstained control sample. Flow cytometric gates were set to select for live cultured cells and to eliminate dead cells and debris.
Statistical analyses
Data were assessed for normality with the D'Agostino Pearson omnibus test, and parametric or nonparametric tests were subsequently used where appropriate. Cell adherence at different IL-1β concentrations was compared with the Kruskal-Wallis test. For wound healing assay results, percentage change in gap closure was compared among exposure times at all concentrations with 2-way ANOVA and the Tukey test. Percentage change in all measures of tubule formation (number of segments, branches, meshes, and junctions; total tubule length; and total mesh area) after IL-1β exposure at all concentrations and exposure times was analyzed with 2-way ANOVA and the Sidak multiple comparison test. Results of the XTT assay were analyzed by use of 2-way ANOVA and linear regression. Statistical softwarek was used for all analyses, and values of P < 0.05 were considered significant.
Results
Wound healing experiment
Continuous challenge of ECFCs with recombinant equine IL-1β for 54 hours resulted in less wound closure than achieved with 6 hours of IL-1β exposure (Figure 1; P = 0.02).
Graph (A) and representative photomicrographs (B through G) showing changes in the area of a scratch-induced in vitro wound for ECFCs derived from peripheral blood samples of 3 horses. A—Mean ± SD percentage change in wound area (ie, gap closure) from that for untreated control cells for ECFCs exposed (exp) to recombinant equine IL-1β at 0.1 ng/mL (black bars), 1 ng/mL (white bars), or 10 ng/mL (gray bars) for 6, 24, or 54 hours (3 replicates/condition). At 48 hours after cell seeding, a scratch was created in all cell cultures with a pipette tip. Gap closure was measured 6 hours after scratch creation. Sets of values with different letters are significantly (P < 0.05) different. B through G—Examples of cells in these conditions immediately (0 hours; B, D, and F) and 6 hours after (C, E, and G) scratch creation. Bar = 100 μm in panels B through G.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Graph (A) and representative photomicrographs (B through G) showing changes in the area of a scratch-induced in vitro wound for ECFCs derived from peripheral blood samples of 3 horses. A—Mean ± SD percentage change in wound area (ie, gap closure) from that for untreated control cells for ECFCs exposed (exp) to recombinant equine IL-1β at 0.1 ng/mL (black bars), 1 ng/mL (white bars), or 10 ng/mL (gray bars) for 6, 24, or 54 hours (3 replicates/condition). At 48 hours after cell seeding, a scratch was created in all cell cultures with a pipette tip. Gap closure was measured 6 hours after scratch creation. Sets of values with different letters are significantly (P < 0.05) different. B through G—Examples of cells in these conditions immediately (0 hours; B, D, and F) and 6 hours after (C, E, and G) scratch creation. Bar = 100 μm in panels B through G.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Graph (A) and representative photomicrographs (B through G) showing changes in the area of a scratch-induced in vitro wound for ECFCs derived from peripheral blood samples of 3 horses. A—Mean ± SD percentage change in wound area (ie, gap closure) from that for untreated control cells for ECFCs exposed (exp) to recombinant equine IL-1β at 0.1 ng/mL (black bars), 1 ng/mL (white bars), or 10 ng/mL (gray bars) for 6, 24, or 54 hours (3 replicates/condition). At 48 hours after cell seeding, a scratch was created in all cell cultures with a pipette tip. Gap closure was measured 6 hours after scratch creation. Sets of values with different letters are significantly (P < 0.05) different. B through G—Examples of cells in these conditions immediately (0 hours; B, D, and F) and 6 hours after (C, E, and G) scratch creation. Bar = 100 μm in panels B through G.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Tubule formation experiment
Unchallenged equine ECFCs from all 3 lines formed vascular tubules by 5 hours of seeding onto the basement membrane and began to regress by 24 hours. Challenge of ECFCs with recombinant equine IL-1β for 48 hours (vs 6 hours) at all concentrations (0.1, 1, and 10 ng/mL) profoundly decreased tubule formation as assessed by the percentage change from untreated control cell values, with significant differences noted in numbers of segments (P < 0.001), branches (P < 0.001), meshes (P < 0.001), and junctions (P < 0.001); tube length (P < 0.001); and mesh area (P = 0.002; Figures 2 and 3). The IL-1β concentration and the interaction between concentration and exposure time did not significantly affect tubule formation in the overall statistical model. Tubule formation was not significantly affected by IL-1β concentration or the interaction of concentration with exposure time as gauged by the percentage change from untreated control cells in numbers of segments (P = 0.54 and P = 0.44, respectively), branches (P = 0.71 and P = 0.11), meshes (P = 0.53 and P = 0.46), and junctions (P = 0.51 and P = 0.38); tube length (P = 0.56 and P = 0.28); and mesh area (P = 0.58 and P = 0.48).
Mean ± SD percentage change in the number of segments (A) and meshes (B) and in total tubule length (C) for 3 lines of equine ECFCs (2 replicates/condition) after 6 and 48 hours of exposure to various concentrations of recombinant equine IL-1β, relative to values for untreated control cells. Cells were seeded onto a basement membrane matrix after exposure to IL-1β or control conditions (untreated cells). Vascular tubule formation was assessed 5 hours later. *Indicated set of values differs significantly (P < 0.05) from the set of values for 6 hours. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Mean ± SD percentage change in the number of segments (A) and meshes (B) and in total tubule length (C) for 3 lines of equine ECFCs (2 replicates/condition) after 6 and 48 hours of exposure to various concentrations of recombinant equine IL-1β, relative to values for untreated control cells. Cells were seeded onto a basement membrane matrix after exposure to IL-1β or control conditions (untreated cells). Vascular tubule formation was assessed 5 hours later. *Indicated set of values differs significantly (P < 0.05) from the set of values for 6 hours. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Mean ± SD percentage change in the number of segments (A) and meshes (B) and in total tubule length (C) for 3 lines of equine ECFCs (2 replicates/condition) after 6 and 48 hours of exposure to various concentrations of recombinant equine IL-1β, relative to values for untreated control cells. Cells were seeded onto a basement membrane matrix after exposure to IL-1β or control conditions (untreated cells). Vascular tubule formation was assessed 5 hours later. *Indicated set of values differs significantly (P < 0.05) from the set of values for 6 hours. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Representative photomicrographs of the ECFCs of Figure 2 showing in vitro vascular tubule formation at 5 hours after seeding onto basement membrane matrix. A and B—Cells exposed (exp) to IL-1β at a concentration of 10 ng/mL for 48 hours (B) had less tubule formation than did cells with no such exposure (A). C and D—No differences in tubule formation were observed after 6 hours of cell exposure to IL-1β at a concentration of 10 ng/mL (D), compared with no such exposure (C). Bar = 200 μm in all panels.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Representative photomicrographs of the ECFCs of Figure 2 showing in vitro vascular tubule formation at 5 hours after seeding onto basement membrane matrix. A and B—Cells exposed (exp) to IL-1β at a concentration of 10 ng/mL for 48 hours (B) had less tubule formation than did cells with no such exposure (A). C and D—No differences in tubule formation were observed after 6 hours of cell exposure to IL-1β at a concentration of 10 ng/mL (D), compared with no such exposure (C). Bar = 200 μm in all panels.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Representative photomicrographs of the ECFCs of Figure 2 showing in vitro vascular tubule formation at 5 hours after seeding onto basement membrane matrix. A and B—Cells exposed (exp) to IL-1β at a concentration of 10 ng/mL for 48 hours (B) had less tubule formation than did cells with no such exposure (A). C and D—No differences in tubule formation were observed after 6 hours of cell exposure to IL-1β at a concentration of 10 ng/mL (D), compared with no such exposure (C). Bar = 200 μm in all panels.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
ECFC adhesion experiment
Treatment of ECFCs with recombinant equine IL-1β had no effect on adherence of the cells to a collagen-coated culture plate at any IL-1β concentration (0.1, 1, and 10 ng/mL; P = 0.10) or exposure time (6 or 48 hours; P = 0.22; Figure 4).
Graph (A) and representative photomicrographs (B and C) showing the number of cells from 3 lines of equine ECFCs adhered to tissue culture polystyrene at various points. A—Cells were exposed (exp) to various concentrations of IL-1β for 6 (black bars) or 48 (gray bars) hours and subcultured to evaluate cell adherence. The mean ± SD number of cells adhered at 1 hour after subculture is shown. No differences in the number of cells adhered at 1 hour were observed at any IL-1β concentration or exposure time. B—Adhered cells (solid arrow) and nonadhered cells (open arrow) at 1 hour for untreated control cells. C—Cells exposed to IL-1β at 10 mg/mL for 48 hours. Bar = 100 μm in panels B and C.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Graph (A) and representative photomicrographs (B and C) showing the number of cells from 3 lines of equine ECFCs adhered to tissue culture polystyrene at various points. A—Cells were exposed (exp) to various concentrations of IL-1β for 6 (black bars) or 48 (gray bars) hours and subcultured to evaluate cell adherence. The mean ± SD number of cells adhered at 1 hour after subculture is shown. No differences in the number of cells adhered at 1 hour were observed at any IL-1β concentration or exposure time. B—Adhered cells (solid arrow) and nonadhered cells (open arrow) at 1 hour for untreated control cells. C—Cells exposed to IL-1β at 10 mg/mL for 48 hours. Bar = 100 μm in panels B and C.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Graph (A) and representative photomicrographs (B and C) showing the number of cells from 3 lines of equine ECFCs adhered to tissue culture polystyrene at various points. A—Cells were exposed (exp) to various concentrations of IL-1β for 6 (black bars) or 48 (gray bars) hours and subcultured to evaluate cell adherence. The mean ± SD number of cells adhered at 1 hour after subculture is shown. No differences in the number of cells adhered at 1 hour were observed at any IL-1β concentration or exposure time. B—Adhered cells (solid arrow) and nonadhered cells (open arrow) at 1 hour for untreated control cells. C—Cells exposed to IL-1β at 10 mg/mL for 48 hours. Bar = 100 μm in panels B and C.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Cell survival and proliferation experiment
Throughout every XTT assay, ECFCs from all 3 lines grew at the expected rate, growth was consistent between lines and with recombinant equine IL-1β challenge, and no differences in the number of dead cells were observed. Cell exposure to increasing concentrations of IL-1β (0.1, 1, and 10 ng/mL) had no significant (P = 0.66) effect on cell proliferation and did not reduce viability (ie, the slope of the linear regression line was positive; P = 0.038; Figure 5).
Mean ± SD absorbance results (450 to 640 nm) for an XTT assay following exposure of 3 lines of equine ECFCs to increasing concentrations of recombinant equine IL-1β (0 ng/mL [black triangles], 0.1 ng/mL [circles], 1 ng/mL [squares], or 10 ng/mL [white triangles]). No effect of IL-1β on cell viability (A) or proliferation (B) was observed.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Mean ± SD absorbance results (450 to 640 nm) for an XTT assay following exposure of 3 lines of equine ECFCs to increasing concentrations of recombinant equine IL-1β (0 ng/mL [black triangles], 0.1 ng/mL [circles], 1 ng/mL [squares], or 10 ng/mL [white triangles]). No effect of IL-1β on cell viability (A) or proliferation (B) was observed.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Mean ± SD absorbance results (450 to 640 nm) for an XTT assay following exposure of 3 lines of equine ECFCs to increasing concentrations of recombinant equine IL-1β (0 ng/mL [black triangles], 0.1 ng/mL [circles], 1 ng/mL [squares], or 10 ng/mL [white triangles]). No effect of IL-1β on cell viability (A) or proliferation (B) was observed.
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
DiI-Ac-LDL uptake experiment
No significant (P = 0.48) difference in DiI-Ac-LDL uptake was observed between equine ECFCs from 1 cell line exposed to recombinant equine IL-1β and untreated control cells at any IL-1β concentration (0.1, 1, and 10 ng/mL). Uptake of DiI-Ac-LDL by all groups of cells was between 98% and 99% (Figure 6).
Histogram overlay of flow cytometric data showing uptake of DiI-Ac-LDL by 3 lines of equine ECFCs following exposure or no exposure to recombinant equine IL-1β at 10 ng/mL for 24 hours. Red lines represent unstained control cells, blue lines represent untreated control cells, and black lines represent cells exposed to IL-1β. The x-axis represents relative fluorescence by the fluorochrome fluorescein isothiocyanate (FITC).
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Histogram overlay of flow cytometric data showing uptake of DiI-Ac-LDL by 3 lines of equine ECFCs following exposure or no exposure to recombinant equine IL-1β at 10 ng/mL for 24 hours. Red lines represent unstained control cells, blue lines represent untreated control cells, and black lines represent cells exposed to IL-1β. The x-axis represents relative fluorescence by the fluorochrome fluorescein isothiocyanate (FITC).
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Histogram overlay of flow cytometric data showing uptake of DiI-Ac-LDL by 3 lines of equine ECFCs following exposure or no exposure to recombinant equine IL-1β at 10 ng/mL for 24 hours. Red lines represent unstained control cells, blue lines represent untreated control cells, and black lines represent cells exposed to IL-1β. The x-axis represents relative fluorescence by the fluorochrome fluorescein isothiocyanate (FITC).
Citation: American Journal of Veterinary Research 82, 4; 10.2460/ajvr.82.4.318
Discussion
To the authors’ knowledge, the present study was the first to investigate the effects of inflammation on function of equine ECFCs or, more specifically, the effects of the proinflammatory cytokine IL-1β on equine ECFC function in vitro. On the basis of the limited available research13 in other species, we hypothesized that challenge of equine ECFCs with recombinant equine IL-1β would increase cell migration, tubule formation, adhesion, and proliferation. Results indicated that exposure to IL-1β for longer times instead had an inhibitory effect on migration of and tubule formation by equine ECFCs. These findings highlighted the importance of chronicity in the pathophysiology of many ischemic disease processes, particularly in chronic distal limb wounds in horses. Protracted inflammation is one of the causes of exuberant granulation tissue in such wounds.
Although distal limb wounds have an abundant vascular supply, these blood vessels are frequently dysfunctional and occluded.22 Indeed, when compared with new microvessels in thoracic wounds of horses, microvessels within exuberant granulation tissue in distal limb wounds were 3.37 times as likely to be occluded in a previous study.22 In humans, a similar fibroproliferative disorder known as hypertrophic scars or keloids exists. Such conditions are characterized by microvascular occlusion due to endothelial hyperplasia or hypertrophy.23 Hypoxia from this occlusion may promote excessive collagen production by fibroblasts and myofibroblasts.24 In another study25 involving distal limb wounds in horses, microvessel occlusion within wounds was associated with endothelial cell hypertrophy. Given the results of the present study, it is possible that some of these changes are due to dysfunctional endothelium secondary to chronic inflammation.
The cellular microenvironment in both healthy and diseased tissue is highly complex. In addition to soluble signals, cell-cell communications, cell-matrix interactions, and mechanical cues all play a role in guiding response. Some cues may only stimulate a response when present along with other cues.26 In the present study, only responses of ECFCs to an individual soluble signal (IL-1β) were evaluated, without accounting for the effect of this cytokine on function in combination with other cues. Additionally, competition for cytokine binding is an important mechanism for maintenance of homeostasis within the immune system.27 This competition likely plays an important role in the regulation of response to injury, with cellular response being dependent on the available cytokine pool. Understanding the dynamics of cytokine production and consumption could be invaluable for understanding cell signaling that governs this response. Although concentrations of IL-1β in medium were not measured at the end of the exposure times within each experiment of the present study, only 1 cell population was studied and cytokine competition is less likely in a monoculture versus coculture environment.27 Therefore, the ECFCs in this study were less likely to have had altered functional capacity related to cytokine consumption.
Decreased migration and tubule formation were demonstrated in equine ECFCs following challenge with recombinant equine IL-1β; however, no mechanism was identified for these changes. Given the results of the XTT proliferation assay, the decreased function observed in the wound healing and tubule formation assays did not appear to have been due to changes in proliferative capacity of the ECFCs. Uptake of DiI-Ac-LDL by equine ECFCs was measured by flow cytometric evaluation of fluorescence, and DiI-Ac-LDL uptake was unchanged by all concentrations of IL-1β, confirming that this aspect of the ECFC phenotype was unchanged by IL-1β. Additional investigation is warranted into the mechanisms of this decreased function. One possible mechanism involves inhibition of endogenous nitric oxide formation. In vitro inhibition of endothelial nitric oxide synthase in bovine aortic endothelial cells results in decreased cell migration but not proliferation.28
The results of the study reported here could have important implications for research involving the treatment of ischemic diseases in horses. Effective in vivo ECFC-based treatments will likely require delivery of functional cells within a supportive scaffold. In addition to promoting successful delivery of cells and mechanical support to diseased tissue, a scaffold can be used to provide physiologic signals to promote wound repair. Our laboratory has already demonstrated effective in vivo delivery of ECFCs encapsulated in poly(ethylene glycol)-fibrinogen hydrogel microspheres into full-thickness distal limb wounds of adult horses.29,30 Understanding how local inflammatory stimuli affect ECFC function will be key in designing local microenvironments for novel therapeutic strategies. Preventing decline in cell function following exposure to proinflammatory cytokines will help optimize cell-based treatments.
The present study demonstrated that recombinant equine IL-1β has a negative impact on equine ECFC migration and tubule formation in vitro. This decreased functional capacity was only evident at longer IL-β1 exposure times. Results indicated that IL-1β likely plays a role in the development of dysfunctional endothelium in horses. The role of IL-1β and potentially other inflammatory cues should be considered when designing microenvironments to optimize the therapeutic potential of ECFCs used to treat ischemic disease.
Acknowledgments
Funded by the Animal Health and Disease Research Funds at Auburn University.
The authors declare that there were no conflicts of interest.
The authors thank Qiao Zhong for technical assistance.
Abbreviations
| ECFC | Endothelial colony-forming cell |
| DiI-Ac-LDL | 1,1′-dioctadecyl-3,3,3′,3′ tetramethylindocarbocyanine perchlorate–labeled acetylated low-density lipoprotein |
| IL | Interleukin |
| XTT | 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide |
Footnotes
EGM-2 with Bullet Kit, Lonza, Visp, Switzerland.
TrypLE, Gibco, Carlsbad, Calif.
IL-1β/IL-1F2, R&D Systems, Minneapolis, Minn.
MRI wound healing tool from Image J, NIH, Bethesda, Md.
BD Matrigel basement membrane matrix, BD Biosciences, Bedford, Mass.
Angiogenesis analyzer for ImageJ, NIH, Bethesda, Md.
XTT cell viability kit, Biotium, Fremont, Calif.
Alexa Fluor 488 AC LDL, Invitrogen, Carlsbad, Calif.
Accumax, Stemcell Technologies, Cambridge, Mass.
BD Accuri C6 Software, BD Biosciences, Brea, Calif.
Prism, version 6, GraphPad Software, San Diego, Calif.
References
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