Peripheral Blood-Derived Mesenchymal Stromal Cells Promote Angiogenesis via Paracrine Stimulation of Vascular Endothelial Growth Factor Secretion in the Equine Model

Abstract

Mesenchymal stromal cells (MSCs) have received much attention as a potential treatment of ischemic diseases, including ischemic tissue injury and cardiac failure. The beneficial effects of MSCs are thought to be mediated by their ability to provide proangiogenic factors, creating a favorable microenvironment that results in neovascularization and tissue regeneration. To study this in more detail and to explore the potential of the horse as a valuable translational model, the objectives of the present study were to examine the presence of angiogenic stimulating factors in the conditioned medium (CM) of peripheral blood-derived equine mesenchymal stromal cells (PB-MSCs) and to study their in vitro effect on angiogenesis-related endothelial cell (EC) behavior, including proliferation and vessel formation. Our salient findings were that CM from PB-MSCs contained significant levels of several proangiogenic factors. Furthermore, we found that CM could induce angiogenesis in equine vascular ECs and confirmed that endothelin-1, insulin growth factor binding protein 2, interleukin-8, and platelet-derived growth factor-AA, but not urokinase-type plasminogen activator, were responsible for this enhanced EC network formation by increasing the expression level of vascular endothelial growth factor-A, an important angiogenesis stimulator.

Keywords: Angiogenesis; Mesenchymal stromal cells; Secretome; Vascular endothelial growth factor.

Figure 1.

Peripheral blood-derived equine mesenchymal stromal cells (MSCs) are positive for MSC markers, negative for differentiated blood cell markers, and capable of undergoing trilineage differentiation. Two-laser flow cytometry was performed with four MSC markers (CD29, CD44, CD90, and CD105) (A) and four negative markers (CD45, CD79α, major histocompatibility complex II, and a monocyte/macrophage marker) (B). Representative histograms illustrate relative numbers of cells versus mean fluorescence intensity. The light and dark gray histograms represent relevant isotype control and marker antibody staining, respectively, with the corresponding mean percentage of positive cells ± SD. (C): Representative microscopic images of alizarin red S, Alcian blue, and Oil Red O staining to confirm osteogenic, chondrogenic, and adipogenic differentiation, respectively. Negative control cells are also presented. Scale bars = 50 μm.

Figure 2.

CM of equine PB-MSCs promotes the proliferation of equine ECs and stimulate endothelial tube-like formation in vitro. (A): A bromodeoxyuridine (BrdU) proliferation assay was performed to evaluate the proliferation activity of ECs after incubation with CM of PB-MSCs (black) or with MSC medium (white). BrdU incorporation was measured by determining the optical density at 450 nm on a Multiskan EX microplate reader using Ascent software (Thermo Scientific). (B): ECs were seeded on an extracellular matrix gel in the presence or absence of CM of PB-MSCs to evaluate the tube-like formation capacity in vitro. After 3 days of culture, ECs were stained with 10 μl of 10× cell-based calcein. JNJ inhibitor was used as a negative control. Fluorescent and bright-field photographs were taken using an Eclipse TE2000-U inverted fluorescence microscope (Nikon). Arrows indicate hollow tube-like structure formation. Scale bars = 100 μm. Abbreviations: CM, conditioned medium; ECs, endothelial cells; PB-MSCs, peripheral blood-derived equine mesenchymal stromal cells.

Figure 3.

CM of equine PB-MSCs contains multiple proangiogenic factors. (A): A human angiogenesis proteome profiler array was used to evaluate the presence of angiogenic factors in the CM of PB-MSCs (black bars indicate untreated; dashed line bars, treated with CoCl₂; white bars, treated with IFNγ; dotted bars, treated with linoleic acid). The average pixel density is proportional to the amount of phosphoprotein bound by each capture antibody and was calculated for each array spot. The dotted line indicates 10,000 pixel density (B). The presence of IL-6 in the CM of PB-MSCs was determined using an equine specific IL-6 enzyme-linked immunosorbent assay. PBMCs were included as a positive control. Absorbance was measured at 450 nm on a Multiskan EX microplate reader using Ascent software (Thermo Fisher Scientific). Abbreviations: CM, conditioned medium; IFNγ, interferon-γ; IGFBP2, insulin growth factor binding protein 2; IL, interleukin; LA, linoleic acid; LLD, lower limit of detection; PBMC, peripheral blood mononuclear cell; PB-MSC, peripheral blood-derived equine mesenchymal stromal cell; PDGF-AA, plasminogen dependent growth factor AA; uPA, urokinase plasminogen activator; VEGF, vascular endothelial growth factor.

Figure 4.

ET1, IL-8, PDGF-AA, and IGFBP2 stimulate endothelial tube-like formation in vitro. (A): Traditional reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate whether vascular endothelial cells (ECs) express receptors for the angiogenic factors detected in the peripheral blood-derived equine mesenchymal stromal cell-derived conditioned medium. RT-PCR products were run on a 1.5% agarose gel. (B): Equine ECs were seeded on an extracellular matrix gel in the presence of different recombinant proteins to evaluate the effect of these proteins on the tube-like formation capacity of ECs in vitro. After 1 day of culture, ECs were stained with 10 μl of 10× cell-based calcein. JNJ inhibitor was used as a negative control. Photographs were taken using an Eclipse TE2000-U inverted fluorescence microscope (Nikon). Arrows indicate hollow tube-like structure formation. Scale bars = 100 μm. Abbreviations: B2M, β2-microglobulin; CXCR2, C-X-C chemokine receptor type 2; EDNRA, endothelin receptor type A; ET1, endothelin-1; IGF1R, insulin-like growth factor 1 receptor; IL, interleukin; PDGFRA, platelet-derived growth factor receptor α; PDGF-AA, platelet-derived growth factor AA; PLAUR, plasminogen activator urokinase receptor; uPA, urokinase plasminogen activator.

Figure 5.

CM of equine PB-MSCs stimulates angiogenesis in vitro through the stimulation of VEGF-A production via the secreted factors ET1, IL-8, PDGF-AA, and IGFBP2. (A): Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate whether recombinant uPA, ET1, IL-8, PDGF-AA, or IGFBP2 could change the expression levels of VEGF-A mRNA in ECs. (B): An equine-specific VEGF enzyme-linked immunosorbent assay was used to determine the levels of VEGF secreted by ECs when exposed to PB-MSC-derived CM. Control medium and EC-derived CM and NBL6-derived CM were included as controls. Absorbance was measured at 490 nm on an Infinite M200 Pro microplate reader using i-control software (Tecan). (C): ECs were seeded on an extracellular matrix gel in the presence of VEGF-A to confirm the positive effect of this recombinant protein on the tube-like formation capacity of ECs in vitro. After 1 day of culture, ECs were stained with 10 μl of 10× cell-based calcein. Arrows indicate hollow tube-like structure formation. Scale bars = 100 μm. (D): Quantitative RT-PCR was performed to evaluate whether CM of PB-MSCs changed the expression levels of VEGFR mRNA in ECs. (E): Western blotting was performed to evaluate whether CM of PB-MSCs changed the expression levels of VEGFR protein in ECs. Lane 1 represents ECs incubated in MSC expansion medium; lane 2, ECs in CM of ECs; lane 3, ECs in CM of NBL-6; and lanes 4-6, ECs in CM of PB-MSCs obtained from 3 different horses. Abbreviations: CM, conditioned medium; EC, endothelial cell; ET1, endothelin-1; IGFBP2, insulin-like growth factor binding protein 2; IL, interleukin; PB-MSC, peripheral blood-derived equine mesenchymal stromal cell; PDGF-AA, platelet-derived growth factor AA; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; uPA, urokinase plasminogen activator.

Figure 6.

Schematic overview depicting how PB-MSC-derived CM stimulates angiogenesis of equine ECs in vitro. The secretome of equine PB-MSCs contains a variety of secreted bioactive factors, including the angiogenic factors ET1, IL-8, PDGF-AA, IGFBP2, and VEGF-A. Once secreted, these paracrine factors will bind to their receptors present on the cellular membrane of ECs. On receptor-ligand binding, ECs will increase the secretion of VEGF-A. This secreted VEGF-A will further enhance angiogenesis through autocrine and paracrine signaling. Abbreviations: CXCR2, C-X-C chemokine receptor type 2; EDNRA, endothelin receptor type A; ET1, endothelin-1; IGF1R, insulin-like growth factor 1 receptor; IL, interleukin; PDGFRA, platelet-derived growth factor receptor α; PDGF-AA, platelet-derived growth factor AA.