Upregulation of vascular endothelial growth factor receptor-3 in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis

Abstract

We investigated the spatiotemporal expression of vascular endothelial growth factor receptor-3 (VEGFR-3) in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. VEGFR-3 mRNA and protein were constitutively expressed in gray matter neurons and in a few white matter astrocytes. Induction of VEGFR-3 occurred predominantly in perivascular infiltrated macrophages in the spinal cord white matter during the inductive phase of EAE. VEGFR-3 expression was also induced in activated microglial cells in the gray and white matter, mainly in the peak phase. In addition, reactive astrocytes in the white matter, but not in the gray matter, expressed VEGFR-3 as disease severity increased. These data suggest that VEGFR-3 is involved in the recruitment of monocytic macrophages and in glial reactions during EAE.

Figure 1.

Quantitative real-time reverse transcriptase–polymerase chain reaction for (A) vascular endothelial growth factor–C (VEGF-C) and (B) vascular endothelial growth factor receptor–3 (VEGFR-3) in the lumbar spinal cord white matter of complete Freund’s adjuvant (CFA) controls and experimental autoimmune encephalomyelitis (EAE)–affected rats. The expression level of VEGF-C and VEGFR-3 was calculated using the comparative threshold cycle method (2^−ΔΔCt) with GAPDH as the control gene. The expression level of VEGF-C and VEGFR-3 in the CFA control was set to 1. Both VEGF-C and VEGFR-3 expression increased and reached a peak during the peak stage of EAE and then declined at the recovery stage. *p<0.05 compared with CFA controls.

Figure 2.

Spatial and temporal expression of vascular endothelial growth factor receptor–3 (VEGFR-3) mRNA (A–H) and protein (I–N) in the lumbar spinal cords of control and experimental autoimmune encephalomyelitis (EAE)–affected rats. (A, E) In control sections, hybridization signals for VEGFR-3 were observed in gray matter neurons and some scattered cells in the white matter. Upregulation of VEGFR-3 mRNA expression was induced in the spinal cords of EAE-affected rats from the onset stage (B, F) and significantly increased at the peak stage (C, G). (D, H) During the recovery stage, expression of VEGFR-3 mRNA was decreased but still more evident than that in the controls. (E–H) Higher magnification views of the boxed areas in A–D, respectively. Changes in VEGFR-3 immunoreactivity in the spinal cord of controls (I, M) and EAE rats at onset (J), peak (K, N), and recovery stages (L) closely matched those of VEGFR-3 mRNA expression. (M, N) Higher magnification views of the boxed areas in I and K, respectively. (O) Representative results of immunoblot analysis of VEGFR-3 expression showing that VEGFR-3, detected at 125 kDa and 170 kDa, in rat spinal cord seemed to increase at the peak stage of EAE and then declined, but remained at a high level, at the recovery stage compared with normal controls. Scale bars = 500 µm for A–D, I–L; 200 µm for E–H, M, N.

Figure 3.

Spatiotemporal relationship of vascular endothelial growth factor receptor–3 (VEGFR-3) mRNA and protein in the lumbar spinal cords of experimental autoimmune encephalomyelitis (EAE)–affected rats at onset (A–H) and peak stages (I–P). Note that VEGFR-3 mRNA and protein are generally co-localized in the same population of neurons and glia-like cells located in the gray and white matter in the spinal cords of EAE rats. Also note that rounded cells resembling brain macrophages adjacent to rat endothelial cell antigen–1 (RECA-1)–positive blood vessels expressed both VEGFR-3 mRNA and protein in EAE rats. (B–D, F–H, J–L, N–P) Higher magnification views of the boxed areas in A, E, I, and M, respectively. Scale bars = 200 µm for A, I; 100 µm for E, M; 50 µm for B–D, F–H, J–L, N–P.

Figure 4.

Identification of phenotypes of vascular endothelial growth factor receptor–3 (VEGFR-3)–expressing cells in the lumbar spinal cords of normal controls. (A–C) Low-magnification views of control spinal cord showing triple labeling for VEGFR-3, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba1). (D–F) Higher magnification views of the white matter of control spinal cord. Note that a subset of astrocytes in the white matter (arrows), especially near the surface of the spinal cord, showed VEGFR-3 expression, whereas Iba1-positive microglia with ramified morphology (arrowheads) were devoid of specific VEGFR-3 expression. (G–L) Higher magnification views of the gray matter of control spinal cord. (G–I) Note that neither astrocytes with thin astroglial processes (arrows) nor microglia with ramified morphology (arrowheads) showed VEGFR-3 expression. (J–L) Also note that intense labeling for VEGFR-3 was observed in gray matter neurons showing the neuronal cell marker NSE (neuron-specific enolase). Scale bars = 200 µm for A–C; 50 µm for D–L.

Figure 5.

Identification of phenotypes of vascular endothelial growth factor receptor–3 (VEGFR-3)–expressing cells in the lumbar spinal cords at the onset stage of experimental autoimmune encephalomyelitis (EAE)–affected rats. (A–H) Triple labeling with VEGFR-3, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba1) showing that virtually all of the perivascular cells expressing VEGFR-3 in the white matter were indeed Iba1-immunoreactive microglia/macrophages. Note that some astrocytes (arrows in E–H) showed VEGFR-3 expression. (E–H) Higher magnification views of the boxed areas in A–D, respectively. (I–P) Triple labeling with VEGFR-3, Iba1, and ED1 showing that VEGFR-3 expression was localized in Iba1/ED1 double-labeled cells. Note that most Iba1⁺/ED1⁻ cells were devoid of specific labeling for VEGFR-3. (M–P) Higher magnification views of the boxed areas in I–L, respectively. Scale bars = 200 µm for A–D; 50 µm for E–L; 20 µm for M–P.

Figure 6.

Identification of phenotypes of vascular endothelial growth factor receptor–3 (VEGFR-3)–expressing cells in the lumbar spinal cords at the onset stage of experimental autoimmune encephalomyelitis (EAE)–affected rats. (A–F) Triple labeling with VEGFR-3, ED1, and laminin showing that ED1-positve macrophages expressing VEGFR-3 (asterisks in E and F) were often associated with laminin-positive vascular profiles. (E, F) Higher magnification views of the boxed areas in A–C, respectively. (G–J) Double labeling using VEGFR-3 and CD45 showing that nearly all of CD45-positive cells coexpressed VEGFR-3. (H–J) Higher magnification views of the boxed area in G. Scale bars = 100 µm for A–D, G; 50 µm for H–J; 20 µm for E, F.

Figure 7.

Identification of phenotypes of vascular endothelial growth factor receptor–3 (VEGFR-3)–expressing cells in the lumbar spinal cords at the peak stage of experimental autoimmune encephalomyelitis (EAE)–affected rats. (A–C) Low-magnification views of EAE-affected spinal cords showing triple labeling for VEGFR-3, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba1). (D–E) Higher magnification views of the white matter. (F) Higher magnification view of the boxed areas in D and E. Three-dimensional confocal analysis demonstrated that hybridization signals for VEGFR-3 were observed in reactive astrocytes (arrow) and microglia/macrophages (arrowhead). (G–I) Higher magnification views of the boxed areas in the gray matter in A–C, respectively. Note that intense labeling for VEGFR-3 was observed in neurons (asterisks in G) and activated stellate microglial cells with thick and short processes (arrowheads) but not in reactive astrocytes (arrows). Scale bars = 200 µm for A–C; 50 µm for D, E, G–I; 20 µm for F.