Dual Role of Thrombospondin-1 in Flow-Induced Remodeling

Abstract

(1) Background: Chronic increases in blood flow, as in cardiovascular diseases, induce outward arterial remodeling. Thrombospondin-1 (TSP-1) is known to interact with matrix proteins and immune cell-surface receptors, but its contribution to flow-mediated remodeling in the microcirculation remains unknown. (2) Methods: Mesenteric arteries were ligated in vivo to generate high- (HF) and normal-flow (NF) arteries in wild-type (WT) and TSP-1-deleted mice (TSP-1^-/-). After 7 days, arteries were isolated and studied ex vivo. (3) Results: Chronic increases in blood flow induced outward remodeling in WT mice (increasing diameter from 221 ± 10 to 280 ± 10 µm with 75 mmHg intraluminal pressure) without significant effect in TSP-1^-/- (296 ± 18 to 303 ± 14 µm), neutropenic or adoptive bone marrow transfer mice. Four days after ligature, pro inflammatory gene expression levels (CD68, Cox2, Gp91phox, p47phox and p22phox) increased in WT HF arteries but not in TSP-1^-/- mice. Perivascular neutrophil accumulation at day 4 was significantly lower in TSP-1^-/- than in WT mice. (4) Conclusions: TSP-1 origin is important; indeed, circulating TSP-1 participates in vasodilation, whereas both circulating and tissue TSP-1 are involved in arterial wall thickness and diameter expansion.

Keywords: blood flow; immune cells; remodeling; resistance arteries; thrombospondin-1.

Figure 2

Remodeling study after seven days of chronic increase in blood flow and BMC transfer experiments. Graphs show (a) the passive diameter of HF mesenteric arteries in percent of NF mesenteric arteries diameter, (b) histomorphometry analysis of these arteries as media-to-lumen ratio. TSP-1^−/− BMC in WT, n = 9 and WT BMC in TSP-1^−/−, n = 9. The mean fluorescence intensity (MFI) of the peri arterial zone representing the total immune cells (c), the macrophages (d) and the neutrophils (e) is measured after four days of ligature in TSP-1^−/− BMC in WT (n = 5) and in WT BMC in TSP-1^−/− (n = 7). TSP-1^−/− BMC in WT arteries are represented in black and the WT BMC in TSP-1^−/− in white. All results are expressed in means ± sem. * p < 0.05, ** p < 0.01, TSP-1^−/− BMC in WT vs. WT BMC in TSP-1^−/−.

Figure 1

Remodeling of TSP-1^−/− and WT mesenteric arteries after seven days of chronic increase in blood flow. Graphs show (a) the passive diameter of HF arteries in percent of NF arteries and (b) the histomorphometry analysis as media-to-lumen ratio. TSP-1^−/− (n = 7) mesenteric arteries are represented in white and WT (n = 5) in black. (c) shows representative images (×10 magnification, scale bar = 100 µm). All results are expressed in means ± sem. * p < 0.05, WT vs. TSP-1^−/−.

Figure 3

Remodeling study after seven days of chronic increase in blood flow and hydralazine treatment. Graphs show (a) the passive diameter of the HF mesenteric arteries in percentage of the NF mesenteric arteries diameter, (b) histomorphometry analysis of these mesenteric arteries as media-to-lumen ratio. (c) shows representative images (×10 magnification, scale bar = 100 µm). TSP-1^−/− (n = 10) mesenteric arteries are represented in white and WT (n = 6) in black. All results are expressed in means ± sem. * p < 0.05, *** p < 0.001, WT vs. TSP-1^−/−.

Figure 4

Immune cells recruitment in WT and TSP-1^−/− mesenteric arteries after four days of chronic increase in blood flow. Immune cells recruitment study with flow cytometry experiment (WT n = 2, TSP-1^−/− n = 6) results showing neutrophils (a) and monocyte and macrophages (b) proportion. Mean fluorescence intensity (MFI) quantification of neutrophils (c) and macrophages (d). (e) Representative pictures of mesenteric arteries section immunostained with anti F4/80-PE, anti Ly6G-PE, anti CD31-FITC antibodies and DAPI, magnification ×10, scale bar = 100 µm (WT n = 7, TSP-1^−/− n = 7). In all results, WT are represented in black and TSP-1^−/− in white. All results are expressed in means ± sem. * p < 0.05, WT vs. TSP-1^−/−.

Figure 5

Remodeling study after seven days of chronic increase in blood flow and neutrophils depletion. Graphs show (a) the passive diameter of HF mesenteric arteries is percent of NF mesenteric arteries diameter and (b) the histomorphometry analysis of these mesenteric arteries as media-to-lumen ratio. (c) shows representative mesenteric arteries images (×10 magnification, scale bar = 100 µm). Neutrophil depleted mice (n = 8) are represented in white and isotype controls (n = 5) in black. (d) shows mesenteric arterial zone MFI measured of cleaved caspase-3 after four days of ligature in mesenteric arteries neutrophils depleted mice (n = 6) and isotype controls mice (n = 4). All results are expressed in means ± sem. * p < 0.05, ** p < 0.01 isotype controls vs. neutrophil depletion. ^## p < 0.01 NF vs. HF.

Figure 6

Gene expression study after qRT PCR experiments performed on WT and KO mesenteric arteries after 4 days of ligature. WT arteries (n = 6) are represented in black and TSP-1^−/− arteries (n = 5) in white. The genes studied are cd68, ptgs1, ptgs2, cybb, cyba, ncf1. Results are normalized to the hprt housekeeping gene and expressed as relative level of expression in means ± sem. * p < 0.05, WT vs. TSP-1^−/−, ^# p < 0.05, NF vs. HF.

Figure 7

Schema of TSP-1 implication in mesenteric resistance arteries remodeling induced by a chronic increase in blood flow.