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. 2009 Sep;13(9B):3463-74.
doi: 10.1111/j.1582-4934.2009.00735.x. Epub 2009 Mar 6.

Endothelial glycocalyx dimensions are reduced in growing collateral arteries and modulate leucocyte adhesion in arteriogenesis

Sebastian Grundmann  1 , Stephan H Schirmer, Liesbeth H P Hekking, Jan Andries Post, Mihaela G Ionita, Daphne de Groot, Niels van Royen, Bernard van den Berg, Hans Vink, Martin Moser, Christoph Bode, Dominique de Kleijn, Gerard Pasterkamp, Jan J Piek, Imo E Hoefer
Affiliations

Endothelial glycocalyx dimensions are reduced in growing collateral arteries and modulate leucocyte adhesion in arteriogenesis

Sebastian Grundmann et al. J Cell Mol Med. 2009 Sep.

Abstract

During collateral artery growth, monocytes adhere to the endothelium and secrete cytokines from the perivascular space promoting arteriogenesis. Recently, the endothelial glycocalyx has been shown to modulate leucocyte infiltration in atherogenic regions. The role of this endothelial surface coating in arteriogenesis, however, has not been investigated so far. We now report that local plasma levels of hyaluronic acid are specifically increased in collateral arterial blood of coronary artery disease patients and hypothesized that components of the endothelial glycocalyx are shed during arteriogenesis, resulting in decreased glycocalyx dimensions and an increased leucocyte extravasation. In a rabbit model of femoral artery ligation, electron microscopy revealed a decrease in glycocalyx dimensions in collateral arteries compared with quiescent anastomoses (67.5 +/- 47.2 nm versus 101.0 +/- 11.3 nm; P < 0.001). This decrease was correlated with a higher number of perivascular macrophages around collateral arteries. The additional glycocalyx perturbation by local hyaluronidase infusion almost completely removed the endothelial surface layer and temporarily stimulated leucocyte accumulation in the perivascular space. However, complete perturbation of the glycocalyx by hyaluronidase infusion resulted in a significant attenuation of collateral artery growth assessed by microsphere-based perfusion measurements (ml/min/100 mmHg: hyaluronidase: 27.5 +/- 3.5;

Controls: 47.1 +/- 3.83; P < 0.001) and a lower percentage of actively proliferating vascular smooth muscle cells. A decreased expression of the shear-stress regulated pro-arteriogenic genes eNOS and TGF-beta1 suggests an impaired mechanotransduction as the underlying mechanisms. For the first time, we describe the role of the endothelial glycocalyx in collateral artery growth. Although complete abrogation led to attenuated arteriogenesis, shedding of glycocalyx components is observed during collateral artery growth.

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Figures

Figure 1
Figure 1
Schematic drawing of experimental set-up of collateral flow index measurements and blood sample aspiration during coronary angiography in coronary artery disease patients (A). Blood samples aspirated via the collateral network contained significantly higher levels of hyaluronic acid than samples from the proximal site (B).
Figure 2
Figure 2
Electron microscopic imaging of Alcian blue 8GX stained rabbit arteries from the quadriceps muscle at 39,000× (A, B) and 93,000× magnification (C, D). The glycocalyx on the luminar surface as well as in the endothelial cell caveolae stains dark and is reduced in growing collateral arteries (B, D) compared with quiescent anastomoses from the unoccluded hind limb (A, C).
Figure 3
Figure 3
Quantification of glycocalyx thickness in collateral arteries and quiescent anastomoses. Infusion of inactivated hyaluronidase had not significant effect on the glycocalyx width (B), whereas the active enzyme almost completely removed the endothelial surface layer (A).
Figure 4
Figure 4
Macrophage accumulation around growing collateral arteries from the quadriceps muscle (400× magnification) shown in a double staining for the macrophage marker CD68 (red) and alpha-smooth muscle actin (green). Macrophages were not regularly present in the perivascular space of unrecruited anastomoses (A, B). Following femoral artery ligation, macrophages are accumulating around growing collateral arteries at day 3 after the induction of arteriogenesis, with a significantly higher number of infiltrating cells in animals treated with hyaluronidase (C), compared to animals treated with the inactivated enzyme (D). At day 7, this difference between hyaluronidase treated (E) and control animals (F) was no longer statistically significant (G). Glycocalyx perturbation resulted in a significant reduction of the expression of the shear stress–regulated pro-arteriogenic genes eNOS and TGF-β1 (H).
Figure 5
Figure 5
(A) Haemodynamic assessment of collateral conductance. Microsphere-based perfusion measurements demonstrated a significant reduction in the conductance of collateral arteries at 7 days following femoral artery ligation in the hyaluronidase-treated group compared with the control animals. (B) Proliferation of vascular smooth muscle cells in collateral arteries (400× magnification). Using a double staining for the proliferation marker Ki67 (red) and alpha-smooth muscle actin (green), the percentage of Ki67-positive nuclei of all nuclei (blue) of vascular smooth muscle cells was calculated. Hyaluronidase infusion resulted in a significantly lower number of actively proliferating cells (C) in collateral arteries compared with control animals treated with inactive enzyme (D). Unrecruited anastomoses from the unoccluded hind limb show almost no smooth muscle cell proliferation in both the hyaluronidase (E) and the control group (F).
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