Heparan sulfate Ndst1 regulates vascular smooth muscle cell proliferation, vessel size and vascular remodeling

Abstract

Heparan sulfate proteoglycans are abundant molecules in the extracellular matrix and at the cell surface. Heparan sulfate chains are composed of groups of disaccharides whose side chains are modified through a series of enzymatic reactions. Deletion of these enzymes alters heparan sulfate fine structure and leads to changes in cell proliferation and tissue development. The role of heparan sulfate modification has not been explored in the vessel wall. The goal of this study was to test the hypothesis that altering heparan sulfate fine structure would impact vascular smooth muscle cell (VSMC) proliferation, vessel structure, and remodeling in response to injury. A heparan sulfate modifying enzyme, N-deacetylase N-sulfotransferase1 (Ndst1) was deleted in smooth muscle resulting in decreased N- and 2-O sulfation of the heparan sulfate chains. Smooth muscle specific deletion of Ndst1 led to a decrease in proliferating VSMCs and the circumference of the femoral artery in neonatal and adult mice. In response to vascular injury, mice lacking Ndst1 exhibited a significant reduction in lesion formation. Taken together, these data provide new evidence that modification of heparan sulfate fine structure through deletion of Ndst1 is sufficient to decrease VSMC proliferation and alter vascular remodeling.

Figure 1

Characterization of Ndst1 deficient mouse models. (a) Ndst1 transcript abundance in adult femoral arteries of control, SMMHC-cre⁺Ndst1^−/− and SM22α-cre⁺Ndst1^−/− mice (n=5/group) as normalized to HPRT1. The control mice used in this experiment and throughout the study were cre⁻Ndst1^+/+. (b) Western analysis of Ndst1 protein in aorta from (1) SM22α-cre⁺Ndst1^−/−, (2) control, (3) SMMHC-cre⁺Ndst1^−/− and in VSMC from (4) control and (5) SM22α-cre⁺Ndst1^−/−. (c) Heparan sulfate disaccharide analysis of Ndst1 deficient mouse models. Bars represent mean ± SE of percent total sulfated, N-sulfated, 2-O sulfated, and 6-O sulfated disaccharides in aortae from control (n = 6), SMMHC-cre⁺Ndst1^−/− (n=2) and SM22α-cre⁺Ndst1^−/− (n = 3) mice. (d) Schematic representation of a native sulfated heparan sulfate chain and the loss of N-sulfated disaccharides as a result of cre mediated deletion of Ndst1. The heparan sulfate chains are depicted using standard symbol nomenclature. Dashed lines represent the disaccharide units generated by heparinases, which are then separated according to the number and pattern of sulfate groups.

Figure 2

Heart function is not altered in Ndst1 deficient mice (a) Systolic pressure and (b) LVEDP were not significantly different at baseline in SM22α-cre⁺Ndst1^−/− mice (n=4) as compared to control mice (n=4, p=ns). Remaining heart function data is within the text. (d) Medial cell number is significantly reduced in neonatal and adult SM22α-cre⁺Ndst1^−/− mice. Bars represent mean ± SE of SMα-actin positive medial nuclei from sections of neonatal femoral artery from control (n=5) and SM22α-cre⁺Ndst1^−/− (n=4) and adult femoral arteries from control (n=6) and SM22α-cre⁺Ndst1^−/− mice (n=5) (e) Proliferation in neonatal SM22α-cre⁺Ndst1^−/− femoral arteries (n=4) compared to control (n=3). Sections were stained with anti-Ki67 and anti-SMα-actin. Bars represent mean ± SE of Ki67 positive VSMC out of total number of VSMC in femoral arteries from both cohorts.

Figure 3

Loss of Ndst1 decreases intima/media ratio in response to vascular injury in femoral artery in adult mice. (a–c) Representative photomicrographs of femoral arteries 28 days post injury with Verhoeff-Van Gieson staining in control, SMMHC-cre⁺Ndst1^−/− and SM22α-cre⁺Ndst1^−/− mice. Scale bar = 25μm. (d) Histogram depicting morphometry measurements of intima/media ratios from control (n=9), SMMHC-cre⁺Ndst1^−/− (n=9) and SM22α-cre⁺Ndst1^−/− (n=5) femoral arteries 28 days post injury. (e) In vivo assessment of cell proliferation by BrdU incorporation. BrdU incorporation was significantly reduced in SM22α-cre⁺Ndst1^−/− (n=3) as compared to control mice (n=4) 3 days post vascular injury. (f) in vitro inhibition of sulfation decreases proliferation of rat embryonic smooth muscle cells.

Figure 4

(a–c) Representative photomicrographs of femoral arteries 28 days post injury with Masson’s Trichrome staining in control, SMMHC-cre⁺Ndst1^−/− and SM22α-cre⁺Ndst1^−/− mice. Scale bar = 25μm. (d) Ndst1 deficiency leads to reduced collagen content in the intima in response to injury. Bars represent mean ± SE of collagen content in femoral arteries from control (n=6) SMMHC-cre⁺Ndst1^−/− (n=6) and SM22α-cre⁺Ndst1^−/− (n=5) mice at 28 days post injury.