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Vascular smooth muscle cell glycocalyx mediates shear stress-induced contractile responses via a Rho kinase (ROCK)-myosin light chain phosphatase (MLCP) pathway

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ABSTRACT

The vascular smooth muscle cells (VSMCs) are exposed to interstitial flow induced shear stress that may be sensed by the surface glycocalyx, a surface layer composed primarily of proteoglycans and glycoproteins, to mediate cell contraction during the myogenic response. We, therefore, attempted to elucidate the signal pathway of the glycocalyx mechanotransduction in shear stress regulated SMC contraction. Human umbilical vein SMCs (HUVSMCs) deprived of serum for 3–4 days were exposed to a step increase (0 to 20 dyn/cm2) in shear stress in a parallel plate flow chamber, and reduction in the cell area was quantified as contraction. The expressions of Rho kinase (ROCK) and its downstream signal molecules, the myosin-binding subunit of myosin phosphatase (MYPT) and the myosin light chain 2 (MLC2), were evaluated. Results showed that the exposure of HUVSMCs to shear stress for 30 min induced cell contraction significantly, which was accompanied by ROCK1 up-regulation, re-distribution, as well as MYPT1 and MLC activation. However, these shear induced phenomenon could be completely abolished by heparinase III or Y-27632 pre-treatment. These results indicate shear stress induced VSMC contraction was mediated by cell surface glycocalyx via a ROCK-MLC phosphatase (MLCP) pathway, providing evidence of the glycocalyx mechanotransduction in myogenic response.

No MeSH data available.


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HUVSMCs co-stained with DAPI (nucleus, blue) and ROCK1 antibody (green).Cells were divided into the following 6 groups: control (without treatment and no shear), shear (without treatment but 30-min 20 dyn/cm2 shear stress exposure), Hep.III (0.2 U/ml Hep.III treated and no shear), Hep.III + sh (0.2 U/ml Hep.III treated then exposed to shear), Y-27632 (10 μM Y-27632 treated and no shear), and Y-27632 + sh (10 μM Y-27632 treated then exposed to shear). Scale bar: 50 μm. Arrowheads denote ROCK1 distributed on the cell border.
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f7: HUVSMCs co-stained with DAPI (nucleus, blue) and ROCK1 antibody (green).Cells were divided into the following 6 groups: control (without treatment and no shear), shear (without treatment but 30-min 20 dyn/cm2 shear stress exposure), Hep.III (0.2 U/ml Hep.III treated and no shear), Hep.III + sh (0.2 U/ml Hep.III treated then exposed to shear), Y-27632 (10 μM Y-27632 treated and no shear), and Y-27632 + sh (10 μM Y-27632 treated then exposed to shear). Scale bar: 50 μm. Arrowheads denote ROCK1 distributed on the cell border.

Mentions: Figure 7 illustrates the representative fluorescence images of ROCK1. Under static conditions, ROCK1 uniformly distributed through the whole cell cytoplasm. After exposing HUVSMCs to 20 dyn/cm2 for 30 min, the fluorescence intensity of ROCK1 was increased 2.10 ± 0.10 fold as compared to the static condition (Fig. 8, P < 0.01), and the distribution preferred to concentrating along the cell borders (indicated by arrowheads). Hep.III pre-treatment induced a 1.43 ± 0.09 fold increase of the ROCK1 fluorescence intensity relative to the control group (P > 0.02). On the other hand, Y-27632 pre-treatment induced the ROCK1 fluorescence intensity a little decrease without any statistical significance (Control: 1 ± 0.06 vs. Y-27632: 0.92 ± 0.10, P > 0.05). Interestingly, shear induced ROCK1 re-distribution and fluorescence intensity enhancement were no longer observed in both Hep.III (Hep.III: 1.43 ± 0.09 vs. Hep.III + shear: 1.12 ± 0.07, P > 0.08) and Y-27632 (Y-27632: 0.92 ± 0.10 vs. Y-27632 + shear: 0.90 ± 0.06, P > 0.83) pre-treated cells.


Vascular smooth muscle cell glycocalyx mediates shear stress-induced contractile responses via a Rho kinase (ROCK)-myosin light chain phosphatase (MLCP) pathway
HUVSMCs co-stained with DAPI (nucleus, blue) and ROCK1 antibody (green).Cells were divided into the following 6 groups: control (without treatment and no shear), shear (without treatment but 30-min 20 dyn/cm2 shear stress exposure), Hep.III (0.2 U/ml Hep.III treated and no shear), Hep.III + sh (0.2 U/ml Hep.III treated then exposed to shear), Y-27632 (10 μM Y-27632 treated and no shear), and Y-27632 + sh (10 μM Y-27632 treated then exposed to shear). Scale bar: 50 μm. Arrowheads denote ROCK1 distributed on the cell border.
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Related In: Results  -  Collection

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f7: HUVSMCs co-stained with DAPI (nucleus, blue) and ROCK1 antibody (green).Cells were divided into the following 6 groups: control (without treatment and no shear), shear (without treatment but 30-min 20 dyn/cm2 shear stress exposure), Hep.III (0.2 U/ml Hep.III treated and no shear), Hep.III + sh (0.2 U/ml Hep.III treated then exposed to shear), Y-27632 (10 μM Y-27632 treated and no shear), and Y-27632 + sh (10 μM Y-27632 treated then exposed to shear). Scale bar: 50 μm. Arrowheads denote ROCK1 distributed on the cell border.
Mentions: Figure 7 illustrates the representative fluorescence images of ROCK1. Under static conditions, ROCK1 uniformly distributed through the whole cell cytoplasm. After exposing HUVSMCs to 20 dyn/cm2 for 30 min, the fluorescence intensity of ROCK1 was increased 2.10 ± 0.10 fold as compared to the static condition (Fig. 8, P < 0.01), and the distribution preferred to concentrating along the cell borders (indicated by arrowheads). Hep.III pre-treatment induced a 1.43 ± 0.09 fold increase of the ROCK1 fluorescence intensity relative to the control group (P > 0.02). On the other hand, Y-27632 pre-treatment induced the ROCK1 fluorescence intensity a little decrease without any statistical significance (Control: 1 ± 0.06 vs. Y-27632: 0.92 ± 0.10, P > 0.05). Interestingly, shear induced ROCK1 re-distribution and fluorescence intensity enhancement were no longer observed in both Hep.III (Hep.III: 1.43 ± 0.09 vs. Hep.III + shear: 1.12 ± 0.07, P > 0.08) and Y-27632 (Y-27632: 0.92 ± 0.10 vs. Y-27632 + shear: 0.90 ± 0.06, P > 0.83) pre-treated cells.

View Article: PubMed Central - PubMed

ABSTRACT

The vascular smooth muscle cells (VSMCs) are exposed to interstitial flow induced shear stress that may be sensed by the surface glycocalyx, a surface layer composed primarily of proteoglycans and glycoproteins, to mediate cell contraction during the myogenic response. We, therefore, attempted to elucidate the signal pathway of the glycocalyx mechanotransduction in shear stress regulated SMC contraction. Human umbilical vein SMCs (HUVSMCs) deprived of serum for 3&ndash;4 days were exposed to a step increase (0 to 20&thinsp;dyn/cm2) in shear stress in a parallel plate flow chamber, and reduction in the cell area was quantified as contraction. The expressions of Rho kinase (ROCK) and its downstream signal molecules, the myosin-binding subunit of myosin phosphatase (MYPT) and the myosin light chain 2 (MLC2), were evaluated. Results showed that the exposure of HUVSMCs to shear stress for 30&thinsp;min induced cell contraction significantly, which was accompanied by ROCK1 up-regulation, re-distribution, as well as MYPT1 and MLC activation. However, these shear induced phenomenon could be completely abolished by heparinase III or Y-27632 pre-treatment. These results indicate shear stress induced VSMC contraction was mediated by cell surface glycocalyx via a ROCK-MLC phosphatase (MLCP) pathway, providing evidence of the glycocalyx mechanotransduction in myogenic response.

No MeSH data available.


Related in: MedlinePlus