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. 2015 Sep 18;48(12):3044-51.
doi: 10.1016/j.jbiomech.2015.07.029. Epub 2015 Aug 7.

Vascular smooth muscle cell functional contractility depends on extracellular mechanical properties

Kerianne E Steucke  1 , Paige V Tracy  1 , Eric S Hald  1 , Jennifer L Hall  2 , Patrick W Alford  3
Affiliations

Vascular smooth muscle cell functional contractility depends on extracellular mechanical properties

Kerianne E Steucke et al. J Biomech. .

Abstract

Vascular smooth muscle cells' primary function is to maintain vascular homeostasis through active contraction and relaxation. In diseases such as hypertension and atherosclerosis, this function is inhibited concurrent to changes in the mechanical environment surrounding vascular smooth muscle cells. It is well established that cell function and extracellular mechanics are interconnected; variations in substrate modulus affect cell migration, proliferation, and differentiation. To date, it is unknown how the evolving extracellular mechanical environment of vascular smooth muscle cells affects their contractile function. Here, we have built upon previous vascular muscular thin film technology to develop a variable-modulus vascular muscular thin film that measures vascular tissue functional contractility on substrates with a range of pathological and physiological moduli. Using this modified vascular muscular thin film, we found that vascular smooth muscle cells generated greater stress on substrates with higher moduli compared to substrates with lower moduli. We then measured protein markers typically thought to indicate a contractile phenotype in vascular smooth muscle cells and found that phenotype is unaffected by substrate modulus. These data suggest that mechanical properties of vascular smooth muscle cells' extracellular environment directly influence their functional behavior and do so without inducing phenotype switching.

Keywords: Arterial mechanics; Atherosclerosis; Extracellular matrix; Hypertension; Vascular muscular thin film.

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Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Figures

Figure 1
Figure 1
Variable Modulus vMTF Fabrication.
Figure 2
Figure 2
Variable modulus vascular muscular thin film structure. A. Side view of each layer in the three layer MTF. B. Film release yields a curved beam. The radius of curvature (R) is used to calculate the stress in the tissue layer. Inset: Transmural stress distribution.
Figure 3
Figure 3
Schematic representation of volumetric pseudo-contraction applied to variable modulus vMTF to calculate cell stress. F is the deformation gradient tensor. F* is the elastic deformation tensor. A is the active deformation tensor.
Figure 4
Figure 4
Engineered arterial lamella tissue mimic structural characterization. A. Representative images of engineered arterial lamella tissue mimics on each substrate modulus. Scale bar: 50μm. Green: F-actin Blue: Nuclei B. F-actin and nuclear alignment for each substrate modulus. Mean±SD (n = 3) C. Nuclear eccentricity on each substrate modulus. (n = 3) D. Representative images of focal adhesions on each substrate modulus. Scale bar: 10μm. Orange: Paxillin E. Focal adhesion density on each substrate modulus. Mean±SD (n = 3). F. Focal adhesion size on each substrate modulus. Mean±SD (n = 3).
Figure 5
Figure 5
VSMC functional contractility increases with increasing substrate modulus. A. Bright field images show corresponding time points of variable modulus vMTF film. Red bar indicates film projection length. B. Representative temporal stress curve for 50kPa film. C. Basal tone for varying substrate moduli. Mean±SEM (n = 18, 12, 21, 20, respectively) p<0.05 compared to 1000kPa. D. Induced contractility for varying substrate moduli. Mean±SEM (n = 18, 13, 17, 14 [n = 20, 13, 21, 20], respectively [n for 50nM]). Significant with respect to substrate modulus with matching asterix color (p<0.05).
Figure 6
Figure 6
VSMC phenotype is not dependent on substrate modulus. A. Representative images of smooth muscle alpha-actin in engineered arterial lamella tissue mimics on each substrate. Scale bar: 50μm Green: Smooth muscle alpha-actin Blue: Nuclei B. Representative Western blots of contractile phenotype markers; smoothelin, calponin, and caldesmon for VSMC tissues exposed to either a substrate modulus of 10kPa, 50kPa, 100kPa, or 1000kPa. C. Protein expression quantification of contractile phenotype markers for VSMC tissues exposed to either a substrate modulus of 10kPa, 50kPa, 100kPa, 1000kPa. Fold change is with respect to VSMC tissues exposed to a substrate modulus of 10kPa (n = 5, 10, and 3, respectively). Mean±SEM.
Figure 7
Figure 7
Substrate modulus sensitivity of VSMC contractility is not affected by N-cadherin. A. Representative images of N-cadherin for engineered arterial lamella tissue mimics on each substrate. Scale bar: 10μm. Red: N-cadherin B. 50nM ET-1 induced contraction for engineered arterial lamella tissue mimics with either neutralizing anti-N-cadherin or IgG on 10kPa and 1000kPa modulus constructs (n = 11, 12, 13, and 10 for 10kPa IgG, 10kPa anti-N-cadherin, 1000kPa IgG, and 1000kPa anti-N-cadherin, respectively). Mean±SEM. C. Basal tone for engineered arterial lamella tissue mimics with either neutralizing anti-N-cadherin or IgG on 10kPa and 1000kPa modulus constructs (n = 11, 12, 13, and 10 for 10kPa IgG, 10kPa anti-N-cadherin, 1000kPa IgG, and 1000kPa anti-N-cadherin, respectively). Mean±SEM. * = p<0.05.
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