Abstract
Efficient mechanotransduction in vascular smooth muscle cells (VSMCs) is intimately coupled to physical coupling of the cell to extracellular matrix proteins (ECM) by integrins. Integrin adhesion receptors are essential for normal vascular function and defective integrin signaling is associated with cardiovascular disease. However, less is known about the mechanism of integrin activation in VSMCs in relation to vasoregulation. Our laboratory previously demonstrated that the vasoconstrictor Angiotensin II increases VSMC stiffness in concert with enhanced adhesion to fibronectin (FN), indicating an important role for adhesion in contraction. However, the mechanism of this coordination remains to be clarified. In this study, intracellular Ca2+ ([Ca2+]i) was hypothesized to link integrin activation through inside-out signaling pathways leading to enhanced adhesion in response to AII. By using atomic force microscopy (AFM) with an anti-α5 antibody coated AFM probe, we confirmed that cell stiffness was increased by AII, while we observed no change in adhesion to an α5 integrin antibody. This indicated that increases in cell adhesion to FN induced by AII were occurring through an integrin activation process, as increased membrane integrin expression/receptor density would have been accompanied by increased adhesion to the anti-α5 antibody. Further studies were performed using either KCl or BAPTA-AM to modulate the level of [Ca2+]i. After KCl, VSMCs showed a rapid transient increase in cell stiffness as well as cell adhesion to FN, and these two events were synchronized with superimposed transient increases in the level of [Ca2+]i, which was measured using the Ca2+ indicator, fluo-4. These relationships were unaffected in VSMCs pretreated with the myosin light chain kinase inhibitor, ML-7. In contrast, unstimulated VSMCs incubated with an intracellular calcium chelator, BAPTA-AM, showed reduced cell adhesion to FN as well the expected decrease in [Ca2+]i. These data suggest that in VSMCs, integrin activation is linked to signaling events tied to levels of [Ca2+]i while being less dependent on events at the level of contractile protein activation. These findings provide additional evidence to support a role for adhesion in VSMC contraction and suggest that following cell contractile activation, that adhesion may be regulated in tandem with the contractile event.
Highlights
The biomechanical properties of blood vessels are important determinants of cardiovascular function, and changes in these properties are characteristic of many cardiovascular diseases including hypertension, stroke, aneurysms, atherosclerosis and heart disease (Evans et al, 2016)
To determine whether the increased cell adhesion during Angiotensin II (AII) treatment was due to enhanced membrane expression of integrin α5β1, we used atomic force microscopy (AFM) probes coated with anti-α5 antibody, since β1 integrin would be present in other integrin dimers
Average cell stiffness was 6.04 ± 0.26 kPa, and which increased to 7.47 ± 0.21 kPa after AII representing a statistically significant increase by 24% (Figure 1B)
Summary
The biomechanical properties of blood vessels are important determinants of cardiovascular function, and changes in these properties are characteristic of many cardiovascular diseases including hypertension, stroke, aneurysms, atherosclerosis and heart disease (Evans et al, 2016). In addition to the ECM, it has been recently established that vascular smooth muscle cells (VSMCs), as the main cell type within the vascular wall, can exhibit increased stiffness that impacts the stiffness of the blood vessel wall. Cytoskeletal proteins and the contractile state have been associated with cell stiffness in VSMCs (Hong et al, 2012, 2014; Lim et al, 2012) In these studies, VSMC adhesion to ECM proteins was shown to be enhanced in stiffer VSMCs. Fibronectin (FN) is a major ECM protein that interacts with integrin receptors on VSMCs and it is reported to accumulate in diseased artery walls (Pickering et al, 2000). The α5β1 integrin is recognized as a major FN receptor and plays an important role in VSMCs mechanoreception linking it to vascular regulation (Francis et al, 2002; Sun et al, 2005)
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