Nanonet force microscopy for measuring forces in single smooth muscle cells of the human aorta

Abstract

A number of innovative methods exist to measure cell-matrix adhesive forces, but they have yet to accurately describe and quantify the intricate interplay of a cell and its fibrous extracellular matrix (ECM). In cardiovascular pathologies, such as aortic aneurysm, new knowledge on the involvement of cell-matrix forces could lead to elucidation of disease mechanisms. To better understand this dynamics, we measured primary human aortic single smooth muscle cell (SMC) forces using nanonet force microscopy in both inside-out (I-O intrinsic contractility) and outside-in (O-I external perturbation) modes. For SMC populations, we measured the I-O and O-I forces to be 12.9 ± 1.0 and 57.9 ± 2.5 nN, respectively. Exposure of cells to oxidative stress conditions caused a force decrease of 57 and 48% in I-O and O-I modes, respectively, and an increase in migration rate by 2.5-fold. Finally, in O-I mode, we cyclically perturbed cells at constant strain of varying duration to simulate in vivo conditions of the cardiac cycle and found that I-O forces decrease with increasing duration and O-I forces decreased by half at shorter cycle times. Thus our findings highlight the need to study forces exerted and felt by cells simultaneously to comprehensively understand force modulation in cardiovascular disease. INTRODUCTIONSmooth muscle cells (SMCs) receive mechanical and chemical stimuli from the extracellular matrix (ECM) via integrin-mediated focal adhesions (Moiseeva, 2001). For a vascular SMC, this interaction plays an important role in modulating vascular resistance and tone, thereby affecting the resistance of a vessel. SMCs generate forces via actomyosin contractions, which impart a mechanical force on the surrounding ECM (Gunst and Zhang, 2008). This leads to vasoconstriction or dilatation of vessels, affecting overall systemic vascular resistance. Furthermore, in the arterial system, particularly in the aorta, there is an ECM-directed force generated by contraction in the cardiac cycle, which is experienced by the SMCs. The pulsatility causes the collagen and elastin microarchitecture to stretch, and the resulting stretch force is transmitted through the focal adhesions to the cytoskeletal network.Establishing a contextually relevant fibrous platform to understand cell-generated (inside-out [I-O]) and ECM-generated (outsidein [O-I]) forces is integral to the study of disease states. At the tissue level, for example, characteristic histopathological features defining the pathophysiology of ascending thoracic aortic aneurysms include degeneration of the elastin matrix, noninflammatory loss of SMCs, and biomechanical weakening of the aortic wall (Nataatmadja et al., 2003). In this regard, our previous work revealed distinctions between the aneurysmal patient cohorts and nonaneurysmal, healthy aortic specimens at the cell, ECM, and tissue levels. Specifically, aneurysmal aortic specimens exhibited higher tensile strength, lower delamination strength, and anisotropic collagen and elastin fiber microarchitecture, and SMC...

Traction Force Measurements of Human Aortic Smooth Muscle Cells Reveal a Motor-Clutch Behavior

“…-and characterized only biological markers of SMC contractility such as Alpha Smooth Muscle Actin (α-SMA), myosin light chain, or calcium entry and release [20,23,39,46,47]. The only study ever published to our best knowledge on mechanical quantification of contractility in human primary aortic SMCs is by Hall et al [48].…”

Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds

“…Cells pull directionally, and these forces are then transmitted back to the substrate by focal adhesions (41). Because cells are able to sense and respond to changes in fiber curvature and structural stiffness (45), deflection of these fibers can be converted to a force measurement (18,42,44,49). Our results showed that, even in the presence of energetic inhibition, the cells were able to sustain a similar amount of force, suggesting that tonic C2C12 force production is a low-energy turnover process, perhaps analogous to the "latch state" observed in other muscle types.…”

Review of the Essential Roles of SMCs in ATAA Biomechanics

“…XX) secreting vasoactive agonists, neurotransmitters and GaGs, notably the Heparan Sulfate, which seems to influence the quiescence of the SMCs [91,92]. Further information about this topic can be found in [ [87] or Traction Force Microscopy (TFM) techniques, from common substrate deformation methods [63,76,94] to uncommon specific microdevices [95][96][97]. SMC stiffness is closely linked to their contractile state [84,98].…”