Here, we examine how external force on platelet endothelial cell adhesion molecule (PECAM)-1 and extracellular matrix cues regulate the mechanical properies of aortic endothelial cells in vitro and in vivo.
Endothelial cells (ECs) lining the walls of the vascular system sense external forces, such as shear stress dues to blood flow and tension due to blood pressure, with mechanosenor proteins such as integrins (Jalali, 2001)(Chachisvilis et al., 2006) and (PECAM)-1 (Tz- ima et al., 2005)(Collins et al., 2012)(Collins et al., 2014). In addition to mechanical signals, ECs also receive cues from the extracellular matrix (ECM). Although most of the vasculature is dominated by collagen (CL) and laminins, distinct regions have con- centrated amounts of fibronectin (FN). In response to force, these variations in ECM composition have been shown to induced ECM-specific intracellular signaling cascades in ECs in different regions of the vascular system. For example, shear stress has been shown to activate protein kinase A (PKA) in ECs adherent to CL, whereas PKA ac- tivation is unaffected in ECs under force when adherent to FN (Funk et al., 2010). Here, we examine how ECM composition and external force on PECAM-1 regulate the mechanical phenotype of aortic ECs.
To examine the role of ECM composition on the stiffness response to force on PECAM-1in vitro, we used magnetic tweezers to apply force to anti-PECAM-1 coated beads which were incubated over cells plated on FN or CL. We find that ECs plated on FN exhibit a stiffness response (indicated by decreased bead displacement) after
approximately 40 sec of pulsatile force (Fig. 4.1 A). In contrast, bead displacement of ECs plated on CL were unaffected after pulsatile force (Fig. 4.1 A). These results indicate that ECM composition plays an integral role in mechanoresponse to force on PECAM-1.
Figure 4.1: ECM composition determines stiffness response to force on PECAM-1. (A) Using magnetic tweezers, a 2 sec, 100 pN pulse of force, followed by a 10 sec period of rest, was applied over a 2 min time course. The average relative displacement (from first pulse) was measured for 2.8µm anti-PECAM-1 coated beads on ECs on either FN or CL (n > 30 per condition from 3 independent experiments; Error is SEM, p < 0.5). (B) Average relative displacement for anti-PECAM-1 coated beads on ECs pretreated with PKI (20µM) or vehicle control for 1 hr (n > 20 per condition from 3 independent experiments; Error is SEM, p < 0.5). Data: Caitlin Collins.
Previously, PECAM-1-mediated stiffening response of ECs on FN was shown to be dependent on phosphoinositide 3-kinase (PI3K) regulation of basal integrin acti- vation and activation of RhoA (Collins et al., 2012). Because RhoA is known to be important for the stiffening response to force in other model systems (Matthews et al.,
Figure 4.2: Experimental setup for en face mechanical characterization of aortic ECs. (A) Schematic showing the aortic arch (rich in FN) and descending aorta (dominated by CL) regions of the tissue. Cartoon from (Collins et al., 2014). (B) The aorta was freshly isolated, cut longitudinally and mounteden face on a glass cover slip with the endothelium facing up. To anchor the aorta to the cover slip for experiments, a thin sheet (20 x 40 x 2 mm) of polydimethylsiloxane (PDMS) rubber was positioned over the tissue. A small section (3 x 5 mm) of the PDMS sheet was removed before anchorage to serve as a media reservoir for the region of interest (here, the descending aorta). 4.5µmFN-coated beads were incubated over the endothelium for 20 min at 37 C.
2006)(Guilluy et al., 2011), we hypothesized RhoA activity may be attenuated when ECs are on CL. Given that RhoA activation is known to negatively regulated by PKA through phosphorylation, we treated ECs on CL with the PKA inhibitor (PKI) and tested for RhoA activation in response to force from a permanent magnet; we found PKI to reverse PKA suppression of RhoA activity (Collins et al., 2014). To test the effect of PKI treatment on stiffness response, we used magnetic tweezers to apply force to anti-PECAM-1 coated beads which were incubated over cells plated on CL. We find that ECs plated on CL exhibit a stiffening response with PKI treatment compared to the vehicle control (Fig. 4.1 B).
Figure 4.3: Imaging through layers of aortic tissue. (A) Adipose cells are found in the outer most layers. (B) Collagen I and IV dominate the ECM composition of the descending aorta, shown here 20µm from the adipose cells. Fibroblast cells exist in this layer. (C) Monolayer of endothelial cells over a thin ECM layer and smooth muscle cells, shown here 70µm from the adipose cells.
To determine the physiological relevance of these findings, we implemented external PBR to determine whether PKA plays a role in defining EC stiffness in vivo. The descending aorta (rich in CL composition) was freshly isolated from control or PKI-
treated mice two hours after injection and prepareden face to expose the endothelium for passive microbead rheology measurements (Fig. 4.2 A,B). The descending aorta was incubated with FN-conjugated beads in order to establish integrin-mediated at- tachments with the cortical actin cytoskeleton. Figure 4.3 shows the monolayer of endothelial cells above a layer of adipose cells and a layer rich in CL. Passive motion of the beads were tracked and the resulting mean-squared displacement (MSD) was calculated for control or PKI-treated aortas (Fig. 4.4 A, B). Because ourin vitro data showed that force on PECAM-1 resulted in a stiffness response on FN, and that cells did not stiffen in response to force on CL, we hypothesized that ECs located in the aortic arch (rich in FN composition) would exhibit increased stiffness compared to ECs in the descending aorta. Thus, we repeated the PBR measurement for ECs in the aortic arch (Fig. 4.4 D). Ensemble-averaging of the bead populations revealed a significant decrease in the MSD of beads on PKI-treated aortas compared to control aortas and towards that of the MSD of beads on the aortic arch (Fig. 4.4 E,F). These data indi- cate that ECs of the FN-rich aortic arch are stiffer than ECs of the CL-rich descending aorta, and that PKA plays a role in defining EC compliance in the descending aorta.
The results of this study suggest that cells integrate external mechanical and bio- chemical cues to modulate their own mechanical propertiesin vivo. Because the FN-rich aortic arch is prone to inflammation and atherosclerosis, and the CL-rich descending aorta is atheroresistant, these results identify PKA as a potential atheroprotective mechanism.
Figure 4.4: The PKA pathway promotes EC compliance in atheroresistant regions of the aorta. (A,B,D) MSD trajectories of FN coated, 4.5µm beads at- tached to (A) the atheroresistant/descending region, (B) the descending region from PKI-treated mice, and (D) the atheroprone/aortic arch region of the aorta. MSDs of individual curves (n > 350 per condition, aggregated from 3 mice, p < 0.0001) are shown in light color and the ensemble average is represented by the dark curve with SEM shown for the indicated timescales. (C)Schematic showing ECM hetergeneity in