Cell-cell junctions are not required for shear-induced endothelial alignment
Due to the importance of endothelial mechano-responses in the arterial circulation, we used primarily an immortalized line of human aortic endothelial cells (telo-HAEC, or tHAEC) for our analyses. To investigate the mechanisms underlying their responses to shear, we examined the behavior of cells grown in collagen-coated microfluidic chambers and subjected to a constant shear stress of 15 dynes/cm2, mimicking conditions encountered in portions of the arterial circulation (Doucette et al., 1992). Using this system, we evaluated changes in endothelial morphology in response to applied shear by fixing and staining the cells with phalloidin to visualize F-actin (Fig. 1A). Consistent with previous studies, if grown under static conditions, endothelial cells in confluent monolayers had actin stress fibers that were oriented randomly (Fig. 1A). After brief (30 min) exposure of the monolayer to flow, the cells elongated in the direction of the applied stress, which was coincident with the parallel orientation of their actin stress fibers (Fig. 1A). We developed an analysis pathway to quantify the degree of actin stress fiber alignment from these images by first segmenting individual fibers using a Hessian-based multiscale filter to identify tubular structures, or “filaments”. The angle between the longest axis (the maximum Feret diameter) of each segment and the direction of applied flow was then calculated (Fig. 1A). The interquartile range of the distribution of segmented filament orientations was used to quantify the variability between conditions and revealed significantly less variation in the F-actin orientations – which we interpreted as alignment – in cells exposed to shear when compared with those in static culture (Fig. 1A).
Intriguingly, the equivalent treatment of sub-confluent endothelial cultures revealed that isolated cells lacking significant intercellular contacts retained the ability to elongate and align following exposure to shear stress, mimicking the behavior observed for endothelial monolayers (Fig. 1A). This occurred despite the internalization of PECAM-1 from the surface of single cells (Fig. 1B). Sequestration of these proteins into intracellular compartments is expected to preclude their mechanosensory functions. These observations therefore imply that the junctional mechanosensory complex plays a dispensable role in the alignment response of endothelial cells to shear. It was likely that other mechanosensors mediated endothelial alignment in response to shear.
Junction-independent responses to shear require hyaluronic acid
We considered the contributions of other proposed mechano-sensors to junction-independent responses to shear. We proceeded to examine the role of HA in mediating shear-induced F-actin alignment and Ca2+ signaling. Endothelial cells respond to acute changes in their mechanical environment with rapid increases in their cytosolic concentration of free calcium ([Ca2+]cyto) (Buga et al., 1991; Shen et al., 1992), which are in fact critical in orchestrating cellular alignment and NO production by the endothelium (Buga et al., 1991; Lückhoff et al., 1988). During these signaling events, [Ca2+]cyto increases proportionally to the magnitude of the applied force (Hong et al., 2006; Uematsu et al., 1995), thus serving as a clear indicator of shear sensing.
We first confirmed that the enzymatic degradation of surface HA with hyaluronidase (HAase) resulted in the loss of shear-induced F-actin alignment of endothelial cell monolayers (Fig. 1C). To examine the mechanisms governing the elevation of endothelial [Ca2+]cyto in response to shear, we monitored changes in the fluorescence of the genetically-encoded cytosolic Ca2+ indicator, GCaMP6s in response to various stimuli. Consistent with previous studies (Mochizuki et al., 2003), the application of flow-induced shear stress to otherwise untreated (control) cells was associated with a rapid, pronounced increase in cytosolic [Ca2+] (indicated by an increase in GCaMP6s fluorescence) that returned to near-baseline levels within minutes despite continuous exposure to fluid flow (Fig. 1D). Remarkably, pretreating the cells with HAase virtually eliminated the shear-induced [Ca2+]cyto transient (Fig. 1D).
To investigate whether HA also regulates shear-sensing in isolated endothelial cells, we first evaluated whether HA was retained at the surface of cells lacking intercellular contacts. We visualized the distribution of HA on the endothelial surface using a biotinylated HA-binding complex (HABC) labelled with fluorophore-conjugated streptavidin (Fig. 1E, 1F). Analysis of live, subconfluent cells revealed extensive regions with dense HABC staining (Fig. 1E). Furthermore, as in confluent cells, HABC bound predominantly to the apical surface of isolated endothelial cells (Fig. 1F). HA was thus localized to the surface of endothelial cells that most directly experiences shear. Indeed, HAase treatment eliminated the shear-induced alignment of sparsely distributed cells to the same extent as it inhibited the alignment of confluent monolayers (Fig. 1C,G). The isolated cells also required HA for the application of shear stress to elicit a [Ca2+]cyto elevation (Fig. 1H). We thus concluded that in addition to its previously described roles in confluent endothelial tissues, HA is able to mediate junction-independent cellular responses to fluid flow. It can therefore be considered a critical regulator of vascular responses to shear stress.
CD44, the primary HA receptor, regulates shear-induced alignment in vitro
When exerted on the glycocalyx, forces like fluid shear are expected to rapidly distribute throughout this gel-like substance (Weinbaum et al., 2007). Core glycoproteins and glycosaminoglycans that are embedded in this layer are displaced by this force and can then, in theory, deliver information about the direction and magnitude of the applied stress to other membrane-associated proteins (Davies, 1995). Because HA is not directly attached to the plasma membrane, the previous observations implied that receptors connecting the glycopolymer to the endothelium may be important in relaying the mechanical signal. We were particularly interested in CD44, the primary receptor for HA in most tissues (Aruffo et al., 1990). By immunofluorescence, we observed that CD44 accumulated in the apical plasma membrane of sparsely distributed endothelial cells (Fig. 2A), as found earlier in confluent monolayers. It was therefore plausible that it may help establish and maintain a sufficient density of HA on the surface of isolated cells (Fig. 1E, 1F). Accordingly, genetic depletion of CD44 expression by RNA interference (S. Figure 1) was associated with defective responses to shear (Fig. 2B). This indicated that CD44 was required for normal shear responses in vitro. More broadly, it appeared that proteins that bridge glycocalyx components to the endothelial surface play an important role in its mechanosensory behavior.
Spectrin is required to maintain HA on the luminal endothelium
It remained unclear how transmembrane glycoproteins like CD44 activate downstream signaling cascades. We had previously observed that the apical retention and immobilization of CD44 are regulated by the spectrin cytoskeleton (Mylvaganam et al., 2020), which is predominantly localized to the apical surface in endothelial cells (Fig. 2C). As an extensive cytoskeletal network that spans the apical plasma membrane (Fig. 2C), we considered the possibility that spectrin might facilitate communication between the glycocalyx and other plasmalemmal molecules.
We investigated the potential relationship between HA and spectrin by studying whether HA abundance is normal on the surface of b-spectrin-knockdown endothelial cells (Fig. 2D). As noted, α/β spectrin heterodimers are the functional units of the spectrin cytoskeleton and depleting either subunit is therefore an effective strategy for preventing the assembly of these networks. Wild-type endothelial cells grown in culture had appreciable surface HA that was detectable by enzyme-linked immunosorbent assay (ELISA) and was virtually eliminated by HAase (Fig. 2E). Importantly, spectrin-depletion by RNA interference was associated with a pronounced decrease in the surface density of HA (Fig. 2E). These experiments thus provided suggestive evidence that spectrin may regulate endothelial functions associated with HA.
Spectrin is required for normal endothelial responses to shear
To perform a systematic analysis of the role of spectrin in mediating endothelial responses to shear, we edited the tHAEC cell line to effectively knock out bII-spectrin using CRISPR-Cas9 (Fig. 3A). bII-spectrin-knockout (KO) endothelial cells did not exhibit any overt morphological defects and the distribution of junctional-complex proteins to intercellular contacts in confluent monolayers was comparable to that observed in wild-type cells (Fig. 3B). Ratios of VE-cadherin fluorescence intensity in the junctional membrane to that in the cytosol were also similar between the KO clones and the parental wild-type cells, indicating that the loss of bII-spectrin was not associated with overt defects in junctional integrity (S. Figure 2A).
Remarkably, following treatment with fluid flow for 30 min, stress fiber alignment failed to occur in bII-spectrin-KO cells (Fig. 3C, 3D). Instead, the architecture of F-actin networks in these cells more closely resembled that observed in wild-type cells in static culture (Fig. 3C, 3D). Moreover, in addition to alignment, shear is also known to induce the elongation of endothelial cells along the flow axis (Dewey et al., 1981). Changes in cell shape were assessed by immunostaining the junctional protein VE-cadherin in cells subjected to static or shear-stress conditions. Cellular aspect ratio –the ratio of the long axis to the short axis of each cell– was then analyzed from these images. As expected, aspect ratios of wild-type tHAEC monolayers following exposure to shear stress were significantly higher than those of cells maintained in static conditions, indicating a more elongated phenotype (Fig. 3E). Aspect ratios of bII-spectrin-KO cells exposed to shear stress were comparatively lower, indicating a failure to elongate (Fig. 3E). Furthermore, no appreciable changes in [Ca2+]cyto were detected in bII-spectrin-KO cells in response to shear (Fig. 3F, 3G). On the other hand, shear-induced increases in tyrosine phosphorylation of junctional proteins were comparable between wild-type and bII-spectrin-KO cells (S. Figure 2B, 2C). We concluded that spectrin was required for shear-induced Ca2+ responses and the associated changes in endothelial morphology, independently of junctional complex proteins.
Piezo1 activation is regulated by spectrin
The shear-induced elevation of [Ca2+ ]cyto in endothelial cells requires extracellular Ca2+ (Schwarz et al., 1992; Shen et al., 1992), implicating channels localized to the plasma membrane that are directly or indirectly mechanosensitive. Multiple shear-sensitive Ca2+ channels have been proposed to participate. Shear triggers the release of ATP, which then acts in an autocrine manner to induce Ca2+ entry. P2X4 purinoceptors, that are ATP-operated cation channels (Yamamoto et al., 2000), and P2Y receptors, a family of nucleotide-activated G-protein coupled receptors (Wang et al., 2015), have both been implicated in endothelial mechanotransduction. However, we found that ATP-induced Ca2+ transients in bII-spectrin-KO cells were comparable to those observed in wild-type cells (Fig. 4A). This indicated that the defective responses to shear in spectrin-deficient cells were not attributable to impaired purinergic receptor activity.
Notably, however, responses to other mechanical stimuli were also impaired in bII-spectrin-KO cells (Fig. 4B, 4C). Increasing plasma membrane tension by inducing osmotic swelling applying hypotonic stress resulted in a significant increase in [Ca2+]cyto in wild-type but not in bII-spectrin-KO cells (Fig. 4B, 4C). Importantly, swelling-induced [Ca2+]cyto was regulated by stretch-activated ion channels since pre-treatment with the small peptide GsMTx4, an amphipathic peptide that inhibits cationic mechanosensitive currents (Bae et al., 2011), eliminated this response (S. Figure 3 and Fig. 4C). This strongly suggested that the responsiveness of mechanosensitive ion channels was impaired in bII-spectrin-KO cells.
It has been demonstrated that the stretch-activated, non-selective cation channel Piezo1 mediates (at least part of) the shear-induced Ca2+ increases in endothelial monolayers and their alignment in the direction of flow (Li et al., 2014). Consistent with this report, pre-treatment of tHAEC cells with GsMTx4 was sufficient to abrogate the shear-evoked [Ca2+] elevation in confluent monolayers (Fig. 4D). Importantly, this treatment also blunted the Ca2+ responses normally seen in single cells (Fig. 4E). The pan-inhibitor of stretch-activated ion channels also inhibited the alignment of sparsely distributed endothelial cells in response to shear (Fig. 4F). These observations further indicated that the indispensable role of mechanosensitive ion channels in endothelial responses to fluid flow is regulated independently of junctional complexes.
The involvement of the Piezo1 channel was then probed more precisely using the highly specific, small molecule agonist, Yoda1. In static culture, treatment of wild-type cells with Yoda1 induced increases in [Ca2+]cyto that strongly resembled the dynamics observed in response to flow (compare Fig. 4G to 3G). In contrast, Yoda1 did not elicit any significant [Ca2+]cyto changes in bII-spectrin-KO cells (Fig. 4G). From this, we concluded that the apical bII-spectrin network regulates the activity of Piezo1.
Plasma membrane expression of Piezo1 is not regulated by spectrin
The unresponsiveness of bII-spectrin-KO cells to Yoda1 suggested two possibilities: either (1) spectrins are required for the plasma membrane retention of Piezo1; or (2) Piezo1 activity at the cell surface is somehow regulated by the spectrin cytoskeleton. To test the first possibility, we examined the expression and cellular distribution of Piezo1. Immunoblotting experiments revealed comparable concentrations of Piezo1 protein in wild-type and bII-spectrin-KO cells (Fig. 5A). By immunofluorescence, we observed that in wild-type cells, Piezo1 appeared as punctate structures that concentrated on the apical surface of the endothelial cells, consistent with its shear-sensing functions (Fig. 5B). This distribution was largely preserved in bII-spectrin-KO cells, where numerous plasmalemma-localized punctate structures were observed (Fig. 5B). It is therefore unlikely that the alterations in Piezo1 responsiveness reported in spectrin-deficient cells are attributable to changes in the expression or localization of the channel.
Spectrin stabilizes endothelial caveolae which regulate Piezo1
We proceeded to study how the spectrin-based membrane skeleton might directly impact Piezo1 function. Piezo1 expression is associated with local distortion of the lipid bilayer, often resulting in the preferential localization of the channels to curved membrane domains (Buyan et al., 2019; Diem et al., 2020; Liang and Howard, 2018). Localization to these domains is thought to confer a high degree of sensitivity to the channel, which responds to changes in membrane curvature that result from alterations in membrane tension (Diem et al., 2020; Liang and Howard, 2018).
Indeed, endothelial cells are uniquely enriched in curved membrane microdomains termed caveolae (Parton and Simons, 2007; Simionescu et al., 1981). Caveolae unfurl or flatten in response to application of force (Sinha et al., 2011). Of note, localization of Piezo1 to caveolar structures has previously been reported in other cells (Diem et al., 2020; Ridone et al., 2020). Accordingly, in wild-type endothelial cells, immunofluorescence revealed significant colocalization of Piezo1 and caveolin-1, a primary component of caveolae (Fig. 5C). We therefore investigated the effects of spectrin on caveolae, since these might indirectly influence Piezo1 activity.
Similar concentrations of total caveolin-1 protein were detected in wild-type and bII-spectrin-KO endothelial cells (Fig. 5D). When immunostained in wild-type endothelial cells, caveolin-1 formed punctate arrays that were enriched on the apical surface, consistent with the reported abundance of caveolae (Fig. 5E, F) (Drab et al., 2001; Oh et al., 2007). This polarized distribution was lost in bII-spectrin-KO cells, where a greater fraction of caveolin-1 was found in the basal and internal membranes (Fig. 5E). By analyzing the mean fluorescence intensities of the conjugated secondary antibodies within individual puncta on the apical cell surface, the number of caveolin-1 monomers in each cluster was also found to be significantly decreased in bII-spectrin-KO cells (Fig. 5F).
In addition to the observed changes in caveolin-1 distribution and clustering, we had previously observed that spectrin regulates the mobility of caveolar components in the plane of the membrane (Mylvaganam et al., 2020). However, it remained unclear whether the behavior of caveolin-1 reflected the number or appearance of the caveolae themselves. This was addressed by performing transmission electron microscopy (TEM) and analyzing sagittal sections of endothelial cells. In wild-type cells, TEM revealed a high apical density of uncoated, bulb-shaped plasmalemmal structures which we identified as caveolae (Fig. 5G, 5H). While still present, markedly fewer caveolae were observed on the surface of bII-spectrin-KO cells (Fig. 5G, 5H).
Altered membrane tension in spectrin-deficient cells
Since caveolae play an important role in calibrating plasma membrane tension (Sinha et al., 2011), and given the relationship between spectrin expression and caveolin density, we wondered whether the resting membrane tension was altered in bII-spectrin-KO cells. To assess membrane tension, we performed fluorescence lifetime imaging microscopy (FLIM) using Flipper-TR, a membrane tension probe. The fluorescence lifetime of Flipper-TR increases at higher membrane tensions (Colom et al., 2018), which we confirmed by inducing osmotic swelling of wild-type cells with hypotonic (150 mOsm) medium (Fig. 6A). In isotonic conditions at rest, the fluorescence lifetime of flipper-TR in the plasma membrane of bII-spectrin-KO cells was significantly longer than the lifetime measured in their wild-type counterparts (Fig. 6B). This revealed that as in erythrocytes, spectrins maintain the flexibility of the endothelial plasma membrane. Elevated surface tension at rest in bII-spectrin-KO cells could account for the disappearance of caveolae, causing their flattening, and/or for a loss of the control of membrane tension afforded by caveolar structures. It could also account for the unresponsiveness of Piezo1 channels, which are known to desensitize when subjected to sustained increases in resting tension (Lewis and Grandl, 2015).
Spectrin and HA regulate shear-induced changes in membrane tension
We then studied the effects of shear on membrane tension. Shear stress acutely induced a significant increase in Flipper-TR fluorescence lifetime in wild-type cells, revealing an increase in plasma membrane tension (Fig. 6B). These changes were observed across the entire apical cell surface and did not appear to localize preferentially to any specific regions. In spectrin-KO cells, while shear induced an increase in Flipper-TR fluorescence lifetime (Fig. 6B), the magnitude of this increase was markedly lower than that observed in wild-type cells (Fig. 6B). Conceivably, this more modest increase in membrane tension was insufficient to activate Piezo1, which may already be rendered less sensitive/desensitized due to the higher resting tension in these cells.
While clearly important, spectrin is unlikely to directly sense the apical shear flow in order to increase membrane tension. Given its high extracellular density and established role in mediating shear-induced Ca2+ signaling events, it seemed more conceivable that HA, via its receptors, might instead transmit forces from the vascular lumen to the spectrin cytoskeleton to then induce and distribute changes in tension. Indeed, while treatment with HAase had no appreciable effect on membrane tension of cells in static culture, it greatly inhibited the shear-induced increase in the lifetime of Flipper-TR (Fig. 6B).
Shear induced production of nitric oxide is regulated by spectrins
The preceding experiments indicated that spectrin behaves as a central integrator for endothelial signaling responses to flow. We therefore decided to further examine the importance of the cytoskeletal protein in general vascular functions. As mentioned, shear-induced production of NO is essential to maintain vascular health; NO both regulates changes in vascular tone and has important anti-coagulant properties. In the endothelium, NO is primarily produced by endothelial nitric oxide synthase (eNOS) which under basal conditions, interacts with caveolin-1. Caveolin-1 maintains the enzyme in an inactive state by sequestering it within caveolae. Increases in [Ca2+]cyto result in the activation of calmodulin which, in its Ca2+-complexed form, can bind eNOS, disrupting its inhibitory interaction with caveolin-1 (Michel et al., 1997). Once dissociated from caveolae, eNOS can be activated through phosphorylation by multiple kinases (Bir et al., 2012; Calvert et al., 2008; Dimmeler et al., 1999; Fleming et al., 2001). Given that spectrin regulates both shear-induced [Ca2+]cyto increases and the stability of caveolin-1/caveolae, it was likely that NO production might also be regulated by this network. This was tested by immunoblotting cell lysates with specific antibodies that detect the phosphorylation of Ser1177 of eNOS. As shown in Fig. 7A, while application of shear (applied by vigorous rotary agitation) induced a distinct increase in eNOS phosphorylation in wild-type cells, no significant phosphorylation changes were recorded in bII-spectrin-KO cells.
To examine whether the impaired phosphorylation of eNOS translated into a functionally significant reduction in NO production, we examined the interaction between platelets and endothelial cells under flow. Very few platelets adhered to wild-type endothelial cells, consistent with the ability of NO to inhibit the attachment of platelets to the vessel wall. However, the number of platelets that adhered firmly to bII-spectrin-KO endothelial monolayers increased markedly, primarily along endothelial cell borders (Fig. 7B). These findings revealed that spectrin contributes to the anti-thrombotic properties of the endothelium.
We wondered whether changes in spectrin expression are associated with endothelial dysfunction in certain pathological contexts. Previous studies revealed a relationship between decreased expression of spectrins and atherosclerotic plaque severity (Rademakers et al., 2018). Increased activity of the protease calpain, which cleaves spectrin subunits leading to network disassembly, is also associated with early stages of atherosclerosis (Miyazaki et al., 2011). However, it remains unclear whether these changes in spectrin expression were reflective of the endothelium or other cells in the artery. To investigate this possibility, we examined the endothelial expression of bII-spectrin in en face preparations of descending thoracic aortas. We immunostained aortas of wild-type and Ldlr−/− mice that had been placed on a high-fat diet for four weeks to promote atheroma formation, according to previously established protocols. Consistent with previous observations, in contrast to their wild-type counterparts, diseased descending aortas were associated with a loss of endothelial cell alignment (Chien, 2007; Hahn and Schwartz, 2009). Interestingly, the aortic endothelium of wild-type mice expressed high levels of bII-spectrin (Fig. 7C-D). However, in Ldlr−/− aortas where plaques had formed, loss of alignment was coincident with a significantly lowered expression of bII-spectrin (Fig. 7C-D). These observations are generally consistent with a role of spectrin in promoting the normal atheroprotective functions of the endothelium and implicate its dysregulation in the sequelae of atherosclerosis.