The vascular endothelium is a pivotal determinant of cardiovascular health. Risk factors including diabetes, hypertension and obesity along with altered shear stress along the vascular wall promote endothelial dysfunction (De Vriese et al., 2000; Barton, 2010). Characterized by impaired endothelium dependent vasodilation and increased oxidative stress, endothelial dysfunction is a harbinger of serious cardiovascular disease (CVD). Atherosclerosis is a common progressive disease process that involves deposition of cholesterol-rich plaque in affected conduit arteries and obstructive remodeling of the arterial wall, including inward growth of neointima into the vascular lumen (Gimbrone and García-Cardeña, 2016). Within major conduit arteries (e.g., carotid, coronary and arteries in the legs), narrowing of the lumen and thrombosis can severely compromise blood flow and ultimately cause life-threatening ischemia or loss of limbs. Indeed, coronary and peripheral artery disease (PAD) remain major health care challenges despite interventional strategies aimed at reducing risk factors, preventing thrombosis and reestablishing blood flow through angioplasty (Wolf and Hunziker, 2020). Moreover, vessels downstream of obstructive atheromas may be impacted by chronic low-flow inflammatory conditions. New strategies aimed at understanding and preserving endothelial function are warranted.

Maintaining normal Ca²⁺-dependent signaling along the endothelium is crucial for many processes that preserve vascular function, including nitric oxide signaling, secretion, and nuclear transcription (Falcone, 1995; Filippini et al., 2019). Disruption of these processes under inflammatory conditions such as diabetes is known to contribute to endothelial dysfunction and progressive vascular disease (De Vriese et al., 2000; Fitzgerald et al., 2007; Park et al., 2008). Specific changes in signaling along the vascular intima associated with atherosclerosis remain poorly understood. We and others have previously described distinctive profiles of basal dynamic Ca²⁺ signals in the arterial endothelium of rodents and pigs that occur even in the absence of exogenous stimulation and underpin inherent homeostatic Ca²⁺ signatures of the arterial intimal network (Ledoux et al., 2008; Sonkusare et al., 2012; Sullivan and Earley, 2013; Qian et al., 2014; Francis et al., 2016; Wilson et al., 2020). Within these signatures, discrete changes in the specific Ca²⁺ events (i.e., amplitude, duration and spatial spread or area) correspond to vascular function, including vasodilator and shear stress responses. Although effects of chronic pathologic signaling on endothelial Ca²⁺ signatures remain unknown, we previously reported that 2 weeks of reduced blood flow (in a partial ligation mouse model reducing carotid artery flow by >90%) substantially restricts the size and range of Ca²⁺ dynamics along the endothelial network (Taylor et al., 2017). One aspect of this adaptive shift may involve impaired expansion of locally triggered Ca²⁺ signals along the plasma membrane (e.g., Ca²⁺ sparklets occurring through type four vanilloid transient receptor potential channels, TRPV4). Indeed, TRPV4 channels have surfaced as particularly pivotal conduits for dynamic Ca²⁺ signaling in the endothelium (Bagher et al., 2012; Bubolz et al., 2012; Filosa et al., 2013; Liu et al., 2021) and altered TRPV4 signaling has been implicated in progressive cardiovascular disease (Nishijima et al., 2014; Zhang et al., 2018).

Dynamic Ca²⁺ signaling signatures along the intima of human arteries have not been studied. Here, we use confocal imaging and a custom analysis algorithm to characterize Ca²⁺ signaling profiles of peripheral arteries isolated from the amputated lower limbs of patients with or without existing CVD (peripheral artery disease/diabetes). We describe a robust dynamic Ca²⁺ signature along the intima of human arteries and a distinctive restriction of Ca²⁺ dynamics associated with existing CVD.

While distinct functional patterns of endothelial Ca²⁺ dynamics are beginning to emerge in various animal models and different vascular beds, the base Ca²⁺ signaling patterns of human arteries have not been characterized. The primary goal of the current work was to assess the prevailing intimal Ca²⁺ signaling signature in human arteries and provide insight on whether this signaling might be impacted by CVD. We analyzed Ca²⁺ signals along the intima of peripheral arteries isolated from the limbs of patients who had undergone amputation due to either severe cardiovascular disease or non-cardiovascular trauma. We identified a distinct dynamic Ca²⁺ signaling profile along the human arterial intima composed of a broad range of basal Ca²⁺ transients. This profile is notably muted in CVD arteries compared to nCVD arteries, with CVD arteries exhibiting a predominance of smaller focal events and fewer whole-cell waves. Stimulation of TRPV4 channels expanded intimal Ca²⁺ signals, particularly spatial area, in both CVD and nCVD arteries, but the overall signal profile remained reduced in CVD arteries. The current findings reveal an intrinsic Ca²⁺ signaling signature along the arterial intima of human arteries that is restricted in CVD.

Studies over the past decade have exposed the crucial impact of dynamic endothelial Ca²⁺ signals in tuning vascular responses including endothelium derived hyperpolarization and nitric oxide mediated vasodilation (Kansui et al., 2008; Ledoux et al., 2008; Qian et al., 2013; Francis et al., 2016). While endothelial dysfunction is known to play a crucial role in progressive CVD, particularly in the context of diabetes and atherosclerosis, recent findings indicate that early disruption of inherent signaling patterns along the endothelial network is pivotal in this transition (Taylor et al., 2017; Wilson et al., 2020). For instance, constrained or redirected Ca²⁺ signal communication may alter nitric oxide production and favor production of reactive oxygen species. The inherent signals underlying endothelial function in human arteries have not been well-studied due to limited tissue access and difficulties associated with measurement and quantification. Our lab and others have recently begun to apply imaging and analysis approaches to decipher complex dynamic signaling patterns in intact tissues. In the current study, we applied a next-generation version of our custom algorithm LC_Pro that has the distinct advantage of tracking events without applying fixed regions of interest (ROIs). This approach allows for detection and isolation of a wide range of events as well as the characterization of each event with space and time. Such continuous spatio-temporal tracking distinguishes events that might otherwise be coincidently sampled and misinterpreted by fixed ROIs, providing a valuable approach for future elucidation of signals within complex networks.

It is important to note that in the current study we only evaluated arteries with intact endothelia in order to capture continuous intimal Ca²⁺ profiles. This also ensured comparable readouts for CVD and nCVD vessels, allowing us to assess altered signaling in the diseased circulation, even where the intima remained intact. Indeed, we observed that while many arterial branches from CVD patients exhibited some degree of neointima (quite distal from the established upstream atheroma), the endothelium was usually intact. For Ca²⁺ signal quantification, we have found that tracking frequency as well as maximal amplitude, duration and area of detected events provides a useful assessment of signal profile. We noted a trend of increased event frequencies in CVD arteries although additional sampling in future studies will be needed to determine if this is a common feature of CVD or whether it may be specific to a condition, disease, age or some other variable. Analysis of Ca²⁺ parameter profiles showed that CVD artery distributions for amplitude and area were consistently left-shifted compared to nCVD arteries, suggesting the arterial intima of CVD patients elicit truncated Ca²⁺ signals compared to those of non-CVD patients. Overall, analysis of distribution differences revealed that CVD arteries have a higher density of events with amplitudes of one to three ΔF, durations of 2.5–5 s, and areas of 4–16 μm², and a lower density of events with amplitudes of 5–10 ΔF, durations of 10–40 s, and areas of 30–250 μm² compared to nCVD. This truncation of Ca²⁺ event size is similar to the effect of chronic low flow on mouse carotid arteries wherein 2 weeks of reduced blood flow resulted in endothelial dysfunction and significant arterial wall remodeling, including neointima formation (Taylor et al., 2017). We observed variable degrees of neointima in peripheral arteries from CVD patients in the current study. It will be useful in future studies to determine whether general endothelial dysfunction (impaired vasodilation) and remodeling of the peripheral vasculature (distal to obstructive atherosclerosis) correspond to the degree of endothelial Ca²⁺ signal disruption. In the current study, arteries from patients with severe PAD and/or diabetes were grouped into the CVD cohort. Extended studies should allow discrimination of functional Ca²⁺ signaling profiles between these groups and among other variables.

TRPV4 cation channels elicit focal Ca²⁺ influx events at the plasma membrane (Ca²⁺ sparklets), allowing for localized signal targeting and providing a trigger for larger Ca²⁺-induced Ca²⁺ release events (i.e., from the endoplasmic reticulum) (Sullivan et al., 2012; Lin et al., 2015; Heathcote et al., 2019; McFarland et al., 2020). Growing evidence supports involvement of these channels in shear stress and agonist-mediated endothelial signaling, and changes in TRPV4 signaling are linked to disease states including hypertension, diabetes and pulmonary edema (Hartmannsgruber et al., 2007; Mendoza et al., 2010; Sonkusare et al., 2012; Ma et al., 2013; Monaghan et al., 2015; Zhang et al., 2018; Daneva et al., 2021; Rajan et al., 2021). In the current study, we observed TRPV4 channels expressed in the intima as well as the media of peripheral arteries, and even within neointima of arteries from CVD patients. Our data suggest that stimulation of these channels expand Ca²⁺ signals along the intima, particularly the spatial area of events. However, while TRPV4 stimulation substantially increased the magnitude and reach of Ca²⁺ events in CVD arteries, these events remained diminished compared to nCVD arteries. This reduced signaling capacity in peripheral arteries of CVD patients suggests the channels generate initial Ca²⁺ transients but the amplification or spread these signals is impaired. We previously reported that spatially restricted endothelial Ca²⁺ events in mouse carotid arteries exposed to chronic low flow similarly failed to expand normally following acetylcholine stimulation. One possibility is that ion channels such as Ca²⁺-activated K⁺ channels that normally amplify these local signals and allow them to trigger internal Ca²⁺ store release are impaired or decoupled from the TRPV4 channels in the plasma membrane. Such decoupling of TRPV4 and K_Ca2.3 channels has been implicated in vascular dysfunction associated with hypertension (Ma et al., 2013). The implication is that changes due to low flow and/or chronic inflammation reduce the capacity for expanding Ca²⁺ transients (e.g., Ca²⁺ sparklets). Extended investigation of the mechanism driving this shift and the implications for TRPV4 mediated signaling under conditions of shear stress in CVD is warranted.

The current findings underscore the complexity of biological Ca²⁺ profiles and the importance of discerning specific Ca²⁺ signal distributions. While mean parameter values can provide basic information about the events, assessment of full distributions is needed to characterize the disparate nature and range of signals. This will be particularly important for distinguishing Ca²⁺ parameter shifts with specific perturbations (e.g., receptor agonists, shear stress) and determining distinct physiologic and pathologic impacts. Indeed, our data suggest TRPV4 channel stimulation has a complex effect on the endothelial Ca²⁺ signal profile. While spatial area of the signals expands, as evidenced by a general right shift in the event area distribution, the profile for event duration appears to flatten, essentially redistributing to the left and right. This may reflect the widespread triggering of focal Ca²⁺ entry events and subsequent spread of many of these events as internal Ca²⁺ stores are recruited. Notably, this expanded duration profile is not evident in CVD arteries. A key point is that shifts in a parameter distribution may provide important information not reflected by mean values. Overall, our current findings suggest CVD limits the capacity of TRPV4 signaling. Specific aspects of these changes, including the mechanism underlying redirection of Ca²⁺ signals and the direct functional consequences remain to be determined.

It should be noted that cells other than endothelial cells were sometimes captured in our Ca²⁺ imaging studies. These are likely smooth muscle cells just below the internal elastic lamina or part of the neointima of CVD arteries. As these cells were part of the continuous cell population and we could not definitively identify and remove specific cells from analysis, they were included. Overall, we estimate these cells contributed <5% of the signals analyzed and had little impact on the net assessment. Future iterations of the imaging and analysis applied here should allow for discrete discrimination of the vascular intima as well as the specific impact of the neointima itself. This should also include assessment of endothelial status and the relative impact of apoptotic or necrotic cells on the prevailing Ca²⁺ profile. While the current study was limited by the modest number of tissue samples, the findings serve as a valuable foundation for future mechanistic investigations and development of new approaches for preserving homeostatic endothelial signaling.