Sitting leg vasculopathy: potential adaptations beyond the endothelium

Abstract

Increased sitting time, the most common form of sedentary behavior, is an independent risk factor for all-cause and cardiovascular disease mortality; however, the mechanisms linking sitting to cardiovascular risk remain largely elusive. Studies over the last decade have led to the concept that excessive time spent in the sitting position and the ensuing reduction in leg blood flow-induced shear stress cause endothelial dysfunction. This conclusion has been mainly supported by studies using flow-mediated dilation in the lower extremities as the measured outcome. In this review, we summarize evidence from classic studies and more recent ones that collectively support the notion that prolonged sitting-induced leg vascular dysfunction is likely also attributable to changes occurring in vascular smooth muscle cells (VSMCs). Indeed, we provide evidence that prolonged constriction of resistance arteries can lead to modifications in the structural characteristics of the vascular wall, including polymerization of actin filaments in VSMCs and inward remodeling, and that these changes manifest in a time frame that is consistent with the vascular changes observed with prolonged sitting. We expect this review will stimulate future studies with a focus on VSMC cytoskeletal remodeling as a potential target to prevent the detrimental vascular ramifications of too much sitting.

INTRODUCTION

Sedentary behavior, defined as low energy expenditure (≤1.5 METs) while in a seated, reclined, or lying position during waking hours (1), is on the rise worldwide and particularly prevalent in modern societies (2–4). Sedentary behavior represents an independent risk factor for all-cause and cardiovascular disease mortality (2–10), and emerging epidemiological evidence suggests that this increased cardiovascular risk associated with sedentary behavior exists even independently of time spent in physical activity and structured exercise (11–18). However, the mechanisms linking sedentary behavior and cardiovascular disease risk remain largely unknown. Sitting is the most common form of sedentary behavior and, over the last decade, we and others have consistently demonstrated that prolonged, uninterrupted sitting (e.g., from 1 to 6 h) causes a transient reduction in leg vascular function (19–32), referred to as sitting leg vasculopathy (33). Recurring and extended episodes of leg vascular dysfunction associated with sitting may contribute to the genesis of leg peripheral artery disease (PAD) (33–35). Consistent with this idea, epidemiological data are available showing that sedentary time is associated with low levels of ankle-brachial index, a noninvasive clinical predictor of lower-extremity PAD (36).

Most studies documenting the detrimental vascular effects of prolonged sitting have used the Doppler ultrasound-based flow-mediated dilation (FMD) technique, a noninvasive approach to assess conduit artery function (19–25, 37–44). Because FMD is endothelial dependent (45–49), its assessment is commonly used as a barometer of endothelial function. As a result, the conviction is that prolonged sitting causes endothelial dysfunction and that this is the predominant feature of sitting leg vasculopathy (33). The purpose of this review is to challenge this current view by aggregating evidence from basic science and clinical studies that collectively support the possibility that prolonged sitting-induced leg vascular dysfunction is likely also attributable to adaptations beyond the endothelium, e.g., changes occurring in the underlying vascular smooth muscle cells (VSMCs).

EVIDENCE THAT PROLONGED SITTING BLUNTS REACTIVE HYPEREMIA, PASSIVE LIMB MOVEMENT-INDUCED HYPEREMIA, AND ENDOTHELIUM-INDEPENDENT VASODILATION IN THE LOWER LIMBS

The assessment of FMD involves a transient suprasystolic occlusion of the lower limb to generate a reactive hyperemic shear stress response that serves as a stimulus for vasodilation, which can be detected at the level of the conduit artery (e.g., femoral, popliteal, or tibial artery) via ultrasound. Notably, although FMD is reflective of conduit artery endothelial function (50–54), the magnitude of reactive hyperemia elicited by transient limb ischemia reflects the amount of dilation in downstream resistance arteries and, as such, it can serve as an indicator of resistance vessel function (55).

In the forearm, the significance of postocclusive reactive hyperemia was first documented by Mitchell et al. (56). They showed that cardiovascular risk factors are more closely related to reactive hyperemia than to FMD, a finding corroborated by a subsequent study (57). Evidence is also available suggesting that forearm reactive hyperemia is an independent predictor (i.e., beyond other risk factors) of adverse cardiovascular events and a measure that can discriminate subjects with increased cardiovascular disease risk (58–64). Despite its prognostic value, forearm reactive hyperemia is minimally dependent on nitric oxide (NO). Indeed, intra-arterial blockade of NO synthase using N^G-monomethyl-l-arginine (l-NMMA) does not influence forearm reactive hyperemia or it only marginally blunts it (65–69). Work by Crecelius et al. (69) demonstrated that activation of inwardly rectifying potassium channels and Na⁺/K⁺-ATPase, largely expressed in VSMCs of resistance arteries (70), are the primary determinants of the reactive hyperemic response. Based on the above, it is prudent to deduce that, unlike FMD, postocclusive reactive hyperemia is not an endothelium-specific test (66, 69, 71–75) or at minimum not an endothelium NO-dependent phenomenon. Reactive hyperemia is rather likely mediated by a myriad of vasodilator substances (e.g., metabolites). More specifically, the magnitude of the hyperemic response is dependent on the amount of tissue hypoxia generated during occlusion and consequent release of vasodilators, as well as dependent on the capacity of the VSMCs to relax and effectively increase lumen diameter of resistance arteries (55, 76, 77).

Although most vascular sitting studies have primarily focused on the FMD response as the main outcome variable, a consistent observation by our group and others is that lower limb reactive hyperemia is also markedly blunted after prolonged sitting (21, 23, 40, 78–80) (Fig. 1). Because reduced leg FMD following prolonged sitting persists even after statistically controlling for the reduction in hyperemic shear stress stimulus (19–22, 43), most of such papers arrived at the conclusion that prolonged sitting impairs lower limb endothelial function. Accordingly, as mentioned earlier, the prevailing belief in the field is that impaired leg vascular function after prolonged sitting is attributable to endothelial defects. However, through the lenses of this review, we consider the consistent finding that prolonged sitting blunts reactive hyperemia (a largely endothelium-independent response) in the lower limbs as evidence to support the possibility that other factors beyond changes in endothelial function may also occur with sitting.

Figure 1.

Prolonged sitting is associated with pronounced reduction in leg blood flow (A), impaired reactive hyperemia (B), and blunted flow-mediated dilation (FMD; C) in the popliteal artery in healthy young adults. Values are presented as means ± SE. *P < 0.05 vs. presitting. Figure redrawn from Restaino et al. (21) with permission.

An important consideration here is that, as already stated, the extent of vasodilators released during occlusion is likely proportional to the magnitude of tissue hypoxia (i.e., oxygen deficit) that is achieved. This is particularly relevant because inactivity associated with sitting reduces the skeletal muscle metabolic rate, which in turn reduces the oxygen deficit during occlusion (based on measurements using near-infrared spectroscopy) and thus the stimulus for vasodilators to be released (81–84). Therefore, the blunted reactive hyperemia after prolonged sitting may be mediated, at least in part, by the reduced oxygen deficit (76). As recently and nicely put forth by Anderson and Park (76), future studies should examine whether dampened postocclusive reactive hyperemia after prolonged sitting remains observed when the amount of oxygen deficit is experimentally matched, e.g., by manipulating the duration of cuff occlusion (77).

Passive limb movement (PLM) is another approach to induce hyperemia, which can be captured using Doppler ultrasound, and used to assess resistance vessel function. Extensive reviews and guidelines describing this technique exist (55, 85, 86). In brief, PLM involves the manipulation of a limb (e.g., the leg) without voluntary muscle contraction; thus, the increase in skeletal muscle activation and metabolism is negligeable. The hyperemic response is elicited by mechanical deformation and stretch of the skeletal muscle, resulting in a cascade of vasodilatory events in the resistance arteries. Unlike reactive hyperemia, intra-arterial l-NMMA studies in the leg demonstrate that PLM-induced hyperemia is ∼80% NO dependent (87, 88). Given the NO dependency of this hyperemic response, this maneuver is increasingly used to assess endothelial function of the skeletal muscle resistance arteries. Notably, and relevant to the present review, a series of studies led by Garten et al. (27–29) demonstrate that PLM-induced hyperemia is blunted following 1.5 to 3 h of sitting (Fig. 2), again further emphasizing the detrimental effects of prolonged sitting on the vasculature of the lower limbs. No studies have yet been performed to determine whether the magnitude of l-NMMA-induced suppression (i.e., the NO contribution) of hyperemia during PLM is affected by prolonged sitting. Stated differently, it remains unknown whether the prolonged sitting-related reduction in vasodilation to PLM is indeed due to diminished NO bioavailability, for example, as it has been demonstrated with aging (89, 90). It is possible that NO production by endothelial cells during PLM remains intact but that the underlying VSMCs of resistance arteries become less responsive to NO and/or that resistance arteries have inwardly remodeled. Although more work is needed to examine these questions, information presented below reinforces the possibility of these scenarios.

Figure 2.

Evidence that sitting for 3-h blunts passive limb movement (PLM)-induced hyperemia in the leg of young healthy aerobically untrained adults. Values are presented as means ± SE. *P < 0.05 vs. presitting. Figure redrawn from Garten et al. (27) with permission.

With the impetus to better understand the vascular endothelium-independent effects of prolonged sitting, Liu et al. (30) recently conducted a much-needed study to examine whether prolonged sitting (3 h) influences the sensitivity of VSMCs to NO using the well-established nitroglycerin test. The authors reported that prolonged sitting not only impairs popliteal artery FMD, but it also blunts its vasodilation response to nitroglycerin (Fig. 3). Interestingly, data from a prior study by the same group provided evidence that popliteal artery FMD and nitroglycerin-induced vasodilation were positively correlated with the amount of moderate-to-vigorous physical activity (r = 0.85 and r = 0.59, respectively) assessed over a 5-day period in a small cohort of older men and women (91). When sedentary time was used instead as the outcome variable, these correlations (although inversely related as expected; r = −0.15 and −0.18) did not reach statistical significance (91), suggesting findings from laboratory-based sitting studies may not always align with observations from free-living conditions. Nevertheless, taken all together, it can be concluded that endothelium-independent mechanisms likely contribute to the diminished leg vasodilatory capacity following prolonged sitting.

Figure 3.

Nitroglycerin-mediated dilation (NMD), an assessment of endothelium-independent vasodilation, is impaired in the popliteal artery after 3 h of sitting in healthy young adults. Values are presented as means ± SE. *P < 0.05 vs. presitting. Figure redrawn from Liu et al. (30) with permission.

EVIDENCE THAT SITTING IS ASSOCIATED WITH VASOCONSTRICTION OF RESISTANCE ARTERIES AND REDUCED LEG BLOOD FLOW

Another consistent finding by us and others is that leg blood flow is notably reduced during sitting (19–23, 25). In fact, given that blood flow-induced shear stress is an important signal for maintaining optimal endothelial health, in prior studies we have ascribed the impairment in popliteal artery endothelial function after prolonged sitting to the sustained reduction in shear stress to which the vasculature is exposed during sitting (20, 21, 23, 33, 37, 43).

Several factors may contribute to the vasoconstriction of resistance arteries and suppressed leg blood flow during sitting. An evident mechanism is the low metabolic rate of inactive skeletal muscle (40, 77), as metabolic demand is matched by perfusion. However, other mechanisms specific to the sitting posture contribute. For example, it is plausible that, during sitting, increased hydrostatic pressure within the leg vasculature causes blood pooling within the venous circulation. Indices of augmented lower limb venous congestion, including increased calf (21, 27, 29, 39, 42, 79) and ankle (22) circumference, have been documented. This is likely aggravated by reduced skeletal muscle activity during sitting and, consequently, the loss of muscle pump effect that facilitates venous return to the heart (92). It is also possible that during sitting, venous return is further limited by the pressure exerted on the back of the thighs. Therefore, physical compression, venous distension-induced arterial constriction, and increased hydrostatic pressure-induced myogenic constriction (93) are likely key mechanisms underlying increased leg vascular resistance during sitting.

Furthermore, given that muscle sympathetic nerve activity (MSNA) is increased in the upright position (94), α-adrenergic vasoconstriction may also contribute to leg vascular resistance during sitting. In support of this, most studies provide evidence that blood pressure is increased during sitting, relative to the supine position (20, 22, 27, 29, 39, 95, 96); however, this is not a universal finding (97). The increase in blood pressure does not appear to be associated with an increase in cardiac output (95). Finally, we have also provided evidence that flexion of the hips and knees with sitting, and associated arterial bending, hinders blood flow to the lower limbs (20, 24, 33, 98). Decreased blood flow owing to arterial angulations or to other factors described earlier results in a reduction of shear stress stimulus in the leg vasculature, likely leading to reduced endothelium-derived NO production and consequently increased vascular tone. The next section discusses the vascular ramifications of prolonged vasoconstriction.

EVIDENCE THAT PROLONGED VASOCONSTRICTION CAUSES INWARD EUTROPHIC REMODELING

The purpose of this section is to present evidence that prolonged constriction of resistance arteries can lead to modifications in the structural characteristics of the vascular wall in a time frame that is consistent with vascular changes observed after prolonged sitting. The notion that structural changes in the vascular wall can occur within hours is largely underappreciated and this is what, in part, motivated the writing of this review. Arteries that exhibit inward eutrophic remodeling are distinguished by reduced passive luminal diameter with no change in wall cross-sectional area (99). A rapid reduction in vascular diameter is achieved by contraction of VSMCs via calcium-calmodulin-dependent phosphorylation of the regulatory myosin light chains and subsequent actomyosin cross-bridge cycling (100). Evidence indicates that calcium-independent processes are also in place in the acute control of vascular diameter, including calcium sensitization (101) and actin filament remodeling (102). These processes highlight the malleable nature of the VSMC machinery that regulates the structural and active diameters of resistance arteries.

Although the mechanisms implicated in the transition from acute to longer-term (minutes to hours) control of vascular diameter are less understood than the mechanisms controlling rapid changes in diameter, we provide compelling evidence that when arteries are exposed to neurohumoral vasoconstrictors, e.g., norepinephrine (NE) and angiotensin II (ANG II), for an extended period, they maintain a reduced diameter despite removal of the vasoconstrictor agonists and exposure to endothelium-independent vasodilators (103–105) (Fig. 4). This narrowing of the lumen is indicative that longer lasting changes in some cellular and/or extracellular determinants of vascular wall structure have occurred. Of significance, inward eutrophic remodeling of resistance arteries (e.g., mesenteric and cremasteric) is the most common structural change in hypertension (106–112) and its presence is predictive of life-threatening cardiovascular events (113–116).

Figure 4.

Prolonged exposure to norepinephrine and angiotensin II (NE + ANG II) causes inward eutrophic remodeling in rat (male Sprague–Dawley; weight range, 250–350 g)-isolated cremaster arterioles. Luminal diameter of arterioles exposed to NE (10^−5.5 M) + ANG II (10⁻⁷ M) and their maximal relaxation responses under Ca²⁺-free conditions before and after exposure to NE + ANG II for 4 h. Mean trace is illustrated. *P < 0.05 vs. before agonist exposure. Figure redrawn from Martinez-Lemus et al. (104) with permission.

The precise mechanisms that control the initial stages of inward eutrophic remodeling are not fully elucidated; however, evidence indicates changes occur mainly at the level of the actin cytoskeleton and are associated with actin polymerization pathways (111, 117) in conjunction with the repositioning and stiffening of VSMCs in resistance vessels (103–105). It has been reported that during prolonged agonist-induced vasoconstriction, some but not all VSMCs reelongate from their constricted length and increase their degree of overlap by sliding over one another (105, 118). Why some cells reposition while others do not, remains to be determined. A potential explanation is that not all cells in the medial layer of arterioles are fully differentiated VSMCs. It has been well documented, particularly in the cerebral circulation, that different phenotypes of the cells known as pericytes populate the wall of blood vessels from the terminal arterioles to the capillaries and venules (119, 120). The presence of diverse morphologies of mural cells in arterioles suggest that pericyte-like mural cells may be present in the arteriolar wall and as such may respond differently to short- and long-term vasoconstrictor stimuli. Nonetheless, repositioning and stiffening processes of VSMCs likely involve the formation of more permanent actin cytoskeletal structures and new and stronger focal adhesion sites as the cell becomes repositioned in the arteriolar wall of resistance vessels (121–123). Indeed, evidence indicates that VSMCs exposed to vasoconstrictor agonists stiffen while their adhesion to the substrate is also strengthened (124, 125). We posit that these cellular changes prevent the resistance vessel from dilating to its original maximal passive diameter as alluded earlier (Fig. 4). In that regard, we have reported that during the initial stages of inward eutrophic remodeling, most of the reduction in passive diameter observed in isolated resistance arteries following prolonged (i.e., 4 h) vasoconstriction is reverted with actin cytoskeletal disruption (111) (Fig. 5). This finding contributes to the evidence that actin polymerization in VSMCs mediates vascular inward eutrophic remodeling caused by prolonged vasoconstriction in resistance vessels. The next section highlights some of the molecular mechanisms that participate in the regulation of VSMC actin polymerization and stiffening, as well as vascular inward remodeling.

Figure 5.

Disruption of the actin cytoskeleton reverts the inward remodeling caused by prolonged exposure to norepinephrine (NE) and angiotensin-II (ANG II) in rat (male Sprague–Dawley; weight range, 250–350 g)-isolated cremaster arterioles. Before and after the 4-h incubation with NE (10^−5.5 M) + ANG II (10⁻⁷ M), arterioles were allowed to develop spontaneous myogenic tone and were exposed to Ca²⁺-free conditions to assess maximal relaxation. After 5 min under the second exposure to Ca⁺²-free conditions, arterioles were treated (1 h) with vehicle control or mycalolide-B (2 µM) to depolymerize actin fibers. Mean traces are illustrated. *P < 0.05 vs. control. Figure redrawn from Staiculescu et al. (111) with permission.

MOLECULAR MECHANISMS IMPLICATED IN ACTIN POLYMERIZATION AND STIFFENING OF VSMCs AND VASCULAR INWARD REMODELING

Work by our group demonstrated that inward eutrophic remodeling in resistance arteries exposed to vasoconstrictor agonists (i.e., NE and ANG II) is accompanied by the formation of reactive oxygen species (ROS) and activation of metalloproteinases (MMPs) (110) (Fig. 6). It should be noted that the process of inward remodeling can start early, even within 1 h of exposure to vasoconstrictors (108), and it remains sustained with longer exposures (i.e., from hours to days) (109). Of note, broad MMP inhibition did not influence the production of ROS but prevented the remodeling induced by prolonged vasoconstriction. Conversely, inhibition of ROS (using apocynin or tempol) prevented both the activation of MMP and the inward remodeling, indicating that activation of MMP induced by ROS contributes to the remodeling process associated with vasoconstriction (110).

Figure 6.

Prolonged exposure to norepinephrine and angiotensin-II [NE (10^−5.5 M) + ANG II (10⁻⁷ M), 4 h] increases reactive oxygen species (ROS) formation and matrix metalloproteinases (MMPs) activation in rat (male Sprague–Dawley; weight range, 250–350 g)-isolated cremaster arterioles. A: arterioles with or without NE + ANG II were coincubated with 5- (and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFH; 30 µM) and dihydroethidium (DHE; 5 µM) and imaged with a multiphoton microscope. Bar graphs represent percent changes in DCFH and DHE fluorescence intensities from 5 min to 4 h of incubation. B: arterioles with or without NE + ANG II were subjected to gel zymography. Bands representing gelatinolytic activity were analyzed by densitometry and expressed as fold changes from control for the activity of latent (72 kDa) and active (64 kDa) forms of MMP2. Values are presented as means ± SE. *P < 0.05 vs. control. Figure redrawn from Martinez-Lemus et al. (110) with permission.

Although MMP activity is typically associated with extracellular matrix (ECM) degradation, there are several mechanisms by which MMP may modulate cytoskeletal structures. For example, degradation of ECM structures by MMP creates protein fragments with exposed cryptic sites that activate integrins (126). Activation of integrins in turn activates intracellular signals that lead to cytoskeletal remodeling. In this regard, evidence exists indicating that activation of specific integrins also contributes to inward eutrophic remodeling (107). MMP can also induce cytoskeletal modifications by transactivation of the epidermal growth factor (EGF), which occurs upon stimulation with vasoconstrictor agonists such as NE and ANG II. After the shedding of EGF by MMP, EGF binds to the epidermal growth factor receptor (EGFR) and phosphorylates tyrosine kinase. This process leads to the downstream activation of several kinases (i.e., p38 MAPK and ERK1/2) that will be translocated to the nucleus to bind and phosphorylate nuclear transcription factors, stimulating gene transcription, protein synthesis, and cell growth. Among these kinases, p38 MAPK is known to participate in the signaling events, causing vasoconstriction-mediated EGFR activation (127, 128).

Another important enzyme in VSMC cytoskeletal remodeling is tissue transglutaminase (TG2) (129). Although TG2 is most known for its function as a crosslinker of the ECM, TG2 also has the capacity to function as G protein and cellular receptor scaffold (129, 130). TG2 activation stimulates downstream intracellular pathways in VSMCs (131), including the small GTPase Ras homolog family member A (RhoA), and its downstream effector Rho-associated protein kinase (ROCK) (130, 132–134). ROCK activates LIM kinase (LIMK), which is a critical regulator of actin dynamics and VSMC stiffness (134–139). Indeed, LIMK phosphorylates and inactivates cofilin, and as cofilin’s primary role is to sever F-actin stress fibers, cofilin inactivation enhances actin polymerization (140, 141). In this regard, we have shown that TG2 activation causes actin polymerization and stiffening in VSMCs and inward remodeling in isolated resistance arteries, which can be prevented by actin depolymerization (142) (Fig. 7). LIMK inhibition prevents VSMC stiffening and vascular inward remodeling induced by vasoconstrictor agonists, and these effects are accompanied by a reduction in phosphorylated cofilin (134, 139) and F-actin content (134). In vivo, ANG II infusion-induced inward remodeling is also prevented by LIMK inhibition (134). It should be noted that actin dynamics as modulated by the activity of cofilin have diverse effects on different cell types (134, 143, 144). As such, cofilin phosphorylation can promote both cell stiffening and cell migration in addition to modulating cellular phenotype switching (143), which is highly compatible with the reports indicating arteriolar inward remodeling involves both VSMC repositioning and stiffening.

Figure 7.

Actin depolymerization reverts tissue transglutaminase (TG2) activation-induced inward remodeling in rat-isolated cremaster arterioles. A: confocal images of isolated arterioles incubated with Alexa Fluor 488-cadaverine (green) and exposed for 4 h to vehicle control (left), to 200 µM of TG2 activator [dithiothreitol (DTT), middle], or TG2 activator in the presence of 1 mM of TG2 inhibitor cystamine (right). B: passive pressure diameter curves of arterioles before and after exposure to 200 µM of TG2 activator and cystamine (1 mM) for 4 h. *P < 0.05 vs. before TG2 activator; #P < 0.05 vs. TG2 activator. C: confocal images of isolated arterioles exposed to vehicle control (left) or 2 µM mycalolide-B (right) and subsequently stained with phalloidin-Alexa 546 to visualize the actin cytoskeleton. D: pressure-diameter curves of TG2 activator inwardly remodeled arterioles before and after exposure to 2 µM of mycalolide-B or its vehicle control for 1 h. *P < 0.05 vs. remodeled + mycalolide-B or remodeled + vehicle. Figure redrawn from Castorena-Gonzalez et al. (112) with permission.

Prior work indicates that NO is a key downregulator of TG2 activity (145–147). The endothelium is the primary source of NO in the vasculature and its production is largely stimulated by flow-induced shear stress (148–151). In accordance, previous preclinical studies show that TG2 contributes to inward remodeling induced by prolonged low blood flow (108, 142, 151). Along these lines, in a scenario of reduced blood flow as it occurs in the leg vasculature during sitting, it is plausible that diminished endothelium-derived NO production and associated increases in VSMC TG2 activity also contribute to actin polymerization and stiffening of VSMCs, as well as vascular inward remodeling. However, this hypothesis remains to be tested. Other common risk factors for PAD, including aging, obesity, and hypertension, are also associated with increased actin polymerization and stiffening in VSMCs, and TG2 and LIMK activation are key molecular events implicated in this process (134, 152, 153). As such, it is possible that an overabundance of sitting coupled with other cardiovascular risk factors accelerates VSMC cytoskeletal remodeling in the leg circulation of conduit and resistance vessels, thus increasing the susceptibility for PAD development in the lower extremities.

CONCLUSIONS AND PERSPECTIVES

The prevailing dogma is that impaired leg vascular function with prolonged sitting is attributable to endothelial defects. Work by our group has significantly contributed to this view. Without discrediting this prior work, herein we summarize evidence from classic studies and more recent ones that collectively support the notion that prolonged sitting-induced leg vascular dysfunction is likely also attributable to changes occurring in VSMCs (Fig. 8). In that context, evidence is provided that prolonged constriction of resistance arteries can lead to modifications in the structural characteristics of the vascular wall, including polymerization of actin filaments in VSMCs and inward remodeling, and that these changes occur in a time frame that is consistent with the micro- and macrovascular changes observed with prolonged sitting. In fact, it is conceivable that impaired postocclusion reactive hyperemia and PLM-induced hyperemia in the lower limb after uninterrupted sitting are reflective of inward remodeling of resistance arteries induced by prolonged leg vasoconstriction. Because actin polymerization stiffens the cell, it is also likely that VSMC stiffening contributes to the impaired vasorelaxation responses associated with sitting. Studies are now needed to mechanistically determine whether structural modifications in VSMCs (on both conduit and resistance arteries) contribute to the impaired vasodilatory capacity of the leg vasculature caused by prolonged sitting. Ultimately, the goal is to identify therapeutic targets for intervention that can alleviate the vascular burden of excessive sitting and thus prevent risk of PAD development in the lower limbs. Some dietary strategies (e.g., vitamin C and fish oil supplementation) have already been attempted with mixed results (29, 31, 154). We expect pharmacological studies targeting sitting leg vasculopathy are also forthcoming. However, interventions designed to reduce sitting behavior (i.e., removal of the “insult”) are likely to always be the most effective and sustainable.

Figure 8.

Summary of proposed vascular smooth muscle cell (VSMC)-related mechanisms contributing to leg vascular dysfunction with prolonged sitting and likely amplified with superimposition of cardiovascular disease risk factors such as advanced age, obesity, and hypertension. EC, endothelial cell; ECM, extracellular matrix; Kir, inwardly rectifying potassium channel; NE, norepinephrine; ANG II, angiotensin II; ROS, reactive oxygen species; MMP, matrix metalloproteinase; NO, nitric oxide; TG2, tissue transglutaminase; RhoA, Ras homolog family member A; LIMK, LIM kinase; ATR, angiotensin receptor; ADR-α, α-adrenergic receptor.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01HL137769 (to J.P.), R01HL151384 (to L.A.M.-L. and J.P.), and R01HL153264 (to L.A.M.-L. and J.P.) and University of Missouri-Columbia Research Excellence Program (to L.F.-S.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.F.-S., L.A.M.-L., and J.P. conceived and designed research; L.F.-S. and J.P. prepared figures; L.F.-S. and J.P. drafted manuscript; L.F.-S., L.A.M.-L., and J.P. edited and revised manuscript; L.F.-S., L.A.M.-L., and J.P. approved final version of manuscript.