Lower Extremity Peripheral Artery Disease: Atherosclerosis, Decreased Nitric Oxide, and Vascular Arterial Stiffening

1. Introduction

Age-related peripheral artery disease (PAD) and atherosclerosis with vascular ossification-calcification (VOC) have been around since the times of ancient Egyptians that were discovered by the studies of mummies via computed tomography scanning, which reveled vascular calcifications [1,2,3]. Globally, PAD affects 200-250 million people [4,5]. Currently, we are living in one of the oldest living populations in history [6,7,8] and therefore, we can expect to experience an even higher occurance of symptomatic PAD, atherosclerosis, VOC, hypertension (specifically systolic hypertension) with increased injurious pulsatile pulse pressure (pp) and cardiovascular disease (CVD) [4,5,9,10].

The presence of PAD is associated with a marked increase in the prediction of cerebrocardiovascular disease (CCVD) events such as myocardial infarction and stroke with increased morbidity and mortality [11]. Further, Grenon et al. were able to demonstrate that coronary artery disease (CAD) plus PAD had greater risk for cerebrocardiovascular disease (CCVD) events such as myocardial infarction and stroke with increased morbidity and mortality than those individuals with just CAD alone [12]. Indeed, PAD should be considered a red flag warning in that it serves as a biomarker for the increased risk, morbidity and mortality of CCVD events [11,12].

The vascular endothelial cell(s) (ECs) with their protruding cell surface protective endothelial glycocalyx (ecGCx) are the first cells and surface layer that are exposed to the circulating blood plasma, red blood cells, leukocytes, and multiple injurious species that are capable of triggering EC activation and dysfunction (ECact/dys) (Figure 1 and Figure 2) [13,14,15,16,17,18,19,20,21].

ECact/dys results in a proinflammatory surface with attenuation and/or loss of the ecGCx (activation), while ECdys results in a decrease in bioavailable nitric oxide (NO) [9]. Indeed, EC dysfunction with decreased bioavailability of NO may sound an alarm within the cells of the vessel wall (Figure 3) [13,14,15,16,17,18,19,20,21,22].

Interestingly, the decrease in the bioavailability of NO may not only sound an alarm but also may act to activate an alarmin system also referred to as damage-associated molecular patterns (DAMPs), wherein multiple response to injury inflammatory immune signals interact with pattern recognition receptors such as Toll-like receptors (TLRs) [23,24]. A brief list of EC alarmins involved with atherosclerosis include: high mobility group box 1(HMGB1), Fas ligand, heat shock proteins (HSP70-90), S100AB, and S100A9 [23,24].

PAD results primarily from chronic and progressive atherosclerotic disease characterized by narrowing of the peripheral arteries and reduced blood flow to the lower extremities [25]. Vascular arterial stiffening (VAS) of the extremities is closely related to the continued development of atherosclerosis and its progression. As atherosclerotic plaque burden progresses and extracellular matrix accumulate, the arteries and particularly the arterioles will have less lumen diameter and stiffening will continue to progess with less ability to dilate and carry blood oxygen and nutrients resulting in ischemia, ischemic pain and neuropathy in the extremities with intermittent claudication (IC) that may progress to resting pain with ischemic ulceration and or limb loss with critical limb ischemia (CLI) [26].

The primary aim of this narrative review is to increase the database of knowledge regarding the development and progression of PAD in order to decrease its increasing morbidity and mortality. In the following sections, author will discuss PAD and the role of atherosclerosis, aging and HTN, metabolic syndrome and type 2 diabetes mellitus. Also addressed, is the importance of bioavailable NO and its pathologic injurious structural remodeling roles and functional abnormalities in PAD that associate with decreased bioavailability in the presence of ECact/dys as in Figure 3. Additionally, VAS and vascular ossification calcification (VOC), microvessel remodeling in skeletal muscle, and ischemic neuropathy with neuronal remodeling in the lower extremities and the perception of pain will be discussed along with concluding remarks and future directions.

2. Peripheral Arterial Disease (PAD)

PAD results from a chronic progressive accumulation of atherosclerotic lesions that is associated with increased VAS, which is associated with increasing injurious pulsatile pulse pressure (pp) to the endothelium and vessel wall. PAD also associates with decreasing NO bioavailability (as a result of ECact/dys), which results in varying degrees of vascular obstruction and determines the clinical ischemic symptoms of the involved extremities. Further, PAD is often described as a partial or complete occlusion of circulating blood flow to the extremities due to a decrease in patency as a result of not only a reduction in elasticity associated with VAS but also a reduction in the diameter (due to atherosclerosis with enlarging atherosclerotic plaques with or without thrombosis) of the extremity vessels [11,12].

2.1. Atherosclerosis in PAD

Atherosclerosis is a systemic, focal occurring, chronic, progressive, fibroprolifertive, multifactorial, and dysfunctional endothelial-intimal disease that is also a primary underlying and significant cause of VAS and PAD. Further, atherosclerosis is caused by the retention of modified low density lipoprotein-cholesterol (modLDL-C), hemodynamic, and oxidative redox stress (OxRS), which is a term that is inclusive of reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive oxygen, nitrogen and sulfur species (RONSS) and the reactive species interactome (RSI) that results in remodeling of the arterial vessel wall (Figure 4) [1,21,25,27,28,29,30]

[1,21,25,27,28,29,30].

Atherosclerosis is known to be the leading cause of vascular disease (cardiovascular, cerebrovascular, and peripheral artery disease) globally and is significantly associated with dementia when utilizing carotid intima-media thickness to assess the degree of atherosclerosis [31,32]. It initially occurs at areas of turbulent blood flow and low shear stress such as bifurcations and side branches of arteries such as iliac; femoral bifurcations distally and progresses in a cephalad manner to involve the coronaries, carotids and extracranial arteries [1,27,33,34]. While atherosclerosis is a systemic disease it is also focal occurring in regions where there is low shear stress and turbulent blood flow such as bifurcations and side branches (known as atherosclerotic prone regions of the arterial tree). Additionally, atherosclerosis is usually absent in arterial segments with high shear stress and laminar blood flow such as the internal mammary arteries (referred to as atherosclerotic resistant regions) [34]. Importantly, atherosclerosis is not only a fibroproliferative, angiogenic, prothrombotic, multifactorial disease but also a chronic inflammatory disease that begins in the intima. This intimal disease is facilitated by the multiple injurious species as in Figure 1 and Figure 2 (the dirty dozen) and especially the penetration and retention of atherogenic modified-oxidized (modLDL-C/small dense LDL-C or the atherogenic lipid triad phenotype (high triglycerides, low high-density lipoprotein (HDL) and increased small dense lipoprotein-cholesterol (sdLDL-C [35]. Further, this intimal retention of modified and atherogenic lipids are associated with the monocyte to macrophage transformation and the formation of the macrophage foam cell due to the engorgement of modified intimal atherogenic lipids [36,37,38].

Atherosclerosis has been defined by eight major types of plaque lesions by light microscopy histopathology and more recently by magnetic resonance imaging (MRI) (Box 1) [34,39].

Box 1. Comparison of plaque types (I-VIII) by histopath microscopic images to the emerging modified American Heart Association classification for MRI. Note the importance dashed outlined type VI lesions that are known to be vulnerable and prone to rupture or undergo erosion with major and dire clinical consequences. Panel A depicts thrombo-erosion in a type 2 diabetic individual and panel B depicts plaque rupture with thrombosis. Both panels A and B were originally stained with hematoxylin and eosin; however, panel B was pseudo-colored with blue coloring in Microsoft paint to emphasize rupture as compared to erosion (see Figure 5).

Importantly, the American Heart Association classification of type VI atherosclerotic plaques is considered the most prone to rupture because they are defined as "complicated lesions" with features like surface defects, hematoma, hemorrhage, and thrombus formation, indicating a high risk of plaque instability and potential for rupture or even erosion [40]. Additionally, note that type VII plaques are also clinically important, because they allow clinicians to diagnose subclinical coronary artery disease and also aid in predicting those individuals at high risk for future cardiovascular clinical events. Type VII calcified plaques provide the substrate for the indentification of the calcification score, which assist in the evaluation of the severity of atherosclerotic cardiovascular disease [41].

Evidence that T2DM, MetS, and obesity are associated with greater arterial stiffness as compared to non-diabetic individuals is convincing according to Stehouwer et al. and he adds at least 16 supportive references [42]. This increased VAS also helps to explain the increased CVD risk in individuals with diabetes and the MetS (Figure 5) [42,43].

Figure 5. Comparison of type 2 diabetes mellitus (T2DM) culprit thrombo-erosive type VI lesion and sudden death due to arrhythmia to non-diabetic culprit type VI plaque rupture with thrombosis and death from drowning and acute coronary syndrome. Panel A depicts a thrombo-erosion type VI plaque with complete occlusion of lumen. Note that this plaque type is associated with a marked monocytic infiltrate in the adventitia (asterisks) and vasa vasorum (Vv) microvessels. While each of these type VI vulnerable plaques associate with increased vascular arterial stiffness they may result in similar clinical outcomes, i.e. sudden death due to atherothrombosis via different vulnerable plaque morphology. Non-diabetic plaques are more prone to rupture while T2DM individuals are more prone to develop thrombo-erosion and result in sudden death. Additionally, T2DM individuals are known to have increased vascular stiffness as compared to non-diabetic individuals. Panels A and A’ are hematoxylin and eosin (H&E) stained and Panel B is H&E stained that was pseudo-colored blue in Microsoft paint color program in order to provide contrasting colors with A and A’. Modified images provided with permission by CC 4.0 [43]. Phox 22, underlined in red is the light chain component of the reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPHOx) enzyme; its positive staining strongly suggests increased oxidative stress within the plaque and the adventitia that may contribute to plaque instability and erosion in T2DM.

Figure 5. Comparison of type 2 diabetes mellitus (T2DM) culprit thrombo-erosive type VI lesion and sudden death due to arrhythmia to non-diabetic culprit type VI plaque rupture with thrombosis and death from drowning and acute coronary syndrome. Panel A depicts a thrombo-erosion type VI plaque with complete occlusion of lumen. Note that this plaque type is associated with a marked monocytic infiltrate in the adventitia (asterisks) and vasa vasorum (Vv) microvessels. While each of these type VI vulnerable plaques associate with increased vascular arterial stiffness they may result in similar clinical outcomes, i.e. sudden death due to atherothrombosis via different vulnerable plaque morphology. Non-diabetic plaques are more prone to rupture while T2DM individuals are more prone to develop thrombo-erosion and result in sudden death. Additionally, T2DM individuals are known to have increased vascular stiffness as compared to non-diabetic individuals. Panels A and A’ are hematoxylin and eosin (H&E) stained and Panel B is H&E stained that was pseudo-colored blue in Microsoft paint color program in order to provide contrasting colors with A and A’. Modified images provided with permission by CC 4.0 [43]. Phox 22, underlined in red is the light chain component of the reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPHOx) enzyme; its positive staining strongly suggests increased oxidative stress within the plaque and the adventitia that may contribute to plaque instability and erosion in T2DM.

Glucose elevation in T2DM causes an increase in advanced glycation products (AGE) over time, which then promotes collagen crosslinking and interacts with its receptor RAGE and this interaction of AGE/RAGE gives rise to increased OxRS that activate matrix metalloproteinases (MMPs), which result in increased fragmentation of elastin within the media to promote stiffening of the vessel wall. Concurrently, this increase in glucose and AGE instigate a ‘phenotypic switch’ in vascular smooth muscle cell(s) (VSMCs) of the media to form a synthetic type of cell as compared to the normal contractile type that increases the synthesis of stiffer collagen and also the stiff ECM hyaluronan-glycosaminoglycan components as in Figure 4 and Figure 5 to result in increased VAS [44].

While atherosclerosis is defined as being a chronic progressive intimal disease, it is important to note that even when there is continued atheroma lipid, PGN, hyaluronan expansion that these lesions do not usually become clinically symptomatic unless there are superimposed progressive remodeling changes. These changes include the deposition of thrombotic material (intraplaque hemorrhage), inflammation, calcification, and malignant-like invasion via angiogenesis of the atheromatous plaque with plaque hemorrhage and rupture [27,34,43]. The malignant-like invasion of the plaque by the adventitial-derived vasa vasorum (Vv) vessels [27,45] allow for the direct delivery of inflammatory cells. Also, these thin walled Vv vessels are prone to rupture and result in intraplaque hemorrhage with the subsequent release of red blood cels into the lipid core and supply a rich source of membranous lipid material to increase free cholesterol into the plaque. Additionally, these changes are associated necrosis/apoptosis of macrophage foam cells and VSMCs to further increase inflammation and thinning of the luminal surface plaque cap to create a thin-cap atheroma that is prone to rupture [46]. At the shoulders of these thin-cap atheroma, there is an accumulation of inflammatory macrophages resulting in hot plaques [42]. These macrophages are known to result in the activation of the proteolytic matrix metalloproteinases (MMPs) to result in thinning and eventual rupture of these vulnerable plaques with devastating acute coronary syndromes, which present clinically as luminal thrombosis and myocardial infarction in the heart or stroke in the brain (Figure 6) [46].

Notably, these hot plaques with increased temperature represent calor or heat of the four cardinal signs of inflammation: rubor (increased blood flow), dolar (pain), calor (heat), and tumor (swelling) were determined to be an independent predictor in regards to the clinical outcomes of individuals undergoing coronary interventions [47].

When thoughts turn to the realm of PAD and its transition to chronic limb-threatening ischemia (CLTI) or CLI that is considered to be the end-stage of PAD, one has to think about how differently the role of atherosclerotic plaques may differ from the coronary regional beds and the iliac, femoral-popliteal, and the infra-popliteal regional arterial beds (Figure 7).

For example, Narula et al. were able to demonstrate that luminal occlusion by thrombosis associated with insignificant atherosclerotic plaques in CLI, especially in the infra-popliteal (INFRA-POP) regions as in Figure 7 [48]. Also, they were able to show that pathological remodeling of PAD resulted from thrombosis regardless of the extent of atherosclerotic plaques and further that 75% of distal infra-popliteal arteries with stenosis had thrombi even in the presence of insignificant atherosclerotic plaques [49]. These findings by Narula et al. in 2018 and 2020 strongly suggest the need for both a preventive and therapeutic course of dual antiplatelet therapeutic regimens in order to decrease thrombosis, especially in the INFRA-POP regions of the extremities [48,49].

2.2. Advancing Age and essential hypertension (eHTN-HTN) in PAD

eHTN-HTN (also known as primary or idiopathic HTN) is the chronic elevation of blood pressure without a definite identifiable cause such as occurs in secondary reno-vascular hypertension with a known cause. Advancing chronological age (aging) is strongly associated with PAD and HTN, plus they are well-known risk factors for atherosclerosis that is the most common cause of PAD (as discussed in section 2.1.), VAS, and eHTN [50]. Also, many feel that VAS may precede the development and progression of atherosclerosis, PAD, and importantly HTN [50,51,52]. Importantly, aging is thought to be the strongest risk factor for the development of VAS [53]. Indeed, the fatigue and failure of the vessel wall due to the increased injurious pp with chronic wall stress that also associates with stiffening of endothelial cells, internal elastic lamina fragmentation or loss, and dysfunction, loss of elastin lamellar units, and collagen cross-linking with stiffening contribute to the development and progression of VAS and PAD. Also, there is VSMC stiffening and accumulation of advanced glycation end product(s) (AGEs) that bind to its receptor (RAGE) known to increase OxRS with increased MMP production [54]. Notably, HTN is the second most common cause of VAS following aging [54].

HTN is known to associate with and contribute to the development and progression of atherosclerosis, PAD, and VAS [55] as well as acting as one of the leading causes of atherosclerotic plaque formation (Figure 8) [11,56,57].

Despite the enormous efforts put forth in the treatment of HTN, only 53% of those with documented HTN have their blood pressure controlled according to current guidelines [58] and this is precisely why the wheel icon in Figure 8 was chosen as an icon since HTN just keeps on turning and allows for the negative interaction between HTN and the multiple clinical diseases including PAD.

Many decades of research have implicated HTN and atherosclerosis as potential modifiable risk factors in the development of VAS, cerebrocardiovascular disease and PAD [57,59]. Indeed, both atherosclerosis and HTN are implicated in the development and progression of VAS, CCVD, and small vessel disease (SVD); however, they frequently co-exist and may be present simultaneously especially in older individuals [55]. Also, multiple risk factors for the development of atherosclerosis and eHTN including the MetS play an important role (Figure 9) [59,60,61].

2.3. The Metabolic Syndrome (MetS) and Type 2 Diabetes Mellitus (T2DM) in PAD

The MetS is a cluster of multiple risk factors including the four “Hs” (horsemen): I. Hyperlipidemia (obesity); II. Hyperinsulinemia (IR); III. HTN-isolated systolic hypertension (ISH). IV. Hyperglycemia (impaired glucose tolerance (IGT) with or without T2DM and associated variables [21,57]. In addition to paralleling the obesity epidemic, the MetS associates with an increased risk for the development of T2DM and cerebrocardiovascular disease [57]. Each of the previous four Hs (horsemen) that forms the risk factor clustering, in addition to the MetS itself and the other variables, play an important role in the development of both VAS and PAD [62,63,64]. MetS is a strong predictor of CCVD disease, VAS, and PAD, which is increasingly being recognized as a CCVD risk factor. Additionally, other components/variables of the MetS are capable of interacting synergistically to impact carotid intimal media thickness, extracranial VAS, and PAD (Figure 10) [57,62,63,64,65].

Importantly, VAS has been determined to be independently associated with PAD even when adjusting for several atherosclerotic risk factors including HTN [26]. Also, MetS has a 1.76-fold increased prevalence of PAD as compared to non-MetS individuals [66].

Diabetes (specificallyT2DM) has been increasing globally in the past decade with the International Diabetes Federation indicating that 537 million people worldwide had diabetes in 2021 with a projection of in excess of one billion by 2050 with approximately 90-95% having T2DM [67]. These numbers are huge and with T2DM playing a major role in the development of atherosclerosis and a two-fold increased prevalence of PAD [66] it is important to better understand the relation between T2DM and the development of progression of PAD since T2DM is known to be associated with accelerated atherosclerosis (Figure 11) [1,67,68,69].

2.4. Tobacco smoking, oxidative redox stress (OxRS), and accelerated atherosclerosis in PAD

Smoking is considered to be one the four most common risk factors (smoking, HTN, hyperlipidemia/hypercholesterolemia, and T2DM) for the development of cardiovascular diseases and PAD [70]. Importantly, smoking is one of the most preventable causes of PAD. Indeed, cigarette smoking has a very high risk of developing PAD primarily due to increased OxRS, which induces macrovascular atherosclerosis as a result of at least 4,000 chemicals found in cigarette smoke (highly addictive nicotine, thiocyanates, aldehydes, benzenes, nitrosamines, and polycyclic aromatic hydrocarbons to name a few), and a very large number of highly unstable and damaging free radicals that promote OxRS with depletion of antioxidant reserves. Further, smoking with its known increased OxRS is capable of inducing ECact/dys with increased vascular inflammation and decreased nitric oxide (NO) due to eNOS enzyme uncoupling. This promotes monocyte to macrophage transformation, which instigates VSMC remodeling and a phenotypic switch from a contractile phenotype to a synthetic phenotype that predisposes to migration and the development of vascular ossification-calcification (VOC), and the initiation of thrombo-inflammation [71,72]. Importantly, smoking also associates with impaired folate one-carbon metabolism that is associated with elevated levels of homocysteine (Hcy) and decreased levels of folate (vitamin B9), vitamin B6, and B12 [73]. Elevations of Hcy are a known biomarker of impaired folate one-carbon metabolism, which is associated with increased OxRS that contributes to ECact/dys to increase endothelial inflammation and decrease endothelial-derived bioavailable NO, which results in accelerated atherosclerosis and increased cardiovascular diseases including VAS and PAD [74,75]. Unfortunately, smoking/nicotine still remains extremely addictive and remains difficult for many individuals to quit even with its known perceived damaging effects to the vessels. As a result, many individuals are unable to quit smoking even with the supportive assistance and encouragement by health care providers.

3. Decreased Bioavailability of Endothelial Cell-Derived Nitric Oxide in PAD

When the endothelium is exposed to the multiple injurious species as presented in figures one and two in the introduction, it becomes activated and dysfunctional. ECact/dys associates with a proinflammatory EC (activation) and decreased NO bioavailability (dysfunction). This ECact/dys plays an important role in the development of CCVDs, which includes PAD. Indeed, decreased NO bioavailability plays an important role in the development of atherosclerosis, HTN, VAS and PAD [76,77,78].

In health, vascular EC-derived NO (one of three EC gasotransmitters along with carbon monoxide and hydrogen sulfide) is known to be responsible for numerous pleiotropic and vasculoprotective functions. These protective functions include its anti-inflammatory, antioxidant, antithrombotic, antiatherosclerotic, antiplatelet activation (decreasing adherence and aggregation) with potent vasodilation properties [75,79,80,81,82]. NO synthesis is dependent on its substrate L-arginine oxidation with NADPH and oxygen serving as co-substrates and the endothelial nitric oxide synthase enzyme (eNOS) with its essential cofactor tetrahydrobiopterin (BH4) that must be in its totally reduced form in order to run the eNOS reaction to produce NO and L-citrulline [83]. Unfortunately, when there is increased OxRS, BH4 will be oxidized to BH2 and the eNOS reaction will not run to produce NO. The eNOS reaction is very sensitive to detrimental effects of OxRS since this not only interferes with the total reduction of BH4 but also associates with increased asymmetrical dimethyl arginine (ADMA) that competes with L-arginine for NO production [77]. Also, the known decrease in NO due to the excessive and rapid oxidation of NO to nitrite (NO₂⁻), peroxynitrite, and nitrate (NO₃⁻) via increased OxRS also contributes to decreased NO bioavailability [84]. Unfortunately, when there develops a decreased in NO bioavailability due to ECact/dys as in PAD in Figure 3, a vascular alarm is sounded and this results in at least seven detrimental cellular effects of the arterial vessel wall that promotes not only atherogenesis but also VAS and PAD.

Nuclear factor-kappa B (NF-κB) is an important mediator between the vicious cycle of inflammation and OxRS wherein one instigates the other bidirectionally. Additionally, homeostatic levels of NO are known to directly inhibit NF-κB activation by preventing the phosphorylation and degradation of IκB-α, a protein that keeps NF-κB inactive in the cytoplasm and this relationship results in the findings that when NO is decreased it associates with increased activity of NF-κB [85]. Interestingly, the other gasotransmitter hydrogen sulfide (H2S) is capable of NO production via a cascade of phosphorylation events that starts with p38 mitogen activated protein kinase (MAPK) and Akt to eNOS, to form NO [86,87]. Indeed, EC-derived NO via the eNOS enzyme plays a critical role in the maintenance of vascular homeostasis (Figure 12) [88].

4. Vascular Arterial Stiffening (VAS) in PAD

VAS and PAD are clinically determined by the careful examination of individuals for decreased or asymmetrical pulsations of distal lower extremity arteries and elevated systolic blood pressure-isolated systolic hypertension (ISH) with an increase in pulse pressure. Additional evaluation for VAS includes the more precise and quantitative measurement of the ankle-brachial index (ABI) or the gold standard, carotid-femoral pulse wave velocity (cfPWV) [89]. Importantly, the finding of VAS is well known to be an independent predictor of CVD morbidity and mortality [90,91]. Multiple risk factors for the development of VAS include: aging [62,92,93,94]; MetS [62]; T2DM and insulin resistance [95,96,97]; obesity [98,99]. Also, lifestyle risks are involved such as: smoking [100], physical inactivity-sedentary lifestyle [101], and high salt, high caloric intake as occurs with the diet-induced obesity Western style diets [102,103]. Risk factors for the development of VAS and PAD closely parallel those of CVD [104,105] and importantly, VAS may even begin in youth or middle age with some reports that VAS may precede the development of HTN and metabolic risks [106].

The pathobiology mechanisms for the development and progression of VAS are currently thought to be initiated by injuries to the endothelium by a number of injurious stimuli as previously listed in Figure 2 as the dirty dozen [21]. Once injured, ECact/dys develops, which results in a proinflammatory endothelium with increased OxRS and decreased NO bioavailability. This functional change in the activated and dysfunctional endothelium results in a response to injury wound healing mechanism that becomes chronically activated along with the renin-angiotensin-aldosterone system (RAAS) and the direct damaging effects of AngII and aldosterone induced OxRS and transforming growth factor-beta one (TGF-β1). Following the activation and dysfunction of the endothelium there is an activation of MMPs and various elastases to result in the degradation, fragmentation, and splitting of elastin and loss of compliance and recoil upon stretching. These remodeling changes of elastin give rise to elastin-derived fragments (EDFs) that are a rich source of elastin-derived peptides (EDPs) and along with OxRS are known to result in a vascular smooth muscle cell(s) (VSMC(s) “phenotype switch” (from a contractile phenotype to a synthetic phenotype) in the media. Additionally, in the proliferative phases of the wound healing mechanism the cytokine growth factor TGF-β1 is known to be increased and also stimulates these reactive VSMCs to produce stiffer organized collagen, i.e. fibrosis. In turn, these reactive, dedifferentiated, synthetic, proliferative VSMCs are known to be highly synthetic (with increased production of stiffer collagen (types I) and proteoglycans (PGNs) within the media, and a heightened capability for migration to the intima of the vessel wall to result in and contribute to the neogenesis of the intimal space. These structural and functional remodeling changes to the ECM proteins elastin and collagen result in a decreased elastin/collagen ratio with ensuing increased vascular stiffness (Figure 13 and Figure 14) [21,98,105,107,108,109,110].

T2DM with its accelerated atherosclerosis and HTN frequently co-occur in aging [111,112]. Therefore, it is important to compare some of the effects of atherosclerosis and HTN in the development of VAS in PAD (Box 2).

Box 2. Comparison of atherosclerosis and hypertension (HTN) in the development of vascular arterial stiffening (VAS) and peripheral artery disease (PAD). Notably, atherosclerosis is an intimal disease that associates primarily with PAD and that HTN is a disease that associates with the media of the arterial vessel wall that associates primarily with VAS. However, note that many of the pathologic mechanisms of HTN and atherosclerosis may be associated with either VAS or PAD. Reproduced image provided with permission by CC 4.O [21]. ACE, angiotensin converting enzyme; EC(s), endothelial cells; EDPs, elastin-derived peptides; MMP(s), matrix metalloproteinases; SECact/dys, systemic endothelial cell activation/dysfunction; VSMC(s), vascular smooth muscle cells.

4.1. Bioactive Elastin-Derived Peptides (EDPs) and Transglutaminase 2 (TG2)

The degradation of the internal elastic lamina (IEL) and elastin lamella result in the formation/production of elastin-derived fragments (EDFs) that are a rich source of elastin-derived peptides as visualized in Figure 14 in panels A-C; however, these similar remodeling changes are also found to be present in hypertension (not shown). These bioactive EDPs (also termed matrikines or elastokines) interact with their receptors the elastin receptor complex and are capable of instigating multiple interactions that contribute to the development and progression of VAS in PAD. EDPs are known to promote inflammation and react with RAGE to increase OxRS, NFkappa B, MMP activation, cytokine activation including TNFa and IL-6, increased crosslinking of collagen and elastin to collagen, and importantly to promote phenotypic switching from contractile VSMCs to a synthetic, proliferative, migratory phenotype, that increases the synthesis of collagen and proteoglycans. Indeed, EDPs contribute to VSMC remodeling and further amplify elastin degradation to promote VAS in both atherosclerosis and hypertension that may be present in the development of PAD [113,114,115].

Importantly, transglutaminase 2 (TG2) is an enzyme that is ubiquitously expressed in the vasculature, which promotes the synthesis and crosslinking of extracellular matrix collagen that is also important in the development of VAS. Increased TG2 is a known collagen crosslinker that associates with VAS and affects the extracellular matrix by increasing collagen deposition, and crosslinking (in a calcium-dependent manner) that has been long recognized as critical player in vascular stiffening [102,116]. Herein, it is notable that Ramirez-Perez et al. were able to demonstrate that cystamine (a nonspecific TG2 inhibitor) reduced vascular stiffness in Western diet-fed female mice; however, they did not investigate cysteamines’ role as also being an antioxidant that could additionally contribute to the vascular destiffening effects [102].

The multiple functional and structural remodeling changes discussed in this section are multifactorial and complicated to say the least. However, it is possible to create a more simplified possible sequence of events that help to understand the development and progression of VAS in the development and progression of PAD, which can supplement the recent findings by the multiorganizational bodies for the recommended reading of the management of lower extremity PAD (Figure 15) [117].

Importantly, it has been demonstrated that VAS with its damaging increased pulsatile pulse pressure (an injurious stimuli to the endothelium) is independently associated with PAD even after adjusting for several atherosclerotic risk factors and that there is a significant association between VAS and the onset of PAD regardless of blood pressure status [26,118].

4.2. Vascular Ossification Calcification (VOC) in Type VII Plaques

VOC is the term utilized to define the process of calcium phosphate complexes deposition in the form of hydroxyapatite (Ca10(PO4)6(OH)2) in the media of the vasculature, which also results in increased VAS [21,118,119,120,121,122]. Arterial VOC is a definite characteristic of vascular aging [123,124] and is also increased in clinical diseases such as diabetes, HTN, chronic kidney disease (CKD), and hereditary disorders (Figure 16) [21,107,125,126].

VOC occurs at different locations, e.g. in HTN VOC occurs primarily in the tunica media regions and in diabetes it occurs primarily in the tunica intima; however, in certain situations VOC may co-occur in both regions since these two clinical diseases often co-occur [119,127]. Notably, VOC is no longer thought to be a passive process that associates with aging, but involves a reprogramming of tunica media VSMC from its contractile phenotype to a synthetic and proliferative, migratory phenotype that is capable of becoming an osteogenic cell [108,128]. Indeed, local VSMC cues are different for medial calcification that associates with aging, HTN, senescence, and uremia of renal failure with multiple elevations of uremic toxins and specifically elevated calcium and phosphate levels in contrast to atherosclerosis-related aortic tunica intima calcification, which involves inflammation, OxRS, and apoptosis [99,128].

Also, elastin degradation/fragmentation as depicted in Figure 14C results in the release of bioactive EDPs, which can readily induce an osteogenic phenotype switch along with OxRS from an activated endothelium to induce a contractile VSMC to a synthetic phenotype. Once this phenotype switch occurs these VSMC are capable of producing calcification similar to osteogenic cells in bone in the media that have a predilection to adhere to the damaged elastin lamella within the media or to the internal elastic lamella of the intima [129]. While VOC was not included in the six-steps regarding the development of PAD in Figure 15, it is nevertheless a very important step in its progression.

4.3. PAD involves arterial vessels large and small: Microvessel (Mv) remodeling in skeletal muscle (SkM)

Lower extremity PAD is caused by the initial progression and expansion of atherosclerosis of the large conduit arteries that develop VAS and PAD with ischemia due to progressive luminal narrowing and or thrombosis. However, it is the microvessels (arterioles, capillaries, and venules with lumen diameters ranging from 5 to 500 micrometers) that undergo structural remodeling and functional changes that contribute to the horrendous and horrifying pain associated with claudication, nocturnal leg cramps (lower extremity myalgias and myopathy), and the stabbing-like pain of ischemic peripheral neuropathy [130,131].

Mv remodeling is extensive in PAD and consists of i) basement membrane thickening (primarily due to increased type IV collagen; ii) perivascular collagen I and type III fibrosis due to the initial response to injury to acute or early critical ischemia with an initial increased pericyte coverage of Mv capillary ECs and their capability to transition to myofibroblasts as a result of increased transforming growth factor-beta 1 (TGF-β1) chronically; iii) endothelial cell remodeling, which includes endothelial activation that is proinflammatory and endothelial dysfunction that associates with decreased protective bioavailable NO primarily due to eNOS uncoupling; iv) increased OxRS that is central to the peripheral neuronal unit (PNU), neuromuscular junction (NMJ), and the skeletal muscle myopathy aberrant remodeling of PAD [130,131]. Importantly, there is evidence that decreased lower extremity skeletal muscle microvascularization is primarily responsible for exercise intolerance in PAD [132] and that microvascular disease potentiates the risk of amputation in PAD [133]. Further, the initial response to regional hypoxia due to lower extremity PAD would be increased angiogenesis due to ischemia-induced hypoxia inducible factor-1alpha (HIF-1α) and activation of vascular endothelial growth factor (VEGF) to result in angiogenesis and capillary expansion and density. However, over time this initial angiogenesis may be complicated by capillary loss with the development of string-like microvessels and capillary rarefaction due to the ongoing chronic ischemia induced ECact/dys, OxRS, inflammation, and decreased NO (Figure 17) [131,132,133].

Lower extremity PAD impacts skeletal muscle by increased OxRS damage, inflammation, aberrant mitochondria dysfunction/loss, and decreased adenosine triphosphate (ATP) production, loss of number and function of myofibers, dysfunction and loss of capillary density, increased proteolysis and apoptosis. Although ischemia/ischemia reperfusion injury cycles are considered the principal cause of skeletal muscle pathophysiology in PAD, the impact of other comorbidities such as hyperlipidemia on endothelial dysfunction and skeletal muscle pathophysiology should not be overlooked [135].

In addition to microvessel remodeling there is a co-occurance of skeletal muscle (SkM) remodeling that includes skeletal muscle loss (atrophy/ischemia-induced sarcopenia), endomysial fibrosis, myocyte lipid infiltration, and aberrant mitochondria with loss of pericapillary skeletal muscle mitochondrial mounding and loss of subsarcolemmal mitochondria with reduced ATP production in PAD [136].

Thus, as PAD progresses from chronic atherosclerotic macrovessel disease it results in the development of hypoperfusion and hypometabolism to the lower extremity with microvessel remodeling that transition from the early symptoms of IC, which often progresses to CLI. This results, in structural remodeling in three critical regional tissue beds that are also involved with aberrant functional changes, which include microvessel (Mv), peripheral neuronal unit (PNU), neuromuscular junction (NMJ), and lower extremity SkM regions (Figure 18).

While VAS describes a condition or a process of stiffening of the arterial vessel wall due to a loss of elastin and an increase in stiffer collagen, PAD describes a specific vascular disease associated with the accumulation of intimal atherosclerotic plaques that eventually result in a narrowing of the arterial vessel lumen resulting in ischemia to peripheral arteries as well as an increased incidence of thrombosis and embolism to the distal extremities. Indeed, while VAS and PAD are each important in the development and progression of cerebrocardiovascular diseases, when compared side by side what stands out is how they co-develop, co-occur and share so many commonalities (Box 3) [26,51,70,90,91,137].

Box 3. A simplistic comparison of vascular arterial stiffening (VAS) and peripheral arterial disease (PAD). ACE, angiotensin converting enzyme; CT, computed tomography; MRA, magnetic resonance angiography; MRI, magnetic resonance imaging.

At this juncture, it is noteworthy to point out that PAD may be considered a spectrum disorder/disease as portrayed in following image (Figure 19) [131,133,138].

The clinical identification of PAD is important and ankle brachial index (ABI) and/or toe brachial index (TBI) measurements will allow for its diagnosis that is more sensitive than the clinical findings of intermittent claudication since only 50% of individuals with PAD have symptoms of IC, while the other half with PAD still remain asymptomatic. Additionally, microvessel remodeling is associated with elevations of vascular endothelial growth factor (VEGF) due to hypoxia induced factor alpha (HIF-1α) that induces VEGF.

5. Ischemia and Ischemia/Reperfusion (IR) Injury, Microvessel (Mv) Remodeling, Ischemic Neuropathy, Ischemic Skeletal Muscle (SkM) Remodeling with Myopathy and Myalgias, and Pain Perception

Progressive atherosclerotic macrovascular remodeling changes result in luminal narrowing and additionally contribute to Mv remodeling with ECact/dys and decreased NO that contribute to neuronal and SkM ischemia and ischemia/reperfusion injury. Initially ischemia results in hypoperfusion with hypometabolism and hypoxia to tissues. This combined energy and oxygen deficiency or deficit contribute to both Mt dysfunction and inflammation in SkM. Notably, this brings into play the ROS-inflammation ongoing damaging vicious cycle. Mt dysfunction results in increased OxRS–ROS, which overtime depletes the natural antioxidant reserves. Anerobic metabolism contributes to increased lactic acid, H+ proton accumulation, and the initial decrease in ATP to supply cellular energy; however, over time increased ATP leakage via the opening of Mt transition pores and liberation of ATP upon cellular death allow for the signaling of afferent sensory neurons. These abnormalities along with increased cytokines and chemokines of inflammation also contribute to cellular damage and death via apoptosis-necrosis. Thus, IR injury is largely due to mitochondrial dysfunction and increased mtROS production with increased OxRS and inflammation to result in SkM remodeling (Figure 20) [13,139,140].

Also, Mv remodeling as previously discussed in subsection 4.3. and Figure 18 and Figure 19 is accompanied with a co-occurance of peripheral neuronal unit remodeling in the lower extremities in regards to the development and progression of ischemic neuropathy that occurs in intermittent claudication as it progresses to CLI in lower extremity PAD (Figure 21) [141,142,143].

Ischemic neuropathy is tightly associated with Mv remodeling and subsequent ischemia to the PNU and associated remodeling as in Figure 19, Figure 20 and Figure 21. The perception of pain in ischemic neuropathy of PAD is initially instigated via the ischemic injury, which results in the accumulation of toxic metabolic stimuli such as increased lactic acid, decreased pH, increased hydrogen protons, and ATP. Additionally, this ischemic injury is capable of instigating an activation of mast cells since they serve as the first responders to injury to create a cellular inflammatory response to injury and pain perception. In turn, these activated mast cells are capable of forming a neuroimmune axis with the sensory afferent neurons within the SkM interstitial or endomysial spaces between SkM myocytes (Figure 22) [144].

SkM remodeling in PAD was specifically discussed in section 4.3. Figure 17 and this section Figure 21 and consisted primarily of skeletal muscle loss (atrophy/ischemic sarcopenia), endomysial fibrosis, myocyte lipid infiltration, and aberrant mitochondria with loss of pericapillary skeletal muscle mitochondrial mounding and loss of subsarcolemmal mitochondria with reduced ATP production [136]. Once the lower extremity SkM is injured and remodeled due to chronic atherosclerotic macrovascular disease progression with continued luminal narrowing and Mv remodeling with ischemia occur or acute peripheral embolic events occur, there develops a sequence of signaling events, which result in the perception of pain (Figure 23) [145].

Incidently, these same SkM groups III and IV sensory afferents as depicted in Figure 22 also form the sensory components of the exercise pressor reflex that involves the increase in heart rate and blood pressure following muscle contraction during casual walking and exercise [146,147,148].

The two most common forms of neuropathy are diabetic peripheral neuropathy (DPN) of diabetes (specifically T2DM) and ischemic neuropathy of PAD as discussed in subsection 2.3. and illustrated in Figure 11. Therefore, it is appropriate to compare these two most common forms of neuropathy, especially since diabetes has a 2-fold increase in the prevalence of PAD (Figure 24) [67].

6. Conclusions and Future Directions

PAD is the third most common manifestation of atherosclerosis after coronary artery (CAD) and cerebrovascular disease (CVD) that currently affects between 200-250 million individuals globally, and it is considered to be an aging-associated disease [4,5]. Since we are now living in one of the oldest living populations in history, we can only expect to experience an even greater number of individuals to develop PAD as the global aging population expands along with the major risk factors associated with the development and progression of PAD. Further, numerous authors have pointed out that PAD is underappreciated, understudied, underdiagnosed, and undertreated [150,151,152,153,154] and in the past some have even pointed out that PAD may be the last major pandemic of cardiovascular disease [4,5,150]. Indeed, the diagnosis of PAD should be considered a ‘red flag’ in that it serves as a biomarker for the increased risk, morbidity, and mortality of cardiovascular and cerebrovascular disease that should merit earlier detection and a more aggressive and sustained treatment plan [150].

This narrative review has examined the background of PAD and how it is associated with ECact/dys due to multiple injurious species, atherosclerosis development and progression, aging and HTN, MetS and T2DM, and tobacco smoking. Also discussed, was how ECact/dys with eNOS uncoupling associated with the decrease in vasculoprotective bioavailable nitric oxide in the development and progression of PAD. Further, the role of VAS with elastin loss and associated increase in collagen and proteoglycans, the role of transglutaminase 2, and vascular ossification and calcification and how they are involved with the development and progression of PAD was presented. Then, PAD progression via progressive macrovascular atherosclerosis accumulation with lumen encroachment to limit blood flow to the lower extremities was presented. Also, Mv remodeling as a result of increased VAS and increased pulsatile pp and impaired vasodilation with the persistence of ECact/dys and decreased NO bioavailability, which resulted in ischemia, ischemia/reperfusion (IR) injury was discussed. Finally, IR injury and its effects not only on Mv remodeling but also its effects on the development of ischemic neuropathy with remodeling of the PNU and NMJ, ischemic SkM remodeling. and pain perception were presented.

In regards to future directions, we may need to focus more on the microvessel disease associated with VAS and PAD in regards to the development ischemic neuropathy and the skeletal muscle remodeling in the lower extremities. Especially, since these remodeling changes along with macrovascular remodeling changes are responsible for the horrific debilitating pain that control individual lives every day if they are not eligible for surgical revascularization.

Indeed, future directions should include methods to increase the awareness of PAD not only to individuals but also to their health care providers, especially since the study by Hirsch et al. reported that only 49% of caring physicians were aware that their patients had PAD that were identified by ankle-brachial index (ABI) studies in their large reported study with 6,979 individuals [150]. Therefore, creative planning by primary care provider offices may be necessary in order to overcome the findings that limited time by staff and limited reimbursement were thought to be roadblocks to perform ABI testing [155]. In the future, clinicians might consider annual or new patient vascular health questioners for their patients to fill out prior to examination. Also, regional hospitals that serve multiple out-patient clinics might consider having outpatient vascular health screening clinics that include BP recordings, blood tests to access for hyperlipidemia, and ABI measurements to overcome these concerns regarding underappreciation and underdiagnosis concerns by multiple authors in this field of study.

Because PAD is associated with increased morbidity and mortality and its association with increased prevalence of risk for morbid, mortal CVDs, newer treatment strategies for the prevention and delaying the progression in order to extend life expectancy and prevention of disability, pain, and limb loss are needed. Further, Arabzadeh et al. have discussed the possibility of utilizing strategies like regenerative medicine, cell-based therapies, nano-therapy, gene therapy, and targeted therapy, besides other traditional drug combination therapy for PAD that may be considered promising novel therapies [156]. Also, novel or innovative treatments might be considered regarding the treatment of PAD in addition to aggressive therapy with antihypertensive medications to lower blood pressure (specifically angiotensin converting enzyme inhibitors and angiotensin receptor blockers), lipid lowering strategies with the use of statins and/or monoclonal antibodies along with diet to slow the progression of atherosclerosis, dual antiplatelet therapies to prevent thromboembolism, emphasizing smoking cessation, and if possible regular exercise that are already recommended.

In this regard, author has recently proposed utilizing intravenous sodium thiosulfate (STS) as an innovative, multi-targeting, and repurposed therapeutic approach for the treatment of late-onset Alzheimer’s disease [156] and has also previously suggested STS treatment for its novel antioxidant potential in the treatment of diabetic peripheral neuropathy [142]. Because STS has been successfully and widely utilized in the global treatment of calciphylaxis [127,157], it is felt that STS might also be utilized as an innovative treatment modality for PAD. PAD like late-onset Alzheimer’s disease is also multifactorial in its development and progression, which involves oxidant stress, inflammation, excess calcium deposition in the development of VOC, and ECact/dys with decreased bioavailability of NO due to excessive oxidation and dysfunction of the essential coenzyme tetrahydrobiopterin (BH4) that allows the eNOS enzyme to produce NO may also benefit with the use of STS treatment (Figure 25) [157].

While STS has not yet been approved for the treatment of calciphylaxis in humans, it has now been sucessfully used off-label to treat calciphylaxis for just over two decades and results have been reported in the literature primarily as case reports, small case series, and reviews of calciphylaxis with discussions of STS embedded within these reviews [127,158,159,160,161,162,163,164,165,166,167]. Additionally, STS has been utilized to prevent the excessive oxidative stress associated with cisplatin induced hearing loss for solid tumors and head and neck cancers [168,169] and also recently approved for the treatment in pediatric age populations [170]. Also, STS has been shown to improve cardiac dysfunction and the extracellular matrix remodeling in a heart failure induced arterial venous fistula mouse model, in part, by increasing ventricular H₂S generation [171]. Additionally, Wen et al. found that STS attenuated the progression of vascular calcification and importantly vascular arterial stiffness (VAS) in hemodialysis treated individuals [172]. These findings by Wen et al. are pertinent in that VAS plays an important role in the development and progression of PAD. Further, ischemia and IR injury to skeletal muscle has been shown to be associated with increased OxRS and decreased H2S with SkM dysfunction and weakness. Recently, Du et al were able to demonstrate in rat models that H2S treatment was able to significantly protect rat SkM against ischemia and IR injury [173]. These findings are important since we now know that STS serves as an H2S donor/mimetic as presented in Figure 25 and may be able to restore SkM dysfunction and possibly weakness.

Notably, STS treatment has been consistently associated with a rapid improvement of the neuritic/ischemic pain associated with the vascular calcification of calciphylaxis in human individuals and this may be due to a restoration of eNOS enzyme function and NO production [126,174]. Indeed, STS has U.S. Food and Drug Administration (FDA) approved medical uses, off-label uses, side effects, and possible future uses in clinical diseases (Figure 26) [157].

Of great concern to suffering individuals and their health care providers is that a full understanding of the pathobiology for the development of nocturnal leg cramping and rest pain as occur in critical limb ischemia (CLI) or chronic limb-threatening ischemia (CLTI) still remain uncertain and therefore poses a treatment problem [175]. While Figure 19 provides somewhat of a spectrum and a roadmap for the development and progression of PAD, this image also points to the importance of microvessel remodeling and its relation to the development of ischemic neuropathy and ischemia-related myopathy and myalgias. Author’s personal observations have concluded that a personalized approach of utilizing a walker to prevent falling and stretching the involved cramping muscle(s) by standing upright from the supine nocturnal sleeping position to literally walk away from this horrendous cramping pain of the lower extremity myalgias. While this may not prevent future nocturnal cramping that may occur later during the night, it at least helps to prevent the active cramping at that moment in time. Additionally, author highly recommends the algorithm created by Allen and Kirby for the evaluation and treatment of nocturnal leg cramps [175].

Over the years we have been taught that PAD-associated atherosclerosis begins distally in the abdominal aorta at the iliac artery bifurcations while concurrently developing VAS that progresses cephalad initially to the coronary arteries resulting in CVD, CAD, and congestive heart failure and then to the extracranial carotid arteries to involve the brain’s microvessels with brain ECact/dys of the neurovascular unit to result in the co-occurance of vascular dementia and VCID that co-occurs with neurodegeneration resulting in mixed dementias (cerebrovascular disease) and stroke [21]. In reality, while atherosclerosis is progressing cephalad it is concurrently progressing caudad to the distal lower extremities via the femoral, popliteal, peroneal, tibial, and dorsalis pedis arteries on the road to PAD and perdition. PAD frequently presents clinically with asymptomatic stenosis; however, it is known to progress from intermittent claudication to the most severe form of critical limb ischemia that leads to severe and debilitating pain due to ischemic neuropathy and SkM myopathy with the development of ischemic skin ulcerations, gangrene, and eventual limb amputation that also associates with a high risk of morbidity and mortality [176]. Indeed, we are as old as our arteries [177].