Evaluation of the Role of Microvascular Pathology on Peripheral Artery Disease

Background: Peripheral Artery Disease (PAD) begins with atherosclerotic narrowing of arteries, including those that supply the legs. Individuals with PAD experience pain during walking, which becomes increasingly limiting. Studies from our group and others have shown that a myopathy is present in the skeletal muscle of PAD patients, and is characterized by myofiber degeneration, fibrosis, and remodeling of vessels ranging from 50 – 150 mm in diameter. However, microvascular pathology, particularly of the smallest microvessels (5 – 15 mm in diameter) remains poorly characterized. Furthermore, little is known about the relationships between microvascular architecture, microperfusion, and patient walking performance. We hypothesize that microvascular pathology is present in the terminal microvasculature of PAD muscle compared to control and worsens with PAD severity. Additionally, we hypothesize that microvascular architecture is associated with deficits in micro- and macro- perfusion and walking performance in PAD patients with intermittent claudication (IC). Methods: Gastrocnemius biopsy specimens were collected from control, PAD patients with IC, and PAD patients with critical limb ischemia. Microvascular architecture, microvascular fibrosis, total collagen, and the abundance and phenotype of pericytes were quantified. Microvascular perfusion was assessed by Contrast Enhanced Ultrasonography (CEU). Gardner walking protocols were used to assess claudication onset time (COT) and peak walking time (PWT). Patients also completed the Walking Impairment Questionnaire (WIQ). Results: Microvascular pathology increased with advancing PAD severity and included progressive increases in basement membrane thickening, abundance of aSMA+ pericytes, and microvessel density. In advanced PAD muscle, increases were observed in total fibrotic burden and peri-microvascular Collagen I and IV deposition. aSMA+ pericytes expressed TGF-b1. Relationships were observed between microvascular architecture and microperfusion both at rest and after ischemic stress. Microvascular architecture was associated with macrovascular hemodynamic restrictions. Microvascular architecture was associated with COT, PWT, and patient self-reports of walking speed, walking distance, and stair climbing ability. Conclusions: Microvascular pathology worsens with PAD severity in association with fibrosis. Alteration of microvascular architecture contributes to microperfusion deficits and walking limitations in PAD

Quantification of Daily Physical Activity and Sedentary Behavior of Claudicating Patients

Background Claudication is the most common manifestation of peripheral artery disease (PAD), producing significant ambulatory compromise. Limited information exists on the routine physical activity of claudicating patients. Our objective was to record the intensity/time profiles of physical activity and the timing and duration of sedentary behavior of a sample of community-dwelling claudicating patients. Methods Forty-four claudicating patients referred to our vascular clinic were recruited. Physical activity was recorded using the ActiGraph GT1M activity monitor. The Actigraph monitor is a lightweight instrument designed to measure human movement through changes in acceleration, measured as counts over 1-minute time periods. Data from 7 consecutive days were used for the calculations. We processed the data using the ActiLife software program. Results The average daily activity of the claudicating patients shows a steady increase beginning approximately 05:30 AM until a peak plateau from approximately 10:00 AM to 01:30 PM followed by a steady decrease until approximately 09:30 PM, when a sustained period of inactivity begins. The average claudicating patient takes 3586 steps per day at an average intensity of 1.77 metabolic equivalents of task (METs, a physiological measure expressing the energy cost of physical activities). Average physical activity intensity and peak intensity fluctuate very little during the day, and they rarely exceed the level of light activity (light = \u3c3 METs maximum effort, such as casual walking or light housework). During awake time, approximately 7 hours are spent in sedentary behaviors (\u3c1.5 METs), and sedentary time is spread throughout the day mostly in short intervals between periods of low-energy activity. Conclusions Our study objectively demonstrates the reduced physical activity of claudicating patients and documents physical activity/duration profiles throughout the day. The intensity of the physical activity of the average claudicating patient fluctuates very little during the day and rarely exceeds a light intensity level. Claudicating patients spend approximately half of their awake time in sedentary behavior and when they walk they do it in short bursts followed by several minutes of rest. We anticipate that changes in routine physical activity/duration profiles of patients with PAD will provide relevant, sensitive, and direct measures of the effectiveness of therapeutic interventions

Cytokine signature of inflammation mediated by autoreactive Th-cells, in calf muscle of claudicating patients with Fontaine stage II peripheral artery disease

Peripheral artery disease (PAD), a severe atherosclerotic condition primarily of the elderly, afflicts 200 million individuals, worldwide, and is associated with lower extremity myopathy. Circulating markers of inflammation have been linked to risk and severity of PAD but the contribution of local inflammation to myopathy remains unknown. We evaluated, by ELISA, calf muscle of PAD patients (N = 23) and control subjects (N = 18) for local expression of inflammatory cytokines including Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF), Interleukin 17A (IL-17A), Interferon ϒ (IFN-ϒ), tumor necrosis factor α (TNF-α), and Interleukin 6 (IL-6). One or more of these cytokines were expressed in nineteen patients and 2 controls and coordinated expression of GM-CSF, IL-17A, IFN-ϒ, and TNF-α, a signature of activated, MHC Class II dependent autoreactive Th-cells, was unique to 11 patients. GM-CSF is the central driver of tissue-damaging myeloid macrophages. Patients with this cytokine signature had a shorter (P= 0.017) Claudication Onset Distance (17 m) compared with patients lacking the signature (102 m). Transforming Growth Factor β1 (TGFβ1) and Chemokine Ligand 5 (CCL5) were expressed coordinately in all PAD and control muscles, independently of GM-CSF, IL-17A, IFN-ϒ, TNF-α, or IL-6. TGFβ1 and CCL5 and their gene transcripts were increased in PAD muscle, consistent with increased age-associated inflammation in these patients. Serum cytokines were not informative of muscle cytokine expression. We have identified a cytokine profile of autoimmune inflammation in calf muscles of a significant proportion of claudicating PAD patients, in association with decreased limb function, and a second independent profile consistent with increased “inflammaging” in all PAD patients

Altered Metabolomic Profile in Patients with Peripheral Artery Disease

Peripheral artery disease (PAD) is a common atherosclerotic disease characterized by narrowed or blocked arteries in the lower extremities. Circulating serum biomarkers can provide significant insight regarding the disease progression. Here, we explore the metabolomics signatures associated with different stages of PAD and investigate potential mechanisms of the disease. We compared the serum metabolites of a cohort of 26 PAD patients presenting with claudication and 26 PAD patients presenting with critical limb ischemia (CLI) to those of 26 non-PAD controls. A difference between the metabolite profiles of PAD patients from non-PAD controls was observed for several amino acids, acylcarnitines, ceramides, and cholesteryl esters. Furthermore, our data demonstrate that patients with CLI possess an altered metabolomic signature different from that of both claudicants and non-PAD controls. These findings provide new insight into the pathophysiology of PAD and may help develop future diagnostic procedures and therapies for PAD patients

Correction: Premature aortic smooth muscle cell differentiation contributes to matrix dysregulation in Marfan Syndrome.

[This corrects the article DOI: 10.1371/journal.pone.0186603.]

Premature aortic smooth muscle cell differentiation contributes to matrix dysregulation in Marfan Syndrome.

Thoracic aortic aneurysm and dissection are life-threatening complications of Marfan syndrome (MFS). Studies of human and mouse aortic samples from late stage MFS demonstrate increased TGF-β activation/signaling and diffuse matrix changes. However, the role of the aortic smooth muscle cell (SMC) phenotype in early aneurysm formation in MFS has yet to be fully elucidated. As our objective, we investigated whether an altered aortic SMC phenotype plays a role in aneurysm formation in MFS. We describe previously unrecognized concordant findings in the aortas of a murine model of MFS, mgR, during a critical and dynamic phase of early development. Using Western blot, gelatin zymography, and histological analysis, we demonstrated that at postnatal day (PD) 7, before aortic TGF-β levels are increased, there is elastic fiber fragmentation/disorganization and increased levels of MMP-2 and MMP-9. Compared to wild type (WT) littermates, aortic SMCs in mgR mice express higher levels of contractile proteins suggesting a switch to a more mature contractile phenotype. In addition, tropoelastin levels are decreased in mgR mice, a finding consistent with a premature switch to a contractile phenotype. Proliferation assays indicate a decrease in the proliferation rate of mgR cultured SMCs compared to WT SMCs. KLF4, a regulator of smooth muscle cell phenotype, was decreased in aortic tissue of mgR mice. Finally, overexpression of KLF4 partially reversed this phenotypic change in the Marfan SMCs. This study indicates that an early phenotypic switch appears to be associated with initiation of important metabolic changes in SMCs that contribute to subsequent pathology in MFS

Abnormal Microvascular Architecture, Fibrosis, and Pericyte Characteristics in the Calf Muscle of Peripheral Artery Disease Patients with Claudication and Critical Limb Ischemia

Work from our laboratory documents pathological events, including myofiber oxidative damage and degeneration, myofibrosis, micro-vessel (diameter = 50–150 μm) remodeling, and collagenous investment of terminal micro-vessels (diameter ≤ 15 µm) in the calf muscle of patients with Peripheral Artery Disease (PAD). In this study, we evaluate the hypothesis that the vascular pathology associated with the legs of PAD patients encompasses pathologic changes to the smallest micro-vessels in calf muscle. Biopsies were collected from the calf muscle of control subjects and patients with Fontaine Stage II and Stage IV PAD. Slide specimens were evaluated by Quantitative Multi-Spectral and Fluorescence Microscopy. Inter-myofiber collagen, stained with Masson Trichrome (MT), was increased in Stage II patients, and more substantially in Stage IV patients in association with collagenous thickening of terminal micro-vessel walls. Evaluation of the Basement Membrane (BM) of these vessels reveals increased thickness in Stage II patients, and increased thickness, diameter, and Collagen I deposition in Stage IV patients. Coverage of these micro-vessels with pericytes, key contributors to fibrosis and BM remodeling, was increased in Stage II patients, and was greatest in Stage IV patients. Vascular pathology of the legs of PAD patients extends beyond atherosclerotic main inflow arteries and affects the entire vascular tree—including the smallest micro-vessels

Overexpression of KLF4 in mgR SMCs normalized expression of contractile markers.

<p>a) KLF4 mRNA levels were lower in the mgR SMCs compared to WT SMCs derived from PD7 aortic tissue. Both α-actin and calponin mRNA were significantly higher in mgR SMCs compared to WT SMCs. Values represent the mean relative qualification of three cell lines per group (*p< 0.05 WT vs. mgR). This was consistent with difference in protein expression shown by Western Blot (right panel). b) Bar graph demonstrates KLF4, α-actin, and calponin mRNA expression after adenoviral transduction of mgR SMCs. Dotted line represents WT SMC expression. Adenoviral transduction with KLF4 expressing vector resulted in increased KLF4 and decreased α-actin and calponin expression (mean ± SEM, n = 3. * p < 0.05 vs. no infection and # p < 0.05 vs. AdEmpty control infection. 18s was used to normalize samples. Right panel represents a Western Blot showing downregulation of α-actin, and calponin protein expression after adenoviral KLF4 overexpression. c) FACS analysis of aortic SMC proliferation using EdU. Aortic SMCs were isolated from WT and mgR mice. SMCs from mgR mice were infected with adenovirus containing KLF4 (AdKLF4). As a negative control, SMCs from mgR mice were infected with empty vector adenovirus (AdEmpty). Cells from WT and mgR mice without adenoviral infection were baseline controls.</p