Targeting Anti-Angiogenic VEGF165b-VEGFR1 Signaling Promotes Nitric Oxide Independent Therapeutic Angiogenesis in Preclinical Peripheral Artery Disease Models.

Nitric oxide (NO) is the critical regulator of VEGFR2-induced angiogenesis. Neither VEGF-A over-expression nor L-Arginine (NO-precursor) supplementation has been effective in helping patients with Peripheral Artery Disease (PAD) in clinical trials. One incompletely studied reason may be due to the presence of the less characterized anti-angiogenic VEGF-A (VEGF165b) isoform. We have recently shown that VEGF165b inhibits ischemic angiogenesis by blocking VEGFR1, not VEGFR2 activation. Here we wanted to determine whether VEGF165b inhibition using a monoclonal isoform-specific antibody against VEGF165b vs. control, improved perfusion recovery in preclinical PAD models that have impaired VEGFR2-NO signaling, including (1) type-2 diabetic model, (2) endothelial Nitric oxide synthase-knock out mice, and (3) Myoglobin transgenic mice that have impaired NO bioavailability. In all PAD models, VEGF165b inhibition vs. control enhanced perfusion recovery, increased microvascular density in the ischemic limb, and activated VEGFR1-STAT3 signaling. In vitro, VEGF165b inhibition vs. control enhanced a VEGFR1-dependent endothelial survival/proliferation and angiogenic capacity. These data demonstrate that VEGF165b inhibition induces VEGFR1-STAT3 activation, which does not require increased NO to induce therapeutic angiogenesis in PAD. These results may have implications for advancing therapies for patients with PAD where the VEGFR2-eNOS-NO pathway is impaired.

Electromagnetic energy (670 nm) stimulates vasodilation through activation of the large conductance potassium channel (BKCa).

INTRODUCTION: Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC. METHODS: Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique. RESULTS: R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening. CONCLUSION: R/NIR vasodilation requires indirect activation of the BKca channel.

Effect of Creatine Supplementation on Functional Capacity and Muscle Oxygen Saturation in Patients with Symptomatic Peripheral Arterial Disease: A Pilot Study of a Randomized, Double-Blind Placebo-Controlled Clinical Trial.

The aim of the study was to verify the effects of creatine (Cr) supplementation on functional capacity (walking capacity; primary outcome) and calf muscle oxygen saturation (StO2) (secondary outcome) in symptomatic peripheral arterial disease (PAD) patients. Twenty-nine patients, of both sexes, were randomized (1:1) in a double-blind manner for administration of placebo (PLA, n = 15) or creatine monohydrate (Cr, n = 14). The supplementation protocol consisted of 20 g/day for 1 week divided into four equal doses (loading phase), followed by single daily doses of 5 g in the subsequent 7 weeks (maintenance phase). Functional capacity (total walking distance) was assessed by the 6 min walk test, and calf muscle StO2 was assessed through near infrared spectroscopy. The measurements were collected before and after loading and after the maintenance phase. The level of significance was p < 0.05. No significant differences were found for function capacity (total walking distance (PLA: pre 389 ± 123 m vs. post loading 413 ± 131 m vs. post maintenance 382 ± 99 m; Cr: pre 373 ± 149 m vs. post loading 390 ± 115 m vs. post maintenance 369 ± 115 m, p = 0.170) and the calf muscle StO2 parameters (p > 0.05). Short- and long-term Cr supplementation does not influence functional capacity and calf muscle StO2 parameters in patients with symptomatic PAD.

Protection against peripheral artery disease injury by Ruan Jian Qing Mai formula via metabolic programming.

Ruan Jian Qing Mai formula (RJQM), a multicomponent herbal formula, has been widely used to treat peripheral arterial disease (PAD) in China. However, its active compounds and mechanisms of action are still unknown. First, RNA sequencing analysis of 15 healthy and 16 PAD samples showed that 524 PAD differential genes were significantly enriched in Go Ontology (ribonucleotide metabolic process, oxidoreductase complex, and electron transfer activity), Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA pathways (OXPHOS and TCA cycle), miRNA (MIR183), and kinase (PAK6). Fifty-three active ingredients in RJQM had similar structures to the seven drug molecules in CLUE. Then, network topology analysis of the 53 components-target-pathway-disease network yielded 10 active ingredients. Finally, computational toxicity estimations showed that the median lethal dose (LD50) of the 10 active ingredients was above 1000 mg/kg, and eight of them did not cause hepatotoxicity, mutagenicity, carcinogenicity, cytotoxicity, and immunotoxicity nor activate 12 toxic pathways. In conclusion, RJQM has a protection effect on PAD by regulating a complex molecular network. Part of the mechanism is associated with the regulation of OXPHOS by 10 active components, which may alleviate mitochondrial dysfunction and pathological metabolic programming.

Effects of Valsartan on Restenosis in Patients with Arteriosclerosis Obliterans of the Lower Extremities Undergoing Interventional Therapy: A Prospective, Randomized, Single-Blind Trial.

BACKGROUND The aim of this study was to further clarify the effects of valsartan on restenosis in patients with arteriosclerosis obliterans of the lower extremities. MATERIAL AND METHODS Patients with arteriosclerosis obliterans of the lower extremities undergoing continuous stent implantation in the superficial femoral artery were enrolled and randomly divided into an ARB group and a control group. Patients in the ARB group received valsartan orally in a single-blind manner and were followed up for 6 months. An evaluation was performed based on the criteria for clinical efficacies designed by the Committee of Vascular Disease, Chinese Association of Integrative Medicine. The total clinical effective rate was calculated, and ankle brachial index (ABI) of the patients was assessed. The concentrations of interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hs-CRP) were measured using enzyme-linked immunosorbent assay. The in-stent restenosis of patients was examined by angiography. RESULTS One patient in the control group died due to acute cerebral hemorrhage 4 months after enrollment, and 1 patient was lost to follow-up due to acute myocardial infarction during follow-up 5 months after enrollment. Age, sex, Fontaine stage, and underlying diseases were comparable between the 2 groups. Hs-CRP (3.93±1.43) and IL-6 (11.26±2.29) levels were significant different in the ARB group compared with the control group. The postoperative follow-up showed that ABI was 0.98±0.20 in the ARB group and 0.62±0.48 in the control group. CONCLUSIONS Valsartan inhibited the increase in hs-CRP and IL-6 levels, improved clinical efficacies, increased ABI, and decreased the restenosis rate after the interventional therapy in patients with arteriosclerosis obliterans of the lower extremities.

Pre-Clinical Investigation of Keratose as an Excipient of Drug Coated Balloons.

BACKGROUND: Drug-coated balloons (DCBs), which deliver anti-proliferative drugs with the aid of excipients, have emerged as a new endovascular therapy for the treatment of peripheral arterial disease. In this study, we evaluated the use of keratose (KOS) as a novel DCB-coating excipient to deliver and retain paclitaxel. METHODS: A custom coating method was developed to deposit KOS and paclitaxel on uncoated angioplasty balloons. The retention of the KOS-paclitaxel coating, in comparison to a commercially available DCB, was evaluated using a novel vascular-motion simulating ex vivo flow model at 1 h and 3 days. Additionally, the locoregional biological response of the KOS-paclitaxel coating was evaluated in a rabbit ilio-femoral injury model at 14 days. RESULTS: The KOS coating exhibited greater retention of the paclitaxel at 3 days under pulsatile conditions with vascular motion as compared to the commercially available DCB (14.89 ± 4.12 ng/mg vs. 0.60 ± 0.26 ng/mg, p = 0.018). Histological analysis of the KOS-paclitaxel-treated arteries demonstrated a significant reduction in neointimal thickness as compared to the uncoated balloons, KOS-only balloon and paclitaxel-only balloon. CONCLUSIONS: The ability to enhance drug delivery and retention in targeted arterial segments can ultimately improve clinical peripheral endovascular outcomes.

Vitamin D as A Protector of Arterial Health: Potential Role in Peripheral Arterial Disease Formation.

Atherosclerotic occlusive diseases and aneurysms that affect large and medium-sized arteries outside the cardiac and cerebral circulation are collectively known as peripheral arterial disease (PAD). With a rise in the rate of aging population worldwide, the number of people diagnosed with PAD is rapidly increasing. The micronutrient vitamin D is an important steroid hormone that acts on many crucial cellular mechanisms. Experimental studies suggest that optimal levels of vitamin D have beneficial effects on the heart and blood vessels; however, high vitamin D concentrations have been implicated in promoting vascular calcification and arterial stiffness. Observations from various clinical studies shows that deficiency of vitamin D has been associated with a greater risk of PAD. Epidemiological studies have often reported an inverse relation between circulating vitamin D status measured in terms of 25-hydroxivitamin D [25(OH)D] levels and increased cardiovascular disease risk; however, randomized controlled trials did not show a consistent positive effect of vitamin D supplementation on cardiovascular disease risk or events. Even though PAD shares all the major risk factors with cardiovascular diseases, the effect of vitamin D deficiency in PAD is not clear. Current evidence suggests a strong role of vitamin D in promoting genomic and epigenomic changes. This review summarises the current literature that supports the notion that vitamin D deficiency may promote PAD formation. A better understanding of underlying pathological mechanisms will open up new therapeutic possibilities which is the main unmet need in PAD management. Furthermore, epigenetic evidence shows that a more holistic approach towards PAD prevention that incorporates a healthy lifestyle, adequate exercise and optimal nutrition may be more effective in protecting the genome and maintaining a healthy vasculature.

Peripheral Artery Disease Is Associated with a Deficiency of Erythrocyte Membrane n-3 Polyunsaturated Fatty Acids.

Population-based data suggest that individuals who consume large dietary amounts of n-3 polyunsaturated fatty acids (PUFA) have lower odds of peripheral artery disease (PAD); however, clinical studies examining n-3 PUFA levels in patients with PAD are sparse. The objective of this study is to compare erythrocyte membrane fatty acid (FA) content between patients with PAD and controls. We conducted a cross-sectional study of 179 vascular surgery outpatients (controls, 34; PAD, 145). A blood sample was drawn and the erythrocyte FA content was assayed using capillary gas chromatography. We calculated the ratio of the n-3 PUFA eicosapentaenoic acid (EPA) to the n-6 PUFA arachidonic acid (ARA) as well as the omega-3 index (O3I), a measure of erythrocyte content of the n-3 PUFA, EPA, and docosahexaenoic acid (DHA), expressed as a percentage of total erythrocyte FA. Compared with controls, patients with PAD smoked more and were more likely to have hypertension and hyperlipidemia (p < 0.05). Patients with PAD had a lower mean O3I (5.0 ± 1.7% vs 6.0 ± 1.6%, p < 0.001) and EPA:ARA ratio (0.04 ± 0.02 vs 0.05 ± 0.05, p < 0.001), but greater mean total saturated fats (39.5 ± 2.5% vs 38.5 ± 2.6%, p = 0.01). After adjusting for several patient characteristics, comorbidities, and medications, an absolute decrease of 1% in the O3I was associated with 39% greater odds of PAD (odds ratio [OR] 1.39, 95% confidence interval [CI] 1.03-1.86, and p = 0.03). PAD was associated with a deficiency of erythrocyte n-3 PUFA, a lower EPA:ARA ratio, and greater mean total saturated fats. These alterations in FA content may be involved in the pathogenesis or development of poor outcomes in PAD.

Ultrasound imaging and on-demand therapy of peripheral arterial diseases using H2O2-Activated bubble generating anti-inflammatory polymer particles.

Muscles of peripheral artery disease (PAD) patients are under oxidative stress associated with a significantly elevated level of reactive oxygen species (ROS) including hydrogen peroxide (H2O2). Curcumin is a major active constituent of turmeric and is well known for its highly potent antioxidant, anti-inflammatory and angiogenic effects. We previously reported antioxidant vanillyl alcohol-incorporated copolyoxalate (PVAX) which is designed to rapidly scavenge H2O2 and release bioactive vanillyl alcohol and CO2 in a H2O2-triggered manner. In this work, we developed curcumin-loaded PVAX (CUR-PVAX) nanoparticles as contrast-enhanced ultrasound imaging agents as well as on-demand therapeutic agents for ischemic injuries based on the hypothesis that PVAX nanoparticles generate echogenic CO2 bubbles through H2O2-triggered oxidation of peroxalate esters and the merger of curcumin and PVAX exerts H2O2-activatable synergistic therapeutic actions. CUR-PVAX nanoparticles also displayed the drastic ultrasound signal in ischemic areas by generating CO2 bubbles. CUR-PVAX nanoparticles exhibited significantly higher antioxidant and anti-inflammatory activities than empty PVAX nanoparticles and equivalent curcumin in vascular endothelial cells. A mouse model of ischemic injury was used to evaluate the potential of CUR-PVAX nanoparticles as ultrasound imaging agents and on-demand therapeutic agents. CUR-PVAX nanoparticles significantly suppressed the expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß). Moreover, CUR-PVAX nanoparticles significantly enhanced the level of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (PECAM-1, also known as CD31), leading to blood perfusion into ischemic tissues. We, therefore, believe that CUR-PVAX nanoparticles hold great translational potential as novel theranostic agents for ischemic diseases such as PAD.

Kidney function, bone-mineral metabolism markers, and future risk of peripheral artery disease.

BACKGROUND AND AIMS: Reduced kidney function is a risk factor for lower-extremity peripheral artery disease (PAD). However, the associations of novel filtration markers with PAD are yet to be quantified. Moreover, little is known on whether bone-mineral metabolism (BMM) markers are related to incident PAD beyond kidney function. METHODS: Using data from 12,472 participants at baseline (1990-1992) of the Atherosclerosis Risk in Communities (ARIC) study, we comprehensively quantified the associations of kidney related markers with incident PAD (defined as hospitalizations with diagnosis of lower-extremity atherosclerosis, revascularization, or amputation). Kidney related markers of interest included estimated glomerular filtration rate (eGFR) (based on creatinine, cystatin C, and both), cystatin C, beta-2 microglobulin (B2M), and BMM markers (serum fibroblast growth factor 23, parathyroid hormone, calcium, and phosphorus). RESULTS: During a median follow-up of 21 years, 471 participants developed incident PAD. Low eGFR was significantly associated with future PAD risk, with slightly stronger relationship when cystatin C was used (adjusted hazard ratio [HR] 6.3-8.3 for eGFR <30 and 2.4-3.5 for eGFR 30-59 vs. eGFR ≥90 mL/min/1.73 m2). Among all filtration markers, B2M had the strongest association with incident PAD (HR for top vs. bottom quartile 2.60 [95% CI: 1.91-3.54] for B2M vs. 1.20 [0.91-1.58] for creatinine-based eGFR). Among BMM markers, only phosphorus remained significant for PAD risk beyond potential confounders, including kidney function (HR 1.47 [1.11-1.94] in top quartile). CONCLUSIONS: Kidney dysfunction was independently associated with future PAD risk, particularly when assessed with cystatin C and B2M. Among the BMM markers tested, phosphorus was most robustly associated with incident PAD beyond kidney function. Our results suggest the potential usefulness of novel filtration markers for PAD risk assessment and the possible role of phosphorus in the pathophysiology of PAD.

Coordinated Activation of VEGF/VEGFR-2 and PPARδ Pathways by a Multi-Component Chinese Medicine DHI Accelerated Recovery from Peripheral Arterial Disease in Type 2 Diabetic Mice.

Diabetic mellitus (DM) patients are at an increased risk of developing peripheral arterial disease (PAD). Danhong injection (DHI) is a Chinese patent medicine widely used for several cardiovascular indications but the mechanism of action is not well-understood. We investigated the therapeutic potential of DHI on experimental PAD in mice with chemically induced as well as genetic (KKAy) type 2 DM and the overlapping signaling pathways regulating both therapeutic angiogenesis and glucose homeostasis. Compared with normal genetic background wild type (WT) mice, both DM mice showed impaired perfusion recovery in hind-limb ischemia (HLI) model. DHI treatment significantly accelerated perfusion recovery, lowered blood glucose and improved glucose tolerance in both DM models. Bioluminescent imaging demonstrated a continuous ischemia-induced vascular endothelial growth factor receptor 2 (VEGFR-2) gene expressions with a peak time coincident with the maximal DHI stimulation. Flow cytometry analysis showed a DHI-mediated increase in endothelial progenitor cell (EPC) mobilization from bone marrow to circulating peripheral blood. DHI administration upregulated the expression of vascular endothelial growth factor A (VEGF-A) and VEGF receptor-2 (VEGFR-2) in ischemic muscle. A cross talk between ischemia-induced angiogenesis and glucose tolerance pathways was analyzed by Ingenuity Pathway Analysis (IPA) which suggested an interaction of VEGF-A/VEGFR-2 and peroxisome proliferator-activated receptor δ (PPARδ)/peroxisome proliferator-activated receptor γ (PPARγ) genes. We confirmed that upregulation of VEGF-A/VEGFR-2 by DHI promoted PPARδ gene expression in both type 2 diabetic mice. Our findings demonstrated that a multi-component Chinese medicine DHI effectively increased blood flow recovery after tissue ischemia in diabetic mice by promoting angiogenesis and improving glucose tolerance through a concomitant activation of VEGF-A/VEGFR-2 and PPARδ signaling pathways.

Chronic inflammatory disease and osteopathy: a systematic review.

BACKGROUND: Chronic inflammatory diseases (CID) are globally highly prevalent and characterized by severe pathological medical conditions. Several trials were conducted aiming at measuring the effects of manipulative therapies on patients affected by CID. The purpose of this review was to explore the extent to which osteopathic manipulative treatment (OMT) can be benefi-cial in medical conditions also classified as CID. METHODS: This review included any type of experimental study which enrolled sub-jects with CID comparing OMT with any type of control procedure. The search was conducted on eight databases in January 2014 using a pragmatic literature search approach. Two independent re-viewers conducted study selection and data extraction for each study. The risk of bias was evaluated according to the Cochrane methods. Heterogeneity was assessed and meta-analysis performed where possible. RESULTS: 10 studies met the inclusion criteria for this review enrolling 386 subjects. The search identified six RCTs, one laboratory study, one cross-over pilot studies, one observation-al study and one case control pilot study. Results suggest a potential effect of osteopathic medicine on patients with medical pathologies associated with CID (in particular Chronic Obstructive Pul-monary Disease (COPD), Irritable Bowel Syndrome, Asthma and Peripheral Arterial Disease) com-pared to no treatment or sham therapy although data remain elusive. Moreover one study showed possible effects on arthritis rat model. Meta-analysis was performed for COPD studies only show-ing no effect of any type of OMT applied versus control. No major side effects were reported by those receiving OMT. CONCLUSION: The present systematic review showed inconsistent data on the effect of OMT in the treatment of medical conditions potentially associated with CID, however the OMT appears to be a safe approach. Further more robust trials are needed to determine the direction and magnitude of the effect of OMT and to generalize favorable results.

Pycnogenol® and Centella asiatica in the management of asymptomatic atherosclerosis progression.

AIM: The aim of the study was to evaluate the effect of the nutritional supplements Pycnogenol® and total triterpenic fraction of Centella asiatica (TTFCA) on atherosclerosis progression in low-risk asymptomatic subjects with carotid or femoral stenosing plaques. METHODS: This was an observational pilot, substudy of the San Valentino epidemiological cardiovascular study. The study included 824 subjects aged 45-60 without any conventional risk factors who had a stenosing atherosclerotic plaque (>50-60%) in at least one carotid or common femoral bifurcation, allocated into 6 groups: Group 1 (Controls): management was based on education, exercise, diet and lifestyle changes. This same management plan was used in all other groups; group 2: Pycnogenol® 50 mg/day; group 3: Pycnogenol® 100 mg/day; group 4: Aspirin® 100 mg/day or ticlopidine 250 mg/day if intolerant to aspirin; group 5: Aspirin® 100 mg/day and Pycnogenol® 100 mg/day; group 6: Pycnogenol® 100 mg/day plus TTFCA 100 mg/day. The follow-up lasted 42 months. Plaque progression was assessed using the ultrasonic arterial score based on the arterial wall morphology and the number of plaques that progressed and on the number of subjects that had cardiovascular events. A secondary endpoint was to evaluate the changes in oxidative stress at baseline and at 42 months. RESULTS: The ultrasonic score increased significantly in groups 1, 2, and 4 (>1%) but not in groups 3, 5 and 6 (<1%) suggesting a beneficial effect of Pycnogenol® 100 mg. Considering the percent of patients that progressed from class V (asymptomatic) to VI (symptomatic) there was a progression of plaques in 48.09% of controls. In the Pycnogenol® 100 (group 3, 10.4%) and in the Aspirin®+ Pycnogenol® (group 5, 10.68%) progression was half of what observed with antiplatelet agent (group 4, 20.93%); in the TTFCA+ Pycnogenol®group (group 6) progression was 7.4 times lower than in controls; 3.22 times lower than in the antiplatelet agents group (4). Events (hospital admission, specialized care) were observed in 16.03% of controls; there were 8.83% of subjects with events with Pycnogenol® 50 mg and 8% in group 3 (Pycnogenol® 100 mg). In group 4 (antiplatelets), 8.52% of subjects had events; in group 5, 6.87% of subjects had events and in group 6 (TTFCA+ Pycnogenol®) only 4.41% had events (this was the lowest event rate; P<0.05). All treatment groups had a significantly lower event rate (P<0.05) in comparison with controls. Considering treatments groups 2, 3, 5, 6 had a lower number (P<0.05) of subjects in need of cardiovascular management in comparison with controls. The need for risk factor management was higher in controls and lower in group 6 (P<0.05). In groups 2 to 6 the need for risk factor management was lower than in controls (P<0.05). Including all events (hospital admission, need for treatment or for risk management) 51.9% of controls were involved. In the other groups there was a reduction (from a -9.28% reduction in group 2 to a -26% in group 6) (P<0.002). The most important reduction (higher that in all groups; P<0.05) was in group 6. At 42 months, oxidative stress in all the Pycnogenol® groups was less than in the control group. In the combined group of Pycnogenol® and TTFCA the oxidative stress was less than with Pycnogenol® alone (P<0.001). CONCLUSION: Pycnogenol® and the combination of Pycnogenol® +TTFCA appear to reduce the progression of subclinical arterial plaques and the progression to clinical stages. The reduction in plaque and clinical progression was associated with a reduction in oxidative stress. The results justify a large, randomized, controlled study to demonstrate the efficacy of the combined Pycnogenol® and TTFCA prophylactic therapy in preclinical atherosclerosis.

Techniques to harvest diseased human peripheral arteries and measure endothelial function in an ex vivo model.

OBJECTIVE: Endothelial dysfunction has been studied in animal models. However, direct evidence of endothelial function from human vessels is limited. Our objectives were to optimize methods in harvesting human arteries from amputation specimens, determine endothelial function, and measure responsiveness to l-arginine, a nitric oxide precursor. METHODS: Fresh amputation specimens were transferred expeditiously from the operating room to the bench laboratory for dissection and arterial harvest in an Investigational Review Board-approved protocol. Popliteal and tibial vessels were examined in pilot experiments leading to the use of the anterior tibial artery in consecutive experiments. Human lower extremity anterior tibial artery segments were harvested from 14 amputation specimens. Specimens were rapidly collected and divided for endothelial-dependent relaxation (EDR) studies in a tissue bath apparatus, immunohistochemistry, and intravascular ultrasound-derived virtual histology. A total of 47 ring segments were studied. The data were compared with two-way analysis of variance. RESULTS: Human lower extremity arteries exhibited low responsiveness to acetylcholine (EDR, 24.9%; acetylcholine, 10(-4)). L-arginine supplementation enhanced EDR by 38.5% (P < .0001). N-nitro-L-arginine methyl ester abrogated EDR (P < .0001) in vessels exposed to L-arginine. Arterial responsiveness was intact in all vessels (endothelial independent relaxation to sodium nitroprusside, 113.2% ± 28.1%). Histology and immunohistochemistry confirmed intact endothelium by morphometric analysis, cluster of differentiation 31, endothelial nitric oxide synthase, and arginase II staining. Intravascular ultrasound-derived virtual histology indicated atheroma burden was 11.9 ± 4.7 mm(3)/cm, and plaque stratification indicated fibrous morphology was predominant (59.9%; necrotic core, 16.9%; calcium, 11.2%). Variations in plaque morphology did not correlate with endothelial function or responsiveness to L-arginine. CONCLUSIONS: Human lower extremity arteries demonstrate low baseline endothelial function in patients requiring amputation. Endothelial dysfunction is improved by L-arginine supplementation in an ex vivo model. These results support strategies to increase local levels of nitric oxide in human vessels.

Polyunsaturated fatty acids and peripheral artery disease.

There is substantial evidence that polyunsaturated fatty acids (PUFAs) such as n-3 and n-6 fatty acids (FAs) play an important role in prevention of atherosclerosis. In vitro and in vivo studies focusing on the interactions between monocytes and endothelial cells have explored the molecular effects of FAs on these interactions. Epidemiological surveys, followed by large, randomized, control trials have demonstrated a reduction in major cardiovascular events with supplementation of n-3 FAs in secondary prevention settings. The evidence of beneficial effects specific to patients with peripheral artery disease (PAD) remains elusive, and is the focus of this review.