Skeletal muscle expression of adipose-specific phospholipase in peripheral artery disease.

Flow-limiting atherosclerotic lesions of arteries supplying the limbs are a cause of symptoms in patients with peripheral artery disease (PAD). Musculoskeletal metabolic factors also contribute to the pathophysiology of claudication, which is manifest as leg discomfort that impairs walking capacity. Accordingly, we conducted a case-control study to determine whether skeletal muscle metabolic gene expression is altered in PAD. Calf skeletal muscle gene expression of patients with PAD and healthy subjects was analyzed using microarrays. The top-ranking gene differentially expressed between PAD and controls (FDR < 0.001) was PLA2G16, which encodes adipose-specific phospholipase A2 (AdPLA) and is implicated in the maintenance of insulin sensitivity and regulation of lipid metabolism. Differential expression was confirmed by qRT-PCR; PLA2G16 was downregulated by 68% in patients with PAD (p < 0.001). Expression of Pla2g16 was then measured in control (db/+) and diabetic (db/db) mice that underwent unilateral femoral artery ligation. There was significantly reduced expression of Pla2g16 in the ischemic leg of both control and diabetic mice (by 51%), with significantly greater magnitude of reduction in the diabetic mice (by 79%). We conclude that AdPLA is downregulated in humans with PAD and in mice with hindlimb ischemia. Reduced AdPLA may contribute to impaired walking capacity in patients with PAD via its effects on skeletal muscle metabolism. Further studies are needed to fully characterize the role of AdPLA in PAD and to investigate its potential as a therapeutic target for alleviating symptoms of claudication.

NT5E Genetic Mutation Is a Rare But Important Cause of Intermittent Claudication and Chronic Limb-Threatening Ischemia.

BACKGROUND: NT5Egenetic mutations are known to result in calcification of joints and arteries (CALJA), and worldwide, 14 patients from 7 families have been reported.Methods and Results:A total of 5 patients from 2 independent families with CALJA were found in Japan. Of them, 3 complained of intermittent claudication (IC), and 1 suffered from bilateral chronic limb-threatening ischemia (CLTI). Whole-exome sequencing analysis revealed an identical mutation pattern (c.G3C on the exon 1 start codon) that was unique compared withNT5Emutations reported in other countries. CONCLUSIONS: Vascular specialists need to recognize CALJA as a rare cause of ischemic IC and CLTI.

Matrix metalloproteinase 10 is associated with disease severity and mortality in patients with peripheral arterial disease.

OBJECTIVE: Peripheral arterial disease (PAD) is associated with poor prognosis in terms of cardiovascular (CV) morbidity and mortality. Matrix metalloproteinases (MMPs) contribute to vascular remodeling by degrading extracellular matrix components and play a role in atherosclerosis as demonstrated for MMP-10 (stromelysin-2). This study analyzed MMP-10 levels in PAD patients according to disease severity and CV risk factors and evaluated the prognostic value of MMP-10 for CV events and mortality in lower limb arterial disease after a follow-up period of 2 years. METHODS: MMP-10 was measured by enzyme-linked immunosorbent assay in 187 PAD patients and 200 sex-matched controls. RESULTS: PAD patients presented with increased levels of MMP-10 (702 ± 326 pg/mL control vs 946 ± 473 pg/mL PAD; P < .001) and decreased levels of tissue inhibitor of matrix metalloproteinase 1 (312 ± 117 ng/mL control vs 235 ± 110 ng/mL PAD; P < .001) compared with controls. Among PAD patients, those with critical limb ischemia (n = 88) showed higher levels of MMP-10 (1086 ± 478 pg/mL vs 822 ± 436 pg/mL; P < .001) compared with those with intermittent claudication (n = 99), whereas the MMP-10/tissue inhibitor of matrix metalloproteinase 1 ratio remained similar. The univariate analysis showed an association between MMP-10, age (P = .015), hypertension (P = .021), and ankle-brachial index (P = .006) in PAD patients that remained significantly associated with PAD severity after adjustment for other CV risk factors. Patients with the highest MMP-10 tertile had an increased incidence of all-cause mortality and CV mortality (P < .03). CONCLUSIONS: Our results suggest that MMP-10 is associated with severity and poor outcome in PAD.

Angiotensin-converting enzyme inhibitors for intermittent claudication associated with peripheral arterial disease.

OBJECTIVE: To review published literature regarding the effectiveness of angiotensin-converting enzyme (ACE) inhibitors for managing intermittent claudication (IC) associated with peripheral arterial disease (PAD). DATA SOURCES: A search of MEDLINE/PubMed (1966-July 2013) using the MeSH terms intermittent claudication and angiotensin-converting enzyme inhibitors was conducted. Limits included articles written in English with human participants. Additional data were identified through bibliographic reviews. STUDY SELECTION AND DATA EXTRACTION: All English-language articles identified from the data sources were evaluated. Clinical trials and meta-analyses were included if they evaluated the efficacy of ACE inhibitors for improving functional capacity of patients with PAD with IC. In all, 9 clinical trials and 1 meta-analysis were identified and included for review. ACE inhibitors evaluated in the studies were captopril, lisinopril, perindopril, quinapril, and ramipril. DATA SYNTHESIS: Current medications approved for treating the symptoms and improving function in PAD with IC have limited efficacy. It has been suggested that ACE inhibitors may be effective in PAD with IC. Though data evaluating ACE inhibitors as a class in this patient population are conflicting, results of the largest and longest trial reported that ramipril increases maximum walking time and pain-free walking time and improves quality of life in patients with PAD with IC. CONCLUSIONS: ACE inhibitors may provide some relief of IC symptoms when used in patients with PAD. The greatest functional benefit has been seen with ramipril; it is unknown whether other agents in the class would show similar results. Well-controlled and designed studies with sufficient power and using diverse patient populations are needed.

A phase II dose-ranging study of the phosphodiesterase inhibitor K-134 in patients with peripheral artery disease and claudication.

BACKGROUND: Phosphodiesterase inhibitors have been shown to improve claudication-limited exercise performance in patients with peripheral artery disease. K-134, a novel phosphodiesterase inhibitor, was evaluated in a phase II trial incorporating an adaptive design to assess its safety, tolerability, and effect on treadmill walking time. DESIGN: Patients with peripheral artery disease were randomized to receive placebo (n = 87), K-134 at a dose of 25 mg (n = 42), 50 mg (n = 85), or 100 mg (n = 84), or cilostazol at a dose of 100 mg (n = 89), each twice daily for 26 weeks. Peak walking time (PWT) was assessed using a graded treadmill protocol at baseline and after 14 and 26 weeks of treatment. A Data and Safety Monitoring Board-implemented adaptive design was used that allowed early discontinuation of unsafe or minimally informative K-134 arms. RESULTS: As determined by the prospectively defined adaptive criteria, the 25-mg K-134 arm was discontinued after 42 individuals had been randomized to the arm. During the 26-week treatment period, PWT increased by 23%, 33%, 37%, and 46% in the placebo, 50-mg K-134, 100-mg K-134, and cilostazol arms, respectively (primary analysis placebo vs 100-mg K-134 arm not statistically significant, P = .089). Secondary analyses showed that cilostazol significantly increased PWT after 14 weeks of treatment and that the 100-mg K-134 dose and cilostazol both increased PWT vs placebo after 14 and 26 weeks in those individuals who completed the 26-week trial and were compliant with the study drug, or when the data were analyzed using a mixed-effects model incorporating all time points. K-134 had tolerability and adverse effect profiles similar to that of cilostazol. Both drugs were associated with an increase in withdrawals before study completion due to adverse events compared with placebo. CONCLUSIONS: K-134 was generally well tolerated. K-134 at a dose of 100 mg twice daily did not affect PWT according to the primary analysis, but K-134 and cilostazol both increased PWT when analyzed using a mixed-effects model and in the per-protocol population.

High MAPK p38 activity and low level of IL-10 in intermittent claudication as opposed to stable angina.

AIM: The aim of the present pilot study was to relate the activity of MAPK p38 with the levels of pro- and anti-inflammatory cytokines in a small cohort of patients with either stable angina (N=5) or intermittent claudication (N=5) compared to healthy controls (N=10). METHODS: The activity of MAPK p38 was determined in peripheral blood mononuclear cells, isolated from whole blood by western blot using phospho-specific anti-MAPK p38 antibodies. Cytokine levels of 11 pro- and anti-inflammatory cytokines were determined from the serum using flow cytometry. RESULTS: We found a significant elevation of the MAPK p38 activity in the intermittent claudication group (P=0.0027) compared with the healthy control group whereas the stable angina group showed similar MAPK p38 activity as the healthy control group. The IL-10 level in serum found in the stable angina group was significantly higher compared with both the healthy control group (P=0.0116) and the intermittent claudication group (P=0.0317). CONCLUSION: Our results imply that there is a casual relationship between increased levels of the anti-inflammatory cytokines IL-10 and IL-4 and the activity of the MAPK p38. Possibly has IL-10 a protective role that down-regulates the activity of MAPK p38 and thereby further inflammatory processes in stable angina patients.

Type 3 phosphodiesterase inhibitors may be protective against cerebrovascular events in patients with claudication.

OBJECTIVE: The risk of cerebrovascular events in patients with mild to moderate peripheral vascular disease is significant. Cilostazol is a phosphodiesterase type 3 (PDE3) inhibitor that is effective in the treatment of symptoms of peripheral arterial occlusive disease. The method of action includes antithrombotic, vasodilatory, and antiproliferative effects. METHODS: The Cilostazol: A Study in Long-Term Effects (CASTLE) trial was a prospective randomized double-blinded trial to establish the safety of this PDE3 inhibitor use in 1435 patients with mild to moderate peripheral arterial occlusive disease. A post hoc analysis of the CASTLE trial was undertaken to evaluate cilostazol use on cerebrovascular events. Blinded adjudication of all cerebrovascular events (stroke, transient ischemic attack, and carotid revascularization) in this trial was performed. Kaplan-Meier analysis was used for statistical evaluation. RESULTS: The overall rate of cerebrovascular events was 4.6% (67 of 1435 patients) with a mean follow-up of 515 days. Ischemic vascular events were more common (2.5%) than hemorrhagic events (0.3%; P < .05). The placebo group demonstrated a greater risk for events (6.1%; 43 of 718 patients) versus the cilostazol treated group (3.2%; 24 of 717 patients; P < .05). Cerebrovascular risk factors were similar in both groups. CONCLUSION: The risk of cerebrovascular events in patients with mild to moderate peripheral arterial occlusive disease is 4.6% with a mean follow-up of 515 days. Treatment with PDE3 inhibitors may reduce this risk. Further evaluation of the use of PDE3 inhibitors for prevention of cerebrovascular events should be considered.

Activation of extracellular signal-regulated protein kinase in dorsal horn neurons in the rat neuropathic intermittent claudication model.

Extracellular signal-regulated protein kinase (ERK) is a mitogen-activated protein kinase (MAPK) that mediates several cellular responses to mitogenic and differentiation signals, and activation of ERK in dorsal horn neurons by noxious stimulation is known to contribute to pain hypersensitivity. In order to elucidate the pathophysiological mechanisms of the cauda equina syndrome, secondary to spinal canal stenosis, we evaluated walking dysfunction triggered by forced exercise and activation of ERK in the dorsal horn using a rat model of neuropathic intermittent claudication. Rats in the lumbar canal stenosis (LCS) group showed a shorter running distance from 1 to 14 days after surgery. Two minutes after running on the treadmill apparatus, phosphorylation of ERK was induced in neurons in the superficial laminae in the LCS group but not in the sham group, whereas there was no change in the deeper laminae. Intrathecal administration of the MAPK kinase inhibitor, U0126, 30 min before running, clearly increased the running distance, whereas there was no significant change in the vehicle control group 3 days after surgery. In addition, a prostaglandin E1 analog, OP-1206 alpha-CD, administered orally, improved the walking dysfunction, and further, inhibited activation of ERK following running 7 days after surgery. These findings suggest that intermittent claudication triggered by forced walking might affect the phosphorylation of ERK in the superficial laminae, possibly via transient (partial) ischemia of the spinal cord. ERK activation in the dorsal horn neurons may be involved in the transient pain in the neuropathic intermittent claudication model.

Effect of exercise training on skeletal muscle histology and metabolism in peripheral arterial disease.

Patients with symptomatic peripheral arterial occlusive disease have a claudication-limited peak exercise performance that is improved with exercise training. The effects of training on skeletal muscle metabolism were evaluated in 26 patients with claudication, randomized into a 12-wk program of treadmill training (enhances muscle metabolic activity in normal subjects), strength training (stimulates muscle hypertrophy in normal subjects), or a nonexercising control group. Gastrocnemius muscle biopsies were performed at rest and before and after training. After 12 wk, only treadmill training improved peak exercise performance and peak oxygen consumption. Treadmill training did not alter type I or type II fiber area and did not increase citrate synthase activity but was associated with an increase in the percentage of denervated fibers (from 7.6 +/- 5.4 to 15.6 +/- 7.5%, P < 0.05). Improvement in exercise performance with treadmill training was associated with a correlative decrease in the plasma (r = -0.67) and muscle (r = -0.59) short-chain acylcarnitine concentrations (intermediates of oxidative metabolism). Patients in the strength and control groups had no changes in muscle histology or carnitine metabolism, but strength-trained subjects had a decrease in citrate synthase activity. Thus treadmill training increased peak exercise performance, but this benefit was associated with skeletal muscle denervation and the absence of a "classic" mitochondrial training response (increase in citrate synthase activity). The present study confirms the relationship between skeletal muscle acylcarnitine content and function in peripheral arterial occlusive disease, demonstrating that the response to treadmill training was associated with parallel improvements in intermediary metabolism.

Xanthine oxidase in critically ischaemic and claudicant limbs: profile of activity during early reperfusion.

Xanthine oxidase activity in blood from the ipsilateral femoral vein, and the relationship between xanthine oxidase production and the products of lipid peroxidation, were studied before operation and for 60 min following release of clamps after successful revascularization in two groups of patients with claudication or critical ischaemia. Before revascularization, detectable levels of xanthine oxidase were found only in patients with critical ischaemia. Clamping during bypass surgery led to release of xanthine oxidase in claudicants, but this activity reduced after 60 min. There was no evidence of lipid peroxidation during this time. Xanthine oxidase activity in brachial vein blood was higher than in femoral vein blood in patients with critical ischaemia before revascularization.

Expression of lipoprotein lipase mRNA and secretion in macrophages isolated from human atherosclerotic aorta.

The expression of lipoprotein lipase (LPL) mRNA and the LPL activity were studied in macrophages (CD14 positive) from human atherosclerotic tissue. Macrophages were isolated after collagenase digestion by immunomagnetic isolation. About 90% of the cells were foam cells with oil red O positive lipid droplets. To analyze the mRNA expression, PCR with specific primers for LPL was used. Arterial macrophages were analyzed directly after isolation and the data showed low expression of LPL mRNA when compared with monocyte-derived macrophages. To induce the expression of LPL mRNA in macrophages, PMA was used. When incubating arterial macrophages with PMA for 24 h we could not detect any increase in LPL mRNA levels. Similarly, the cells secreted very small amounts of LPL even after PMA stimulation. In conclusion, these studies show a very low expression of LPL mRNA in the CD14-positive macrophage-derived foam cells isolated from human atherosclerotic tissue. These data suggest that the CD14-positive cells are a subpopulation of foam cells that express low levels of lipoprotein lipase, and the lipid content could be a major factor for downregulation of LPL. However, the cells were isolated from advanced atherosclerotic lesions, and these findings may not reflect the situation in early fatty streaks.

Lack of glutathione transferase activity in intermittent claudication.

Glutathione transferase activity towards trans-stilbene oxide (GT-tSBO), an enzyme involved in the detoxification of many substances such as polycyclic aromatic hydrocarbons, was studied in 77 consecutive patients operated with coronary bypass, 73 patients with intermittent claudication, 78 healthy smokers and in 38 healthy non-smokers. The mean ages of these groups were similar. Lack of GT-tSBO was recorded in 45% of coronary bypass patients, in 39% of smoking coronary bypass patients, in 61% of patients with intermittent claudication, in 41% of healthy smokers and in 31% of healthy non-smokers. The lack of GT-tSBO was significantly more frequent among patients with intermittent claudication compared to healthy non-smokers (p less than 0.01) and healthy smokers (p less than 0.025) and to smoking coronary bypass. It is concluded that the lack of GT-tSBO is found more frequently among patients with intermittent claudication and this might contribute to explain the sensitivity to smoking among these subjects.

Muscle enzyme adaptation in patients with peripheral arterial insufficiency: spontaneous adaptation, effect of different treatments and consequences on walking performance.

1. The activities of phosphofructokinase (PFK), citrate synthetase (CS), lactate dehydrogenase (LDH), 3-hydroxyacyl-CoA dehydrogenase (ACDH) and cytochrome-c oxidase(Cyt-ox) in the calf muscle tissue were compared in subjects with intermittent claudication (n = 38) and controls (n = 20). The activities of CS, ACDH and Cyt-ox were increased and the activity of Cytox was positively correlated to the maximal walking distance (MWD) in the patients. 2. Thirty-three patients with intermittent claudication were randomized to three treatment groups: (1) operative surgery, (2) operative surgery supplemented with physical training and (3) physical training alone. Before and after 6-12 months of treatment, symptom-free walking distance (SFWD), MWD, ankle-brachial blood pressure quotient (ankle index), maximal plethysmographic calf blood flow (MPBF) and the activities of PFK, CS, LDH, ACDH and Cyt-ox were measured. 3. SFWD and MWD increased in all three groups. Ankle index and MPBF increased in groups 1 and 2, but were unchanged in group 3. The activities of Cyt-ox and CS decreased with operation, but the activity of Cyt-ox was further augmented with training in group 3. Overall, the change in ankle index explained 80-90% of the variability in walking performance. In a separate analysis, the increased activity of Cyt-ox in group 3 was positively correlated to, and explained 31% of the variability in, the improvement in SFWD. 4. These findings indicate that both physical activity and a reduced calf blood flow are necessary conditions for the enzymatic adaptation to take place. A causal relationship between metabolic adaptation in the muscle tissue and walking performance is suggested.(ABSTRACT TRUNCATED AT 250 WORDS)

Calf muscle adaptation in intermittent claudication. Side-differences in muscle metabolic characteristics in patients with unilateral arterial disease.

The adaptation of enzyme activities, notably in the oxidative metabolism, and of prerequisites for tissue transport of oxygen in the claudication leg was evaluated by comparing muscle biopsies from the gastrocnemius muscle of the claudication and the symptom-free leg of seven patients with unilateral claudication. The claudication leg had higher activities of a marker enzyme for mitochondrial oxidative capacity, citrate synthase (CS), as well as of the MB and the mitochondrial isoenzyme of creatine kinase (CK), which are considered to be involved in the transfer of high energy phosphate from the mitochondria to the resynthesis of ATP in the cytoplasm. The difference between claudication and healthy leg in activities of these CK isoenzymes were well correlated with the corresponding side difference in CS activity. No significant differences between claudication and healthy leg were found in distribution of muscle fibre types or fibre dimension, capillary density or myoglobin content, nor was there any side difference in phosphofructokinase or lactate dehydrogenase. Side differences tended to be greater in those patients with the most advanced obstructive arterial disease as estimated from non-invasive pressure measurements. It is concluded that in reasonably physically-active patients, the mode of ischaemia to which the claudication leg is subjected leads to a metabolic adaptation characterized by increased activities of enzymes involved in the oxidative metabolism, but no significant adaptation of either the conditions for local oxygen transport, as estimated by myoglobin content, and capillary density, or capacity for anaerobic metabolism.

Biochemical and morphometric properties of mitochondrial populations in human muscle fibres.

Two mitochondrial subpopulations were evaluated with biochemical and morphological techniques in human gastrocnemius muscle of 10 patients with peripheral arterial insufficiency and 12 control individuals. The subsarcolemmal mitochondria were released by gentle homogenization, with a recovery of 32-37%, and the intermyofibrillar by enzymic digestion and further mechanical disintegration, recovery 18-21%. The subsarcolemmal mitochondria were morphologically defined as those located within 2 micron from the sarcolemma membrane and the intermyofibrillar mitochondria as those located in the rest of the fibre. In the controls the intermyofibrillar mitochondria had a lower respiratory ratio than the subsarcolemmal, owing to a higher state II respiration. The subsarcolemmal space, which contained 25% of the mitochondria, had a mitochondrial volume density two- to three-fold that of the intermyofibrillar space in the controls. The patients, who had a 48-64% higher oxidative enzyme capacity in their muscle tissue, had higher respiratory rate and respiratory control index with similar ADP/O ratio in the subsarcolemmal fraction in comparison with the controls. The citrate synthase activity was higher in both mitochondrial fractions of the patients. The volume densities of mitochondria, total as well as for both subpopulations, were also higher in the patients, which was further reflected in higher yields of mitochondrial protein. The results demonstrate that both subpopulations of muscle mitochondria are able to adapt quantitatively and/or qualitatively. Furthermore, they show that the increased oxidative enzyme capacity of the patients is associated with an increased quantity of both mitochondrial populations and a qualitative improvement of the respiratory activity of the subsarcolemmal mitochondria.

Histochemical changes in striated muscle in patients with intermittent claudication.

Biopsy specimens from the gastrocnemius or rectus femoris muscle of 20 patients with intermittent claudication were studied using fresh frozen cryostat sections and histochemical reactions for adenosine triphosphatase, nicotinamide adenine nucleotide dehydrogenase reductase and phosphorylase and modified Gomori trichrome staining. Neuropathic changes, such as fibertype grouping and small group atrophy, were present to some extent in all of the biopsy specimens. Myogenic muscle changes such as necrosis and phagocytosis were seen in approximately one third and various forms of myofibrillar disorganization in approximately two thirds of the specimens. The amount and size of the type I aerobic fibers increased with the increasing severity of the ischemic disease.

Enzyme activities in skeletal muscles from patients with peripheral arterial insufficiency.

The enzyme pattern in gastrocnemius muscle tissue was studied in 39 patients with peripheral arterial insufficiency. Phosphofructokinase and glucose-6-phosphate-dehydrogenase were significantly increased in the skeletal muscles from these patients. The most pronounced changes were found in 3-OH-acyl-CoA-dehydrogenase, citrate synthase, and in cytochrome-c-oxidase. These enzyme activities were increased by 60, 40 and 25 per cent respectively. In patients with claudication as the only symptom, the metabolic capacity was generally increased in skeletal muscles affected by the low blood flow. With increasing severity of arterial insufficiency, all enzyme activities decreased and glycolytic enzymes were affected first. 3-OH-acyl-CoA-dehydrogenase, citrate synthase and cytochrome-c-oxidase activities were still comparatively high in patients with gangrenous foot ulcers, indicating some maintenance of the muscle viability even in situations with very low blood flow.