Nitric oxide manipulation: a therapeutic target for peripheral arterial disease?

Peripheral Arterial Disease (PAD) is a cause of significant morbidity and mortality in the Western world. Risk factor modification and endovascular and surgical revascularisation are the main treatment options at present. However, a significant number of patients still require major amputation. There is evidence that nitric oxide (NO) and its endogenous inhibitor asymmetric dimethylarginine (ADMA) play significant roles in the pathophysiology of PAD. This paper reviews experimental work implicating the ADMA-DDAH-NO pathway in PAD, focussing on both the vascular dysfunction and effects within the ischaemic muscle, and examines the potential of manipulating this pathway as a novel adjunct therapy in PAD.

Toll-like receptors in ischaemia and its potential role in the pathophysiology of muscle damage in critical limb ischaemia.

Toll-like receptors (TLRs) are key receptors of the innate immune system which are expressed on immune and nonimmune cells. They are activated by both pathogen-associated molecular patterns and endogenous ligands. Activation of TLRs culminates in the release of proinflammatory cytokines, chemokines, and apoptosis. Ischaemia and ischaemia/reperfusion (I/R) injury are associated with significant inflammation and tissue damage. There is emerging evidence to suggest that TLRs are involved in mediating ischaemia-induced damage in several organs. Critical limb ischaemia (CLI) is the most severe form of peripheral arterial disease (PAD) and is associated with skeletal muscle damage and tissue loss; however its pathophysiology is poorly understood. This paper will underline the evidence implicating TLRs in the pathophysiology of cerebral, renal, hepatic, myocardial, and skeletal muscle ischaemia and I/R injury and discuss preliminary data that alludes to the potential role of TLRs in the pathophysiology of skeletal muscle damage in CLI.

Further evidence for a role of endothelin-1 (ET-1) in critical limb ischaemia.

Critical limb ischaemia (CLI), due to atherosclerotic arterial occlusion, affects over 20,000 people per year in the United Kingdom with many facing lower limb amputation and early death. A role for endothelin-1 (ET-1) in atherosclerosis is well-established and increased circulating and tissue levels of this peptide have been detected in patients with CLI. ET-1 and its receptors were identified in atherosclerotic popliteal arteries obtained from CLI patients undergoing lower limb amputation. In addition, plasma ET-1 levels were compared with those of non-ischaemic controls. ET-1 was associated with regions of atherosclerotic plaque, particularly in regions with high macrophage content. This peptide was also associated with endothelial cells lining the main vessel lumen as well as adventitial microvessels. ET(A) and ET(B) receptors were located within regions of plaque, adventitial microvessels and perivascular nerves. There was a statistically significant increase (P < 0.001) in plasma ET-1 in CLI patients when compared with controls. These results reveal sources of ET-1 in atherosclerotic popliteal arteries that potentially contribute to increased circulating levels of this peptide. Identification of variable receptor distributions in ischaemic tissue suggests a therapeutic potential of selective receptor targeting in patients with CLI.

Retaining perivascular tissue of human saphenous vein grafts protects against surgical and distension-induced damage and preserves endothelial nitric oxide synthase and nitric oxide synthase activity.

OBJECTIVE: Conventional harvesting of saphenous vein used for coronary artery bypass surgery induces a vasospasm that is overcome by high-pressure distension. Saphenous vein harvested with its cushion of perivascular tissue by a "no touch" technique does not undergo vasospasm and distension is not required, leading to an improved graft patency. The aim of this study is to investigate the effect of surgical damage and high-pressure distension on endothelial integrity and endothelial nitric oxide synthase expression and activity in saphenous vein harvested with and without perivascular tissue. METHODS: Saphenous veins from patients (n = 26) undergoing coronary artery bypass surgery were prepared with and without perivascular tissue. We analyzed the effect of 300 mm Hg distension on morphology and endothelial nitric oxide synthase/nitric oxide synthase activity using a combination of immunohistochemistry, Western blot analysis, reverse transcriptase polymerase chain reaction, and enzyme assay in distended (with and without perivascular tissue) compared with nondistended (with and without perivascular tissue) segments. RESULTS: Distension induced substantial damage to the luminal endothelium (assessed by CD31 staining) and vessel wall. Endothelial nitric oxide synthase expression and activity were significantly reduced by high-pressure distension and removal of, or damage to, perivascular tissue. The effect of distension was significantly less for those with perivascular tissue than for those without perivascular tissue in most cases. CONCLUSION: The success of the saphenous vein used as a bypass graft is affected by surgical trauma and distension. Veins removed with minimal damage exhibit increased patency rates. We show that retention of perivascular tissue on saphenous vein prepared for coronary artery bypass surgery by the "no touch" technique protects against distension-induced damage, preserves vessel morphology, and maintains endothelial nitric oxide synthase/nitric oxide synthase activity.

Alterations in nitric oxide synthase isoforms in acute lower limb ischemia and reperfusion.

Alterations in nitric oxide synthase (NOS) are implicated in ischemia and ischemia-reperfusion injury. Changes in the 3 NOS isoforms in human skeletal muscle subjected to acute ischemia and reperfusion were studied. Muscle biopsies were taken from patients undergoing total knee replacement. Distribution of the specific NOS isoforms within muscle sections was studied using immunohistochemistry. NOS mRNA levels were measured using real-time reverse transcription-polymerase chain reaction and protein levels studied using Western blotting. NOS activity was also assessed using the citrulline assay. All 3 NOS isoforms were found in muscle sections associated with muscle fibers and microvessels. In muscle subjected to acute ischemia and reperfusion, NOS I/neuronal NOS mRNA and protein were elevated during reperfusion. NOS III/endothelial NOS was also upregulated at the protein level during reperfusion. No changes in NOS II/inducible NOS expression or NOS activity occurred. In conclusion, alterations in NOS I and III (neuronal NOS and endothelial NOS) at different levels occurred after acute ischemia and reperfusion in human skeletal muscle; however, this did not result in increased NOS activity. In the development of therapeutic agents based on manipulation of the NO pathway, targeting the appropriate NOS isoenzymes may be important.

The effect of acute ischemia on ET-1 and its receptors in patients with underlying chronic ischemia of the lower limb.

Elevated plasma and tissue endothelin (ET)-1 levels in patients with critical limb ischemia (CLI) has been described. Here the effect of a period of acute ischemia and subsequent reperfusion on plasma ET-1 and tissue ET-1/ET receptors in skeletal muscle biopsies from CLI patients undergoing femoro-distal bypass surgery was studied. Peripheral and "local" blood and muscle biopsies were obtained from patients undergoing femoro-distal bypass surgery, at the start of the procedure (control), after a period of vascular clamping (ischemia), and after clamp release (reperfusion). Plasma ET-1 was determined by enzyme-linked immunosorbent assay. Tissue ET-1 was assessed by counting ET-1 immunostaining cells per unit area, and ET(A)/ET(B) receptors were identified on sections by in vitro autoradiography in which binding was quantitatively assessed by densitometry. There was no significant effect of ischemia or reperfusion on plasma ET-1 levels or on ET(A)/ET(B) receptor binding. However, tissue ET-1 increased during both acute ischemia and reperfusion (P < 0.05). A high proportion of positive ET-1 immunostaining was associated with microvessels and also exhibited a similar distribution to macrophages. Previously, it has been shown that both plasma ET-1 and tissue ET-1/ET receptors are increased in CLI patients compared with atherosclerotic controls. Also, increased muscle ET-1 levels have been described in acute ischemia caused by tourniquet application in nonischemic patients undergoing total knee replacement. In CLI patients, in whom ET-1 is already upregulated, this further increase may exacerbate existing pathologic processes and contribute to ischemia-reperfusion injury. ET-1 antagonists may therefore be useful adjuncts in CLI and other surgical procedures in which ischemia-reperfusion damage occurs.

A role for endothelin-1 in peripheral vascular disease.

Peripheral vascular disease can compromise the blood supply to the lower limb with amputation being necessary in severe cases. Reduced blood flow may be due to arterial occlusive disease or constriction of skeletal microvessels with the resultant ischaemia causing pain, tissue damage, ulceration and gangrene. These events are associated with endothelial damage or dysfunction: endothelin-1 is implicated as a mediator via its constrictor, proinflammatory and proliferative actions. Raised plasma and tissue levels of this peptide have been described in various ischaemic conditions, including peripheral vascular disease. Here, the possible role of endothelin-1 in peripheral vascular disease is discussed and potential therapeutic tools are considered.

Improved patency in vein grafts harvested with surrounding tissue: results of a randomized study using three harvesting techniques.

BACKGROUND: The technique of harvesting the saphenous vein for coronary artery bypass grafting influences the fate of vein grafts. The patency rate of a novel "no-touch" technique in which the vein is harvested with a pedicle of surrounding tissue and not distended was compared with two other techniques. METHODS: One hundred fifty-six patients who underwent coronary artery bypass grafting were randomized to three saphenous vein harvesting groups: group C (conventional)--the vein was stripped, distended, and stored in saline; group I (intermediate)--the vein was stripped, local application of papaverine was used instead of distention, and the vessel was then stored in heparinized blood; and group NT (no-touch)--the vein was harvested with surrounding tissue, not distended, and stored in heparinized blood. Surgical and clinical factors that might influence graft occlusion were recorded. One hundred twenty-seven vein grafts in group C, 116 in group I, and 124 in group NT, as well as 118 left internal mammary artery grafts, were angiographically assessed at 18 months mean follow-up time. RESULTS: The vein graft patency was 88.9% in group C, 86.2% in group I, and 95.4% in group NT. There was a statistically significant difference between the patency of the single-vein grafts in NT and the other two groups (p = 0.025). The higher the flow, the better the patency irrespective of the technique used. A higher attrition rate was found in vein segments taken from the knee area in group I. Poor vein quality affected patency in all groups. Forty-seven of all 51 sequential grafts (92.2%) were patent. The patency of left internal mammary artery grafts was 108 of 118 (91.5%). CONCLUSIONS: We conclude that preservation of the surrounding tissue of the saphenous vein using this no-touch technique abolishes venospasm intraoperatively and plays an important role in maintaining vein graft function and patency.

Endothelin-1 and atherosclerosis: potential complications associated with endothelin-receptor blockade.

Atherosclerotic vascular disease remains the single most prevalent cause of death and morbidity in the western world. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that also possesses mitogenic activity on many cell types, including vascular smooth muscle cells. Raised plasma and tissue levels of ET-1 have been described in atherosclerosis in animal models and in man, suggesting that this peptide plays a pathophysiological role in this condition. Two main ET-1 receptors have been cloned (ET(A) and ET(B)). Mixed ET(A/B) and receptor subtype selective antagonists are now available. Since ET-1 is generally believed to be a 'pathophysiological peptide', we discuss the therapeutic potential of ET-1 antagonists in atherosclerosis and consider whether, at certain sites in this process, ET-1 may play a beneficial role. In such situations ET antagonism may be undesirable.

Localization of nitric oxide synthase in saphenous vein grafts harvested with a novel "no-touch" technique: potential role of nitric oxide contribution to improved early graft patency rates.

OBJECTIVE: The use of the saphenous vein in coronary artery bypass graft surgery is associated with high 1-year occlusion rates of as much as 30%. A new "no-touch" technique of saphenous vein harvesting in which the vein is harvested with a pedicle of surrounding tissue and not distended may result in improved early patency rates. We hypothesize that nitric oxide synthase is better preserved with the no-touch technique, and the aim of this study was the investigation of whether nitric oxide synthase distribution and quantity in saphenous veins harvested with the no-touch technique differ from those veins harvested with the conventional technique. The separate contribution of perivascular tissue removal and distension to alterations in nitric oxide synthase was also studied. METHODS: Segments of 10 saphenous veins were harvested from 10 patients who underwent coronary artery bypass grafting surgery with the no-touch and conventional techniques. Samples were also taken from segments that were stripped of surrounding tissue but not distended. Nitric oxide synthase distribution was studied with reduced nicotinamide adenine dinucleotide phosphate--diaphorase histochemistry, and staining was quantified with image analysis. Immunohistochemistry was used for the identification of specific nitric oxide synthase isoforms, and immunomarkers were used for the identification of associated cell types. RESULTS: Nitric oxide synthase content was higher in no-touch vessels as compared with conventionally harvested vessels (35.5%; P <.05, with analysis of variance). This content was associated with endothelial nitric oxide synthase on the lumen while all three isoforms were present in the media. In the intact adventitia of no-touch vessels, all three isoforms of nitric oxide synthase were also present, associated with microvessels and perivascular nerves. Perivascular tissue stripping and venous distension both contribute to the reduced nitric oxide synthase in conventionally harvested veins. CONCLUSION: The new no-touch technique of saphenous vein harvesting preserves nitric oxide synthase, which suggests that improved nitric oxide availability may be an important mechanism in the success of this technique.