In vivo assessment of endothelial function in human lower extremity arteries.

OBJECTIVE: Endothelial function has been measured in preclinical studies in human brachial and coronary arteries but not in lower extremity arteries affected by atherosclerosis. We describe a novel, first-in-man evaluation of endothelial function of the superficial femoral arteries (SFAs) in patients with peripheral arterial disease (PAD). METHODS: Enrolled were 25 patients with PAD requiring lower extremity angiography. Endothelial-dependent relaxation was measured using intravascular ultrasound (IVUS) imaging and a Doppler flow wire after the infusion of acetylcholine (Ach). IVUS-derived virtual histology of the same vessel was calculated. Endothelial-independent relaxation was measured with an infusion of nitroglycerin (200 µg). Levels of nitric oxide and serum nitric oxide metabolites were determined by laboratory analysis. RESULTS: Patients (48% male; mean age, 62 years) had a history of hypertension (80%), coronary disease (36%), and diabetes (40%). The mean SFA diameter was 5.2 ± 1 mm (range, 3.2-6.9 mm). Patients tolerated Ach infusion with no adverse events. Endothelial-dependent relaxation increased over baseline for all patients with Ach infusion of 10(-6) to 10(-4). At Ach 10(-4), diameter (0.5%) and area (1.8%) changes in the diseased SFAs were modest and insignificant; however, average peak velocity of blood flow significantly increased 26%, 46%, and 63% with an Ach 10(-6) to 10(-4) infusion. Calculations of limb volumetric flow (68% at Ach 10(-4)) were significantly increased after Ach infusion. Lower extremity nitric oxide levels were slightly lower than systemic venous levels (P = .04). Nitroglycerin infusion indicated normal smooth muscle responsiveness (3% diameter, 9% area, and 116% velocity change over baseline). IVUS-virtual histology plaque stratification indicated predominantly fibrous morphology (46%; necrotic core, 29%; calcium, 18%). Atheroma burden was 14.9 ± 5.5 mm(3)/cm and did not correlate with endothelial responsiveness. CONCLUSIONS: Endothelial function can be measured directly in human lower extremity arteries at the sites of vascular disease. Despite extensive atherosclerosis, endothelial function is still intact. These data support the application of regional endothelial-specific biologic therapies in patients with PAD.

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.

Evaluation of peripheral atherosclerosis: a comparative analysis of angiography and intravascular ultrasound imaging.

OBJECTIVE: Angiography remains a critical component for diagnostic imaging and therapeutic intervention in peripheral arterial disease (PAD). The goal of this study was to compare angiography with corresponding intravascular ultrasound (IVUS) imaging of the same vessels in patients with PAD. METHODS: From 2004 to 2008, 93 patients undergoing angiography for PAD were recruited in a prospective observational analysis. At the time of angiography, diseased lower extremities were interrogated using a 10-cm IVUS pullback with registration points. IVUS data were analyzed with radiofrequency techniques for vessel and lumen diameter, plaque volume, plaque composition, and cross-sectional area. Similarly, three vascular surgeons blinded to the IVUS data graded corresponding angiographic images according to vessel diameter, degree of stenosis, degree of calcification, and extent of eccentricity. Statistical analyses of matched IVUS images and angiograms were performed. RESULTS: The distribution of demographic and risk variables were typical for PAD: 54% male, 96% hypertension, 78% hyperlipidemia, 44% diabetic, 87% tobacco history, 65% coronary artery disease, and 10% end-stage renal disease. Symptoms precipitating the angiographic evaluation included claudication (53%), rest pain (18%), and tissue loss (29%). Angiographic and IVUS interpretation were similar for luminal diameters, but external vessel diameter was greater by IVUS imaging (7.0 +/- 0.7 vs 5.2 +/- 0.8 mm, P < .05). The two-dimensional diameter method resulted in a significant correlation for stenosis determination (r = 0.84); however, IVUS determination of vessel area stenosis was greater by 10% (95% confidence interval, 0.3%-21%, P < .05). IVUS imaging indicated that a higher proportion of plaques were concentric. Grading of calcification was moderate to severe in 40% by angiography but in only 7% by IVUS (P < .05). CONCLUSIONS: In the evaluation of PAD, angiography and IVUS imaging provide similar luminal diameters and diameter-reducing stenosis measurements. Determination of overall vessel diameter and interpretation of plaque morphology by angiography are discordant from IVUS-derived data.

Arterial calcification increases in distal arteries in patients with peripheral arterial disease.

The aim of this study was to determine if significant differences in plaque composition exist between the popliteal and tibial vessels in patients with severe peripheral arterial disease (PAD). Forty-four patients with PAD required either above-knee (n = 38), below-knee (n = 5), or through-knee (n = 1) amputation for pedal sepsis/gangrene. The 51 vessels (anterior tibial, n = 9; posterior tibial, n = 10; peroneal, n = 3; popliteal, n = 29) were obtained and underwent intravascular ultrasound (IVUS) evaluation ex vivo within 24 hr of amputation. Sequential IVUS data were obtained at known intervals throughout the vessel length and then analyzed with radiofrequency techniques for quantification of plaque composition, plaque volume, and total vessel volume. Plaque composition was categorized as fibrous, fibro-fatty, necrotic core, and dense calcium. Clinical data were obtained via review of electronic records at the time of amputation. Two-sided t-tests were performed to compare components within each plaque. Results are expressed as mean percentage +/- standard error of the mean. Tibial vessels had more dense calcium within these plaques than popliteal arteries (33.8 +/- 5.6% vs. 10.6 +/- 1.9%, p < 0.001). Consequently, distal vessels had less fibro-fatty and fibrous plaque than popliteal arteries (7.7 +/- 1.4% vs. 13.1 +/- 1.2%, p < 0.005; 42.4 +/- 4.7% vs. 61.4 +/- 2.2%, p < 0.001, respectively). Necrotic core plaque composition was found to be similar when comparing tibial versus popliteal arteries (16.1% vs. 14.9%, p = nonsignificant). Clinical factors including diabetes, hyperlipidemia, and chronic renal insufficiency were not associated with plaque composition differences using a univariate analysis. As we progress distally in the arterial tree of patients with PAD, calcium plaque content increases with decreasing burden of fibro-fatty plaque. Clinical and demographic factors, with the exception of smoking, were not found to be associated with atherosclerotic plaque composition.