Jason N. MacTaggart

Blocking TNF-α Attenuates Aneurysm Formation in a Murine Model

Abdominal aortic aneurysm (AAA) is one of a number of diseases associated with a prominent inflammatory cell infiltrate and local destruction of structural matrix macromolecules. This chronic infiltrate is predominately composed of macrophages and T lymphocytes. Activated macrophages produce a variety of cytokines, including TNF-α. Elevated levels of TNF-α were observed in patients with AAA, suggesting that TNF-α may play a role in the pathogenic mechanisms of AAA. In the present study, we investigated the role of TNF-α in AAA formation. By studying a murine aneurysm model, we found that both mRNA and protein levels of TNF-α were increased in aneurysm tissue compared with normal aortic tissues. Therefore, we tested the response of mice lacking expression of TNF-α. These mice were resistant to aneurysm formation. Our results show that TNF-α deficiency attenuates matrix metalloproteinase (MMP) 2 and MMP-9 expression and macrophage infiltration into the aortic tissue. These data suggest that TNF-α plays a central role in regulating matrix remodeling and inflammation in the aortic wall leading to AAA. In addition, we investigated the pharmacological inhibition of AAA. A Food and Drug Administration-approved TNF-α antagonist, infliximab, inhibited aneurysm growth. Our data also show that infliximab treatment attenuated elastic fiber disruption, macrophage infiltration, and MMP-2 and MMP-9 expression in aortic tissue. This study confirms that a strategy of TNF-α antagonism may be an important therapeutic strategy for treating AAA.

Inhibition of reactive oxygen species attenuates aneurysm formation in a murine model

Reactive oxygen species (ROS) are increased in human abdominal aortic aneurysms (AAA). NADPH oxidases are the predominant source of superoxide anion (O(2)(-)) in the vasculature. Inducible nitric oxide synthase (iNOS) produces a significant amount of nitric oxide (NO) during inflammatory processes. We hypothesized that ROS produced by NADPH oxidases and iNOS played an important role in aneurysm formation. We examined this hypothesis using selective blockade of NADPH oxidases and iNOS in a murine model of AAA. Mice, including C57BL/6, iNOS knockout (iNOS(-/-)) mice, and its background matched control (C57BL/6), underwent AAA induction by periaortic application of CaCl(2). Aortic diameter was measured at aneurysm induction and harvest. Beginning 1 week prior to aneurysm induction and continuing to aortic harvest 6 weeks later, one group of the C57BL/6 mice were treated with orally administered apocynin (NADPH oxidase inhibitor). Control mice were given water. The mean diameter and change in diameter of each group were compared with concurrent controls. Aortic levels of the NO metabolite, NO(x) (NO(2) and NO(3)), were significantly increased in CaCl(2)-treated wild type mice. INOS(-/-) mice were partly resistant to aneurysm induction. This was associated with reduced expression of matrix metalloproteinase (MMP)-2 and MMP-9 and decreased production of NO(x) in the aortic tissues. Inhibition of NADPH oxidase by apocynin also blocked aneurysm formation. In conclusion, both iNOS deficiency and NADPH oxidase inhibition suppressed aneurysm formation in association with decreased NO(x) levels. These studies suggest that both NADPH oxidase and iNOS pathways contribute to ROS production and AAA development.

Membrane-type 1 matrix metalloproteinase regulates macrophage-dependent elastolytic activity and aneurysm formation in vivo

During arterial aneurysm formation, levels of the membrane-anchored matrix metalloproteinase, MT1-MMP, are elevated dramatically. Although MT1-MMP is expressed predominately by infiltrating macrophages, the roles played by the proteinase in abdominal aortic aneurysm (AAA) formation in vivo remain undefined. Using a newly developed chimeric mouse model of AAA, we now demonstrate that macrophage-derived MT1-MMP plays a dominant role in disease progression. In wild-type mice transplanted with MT1-MMP-null marrow, aneurysm formation induced by the application of CaCl2 to the aortic surface was almost completely ablated. Macrophage infiltration into the aortic media was unaffected by MT1-MMP deletion, and AAA formation could be reconstituted when MT1-MMP+/+ macrophages, but not MT1-MMP+/+ lymphocytes, were infused into MT1-MMP-null marrow recipients. In vitro studies using macrophages isolated from either WT/MT1-MMP-/- chimeric mice, MMP-2-null mice, or MMP-9-null mice demonstrate that MT1-MMP alone plays a dominant role in macrophage-mediated elastolysis. These studies demonstrate that destruction of the elastin fiber network during AAA formation is dependent on macrophage-derived MT1-MMP, which unexpectedly serves as a direct-acting regulator of macrophage proteolytic activity.

Passive biaxial mechanical properties and in vivo axial pre-stretch of the diseased human femoropopliteal and tibial arteries.

Surgical and interventional therapies for atherosclerotic lesions of the infrainguinal arteries are notorious for high rates of failure. Frequently, this leads to expensive reinterventions, return of disabling symptoms or limb loss. Interaction between the artery and repair material likely plays an important role in reconstruction failure, but data describing the mechanical properties and functional characteristics of human femoropopliteal and tibial arteries are currently not available. Diseased superficial femoral (SFA, n = 10), popliteal (PA, n = 8) and tibial arteries (TA, n = 3) from 10 patients with critical limb ischemia were tested to determine passive mechanical properties using planar biaxial extension. All specimens exhibited large nonlinear deformations and anisotropy. Under equibiaxial loading, all arteries were stiffer in the circumferential direction than in the longitudinal direction. Anisotropy and longitudinal compliance decreased distally, but circumferential compliance increased, possibly to maintain a homeostatic multiaxial stress state. Constitutive parameters for a four-fiber family invariant-based model were determined for all tissues to calculate in vivo axial pre-stretch that allows the artery to function in the most energy efficient manner while also preventing buckling during extremity flexion. Calculated axial pre-stretch was found to decrease with age, disease severity and more distal arterial location. Histological analysis of the femoropopliteal artery demonstrated a distinct sub-adventitial layer of longitudinal elastin fibers that appeared thicker in healthier arteries. The femoropopliteal artery characteristics and properties determined in this study may assist in devising better diagnostic and treatment modalities for patients with peripheral arterial disease.

Development and validation of a risk calculator for prediction of mortality after infrainguinal bypass surgery.

OBJECTIVE For peripheral arterial disease, infrainguinal bypass grafting (BPG) carries a higher perioperative risk compared with peripheral endovascular procedures. The choice between the open and endovascular therapies is to an extent dependent on the expected periprocedural risk associated with each. Tools for estimating the periprocedural risk in patients undergoing BPG have not been reported in the literature. The objective of this study was to develop and validate a calculator to estimate the risk of perioperative mortality ≤30 days of elective BPG. METHODS We identified 9556 patients (63.9% men) who underwent elective BPG from the 2007 to 2009 National Surgical Quality Improvement Program data sets. Multivariable logistic regression analysis was performed to identify risk factors associated with 30-day perioperative mortality. Bootstrapping was used for internal validation. The risk factors were subsequently used to develop a risk calculator. RESULTS Patients had a median age of 68 years. The 30-day mortality rate was 1.8% (n = 170). Multivariable logistic regression analysis identified seven preoperative predictors of 30-day mortality: increasing age, systemic inflammatory response syndrome, chronic corticosteroid use, chronic obstructive pulmonary disease, dependent functional status, dialysis dependence, and lower extremity rest pain. Bootstrapping was used for internal validation. The model demonstrated excellent discrimination (C statistic, 0.81; bias-corrected C statistic, 0.81) and calibration. The validated risk model was used to develop an interactive risk calculator using the logistic regression equation. CONCLUSIONS The validated risk calculator has excellent predictive ability for 30-day mortality in a patient after an elective BPG. It is anticipated to aid in surgical decision making, informed patient consent, preoperative optimization, and consequently, risk reduction.

Effects of age on the physiological and mechanical characteristics of human femoropopliteal arteries

Surgical and interventional therapies for peripheral artery disease (PAD) are notorious for high rates of failure. Interactions between the artery and repair materials play an important role, but comprehensive data describing the physiological and mechanical characteristics of human femoropopliteal arteries are not available. Fresh femoropopliteal arteries were obtained from 70 human subjects (13-79 years old), and in situ vs. excised arterial lengths were measured. Circumferential and longitudinal opening angles were determined for proximal superficial femoral, proximal popliteal and distal popliteal arteries. Mechanical properties were assessed by multi-ratio planar biaxial extension, and experimental data were used to calculate physiological stresses and stretches, in situ axial force and anisotropy. Verhoeff-Van Gieson-stained axial and transverse arterial sections were used for histological analysis. Most specimens demonstrated nonlinear deformations and were more compliant longitudinally than circumferentially. In situ axial pre-stretch decreased 0.088 per decade of life. In situ axial force and axial stress also decreased with age, but circumferential physiological stress remained constant. Physiological circumferential stretch decreased 55-75% after 45 years of age. Histology demonstrated a thickened external elastic lamina with longitudinally oriented elastin that was denser in smaller, younger arteries. Axial elastin likely regulates axial pre-stretch to help accommodate the complex deformations required of the artery wall during locomotion. Degradation and fragmentation of elastin as a consequence of age, cyclic mechanical stress and atherosclerotic arterial disease may contribute to decreased in situ axial pre-stretch, predisposing to more severe kinking of the artery during limb flexion and loss of energy-efficient arterial function.

Three-dimensional bending, torsion and axial compression of the femoropopliteal artery during limb flexion

High failure rates of femoropopliteal artery reconstruction are commonly attributed to complex 3D arterial deformations that occur with limb movement. The purpose of this study was to develop a method for accurate assessment of these deformations. Custom-made stainless-steel markers were deployed into 5 in situ cadaveric femoropopliteal arteries using fluoroscopy. Thin-section CT images were acquired with each limb in the straight and acutely bent states. Image segmentation and 3D reconstruction allowed comparison of the relative locations of each intra-arterial marker position for determination of the arterys bending, torsion and axial compression. After imaging, each artery was excised for histological analysis using Verhoeff-Van Gieson staining. Femoropopliteal arteries deformed non-uniformly with highly localized deformations in the proximal superficial femoral artery, and between the adductor hiatus and distal popliteal artery. The largest bending (11±3-6±1 mm radius of curvature), twisting (28±9-77±27°/cm) and axial compression (19±10-30±8%) were registered at the adductor hiatus and the below knee popliteal artery. These deformations were 3.7, 19 and 2.5 fold more severe than values currently reported in the literature. Histology demonstrated a distinct sub-adventitial layer of longitudinally oriented elastin fibers with intimal thickening in the segments with the largest deformations. This endovascular intra-arterial marker technique can quantify the non-uniform 3D deformations of the femoropopliteal artery during knee flexion without disturbing surrounding structures. We demonstrate that 3D arterial bending, torsion and compression in the flexed lower limb are highly localized and are substantially more severe than previously reported.

Nonlinear Mechanical Behavior of The Human Common, External, and Internal Carotid Arteries In Vivo

BACKGROUND The mechanical environment and properties of the carotid artery play an important role in the formation and progression of atherosclerosis in the carotid bifurcation. The purpose of this work was to measure and compare the range and variation of circumferential stress and tangent elastic moduli in the human common (CCA), external (ECA), and internal (ICA) carotid arteries over the cardiac cycle in vivo. METHODS Measurements were performed in the surgically exposed proximal cervical CCA, distal ECA, and distal ICA of normotensive patients (n = 16) undergoing carotid endarterectomy. All measurements were completed in vivo over the cardiac cycle in the repaired carotid bifurcation after the atherosclerotic plaque was successfully removed. B-mode Duplex ultrasonography was used for measurement of arterial diameter and wall thickness, and an angiocatheter placed in the CCA was used for concurrent measurement of blood pressure. A semiautomatic segmentation algorithm was used to track changes in arterial diameter and wall thickness in response to blood pressure. These measurements were then used to calculate the variation of circumferential (hoop) stresses, tangent elastic moduli (the slope of the stress-strain curve at specified stresses), and strain-induced stiffness of the arterial wall (stiffening in response to the increase of intraluminal blood pressure) for each patient. RESULTS The diameter and wall thickness of the segments (CCA, ECA, and ICA) of the carotid bifurcation were found to decrease and strain-induced stiffness to increase from proximal CCA to distal ECA and ICA. The circumferential stress from end-diastole (minimum pressure) to peak-systole (maximum pressure) varied nonlinearly from 25 ± 7 to 63 ± 23 kPa (CCA), from 22 ± 7 to 57 ± 19 kPa (ECA), and from 28 ± 8 to 67 ± 23 kPa (ICA). Tangent elastic moduli also varied nonlinearly from end-diastole to peak-systole as follows: from 0.40 ± 0.25 to 1.50 ± 2.05 MPa (CCA), from 0.49 ± 0.34 to 1.14 ± 0.52 MPa (ECA), and from 0.68 ± 0.31 to 1.51 ± 0.69 MPa (ICA). The strain-induced stiffness of CCA and ECA increased more than 3-fold and the stiffness of ICA increased more than 2.5-fold at peak-systole compared with end-diastole. CONCLUSIONS The in vivo mechanical behavior of the three segments of the carotid bifurcation was qualitatively similar, but quantitatively different. All three arteries--CCA, ECA and ICA--exhibited nonlinear variations of circumferential stress and tangent elastic moduli within the normal pressure range. The variability in the properties of the three segments of the carotid bifurcation indicates a need for development of carotid models that match the in vivo properties of the carotid segments. Finally, the observed nonlinear behavior of the artery points to the need for future vascular mechanical studies to evaluate the mechanical factors of the arterial wall over the entire cardiac cycle.

Comparative Analysis of the Biaxial Mechanical Behavior of Carotid Wall Tissue and Biological and Synthetic Materials Used for Carotid Patch Angioplasty

Patch angioplasty is the most common technique used for the performance of carotid endarterectomy. A large number of patching materials are available for use while new materials are being continuously developed. Surprisingly little is known about the mechanical properties of these materials and how these properties compare with those of the carotid artery wall. Mismatch of the mechanical properties can produce mechanical and hemodynamic effects that may compromise the long-term patency of the endarterectomized arterial segment. The aim of this paper was to systematically evaluate and compare the biaxial mechanical behavior of the most commonly used patching materials. We compared PTFE (n  =  1), Dacron (n  =  2), bovine pericardium (n  =  10), autogenous greater saphenous vein (n  =  10), and autogenous external jugular vein (n  =  9) with the wall of the common carotid artery (n  =  18). All patching materials were found to be significantly stiffer than the carotid wall in both the longitudinal and circumferential directions. Synthetic patches demonstrated the most mismatch in stiffness values and vein patches the least mismatch in stiffness values compared to those of the native carotid artery. All biological materials, including the carotid artery, demonstrated substantial nonlinearity, anisotropy, and variability; however, the behavior of biological and biologically-derived patches was both qualitatively and quantitatively different from the behavior of the carotid wall. The majority of carotid arteries tested were stiffer in the circumferential direction, while the opposite anisotropy was observed for all types of vein patches and bovine pericardium. The rates of increase in the nonlinear stiffness over the physiological stress range were also different for the carotid and patching materials. Several carotid wall samples exhibited reverse anisotropy compared to the average behavior of the carotid tissue. A similar characteristic was observed for two of 19 vein patches. The obtained results quantify, for the first time, significant mechanical dissimilarity of the currently available patching materials and the carotid artery. The results can be used as guidance for designing more efficient patches with mechanical properties resembling those of the carotid wall. The presented systematic comparative mechanical analysis of the existing patching materials provides valuable information for patch selection in the daily practice of carotid surgery and can be used in future clinical studies comparing the efficacy of different patches in the performance of carotid endarterectomy.

In vivo three-dimensional blood velocity profile shapes in the human common, internal, and external carotid arteries

OBJECTIVE True understanding of carotid bifurcation pathophysiology requires a detailed knowledge of the hemodynamic conditions within the arteries. Data on carotid artery hemodynamics are usually based on simplified, computer-based, or in vitro experimental models, most of which assume that the velocity profiles are axially symmetric away from the carotid bulb. Modeling accuracy and, more importantly, our understanding of the pathophysiology of carotid bifurcation disease could be considerably improved by more precise knowledge of the in vivo flow properties within the human carotid artery. The purpose of this work was to determine the three-dimensional pulsatile velocity profiles of human carotid arteries. METHODS Flow velocities were measured over the cardiac cycle using duplex ultrasonography, before and after endarterectomy, in the surgically exposed common (CCA), internal (ICA), and external (ECA) carotid arteries (n = 16) proximal and distal to the stenosis/endarterectomy zone. These measurements were linked to a standardized grid across the flow lumina of the CCA, ICA, and ECA. The individual velocities were then used to build mean three-dimensional pulsatile velocity profiles for each of the carotid artery branches. RESULTS Pulsatile velocity profiles in all arteries were asymmetric about the arterial centerline. Posterior velocities were higher than anterior velocities in all arteries. In the CCA and ECA, velocities were higher laterally, while in the ICA, velocities were higher medially. Pre- and postendarterectomy velocity profiles were significantly different. After endarterectomy, velocity values increased in the common and internal and decreased in the external carotid artery. CONCLUSIONS The in vivo hemodynamics of the human carotid artery are different from those used in most current computer-based and in vitro models. The new information on three-dimensional blood velocity profiles can be used to design models that more closely replicate the actual hemodynamic conditions within the carotid bifurcation. Such models can be used to further improve our understanding of the pathophysiologic processes leading to stroke and for the rational design of medical and interventional therapies.