Nikolaos Stergiopulos

Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy

Mechanical properties of the adventitia are largely determined by the organization of collagen fibers. Measurements on the waviness and orientation of collagen, particularly at the zero-stress state, are necessary to relate the structural organization of collagen to the mechanical response of the adventitia. Using the fluorescence collagen marker CNA38-OG488 and confocal laser scanning microscopy, we imaged collagen fibers in the adventitia of rabbit common carotid arteries ex vivo. The arteries were cut open along their longitudinal axes to get the zero-stress state. We used semi-manual and automatic techniques to measure parameters related to the waviness and orientation of fibers. Our results showed that the straightness parameter (defined as the ratio between the distances of endpoints of a fiber to its length) was distributed with a beta distribution (mean value 0.72, variance 0.028) and did not depend on the mean angle orientation of fibers. Local angular density distributions revealed four axially symmetric families of fibers with mean directions of 0°, 90°, 43° and −43°, with respect to the axial direction of the artery, and corresponding circular standard deviations of 40°, 47°, 37° and 37°. The distribution of local orientations was shifted to the circumferential direction when measured in arteries at the zero-load state (intact), as compared to arteries at the zero-stress state (cut-open). Information on collagen fiber waviness and orientation, such as obtained in this study, could be used to develop structural models of the adventitia, providing better means for analyzing and understanding the mechanical properties of vascular wall.

Effect of aging on elastin functionality in human cerebral arteries

Background and Purpose— Aging affects elastin, a key component of the arterial wall integrity and functionality. Elastin degradation in cerebral vessels is associated with cerebrovascular disease. The goal of this study is to assess the biomechanical properties of human cerebral arteries, their composition, and their geometry, with particular focus on the functional alteration of elastin attributable to aging. Methods— Twelve posterior cranial arteries obtained from human cadavers of 2 different age groups were compared morphologically and tested biomechanically before and after enzymatic degradation of elastin. Light, confocal, and scanning electron microscopy were used to analyze and determine structural differences, potentially attributed to aging. Results— Aging affects structural morphology and the mechanical properties of intracranial arteries. In contrast to main systemic arteries, intima and media thicken while outer diameter remains relatively constant with age, leading to concentric hypertrophy. The structural morphology of elastin changed from a fiber network oriented primarily in the circumferential direction to a more heterogeneously oriented fiber mesh, especially at the intima. Biomechanically, cerebral arteries stiffen with age and lose compliance in the elastin dominated regime. Enzymatic degradation of elastin led to loss in compliance and stiffening in the young group but did not affect the structural and material properties in the older group, suggesting that elastin, though present in equal quantities in the old group, becomes dysfunctional with aging. Conclusions— Elastin loses its functionality in cerebral arteries with aging, leading to stiffer less compliant arteries. The area fraction of elastin remained, however, fairly constant. The loss of functionality may thus be attributed to fragmentation and structural reorganization of elastin occurring with age.

Physiological simulation of blood flow in the aorta: comparison of hemodynamic indices as predicted by 3-D FSI, 3-D rigid wall and 1-D models

Interest in patient-specific blood-flow circulation modeling has increased substantially in recent years. The availability of clinical data for geometric and elastic properties together with efficient numerical methods has now made model rendering feasible. This work uses 3-D fluid-structure interaction (FSI) to provide physiological simulation resulting in modeling with a high level of detail. Comparisons are made between results using FSI and rigid wall models. The relevance of wall compliance in determining parameters of clinical importance, such as wall shear stress, is discussed together with the significance of differences found in the pressure and flow waveforms when using the 1-D model. Patient-specific geometry of the aorta and its branches was based on MRI angiography data. The arterial wall was created with a variable thickness. The boundary conditions for the fluid domain were pressure waveform at the ascending aorta and flow for each outlet. The waveforms were obtained using a 1-D model validated by in vivo measurements performed on the same person. In order to mimic the mechanical effect of surrounding tissues in the simulation, a stress-displacement relation was applied to the arterial wall. The temporal variation and spatial patterns of wall shear stress are presented in the aortic arch and thoracic aorta together with differences using rigid wall and FSI models. A comparison of the 3-D simulations to the 1-D model shows good reproduction of the pressure and flow waveforms.

Dissecting abdominal aortic aneurysm in Ang II-infused mice: suprarenal branch ruptures and apparent luminal dilatation.

AIMS In this work, we provide novel insight into the morphology of dissecting abdominal aortic aneurysms in angiotensin II-infused mice. We demonstrate why they exhibit a large variation in shape and, unlike their human counterparts, are located suprarenally rather than infrarenally. METHODS AND RESULTS We combined synchrotron-based, ultra-high resolution ex vivo imaging (phase contrast X-Ray tomographic microscopy) with in vivo imaging (high-frequency ultrasound and contrast-enhanced micro-CT) and image-guided histology. In all mice, we observed a tear in the tunica media of the abdominal aorta near the ostium of the celiac artery. Independently we found that, unlike the gradual luminal expansion typical for human aneurysms, the outer diameter increase of angiotensin II-induced dissecting aneurysms in mice was related to one or several intramural haematomas. These were caused by ruptures of the tunica media near the ostium of small suprarenal side branches, which had never been detected by the established small animal imaging techniques. The tear near the celiac artery led to apparent luminal dilatation, while the intramural haematoma led to a dissection of the tunica adventitia on the left suprarenal side of the aorta. The number of ruptured branches was higher in those aneurysms that extended into the thoracic aorta, which explained the observed variability in aneurysm shape. CONCLUSION Our results are the first to describe apparent luminal dilatation, suprarenal branch ruptures, and intramural haematoma formation in dissecting abdominal aortic aneurysms in mice. Moreover, we validate and demonstrate the vast potential of phase contrast X-ray tomographic microscopy in cardiovascular small animal applications.

Comparison of 4D phase-contrast MRI flow measurements to computational fluid dynamics simulations of cerebrospinal fluid motion in the cervical spine.

Cerebrospinal fluid (CSF) dynamics in the cervical spinal subarachnoid space (SSS) have been thought to be important to help diagnose and assess craniospinal disorders such as Chiari I malformation (CM). In this study we obtained time-resolved three directional velocity encoded phase-contrast MRI (4D PC MRI) in three healthy volunteers and four CM patients and compared the 4D PC MRI measurements to subject-specific 3D computational fluid dynamics (CFD) simulations. The CFD simulations considered the geometry to be rigid-walled and did not include small anatomical structures such as nerve roots, denticulate ligaments and arachnoid trabeculae. Results were compared at nine axial planes along the cervical SSS in terms of peak CSF velocities in both the cranial and caudal direction and visual interpretation of thru-plane velocity profiles. 4D PC MRI peak CSF velocities were consistently greater than the CFD peak velocities and these differences were more pronounced in CM patients than in healthy subjects. In the upper cervical SSS of CM patients the 4D PC MRI quantified stronger fluid jets than the CFD. Visual interpretation of the 4D PC MRI thru-plane velocity profiles showed greater pulsatile movement of CSF in the anterior SSS in comparison to the posterior and reduction in local CSF velocities near nerve roots. CFD velocity profiles were relatively uniform around the spinal cord for all subjects. This study represents the first comparison of 4D PC MRI measurements to CFD of CSF flow in the cervical SSS. The results highlight the utility of 4D PC MRI for evaluation of complex CSF dynamics and the need for improvement of CFD methodology. Future studies are needed to investigate whether integration of fine anatomical structures and gross motion of the brain and/or spinal cord into the computational model will lead to a better agreement between the two techniques.

Biomechanical adaptation of porcine carotid vascular smooth muscle to hypo and hypertension in vitro

Previous research in arterial remodeling in response to changes in blood pressure seldom included both hyper- and hypotension. To compare the effects of low and high pressure on arterial remodeling and vascular smooth muscle tone and performance, we have utilized an in vitro model. Porcine carotid arteries were cultured for 3 days at 30 and 170mmHg and compared to controls cultured at 100mmHg for 1 and 3 days. On the first and last day of culture, pressure-diameter and pressure-wall thickness curves were measured under normal smooth muscle tone using a high-resolution ultrasonic device. Last-day experiments included measurements where vascular smooth muscle was contracted or totally relaxed. From the data wall cross-sectional area, Hudetz elastic modulus and a contraction index related to the diameter reduction under normal smooth muscle tone were calculated. We found that although wall cross-sectional area (indicating wall mass) did not change much, Hudetz elastic modulus was significantly reduced in the 3-day hypotension group. Inspection of the wall contraction index suggests that this is due to a reduction in the vascular smooth muscle tone. Further, the peak of contraction index was found to be shifted to higher pressures in the 3-day 170mmHg group. We conclude that vascular smooth muscle performance adapts to both hypo- and hypertension at short time scales and can alter the biomechanics of the vascular wall in vitro.

Treatment with Angiotensin-(1-7) reduces inflammation in carotid atherosclerotic plaques

Angiotensin (Ang)-(1-7), acting through the receptor Mas, has atheroprotective effects; however, its role on plaque vulnerability has been poorly studied. Here, we investigated the expression of the renin-angiotensin system (RAS) components in stable and unstable human carotid plaques. In addition, we evaluated the effects of the chronic treatment with an oral formulation of Ang-(1-7) in a mouse model of shear stress-determined carotid atherosclerotic plaque. Upstream and downstream regions of internal carotid plaques were obtained from a recently published cohort of patients asymptomatic or symptomatic for ischaemic stroke. Angiotensinogen and renin genes were strongly expressed in the entire cohort, indicating an intense intraplaque modulation of the RAS. Intraplaque expression of the Mas receptor mRNA was increased in the downstream portion of asymptomatic patients as compared to corresponding region in symptomatic patients. Conversely, AT1 receptor gene expression was not modified between asymptomatic and symptomatic patients. Treatment with Ang-(1-7) in ApoE-/- mice was associated with increased intraplaque collagen content in the aortic root and low shear stress-induced carotid plaques, and a decreased MMP-9 content and neutrophil and macrophage infiltration. These beneficial effects were not observed in the oscillatory shear stress-induced plaque. In vitro incubation with Ang-(1-7) did not affect ICAM-1 expression and apoptosis on cultured endothelial cells. In conclusion, Mas receptor is up regulated in the downstream portions of human stable carotid plaques as compared to unstable lesions. Treatment with the oral formulation of Ang-(1-7) enhances a more stable phenotype in atherosclerotic plaques, depending on the local pattern of shear stress forces.

Nicotinamide phosphoribosyltransferase inhibition reduces intraplaque CXCL1 production and associated neutrophil infiltration in atherosclerotic mice

Pharmacological treatments targeting CXC chemokines and the associated neutrophil activation and recruitment into atherosclerotic plaques hold promise for treating cardiovascular disorders. Therefore, we investigated whether FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with anti-inflammatory properties that we recently found to reduce neutrophil recruitment into the ischaemic myocardium, would exert beneficial effects in a mouse atherosclerosis model. Atherosclerotic plaque formation was induced by carotid cast implantation in ApoE-/- mice that were fed with a Western-type diet. FK866 or vehicle were administrated intraperitoneally from week 8 until week 11 of the diet. Treatment with FK866 reduced neutrophil infiltration and MMP-9 content and increased collagen levels in atherosclerotic plaques compared to vehicle. No effect on other histological parameters, including intraplaque lipids or macrophages, was observed. These findings were associated with a reduction in both systemic and intraplaque CXCL1 levels in FK866-treated mice. In vitro, FK866 did not affect MMP-9 release by neutrophils, but it strongly reduced CXCL1 production by endothelial cells which, in the in vivo model, were identified as a main CXCL1 source at the plaque level. CXCL1 synthesis inhibition by FK866 appears to reflect interference with nuclear factor-κB signalling as shown by reduced p65 nuclear levels in endothelial cells pre-treated with FK866. In conclusion, pharmacological inhibition of NAMPT activity mitigates inflammation in atherosclerotic plaques by reducing CXCL1-mediated activities on neutrophils. These results support further assessments of NAMPT inhibitors for the potential prevention of plaque vulnerability.

A method for the quantification of the pressure dependent 3D collagen configuration in the arterial adventitia

Collagen plays an important role in the response of the arterial wall to mechanical loading and presumably has a load-bearing function preventing overdistension. Collagen configuration is important for understanding this role, in particular in mathematical models of arterial wall mechanics. In this study a new method is presented to image and quantify this configuration. Collagen in the arterial adventitia is stained with CNA35, and imaged in situ at high resolution with confocal microscopy at luminal pressures from 0 to 140mm Hg. The images are processed with a new automatic approach, utilizing techniques intended for MRI-DTI data. Collagen configuration is quantified through three parameters: the waviness, the transmural angle and the helical angle. The method is demonstrated for the case of carotid arteries of the white New Zealand rabbit. The waviness indicated a gradual straightening between 40 and 80mm Hg. The transmural angle was about zero indicating that the fibers stayed within an axial-circumferential plane at all pressures. The helical angle was characterized by a symmetrical distribution around the axial direction, indicating a double symmetrical helix. The method is the first to combine high resolution imaging with a new automatic image processing approach to quantify the 3D configuration of collagen in the adventitia as a function of pressure.

An oral formulation of angiotensin-(1-7) reverses corpus cavernosum damages induced by hypercholesterolemia.

INTRODUCTION The renin angiotensin system plays a crucial role in erectile function. It has been shown that elevated angiotensin-II levels contribute to the development of erectile dysfunction (ED). Oppositely, angiotensin-(1-7) (Ang-[1-7]) mediates penile erection by activation of receptor Mas. Recently, we have developed a formulation based on Ang-(1-7) inclusion in cyclodextrin (CyD) [Ang-(1-7)-CyD], which allows for the oral administration of Ang-(1-7). AIM In the present study, we evaluated the effects of chronic treatment with Ang-(1-7)-CyD on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemic mice. METHODS Apolipoprotein(Apo)E-/- mice fed a Western-type diet for 11 weeks received Ang-(1-7)-CyD or vehicle during the final 3 weeks. Collagen content and reactive oxygen species (ROS) production within the corpus cavernosum were evaluated by Sirius red and dihydroethidium staining, respectively. Protein expression of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) subunits (p67-phox and p22-phox), and AT1 and Mas receptors in the penis was assessed by Western blotting. Nitric oxide (NO) production was measured by Griess assay in the mice serum. Cavernosal strips were mounted in an isometric organ bath to evaluate the endothelial function. MAIN OUTCOME MEASURES The effect of Ang-(1-7)-CyD treatment on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemia-induced ED. RESULTS Ang-(1-7)-CyD treatment reduced collagen content in the corpus cavernosum of ApoE-/- mice. This effect was associated with an attenuation of ROS production and a diminished expression of NADPH. Furthermore, Ang-(1-7)-CyD treatment augmented the expression of nNOS and eNOS in the penis and elevated vascular NO production. Importantly, these effects were accompanied by an improvement in cavernosal endothelial function. CONCLUSION Long-term treatment with Ang-(1-7)-CyD reduces penile fibrosis associated with attenuation of oxidative stress. Additionally, cavernosal endothelial function in hypercholesterolemic mice was markedly improved. These results suggest that Ang-(1-7)-CyD might have significant therapeutic benefits for the treatment of erectile dysfunction.