Lewis, Debra A.

Purpose: Endovascular occlusion of aneurysms with the use of detachable platinum coils has proved to be a safe and durable alternative to the traditional surgical clipping. The aim of the present study is to understand the mechanism of apoptosis in aneurysm embolized with platinum coils using rabbit elastase‐induced aneurysm model. Materials & Methods: Elastase‐induced saccular aneurysms were created at the origin of right common carotid artery in thirty rabbits. Aneurysms were allowed to mature for 8 weeks then 20 aneurysms were embolized using platinum coils by endovascular means. Ten uncoiled aneurysms were used as controls. After 2 weeks (n=10) and 4 weeks (n=10) of implantation, aneurysm samples were harvested for apoptotic studies. Results: Expression of pro‐caspases‐3, ‐8 and ‐9 were elevated in coiled aneurysm embolized with platinum coils at both time points when compared with uncoiled aneurysm and control left carotid artery. Cleaved caspases‐3, ‐8 and ‐9 were found to be expressed only at 4 weeks after embolization. Cells within aneurysm cavity were TUNEL positive at 4 weeks only. Expression of tumor necrosis‐α (TNF‐α) was high in coiled aneurysm when compared with controls. There was no significant difference in the expression of Fas‐ligand among groups. Decreased expression of antiapoptotic protein, Bcl‐2, and increased expression of proapoptotic proteins, Bad, Bax and Bid, were observed in coiled aneurysms at both time points. Conclusion: Activation apoptosis in aneurysms after embolization with platinum coils is mediated by both TNF‐α mediated‐extrinsic and Bcl‐2 mediated‐intrinsic pathway.

The purpose of the study was to test the hypothesis that systemic administration of vitamin C, through its action of stimulating collagen synthesis and crosslinking, would improve the healing response in experimental aneurysms treated with platinum coil embolization. Nineteen experimental aneurysms in rabbits were embolized with platinum coils (>30% packing density). Animals were divided into three groups. Group 1 (n=6) rabbits served as controls, Group 2 (n=5) rabbits were fed with a vitamin C‐supplemented feed and Group 3 (n=8) rabbits were medicated with vitamin C pills. Subjects were euthanized at 12 weeks following coil implantation and serum vitamin C levels were measured. Digital subtraction angiography was used to evaluate stability after embolization. Histological samples were examined with a grading system (range, 0–12) based on the neck and dome features. Masson Trichrome was employed to evaluate the collagen deposition. One‐way analysis of variance was used to test the significance in histological healing and angiographic measurements between groups. There were no significant differences between groups in aneurysm size or angiographic findings. Mean serum vitamin C concentration was significantly higher in Group 3 (45.4±9.0 mM) and Group 2 (38.6±8.9 mM) compared to Group 1 (21.1±3.6 mM). There was no statistically significant change in the level of vitamin C between Group 2 and Group 3. There was no significant difference in histological scores or collagen deposition among groups. Our results suggest that vitamin C supplementation following platinum coil embolization may not improve long‐term cerebral aneurysm. This study was supported by the American Heart Association (09SDG2160146‐01).

Estrogen replacement increases risk of venous thrombosis. In this study, we determined responses in vitro to platelets and platelet products in veins from adult male and intact and ovariectomized female pigs. When contracted with prostaglandin F2α, platelets (25,000 platelets/μl) caused relaxation in veins with endothelium. Higher numbers of platelets caused contraction in veins with and without endothelium. In veins without endothelium, contractions were greater in veins from male than in veins from female pigs, and contractions in intact female pig veins were greater than in ovariectomized females pig veins. Platelet products 5-hydroxytryptamine and thromboxane (analog U-46619) caused comparable contractions in all veins; contractions to prostacyclin were less in veins from intact female pigs. ADP caused comparable endothelium-dependent relaxations in all groups. These relaxations were increased by indomethacin in veins from intact males and females; with inhibition of nitric oxide, relaxations were comparable in all groups. These results suggest that venous responses to platelets vary with sex and presence of ovaries in female pigs. These variations reflect differences in type and quantity of substances released from platelets as well as the sensitivity of the smooth muscle to some vasoactive substances. In addition, products of cyclooxygenase may reduce endothelium-dependent relaxations in veins.

Experiments were designed to determine whether normal fluctuations in sex steroid hormones alter gene transcription for endothelial nitric oxide synthase (NOS) and preproendothelin-1 (prepro-ET-1). Aortic endothelial cells were removed from adult, gonadally intact male and female or ovariectomized Yorkshire pigs. Endothelial cells were prepared for Northern blot analysis, Western blot analysis or enzyme activity. Nitric oxide products (NOx) and endothelin-1 (ET-1) in plasma were measured by chemiluminescence and radioimmunoassay, respectively. Northern blot analysis identified single bands corresponding to endothelial NOS and prepro-ET-1. Quantification of the blots showed an increase in expression of mRNA for both endothelial NOS and prepro-ET-1 in ovariectomized pigs compared with gonadally intact male and female pigs. There were no differences in amount of endothelial NOS protein identified by Western blot analysis among groups. On the contrary, plasma concentrations of NOx were significantly decreased in ovariectomized pigs, and there were no differences either in the concentrations of ET-1 in the plasma or extracts from the coronary arteries. These results suggest that expression of endothelial NOS and prepro-ET-1 may be regulated at transcriptional level by ovarian hormones. In addition, the ovarian hormones may regulate production of these endothelium-derived factors at the posttranscriptional level.

Hemodynamic factors are thought to play an important role in the initiation, growth, and rupture of cerebral aneurysms. In-vitro studies have demonstrated a correlation between the magnitude and distribution of wall shear stress (WSS) and biological response of both endothelial cells and smooth muscle cells [1–3]. In elastase induced saccular aneurysms, low WSS (below 0.5 Pa) was found to have a correlation with altered expression of biological markers [4]. Localized regions of rapid aneurysm growth in-vivo have been shown to be associated with regions where WSS is below a critical value of 0.1 Pa [5]. Further, aspect ratio (AR), the ratio of the maximum diameter of the aneurysm to the width of the aneurysm neck, has been correlated with elevated risk of rupture [6]. The purpose of the current study is to explore the possibility of creating elastase induced aneurysms in rabbits with a range of aspect ratios (ratio of aneurysm height/neck) and evaluate the existence of a correlation between aspect ratio and WSS distribution. Aneurysms with ARs from 0.98 to 2.8 were created at the origin of the right common carotid artery (n = 30). Qualitative differences in WSS distribution were found in the high AR aneurysms (HARA) (AR>1.6) and low AR aneurysms (LARA) (AR<1.6) [7].

An intracranial aneurysm (IA) is a pathological condition of cerebral arteries characterized by local enlargements of the arterial wall, typically into a saccular shape. Rupture of the aneurysm sac can result in devastating cerebral hemorrhage. Hemodynamic factors are believed to play an important role in initiation, development and rupture of IAs [1–3]. However, the coupling between hemodynamics and aneurysm pathophysiology is complex and remains poorly understood. Patient specific diagnostics regarding risk of rupture can be substantially advanced by improving our understanding of the in-vivo response of the aneurysm wall to intra-saccular hemodynamic stresses. A mechanism for fundamental studies of the impact of chronically altered WSS on the intact vascular wall is provided by animal models. However, cerebral aneurysms have not been shown to occur naturally in animals. Thus, a number of animal models have been created for studying aneurysm pathogenesis including those in mice, rats, rabbits, canines, swine and primates. To make meaningful use of these models, it is important to evaluate their relevance to human biomechanics and pathophysiology.

Background and purpose To test the hypothesis that systemic administration of vitamin C, through its action of stimulating collagen synthesis and crosslinking, would decrease the recurrence and improve the occlusion of experimental aneurysms treated with platinum coils. Methods Experimental aneurysms were created in female rabbits and were embolized with platinum coils (>30% packing density). The animals were divided into three groups: group 1 (n=6) rabbits served as controls, group 2 (n=5) rabbits were fed with a vitamin C supplemented feed and group 3 (n=8) rabbits were medicated with vitamin C pills. Digital subtraction angiography was used to evaluate stability after embolization. Subjects were euthanized at 12 weeks after coil implantation, and serum vitamin C levels were then measured. Histological samples were examined with a grading system (range 0–12) based on the neck and dome features. Masson Trichrome staining was used to evaluate collagen deposition. Parametric data were analyzed with one way analysis of variance and non-parametric data were examined using a Kruskal–Wallis test. Results There were no significant differences between groups in mean aneurysm size. Mean serum vitamin C concentration was significantly higher in group 3 and group 2 compared with group 1, while vitamin C levels between group 2 and group 3 were statistically comparable. Coil compaction was noted in one of six subjects in group 1 and in three of eight subjects in group 3. All of the remaining aneurysms in the test and control groups showed stable occlusion. There were no significant differences in histological scores or collagen deposition among groups. Conclusions Vitamin C supplementation following platinum coil embolization does not demonstrate improvement of long term occlusion rates of aneurysms.

Abstract —Insulin-like growth factor (IGF)-I stimulates vascular smooth muscle cell (VSMC) migration and proliferation, which are fundamental to neointimal hyperplasia in postangioplasty restenosis. IGF-I action is modulated by several high-affinity IGF binding proteins (IGFBPs). IGFBP-4 is the predominant IGFBP produced by VSMCs and is a potent inhibitor of IGF-I action. However, specific IGFBP-4 proteases can cleave IGFBP-4 and liberate active IGF-I. In this study, we document IGFBP-4 protease produced by human and porcine coronary artery VSMCs in culture as pregnancy-associated plasma protein-A (PAPP-A). This was shown by a distinctive IGFBP-4 cleavage pattern, specific inhibition of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition of PAPP-A by monoclonal antibodies. Furthermore, we found a 2-fold increase in IGFBP-4 protease activity in injured porcine VSMC cultures in vitro ( P <0.05). We also evaluated IGFBP-4 protease/PAPP-A expression in vivo after coronary artery balloon injury. Twenty-five immature female pigs underwent coronary overstretch balloon injury, and vessels were examined at defined time points after the procedure. Abundant PAPP-A expression was observed in the cytoplasm of medial and neointimal cells 7, 14, and 28 days after angioplasty ( P <0.01 vs control). The highest PAPP-A labeling indices were located in the neointima (36.1±2.1%) and the media (31.7±1.2%) 28 days after injury. Western blot analysis confirmed increased PAPP-A in injured vessels. PAPP-A, a regulator of IGF-I bioavailability through proteolysis of IGFBP-4, is thus expressed by VSMCs in vitro and in restenotic lesions in vivo. These results suggest a possible role for PAPP-A in neointimal hyperplasia.

Computational fluid dynamics (CFD) studies provide a valuable tool for evaluating the role of hemodynamics in vascular diseases such as cerebral aneurysms and atherosclerosis. However, such models necessarily only include isolated segments of the vasculature. In this work, we evaluate the influence of geometric approximations in vascular anatomy on hemodynamics in elastase induced saccular aneurysms in rabbits. One representative high aspect ratio (AR—height/neck width) aneurysm and one low AR aneurysm were created at the origin of the right common carotid artery in two New Zealand white rabbits. Three-dimensional (3D) reconstructions of the aneurysm and surrounding arteries were created using 3D rotational angiographic data. Five models with varying extents of neighboring vasculature were created for both the high and low AR cases. A reference model included the aneurysm sac, left common carotid artery (LCCA), aortic arch, and downstream trifurcation/quadrification. Three-dimensional, pulsatile CFD studies were performed and streamlines, wall shear stress (WSS), oscillatory shear index, and cross sectional velocity were compared between the models. The influence of the vascular domain on intra-aneurysmal hemodynamics varied between the low and high AR cases. For the high AR case, even a simple model including only the aneurysm, a small section of neighboring vasculature, and simple extensions captured the main features of the steamline and WSS distribution predicted by the reference model. However, the WSS distribution in the low AR case was more strongly influenced by the extent of vasculature. In particular, it was necessary to include the downstream quadrification and upstream LCCA to obtain good predictions of WSS. The findings in this work demonstrate the accuracy of CFD results can be compromised if insufficient neighboring vessels are included in studies of hemodynamics in elastase induced rabbit aneurysms. Consideration of aspect ratio, hemodynamic parameters of interest, and acceptable magnitude of error when selecting the vascular domain will increase reliability of the results while decreasing computational time.

Background and Purpose— Even though endovascular coils have been widely used for the treatment of intracranial aneurysms, the cellular and molecular responses of aneurysms to the coils after embolization remain poorly understood. The aim of the present study was to understand the mechanism of apoptosis in aneurysms embolized with platinum coils in the rabbit model of elastase-induced aneurysms. Methods— Elastase-induced saccular aneurysms were created at the origin of the right common carotid artery in 30 rabbits. Aneurysms were allowed to mature for 8 weeks, after which 20 aneurysms were embolized with platinum coils by endovascular means. After 2 (n=10) and 4 (n=10) weeks of implantation, aneurysm samples harboring coils were harvested for apoptotic studies. The remaining 10 uncoiled aneurysms were used as controls; additional controls included the left common carotid artery, which had not undergone any surgical procedure. Control samples were harvested at 12 weeks after aneurysm creation. Results— Expression of procaspases-3, -8, and -9 was elevated in coiled aneurysms embolized with platinum coils at both time points when compared with uncoiled aneurysms and the left common carotid artery. Cleaved caspases-3, -8, and -9 were found to be expressed only at 4 weeks after embolization. Cells within the aneurysm cavity were terminal dUTP nick end-labeling–positive at 4 weeks only. These apoptotic cells were identified as smooth muscle actin–positive cells. Expression of tumor necrosis-α was high in coiled aneurysms when compared with controls. There was no significant difference in the expression of Fas ligand among groups. Decreased expression of antiapoptotic proteins Bcl-2 and phospho-Bad, as well as increased expression of proapoptotic proteins Bax and Bid, was observed in coiled aneurysms at both time points. Conclusions— Activation of apoptosis in aneurysms after embolization with platinum coils is induced by both tumor necrosis factor-α–mediated extrinsic and Bcl-2-mediated intrinsic pathways.