Optimal cut-off criteria for duplex ultrasound for the diagnosis of restenosis in stented carotid arteries: Review and protocol for a diagnostic study

Background  

Carotid angioplasty with stenting is a relatively new, increasingly used, less-invasive treatment for the treatment of symptomatic carotid artery stenosis. It is being evaluated in ongoing and nearly finished randomized trials. An important factor in the evaluation of stents is the occurrence of in-stent restenosis. An un-stented carotid artery is likely to have a more elastic vessel wall than a stented one, even if stenosis is present. Therefore, duplex ultrasound cut-off criteria for the degrees of an in-stent stenosis, based on blood velocity parameters, are probably different from the established cut-offs used for un-stented arteries. Routine criteria can not be applied to stented arteries but new criteria need to be established for this particular purpose.     

Light microscopic immunolocation of thrombospondin in human tissues

Affinity-purified antisera against thrombospondin were used to locate the presence of this glycoprotein in frozen sections of several human tissues by immunofluorescence techniques. Immunostaining was observed in the peritubular connective tissue and in basement membrane regions beneath glandular epithelium in skin and lung. Intense immunostaining was observed at the dermal-epidermal junction in skin and in small blood vessels throughout this tissue. Skeletal muscle exhibited positive staining with anti-thrombospondin antisera within interstitial areas. Immunostaining was confined to the luminal portions of large blood vessels such as aorta. In large blood vessels that contained lesions of atherosclerosis, immunostaining was observed throughout the lesion area and was especially prominent surrounding some of the lesion cells. These results indicate that thrombospondin is located within the matrix of a variety of human tissues and supports the suggestion that this glycoprotein is an endogenous component of some extracellular matrices.     

Circumferential vascular deformation after stent implantation alters wall shear stress evaluated with time-dependent 3D computational fluid dynamics models.

The success of vascular stents in the restoration of blood flow is limited by restenosis. Recent data generated from computational fluid dynamics (CFD) models suggest that stent geometry may cause local alterations in wall shear stress (WSS) that have been associated with neointimal hyperplasia and subsequent restenosis. However, previous CFD studies have ignored histological evidence of vascular straightening between circumferential stent struts. We tested the hypothesis that consideration of stent-induced vascular deformation may more accurately predict alterations in indexes of WSS that may subsequently account for histological findings after stenting. We further tested the hypothesis that the severity of these alterations in WSS varies with the degree of vascular deformation after implantation. Steady-state and time-dependent simulations of three-dimensional CFD arteries based on canine coronary artery measurements of diameter and blood flow were conducted, and WSS and WSS gradients were calculated. Circumferential straightening introduced areas of high WSS between stent struts that were absent in stented vessels of circular cross section. The area of vessel exposed to low WSS was dependent on the degree of circumferential vascular deformation and axial location within the stent. Stents with four vs. eight struts increased the intrastrut area of low WSS in vessels, regardless of cross-sectional geometry. Elevated WSS gradients were also observed between struts in vessels with polygonal cross sections. The results obtained using three-dimensional CFD models suggest that changes in vascular geometry after stent implantation are important determinants of WSS distributions that may be associated with subsequent neointimal hyperplasia.     

Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs: comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement

Odontoblasts and osteoblasts synthesize gamma-carboxyglutamatic acid (Gla)-containing proteins which are partially deposited in the mineralizing tissues and partially released into the plasma. Using four immunostaining techniques, we have evaluated the question of whether dentin Gla proteins (DGP) are transported to the mineralization front through the odontoblast processes. Undecalcified sections of rat incisors and molar tooth germs were immunostained with affinity-purified antibodies to DGP using the following methods: indirect immunofluorescence; peroxidase-antiperoxidase (PAP); avidin-biotin-peroxidase complex (ABC-peroxidase); and avidin-biotin-gold complex with silver enhancement (ABC-GSS). The results obtained with these four procedures were compared with respect to the developmental appearance of DGP, staining intensity and presence in odontoblastic processes, predentin, dentin, and blood vessels. Qualitatively, similar results were obtained with the four, with respect to the distribution and developmental appearance of DGP, with two exceptions: indirect immunofluorescence never stained DGP within blood vessels, whereas the other methods occasionally did; and because of its sensitivity, only the ABC-GSS method revealed immunostaining for DGP in odontoblastic processes. All methods revealed weak immunostaining in predentin which was considerably enhanced with hyaluronidase treatment; however, hyaluronidase only moderately increased predentin immunostaining with ABC-GSS. Of these four procedures, ABC-GSS is the most sensitive; however, ABC-GSS appears to detect predominantly antigens at the surface of tissue sections. We conclude that DGP is present in odontoblastic processes but in low amounts; the weak staining was due either to rapid transport of DGP through the process or to the fact that this mode of transport is limited.     

Mechanical and hydrodynamic effects of stent expansion in tapered coronary vessels

Percutaneous coronary intervention (PCI) has become the primary treatment for patients with coronary heart disease because of its minimally invasive nature and high efficiency. Anatomical studies have shown that most coronary vessels gradually shrink, and the vessels gradually become thinner from the proximal to the distal end. In this paper, the effects of different stent expansion methods on the mechanical and hemodynamic behaviors of coronary vessels and stents were studied. To perform a structural-mechanical analysis of stent implantation, the coronary vessels with branching vessels and the coronary vessels with large bending curvature are selected. The two characteristic structures are implanted in equal diameter expansion mode and conical expansion mode, and the stress and mechanical behaviors of the coronary vessels and stents are analyzed. The results of the structural-mechanical analysis showed that the mechanical behaviors and fatigue performance of the cobalt-chromium alloy stent were good, and the different expansion modes of the stent had little effect on the fatigue performance of the stent. However, the equal diameter expansion mode increased distal coronary artery stress and the risk of vascular injury. The computational fluid dynamics analysis results showed that different stent expansion methods had varied effects on coronary vessel hemodynamics and that the wall shear stress distribution of conical stent expansion is more uniform compared with equal diameter expansion. Additionally, the vortex phenomenon is not apparent, the blood flow velocity is slightly increased, the hydrodynamic environment is more reasonable, and the risk of coronary artery injury is reduced.

     

Bifurcation asymmetry of the porcine coronary vasculature and its implications on coronary flow heterogeneity

The branching pattern of the coronary arteries and veins is asymmetric, i.e., many small vessels branch off of a large trunk such that the two daughter vessels at a bifurcation are of unequal diameters and lengths. One important implication of the geometric vascular asymmetry is the dispersion of blood flow at a bifurcation, which leads to large spatial heterogeneity of myocardial blood flow. To document the asymmetric branching pattern of the coronary vessels, we computed an asymmetry ratio for the diameters and lengths of all vessels, defined as the ratio of the daughter diameters and lengths, respectively. Previous data from silicone elastomer cast of the entire coronary vasculature including arteries, arterioles, venules, and veins were analyzed. Data on smaller vessels were obtained from histological specimens by optical sectioning, whereas data on larger vessels were obtained from vascular casts. Asymmetry ratios for vascular areas, volumes, resistances, and flows of the various daughter vessels were computed from the asymmetry ratios of diameters and lengths for every order of mother vessel. The results show that the largest orders of arterial and venous vessels are most asymmetric and the degree of asymmetry decreases toward the smaller vessels. Furthermore, the diameter asymmetry at a bifurcation is significantly larger for the coronary veins (1.7-6.8 for sinus veins) than the corresponding arteries (1.5-5.8 for left anterior descending coronary artery) for orders 2-10, respectively. The reported diameter asymmetry at a bifurcation leads to significant heterogeneity of blood flow at a bifurcation. Hence, the present data quantify the dispersion of blood flow at a bifurcation and are essential for understanding flow heterogeneity in the coronary circulation.     

Compound Ex Vivo and In Silico Method for Hemodynamic Analysis of Stented Arteries

Hemodynamic factors such as low wall shear stress have been shown to influence endothelial healing and atherogenesis in stent-free vessels. However, in stented vessels, a reliable quantitative analysis of such relations has not been possible due to the lack of a suitable method for the accurate acquisition of blood flow. The objective of this work was to develop a method for the precise reconstruction of hemodynamics and quantification of wall shear stress in stented vessels. We have developed such a method that can be applied to vessels stented in or ex vivo and processed ex vivo. Here we stented the coronary arteries of ex vivo porcine hearts, performed vascular corrosion casting, acquired the vessel geometry using micro-computed tomography and reconstructed blood flow and shear stress using computational fluid dynamics. The method yields accurate local flow information through anatomic fidelity, capturing in detail the stent geometry, arterial tissue prolapse, radial and axial arterial deformation as well as strut malapposition. This novel compound method may serve as a unique tool for spatially resolved analysis of the relationship between hemodynamic factors and vascular biology. It can further be employed to optimize stent design and stenting strategies.     

c-kit-immunopositive vascular progenitor cells populate human coronary in-stent restenosis but not primary atherosclerotic lesions

Progress in the treatment of human in-stent restenosis (ISR) is hampered by an imprecise understanding of the nature of the cells that occlude vascular stents. Recent studies suggest that circulating vascular progenitor cells may mediate vascular repair and lesion formation. Moreover, functional endothelial progenitor cells appear to play a protective role in attenuating vascular lesion formation. Hence, we sought to answer two important questions: 1). Are primitive cells found in ISR lesions? 2). Is the abundance of cultured angiogenic cells (CACs) in patients with ISR different from that in patients with non-ISR lesions or normal controls? Human coronary atherectomy tissue from 13 ISR, 6 postangioplasty restenosis (RS), and 14 primary (PR) atherosclerotic lesions, as well as 15 postmortem coronary artery cross sections from young individuals without atherosclerosis, were studied. All 13 ISR and 4 of 6 RS tissue specimens contained cells that immunolabeled for the primitive cell marker c-kit and smooth muscle alpha-actin, whereas the intima and media of PR lesions and normal arteries were devoid of c-kit-immunopositive cells. The abundance of peripheral blood mononuclear cell-derived CACs was assessed in 10 patients with ISR, 6 patients with angiographically verified patent stents, and 6 individuals with no clinical evidence of coronary artery disease. CACs were less abundant in ISR patients than in non-ISR controls (13.9 +/- 3.1 vs. 22.3 +/- 6.7 cells/high-power field, P < 0.05), and both of these groups had fewer CACs than non-coronary artery disease patients (37.6 +/- 3.8, P < 0.05). These findings suggest a unique pathogenesis for ISR and RS lesions that involves c-kit-immunopositive smooth muscle cells. Moreover, the paucity of CACs in patients with ISR may contribute to the pathogenesis of ISR, perhaps because of attenuated reendothelialization.     

冠状动脉弥漫性长病变治疗的研究进展

冠心病目前已经成为全球性关注的健康问题,为当今人类一大灾难性疾病。既往研究表明,冠状动脉弥漫性长病变占冠心病总患者约20%,冠状动脉弥漫性长病变患者其动脉粥样硬化的病变范围更加广泛,病变呈弥漫性,而且更多累及左主干,常常伴有血管直径小,血管成角、钙化、扭曲等特点,而且多发生于高龄、糖尿病患者中,以上特点又决定了冠状动脉弥漫性长病变成为冠心病治疗的又一难题。因此,有效的预防与治疗冠状动脉弥漫性长病变,已成为目前的关注重点与热点,本文概述了冠状动脉弥漫性长病变临床治疗中的常用方法以及各方法的疗效与优劣之势,多年临床实践经验表明PCI治疗仍占有主导位置,虽然目前冠状动脉弥漫性长病变的介入治疗应用药物洗脱支架的已经取得良好的临床效果,在很大程度降低了心血管事件发生率和再次血管重建率,但药物洗脱支架支架治疗冠状动脉弥漫性长病变的远期疗效仍在评估中。     

铜在心血管系统中的有益作用

随着经济水平和生活水平的逐步提高,我国心血管疾病的患者数量也在逐年上升。因此,加深对心血管疾病的认知和预防乃是当务之急。在人体所需的众多微量金属元素中,铜对维护心血管的健康起到了重要的作用。铜的缺乏可能会导致一系列心血管疾病的发生,如冠心病、高血压、心律失常等。究其原因,是由于铜能影响血管的形成以及血管的正常生理功能,同时也参与了众多相关生长因子的调控。所以,人体要保证足量的铜摄入,避免铜元素的缺乏。向生物材料中主动添加铜元素,可以通过释放铜离子起到促进内皮细胞增殖和迁移的作用,进而加速伤口的愈合。目前,治疗高发的冠心病的重要手段是采用冠脉支架植入手术,但是其依旧面临着支架内再狭窄和血栓这两种风险。含铜的金属支架材料通过释放对血管有益的铜离子,有望加快支架植入后的内皮化过程,进而降低支架内再狭窄和血栓的发生率。所以,将来积极开发应用于心血管领域的含铜医用材料是一种缓解和治疗心血管疾病的有效途径。     

Detection of antileukoprotease in connective tissue of the lung

An indirect immunofluorescence technique was applied to frozen sections of central and peripheral human lung tissue to search for extracellular localizations of antileukoprotease (ALP). Two monoclonal anti-ALP antibodies recognizing different epitopes and polyclonal anti-ALP antibodies were used. ALP was found to be localized along elastic fibers in alveolar septa, and also along elastic fibers in the walls of bronchi, bronchioles and blood vessels. Serous cells of bronchial submucosal glands showed labelling as well. In frozen sections of liver and spleen no label was found. Cells and elastic fibers were not labelled when lung tissue sections were processed with polyclonal or monoclonal anti-ALP antibodies, that were blocked with purified ALP before the immunostaining. The association of ALP with elastic fibers of human pulmonary connective tissue is of importance in understanding the role of the inhibitor in the defense of the lung parenchyma against the action of proteolytic enzymes, which is thought to result in emphysema.     

Detection of antileukoprotease in connective tissue of the lung

Summary An indirect immunofluorescence technique was applied to frozen sections of central and peripheral human lung tissue to search for extracellular localizations of antileukoprotease (ALP). Two monoclonal anti-ALP antibodies recognizing different epitopes and polyclonal anti-ALP antibodies were used. ALP was found to be localized along elastic fibers in alveolar septa, and also along elastic fibers in the walls of bronchi, bronchioles and blood vessels. Serous cells of bronchial submucosal glands showed labelling as well. In frozen sections of liver and spleen no label was found. Cells and elastic fibers were not labelled when lung tissue sections were processed with polyclonal or monoclonal anti-ALP antibodies, that were blocked with purified ALP before the immunostaining. The association of ALP with elastic fibers of human pulmonary connective tissue is of importance in understanding the role of the inhibitor in the defense of the lung parenchyma against the action of proteolytic enzymes, which is thought to result in emphysema.     

Microwave irradiation for fixation and immunostaining of endothelial cells in situ

Using microwave irradiation during tissue fixation and immunostaining reduces sample preparation time and facilitates penetration of fixatives and antibody solutions into the tissues. This results in improved fixation and reduction of non-specific binding of antibodies, respectively. Experimental analyses of endothelial cells in blood vessels in situ have been limited because of the difficulty of tissue preparation. We report here a technique using intermittent microwave irradiation for blood vessel fixation and immunostaining the fixed tissues. Intermittent microwave irradiation during fixation reduced blood vessel contraction and resulted in well preserved morphology of blood vessels, especially the endothelial cells. Microwave irradiation also reduced non-specific binding of fluorescein-labeled antibodies. These microwave irradiation-assisted techniques are useful for analysis of endothelial cell function and for pathological study of blood vessels in situ.     

Alterations in wall shear stress predict sites of neointimal hyperplasia after stent implantation in rabbit iliac arteries

Restenosis resulting from neointimal hyperplasia (NH) limits the effectiveness of intravascular stents. Rates of restenosis vary with stent geometry, but whether stents affect spatial and temporal distributions of wall shear stress (WSS) in vivo is unknown. We tested the hypothesis that alterations in spatial WSS after stent implantation predict sites of NH in rabbit iliac arteries. Antegrade iliac artery stent implantation was performed under angiography, and blood flow was measured before casting 14 or 21 days after implantation. Iliac artery blood flow domains were obtained from three-dimensional microfocal X-ray computed tomography imaging and reconstruction of the arterial casts. Indexes of WSS were determined using three-dimensional computational fluid dynamics. Vascular histology was unchanged proximal and distal to the stent. Time-dependent NH was localized within the stented region and was greatest in regions exposed to low WSS and acute elevations in spatial WSS gradients. The lowest values of WSS spatially localized to the stented area of a theoretical artery progressively increased after 14 and 21 days as NH occurred within these regions. This NH abolished spatial disparity in distributions of WSS. The results suggest that stents may introduce spatial alterations in WSS that modulate NH in vivo.     

Hemodynamics in coronary arteries with overlapping stents

Coronary artery stenosis is commonly treated by stent placement via percutaneous intervention, at times requiring multiple stents that may overlap. Stent overlap is associated with increased risk of adverse clinical outcome. While changes in local blood flow are suspected to play a role therein, hemodynamics in arteries with overlapping stents remain poorly understood. In this study we analyzed six cases of partially overlapping stents, placed ex vivo in porcine left coronary arteries and compared them to five cases with two non-overlapping stents. The stented vessel geometries were obtained by micro-computed tomography of corrosion casts. Flow and shear stress distribution were calculated using computational fluid dynamics. We observed a significant increase in the relative area exposed to low wall shear stress (WSS<0.5 Pa) in the overlapping stent segments compared both to areas without overlap in the same samples, as well as to non-overlapping stents. We further observed that the configuration of the overlapping stent struts relative to each other influenced the size of the low WSS area: positioning of the struts in the same axial location led to larger areas of low WSS compared to alternating struts. Our results indicate that the overlap geometry is by itself sufficient to cause unfavorable flow conditions that may worsen clinical outcome. While stent overlap cannot always be avoided, improved deployment strategies or stent designs could reduce the low WSS burden.     

Hydrodynamic effects of compliance mismatch in stented arteries

Cardiovascular diseases are the number one cause of death in the world, making the understanding of hemodynamics and development of treatment options imperative. The most common modality for treatment of occlusive coronary artery diseases is the use of stents. Stent design profoundly influences the postprocedural hemodynamic and solid mechanical environment of the stented artery. However, despite their wide acceptance, the incidence of stent late restenosis is still high (Zwart et al., 2010, "Coronary Stent Thrombosis in the Current Era: Challenges and Opportunities for Treatment," Current Treatment Options in Cardiovascular Medicine, 12(1), pp. 46-57), and it is most prevailing at the proximal and distal ends of the stent. In this work, we focus our investigation on the localized hemodynamic effects of compliance mismatch due to the presence of a stent in an artery. The compliance mismatch in a stented artery is maximized at the proximal and distal ends of the stent. Hence, it is our objective to understand and reveal the mechanism by which changes in compliance contribute to the generation of nonphysiological wall shear stress (WSS). Such adverse hemodynamic conditions could have an effect on the onset of restenosis. Three-dimensional, spatiotemporally resolved computational fluid dynamics simulations of pulsatile flow with fluid-structure interaction were carried out for a simplified coronary artery with physiologically relevant flow parameters. A model with uniform elastic modulus is used as the baseline control case. In order to study the effect of compliance variation on local hemodynamics, this baseline model is compared with models where the elastic modulus was increased by two-, five-, and tenfold in the middle of the vessel. The simulations provided detailed information regarding the recirculation zone dynamics formed during flow reversals. The results suggest that discontinuities in compliance cause critical changes in local hemodynamics, namely, altering the local pressure and velocity gradients. The change in pressure gradient at the discontinuity was as high as 90%. The corresponding changes in WSS and oscillatory shear index calculated were 9% and 15%, respectively. We demonstrate that these changes are attributed to the physical mechanism associating the pressure gradient discontinuities to the production of vorticity (vorticity flux) due to the presence of the stent. The pressure gradient discontinuities and augmented vorticity flux are affecting the wall shear stresses. As a result, this work reveals how compliance variations act to modify the near wall hemodynamics of stented arteries.     

Contribution of recipient-derived cells in allograft neointima formation and the response to stent implantation

Allograft coronary disease is the dominant cause of increased risk of death after cardiac transplantation. While the percutaneous insertion of stents is the most efficacious revascularization strategy for allograft coronary disease there is a high incidence of stent renarrowing. We developed a novel rabbit model of sex-mismatched allograft vascular disease as well as the response to stent implantation. In situ hybridization for the Y-chromosome was employed to detect male cells in the neointima of stented allograft, and the population of recipient derived neointimal cells was measured by quantitative polymerase chain reaction and characterized by immunohistochemistry. To demonstrate the participation of circulatory derived cells in stent neointima formation we infused ex vivo labeled peripheral blood mononuclear cells into native rabbit carotid arteries immediately after stenting. Fourteen days after stenting the neointima area was 58% greater in the stented vs. non-stented allograft segments (p = 0.02). Male cells were detected in the neointima of stented female-to-male allografts. Recipient-derived cells constituted 72.1+/-5.7% and 81.5+/-4.2% of neointimal cell population in the non-stented and stented segments, respectively and the corresponding proliferation rates were only 2.7+/-0.5% and 2.3+/-0.2%. Some of the recipient-derived neointimal cells were of endothelial lineage. The ex vivo tagged cells constituted 9.0+/-0.4% of the cells per high power field in the stent neointima 14 days after stenting. These experiments provide important quantitative data regarding the degree to which host-derived blood-borne cells contribute to neointima formation in allograft vasculopathy and the early response to stent implantation.     

Corticotropin-releasing factor: immunohistochemical colocalization with adrenocorticotropin and beta-endorphin, but not with Met-enkephalin, in subpopulations of duodenal perikarya of rat

By the use of two different double-staining techniques (simultaneous staining of adjacent serial sections and the double-staining elution method) it was possible to demonstrate that a corticotropin-releasing factor (CRF) immunofluorescence co-existed with an adrenocorticotropin (ACTH) and beta-endorphin (beta-END) immunoreactivity, but not with a Met-enkephalin (Met-ENK) immunostaining, within perikarya subpopulations of both the myenteric and submucousal plexus of the rat duodenum. Not a single Met-ENK-positive neuronal cell body was stained also for CRF, ACTH or beta-END. Even nerve fibres, localized in both the myenteric plexus and closely to submucousal blood vessels (probably arterioles), revealed a CRF immunofluorescence, which is also colocalized with an beta-END staining. These results are quite different to the recent observations in the mammalian hypothalamus, suggesting that some myenteric and submucousal plexus neurons may synthesize CRF as well as beta-END and ACTH, but not Met-ENK. The colocalized peptides might be concomitantly released into the synaptic cleft after terminal stimulation.     

Late thrombosis in cypher stents after the discontinuation of antiplatelet therapy

Drug-eluting stents, since their approval in the United States, have become the treatment of choice for de novo coronary artery narrowing due to their ability to reduce restenosis and the need for repeat revascularization. We present two patients who underwent percutaneous coronary intervention for the treatment of multivessel coronary artery disease; both patients were treated with sirolimus-eluting stents (SES) and bare metal stents (BMS).     

Immunolocalization of collagen types I and III in the arterial wall of the rat

Summary Collagen types I and III were located by immunofluorescence procedures in the aorta and coronary arteries of the rat. Type I collagen was most prevalent in the adventitia of the aorta with only small amounts present in the intima and media. Type III collagen appeared to be a significant component in the media of the aorta and also in the adventitia of both blood vessels. The intima and media of the coronary arteries did not stain strongly for either type I or III collagen. Neither staining procedure was altered with preincubation of the sections with hyaluronidase or chondroitinase ABC. These studies indicate that type III collagen is a major component of the adventitia which has previously not been recognized by immunohistochemical techniques, possibly due to masking of collagen staining with glycosaminoglycans.