Fully coupled fluid–structure interaction model of congenital bicuspid aortic valves: effect of asymmetry on hemodynamics

A bicuspid aortic valve (BAV) is a congenital cardiac disorder where the valve consists of only two cusps instead of three, as in a normal tricuspid valve (TAV). Although 97 % of BAVs include asymmetric cusps, little or no prior studies have investigated the blood flow through a three-dimensional BAV and root. The aim of the present study was to characterize the effect of asymmetric BAV on the blood flow using fully coupled fluid–structure interaction (FSI) models with improved boundary conditions and tissue properties. This study presents four FSI models, including a native TAV, asymmetric BAVs with or without a raphe, and an almost symmetric BAV. Cusp tissue is composed of hyperelastic finite elements with collagen fibres embedded in the elastin matrix. A full cardiac cycle is simulated by imposing the same physiological blood pressures for all the TAV and BAV models. The latter have significantly smaller opening areas compared with the TAV. Larger stress values were found in the cusps of BAVs with fused cusps, at both the systolic and diastolic phases. The asymmetric geometry caused asymmetric vortices and much larger flow shear stress on the cusps which could be a potential initiator for early valvular calcification of BAVs.     

Amyloid substance within stenotic aortic valves promotes mineralization

Audet A, Côté N, Couture C, Bossé Y, Després J‐P, Pibarot P & Mathieu P
(2012) Histopathology  61, 610–619 Amyloid substance within stenotic aortic valves promotes mineralization Aims: Accumulation of apolipoproteins may play an important role in the pathobiology of calcific aortic valve disease (CAVD). We aimed to explore the hypothesis that apolipoprotein‐derived amyloid could play a role in the development of CAVD. Methods and results: In 70 explanted CAVD valves and 15 control non‐calcified aortic valves, we assessed the presence of amyloid by using Congo red staining. Immunohistochemistry was performed to document the presence of apolipoprotein AI (Apo‐AI). Apoptosis was documented by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) studies performed in control and CAVD valves. Control valves were free of amyloid. Deposition of amyloid was detected in all CAVD valves, and the amount was positively correlated with plasma high‐density lipoprotein and Apo‐AI levels. Apo‐AI within CAVD valves co‐localized with intense staining of fibrillar amyloid. In turn, deposition of amyloid co‐localized with apoptosis near mineralized areas. Isolation of amyloid fibrils confirmed that Apo‐AI is a major component of amyloid deposits in CAVD. In vitro, CAVD‐derived amyloid extracts increased apoptosis and mineralization of isolated aortic valvular interstitial cells. Conclusions: Apo‐AI is a major component of amyloid substance present within CAVD valves. Furthermore, amyloid deposits participate in mineralization in CAVD by promoting apoptosis of valvular interstitial cells.     

Valvular endothelial cells regulate the phenotype of interstitial cells in co-culture: effects of steady shear stress

Valvular endothelial cells interact with interstitial cells in a complex hemodynamic and mechanical environment to maintain leaflet tissue integrity. The precise roles of each cell type are difficult to ascertain in a controlled manner in vivo. The objective of this study was to develop a three-dimensional aortic valve leaflet model, comprised of valvular endothelium and interstitial cells, and determine the cellular responses to imposed lumenal fluid flow. Two leaflet models were created using type I collagen hydrogels. Model 1 contained 1 million/mL porcine aortic valve interstitial cells (PAVICs). Model 2 added a seeding of the lumenal surface of Model 1 with approximately 50,000/cm(2) porcine aortic valve endothelial cells (PAVECs). Both leaflet models were exposed to 20 dynes/cm(2) steady shear for up to 96 h, with static constructs serving as controls. Endothelial cell alignment, matrix production, and cell phenotype were monitored. The results indicate that PAVECs align perpendicularly to flow similar to 2D culture. We report that PAVICs in model 1 express vimentin strongly and alpha-smooth-muscle actin (SMA) to a lesser extent, but SMA expression is increased by shear stress, particularly near the lumenal surface. Model 1 constructs increase in cell number, maintain protein levels, but lose glycosaminoglycans in response to shear. Co-culture with PAVECs (Model 2) modulates these responses in both static and flow environments, resulting in PAVIC phenotype that is more similar to the native condition. PAVECs stimulated a decrease in PAVIC proliferation, an increase in protein synthesis with shear stress, and reduced the loss of glycosaminoglycans with flow. Additionally, PAVECs stimulated PAVIC differentiation to a more quiescent phenotype, defined by reduced expression of SMA. These results suggest that valvular endothelial cells are necessary to properly regulate interstitial cell phenotype and matrix synthesis. Additionally, we show that tissue-engineered models can be used to discover and understand complex biomechanical relationships between cells that interact in vivo.     

流动切应力对培养的血管内皮细胞形态的影响

为探索培养的血客内皮细胞承受流体冲击的能力，以便在人工心脏瓣膜表面形成一个抵抗血流切应力作用强的血管内皮细胞层，在我们研制的内皮细胞切应力反应测试装置上，观察了培养在盖玻片表现的牛主动脉内皮细胞形成单层细胞后在切应力分别在１５ｄｆｙｍｅｓ／ｃｍ＾２２４小时和１１５ｄｙｍｓ／ｃｍ＾２８小时作用下细胞的形态张脱落情况。     

大鼠脑微血管内皮细胞应力敏感性钾通道的研究

为探讨脑微血管内皮细胞的电生理学特征,研究了脑微血管内皮细胞钾离子通道的应力反应性.1.建立了开放式切应力作用装置并进行了切应力值的计算; 2.培养大鼠脑微血管内皮细胞并种植到1 cm×1 cm玻片上,采用Axonpatch 200A型膜片钳放大器以及全细胞膜片钳技术记录脑微血管内皮细胞的应力敏感性钾通道.结果表明1.1 dynes/cm2剪切应力能激发脑微血管内皮细胞的应力敏感性钾通道,该通道电流与钳制电压有良好的相关性.脑血管内皮细胞膜上的各种应力反应与细胞膜上的应力敏感性钾通道相关.     

不同条件培养的内皮细胞耐受流体剪切力的比较研究

为提高内皮细胞（EC）与移植间的粘附,研究其可能的机制,我们采用改进的流室装置比较流动培养的EC与静态培养的EC对剪切力的耐受强度,预铺纤维粘连蛋白,层粘连蛋白对其对其粘附的影响,并研究2了EC骨架成分肌动蛋白丝的分布及在剪切力作用下的变化,结果显示流动培养组的细胞残留明显多于相应静态培养组,预辅纤维连接蛋白可显著提高静态培养EC的残留,加入纤维连接蛋白和层粘连蛋白效应更明显,而预铺的基质对流动培养组结果影响较小,剪切力作用下EC的肌动蛋白丝有序排列,并形成张力纤维,提示流动培养可显著提高EC对剪切力的耐受,其机制与剪切力诱导肌动蛋白丝重建,张力纤维形成,细胞形态的变化有关。     

Attachment, morphology and adherence of human endothelial cells to vascular prosthesis materials under the action of shear stress

In an effort to improve the long-term patency of vascular prostheses several groups now advocate seeding autologous endothelial cells (ECs) onto the lumen of the vessel prior to implantation, a procedure that involves pre-treating the prosthesis material with fibrin, collagen and/or other matrix molecules to promote cell attachment and retention. In this study, we examined the degree to which human umbilical venous endothelial cells (HUVECs) adhered to three materials commonly used polymeric vascular prosthesis that had been coated with the same commercial extra cellular matrix proteins, and after exposure to fluid shear stresses representative of femoro-distal bypass in a cone-and-plate shearing device. We quantified cell number, area of coverage and degree of cell spreading using image analysis techniques. The response of cells that adhered to the surface of each material, and following exposure to fluid shear stress, depended on surface treatment, topology and cell type. Whereas collagen coating improved primary cellular adhesion and coverage significantly, the degree of spreading depended on the underlying surface structure and on the application of the shear stress. In some cases, fewer than 30% of cells remained on the surface after only 1-h exposure to physiological levels of shear stress. The proportion of the surface that was covered by cells also decreased, despite an increase in the degree to which individual cells spread on exposure to shear stress. Moreover, the behaviour of HUVECs was distinct from that of fibroblasts, in that the human ECs were able to adapt to their environment by spreading to a much greater extent in response to shear. The quality of HUVEC attachment, as measured by extent of cell coverage and resistance to fluid shear stress, was greatest on expanded polytetrafluoroethylene samples that had been impregnated with Type I/III collagen.     

Biomechanopharmacology in Evaluation of Herbs of Activating Blood Circulation to Remove Blood Stasis

Herbs of activating blood circulation to remove blood stasis （ABCRBS） are a category of over 10% in the modern Chinese Pharmacopoeia. A new borderline discipline, biomechanopharmacology, is shaping by the efforts of applying biomechanics in pharmacological studies of ABCRBS herbs. Biomechanics is involved in modeling of blood stasis syndrome （BSS） with mechanical force induced injury and model evaluation by shear stress monitoring for blood coagulation. Investigations showed that tetramethylpyrazine （TMP） contained in Ligusticum chuanxiong Hort and diallyl trisulfide （DT） extracted from garlic demonstrated inhibiting characteristics on vWF mediated platelet activation and thrombus formation occurring under high shear rates. The effect of TMP on shear-induced platelet aggregation might be due to inhibition of calcium channel activity since it showed significant inhibition on intracellular level of calcium demonstrated by laser confocal microscope. The combined effects of TMP and shear stress on rat cerebral microvascular endothelial cell （rCMEC） were investigated by various doses of TMP incorporated with different levels of shear stress generated by a rotational coneplate rheometer. The results indicated that apoptosis of rCMECs could be restrained by a combination of medial level of shear stress with a suitable dose of TMP. To study the influences of shear stress, pressure and TMP on angiogenesis of vascular endothelial cell, cultured rCMEC was pretreated in a flow chamber with independent adjustment for levels of shear stress and pressure, and then 3D cultured on Matrigel. The results indicate that combined effects of shear stress, pressure and TMP may influence angiogenesis significantly. We believe that research on interactions among blood shear stress, secretion of endothelial cell, and pharmacodynamics will be an interesting area of biomechanopharmacology. Herbs of ABCRBS and their extracts for protecting endothelial cells to maintain their normal functions are expected.     

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.     

The effect of gradually graded shear stress on the morphological integrity of a huvec-seeded compliant small-diameter vascular graft

The premature endothelialization of tissue-engineered grafts had often induced cellular detachment at an early period of implantation in arterial circulation, resulting in occlusion at an early period of implantation. This study was aimed to determine whether gradually increased shear stress applied ex vivo improves cell retention and tissue morphological integrity including cell shape and alignment, actin fiber alignment and expression of vascular endothelial (VE) cadherin. Tissue-engineered grafts used for this study were human umbilical vein endothelial cell (HUVEC)-seeded compliant small-diameter grafts made of poly(L-lactide-co-epsilon-caprolactone) fiber meshes fabricated by electrospinning. The shear stresses applied to grafts, generated using a custom-designed mock circulatory apparatus, were 3.2, 8.7 and 19.6 dyn/cm(2). The grafts completely monolayered prior to shear stress exposure exhibited a polygonal cobblestone morphology with randomly distributed actin fibers and VE cadherin at the continuous peripheral region of adjacent cells. The 24-h-loading of high shear stresses (8.7 and 19.6 dyn/cm(2)) equivalent to those of the arterial circulatory system resulted in severe cellular damage resulting in the complete loss of cells. However, a gradually increased graded exposure from a low (3.2 dyn/cm(2)) to a high shear stress (19.6 dyn/cm(2)) resulted in a markedly reduced cell detachment, a highly elongated cell shape, and orientation or alignment of both cells and actin fibers, which were parallel to the direction of flow. Although VE-cadherin expression was not detected yet, a higher degree of tissue integrity was achieved, which may greatly improve the performance particularly at an early period of implantation.     

Physiological pulsatile flow culture conditions to generate functional endothelium on a sulfated silk fibroin nanofibrous scaffold

Many studies have demonstrated that in vitro shear stress conditioning of endothelial cell-seeded small-diameter vascular grafts can improve cell retention and function. However, the laminar flow and pulsatile flow conditions which are commonly used in vascular tissue engineering and hemodynamic studies are quite different from the actual physiological pulsatile flow which is pulsatile in nature with typical pressure and flow waveforms. The actual physiological pulsatile flow leading to temporal and spatial variations of the wall shear stress may result in different phenotypes and functions of ECs. Thus, the aim of this study is to find out the best in vitro dynamic culture conditions to generate functional endothelium on sulfated silk fibroin nanofibrous scaffolds for small-diameter vascular tissue engineering. Rat aortic endothelial cells (RAECs) were seeded on sulfated silk fibroin nanofibrous scaffolds and cultured under three different patterns of flow conditioning, e.g., steady laminar flow (SLF), sinusoidal flow (SF), or physiological pulsatile flow (PPF) representative of a typical femoral distal pulse wave in vivo for up to 24 h. Cell morphology, cytoskeleton alignment, fibronectin assembly, apoptosis, and retention on the scaffolds were investigated and were compared between three different patterns of flow conditioning. The results showed that ECs responded differentially to different exposure time and different flow patterns. The actual PPF conditioning demonstrated excellent EC retention on sulfated silk fibroin scaffolds in comparison with SLF and SF, in addition to the alignment of cells in the direction of fluid flow, the formation of denser and regular F-actin microfilament bundles in the same direction, the assembly of thicker and highly crosslinked fibronectin, and the significant inhibition of cell apoptosis. Therefore, the actual PPF conditioning might contribute importantly to the generation of functional endothelium on a sulfated silk fibroin nanofibrous scaffold and thereby yield a thromboresistant luminal surface.     

Primary cilia sensitize endothelial cells for fluid shear stress.

Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Non-ciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krüppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress.     

Shear stress and material surface effects on adherent human monocyte apoptosis

Monocytes play a critical role as both phagocytes and mediators of inflammatory responses in the prevention of cardiovascular device-related infections. However, persistent infection of these devices still occurs and may be attributed to deleterious cellular alterations resulting from monocyte interactions with a foreign material in an environment of dynamic flow. Thus, the effects of both shear stress and adhesion to material surfaces on human monocyte apoptosis were investigated. A rotating disk system generated physiologically relevant shear stress levels (0-14 dyn/cm(2)), and shear-related apoptosis occurring in adherent monocytes was characterized. Using annexin V analysis, apoptosis of polyurethane-adherent monocytes under shear for 4 h increased to levels >70% with increasing shear in a near-linear fashion (r2 = 0.713). It was qualitatively confirmed using confocal microscopy that filamentous (F)-actin distribution was altered, that DNA fragmentation occurred, and that activated caspases were involved in shear-induced apoptosis. Static studies determined that spontaneous apoptosis was material-dependent over 72 h by demonstrating marked differences between apoptosis of monocytes adherent to a polyurethane compared to an alkyl-modified glass. Treatment with TNF-alpha augmented this material dependency in a dose-dependent fashion over time. F-actin content of TNF-alpha-treated cells decreased to <62% of untreated cells. We conclude that concomitant effects from both material surfaces and dynamic flow mediate human monocyte apoptosis and may have serious implications in the context of implanted cardiovascular device infection.     

Responsiveness of vascular endothelium to shear stress: potential role of ion channels and cellular cytoskeleton (review).

The frictional forces associated with blood flow expose vascular endothelium in arteries to a complex and highly dynamic shear stress distribution. The ability of endothelial cells to respond to shear stress is essential for arterial vasoregulation in response to acute hemodynamic changes and for vascular wall remodeling following chronic changes in blood flow. Furthermore, endothelial responsiveness to shear stress may play a role in the localization of early atherosclerotic lesions. Shear stress elicits a wide range of humoral, metabolic, and structural responses in endothelial cells. These include activation of ion channels and of G proteins, induction of oscillations in intracellular calcium concentration, alterations in the expression of various important genes, and extensive cytoskeletal reorganization. Mechanisms of shear stress sensing and transmission in endothelium are discussed in light of the complex shear stress distribution to which endothelial cells are exposed in vivo and with particular emphasis on the potentially central role of flow-sensitive ion channels and the cellular cytoskeleton. Finally, the ability of endothelial cells to distinguish among and to respond differentially to different types of shear stress is highlighted.     

体外内皮细胞培养装置的研制与实验研究

目的研制一种创新性的体外内皮细胞培养装置,即基于血液动力学环境的血管内皮细胞体外培养装置,介绍内皮细胞体外培养装置的研制与实验研究。方法运用血液动力学理论与方法,在课题组现有研究基础上设计并研制内皮细胞体外动态培养系统,其流动环境中切应力、正应力和张应力同时存在。并从装置的研制背景、结构与组成、设计原理、理论基础以及实验研究5个方面详细描述该装置的研制和实验研究。结果装置能够准确模拟正常生理水平下内皮细胞所处的力学环境,实现切应力、正应力、张应力分别在0~12 Pa、0~15. 96 k Pa、0~0. 5 MPa范围内的精确调控。结论装置能提供一个更接近人体生理条件的血液动力学环境,为深入探索血管内膜损伤机制提供更理想的实验环境和手段。     

Activation of caspase 3 during shear stress-induced neutrophil apoptosis on biomaterials

Within the complex environment of an implanted cardiovascular device comprised of dynamic flow and foreign materials, phagocytic neutrophils may be ineffective in combating infection due to cellular responses to shear stress. This may be explained, in part, by our recent reports of apoptosis of biomaterial-adherent leukocytes induced through exposure to shear stress. Here we utilize a rotating disk system to generate physiologically relevant shear stress levels (0-18 dynes/cm(2)) at the surface of a polyetherurethane urea (PEUU) and investigate neutrophil intracellular pathways involved in shear-induced apoptosis. In situ detection of activated caspases, the enzymatic mediators of the apoptosis cascade, showed qualitatively that these proteases participate in shear-induced apoptosis and are activated in a shear-dependent manner. The involvement of caspase 3 was confirmed through immunoprecipitation and immunoblotting of extracted neutrophil proteins. Comparative studies with neutrophils adherent under static conditions demonstrated time-dependent activation of caspases in TNF-alpha/cycloheximide-induced apoptosis, for which caspase-3 also was implicated. These findings are the first steps toward elucidation of the mechanisms behind the inappropriate induction of apoptosis by adhesion to biomaterials, which may contribute to the development and persistence of device-related infections.     

血流剪切应力对内皮细胞表达人类凋亡蛋白抑制剂的作用

 目的：本研究目的是阐明血流剪切应力抑制血管内皮细胞凋亡的分子机制。方法: 人脐静脉内皮细胞（HUVECs）培养于DMEM, 当细胞融合时，转染Smac基因，将细胞暴露于20 dyne/cm2 的剪切应力，分别测定人类凋亡蛋白抑制剂(human inhibitor of apoptosis protein-2, HIAP-2） 蛋白表达及细胞凋亡蛋白酶（caspase-3）活性。 结果: 20 dyne/cm2 的剪切应力抑制caspase-3活性的增加，此作用与剪切应力能够明显诱导内皮细胞产生HIAP-2 蛋白表达明显增加有关。 结论：证实生理水平剪切应力能够明显诱导内皮细胞产生HIAP-2 蛋白的表达，这可能是剪切应力抑制内皮细胞凋亡发挥抗动脉粥样硬化作用的机制之一。     

Effect of LVAD outflow conduit insertion angle on flow through the native aorta

BACKGROUND AND AIM: The goal of this study was to evaluate the effect of surgical anastomosis configuration of the aortic outflow conduit (AOC) from a continuous flow left ventricular assist device (LVAD) on the flow fields in the aorta using CFD simulations. The geometry of the surgical integration of the LVAD is an important factor in the flow pattern that develops both in series (aortic valve closed, all flow through LVAD) and in parallel (heart pumping in addition to LVAD). METHODS: CFD models of the AOC junctions simulate geometry as cylindrical tubes that intersect at angles ranging from 30 degrees to 90 degrees. Velocity fields are computed over a range of cardiac output for both series and parallel flow. RESULTS: Our results demonstrate that the flow patterns are significantly affected by the angle of insertion of the AOC into the native aorta, both during series and parallel flow conditions. Zones of flow recirculation and high shear stress on the aortic wall can be observed at the highest angle, gradually decreasing in size until disappearing at the lowest angle of 30 degrees. The highest velocity and shear stress values were associated with series flow. CONCLUSIONS: The results suggest that connecting the LVAD outflow conduit to the proximal aorta at a shallower angle produces fewer secondary flow patterns in the native cardiovascular system.     

Interaction of human malignant melanoma (ST-ML-12) tumor spheroids with endothelial cell monolayers. Damage to endothelium by oxygen-derived free radicals.

Clinical and experimental observations suggest that tumor-induced endothelial cell injury may be one of several initial events in the establishment of tumor metastases. To test this hypothesis, the authors have analyzed the interaction of malignant melanoma (ST-ML-12) multicenter tumor spheroids with endothelial cell monolayers in a three-dimensional coculture system. After 1.5 hours of interaction, the authors observed a toxic effect on endothelial cells in the perispheroid region. The latter was demonstrated by testing membrane integrity with the fluorescent probes acridine orange/ethidium bromide and resulted in sensitivity to shear stress of the damaged cells. The endothelium then underwent a regenerative cycle to replace the denuded halo. Addition of the oxygen radical-scavenging enzyme superoxide dismutase to the culture medium prevented this endothelial cell damage in a dose-dependent manner for up to 12 hours. By contrast, catalase, deferoxamine mesylate, allopurinol, and the proteinase inhibitors soybean trypsin inhibitor and aprotinin were not protective under the same conditions. The endothelial damage was dependent on the attachment of the spheroids. Medium conditioned by ST-ML-12-spheroids proved to be ineffective. A similar, but less prominent, deleterious effect was seen when human peritoneal mesothelial cells were used in place of the human umbilical vein endothelial cells. Spheroids of the uroepithelial cell line HU-609 were used as control. No toxicity was observed in these cocultures. Melanin biosynthesis is associated with the production of oxygen-derived free radicals. The results suggest a possible implication of these free radicals in metastasis formation of malignant melanoma.