Porcine aortic organ culture: A model to study the cellular response to vascular injury

Summary Organ cultures of porcine thoracic aorta were studied to define the characteristics of this system as a model to study the reaction of endothelial cells (ECs) and the underlying smooth muscle cells (SMCs) to injury. Both nonwounded and wounded cultures the latter having had part of the endothelial surface gently denuded with a scalpel blade, were studied over a 7 d period by scanning and transmission electron microscopy. The results showed that the nonwounded ECs underwent a shape change from elongated to polygonal within 24 h in culture. In both nonwounded and wounded explants there was cell proliferation beneath the nondenuded endothelium so that by 7 d several layers of cells were present showing features of the secretory type of SMCs. This proliferation, however, did not occur if the endothelium was totally removed from the aorta. There was also evidence of gaps between the surface ECs, and by 7 d lamellipodia of cells beneath the surface were present in these gaps. Occasionally, elongated cells were seen to be present on the surface of the endothelium. In the wounded organ culture, cell migration and proliferation occurred extending from the wound edge and producing a covering of cells on the denuded area. There were also multilayered cells beneath the surface similar to the nonwounded area. Occasional foam cells were seen in the depth of the multilayered proliferating cells. The results indicate that organ culture of porcine thoracic aorta is a good model to study the reaction of ECs and underlying SMCs to injury. This work was supported by a grant from the Ontario Heart Foundation.     

Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture

When a confluent monolayer of bovine aortic endothelial (BAE) cells is wounded with a razor blade, endothelial cells (ECs) spontaneously move into the denuded area. If bovine pericytes or smooth muscle cells (SMCs) are plated into the denuded area at low density, they block the movement of the ECs. This effect is dependent upon the number of cells plated into the wound area and contact between ECs and the plated cells. Antibodies to transforming growth factor-beta 1 (TGF-beta 1) abrogate the inhibition of BAE cell movement by pericytes or SMCs. TGF-beta 1, if added to wounded BAE cell monolayers, also inhibits cell movement. When cultured separately, BAE cells, pericytes, and SMCs each produce an inactive TGF-beta 1-like molecule which is activated in BAE cell-pericyte or BAE cell-SMC co-cultures. The activation appears to be mediated by plasmin as the inhibitory effect on cell movement in co-cultures of BAE cells and pericytes is blocked by the inclusion of inhibitors of plasmin in the culture medium.     

Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta

The relaxation effect of cilostazol, a phosphodiesterase III inhibitor, on the thoracic aorta was investigated. Cilostazol induced the relaxation of the thoracic aorta precontracted by phenylephrine in a concentration-dependent manner. The concentration-dependent relaxation was shifted to the right in the endothelium denuded aorta compared with that of intact endothelium, suggesting that this relaxation was partly dependent on endothelium. Cilostazol-induced relaxation of thoracic aorta tone was reversed by treatment with N(G)-nitro L-arginine (L-NNA), a competitive inhibitor of nitric oxide (NO) synthase. Cilostazol also significantly increased the NO level in the porcine thoracic aorta. In rats treated with cilostazol, the urinary excretion of nitrites, a stable metabolite of NO, and basal production of NO of the aortic ring were significantly greater than in those without treatment. These findings indicate that cilostazol-induced vasodilation of the rat thoracic aorta was dependent on the endothelium, which released NO from aortic endothelial cells.     

The distribution of centrosomes in endothelial cells of non-wounded and wounded aortic organ cultures

Summary The distribution of centrosomes in porcine vascular endothelial cells of the thoracic aorta maintained in organ culture was determined in en face preparations using immunofluorescence. Rectangular pieces of aorta that had the distal half (with respect to the heart) of their endothelial surface gently denuded with a scalpel blade and pieces with intact endothelium were cultured for up to 96 h. At time 0, centrosomes were found to be preferentially oriented toward the heart, both in the cells of intact monolayers and in cells at the wound edge. This distribution was maintained in the intact monolayers for at least 24 h, but by 72 h the number of centrosomes in the center of the cells exceeded the number oriented toward the heart as the cells changed from a fusiform to a polygonal shape. The centrosomes of most endothelial cells at the wound edge began to redistribute themselves within the first 24 h in culture, moving from a position toward the heart to a position either in the center of the cell or away from the heart. By 72 h, the majority of centrosomes in endothelial cells at the wound edge were oriented away from the heart toward the denuded region. It is concluded that the centrosomes in the endothelial cells maintained in organ culture respond to injury in a manner similar to those grown in monolayer cell culture except that the reorientation of centrosomes occurs more slowly.     

Improved arterial wall model by coculturing vascular endothelial and smooth muscle cells

We have constructed an in vitro arterial wall model by coculturing bovine arterial endothelial cells (ECs) and smooth muscle cells (SMCs). When ECs were seeded directly over SMCs and cocultured in an ordinary culture medium, ECs grew sparsely and did not form a confluent monolayer. Addition of ascorbic acid to the culture medium at concentrations greater than 50 μg/ml increased the production of type IV collagen by the SMCs, and ECs formed a confluent monolayer covering the entire surface of SMCs. Histological studies showed that the thickness of the cell layer composed of ECs and SMCs increased with increasing duration of coculture. This arterial wall model, prepared by our method, may serve as a simple and good in vitro model to study the effects of factors such as biological chemicals and shear stress on cell proliferation and other physiological functions of arterial walls.     

旋转加压种植法在新型生物人工复合血管体外内皮化过程中的实验研究

目的：探讨在新型生物人工复合血管内腔面联合种植平滑肌细胞和内皮细胞的方法,比较研究旋转加压种植与普通灌注种植两种方法的内皮化效果,方法：先制备新型生物人工复合血管及获取培养鉴定平滑肌细胞和内皮细胞,再和旋转加压种植与普通灌注种植两种方法将平滑肌细胞和内皮细胞培养种植于新型复合血管内腔面,以光镜及扫描电镜等观察评价内皮化的效果。结果：旋转加压种植2小时末的复合血管腔内有大量内皮细胞,旋转加压种植9天后已形成完整的内皮细胞单层;普通灌注种植的复合血管内腔有内皮细胞附着,分布不均匀,未形成完整的内皮细胞层,结论：以旋转加压种植法在新型复合血管内腔面联合种植平滑肌细胞和内皮细胞效果满意,基本实现内皮化,可以满足复合血管内皮化的要求。     

Simultaneous isolation of endothelial and smooth muscle cells from human umbilical artery or vein and their growth response to low-density lipoproteins

In the pathogenesis of atherosclerosis the interplay of endothelial cells (ECs) and smooth muscle cells (SMCs) is disturbed. Oxidatively modified low-density lipoproteins (oxLDLs), important stimulators of atherosclerotic plaque formation in vessels, modify the growth response of both cell types. To compare growth responses of ECs and SMCs of the same vessel with oxLDLs, we developed a method to isolate both cell types from the vessel walls of umbilical cords by enzymatic digestion. The method further allowed the simultaneous isolation of venous and arterial cells from a single umbilical cord. In culture, venous ECs showed an elongated appearance compared with arterial ECs, whereas SMCs of artery and vein did not look different. Smooth muscle cells of both vessel types responded to oxLDLs (60 microg/ml) with an increase in their [(3)H]-thymidine incorporation into DNA. On the contrary, ECs of artery or vein decreased [(3)H]-thymidine incorporation and cell number in the presence of oxLDLs (60 microg/ml) of increasing oxidation grade. Thus, human umbilical SMCs and ECs of the same vessel show a disparate growth response toward oxLDLs. But the physiologically more relevant minimal oxLDLs did not decrease proliferation in venous ECs but only in arterial ECs. This difference in tolerance toward minimal oxLDLs should be taken into account while using venous or arterial ECs of umbilical cord for research in atherosclerosis. Further differences of venous and arterial ECs in tolerance toward minimal oxLDLs could be of clinical relevance for coronary artery bypass grafts.     

Protein expression profiles of Bos taurus blood and lymphatic vessel endothelial cells

The endothelium is a metabolically active organ that regulates the interaction between blood or lymph and the vessel or the surrounding tissue. Blood endothelium has been the object of many investigations whereas lymphatic endothelium biology is yet poorly understood. This report deals with a proteomic approach to the characterization and comparative analysis of lymphatic and blood vessel endothelial cells (ECs). By 2-DE we visualized the protein profiles of EC extracts from the thoracic aorta, inferior vena cava, and thoracic duct of Bos taurus. The three obtained electropherograms were then analyzed by specific software, and 113 quantitative and 25 qualitative differences were detected between the three endothelial gels. The cluster analysis of qualitative and quantitative differences evidenced the protein pattern of lymphatic ECs to be more similar to the venous than to the arterial one. Moreover, venous ECs were interestingly found showing a protein expression profile more similar to the lymphatic ECs than to the arterial ones. We also identified 64 protein spots by MALDI-TOF MS and ESI-IT MS/MS and three reference maps of bovine endothelium were obtained. The functional implications of the identified proteins in vascular endothelial biology are discussed.     

Cell movements in a living mammalian tissue: Long-term observation of individual cells in wounded corneal endothelia of cats

Although the cells in tissues are known to be motile under special conditions (e.g., during tissue turnover or wound healing), there are not many reports that polygonal cells covering an area without leaving any gaps are also capable of movement. In the present study, cell movements (cell shifting and rearrangement) in a living mammalian eye tissue were documented by identifying and locating individual cells over intervals as long as 100 days. Cat corneal endothelium, a monolayered cell sheet, was wounded by removing a small number (about 180) of endothelial cells from the internal lining of the cornea. Healing of the wounded tissue was observed with a wide-view specular microscope applied to the outer surface of the cornea, enabling us to identify individual cells for as long as two to three months. Cells surrounding the wound underwent areal enlargement, elongated toward the wound, and shifted to cover the wound surface. During days 4–7, cells became rearranged by changing neighbors in such a way that they retained their enlarged size but recovered their non-elongated, original shape. This pattern of cell rearrangement was interpreted by a computer simulation which assumed that cells shorten their boundary length while maintaining contacts with contiguous cells. After day 7, the enlarged cells adjacent to the wounded area gradually contracted and pulled surrounding cells toward the wounded area. These movements were followed by a temporary halt in cell shifting, then by a recovery of shifting and cell elongation. These movements are interpreted as a result of the contractility of endothelial cell microfilaments.     

The effect of coculture with human smooth muscle cells on the proliferation,the IL‐1 β secretion,the PDGF production and tube formation of human aortic endothelial cells

Endothelial cells (ECs) and smooth muscle cells (SMCs), which are the major component cells of blood vessels, produce various bioactive substances and communicate with each other through them. Although several studies of the interaction between ECs and SMCs have been reported, the effect of coculture with SMCs on ECs is still obscure. To clarify the interaction of ECs and SMCs, we examined the effect of coculture with SMCs on the proliferation, the IL‐1β secretion, the PDGF production and tube formation of ECs, using the coculture model: transferable wells and collagen gel. IL‐1 and PDGF are considered to be related to progression of atherosclerosis. Proliferation and tube formation of ECs are associated with repair of vessels. In the transferable well system coculture with SMCs stimulated the proliferation of ECs, and enhanced the IL‐1β secretion of ECs and in the collagen gel system coculture with SMCs induced the tube formation of ECs, and appeared to enhance the PDGF production of ECs. In conclusion, the effect of coculture with SMCs on ECs has two conflicting aspects: progression of atherosclerosis and angiogenesis. These results suggest that an imbalance of their effects may lead to pathological events. Copyright © 1999 John Wiley & Sons, Ltd.     

内皮细胞生长状态对血管平滑肌细胞增生迁移的影响

实验通过建立细胞共培养体系,探讨内皮细胞生长状态对血管平滑肌细胞增生迁移的影响及机制。检测指标包括~3H-TdR掺入、细胞周期、细胞迁移计数和α-SM-actin mRNA表达。结果显示,融合生长内皮使平滑肌细胞~3H-TdR掺入量明显降低,增加平滑肌细胞停留在G_0/G_1期的比例,上调平滑肌细胞α-SM-actin mRNA表达;而对数生长内皮细胞使平滑肌细胞~3H-TdR掺入量明显升高,促进平滑肌细胞由 G_0/G_1期进入G_2/M和S期,下调平滑肌细胞α-SM-actin mRNA表达。对照组平滑肌细胞在基础状态下存在少量迁移,对数增殖内皮细胞组平滑肌迁移数比对照组增高约4倍(P<0.01),而融合生长内皮细胞组平滑肌迁移数仅为对照组的0.5倍(P<0.05)。结果提示内皮细胞生长状态不同,对平滑肌细胞生物学特性的影响也不同,增殖期内皮明显促进平滑肌细胞增生迁移、下调平滑肌细胞α-SM-actin mRNA表达。     

Morphologic adaptation of arterial endothelial cells to longitudinal stretch in organ culture

Arteries in vivo are subjected to large longitudinal stretch, which changes significantly due to vascular disease and surgery. However, little is known about the effect of longitudinal stretch on arterial endothelium. The aim of this study was to determine the morphologic adaptation of arterial endothelial cells (ECs) to elevated axial stretch. Porcine carotid arteries were stretched 20% more than their in vivo length while being maintained at physiological pressure and flow rate in an organ culture system. The ECs were elongated with the application of the axial stretch (aspect ratio 2.81+/-0.25 versus 3.65+/-0.38, n=8, p<0.001). The elongation was slightly decreased after three days and the ECs recovered their normal shape after seven days, as measured by the shape index and aspect ratio (0.55+/-0.03 versus 0.56+/-0.04, and 2.93+/-0.28 versus 2.88+/-0.20, respectively, n=5). Cell proliferation was increased in the intima of stretched arteries in three days as compared to control arteries but showed no difference after seven days in organ culture. These results demonstrate that the ECs adapt to axial stretch and maintain their normal shape.     

Establishment and characterization of porcine aortic endothelial cell cultures with prolonged replicative lifespan by a non-enzymatic method

The aim of this work was the establishment and characterization of porcine aortic endothelial cell cultures with prolonged lifespan. Endothelial cells where isolated from porcine thoracic aorta and established in primary culture; after sub cultivation, the cells showed typical morphology of endothelial phenotype with cobblestone appearance and growth in monolayer; they were positive against anti-CD31 and anti-CD54 immunostaining and Ac-LDL-Dil uptake. The cells were able to migrate in culture and showed a normal growth curve. The phenotype of our in vitro model of endothelial cells was stable through subcultivation; so, it should be a valuable tool for diverse studies of the endothelial response against physiological and pathological stimulus, particularly it could be useful to study interactions of endothelium with pathogenic bacteria causing diseases in pigs, as they are difficult to study in vivo.     

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 X 106 smooth muscle cells (SMCs) ob-tained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biode-gradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6-8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.     

Differential sensitivity of endothelial cells of various species to apoptosis induced by gene transfer of Fas ligand: role of FLIP levels

BACKGROUND: Fas ligand expression by cells of the vessel wall has been proposed to play a role in normal and pathologic conditions. Genetic engineering of vascularized organs for endothelial cell (EC) expression of FasL could protect the endothelium and underlying tissues from infiltrating Fas+ leukocytes. Nevertheless, the endogenous expression of FasL by ECs of different species and the potential deleterious effects of enforced FasL expression by ECs are largely unknown. In human ECs, levels of FLICE/caspase 8-inhibitory protein (FLIP) have been shown to control apoptosis mediated by Fas. MATERIALS AND METHODS: Cell surface expression of FasL in rat, mouse, human, and pig ECs was obtained using recombinant adenoviruses or transient plasmid transfection assays. FasL expression was evaluated by FACS analysis and cytotoxicity assays. Apoptosis was evaluated using annexin V, TUNEL, and cytotoxicity assays. FLIP levels were evaluated by Western blot analysis and overexpression was obtained by transient transfection. RESULTS: Analysis of ECs from different species showed that FasL was predominantly present in the cytoplasm, and depending on the species, little or no cell surface expression was detected. Enforced cell surface expression of FasL on rat or mouse ECs, either in culture or within the vessel wall resulted in massive apoptosis. In contrast, porcine or human ECs were completely resistant to apoptosis mediated by Fas-FasL interaction. Markedly reduced FLIP levels were observed in rat and mouse ECs compared to human and porcine ECs. Overexpression of FLIP in rat ECs conferred protection against cell surface expression of FasL. CONCLUSIONS: The consequences of FasL overexpression depend on the subcellular compartment and species in which FasL enforced expression is targeted and this is at least partially related to FLIP levels.     

红细胞生成素3‘端增强子在肺动脉平滑肌细胞代氧性增殖调控中的作用

用噻唑蓝比色法（ＭＴＴ法）、Ｈ＾３－胸腺嘧啶核苷（Ｈ＾３－ＴｄＲ）掺入法和流式细胞术,观察红细胞生成素（ＥＰＯ）３’端增强子片段对培养的猪肺动脉平滑肌细胞（ＰＡＳＭＣｓ）的内皮依赖性和非内皮依赖性低氧性增殖的影响。结果为：（１）低氧２４ｈ后ＰＡＳＭＣｓ明显增殖,转入野生型ＥＰＯ３’端增强子片段可被抑制,而转入突变型片段无此作用;（２）肺动脉内皮细胞（ＰＡＥＣｓ）低氧２４ｈ,其条件培养液有明显的促Ｐ     

Macrovascular and microvascular endothelium during long-term hypoxia: alterations in cell growth, monolayer permeability, and cell surface coagulant properties

In bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose-dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a approximately 3-fold increase in the total number of high-affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia-mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin-based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro- and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions.     

Changes in the endothelium of the aorta of the rat during regeneration (according to the results of scanning electron microscopy)

In the experiments performed on 53 Kioto-Wistar normotensive rats, the reaction of endothelial cells of the abdominal aorta has been analysed in response to the lesion by freezing or by hypoosmotic effect. At first the deendothelized surface is covered with flatten thrombocytes, and then the continuity of the endothelium is restorted as a result of migration and proliferation of endotheliocytes. This is accompanied with a reconstruction of their cytoskeleton (structurization and redistribution). The immature endothelium is characterized by an increased numerical density of strongly elongated endothelial cells, decreased resistency to collagenase effect and a great adhesiveness to blood leucocytes. No essential differences in the reaction of endotheliocytes are revealed in dependence of deendothelization means.     

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the alpha(5)beta(1)-integrin complex, whereas ECs used either alpha(5)beta(1)- or alpha(v)beta(3)-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC alpha(5)beta(1)-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.