New stent surface materials: The impact of polymer-dependent interactions of human endothelial cells,smooth muscle cells,and platelets

Despite the development of new coronary stent technologies, in-stent restenosis and stent thrombosis are still clinically relevant. Interactions of blood and tissue cells with the implanted material may represent an important cause of these side effects. We hypothesize material-dependent interaction of blood and tissue cells. The aim of this study is accordingly to investigate the impact of vascular endothelial cells, smooth muscle cells and platelets with various biodegradable polymers to identify a stent coating or platform material that demonstrates excellent endothelial-cell-supportive and non-thrombogenic properties. Human umbilical venous endothelial cells, human coronary arterial endothelial cells and human coronary arterial smooth muscle cells were cultivated on the surfaces of two established biostable polymers used for drug-eluting stents, namely poly(ethylene-co-vinylacetate) (PEVA) and poly(butyl methacrylate) (PBMA). We compared these polymers to new biodegradable polyesters poly(l-lactide) (PLLA), poly(3-hydroxybutyrate) (P(3HB)), poly(4-hydroxybutyrate) (P(4HB)) and a polymeric blend of PLLA/P(4HB) in a ratio of 78/22% (w/w). Biocompatibility tests were performed under static and dynamic conditions. Measurement of cell proliferation, viability, glycocalix width, eNOS and PECAM-1 mRNA expression revealed strong material dependency among the six polymer samples investigated. Only the polymeric blend of PLLA/P(4HB) achieved excellent endothelial markers of biocompatibility. Data show that PLLA and P(4HB) tend to a more thrombotic response, whereas the polymer blend is characterized by a lower thrombotic potential. These data demonstrate material-dependent endothelialization, smooth muscle cell growth and thrombogenicity. Although polymers such as PEVA and PBMA are already commonly used for vascular implants, they did not sufficiently meet the criteria for biocompatibility. The investigated biodegradable polymeric blend PLLA/P(4HB) evidently represents a promising material for vascular stents and stent coatings.     

The response to percutaneous transluminal coronary angioplasty: An ultrastructural study of smooth muscle cells and endothelial cells

We investigated the ultrastructure of the repair tissue formed after percutaneous transluminal coronary angioplasty (PTCA). Four hearts (6 coronary arteries) were investigated; 5 arteries after single PTCA and 1 after repeated PTCA. In the earliest lesion (5 days after PTCA), all smooth muscle cells were of a synthetic phenotype, with abundant rough endoplasmic reticulum and few myofilaments, whereas the oldest lesion (259 days after PTCA) was composed of contractile smooth muscle cells, characterized by abundant myofilaments and small amounts of rough endoplasmic reticulum. In lesions taken at intermediate times (74 and 77 days), the smooth muscle cells were of an intermediate phenotype, with rough endoplasmic reticulum and myofilaments present in approximately equal portions. Regenerating endothelial cells observed at 74 days after PTCA were characterized by a rounded shape and a paucity of intercellular connections in the form of simple junctions, which suggested an immature state. At 259 days after PTCA, the endothelial cells were more mature, as indicated by a flat shape and the presence of many tight junctions and a complete basement membrane. The findings suggest a relation between the stage of endothelial cell regeneration and the phenotype of the smooth muscle cells.     

Interaction of foamy cells from experimental hyperlipemia rabbits with smooth muscle cells and the endothelial surface of intima preparation]

In the development of atherosclerosis (AS), circulating monocytes emigrate into and accumulate in the intima as foamy cells. Interaction of the lipid laden macrophage (M phi) with cells of the arterial wall may contribute to the formation of atheromatous plaque. Using subcutaneous and peritoneal foamy cells (FC) collected from diet-induced hyperlipemia rabbits, the authors observed the influence of lipid laden on macrophage's functions relevant to AS lesion formation. In comparison with normal M phi, peritoneal FC were 4.8 and 5.4 fold more adhesive to the endothelial surface of intima preparation and the smooth muscle cells (SMC), while the adhesion rate produced by subcutaneous FC were increased 0.79 and 0.16 fold. SMC migration stimulated by both FC-conditioned medium slowed 5.78 and 5.90 fold increased respectively as in comparing with the control's, whereas normal M phi-conditioned medium only gave a 2.3 fold increase of SMC migration stimulation. In addition, both FC-conditioned medium stimulated SMC growth making 1.96 and 2.59 fold increase, and normal M phi-conditioned medium produced a 1.3 fold increase as compared with the control's. The results suggest that lipid-laden macrophages, may accelerate AS development due to changes of their biological properties. Since more than 95% of peritoneal macrophages as well as macrophages in the pleura, pericardial and synovial cavities from experimental hyperlipidemia rabbits are fully filled with lipid assuming the morphologic characteristics of atheromatous intimal foamy cells, it is considered that these cells will be valuable as the model.     

Different sensitivity of human endothelial cells, smooth muscle cells and fibroblasts to topography in the nano–micro range

Cell adhesion, orientation and migration are influenced by surface topographies in the micrometer and nanometer range. In this work, we demonstrate the stimulation by topographical signals of human fibroblast cells (FCs), endothelial cells (ECs) and smooth muscle cells (SMCs). We systematically quantified the contact guidance alignment and directed migration of FCs, ECs and SMCs adhering to grooved substrates with lateral dimensions of 2–10 μm and depths of 50–200 nm. We found a common quantitative response characteristic of all three cell types: contact guidance significantly increased when the cells were cultured on substrates with smaller lateral dimensions or deeper grooves. Despite their general behavior, the three cell types exhibited a cell-type specific sensitivity to the groove patterns. The minimum groove depth to induce an orientation response and change cell shape was 50 nm for FCs and about two times deeper for ECs and SMCs. The degree of alignment and directed migration of the FCs along the grooves was significantly stronger than for the ECs and SMCs. We demonstrate that ECs and SMCs can be stimulated by topographical signals but are less sensitive than FCs.     

The properties of the inward rectifier potassium currents in rabbit coronary arterial smooth muscle cells

In freshly-isolated, single, smooth muscle cells of rabbit coronary arteries, an inward rectifier K⁺ current [I _K(IR)] was identified using the whole-cell voltage-clamp technique. The current/voltage (I/V) relationship of I _K(IR) showed strong inward rectification with a very small outward current when the smooth muscle cells were dialyzed with a pipette solution containing Mg²⁺. However, dialyzing the cells with a nominally Mg²⁺-free pipette solution revealed a significant outward current hump in the I/V relation of I _K(IR), suggesting that the strong inward rectification of I _K(IR) is partly due to the inhibitory effects of internal Mg²⁺. I _K(IR) was unaffected by tetraethylammonium (1 mM), 4-aminopyridine (1 mM), or glibenclamide (1 μM), but was inhibited by extracellular Ba²⁺ with a concentration of 0.87 μM eliciting half-maximal inhibition at –120 mV. I _K(IR) induced in rabbit coronary smooth muscle cells declined during very negative hyperpolarizing steps, due largely to a block by external Na⁺. I _K(IR) was inhibited by α₁-adrenergic stimuli. Methoxamine, an α₁-adrenergic agonist, concentration dependently inhibited I _K(IR) in the presence of the β-adrenergic antagonist propranolol. The methoxamine concentration required for half-maximal inhibition was 205 μM. We conclude that inward rectifier K⁺ current is present in rabbit coronary smooth muscle cells and that it shares many properties with the inward rectifier K⁺ current described for other cell types. Received: 2 February 1999 / Accepted: 24 March 1999     

Role of agr (RNAIII) in Staphylococcus aureus adherence to fibrinogen,fibronectin, platelets and endothelial cells under static and flow conditions

In the present study, the adherence of Staphylococcus aureus (strain 83254) and its RNAIII mutant (WA400) to immobilised fibrinogen and fibronectin, and to human endothelial cells (EC), was studied. [3H]Thymidine-labelled bacteria in stationary phase were incubated on the test surfaces for 20 min under static or flow (200/s) conditions. The results showed: (a) increased adherence of the RNAIII mutant to fibrinogen under static conditions, and decreased adherence of the mutant to fibronectin and EC under both static and flow conditions compared with the parental strain; (b) stronger ability of the mutant compared with the parental strain to induce platelet aggregation in suspension; (c) greater adherence of the parental strain and the mutant to EC pre-treated with platelet-rich plasma compared with platelet-poor plasma, and to EC pre-treated with platelet-poor plasma compared with control; (d) increased adherence of S. aureus to EC pre-treated with PMA-activated platelets and decreased adherence to EC pre-treated with tirofiban, a platelet glycoprotein IIb-IlIa inhibitor, which paralleled with increased adherence of PMA-activated platelets and decreased adherence of glycoprotein IIb-IIIa-blocked platelets to EC in the absence of bacteria; and (e) adherence of the mutant was more sensitive to pre-treatment of EC with plasma and PMA-activated platelets. In conclusion, RNAIII down-regulates S. aureus adherence to fibrinogen under static condition and up-regulates S. aureus adherence to fibronectin and EC under both static and flow conditions. The potentiating role of activated platelets in the presence of plasma in S. aureus adherence to EC is down-regulated by RNAIII, probably due to down-regulation of adherence to fibrinogen, the important plasma protein bridging S. aureus, platelets and EC.     

血管平滑肌细胞对联合培养内皮祖细胞黏附、增殖与形态的影响

目的：探讨血管平滑肌细胞对联合培养的内皮祖细胞黏附、增殖与分化的影响。方法：从人脐血中分离纯化内皮祖细胞并鉴定；建立内皮祖细胞和平滑肌细胞的联合培养模型；细胞粘附实验测定内皮祖细胞的粘附能力；形态学观察内皮祖细胞的分化与增殖。结果：与血管平滑肌细胞联合培养的内皮祖细胞的粘附能力比单独培养时提高了63％，长梭形细胞数量增加，同时克隆形成单位的数量下降了约60％。结论：血管平滑肌细胞促进了内皮祖细胞的粘附与分化，同时抑制了内皮祖细胞的增殖能力。     

丹参对血管平滑肌细胞抑制内皮细胞分泌的纤溶酶原激活物活性的影响

培养的猪内皮细胞(EC)产生和分泌纤溶酶原激活物(PA),血管平滑肌细胞(SMC)的无血清条件培养液(SMC-CM)与EC的无血清条件培养液(EC-CM)混合后,后者的PA活力明显减弱。EC与SMC复合培养时,其产生的PA活力亦降低(降低70.7%)。以上结果提示SMC产生纤溶酶原激活物抑制剂PAI抑制了EC分泌的PA活性。SMC-CM经SDS-PAGE后作反向纤维蛋白自显影显示PAI的分子量范围在49,000—62,000,与Laug(1985)报道的相似(55,000)。我们研究了中药丹参(Radix Salviae Miltiorrhizae,RSM)对SMC分泌PAI活性的影响。结果表明,培养的SMC经丹参作用后,其CM对尿激酶及内皮细胞产生的PA活性的中和作用明显减弱(P<0.05、P<0.01或P<0.001)。     

In atherogenesis, the apoptosis of endothelial cell itself could directly induce over-proliferation of smooth muscle cells

Over-proliferation of SMC (smooth muscle cell) is one characteristics of atherosclerosis. One well accepted mechanism is that the decrease of ECs (endothelial cells) induced by over apoptosis leads to endothelial dysfunction, which in turn results in over-proliferation of SMC. Obviously, the mechanism works after endothelial apoptosis. Compared with necrosis, apoptosis is time and energy consuming. The question is why the cell ends in the form of apoptosis instead of necrosis. From the evolutionary standpoint, apoptosis has some useful functions other than removing the damaged or unwanted cells. Recent studies showed that cells nearby the apoptotic ones began to proliferate and differentiate before apoptosis and the apoptotic signals could induce the near cells to proliferate without the death of cells. Apparently, some mechanism in apoptosis results in the proliferation of cells. So, we hypotheses that endothelial apoptosis can directly induce the over-proliferation of SMC.     

Studies of the cellular uptake of hydrogel nanospheres and microspheres by phagocytes, vascular endothelial cells, and smooth muscle cells

Intensive research efforts have been placed on the development of nanospheres for targeted drug delivery for treating a variety of diseases, including coronary restenosis, cancer, and inflammatory reactions. Although most of these drug-bearing spheres are delivered via intravenous administration, little is known about the effect of sphere physical characteristics on the responses of vascular and blood cells. To find the answer, this work was aimed to investigate the cellular uptake of nanosized (100 nm) and microsized hydrogel spheres (1 microm) made of poly(N-isopropylacrylamide) by vascular cells and phagocytes under various flow conditions in vitro. We found that the cellular uptake of nanospheres depended on incubation times and sphere concentrations as well as on the introduced shear stress levels of the medium. Measurements of the intracellular-released fluorescence and confocal fluorescence microscopy revealed that nanospheres were internalized by endothelial cells and smooth muscle cells more than microspheres, whereas microspheres were rapidly taken up by phagocytes, especially at high concentration. Our results strongly suggest that hydrogel nanospheres are more effective as an intravascular delivery system compared to microspheres in the terms of vascular cellular uptake and biocompatibility.     

纤维蛋白原对人血管平滑肌细胞株增殖和移行的影响

目的:探讨纤维蛋白原(Fg)、纤维蛋白(Fb)及其降解产物(FDPs)对体外人血管平滑肌细胞(VSMC)增殖与移行的影响。方法:采用细胞计数法和四唑盐(MTT)比色法测定VSMC增殖,通过刮伤实验(woundingmodel)和Transwell嵌套装置观察VSMC的移行。结果:Fg本身对VSMC增殖无促进作用,但可促进VSMC的移行,且呈浓度依赖关系;Fb及FDPs均可明显促进VSMC增殖和移行,Fb的作用还呈现浓度依赖关系,而FDPs作用呈现先增强后减弱的趋势,即在一定浓度范围内作用明显,高于或低于此范围作用较弱。结论:Fb及FDPs通过促VSMC的增殖和移行作用,可能在再狭窄和动脉粥样硬化中起重要作用。     

The role of the sarcoplasmic reticulum in neonatal uterine smooth muscle: enhanced role compared to adult rat

Little is known about contractile activity, response to agonists or excitation-contraction coupling in neonatal smooth muscle. We have therefore investigated 10-day rat uterus to better understand these processes, and compared it to adult uterus to elucidate how control of contractility develops. Spontaneous contractions are present in the 10-day neonatal uterus, although they are not as large or as regular as those present in adult tissues. External Ca²⁺ entry via L-type Ca²⁺ channels is the sole source of Ca²⁺ and is essential for the spontaneous activity. The neonatal uterus was responsive to carbachol or prostaglandin F_2α application; it showed a marked stimulation and a clear dissociation between the force and Ca²⁺ changes. Such sensitization was not apparent in adult rat myometrium. The sarcoplasmic reticulum (SR) had more releasable Ca²⁺ and contributed more to the response to agonists in neonatal compared to adult tissues. Thus, Ca²⁺ entry as opposed to SR Ca²⁺ release contributed much less to the uterine response to agonists in the neonatal, compared to adult tissues. Inhibition of the SR by cyclopiazonic acid also caused a more vigorous increase in Ca²⁺ and contractile activity, particularly frequency, in the neonatal compared to the adult uterus. Taken together these data suggest that: (1) spontaneous activity is already present by day 10, (2) receptor-coupling and excitation-contraction signalling pathways are functional, (3) the SR and Ca²⁺ sensitization mechanisms play a more prominent role in the neonate, and (4) there is a shift to a greater reliance on Ca²⁺ entry and excitability with development of the myometrium.     

A novel system for investigating the ability of smooth muscle cells and fibroblasts to regulate adhesion of flowing leukocytes to endothelial cells

Allergic contact dermatitis is a frequent and increasing health problem. For ethical reasons, the current animal tests used to screen for contact sensitizers should be replaced by in vitro alternatives. Contact sensitizers have been shown to accelerate Langerhans cell (LC) migration from human organotypic skin explant cultures (hOSECs) more rapidly than non-sensitizers and it has been proposed that the hOSEC model could be used to screen for sensitizers. However, chemically induced decreases in epidermal LC numbers need to be accurately quantified if the alterations in epidermal LC numbers are to form the basis of an alternative system for screening contact sensitizers in vitro. As manual counting of LCs is labour intensive and subject to intra- and inter-personal variation we developed an image analysis routine, using the Leica QWin image analysis software, to quantify LCs in situ using immunohistochemically stained skin sections. LCs can be identified using antibodies against the membrane molecule CD1a or the Lag antibody, which recognises cytoplasmic Birbeck granules. Quantification of epidermal LC number using the image analysis software had a much lower inter-person variation than when the same specimens were counted manually, using both the anti-Lag and CD1a antibodies. The software-aided quantification of epidermal LCs provides an accurate method for measuring chemically-induced changes in LC numbers.     

The capsule of the renal cell carcinoma (clear cell phenotype) contains modified smooth muscle cells

The capsule of five renal clear cell carcinoma ranging from 6 to 9 cm in diameter was investigated by light microscopy and thin section electron microscopy. The capsule consists essentially of superimposed layers of collagen fibers and modified smooth muscle cells. The possible origin and development of these cells are briefly discussed.     

The responses of osteoblasts, osteoclasts and endothelial cells to zirconium modified calcium-silicate-based ceramic

In this study we have developed Ca(3)ZrSi(2)O(9) (Baghdadite) ceramics by incorporating Zirconium in Ca-Si system and determined their biological properties. Ca(3)ZrSi(2)O(9) ceramics possess apatite-formation ability in simulated body fluid, indicating their potential bioactivity. The response of human osteoblast like cells (HOB), osteoclast and endothelial cells when cultured on Ca(3)ZrSi(2)O(9) ceramics was investigated. Scanning electron microscopy and immunofluorescence studies demonstrated that this material supports HOB cell attachment with organized cytoskeleton structure. Compared to CaSiO(3), Ca(3)ZrSi(2)O(9) ceramics induced increased HOB proliferation and differentiation as shown by increased methyltetrazidium salt (MTS), alkaline phosphatase activity, and mRNA expression levels of bone-related genes (Collagen type I, alkaline phosphatase, Bone Sialoprotein, receptor activator of NF-kappaB ligand and osteoprotegerin). Ca(3)ZrSi(2)O(9) ceramics supported the fusion of monocytes to form functional osteoclasts with their characteristic features of f-actin ring structures and the expression of alpha(v)beta(3) integrin consistent with functional activity. Osteoclasts cultured on Ca(3)ZrSi(2)O(9) expressed increased levels of osteoclast-related genes; Cathepsin K, Carbonic Anhydrase II, Matrix metalloproteinase-9, receptor activator of NF-kappaB and Calcitonin Receptor, consistent with the formation of functional osteoclasts. In addition to HOB and osteoclasts, Ca(3)ZrSi(2)O(9) supported the attachment of endothelial cells, which expressed the endothelial cell markers; ZO-1 and VE-Cadherin. Results presented here indicate that Ca(3)ZrSi(2)O(9) ceramics have the potential for applications in bone tissue regeneration.     

Hypoxia stimulates the release by bovine pulmonary artery endothelial cells of an inhibitor of pulmonary artery smooth muscle cell growth

The proliferation of smooth muscle cells (SMC) seen in hypoxic pulmonary hypertension is a poorly understood phenomenon but may involve endothelial cell (EC)-SMC interaction. Using bovine pulmonary artery cells, we examined the effect of O2 tension and the role of EC or media conditioned by EC (ECCM) on SMC proliferation. We found no difference in SMC proliferation under 3%, 10%, and 20% O2. EC, co-cultured with SMC in 3% O2, inhibited SMC proliferation consistently by about 40% (versus SMC exposed to hypoxia but not to EC). In normoxia, the degree of inhibition was dependent on EC:SMC ratio. In separate experiments, media from EC exposed to 3% or 20% O2 had a mitogenic activity of 24% and 42%, respectively (compared to 100% mitogenic activity with 5% FCS), on serum-deprived SMC. On the other hand, when SMC were stimulated to grow with FCS, an inhibitory activity (IA) from ECCM on SMC proliferation was observed and was significantly greater in hypoxic versus normoxic ECCM (40% versus 21%, respectively). Amicon concentration showed that the IA was contained in the less than 10 kD fraction of ECCM. Preliminary characterization of this IA indicates that it is unlike any of the known inhibitors of SMC growth, such as lactic acid, prostaglandin derivatives, or heparan sulfate. We conclude that hypoxia causes pulmonary artery EC to release a unique inhibitor of SMC growth.     

Localization of cysteine protease, cathepsin S, to the surface of vascular smooth muscle cells by association with integrin alphanubeta3

Smooth muscle cell (SMC) migration from the tunica media to the intima, a key event in neointimal formation, requires proteolytic degradation of elastin-rich extracellular matrix barriers. Although cathepsin S (Cat S) is overexpressed in atherosclerotic and neointimal lesions, its exact role in SMC behavior remains primarily unresolved. We examined the involvement of Cat S on SMC migration through an extracellular matrix barrier and its localization in SMCs. A selective Cat S inhibitor and the endogenous inhibitor cystatin C significantly attenuated SMC invasion across elastin gel. Western blotting and cell surface biotinylation analysis demonstrated localization of the 28-kd active form of Cat S on the SMC surface, consistent with its role in the proteolysis of subcellular matrices. Treatment with interferon-gamma or interleukin-beta1 significantly augmented the ability of SMC membranes to degrade elastin along with a significant increase in the level of active Cat S compared with controls. Immunofluorescence and confocal microscopy showed a punctuated pattern of Cat S clusters at the periphery of SMCs; further studies demonstrated partial co-localization of Cat S and integrin alphanubeta3 at the cell surfaces. These findings demonstrate that active Cat S co-localizes with integrin alphanubeta3 as a receptor on the SMC surface, playing an important role in the invasive behavior of SMCs.