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.     

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

The prothrombotic mediator thromboxane A₂ is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.     

Tissue inhibitor of metalloproteinases-4 suppresses vascular smooth muscle cell migration and induces cell apoptosis

In a previous study, we have demonstrated that overexpression of the tissue inhibitors of metalloproteinases-4 (TIMP-4) can inhibit the neointima formation in the rat carotid model. To define the functions of tissue inhibitor of metalloproteinases-4 (TIMP-4) in SMCs, we transduced human TIMP-4 cDNA into rat aortic SMCs by using adenoviral vector. Overexpression of TIMP-4 blocked the conversion of pro-MMP-2 to the active form and inhibited basic fibroblast growth factor-induced migration by 56.7% (p < 0.01). Overexpression of TIMP-4 markedly increased apoptotic cell death without changing their proliferation. Importantly, overexpression of human TIMP-4 in the wall of balloon-injured rat carotid artery also increased SMC apoptosis. The percentages of TUNEL-positive cells of total cells increased significantly in AdTIMP-4 infected group compared with AdGFP infected group. These findings demonstrate that TIMP-4 can inhibit SMCs migration and induce apoptosis in vitro and in vivo, which may generate new targets for prevention and treatment of vascular diseases.     

Goniothalamin induces apoptosis in vascular smooth muscle cells

Restenosis represents a major impediment to the success of coronary angioplasty. Abnormal proliferation of vascular smooth muscle cells (VSMCs) has been shown to be an important process in the pathogenesis of restenosis. A number of agents, particularly rapamycin and paclitaxel, have been shown to impact on this process. This study was carried out to determine the mechanisms of cytotoxicity of goniothalamin (GN) on VSMCs. Results from MTT cytotoxicity assay showed that the IC(50) for GN was 4.4 microg/ml (22 microM), which was lower compared to the clinically used rapamycin (IC(50) of 25 microg/ml [27.346 microM]). This was achieved primarily via apoptosis where up to 25.83 +/- 0.44% of apoptotic cells were detected after 72 h treatment with GN. In addition, GN demonstrated similar effects as rapamycin in inhibiting VSMCs proliferation using bromodeoxyuridine (BrdU) cell proliferation assay after 72 h treatment at IC(50) concentration (p > 0.05). In order to understand the mechanisms of GN, DNA damage detection using comet assay was determined at 2h post-treatment with GN. Our results showed that there was a concentration-dependent increase in DNA damage in VSMCs prior to cytotoxicity. Moreover, GN effects were comparable to rapamycin. In conclusion, our data show that GN initially induces DNA damage which subsequently leads to cytotoxicity primarily via apoptosis in VSMCs.     

Shear stress induces apoptosis in vascular smooth muscle cells via an autocrine Fas/FasL pathway

Differentiated melanocytic cells produce melanin, through several redox reactions including tyrosinase-catalyzed DOPA oxidation to DOPA quinone. We now developed a method based on DOPA oxidase in-gel detection and Sypro Ruby fluorometric normalization to investigate induction of specific DOPA oxidase isoforms in response to hydrogen peroxide-mediated stress, and to ask whether this is associated with p53-dependent adaptive responses. This report shows that hydrogen peroxide leads to comparable induction of 60 and 55 kDa DOPA oxidases in poorly pigmented B16 melanoma, in contrast to sole induction of a major 55 kDa DOPA oxidase in their highly pigmented counterparts. In the latter cells, this response also increases p53 concomitant with joint induction of p53-activated proteins like the cell-cycle inhibitor p21WAF1 and pro-apoptotic bax, with no comparable effect on expression of anti-apoptotic bcl-2. Together, these data suggest that response to hydrogen peroxide involves p53-mediated growth-restrictive signaling and unequal induction of specific DOPA oxidases in melanocytic cells with unequal basal pigmentation.     

Shear stress induces endothelial transdifferentiation from mouse smooth muscle cells

Smooth muscle cells (SMCs) under shear stress may alter their gene expression patterns to adapt to a new hemodynamic environment. Their plasticity may play an important role in vascular development, healing, and remodeling as well as vascular lesion formation under abnormal environmental conditions. A mouse vascular SMC line (P53LMACO1) cultured under shear stress significantly increased the mRNA levels of endothelial cell markers including Platelet-endothelial cell adhesion molecule-1 (PECAM-1), von Willebrand factor (vWF), and VE-cadherin, while significantly decreasing the mRNA levels of SMC markers including alpha-smooth muscle actin (alpha-SMA), calponin-1, smooth muscle myosin heavy chain (SMMHC), and transgelin as compared to static control cells. Protein levels of PECAM-1 and vWF were significantly increased, while protein levels of alpha-SMA were substantially decreased in the shear stress-cultured cells. In addition, shear stress-cultured cells showed an enhanced capability to form capillary-like structures on Matrigel. Thus, shear stress may promote endothelial cell transdifferentiation from SMCs.     

AlphaB-crystallin inhibits glucose-induced apoptosis in vascular endothelial cells

Recent studies implicate hyperglycemia as a cause of vascular complications in diabetes. Our study confirmed that high concentration of glucose (30 mM) induces apoptosis in cultures of human umbilical vein endothelial cells. After 5 days of culture TUNEL positive cells in high concentration of glucose were nearly 63% higher when compared to normal concentration of glucose (5 mM). Transfection of pcDNA3-rat alphaB-crystallin into these cells inhibited high glucose-induced apoptosis by approximately 36%, such an effect was not observed when cells were transfected with an empty vector. AlphaB-crystallin transfection inhibited by about 35% of high glucose induced activation of caspase-3. High concentration of glucose enhanced formation of reactive oxygen species (ROS) in these cells but this was significantly (p < 0.001) curtailed by transfection of alphaB-crystallin. Results of our study indicate that alphaB-crystallin effectively inhibits both ROS formation and apoptosis in cultured vascular endothelial cells and provide a basis for future therapeutic interventions in diabetic vascular complications.     

Regulation of vascular smooth muscle cells by micropatterning

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.     

Ultrafast Ca wave in cultured vascular smooth muscle cells aligned on a micropatterned surface

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which propagates as Ca²⁺ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca²⁺ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca²⁺ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca²⁺]_i dynamics and membrane potential events along the network of cells. The rate of [Ca²⁺]_i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca²⁺ channels (VOCCs) inhibition suppressed the ultrafast Ca²⁺ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca²⁺ entry from the external medium through VOCCs, modeled as an ultrafast Ca²⁺ wave. This wave may trigger and drive slower Ca²⁺ waves observed ex vivo and in vivo.     

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.     

Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells

Objective To investigate the pro-apoptotic effect of tissue factor pathway inhibitor (TFPI) gene transfer mediated by adenovirus on vascular smooth muscle cells (VSMCs). Methods Rat VSMCs were infected with recombinant adenovirus containing either the TFPI (Ad-TFPI) or LacZ (Ad-LacZ) gene or DMEM in vitro. TFPI expression was detected by ELISA. Apoptosis of VSMCs was determined by electron microscopy and flow cytometry. The expression of cytochrome c, procaspase-3, cleaved caspase-3, cleaved caspase-9 and inhibitor of apoptosis protein-1(IAP-1) were examined by western blot and RT-PCR. Results TFPI protein was detected in the TFPI group after gene transfer and the peak expression was at the 3rd day. At the 3rd, 5th and 7th day after gene transfer, the apoptotic rates in the TFPI group were 11.95%, 71.96% and 37.83%, respectively, whereas those in the LacZ group were 1.34%, 1.83% and 6.37%, respectively. We observed cell contraction, slight mitochondrial swelling, nuclear pyknosis and apoptotic body formation in TFPI-treated VSMCs using electron microscopy. Cytochrome c, cleaved caspase-3 and cleaved caspase-9, which are all involved in mitochondrial pathway, were detected in the cytoplasm on the 3rd, 5th and 7th day after TFPI gene transfer. Procaspase-3 expression was significantly decreased over time in the TFPI group (each P < 0.05), which were not seen in the Ad-LacZ and DMEM groups. The expression of IAP-1 mRNA in the TFPI group was also decreased compared with the Ad-LacZ and DMEM groups (each P < 0.05) at the 3rd and 7th day after gene transfer. Conclusion The results demonstrated that overexpression of TFPI gene might induce VSMC apoptosis in vitro through the mitochondrial pathway; meanwhile, IAP-1 expression is decreased. These findings indicated that TFPI might inhibit restenosis by inducing apoptosis in VSMCs.     

Succinobucol induces apoptosis in vascular smooth muscle cells

Probucol inhibits the proliferation of vascular smooth muscle cells in vitro and in vivo, and the drug reduces intimal hyperplasia and atherosclerosis in animals via induction of heme oxygenase-1 (HO-1). Because the succinyl ester of probucol, succinobucol, recently failed as an antiatherogenic drug in humans, we investigated its effects on smooth muscle cell proliferation. Succinobucol and probucol induced HO-1 and decreased cell proliferation in rat aortic smooth muscle cells. However, whereas inhibition of HO-1 reversed the antiproliferative effects of probucol, this was not observed with succinobucol. Instead, succinobucol but not probucol induced caspase activity and apoptosis, and it increased mitochondrial oxidation of hydroethidine to ethidium, suggestive of the participation of H(2)O(2) and cytochrome c. Also, succinobucol but not probucol converted cytochrome c into a peroxidase in the presence of H(2)O(2), and succinobucol-induced apoptosis was decreased in cells that lacked cytochrome c or a functional mitochondrial complex II. In addition, succinobucol increased apoptosis of vascular smooth muscle cells in vivo after balloon angioplasty-mediated vascular injury. Our results suggest that succinobucol induces apoptosis via a pathway involving mitochondrial complex II, H(2)O(2), and cytochrome c. These unexpected results are discussed in light of the failure of succinobucol as an antiatherogenic drug in humans.     

Reactive oxygen species and vascular cell apoptosis in response to angiotensin II and pro-atherosclerotic factors

Reactive oxygen species (ROS) are known to induce apoptotic cell death in various cell types. In the vessel wall, ROS can be formed by macrophages within the atherosclerotic plaque or can act on the endothelium after adhesion of monocytes or leucocytes. Moreover, ROS are endogenously synthesized by endothelial and vascular smooth muscle cells by NAD(P)H oxidase. Enhanced ROS production is a very early hallmark in the atherogenic process, suggesting a link between ROS and apoptosis. In endothelial cells, the endogenous generation of ROS is induced by different pro-inflammatory and pro-atherosclerotic factors such as Ang II, oxLDL or TNFalpha, which all promote the execution of programmed cell death. ROS synthesis is thereby causally involved in apoptosis induction, because antioxidants prevent endothelial cell death. The pro-apoptotic effects of endogenous ROS in endothelial cells mechanistically seems to involve the disturbance of mitochondrial membrane permeability followed by cytochrome c release, which finally activates the executioner caspases. In contrast to the pro-apoptotic capacity of ROS in endothelial cells, in vascular smooth muscle cells emerging evidence suggests that endogenous ROS synthesis promotes cell proliferation and hypertrophy and does not affect cell survival. However, high concentrations of exogenous ROS can also stimulate smooth muscle cell apoptosis as shown for other cell types probably via activation of p53. Taken together, the double-edged effects of endogenously derived ROS in endothelial cells versus VSMC may provide a mechanistic clue to the anti-atherosclerotic effects of antioxidants shown in experimental studies, given that the promotion of endothelial survival in combination with inhibition of VSMC proliferation blocks two very important steps in the pathogenesis of atherosclerosis.     

Distinction between smooth muscle,fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

Summary FITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological dedifferentiation to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.This work was supported by the Life Insurance Medical Research Fund of Australia and New Zealand, the National Heart Foundation of Australia, the Deutsche Forschungsgemeinschaft and the Wellcome Trust (London). We thank Janet D. McConnell for excellent technical assistance     

Caveolin-1 sensitizes vascular smooth muscle cells to mildly oxidized LDL-induced apoptosis

Oxidized low-density lipoprotein (oxLDL)-induced apoptosis of vascular cells may participate to plaque instability and rupture. Caveolin-1 has emerged as an important regulator of several signal transduction pathways and processes that play a role in atherosclerosis. In this study we examined the potential role of caveolin-1 in the regulation of oxLDL-induced Ca²⁺ signaling and apoptosis in vascular smooth muscle cells (VSMC). Cells expressing caveolin-1 were more susceptible to oxLDL-induced apoptosis, and this was correlated with enhanced Ca²⁺ entry and pro-apoptotic events. Moreover, caveolin-1 silencing by small interfering RNA decreased the level of apoptotic cells after oxLDL treatment. These findings provide new insights about the potential role of caveolin-1 in the regulation of oxLDL-induced apoptosis in vascular cells and its contribution to the instability of the plaque.     

Physiological effects of androgens on human vascular endothelial and smooth muscle cells in culture

Androgenic hormones are associated with atherosclerotic cardiovascular disease, although the underlying cellular and molecular mechanisms remain unclear. This study examines the impact of androgens on the physiology of human vascular endothelial cells (EC) and smooth muscle cells (SMC) in culture. Cells were incubated with testosterone, dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) at various physiological concentrations (5-50 nM) in the present or absence of an androgen receptor (AR) blocker flutamide (100 nM). Cell growth and death, DNA and collagen synthesis, and gene protein expression were assessed. It was shown that: (1) DHEA protected EC from superoxide injury via AR-independent mechanisms; (2) testosterone induced DNA synthesis and growth in EC via an AR-independent manner with activation of ERK1/2 activity; (3) DHT inhibited DNA synthesis and growth in EC in an AR-dependent manner; (4) testosterone and DHT enhanced ERK1/2 activation and proliferation in SMC via AR-independent and -dependent pathways, respectively; and (5) these androgens did not significantly affect collagen synthesis in SMC. We conclude that androgens possess multiple effects on vascular cells via either AR-dependent or -independent mechanisms. Testosterone and DHEA may be “beneficial” in preventing atherosclerosis by improving EC growth and survival; in contrast, stimulation of VSMC proliferation by testosterone and DHT is potentially “harmful”. The relationship of these in vitro effects by androgens to in vivo vascular function and atherogenesis needs to be further clarified.     

The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells

In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac-cellular matrix upon which cells rest.     

Bortezomib induces autophagic death in proliferating human endothelial cells

The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractory multiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomib has antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomib undergo death to an extent that depends strictly on their activation state. Indeed, while quiescent EC are resistant to Bortezomib, the drug results maximally toxic in EC switched toward angiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, EC activation state deeply influences the death pathway elicited by Bortezomib: after treatment, angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluent EC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmic vacuolization with evidence of autophagosomes at electron microscopy, and conversion of the cytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death. Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability. These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC; interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.