Smooth Muscle Cell Death Research Articles

Labedipinedilol-A prevents lysophosphatidylcholine-induced vascular smooth muscle cell death through reducing reactive oxygen species production and anti-apoptosis

Objective : Labedipinedilol-A, a novel calcium antagonist, has been previously demonstrated to have pleiotropic protective effects in the cardiovascular system. This study aimed to investigate its cytoprotective effects in rat vascular smooth muscle cells (VSMCs) treated with lysophosphatidylcholine (lysoPC), a key lipid component mediating atherogenesis. Methods and results : VSMCs were incubated with lysoPC with or without labedipinedilol-A pretreatment to determine its effects on lysoPC-induced cell death, Ca 2+ influx, oxidative stress, MAPK signaling and apoptosis. Labedipinedilol-A attenuated lysoPC-induced cell death and Ca 2+ influx. It also reduced reactive oxygen species (ROS) production evoked by lysoPC and down-regulated expressions of NAD(P)H oxidase subunits, Nox1 and Rac1. Moreover, it inhibited lysoPC-induced phosphorylation of MAPK including ERK1/2, JNK, and p38. It mitigated the dissipation of mitochondrial transmembrane potential induced by lysoPC. Lastly, labedipinedilol-A inhibited lysoPC-induced apoptosis with attenuation of caspase-3/-9 activations and modulation of Bax/Bcl-2 protein expressions. Conclusion : Labedipinedilol-A can suppress lysoPC-induced VSMCs death via reducing ROS production and anti-apoptosis. These protective effects are potentially mediated through the inhibition of Ca 2+ influx, down-regulation of the NAD(P)H oxidase subunits (Nox1/Rac1) and MAPK signaling, and attenuation of mitochondrial depolarization. Thus, labedipinedilol-A may have a valuable role in the preventing atherosclerosis associated with hyperlipidemia.

A link between smooth muscle cell death and extracellular matrix degradation during vascular atrophy

High blood flow induces neointimal atrophy in polytetrafluoroethylene (PTFE) aortoiliac grafts and a tight external PTFE wrap of the iliac artery induces medial atrophy. In both nonhuman primate models, atrophy with loss of smooth muscle cells and extracellular matrix (ECM) begins at ≤4 days. We hypothesized that matrix loss would be linked to cell death, but the factors and mechanisms involved are not known. The purpose of this study was to determine commonly regulated genes in these two models, which we hypothesized would be a small set of genes that might be key regulators of vascular atrophy. DNA microarray analysis (Sentrix Human Ref 8; Illumina, San Diego, Calif; ∼23,000 genes) was performed on arterial tissue from the wrap model (n = 9) and graft neointima from the graft model (n = 5) 1 day after wrapping or the switch to high flow, respectively. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was also performed. Expression of this vascular atrophy gene set was also studied after Fas ligand-induced cell death in cultured smooth muscle cells and organ cultured arteries. Microarray analysis showed 15 genes were regulated in the same direction in both atrophy models: 9 upregulated and 6 downregulated. Seven of nine upregulated genes were confirmed by qRT-PCR in both models. Upregulated genes included the ECM-degrading enzymes ADAMTS4, tissue plasminogen activator (PLAT), and hyaluronidase 2; possible growth regulatory factors, including chromosome 8 open reading frame 4 and leucine-rich repeat family containing 8; a differentiation regulatory factor (musculoskeletal embryonic nuclear protein 1); a dead cell removal factor (ficolin 3); and a prostaglandin transporter (solute carrier organic anion transporter family member 2A1). Five downregulated genes were confirmed but only in one or the other model. Of the seven upregulated genes, ADAMTS4, PLAT, hyaluronidase 2, solute carrier organic anion transporter family member 2A1, leucine-rich repeat family containing 8, and chromosome 8 open reading frame 4 were also upregulated in vitro in cultured smooth muscle cells or cultured iliac artery by treatment with FasL, which causes cell death. However, blockade of caspase activity with Z-VAD inhibited FasL-mediated cell death, but not gene induction. Seven gene products were upregulated in two distinctly different in vivo nonhuman primate vascular atrophy models. Induction of cell death by FasL in vitro induced six of these genes, including the ECM-degrading factors ADAMTS4, hyaluronidase 2, and PLAT, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die. These genes may be key regulators of vascular atrophy.

Targeting Inflammation in Cardiovascular Diseases. Still a Neglected field?

Prevention and treatment of atherosclerosis is still a clinical challenge in the cardiovascular medicine. The classical belief that atherosclerotic lesion development solely depends on lipid deposition has been replaced by the current concept that activation of immune and inflammatory responses plays a central role in plaque initiation and progression. In this review we summarize studies on human and genetically modified animals describing a finite number of cellular and molecular mechanisms that underlie immunoinflammation in atherosclerotic plaques. We focus on the pro- and antiinflammatory mediators activated during atherogenesis and the intracellular signaling pathways regulating these events. Besides the advances on established pharmacological agents, we propose potential strategies for reduction/stabilization of atherosclerotic plaques based on the clinical data in inflammatory-associated pathologies and on the encouraging studies in experimental models of atherosclerosis. We emphasize the potential of such novel inhibitors comprising receptor antagonists, neutralizing antibodies, kinase inhibitors, peptide-based technologies, and chemicals as emerging antiinflammatory strategies for the treatment of atherosclerotic disease complications.

Autophagy plays a protective role in free cholesterol overload-induced death of smooth muscle cells

Smooth muscle cells (SMC) make up most of the vascular system. In advanced atherosclerotic plaques, dying SMCs undergo a complex death mode. In the present study, we examined the activation of autophagy in SMCs overloaded with excess free cholesterol (FC) and investigated the possible role which autophagy plays during the FC-induced cell death. After incubation with excess FC, a robust expression of autophagic vacuoles (AV) was detected using both fluorescence microscopy and transmission electron microscopy (TEM). The results revealed that FC induced a time-dependent upregulation of microtubule-associated protein-1 light chain 3-II (LC3-II). Inhibition of autophagy by 3-methyladenine (3-MA) enhanced both cell apoptosis and necrosis, while on the contrary, rapamycin inhibited cell death following cholesterol application. Furthermore, the impact of the colocalization of fragmented mitochondria with AVs was observed after cholesterol treatment. Our results also revealed that the modulation of autophagy directly influenced the cellular organellar stress. In conclusion, our findings demonstrated that excess FC induced the activation of autophagy in SMCs as a cellular defense mechanism, possibly through the degradation of dysfunctional organelles such as mitochondria and endoplasmic reticulum.

Osteo-Progenitors in Vascular Calcification

Vascular calcification has been associated with the incidence of cardiovascular events and thus there has been interest in better understanding its pathogenesis. Early theories considered vascular calcification to be a passive process which occurred as a non-specific response to tissue injury or necrosis. More recent theories propose vascular calcification results from loss of molecular inhibitors or via an active cell mediated process. The origin of the cells responsible for vascular calcification is controversial and may vary in different sites and patients. Calcification has been reported as result of apoptosis or death of vascular smooth muscle cells for example. One novel source of cells controlling vascular calcification is from the bone marrow. A circulating immature bone marrow derived population has been identified and a small subset of this bone marrow population has been reported to possess bone forming properties in vitro and hence termed osteo-progenitors. This article reviews evidence supporting the contribution of these naive bone marrow derived circulating osteo-progenitor cells in vascular calcification.

Naringenin Inhibits Neointimal Hyperplasia Following Arterial Reconstruction With Interpositional Vein Graft

Vessels respond to injury by a healing process that includes the development of neointima. Stenosis secondary to neointima formation is the main cause of failure following arterial reconstructions. Vessel wall homeostasis is regulated by proinflammatory cytokines that affect smooth muscle cell proliferation, growth, migration, and death. We assessed the hypothesis that naringenin, a flavinoid possessing anti-inflammatory, antioxidant, and antiproliferative activities, reduces neointimal hyperplasia (NIH) following vascular injury.Arterial injury was created by interposition grafting of autologous right superficial epigastric vein graft into the right femoral artery (FA) in 48 male Sprague-Dawley rats. Following injury, the rats were divided into 4 groups (n = 12). Two groups were treated with naringenin (100 mg/kg intraperitoneal q daily) for 2 and 4 weeks each while 2 control groups received normal saline for the same durations. For Sham group (n = 10), the FA and vein were isolated without any additional procedure. Rats were killed at the end of treatment regimen in all groups, and FAs were harvested. Thickness of intima was measured in histologic sections, and levels of platelet derived growth factor (PDGF)-BB, TNFalpha, and Ki67 labeling index (Ki67 LI) were quantified in immunohistochemical analyses to assess the amount of NIH and mechanisms underlying its formation.Although there was no significant difference between the groups at 2 weeks, neointima thickness was lower in the naringenin treated group at 4 weeks (23.7 +/- 2.3 vs. 35.6 +/- 2.6 microm in control group; P < 0.001). The levels of PDGF-BB, and TNFalpha were lower in naringenin treated groups at both 2 weeks (PDGF-BB [0.21% +/- 0.03% versus 0.39% +/- 0.05% in control group, P < 0.001), TNFalpha (21.2% +/- 0.8% vs. 36.1% +/- 1.9% in control group, P < 0.001]) and 4 weeks (PDGF-BB [0.25% +/- 0.03% vs. 0.57% +/- 0.09% in control group, P < 0.001], TNFalpha [25.5% +/- 1.8% vs. 45.0% +/- 2.9% in control group, P < 0.001]). Ki67 LI was lower in naringenin treated groups at 2 weeks (13.9% +/- 2.8% vs. 18.7% +/- 3.7% in control group, P < 0.05), and at 4 weeks (17.5% +/- 2.6% vs. 31.1% +/- 4.7% in control group, P < 0.001), indicating a lower level of cellular proliferation.Naringenin reduces NIH following arterial reconstruction. This may be mediated by a decrease in PDGF-BB and TNFalpha levels and the resulting down-regulation of smooth muscle cells' migration and proliferation.

Apoptotic cell death and rupture of abdominal aortic aneurysm

The abdominal aortic aneurysm (AAA) development and expansion is characterized by an extracellular matrix degradation and widespread inflammation. In contrast, the processes that characterize the AAA rupture are not completely understood. The results obtained in animal and clinical studies have shown the importance of inflammation, proteolysis, and antioxidant mechanisms in the aortic degeneration and formation of AAA. We hypothesize that the rupture of the AAA could have a similar pathway like an atherosclerotic plaque rupture, and in both the cases the apoptotic cell death of smooth muscle cells could play a significant role. If the apoptotic cell death significantly contributes to the expansion and rupture of aneurysm, the hypothesis is that aggressive medical antiapoptotic treatment with high doses of appropriate drugs could decrease the apoptotic index of smooth muscle cells, reduce the aneurysm expansion and prevent rupture.

Caspase-mediated protein kinase C-δ cleavage is necessary for apoptosis of vascular smooth muscle cells

Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling and various vascular diseases. We have previously shown that protein kinase C-delta (PKC-delta) plays a critical role in SMC apoptosis. In this study, we tested the importance of PKC-delta proteolytic cleavage and tyrosine phosphorylation within the apoptosis pathway. Using hydrogen peroxide as a paradigm for oxidative stress, we showed that proteolytic cleavage of PKC-delta occurred in SMCs that underwent apoptosis, while tyrosine phosphorylation was detected only in necrotic cells. Furthermore, using a peptide (z-DIPD-fmk) that mimics the caspase-3 binding motif within the linker region of PKC-delta, we were able to prevent the cleavage of PKC-delta, as well as apoptosis. Inhibition of PKC-delta with rottlerin or small-interfering RNA diminished caspase-3 cleavage, caspase-3 activity, cleavage of poly (ADP-ribose) polymerase, cleavage of PKC-delta, and DNA fragmentation, confirming the previously reported role of PKC-delta in initiation of apoptosis. In contrast, z-DIPD-fmk markedly diminished caspase-3 activity, cleavage of PKC-delta, and DNA fragmentation without affecting cleavage of caspase-3 and poly (ADP-ribose) polymerase. Taken together, our data suggest that caspase-3-mediated PKC-delta cleavage underlies SMC apoptosis induced by oxidative stress, and that PKC-delta acts both upstream and downstream of caspase-3.

Optimal proportions of gelatin–resorcin–formalin components in aortic surgery☆☆☆

Gelatin-resorcin-formalin (GRF) glue, a haemostatic agent often employed for aortic surgery, has beneficial effects on early results in surgery for acute aortic dissection but may have late adverse effects, probably due to excess use of the activators such as formaldehyde and glutaraldehyde. The purpose of this study was to determine the optimal proportions of GRF components that minimise toxicity to human aortic smooth muscle cells and elastin with acceptable adhesive strength. (1) The degree of polymerisation was examined at various proportions (activator/gelatin+resorcinol=0%, 2%, 4%, 6%, 8% and 10%) to estimate adhesive strength. (2) (i) The toxicity of the activator was confirmed 24h after its supplementation to human aortic smooth muscle cells in various proportions (activator/human aortic smooth muscle cell=0%, 0.5%, 1%, 1.5%, 2% and 2.5%). (ii) The toxicity of GRF glue to human aortic smooth muscle cells was evaluated 1h and 60 h after its supplementation (activator/gelatin+resorcinol=0%, 2%, 4%, 6%, 8% and 10%). Another set of experiments in the same study was also performed. The only difference was that GRF glues were washed after polymerisation to exclude non-polymerised constituents. (3) Effects of 8%-GRF glue on toxicity to elastin derived from human aortic wall cells were investigated using an immunoblotting method. (1) The polymerisation area increased dose dependently and that of the 10% activator/gelatin+resorcinol mixture was significantly wider than those of 6%, 4%, 2% and 0%, but had no significant difference from that of 8%. (2) (i) Human aortic smooth muscle cell death occurred in all dishes except activator-free dishes. (ii) Sixty hours after exposure to GRF glue, human aortic smooth muscle cell death occurred only in the 10% dish. In a washed GRF glue study, no human aortic smooth muscle cell death occurred in any dishes. (3) Toxicity to elastin was not significantly different between 8%-GRF glue and the control, whereas toxicity of elastase to elastin was significantly higher than for both the glue and the control. An 8%-GRF glue provides lower toxicity to human aortic smooth muscle cells and elastin with an acceptable degree of polymerisation, and thus seems to be an optimal proportion for GRF glue.

Comparison of Inflammatory Response After Implantation of Sirolimus- and Paclitaxel-Eluting Stents in Porcine Coronary Arteries

Although both sirolimus (CYPHER) and paclitaxel (TAXUS) drug-eluting stents have demonstrated efficacy and safety in clinical trials, human autopsy data have raised concerns about long-term healing and the potential for local inflammatory reactions. Overlapping stents (CYPHER drug-eluting stents, Bx SONIC bare metal stents, TAXUS drug-eluting stents, and Liberté bare metal stents) were implanted in noninjured coronary arteries of 58 domestic swine. Histopathological evaluation of proximal, overlapped, and distal stented segments was determined with emphasis on inflammation at 30, 90, and 180 days. Circumferential granulomatous inflammation in all stented segments was defined as inflammation consisting of macrophages, multinucleated giant cells, lymphocytes, and granulocytes, including many eosinophils, adjacent to almost all struts. Circumferential granulomatous inflammation was more prevalent in CYPHER (9 of 23, 39%) compared with TAXUS (1 of 21, 5%; P=0.01) and control bare metal stents (0 of 44) in the combined 90- and 180-day cohorts. Only CYPHER specimens showed marked adventitial inflammation (P=0.0025) and fibrosis (P=0.0055) accompanied by extensive remodeling. Fibrin deposition within neointima and medial smooth muscle cell death were greater (both P<0.001) in TAXUS than CYPHER at 30 days, with more fibrin in TAXUS than CYPHER through 90 days (P<0.05). Although these data cannot be directly extrapolated to humans, the high prevalence in this porcine model of diffuse granulomatous inflammation seen with CYPHER stents, persisting at 180 days and associated with extensive remodeling of the artery, and persistent para-strut fibrin deposition with TAXUS stents emphasize the need for further investigation of biocompatibility with these and other novel combination drug/polymer drug-eluting stents.

Kawasaki Disease

Kawasaki disease (KD) is an acute systemic inflammatory illness that occurs predominately in children <5 years of age. The reported incidence varies widely depending on the ethnicity of the population and the method of case ascertainment. Recent reports would suggest the annual incidence is ≈20 to 25 per 100 000 children <5 years of age in North America, with the highest reported incidence of 188 being in Japan, where the disease was first described in 1967.1 The illness is self-limited and of unknown cause, but is complicated by a systemic vasculitis with a predilection for small- to medium-sized arteries, particularly the coronary arteries. The majority of patients will have either transient coronary artery dilation or no coronary artery luminal changes as noted on echocardiography. Long-term prognosis for these patients is considered to be excellent. Coronary artery aneurysms occur in 25% of patients, but the prevalence is reduced to ≈4% for patients treated with intravenous immunoglobulin infusion within 10 days of illness onset. Aneurysms are associated with an intense inflammatory cell infiltrate, destruction of the internal elastic lamina, and smooth muscle cell death. Coronary artery involvement is usually maximal within 6 to 8 weeks after the acute episode. Regression of aneurysms can occur primarily through myointimal proliferation, although the arterial structure and function remains abnormal,2 and there is an important ongoing risk of stenoses and occlusions.3 Long-term cardiology assessment and management is required, and some of these patients may require revascularization procedures or, rarely, cardiac transplantation. KD has become the most prevalent acquired cardiac disease in children in developed countries. Article see p 60 Although coronary artery complications are the predominant cause of morbidity and mortality, other cardiovascular abnormalities can occur. Valvulitis is a less prevalent complication, and there have been case reports of important long-term aortic and …

Cell Death–associated ADAMTS4 and Versican Degradation in Vascular Tissue

High blood flow through baboon polytetrafluorethylene aorto-iliac grafts increases neointimal vascular smooth muscle cell (SMC) death, neointimal atrophy, and cleavage of versican to generate the DPEAAE neoepitope, a marker of ADAMTS-mediated proteolysis. In this study, we have determined the effect of high blood flow on transcript abundance in the neointima for ADAMTS1, -4, -5, -8, -9, -15, and -20. We found that after 24 hr of flow, the mRNA for ADAMTS4 was significantly increased, whereas that for the other family members was unchanged. Because vascular SMC death is markedly increased in the graft after 24 hr of high flow, we next examined the possibility that the ADAMTS4 induction and the cell death are causally related. The addition of Fas ligand to SMC cultures increased both ADAMTS4 mRNA and cell death approximately 5-fold, consistent with the idea that ADAMTS4-dependent cleavage of versican may be partly responsible for cell death and tissue atrophy under these conditions.

Multifunctional angiogenic factors: add GnRH to the list. Focus on “Gonadotropin-releasing hormone-regulated chemokine expression in human placentation”

the historical view that angiogenesis is governed by the action of a few dominant trophic factors, such as VEGF, FGF, and transforming growth factor-β (TGF-β), has steadily eroded over the past two decades to give rise to the vision of a much more dynamic regulation in which angiogenesis is

T cells and eosinophils in bronchial smooth muscle cell death in asthma

Bronchial smooth muscle cells (SMC) proliferate, express adhesion molecules, secrete cytokines and thus efficiently contribute to the pathogenesis of asthma. The aim of the study was to investigate whether, and by which mechanism, T cells and eosinophils can cause death of airway SMC. The T cell- and eosinophil-induced cell death was analysed in primary human bronchial SMC cultures as well as in bronchial biopsy specimens from non-asthmatic and asthmatic individuals. Bronchial SMC death showed characteristic morphological features of apoptosis in 3-6 days cultures with inflammatory cytokines (IFN-gamma, TNF-alpha), soluble death ligands [sFasL, TNF-related apoptosis-inducing ligand (TRAIL)] and activated T-helper type 1 (Th1) and Th2 cell supernatants. The recombinant eosinophil cationic protein induced SMC necrosis within 1 h. Resting SMC expressed the death receptors TNFR1, TNFR2, Fas, TRAILR1, TRAILR2 and membrane FasL as a death-inducing ligand. IFN-gamma and TNF-alpha up-regulated TNFR1, TNFR2, Fas and membrane FasL on SMC. TNF-alpha up-regulated TRAILR1 and TRAILR2; sFasL up-regulated TNFR2. The intracellular caspase-3 activation in SMC was significantly increased by IFN-gamma, sFasL, TRAIL, Th1 and Th2 cell supernatants. Increased expression of TRAIL in asthmatics, but not in non-asthmatic individuals was demonstrated in situ. The apoptosis receptors TRAILR1 and TRAILR2 were expressed in SMC and epithelial cells both in healthy and asthmatic biopsies. Prominent apoptosis of SMC was observed in fatal asthma, but not intermittent asthma biopsies. The demonstration of bronchial SMC death both by apoptosis and necrosis indicates the essential role of T cells and eosinophils in the bronchial tissue injury particularly in the severe asthma.

Free cholesterol overloading induced smooth muscle cells death and activated both ER- and mitochondrial-dependent death pathway

Objective Smooth muscle cells (SMCs) death promotes atherosclerotic lesion necrosis and plaque destabilization. We investigated the potential mechanisms of rat SMCs death in response to excess free cholesterol (FC). Methods and results Rat aortic SMCs were incubated with “water soluble cholesterol” and acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor Sandoz58035 to establish FC-overloading cell model. Disruption of mitochondrial network and endoplasmic reticulum (ER) was observed after 12 h incubation by transient transfection. After treated for 24 h, enhanced cell death was noted as detected by propidium iodide (PI) staining/flow cytometry ( P < 0.001 vs. control). SMCs death was associated with markedly decreased mitochondrial transmembrane potential (Δ φm), as well as upregulation of cellular reactive oxygen species (ROS) and ER stress. We also investigated possible signaling pathways involved in excess FC-initiated cell death and found that unfolded protein response (UPR) was activated, with increased cellular Bax expression and release of mitochondrial cytochrome c. Conclusion Our findings suggested that FC-overloading might trigger SMCs death. Both ER- and mitochondria-based signals might be implicated in these lethal events.

The Protein Synthesis Inhibitor Anisomycin Induces Macrophage Apoptosis in Rabbit Atherosclerotic Plaques through p38 Mitogen-Activated Protein Kinase

Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH(2)-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.

Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.

Effect of 27-hydroxycholesterol on survival and death of human macrophages and vascular smooth muscle cells

The objective was to compare the effect of a LXR synthetic ligand (T0901317) on cell viability and lysosomal membrane destabilization in human U937 macrophage and aortic smooth muscle cell (HASMC) incubated in the presence of cholesterol or 27-OH and to verify whether the Akt signalling pathway is involved. In U937 macrophages, cholesterol triggered cell survival while 27-OH triggered either survival (low concentration) or a lysosomal independent apoptosis (high concentration). Despite a strong effect of T0901317 on macrophage survival, its effect on cell viability is hampered in cells incubated in the presence of cholesterol or 27-OH. In these cells, cholesterol triggers the phosphorylation of Akt on the Thr308 residue. In HASMC, cholesterol induced apoptosis but no additionnal effect of T0901317 prevented apoptosis. All together, cell survival triggered by LXRs is impaired in the presence of cholesterol or high concentrations of 27-OH in human U937 macrophages and is not effective in HASMC.

Calcium Phosphate Crystals Induce Cell Death in Human Vascular Smooth Muscle Cells

Vascular calcification is associated with an increased risk of myocardial infarction; however, the mechanisms linking these 2 processes are unknown. Studies in macrophages have suggested that calcium phosphate crystals induce the release of proinflammatory cytokines; however, no studies have been performed on the effects of calcium phosphate crystals on vascular smooth muscle cell function. In the present study, we found that calcium phosphate crystals induced cell death in human aortic vascular smooth muscle cells with their potency depending on their size and composition. Calcium phosphate crystals of approximately 1 microm or less in diameter caused rapid rises in intracellular calcium concentration, an effect that was inhibited by the lysosomal proton pump inhibitor, bafilomycin A1. Bafilomycin A1 also blocked vascular smooth muscle cell death suggesting that crystal dissolution in lysosomes leads to an increase in intracellular calcium levels and subsequent cell death. These studies give novel insights into the bioactivity of calcified deposits and suggest that small calcium phosphate crystals could destabilize atherosclerotic plaques by initiating inflammation and by causing vascular smooth muscle cell death.

Macrophage Function and Its Impact on Atherosclerotic Lesion Composition, Progression, and Stability

The role of the macrophage is of fundamental interest in understanding atherosclerotic lesion development and thrombogenicity. After differentiation from blood peripheral monocytes, intimal macrophages incorporate modified lipoproteins through the scavenger receptor pathway.1 This transforms the macrophage into a lipid-rich foam cell which is a hallmark feature of atherosclerosis and leads to lesion expansion. Macrophage activation results in the excretion of proinflammatory and cytotoxic substances,2 including peroxynitrite, an early inducer of atherosclerosis through the endoplasmic reticulum (ER) stress pathway.3 Further, the accumulation of free cholesterol or uptake of oxidized LDL induces macrophage apoptosis.4 Cytokine release from macrophages augments the inflammatory response and increases lesion size. Cytotoxic substances, including peroxynitrite and tumor necrosis factor (TNF)-α, released by the macrophage results in cell death of lesion-resident endothelial and smooth muscle cells, thereby disrupting vessel structure. Macrophages can also alter the extracellular matrix of the vessel by releasing matrix metalloproteinases (MMPs), thereby leading to lesion breakdown and predisposing the lesion to fissure or rupture.5 These aspects of the macrophage in atherosclerotic lesion biology drive the progression of the disease and lead to the decomposition of the arterial wall into atheroma, the ugly gruel remaining after foam cell decomposition. See accompanying articles on pages 1421 and 1429 The macrophage may also have good effects within the blood vessel wall. Macrophages remove debris through the process of phagocytosis.6 One aspect of macrophage phagocytosis, namely efferocytosis, is a process that prevents secondary necrosis and inflammation by removing apoptotic bodies before their decomposition.7 Efferocytosis has been shown to be mediated by 3 tyrosine kinase receptors, Axl, Tyro3, and Mer receptor tyrosine kinase (Mertk) in cells of hematopoietic lineage.8 Mertk is the primary mediator of efferocytosis in macrophages.9 Despite the presence of all 3 receptors on macrophages, Mertk has a …