Mast cell chymase induces smooth muscle cell apoptosis by disrupting NF-kappaB-mediated survival signaling

Chymase released from activated mast cells induces apoptosis of vascular smooth muscle cells (SMCs) in vitro by degrading the pericellular matrix component fibronectin, so causing disruption of focal adhesion complexes and Akt dephosphorylation, which are necessary for cell adhesion and survival. However, the molecular mechanisms of chymase-mediated apoptosis downstream of Akt have remained elusive. Here, we show by means of RT-PCR, Western blotting, EMSA, immunocytochemistry and confocal microscopy, that chymase induces SMC apoptosis by disrupting NF-kappaB-mediated survival signaling. Following chymase treatment, the translocation of active NF-kappaB/p65 to the nucleus was partly abolished and the amount of nuclear p65 was reduced. Pretreatment of SMCs with chymase also inhibited LPS- and IL-1beta-induced nuclear translocation of p65. The chymase-induced degradation of p65 was mediated by active caspases. Loss of NF-kappaB-mediated transactivation resulted in downregulation of bcl-2 mRNA and protein expression, leading to mitochondrial swelling and release of cytochrome c. The apoptotic process involved activation of both caspase 9 and caspase 8. The results reveal that, by disrupting the NF-kappaB-mediated survival-signaling pathway, activated chymase-secreting mast cells can mediate apoptosis of cultured arterial SMCs. Since activated mast cells colocalize with apoptotic SMCs in vulnerable areas of human atherosclerotic plaques, they may participate in the weakening and rupture of atherosclerotic plaques.     

Sortilin Expression Is Essential for Pro-Nerve Growth Factor-Induced Apoptosis of Rat Vascular Smooth Muscle Cells

Background

Sortilin, a member of the Vps10p-domain receptor family, has been demonstrated a key regulator in mediating cellular response to pro-neurotrophins. In the present study, we investigated the role of sortilin in the apoptotic pathway of vascular smooth muscle cells.

Methods and Principal Findings

Immunohistochemistry revealed that sortilin was barely detectable in human and rat normal young vessels, while its expression was increased in human fibroatheromatous plaques. Sortilin immunodetection was also marked in the neointima of the rat aorta fifteen days after ballooning. In vitro, rat aortic intimal cells expressed higher sortilin levels than normal media SMCs; sortilin was distributed in the cytoplasm and in correspondence of the cell membrane. After 48 h, pro-nerve growth factor (proNGF) induced the strong dose-dependent increase of intimal cell apoptosis and the accumulation of sortilin protein. ProNGF was a more potent apoptotic inducer than equimolar or even higher concentration of NGF, whereas brain derived neutrotrophic factor was ineffective. Targeted interfering RNA-mediated sortilin reduction counteracted proNGF-induced apoptosis without affecting p75^NTR expression. ProNGF-induced apoptosis was associated to NF-κB down-regulation and bax increase. Inhibition of NF-κB activity increased intimal cell apoptosis that did not further increase with the addition of proNGF.

Conclusions

Our results indicate that sortilin expression characterizes human atheromatous lesions and rat aortic post-injury neointima, and suggest that sortilin represents an important regulator of proNGF-induced SMC apoptosis and arterial remodeling.     

IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo

The anti-inflammatory cytokine IL-10 inhibits intimal hyperplasia after stent implantation via a powerful inactivation of monocytes. We tested the hypothesis that IL-10 may also inhibit vascular smooth muscle cell (SMC) activation via the inhibition of the NF-kappaB/I-kappaB system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-kappaBalpha and I-kappaBbeta degradation, NF-kappaB activation, as well as the expression of the NF-kappaB-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-kappaB inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.     

Stem cell marker expression,proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.     

Arsenic trioxide induces the apoptosis in vascular smooth muscle cells via increasing intracellular calcium and ROS formation

The present study was designed to investigate whether arsenic trioxide induced the apoptosis in rat mesenteric arterial smooth muscle cells (SMCs), which provides new insights into mechanisms of arsenic-related vascular diseases. Here, we found that arsenic trioxide significantly decreased the viability of SMCs in a dose-dependent manner. In addition, higher level of arsenic trioxide directly caused cellular necrosis. The Hoechst and AO/EB staining demonstrated that apoptotic morphological change was presented in SMCs exposed to arsenic trioxide. The TUNEL assay displayed that more positive apoptotic signal appeared in SMCs treated with arsenic trioxide. The following result showed that ROS formation was markedly increased in arsenic trioxide-treated SMCs. Pretreatment with N-acetylcysteine, an anti-oxidant reagent, obviously attenuated the enhancement of ROS production and the reduction of cell viability induced by arsenic trioxide in SMCs. Arsenic trioxide also enhanced free intracellular Ca²⁺ level in SMCs. BAPTA also significantly prevented the increased intracellular Ca²⁺ and decreased cell viability induced by arsenic trioxide in SMCs. These results suggested that arsenic trioxide obviously induced apoptosis in SMCs, and its mechanism was partially associated with intracellular ROS formation and free Ca²⁺ increasing.     

NF-κB Prevents TNF-α–Induced Apoptosis in an Oligodendrocyte Cell Line

Nuclear factor kappa beta (NF-kappaB) inhibits apoptosis in sensory, hippocampal, and striatal neurons of the central nervous system. Although several apoptotic stimuli have been shown to activate NF-kappaB in oligodendrocytes, the function of NF-kappaB in this cell type remains unknown. In this study, we introduced plasmids expressing either the p50- or p65-subunit of human NF-kappaB into Central Glia-4 (CG-4)--a rat oligodendrocyte precursor cell line-and determined the influence of NF-kappaB function on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Expression of NF-kappaB markedly prevented CG-4 apoptosis, with p50 being more effective than p65. This anti-apoptotic activity was repressed by IkappaB-alpha, an inhibitor of NF-kappaB. These results imply that NF-kappaB acts as a potent inhibitor of TNF-induced apoptosis in oligodendrocytes.     

Reduced Intimal Thickening Following αvβ 3 Blockade is Associated with Smooth Muscle Cell Apoptosis

The adhesion integrin αvβ3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of αvβ 3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of αvβ 3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of αvβ 3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of αvβ 3. At both 2 and 4wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of αvβ 3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing αvβ 3. These findings may explain the reduction in restenosis observed clinically following,93 integrin blockade.     

Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-kappaB activation

The effects of hepatitis C virus (HCV) proteins on anti-Fas (CD95/APO-1) antibody- and tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis in different human cell lines were investigated by magnetic concentration of cells which transiently produced the exogenous protein. HepG2 cells, which produced whole HCV proteins, became resistant to anti-Fas-induced apoptotic cell death. Furthermore, the core protein among HCV proteins had a key role in protecting the various cells from apoptosis mediated by not only anti-Fas but also TNF-alpha. We also found that the core functioned in the activation of nuclear factor kappaB (NF-kappaB) in all cells examined. Deletion analysis of the core revealed that the region required for NF-kappaB activation was closely correlated with that for its antiapoptotic function. In addition, we revealed in some cases that the antiapoptotic effect of the core was restrained by coproduction of the inhibitor of NF-kappaB, IkappaB-alpha protein. These results demonstrated that the core inhibits Fas- and TNF-alpha-mediated apoptotic cell death via a mechanism dependent on the activation of NF-kappaB in particular cell lines.     

Reduced intimal thickening following alpha(v)beta3 blockade is associated with smooth muscle cell apoptosis

The adhesion integrin alpha(v)beta3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of alpha(v)beta3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of alpha(v)beta3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of alpha(v)beta3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of alpha(v)beta3. At both 2 and 4 wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of alpha(v)beta3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing alpha(v)beta3. These findings may explain the reduction in restenosis observed clinically following beta3 integrin blockade.     

Relationship between constitutive nuclear factor-kappaB (NF-kappaB) and inhibitor kappaB-alpha (IkappaB-alpha) in an interferon-alpha-sensitive human Burkitt lymphoma cell line

The human Burkitt lymphoma Daudi cell line expresses constitutively active nuclear factor kappaB (NF-kappaB) in the nucleus in spite of high levels of inhibitor kappaB-alpha (IkappaB-alpha) in the cytoplasm. The antiproliferative response of these cells to interferon-alpha (IFN-alpha) correlated with the inhibition of the constitutive NF-kappaB activity by the cytokine. The present study shows that IFN-alpha caused an increase in p53 level, inhibited cell proliferation by [(3)H]thymidine incorporation, and stimulated cytotoxicity and apoptosis by PARP-cleavage in the Daudi cells. In order to study the relationship between the constitutively active NF-kappaB and IkappaB-alpha, a dominant negative mutant IkappaB-alpha (IkappaB-alphaDN), lacking the N-terminal 36 amino acids required for the activation of NF-kappaB by tumor necrosis factor-alpha (TNF-alpha), was expressed in the Daudi cells. The expression of IkappaB-alphaDN protein did not inhibit the constitutive NF-kappaB activity, but it inhibited cell proliferation, antiproliferative response to IFN-alpha, and phosphorylated mitogen activated protein kinase (p-MAPK) level. Thus, our results suggest that constitutive NF-kappaB activity in the human Burkitt lymphoma Daudi cells is maintained by a mechanism independent of IkappaB-alpha degradation, and that the IkappaB-alpha is involved in the proliferation of these cells, possibly through the MAP kinase pathway. Therefore, in addition to IFN-alpha treatment, both NF-kappaB and IkappaB-alpha may be used as drug targets for inhibiting cell proliferation in the lymphomas.     

Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy

Long-term solid-organ allografts typically develop diffuse arterial intimal lesions (graft arterial disease; GAD), consisting of smooth-muscle cells (SMC), extracellular matrix and admixed mononuclear leukocytes. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although the exact mechanisms are unknown, chronic low-level alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation and matrix synthesis. Although prior work demonstrated that the endothelium and medial SMCs lining GAD lesions in cardiac allografts are donor-derived, the intimal SMC origin could not be determined. They are generally presumed to originate from the donor media, leading to interventions that target donor medial SMC proliferation, with limited efficacy. However, other reports indicate that allograft vessels may contain host-derived endothelium and SMCs (refs. 8,9). Moreover, subpopulations of bone-marrow and circulating cells can differentiate into endothelium, and implanted synthetic vascular grafts are seeded by host SMCs and endothelium. Here we used murine aortic transplants to formally identify the source of SMCs in GAD lesions. Allografts in beta-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of beta-galactosidase--expressing cells into aortic allograft recipients demonstrated that intimal cells included those of marrow origin. Thus, smooth-muscle--like cells in GAD lesions can originate from circulating bone--marrow-derived precursors.     

Role of the IkappaB kinase complex in oncogenic Ras- and Raf-mediated transformation of rat liver epithelial cells

NF-kappaB/Rel factors have been implicated in the regulation of liver cell death during development, after partial hepatectomy, and in hepatocytes in culture. Rat liver epithelial cells (RLEs) display many biochemical and ultrastructural characteristics of oval cells, which are multipotent cells that can differentiate into mature hepatocytes. While untransformed RLEs undergo growth arrest and apoptosis in response to transforming growth factor beta1 (TGF-beta1) treatment, oncogenic Ras- or Raf-transformed RLEs are insensitive to TGF-beta1-mediated growth arrest. Here we have tested the hypothesis that Ras- or Raf-transformed RLEs have altered NF-kappaB regulation, leading to this resistance to TGF-beta1. We show that classical NF-kappaB is aberrantly activated in Ras- or Raf-transformed RLEs, due to increased phosphorylation and degradation of IkappaB-alpha protein. Inhibition of NF-kappaB activity with a dominant negative form of IkappaB-alpha restored TGF-beta1-mediated cell killing of transformed RLEs. IKK activity mediates this hyperphosphorylation of IkappaB-alpha protein. As judged by kinase assays and transfection of dominant negative IKK-1 and IKK-2 expression vectors, NF-kappaB activation by Ras appeared to be mediated by both IKK-1 and IKK-2, while Raf-induced NF-kappaB activation was mediated by IKK-2. NF-kappaB activation in the Ras-transformed cells was mediated by both the Raf and phosphatidylinositol 3-kinase pathways, while in the Raf-transformed cells, NF-kappaB induction was mediated by the mitogen-activated protein kinase cascade. Last, inhibition of either IKK-1 or IKK-2 reduced focus-forming activity in Ras-transformed RLEs. Overall, these studies elucidate a mechanism that contributes to the process of transformation of liver cells by oncogene Ras and Raf through the IkappaB kinase complex leading to constitutive activation of NF-kappaB.     

Tempol therapy attenuates medial smooth muscle cell apoptosis and neointima formation after balloon catheter injury in carotid artery of diabetic rats

Accumulating data support the hypothesis that reactive oxygen species (ROS) play a critical role in the vascular complications observed in diabetes. However, the mechanisms of ROS-mediated vascular complications in diabetes are not clear. We tested the hypothesis that ROS-mediated increase in proapoptotic factor Bax expression leads to medial smooth muscle cell (SMC) apoptosis that is associated with neointima formation. We used a fructose-rich diet for 4 wk to model Type 2 diabetes in rats. SOD mimetic membrane-permeable 4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempol, 1 mM) was administered in drinking water to scavenge superoxide starting 1 day before surgery and continued during the duration of the experiment. Vascular injury resulted in a significant increase in medial SMC apoptosis that was associated with neointima formation. The number of medial SMC positive for Bax immunostaining significantly increased in injured arteries compared with uninjured arteries. Superoxide scavenging by Tempol treatment inhibited both the Bax-positive index as well as the apoptotic index of medial SMC in response to vascular injury. Tempol treatment inhibited apoptotic loss of medial SMC, thus increasing their density in the injured arteries. These alterations in the media were associated with a marked decrease in neointima formation in injured arteries. We conclude that Bax expression may play an important role in vascular SMC apoptosis and, finally, that this regulatory mechanism is redox sensitive.     

Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70–Bax interaction in prostate cancer

Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70–Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy.     

Ethyl isopropyl amiloride inhibits smooth muscle cell proliferation and migration by inducing apoptosis and antagonizing urokinase plasminogen activator activity

Amiloride inhibits activation of the Na(+)-H+ exchanger (NHE), a critical step in smooth muscle cell (SMC) growth. While amiloride treatment reduces SMC proliferation and migration, as well as experimental lesion formation, these effects are not exclusively due to NHE inhibition and remain incompletely understood. The purpose of this study was to examine the mechanisms involved in amiloride-induced attenuation of SMC proliferation and migration, looking specifically at the potential role of apoptosis and urokinase plasminogen activator (uPA) activity in these processes. Rabbit SMCs in tissue culture were exposed to 10-80 microM of the amiloride analogue ethyl isopropyl amiloride (EIPA). Compared with controls, EIPA reduced DNA synthesis, cell number, and mitochondrial respiration, but without toxic effects on quiescent or proliferating cells. In a Boyden chamber assay, EIPA reduced uPA-induced SMC migration. Moreover, in a SMC scratch assay EIPA treatment resulted in a 66% reduction in the number of repopulating cells, a 92% decrease in the number of proliferating cells, and a 37-fold increase in the number of apoptotic cells. SMC apoptosis was frequently localized to the scratch edges, where cell proliferation and bcl-2 expression were absent. Finally, uPA enzymatic activity in the cell culture media was lower for EIPA-treated versus control SMCs. Therefore, EIPA inhibits both SMC proliferation and migration by inducing apoptosis and antagonizing uPA activity, respectively, and requires further study as an agent for reducing vascular lesion formation.