Jak2 tyrosine kinase mediates oxidative stress-induced apoptosis in vascular smooth muscle cells

In vascular smooth muscle cells, Jak2 tyrosine kinase becomes activated in response to oxidative stress in the form of hydrogen peroxide. Although it has been postulated that hydrogen peroxide-induced Jak2 activation promotes cell survival, this has never been tested. We therefore examined the role that Jak2 plays in vascular smooth muscle cell apoptosis following hydrogen peroxide treatment. Here, we report that Jak2 tyrosine kinase activation by hydrogen peroxide is required for apoptosis of vascular smooth muscle cells. Upon treatment of primary rat aortic smooth muscle cells with hydrogen peroxide, we observed laddering of genomic DNA and nuclear condensation, both hallmarks of apoptotic cells. However, apoptosis was prevented by either the expression of a dominant negative Jak2 protein or by the Jak2 pharmacological inhibitor AG490. Moreover, expression of the proapoptotic Bax protein was induced following hydrogen peroxide treatment. Again, expression of a dominant negative Jak2 protein or treatment of cells with AG490 prevented this Bax induction. Following Bax induction by hydrogen peroxide, mitochondrial membrane integrity was compromised, and caspase-9 became activated. In contrast, in cells expressing a Jak2 dominant negative we observed that mitochondrial membrane integrity was preserved, and no caspase-9 activation occurred. These data demonstrate that the activation of Jak2 tyrosine kinase by hydrogen peroxide is essential for apoptosis of vascular smooth muscle cells. Furthermore, this report identifies Jak2 as a potential therapeutic target in vascular diseases in which vascular smooth muscle cell apoptosis contributes to pathological progression.     

Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis

Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are an important cause for VSMC death. However, the precise mechanism of oxidative stress-induced VSMC apoptosis is still poorly understood. Here, we aimed to analyse the role of soluble adenylyl cylclase (sAC). VSMC derived from rat aorta were treated with either H₂O₂ (300 µmol/L) or DMNQ (30 µmol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 µmol/L KH7 (a specific inhibitor of sAC) or by stable sAC-knockdown (shRNA-transfection). A similar effect was found after inhibition of protein kinase A (PKA). Suppression of the sAC/PKA-axis led to a significant increase in phosphorylation of the p38 mitogen-activated protein kinase under oxidative stress accompanied by a p38-dependent phosphorylation/inactivation of the pro-apoptotic Bcl-2-family protein Bad. Pharmacological inhibition of p38 reversed these effects of sAC knockdown on apoptosis and Bad phosphorylation, suggesting p38 as a link between sAC and apoptosis. Analysis of the protein phosphatases 1 and 2A activities revealed an activation of phosphatase 1, but not phosphatase 2A, under oxidative stress in a sAC/PKA-dependent manner and its role in controlling the p38 phosphorylation. Inhibition of protein phosphatase 1, but not 2A, prevented the pro-apoptotic effect of oxidative stress. In conclusion, sAC/PKA-signaling plays a key role in the oxidative stress-induced apoptosis of VSMC. The cellular mechanism consists of the sAC-promoted and protein phosphatase 1-mediated suppression of p38 phosphorylation resulting to activation of the mitochondrial pathway of apoptosis.     

CHIP has a protective role against oxidative stress-induced cell death through specific regulation of Endonuclease G

Oxidative stress is implicated in carcinogenesis, aging, and neurodegenerative diseases. The E3 ligase C terminus of Hsc-70 interacting protein (CHIP) has a protective role against various stresses by targeting damaged proteins for proteasomal degradation, and thus maintains protein quality control. However, the detailed mechanism by which CHIP protects cells from oxidative stress has not been demonstrated. Here, we show that depletion of CHIP led to elevated Endonuclease G (EndoG) levels and enhanced cell death upon oxidative stress. In contrast, CHIP overexpression reduced EndoG levels, and resulted in reduced or no oxidative stress-induced cell death in cancer cells and primary rat cortical neurons. Under normal conditions Hsp70 mediated the interaction between EndoG and CHIP, downregulating EndoG levels in a Hsp70/proteasome-dependent manner. However, under oxidative stress Hsp70 no longer interacted with EndoG, and the stabilized EndoG translocated to the nucleus and degraded chromosomal DNA. Our data suggest that regulation of the level of EndoG by CHIP in normal conditions may determine the sensitivity to cell death upon oxidative stress. Indeed, injection of H₂O₂ into the rat brain markedly increased cell death in aged mice compared with young mice, which correlated with elevated levels of EndoG and concurrent downregulation of CHIP in aged mice. Taken together, our findings demonstrate a novel protective mechanism of CHIP against oxidative stress through regulation of EndoG, and provide an opportunity to modulate oxidative stress-induced cell death in cancer and aging.     

Requirement of protein tyrosine phosphatase SHP2 for NO-stimulated vascular smooth muscle cell motility

We have previously reported that nitric oxide (NO) increases the motility of differentiated cultured primary aortic smooth muscle cells from adult rats. There is little information on the role of protein tyrosine phosphatases in vascular biology. One such phosphatase, Src homology 2 phosphatase 2 (SHP2), is essential for motility. We tested the hypothesis that NO increases SHP2 levels via a cGMP-mediated mechanism and that this effect is necessary for NO-stimulated cell motility. Here we report that two different NO donors increased SHP2 protein levels and enzyme activity. This effect was mimicked by several cGMP agonists and blocked by an inhibitor of guanylyl cyclase. Specific decrease of SHP2 protein levels via the use of antisense oligodeoxynucleotides (ODNs), but not several control ODNs attenuated the motogenic effect of NO, which indicates the involvement of SHP2 in NO-elicited motogenesis. S-nitroso-N-acetylpenicillamine failed to increase SHP2 protein levels in subcultured aortic smooth muscle cells. This provides a potential explanation for the lack of effect of NO on cell motility in dedifferentiated subcultured cells. These results support the hypothesis that NO-elicited upregulation of SHP2 via a cGMP-mediated pathway is necessary for NO-induced motogenesis in differentiated aortic smooth muscle cells.     

HRT1 modulates vascular smooth muscle cell proliferation and apoptosis

The Notch signaling pathway plays vital roles in vascular development and homeostasis. However, the functional role of HRT1, a primary downstream effector of Notch signaling in VSMC, is poorly characterized. In the present study, we postulated that HRT1 plays fundamental roles in modulating VSMC fate. To test the hypothesis that HRT1 is coupled to growth regulation, we generated VSMC lines constitutively overexpressing HRT1 (HRT1SMC) and demonstrated an exaggerated growth behavior compared to its control cell line. The lack of cell cycle arrest at confluence in HRT1SMC was associated with an attenuated up-regulation of the cell cycle inhibitor, p21(WAF1/CIP1). We further established that both transient and constitutive HRT1 signaling promoted VSMC survival in response to serum deprivation and pro-apoptotic Fas ligand. Resistance to apoptosis was associated with the induction of Akt expression/activity, a well-described anti-apoptotic mediator. Overall, these findings provide initial evidence that HRT1 functions as a critical determinant of VSMC proliferation and survival.     

Inhibitor-1 phosphatase activity in vascular smooth muscle

The histone-H1 and polylysine stimulated "latent" phosphorylase phosphatase, characterized by a molecular weight of 260,000 in gel filtration and 130,000 in sucrose density gradient centrifugation has been identified as a major inhibitor-1 phosphatase in vascular smooth muscle. Its substrate: protein phosphatase inhibitor-1, was also shown to be present in the same tissue. Following treatment with beta-mercaptoethanol the enzyme dissociates into a lower molecular weight (38,000) form with a higher specific activity.     

Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells

Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis and hypertension, in part by promoting vascular smooth muscle cell (VSMC) growth. We have previously shown that LY83583, a generator of O-(2), activated extracellular signal-regulated kinases (ERK1/2) with early (10 min) and late (2 h) peaks and stimulated VSMC growth. To investigate whether secreted oxidative stress-induced factors (termed SOXF) from VSMC were responsible for late ERK1/2 activation in response to LY83583, we purified putative SOXF proteins from conditioned medium (2 h of LY83583 exposure) by sequential chromatography based on activation of ERK1/2. Proteins identified by capillary chromatography, electrospray ionization tandem mass spectrometry, and data base searching included heat shock protein 90-alpha (HSP90-alpha) and cyclophilin B. Western blot analysis of conditioned medium showed specific secretion of HSP90-alpha but not HSP90-beta. Immunodepletion of HSP90-alpha from conditioned medium significantly inhibited conditioned medium-induced ERK1/2 activation. Human recombinant HSP90-alpha reproduced the effect of conditioned medium on ERK1/2 activation. These results show that brief oxidative stress causes sustained release of protein factors from VSMC that can stimulate ERK1/2. These factors may be important mediators for the effects of reactive oxygen species on vascular function.     

Involvement of phospholipase D in oxidative stress-induced necrosis of vascular smooth muscle cells.

Phospholipase D (PLD) has been associated with necrosis. However, it is not clear whether PLD plays a causative role in this cellular process. We investigated the role of PLD in oxidative stress-induced necrosis of vascular smooth muscle cells (VSMCs). Pervanadate (hydrogen peroxide plus orthovanadate) but not hydrogen peroxide alone activated PLD in a dose- and time-dependent manner. Exposure of VSMCs to pervanadate resulted in necrosis. Pretreatment with butan-1-ol, a PLD inhibitor, attenuated both pervanadate-induced necrosis and increase of intracellular Ca(2+). Removal of extracellular Ca(2+) inhibited pervanadate-induced necrosis by 50%. These results suggest that PLD activation mediates pervanadate-induced necrosis of VSMCs, which is at least partly due to Ca(2+) toxicity.     

Folacin C60 derivative exerts a protective activity against oxidative stress-induced apoptosis in rat pheochromocytoma cells

In the present study, we describe the synthesis and characterization of a novel folacin C₆₀ derivative. The compound was analyzed by FT-IR, ¹H NMR, ¹³C NMR, LC–MS and elemental analysis. This water soluble fullerene derivative was able to scavenge both superoxide and hydroxyl radical with biocompatibility. Rat pheochromocytoma (PC12) cells treated with hydrogen peroxide underwent cytotoxicity and apoptotic death determined by MTT assay and flow cytometry analysis. As a novel derivative of C₆₀, the folacin C₆₀ derivative self-assembled to form spherical aggregates in H₂O. Because the compound was amphiphilic, it could penetrate the cell membrane and play its distinguished role in protecting PC12 cells against hydrogen peroxide-induced cytotoxicity. The results suggest that folacin C₆₀ derivative has the potential to prevent oxidative stress-induced cell death without evident toxicity.     

Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

A spontaneously active (Mr greater than 350,000) and an ATPMg-dependent phosphatase (Mr congruent to 140,000) were identified in bovine aortic smooth muscle. The spontaneously active phosphatase was effective in dephosphorylating both phosphorylase a (240nmol32P/min/mg) and phosphorylated myosin light chains (1000nmol32P/min/mg). In contrast, the ATPMg-dependent phosphatase was only effective in dephosphorylating phosphorylase a (400nmol32P/min/mg). Phosphorylase phosphatase activity of the ATPMg-dependent enzyme was suppressed by the well-characterized modulator protein (inhibitor-2), whereas the activity of the spontaneously active enzyme was unaffected. The aortic spontaneously active phosphatase did not convert to an ATPMg-dependent form when it was stored at 4 degrees or incubated at 30 degrees C in either the presence or absence of modulator protein. These findings suggest that spontaneous and ATPMg-dependent phosphatase activities described in these studies are probably ascribable to different enzymes. Since both phosphorylase and myosin light chains are phosphorylated when smooth muscle contracts these phosphatases may participate in coordinating arterial contractility and metabolism.     

Modulation of latent protein phosphatase activity from vascular smooth muscle by histone-H1 and polylysine

An apparently latent phosphatase which migrated as a protein of Mr 130,000 during sucrose density centrifugation, and a spontaneously active phosphatase (Mr 68,000) were isolated from bovine aortic smooth muscle. Basal phosphorylase phosphatase activity of the latent preparations was stimulated 12 fold by low concentrations of lysine-rich histone-H1 (30 micrograms/ml) and 6 fold by polylysine (Mr 17,000; 12 micrograms/ml), whereas the spontaneously active enzyme was only slightly affected. The enzymatic activity of the spontaneously active preparation was completely destroyed by beta-mercaptoethanol. In contrast, the apparently latent enzyme was converted to a more active form of lower molecular weight (Mr 86,000) following treatment with beta-mercaptoethanol and this form of the enzyme was still stimulateable by histone-H1. These findings show that the aortic spontaneous and apparently latent phosphatase actives are ascribable to separate enzymes and they suggest that the activity of latent phosphatase in living cells may be modulated by cationic proteins such as histones or similar effector molecules.     

C60 carboxyfullerene exerts a protective activity against oxidative stress-induced apoptosis in human peripheral blood mononuclear cells

C60 carboxyfullerene is a novel buckminsterfullerene-derived compound that behaves as a free-radical scavenger. In the present report, we investigated whether this drug exerts a protective activity against oxidative stress-induced apoptosis. Human peripheral blood mononuclear cells (PBMCs) were challenged by 2-deoxy-d-ribose (dRib) or TNF-alpha plus cycloheximide as agents that trigger apoptosis by interfering with the redox status of cell and mitochondrial membrane potential. We found that carboxyfullerene was able to protect quiescent PBMCs from apoptosis caused either by 2-deoxy-d-ribose or TNF-alpha plus cycloheximide by a mechanism partially involving the mitochondrial membrane potential integrity, known to be associated with early stages of apoptosis. These results represent the first indication for a target activity of buckminsterfullerenes on cells of the immune system and their mitochondria.     

Identification of the linker histone H1 as a protein kinase Cepsilon-binding protein in vascular smooth muscle.

A variety of anchoring proteins target specific protein kinase C (PKC) isoenzymes to particular subcellular locations or multimeric signaling complexes, thereby achieving a high degree of substrate specificity by localizing the kinase in proximity to specific substrates. PKCepsilon is widely expressed in smooth muscle tissues, but little is known about its targeting and substrate specificity. We have used a Far-Western (overlay) approach to identify PKCepsilon-binding proteins in vascular smooth muscle of the rat aorta. Proteins of approximately 32 and 34 kDa in the Triton-insoluble fraction were found to bind PKCepsilon in a phospholipid/diacylglycerol-dependent manner. Although of similar molecular weight to RACK-1, a known PKCepsilon-binding protein, these proteins were separated from RACK-1 by SDS-PAGE and differential NaCl extraction and were not recognized by an antibody to RACK-1. The PKCepsilon-binding proteins were further purified from the Triton-insoluble fraction and identified by de novo sequencing of selected tryptic peptides by tandem mass spectrometry as variants of the linker histone H1. Their identity was confirmed by Western blotting with anti-histone H1 and the demonstration that purified histone H1 binds PKCepsilon in the presence of phospholipid and diacylglycerol but absence of Ca(2+). The interaction of PKCepsilon with histone H1 was specific since no interaction was observed with histones H2A, H2S or H3S. Bound PKCepsilon phosphorylated histone H1 in a phospholipid/diacylglycerol-dependent but Ca(2+)-independent manner. Ca(2+)-dependent PKC was also shown to interact with histone H1 but not other histones. These results suggest that histone H1 is both an anchoring protein and a substrate for activated PKCepsilon and other PKC isoenzymes and likely serves to localize activated PKCs that translocate to the nucleus in the vicinity of specific nuclear substrates including histone H1 itself. Since PKC isoenzymes have been implicated in regulation of gene expression, stable interaction with histone H1 may be an important step in this process.     

Cks1 mediates vascular smooth muscle cell polyploidization

Vascular smooth muscle cells (VSMC) at capacitance arteries of hypertensive individuals and animals undergo dramatic polyploidization that contributes toward their hypertrophic phenotype. We report here the identification of a defective mitotic spindle cell cycle checkpoint in VSMC isolated from capacitance arteries of pre-hypertensive rats. These cells demonstrated a high predisposition to polyploidization in culture and failed to maintain cyclin B protein levels in response to colcemid, a mitotic inhibitor. Furthermore, this altered mitotic spindle checkpoint status was associated with the overexpression of Cks1, a Cdc2 adapter protein that promotes cyclin B degradation. Cks1 up-regulation, cyclin B down-regulation, and VSMC polyploidization were evidenced at the smooth muscle of capacitance arteries of genetically hypertensive and Goldblatt-operated rats. In addition, angiotensin II infusion dramatically increased Cks1 protein levels at capacitance arteries of normotensive rats, and angiotensin II treatment of isolated VSMC abrogated their ability to down-regulate Cks1 and maintain cyclin B protein expression in response to colcemid. Finally, transduction of VSMC from normotensive animals with a retrovirus that drives the expression of Cks1 was sufficient to alter their mitotic spindle cell cycle checkpoint status and promote unscheduled cyclin B metabolism, cell cycle re-entry, and polyploidization. These data demonstrate that Cks1 regulates cyclin B metabolism and ploidy in VSMC and may contribute to the understanding of the phenomena of VSMC polyploidization during hypertension.     

Essential role of the low density lipoprotein receptor-related protein in vascular smooth muscle cell migration

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor highly expressed in human aortic smooth muscle cells. In the present study, we used the short interfering RNA (siRNA) technique to explore the role of LRP in smooth muscle cell migration. We identified an LRP-specific siRNA that selective silences LRP expression in human aortic smooth muscle cells. As a consequence, LRP-mediated ligand degradation was significantly reduced. More important, we found that platelet-derived growth factor-dependent cell migration was inhibited in cells transfected with LRP siRNA. These results demonstrate an important role of LRP in smooth muscle cell migration.     

Circadian misalignment promotes vascular smooth muscle cell apoptosis via defective autophagy

Journal of Molecular Histology - Defective autophagy in vascular smooth muscle cells (VSMCs) in response to oxidative stress can lead to cellular apoptosis and plaque instability. Previous studies...