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Biomedical subjects

Arnold G Herman

Publications and source records attributed to Arnold G Herman.

At least 19 recordsLinked to original sources
PubMed2006

Selective clearance of macrophages in atherosclerotic plaques by the protein synthesis inhibitor cycloheximide.

Macrophages are an essential component of unstable atherosclerotic plaques and play a pivotal role in the destabilization process. We have demonstrated previously that local delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus selectively clears macrophages in rabbit plaques. Because mTOR controls mRNA translation, inhibition of protein synthesis might induce selective macrophage cell death. We therefore investigated in the present study the effect of the protein synthesis inhibitor cycloheximide on macrophage and smooth muscle cell (SMC) viability. In vitro studies with cultured macrophages and SMCs showed that cycloheximide induced selective apoptosis of macrophages in a concentration- and time-dependent manner. Moreover, macrophages could be selectively depleted in rabbit carotid artery rings with collar-induced atherosclerotic plaques after in vitro treatment with cycloheximide. Local in vivo administration of cycloheximide via osmotic minipumps to rabbit carotid arteries with collar-induced atherosclerotic plaques significantly reduced the macrophage but not the SMC content. Cycloheximide-treated plaques showed signs of apoptosis (increased terminal deoxynucleotidyl transferase end labeling and fluorescein isothiocyanate-Val-Ala-dl-Asp(O-methyl)-fluoromethylketone labeling) that did not colocalize with SMCs. Organ chamber studies demonstrated that the functionality of SMCs and the endothelium were not influenced by cycloheximide treatment. All together, these findings demonstrate that cycloheximide decreases the macrophage load in atherosclerotic plaques by induction of apoptosis without changing SMC content or contractility.

Animals↗
PubMed2006

z-VAD-fmk-induced non-apoptotic cell death of macrophages: possibilities and limitations for atherosclerotic plaque stabilization.

Macrophages play a pivotal role in atherosclerotic plaque destabilization in contrast to smooth muscle cells (SMCs). As a consequence, removal of macrophages from plaques via selective induction of cell death represents a promising approach to stabilize non-obstructive, rupture-prone atherosclerotic lesions. However, the mechanisms to initiate cell death in macrophages but not in other cell types of the plaque, in particular SMCs, are unknown. Recently, we have shown that the pan-caspase inhibitor z-VAD-fmk induces autophagy and necrotic cell death in J774A.1 and RAW264.7 macrophages as well as in IFN-gamma primed primary mouse peritoneal macrophages, but not in vascular SMCs or C2C12 myoblasts. The different sensitivity to z-VAD-fmk is largely based on differential expression of receptor-interacting protein 1 (RIP1). This finding suggests that caspase inhibition activates RIP1 which in turn initiates autophagy, although other explanations should be taken into account. z-VAD-fmk-treated J774A.1 macrophages overexpress and secrete several chemokines and cytokines, including TNFalpha. The combination of z-VAD-fmk and TNFalpha, but not TNFalpha alone, induces SMC necrosis. In this regard, z-VAD-fmk is detrimental and not beneficial for atherosclerotic plaque stability due to stimulation of inflammatory responses and indirect induction of SMC death. Future work is needed to determine the mechanism(s) that selectively trigger non-apoptotic cell death in plaque macrophages without evoking inflammation and SMC death.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Phagocytosis in atherosclerosis: Molecular mechanisms and implications for plaque progression and stability.

Macrophages play a key role in atherosclerotic plaque destabilization and rupture. In this light, selective removal of macrophages may be beneficial for plaque stability. However, macrophages are phagocytic cells and thus have an important additional role in scavenging of modified lipoproteins, unwanted or dead cells and cellular debris via phagocytosis. The concept of phagocytosis as well as the underlying mechanisms is well defined but the effect of phagocytosis in terms of plaque stability remains poorly understood. Recent findings point towards a complex role of macrophage phagocytosis in atherogenesis. Macrophages are necessary for removal of apoptotic cells from plaques, but exert strong proatherogenic properties upon phagocytosis of lipoproteins, erythrocytes and platelets. Apart from heterophagy, autophagocytosis better known as autophagy may occur in advanced atherosclerotic plaques. Several lines of evidence indicate that autophagy is initiated in plaque smooth muscle cells as a result of cellular distress. Since autophagy is well recognized as a survival mechanism, autophagic smooth muscle cells in the fibrous cap may reflect an important feature underlying plaque stability. All together, phagocytosis is a crucial process involved in atherogenesis that may significantly affect the stability of the atherosclerotic plaque.

Animals↗
PubMed2006

The role of tachykinins in circular muscle contractility of the murine ileum: a functional investigation.

We investigated the participation of different tachykinin receptors in contractility of circular muscle strips of the mouse ileum using selective NK receptor agonists and antagonists. The NK1 receptor agonist septide (1-100 nM) induced dose-dependent contractions which were reduced by atropine and augmented by L-NNA. L-NNA increased and TTX consecutively reduced contractions to the NK2 receptor agonist beta-A-NKA (1-100 nM). Senktide, agonist of NK3 receptors, failed to induce contractions. NANC contractions to EFS were decreased after NK1 receptor blockade with RP67580. This inhibitory effect was more pronounced after additional blockade of NK2 and NK3 receptors. NK3 receptor antagonism alone reduced contractions at higher frequencies of stimulation. When the duration of the EFS stimulus was increased, the participation of all NK receptor subtypes became more evident. Our results suggest that excitatory NANC transmission in the circular muscle layer of the mouse ileum is mediated by tachykinins acting principally on NK1 receptors on cholinergic nerves and smooth muscle cells. Also NK2 receptors, located on smooth muscle cells and nitrergic neurons, and NK3 receptors on enteric neurons are involved.

Animals↗
PubMed2006

Macrophages but not smooth muscle cells undergo benzyloxycarbonyl-Val-Ala-DL-Asp(O-Methyl)-fluoromethylketone-induced nonapoptotic cell death depending on receptor-interacting protein 1 expression: implications for the stabilization of macrophage-rich atherosclerotic plaques.

Several lines of evidence suggest that macrophages play a key role in atherosclerotic plaque destabilization and rupture. Therefore, selective removal of macrophages from plaques via pharmacological therapy could represent a promising approach to stabilize "vulnerable," rupture-prone lesions. Yet, how macrophages can be eliminated from plaques without influencing other cell types, including smooth muscle cells (SMCs), is unknown. In the present study, we report that benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk), a caspase inhibitor with broad specificity, induces nonapoptotic cell death of J774A.1 and RAW264.7 macrophages but not of SMCs. Cell death was characterized by bulk degradation of long-lived proteins, processing of microtubule-associated protein light chain 3, and cytoplasmic vacuolization, which are all markers of autophagy. However, necrosis also occurred, and the number of necrotic cells rapidly increased during z-VAD-fmk treatment. Primary mouse peritoneal macrophages were resistant to z-VAD-fmk-mediated cell death, but unlike SMCs, they underwent z-VAD-fmk-mediated necrosis after pretreatment with interferon-gamma. Further evidence indicated that the expression level of receptor-interacting protein 1 (RIP1) mediates the sensitivity to z-VAD-fmk. Importantly, upon z-VAD-fmk treatment, J774A.1 macrophages overexpressed and secreted several chemokines and cytokines, including tumor necrosis factor (TNF) alpha. The combination of z-VAD-fmk and TNFalpha, but not TNFalpha alone, induced SMCs necrosis via a mechanism that required RIP1 expression. These results suggest that z-VAD-fmk, despite its selective cell death inducing capacity, would be detrimental for the stability of atherosclerotic plaques due to enlargement of the necrotic core, stimulation of inflammatory responses, and indirect induction of SMC death.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Detection of autophagy in tissue by standard immunohistochemistry: possibilities and limitations.

Transmission electron microscopy (TEM) is currently the standard method to monitor autophagy in tissue. Because TEM is labor intensive, we recently questioned whether marker proteins could be found for unambiguous detection of autophagy in tissue using standard immunohistochemical techniques. Our findings indicated that the identification of autophagy-specific biomarkers for tissue is highly compromised due to lack of differential gene expression. In this respect, TEM remains an indispensable technique for evaluation of autophagy in situ. Nevertheless, immunohistochemical staining of microtubule-associated protein 1 light chain 3 (LC3) appeared to be a valuable technique to detect autophagosome formation in tissue but only when this protein is overexpressed, e.g., in GFP-LC3 transgenic animals. Furthermore, demonstration of granular cytoplasmic ubiquitin inclusions by immunohistochemistry may be an attractive technique to measure autophagic cell degeneration in some human pathologies such as neurodegenerative diseases, heart failure and atherosclerosis.

Animals↗
PubMed2005

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum.

OBJECTIVES: Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. RESULTS: Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK(1), NK(2) or NK(3) receptors significantly reduced contractions to EFS. The NK(1) receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK(2) and NK(3) receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK(1) and NK(2) receptor agonists, septide and [beta-ala(8)]-NKA 4-10 (beta-A-NKA), respectively, but not the NK(3) receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to beta-A-NKA were insensitive to atropine but augmented by l-NNA. CONCLUSIONS: Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK(1) receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK(2) and NK(3) receptors, most likely located presynaptically.

Action Potentials↗
PubMed2005

In situ detection of starvation-induced autophagy.

Autophagy is a regulated bulk degradation process involved in many different human pathologies. Transmission electron microscopy (TEM) is currently the only reliable method for monitoring autophagy in situ. Because TEM is labor intensive, we questioned whether useful marker proteins can be found for unambiguous detection of autophagy in tissue via routinely used colorimetric, immunohistochemical, or fluorescent techniques. Starved HepG2 hepatocytes and nutrient deprived liver tissue were used as a model for the initiation of autophagy. Our findings indicate that starvation-induced autophagy in HepG2 cells was associated neither with differential mRNA gene expression nor with changes in the expression level of known autophagy-related proteins. On the contrary, both transcription and translation were inhibited, suggesting that the identification of autophagy-specific biomarkers for tissue is highly compromised. Light chain 3 (LC3) protein, which is an attractive marker of autophagosomes, revealed a relatively low expression level in tissue and cultured cells, but could be detected via immunohistochemistry in liver from GFP-LC3 transgenic mice. The number of LC3 immunopositive dot-like structures was significantly upregulated in liver tissue from nutrient-deprived GFP-LC3 mice as compared with nonstarved control tissue. Our results suggest that LC3 immunostaining can be used as an alternative detection method for autophagy in situ, but only when this protein is overexpressed.

Animals↗
PubMed2005

Therapeutic potential of nitric oxide donors in the prevention and treatment of atherosclerosis.

Well-known risk factors for atherosclerosis include hypercholesterolaemia, hypertension, diabetes, and smoking. These conditions are associated with endothelial dysfunction, which itself is associated with reduced endothelial generation of nitric oxide (NO). This is an overview of the implications of NO generation in atherosclerosis and of the potential therapeutic benefit of drugs which donate NO, such as organic nitrates, nicorandil, and sydnonimines, or those which increase the availability of endogenous NO, such as statins, angiotensin-converting enzyme inhibitors, L-arginine, and tetrahydrobiopterin.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2005

Phagocytosis of apoptotic cells by macrophages is impaired in atherosclerosis.

OBJECTIVE: Apoptotic cell death has been demonstrated in advanced human atherosclerotic plaques. Apoptotic cells (ACs) should be rapidly removed by macrophages, otherwise secondary necrosis occurs, which in turn elicits inflammatory responses and plaque progression. Therefore, we investigated the efficiency of phagocytosis of ACs by macrophages in atherosclerosis. METHODS AND RESULTS: Human endarterectomy specimens and human tonsils were costained for CD68 (macrophages) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) (apoptosis). Free and phagocytized ACs were counted in both tissues. The ratio of free versus phagocytized AC was 19-times higher in human atherosclerotic plaques as compared with human tonsils, indicating a severe defect in clearance of AC. Impaired phagocytosis of AC was also detected in plaques from cholesterol-fed rabbits and did not further change with plaque progression. In vitro experiments with J774 or peritoneal mouse macrophages showed that several factors caused impaired phagocytosis of AC including cytoplasmic overload of macrophages with indigestible material (beads), free radical attack, and competitive inhibition among oxidized red blood cells, oxidized low-density lipoprotein and ACs for the same receptor(s) on the macrophage. CONCLUSIONS: Our data demonstrate that phagocytosis of ACs is impaired in atherosclerotic plaques, which is at least partly attributed to oxidative stress and cytoplasmic saturation with indigestible material.

Aged↗
PubMed2005

mRNA but not plasmid DNA is efficiently transfected in murine J774A.1 macrophages.

Previous studies demonstrated that macrophages are difficult to transfect. In the present study, we investigated whether J774A.1 macrophages can be efficiently transfected using nucleofector technology. Nucleofection of J774A.1 macrophages with mRNA resulted in transfection efficiencies up to 75% without cell death as compared to control pulsed macrophages. In contrast, introduction of DNA into J774A.1 cells caused apoptosis without expression of the gene of interest. Our results show that mRNA nucleofection is a new high-speed transfection method for macrophages.

Animals↗
PubMed2005

Long-term treatment with the NO-donor molsidomine reduces circulating ICAM-1 levels in patients with stable angina.

Recent clinical evidence has indicated that the severity of atherosclerosis is correlated with the level of soluble ICAM-1 (sICAM-1). Nitric oxide (NO) donors are used to treat patients with stable angina pectoris, and the aim of this study was to investigate the short- and long-term effect of molsidomine on the level of this circulating biochemical marker of endothelial function. We included 172 patients and examined the effect of the NO donor treatment on angina related parameters and on sICAM-1 levels after a 4-week- and a 1-year treatment period. After 4 weeks, angina attacks and sublingual (s.l.) isosorbide dinitrate tablet (ISDN) consumption frequency was significantly (p<0.0001) reduced without altering sICAM-1 levels when compared to the baseline values. The anti-anginal effect of molsidomine 16 mg once a day (o.a.d.) was sustained (s.l. ISDN consumption) or improved (angina attacks frequency; p<0.002) during the following year and a significant decrease in sICAM-1 levels (p<0.0001) was observed. When the sICAM-1 changes during the 1-year treatment period were distributed in four categories (quartiles of the distribution), it was demonstrated that the decrease in s.l. ISDN consumption between the start and the end, was most pronounced in the group with the largest sICAM-1 decrease (fourth quartile of distribution; p=0.038). In conclusion, the reduction in the pro-inflammatory marker sICAM-1 after 1-year daily treatment with molsidomine may indicate that this NO donor besides its anti-anginal function, promotes a less activated state of the endothelium in patients with stable angina.

Angina Pectoris↗
PubMed2005

RNA damage in human atherosclerosis: pathophysiological significance and implications for gene expression studies.

RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. Here we summarize recent evidence that loss of RNA integrity and 7,8-dihydro-8-oxo-2'-guanosine (8-oxoG) oxidative RNA modifications frequently occur in advanced human atherosclerotic plaques. At least two features of advanced human plaques, namely oxidative stress and intraplaque hemorrhage followed by iron deposition, may be involved in the process of RNA degradation. Although speculative, RNA damage may lead to destabilization and rupture of atherosclerotic plaques by interfering with protein synthesis and stimulation of cell death. Moreover, RNA damage may affect in vitro transcript quantification, thereby influencing data from gene expression studies.

Animals↗
PubMed2005

Amino acid deprivation induces both apoptosis and autophagy in murine C2C12 muscle cells.

Apoptosis and autophagy are closely interconnected types of programmed cell death. In the present study, mouse C2C12 muscle cells were starved in Earle's Balanced Salt Solution or treated with TNF-alpha and cycloheximide to induce autophagy and apoptosis, respectively. The majority of starved C2C12 cells underwent autophagy, as shown by LC3 processing, formation of autophagic vesicles and bulk degradation of long-lived proteins. However, some cells showed features of apoptosis including caspase-3 cleavage, chromatin condensation, DNA fragmentation and annexin V labeling. Caspase-3 cleavage was also induced in culture medium without serum, suggesting that serum withdrawal rather than amino acid deprivation triggered apoptosis. Starvation eliminated multiple pro-apoptotic proteins, but upregulated caspase-8, and rendered starved C2C12 cells much more susceptible to TNF-alpha/cycloheximide-induced apoptosis than non-starved cells. Our data suggest that amino acid deprivation of C2C12 cells induces a complex form of cell death with hallmarks of both apoptosis and autophagy.

Amino Acids↗
PubMed2005

Gastrointestinal function regulation by nitrergic efferent nerves.

Gastrointestinal (GI) smooth muscle responses to stimulation of the nonadrenergic noncholinergic inhibitory nerves have been suggested to be mediated by polypeptides, ATP, or another unidentified neurotransmitter. The discovery of nitric-oxide (NO) synthase inhibitors greatly contributed to our understanding of mechanisms involved in these responses, leading to the novel hypothesis that NO, an inorganic, gaseous molecule, acts as an inhibitory neurotransmitter. The nerves whose transmitter function depends on the NO release are called "nitrergic", and such nerves are recognized to play major roles in the control of smooth muscle tone and motility and of fluid secretion in the GI tract. Endothelium-derived relaxing factor, discovered by Furchgott and Zawadzki, has been identified to be NO that is biosynthesized from l-arginine by the constitutive NO synthase in endothelial cells and neurons. NO as a mediator or transmitter activates soluble guanylyl cyclase and produces cyclic GMP in smooth muscle cells, resulting in relaxation of the vasculature. On the other hand, NO-induced GI smooth muscle relaxation is mediated, not only by cyclic GMP directly or indirectly via hyperpolarization, but also by cyclic GMP-independent mechanisms. Numerous cotransmitters and cross talk of autonomic efferent nerves make the neural control of GI functions complicated. However, the findingsrelated to the nitrergic innervation may provide us a new way of understanding GI tract physiology and pathophysiology and might result in the development of new therapies of GI diseases. This review article covers the discovery of nitrergic nerves, their functional roles, and pathological implications in the GI tract.

Aging↗
PubMed2004

7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling.

OBJECTIVE: 7-Ketocholesterol, a major oxysterol in oxidized low-density lipoproteins in advanced atherosclerotic plaques, induces vascular smooth muscle cell (SMC) death. We investigated whether cytochrome c release participated in SMC death induced by 7-ketocholesterol and whether the processes were reversible. METHODS: SMC cultures derived from the rabbit aorta were exposed to 25 microM 7-ketocholesterol. Cytochrome c and Bax were studied by means of immunofluorescence and immunoblotting, apoptosis by the TUNEL technique and mitochondrial structure by transmission electron microscopy. RESULTS: 7-Ketocholesterol induced rapid upregulation of the proapoptotic protein Bax and its translocation from cytosol into the mitochondria (4 h). This was followed by mitochondrial cytochrome c release (65% at 8 h) into the cytosol, which was almost complete at 16 h. The mitochondria became spherical and ultracondensed, without showing signs of lysis. They clustered around the nucleus and were wrapped by wide cisternae of the rough endoplasmic reticulum. Cytochrome c release was not blocked by the pan-caspase inhibitor zVAD-fmk, in contrast to DNA fragmentation and SMC loss. Interestingly, upon removal of 7-ketocholesterol after 16 h and re-exposure to serum for 24 h, the mitochondrial cytochrome c content, their transmembrane potential and TUNEL labelling normalised and SMC loss decreased. However, none of these cell death markers was rescued when the SMCs had been exposed to the oxysterol for 24 h. CONCLUSION: The results indicate that cytochrome c release during oxysterol-induced SMC apoptosis is not caspase-dependent and occurs as a result of a reversible mitochondrial conformational change rather than swelling and rupture of the outer membrane. The reversibility of these events suggests that the apoptotic cascade could be arrested before a point of no return.

Animals↗
PubMed2004

7-ketocholesterol induces protein ubiquitination, myelin figure formation, and light chain 3 processing in vascular smooth muscle cells.

OBJECTIVE: Oxysterols such as 7-ketocholesterol (7-KC) are important mediators of cell death in atherosclerosis. Therefore, in vitro studies of human smooth muscle cell (SMC) death in response to 7-KC were undertaken to investigate the potential mechanisms. METHODS AND RESULTS: Human aortic SMCs treated with 7-KC showed enhanced immunoreactivity for the oxidative stress marker 4-hydroxy-2-nonenal and upregulated several stress genes (70-kDa heat shock protein 1, heme oxygenase 1, and growth arrest and DNA damage-inducible protein 153) at the mRNA but not at the protein level. 7-KC-treated SMCs rapidly underwent cell death as determined by neutral red, counting of adherent cells, and depolarization of the mitochondrial inner membrane. Cell death was associated with upregulation of ubiquitin mRNA and ubiquitination of cellular proteins. Inhibition of the proteasome by lactacystin potentiated considerably the toxicity of 7-KC. Transmission electron microscopy revealed formation of myelin figures, extensive vacuolization, and depletion of organelles. Formation of autophagosomes was suggested by labeling cells with LysoTracker and monitoring processing of microtubule-associated protein 1 light chain 3 (LC3). Analogous to our in vitro studies, human atherosclerotic plaques showed signs of ubiquitination in SMCs. CONCLUSIONS: 7-KC activates the ubiquitin-proteasome system and induces a complex mode of cell death associated with myelin figure formation and processing of LC3 evocating autophagic processes.

Aorta↗