The apoptotic regulatory protein ARC (apoptosis repressor with caspase recruitment domain) prevents oxidant stress-mediated cell death by preserving mitochondrial function.

ARC is an apoptotic regulatory protein expressed almost exclusively in myogenic cells. It contains a caspase recruitment domain (CARD) through which it has been shown to block the activation of some initiator caspases. Because ARC also blocks caspase-independent events associated with apoptosis, such as hypoxia-induced cytochrome c release, we examined its role in cell death triggered by exposure to hydrogen peroxide (H(2)O(2)) in the myogenic cell line, H9c2. Cell death in this model was caspase-independent and characterized by dose-dependent reduction in ARC expression accompanied by disruption of the mitochondrial membrane potential (Delta psi(m)) and loss of plasma membrane integrity, typical of necrotic cell death. Ectopic expression of ARC prevented both H(2)O(2)-induced mitochondrial dysfunction and cell death without affecting the stress kinase response, suggesting that ARCs protective effects were downstream of early signaling events and not due to quenching of H(2)O(2). ARC was also effective in blocking H(2)O(2)-induced loss of membrane integrity and/or disruption of Delta psi(m) in two human cell lines in which it is not normally expressed. These results demonstrate that, in addition to its ability to block caspase-dependent and -independent events in apoptosis, ARC also prevents necrosis-like cell death via the preservation of mitochondrial function.

The beta(2)-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G(i)-dependent coupling to phosphatidylinositol 3'-kinase.

Recent studies have shown that chronic beta-adrenergic receptor (beta-AR) stimulation alters cardiac myocyte survival in a receptor subtype-specific manner. We examined the effect of selective beta(1)- and beta(2)-AR subtype stimulation on apoptosis induced by hypoxia or H(2)O(2) in rat neonatal cardiac myocytes. Although neither beta(1)- nor beta(2)-AR stimulation had any significant effect on the basal level of apoptosis, selective beta(2)-AR stimulation protected myocytes from apoptosis. beta(2)-AR stimulation markedly increased mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) activation as well as phosphatidylinositol-3'-kinase (PI-3K) activity and Akt/protein kinase B phosphorylation. beta(1)-AR stimulation also markedly increased MAPK/ERK activation but only minimally activated PI-3K and Akt. Pretreatment with pertussis toxin blocked beta(2)-AR-mediated protection from apoptosis as well as the beta(2)-AR-stimulated changes in MAPK/ERK, PI-3K, and Akt/protein kinase B. The selective PI-3K inhibitor, LY 294002, also blocked beta(2)-AR-mediated protection, whereas inhibition of MAPK/ERK activation at an inhibitor concentration that blocked agonist-induced activation but not the basal level of activation had no effect on beta(2)-AR-mediated protection. These findings demonstrate that beta(2)-ARs activate a PI-3K-dependent, pertussis toxin-sensitive signaling pathway in cardiac myocytes that is required for protection from apoptosis-inducing stimuli often associated with ischemic stress.

ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells.

Ischemia induces apoptosis as well as necrosis of cardiac myocytes. We recently reported the cloning of a cDNA that encodes an apoptotic inhibitor, ARC, that is expressed predominantly in cardiac and skeletal muscle. In the present study, we examined the ability of ARC to protect rat embryonic heart-derived H9c2 cells from apoptosis induced by hypoxia, a component of ischemia. We found that H9c2 cells express ARC and that exposure to hypoxia substantially reduces ARC expression while inducing apoptosis. Transfected H9c2 cells in which cytosolic ARC protein levels remain elevated during hypoxia were significantly more resistant to hypoxia-induced apoptosis than parental H9c2 cells or H9c2 cells transfected with a control vector. Loss of endogenous ARC in the cytosol of H9c2 cells was associated with translocation of ARC from the cytosol to intracellular membranes, release of cytochrome c from the mitochondria, activation of caspase-3, poly(ADP-ribose)polymerase (PARP) cleavage, and DNA fragmentation. All of these events were inhibited in H9c2 cells overexpressing ARC when compared with control cells. In contrast, caspase inhibitors prevented PARP cleavage but not cytochrome c release, suggesting that exogenously expressed ARC acts upstream of caspase activation in this model of apoptosis. These results demonstrate that ARC can protect heart myogenic H9c2 cells from hypoxia-induced apoptosis, and that ARC prevents cytochrome c release by acting upstream of caspase activation, perhaps at the mitochondrial level.

Age-related changes in the signaling and function of vascular smooth muscle cells.

Aging is an independent risk factor for the development of atherosclerosis, a vascular abnormality that plays a significant role in the development of many cardiovascular disorders. Animal experiments have demonstrated that aging predisposes the vasculature to advanced atherosclerotic disease and vessel injury and that this predisposition is a function of age-associated changes in the vessel wall itself. Because vascular smooth muscle cells play important roles in the pathogenesis of many vascular disorders, identifying age-associated differences in the way these cells respond to extracellular clues has been an area of active research. Currently, the most remarkable differences in intracellular signaling between vascular smooth muscle cells isolated from young and old animals are related to the control of cell migration through the CamKII pathways and the accelerated transition of older vascular smooth muscle cells from the contractile to the synthetic phenotype. These differences may be due to alternative signaling pathways revealed by the inability of older cells to respond to inhibitors, such as transforming growth factor (TGF)-beta1, or to altered interactions with the extracellular matrix resulting from age-associated shifts in integrin expression or changes in the matrix composition of blood vessels. The exact role that these alterations have in explaining age-associated differences in the response of the vessel wall to injury and its increased susceptibility to developing advanced atherosclerotic lesions remains to be determined but will be guided by studies on intracellular signaling mechanisms.

Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways.

Platelet-derived growth factor BB (PDGF BB) activation of the mitogen-activated protein kinases (MAPK), ERK1 and ERK2, has been shown to be necessary for mitogen-stimulated proliferation, but its role in regulating cell migration and its relationship to other chemotactic signaling events, such as CamKII activation, has not been defined. Using a modified Boyden chamber apparatus, we tested the effects of a selective inhibitor of the upstream activator of ERK1/2, MEK1, on PDGF-stimulated rat aortic vascular smooth muscle cells (VSMCs) alone and in combination with KN62, a selective inhibitor of CamKII. The MEK1 inhibitor, PD98059, caused a dose-dependent reduction in ERK2 activity that paralleled a decrease in migration up to 60%. This inhibition of migration was similar to that seen with KN62 and the combined effects of both inhibitors were non-additive. Although KN62 did not affect ERK2 activity in response to PDGF, PD98059 markedly inhibited PDGF-stimulated CamKII activity, suggesting that activation of CamKII by PDGF was dependent on ERK activity and that the effects of ERK inhibition on migration may be mediated through its ability to inhibit CamKII activity. To directly test this, VSMCs were infected with a recombinant adenovirus expressing constitutively activated CamKII. Infection reversed the inhibitory effects of KN62 on migration, but had no effect on the inhibition of migration seen with PD98059. These results suggest that while MAPK may act upstream of CamKII to control its activation in response to PDGF, it also regulates migration independently of CamKII activation.

p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes.

Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes.

Transfection with c-Ha rasEJ modulates alpha-actin and alpha 1B-adrenoceptor gene expression in vascular smooth muscle cells.

Co-ordinate down-regulation of smooth muscle-specific genes and acquisition of unregulated proliferative characteristics have been proposed as hallmarks of the atherosclerotic process. In the present study, we have evaluated this reciprocal relationship by examining the impact of c-Ha-rasEJ oncogene transfection on alpha-smooth muscle (SM) actin and alpha 1B-adrenoceptor (ADR) gene expression in vascular (aortic) smooth muscle cells (SMCs), c-Ha-rasEJ transfection of SMCs by lipofection (LF-1) was associated with enhanced DNA synthetic rates relative to vector controls and a significant reduction in alpha-SM actin and beta/gamma-actin mRNAs. Incubation of ras- and neo-LF-1 SMCs in a restrictive serum concentration (0.1%) for 72 h inhibited DNA synthesis in both cell types, but differentially influenced the pattern of alpha-actin gene expression. While neo-LF-1 cells incubated in 0.1% exhibited increased alpha-SM actin mRNA levels relative to 10% serum, slight decreases in alpha-SM actin were observed in ras-LF-1 cells under the same conditions. Cyclical stretch of randomly cycling cells, seeded on a flexible elastin substrate at a rate of 100 cycles/min for 72 h, did not significantly influence the pattern of alpha-SM or beta/gamma-actin mRNA expression in neo-LF-1 or ras-LF-1 cells. Steady-state mRNA levels of alpha 1B-ADR were higher in ras-LF-1 SMCs relative to neo-LF-1 cells, and stretch increased alpha 1B-ADR mRNA levels in neo-LF-1, but not ras-LF-1 cells. Stretch inhibited [1H]thymidine incorporation into DNA in both neo- and ras-LF-1 cells relative to unstretched counterparts. These results demonstrate that c-Ha-rasEJ transfection is associated with alterations in the expression of genes associated with muscle-specific functions in vascular SMCs and implicate c-Ha-ras in the regulation of phenotypic expression in SMCs.

Actin isoform and alpha 1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch.

The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and alpha 1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of alpha 1B-adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in alpha 1B-adrenoceptor or beta/gamma-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.

c-Ha-rasEJ transfection of rat aortic smooth muscle cells induces epidermal growth factor responsiveness and characteristics of a malignant phenotype.

Although the role of several protooncogenes, including sis, myc, and myb in the regulation of growth and differentiation of vascular cells has been examined in some detail, limited information is available on the contribution of ras genes to these processes. In the present studies the influence of oncogenic ras transfection on the phenotypic expression of rat aortic smooth muscle cells (SMCs) was examined. Cultured rat aortic SMCs during early passage (P4) were transfected by lipofection with c-Ha-rasEJ in a pSV2 neo vector or with pSV2 neo vector alone. Stable transfectants were selected in G418 over a 6-week period. Oncogene-transfected cells (ras-LF-1) exhibited differences in morphology and growth pattern relative to vector controls (neo-LF-1), or naive SMCs, including the development of prominent processes and the appearance of focal cellular arrangements giving rise to latticelike structures. Southern analysis revealed multiple integration of oncogenic ras in ras LF-1 cells. Transfection of c-Ha-rasEJ was associated with a twofold increase in p21 levels relative to pSV2 vector controls demonstrating that exogenous ras was expressed in these cells. Overexpression of ras p21 afforded SMCs a lower serum requirement for growth compared to vector controls, anchorage independent growth on soft agar, and acquisition of epidermal growth factor (EGF) responsiveness. Stimulation of serum-deprived SMCs with 5% fetal bovine serum (FBS) increased steady-state levels of c-Ha-ras mRNA in both ras-LF-1 and neo-LF-1 but ras induction was more pronounced in ras-transfected cells. alpha-smooth muscle (SM) actin gene expression was markedly reduced in ras-transfected cells relative to vector controls. These results show that transfection of c-Ha-rasEJ into aortic SMCs induces an altered phenotypic state characterized by alterations in growth factor-related signal transduction and tumorigenic potential.

Demonstration of transient bacterobilia by foreign body implantation in feline biliary tract.

The biliary tract of cats is known to be free of autochthonous bacteria above the sphincter of Oddi. In this experiment we investigated whether transient bacterobilia occurs in the biliary system under normal conditions. Polyethylene tubes and human cholesterol stones were implanted surgically into the gallbladder of cats. Sham cholecystostomy was performed as control operation. These cats were euthanized at two, six, and 12 weeks, and the implants were removed, cultured, and studied by scanning electron microscopy (SEM). Cultures and SEM also were undertaken for material scraped from the mucosal surface of the biliary tract from these animals. Colonization of bacteria on the polyethylene tubes and the gallstones was found six and 12 weeks after implantation. Adherent bacterial biofilms were demonstrated on the surfaces of these implants. This experiment showed that transient bacterobilia exists in the feline biliary tract. The foreign body implants have facilitated the adhesion of planktonic bacteria in the bile onto their surfaces and have initiated the formation of adherent biofilms within which these bacteria persisted until the system was sampled.

Peripheral lymphocyte depletion in gold sodium thiomalate-treated rheumatoid arthritis patients.

It has been reported that gold sodium thiomalate treatment of rheumatoid arthritis reduces the number of peripheral blood lymphocytes. In a prospective, double-blind, randomized trial comparing gold sodium thiomalate, auranofin, and placebo in the treatment of rheumatoid arthritis, we investigated absolute lymphocyte counts. We found that peripheral lymphocyte counts decreased, but there was no correlation with clinical change in disease activity.

Isolation of Haemophilus agni from Six Alberta Ram Lambs with Septicemia.

Six ram lambs were submitted to a veterinary diagnostic laboratory for necropsy. Clinical signs included sudden illness or death with or without observed depression, reluctance to move, scours or fever. Gross findings and histopathology revealed evidence of bacterial septicemia. Haemophilus agni was isolated from brain, spleen, lung and lymph node.

Sister-chromatid exchange frequency in lymphocytes of smoking and nonsmoking mothers and their newborn infants.

Sister-chromatid exchange (SCE) frequency in lymphocytes of 8 smoking mothers and their 9 newborns (one subject bearing twins) was compared to 6 mothers who had never smoked and their 6 newborn infants. Mothers in the first group were required to have smoked throughout pregnancy and to have a minimum of 15 pack-years smoking history. Results confirm our earlier smoking effect reported for adults, deny an effect on the newborn, and concur with other studies that show neonates have consistently lower SCE frequencies than adults. Overall, results are consistent with the idea that toxic substances in tobacco smoke interact with chromosomal DNA of circulating human lymphocytes.