Positive TUNEL Research Articles

Glycoxidation of Low‐Density Lipoprotein Increases TUNEL Positivity and CPP32 Activation in Human Coronary Cells

Apoptosis of arterial cells induced by oxidized low-density lipoprotein (oxLDL) is thought to contribute to the progression of vascular dysfunction and atherogenesis. It is well established that diabetes mellitus is accompanied by both glycosylation and oxidation of LDL (glc-oxLDL), but the biological effects of these modified lipoproteins are poorly understood. We demonstrate here for the first time that glc-oxLDL increases TUNEL positivity and caspase-3 activation (by Western blot and immunocytochemistry) of human coronary smooth muscle cells. Overall, these effects induced by glc-oxLDL were greater than those achieved with oxLDL. Thus, glc-oxLDL activated downstream apoptotic signaling. This may influence the evolution of atherogenesis and vascular complications in diabetes.

Cariporide (HOE642), a selective Na+-H+ exchange inhibitor, inhibits the mitochondrial death pathway.

The Na+-H+ exchanger figures prominently in cardiac ischemia-reperfusion injury. Several experimental and clinical studies have demonstrated a cardioprotective effect of Na+-H+ exchanger inhibition; however, the precise mechanisms have not been established. We examined the effects of cariporide (HOE642, 10 micromol/L) on cell death induced by oxidative stress (H2O2, 100 micromol/L) in cultured neonatal rat cardiomyocytes. Cariporide significantly suppressed markers of cell death, such as TUNEL positivity and caspase-3 cleavage, at 8 or 16 hours after H2O2. The early phase of cell death, reported by increases in phosphatidylserine exposure and propidium iodide uptake, was also inhibited by cariporide. To explore the mechanisms of cell protection, we examined the effects of cariporide on increases in intracellular Na+ and Ca2+ induced by oxidative stress. Cariporide remarkably suppressed cytosolic Na+ and Ca2+ accumulation. Next, we investigated the effects of cariporide on mitochondria-associated death process. Mitochondrial Ca2+ overload induced by H2O2 was remarkably suppressed by cariporide. Loss of mitochondrial membrane potential is a critical step of the death pathway; cariporide prevented mitochondrial membrane potential loss induced by H2O2. Cariporide protects cardiomyocytes against oxidant-induced cell death by preserving intracellular ion homeostasis and mitochondrial integrity.

Cell Death in MMTV-c-myc Transgenic Mouse Mammary Tumors May Not Be Typical Apoptosis

Enforced expression of c-myc has been shown to serve as an apoptotic stimulus in cultured cells. Prior studies have also demonstrated that several tissues expressing c-myc transgene display a large number of dead cells, although a morphologic or biochemical verification of apoptosis in these tissues has actually not been presented. In the present study, we examined the morphologic properties of cell death in the mammary tumors developed from MMTV-c-myc transgenic mice. We found that c-myc–expressing mammary tumor cells exhibited malformation of mitochondria, characterized by an amorphous matrix with very few cristae. The mitochondria were also frequently degenerated by lysis of the matrix and cristae. The protein level of cytochrome c was much lower in the areas of c-myc–expressing tumor cells compared with the adjacent tumor foci, which was previously shown to have decreased expression of c-myc, reduced frequencies of cell death, and increased frequencies of proliferating cells. In the c-myc–expressing tumor areas, there were many dying or dead cells organized in clusters, termed “dead cell islands.” These cells exhibited shrinkage, DNA breakage as indicated by a positive TUNEL staining, and nuclear localization of apoptosis-inducing factor, but a lack of typical apoptotic morphology, such as nuclear condensation and formation of cell membrane blebs and apoptotic bodies. Many macrophages infiltrated into these dead cell islands, engulfing the dying or dead tumor cells. In the total tumor tissue, the protein level of caspase-3 was very low, and the poly(ADP)-ribose polymerase was present mainly as the unprocessed, inactive form. Collectively, these results suggest that programmed cell death in the c-myc transgenic mammary tumor tissue may not be typical apoptosis and may involve a caspase-independent mechanism.

The onset of p53-dependent apoptosis plays a role in terminal differentiation of human normoblasts.

The p53 tumor suppressor gene was found to play a role in the differentiation of several tissue types. We report here that p53-dependent apoptosis plays a role in the final stages of physiological differentiation of normoblasts, resulting in nuclear condensation and expulsion without cell death. Blood samples of healthy newborns, cord blood as well as bone marrow, were analysed for apoptosis by TUNEL and p53 expression by immunostaining. While some samples exhibited simultaneously several distinct patterns of apoptosis, such as perinuclear, diffused nuclear or nuclear apoptotic bodies, others presented a single defined pattern. Overexpression of p53 protein was detected in normoblasts exhibiting either perinuclear or diffused nuclear p53, corresponding to the nuclear apoptotic pattern in the same sample. Similar results were also evident with colonies cultivated for 12-14 days in culture. Differentiated erythroid colonies exhibited overexpression of p53 and positive TUNEL staining only in the normoblasts. We further examined the state of caspase 3/7 and observed a decrease of this activated enzyme during erythroid differentiation in culture. This study suggests a novel role for apoptosis in normoblast differentiation where nuclear degradation occurs with a delay in the actual cell death. A pivotal role for the p53-dependent apoptosis in the erythroid lineage development is implied. However, this apoptotic process is not fully executed because of the exhaustion in caspase 3/7 and thus cells are diverted towards final stages of differentiation.

Uncoupling protein-2 overexpression inhibits mitochondrial death pathway in cardiomyocytes.

Uncoupling proteins (UCPs) are located in the mitochondrial inner membrane and partially dissipate the transmembrane proton electrochemical gradient. UCP2 is expressed in various human and rodent tissues, including the heart, where its functional role is unknown. In the present study, we tested the hypothesis that UCP2 overexpression could protect cardiomyocytes from oxidative stress-induced cell death by reducing reactive oxygen species (ROS) production in mitochondria. Using an adenoviral vector containing human UCP2, we investigated the effects of UCP2 overexpression on the mitochondrial death pathway induced by oxidative stress (100 micromol/L H2O2) in cultured neonatal cardiomyocytes. UCP2 overexpression significantly suppressed markers of cell death, including TUNEL positivity, phosphatidylserine exposure, propidium iodide uptake, and caspase-3 cleavage. Furthermore, UCP2 remarkably prevented the catastrophic loss of mitochondrial inner membrane potential induced by H2O2, which is a critical early event in cell death. Ca2+ overload and the production of ROS in mitochondria, both of which contribute to mitochondrial inner membrane potential loss, were dramatically attenuated by UCP2 overexpression. Thus, overexpression of UCP2 attenuates ROS generation and prevents mitochondrial Ca2+ overload, revealing a novel mechanism of cardioprotection.

Apoptosis and in vitro development of preimplantation porcine embryos derived in vitro or by nuclear transfer.

Apoptosis occurs during preimplantation development in both in vivo- and in vitro-produced embryos, and it may contribute to embryonic loss. The present study investigated the development of porcine nuclear transfer (NT) embryos reconstructed by using fetal fibroblasts as compared to embryos produced by in vitro fertilization (IVF). The onset and the frequency of apoptosis in NT and IVF embryos were examined via morphological and nuclear changes and TUNEL assay. The NT blastocysts had a similar number of nuclei as compared to IVF blastocysts and appeared to be morphologically similar. Relative to IVF embryos, the NT embryos had a lower cleavage rate (42.7% vs. 71.0%) and a lower developmental rate (11.1% vs. 28.6%) to the blastocyst stage. The earliest positive TUNEL signals were detected in the NT embryos on Day 5 of culture. The percentage of cells undergoing apoptosis in the NT embryos was higher than that of the IVF embryos and increased with time in vitro. Some of the abnormal morphological changes observed during early development related to apoptosis. Cytoplasmic fragmentation, developmental arrest, and nuclear condensation were typical characteristics of embryos undergoing apoptosis. Some mechanisms of the apoptotic pathway were triggered by changes in the NT embryos. The developmental rates of NT embryos might be improved by identifying specific apoptotic pathways and then intervening in these pathways to improve development.

Increased islet apoptosis in Pdx1+/- mice.

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.

Increased islet apoptosis in Pdx1+/– mice

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1+/– mice compared with Pdx1+/+ littermate controls. Glucose sensing and islet Ca2+ responses were also normal. Depolarization-evoked exocytosis and Ca2+ currents in single Pdx1+/– cells were not different from controls, arguing against a ubiquitous β cell stimulus-secretion coupling defect. However, isolated Pdx1+/– islets and dispersed β cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1+/+ islets. BclXL and Bcl-2 expression were reduced in Pdx1+/– islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both β cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and β cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.

Wnt in liver development: Role in biliary cell survival and proliferation

Wnt/13-catenni pathway plays a sigmficant role during development and carcinogenesis. We have earlier detected presence of Wnt-1 as early as in embryonic day 10 livers (El0). We have also reported effect of 13-catenin inhibition on liver growth and lineage specification during liver development. In the present study we investigate direct effect of Wnt on liver development utilizing embryonic liver cultures and Wnt conditioned media. (Wnt-3a). El0 livers were isolated and cultured in an organ culture system. Livers were cultured in conditioned medium from control L929 cells or Win-conditioned media (L cells transfected with Wnt-3a) alone or in presence of Hepatocyte growth factor (HGF). At least 3 livers were cultured for each growth condition. Embryonic liver cultures grown in DMEM supplemented with 10% fetal calf serum (FCS) were utilized as positive controls. After 72 hours all organ cultures were harvested, llxed and processed and finally analyzed for histology, proliferation, apoptosis and lineage. Negative conditioned media grown embryonic liver cultures demonstrated loss of architecture, enhanced apoptosia and deficit proliferation. There were almost no viable cells observed in these cultures. In the presence of Wnt-conditioned media, embryonic liver cultures showed several islands of survived cells and demonstrated a ductular morphology. These cells displayed proliferation as seen by PCNA and Ki-67 posinvity. Also this growth condition displayed an overall decrease in TUNEL positivity especially in the ductular structures. These cells were strongly positive for CK-19 and negative for cr ein and c-kit. Some CK-19 positive cells were also positive for albumin. Embryonic livers cultured in presence of Wnt and HGF show phenotype resembling the positive control (cultures grown in 10% FCS). These cultures exhibited normal cellular architecture with active proliferation and decreased apoptosis and displaying stem cells, hepatocytes (immature & mature) and primitive bile ducts. We conclude that Wnt plays an important role in bihary commitment of the bipotential stem cells by providing them a survival advantage. It also induces transdifferentiation of mature hepatocytes into biliary cells as shown by the presence of some hepato-biliary intermediates. HGF and Wnt seem to be sufficient to maintain a normal embryonic liver culture growth and differentiation supporting stem cells, hepatocytes & bile ducts. Our results suggest an existence of a precise balance of these growth factors for normal liver development

Apoptosis of murine cultured biliary epithelial cells induced by glycochenodeoxycholic acid involves Fas receptor and its ligand

Fas-mediated apoptosis of biliary epithelial cells (BECs) is suggested as a main effector process in immune-mediated biliary diseases. Glycochenodeoxycholic acid (GCDCA), a conjugated hydrophobic bile acid, is known to cause apoptosis of hepatocytes via the direct activation of Fas receptor (FasR). In this study, we investigated the apoptotic effect of GCDCA on cultured murine intrahepatic BECs. It was found that GCDCA induced apoptosis of BECs derived from BALB/c mice in a dose- and incubation time-dependent manner. The morphology and TUNEL positivity for the apoptotic process induced by GCDCA were similar to those induced by beauvericin (BV) which is known to cause apoptosis by a direct activation of caspase-3, a member of the caspase family, at its downstream. The GCDCA-induced apoptosis of BECs was accompanied by an up-regulation of FasR, Fas ligand (FasL), and also caspase-3 expression. GCDCA did not induce the apoptosis of BECs derived from C3H · MRL-Fas lpr mice possessing a non-functioning FasR, supporting that the GCDCA-induced apoptosis of BECs in BALB/c involves the Fas system. Furthermore, GCDCA induced an increase in interleukin-18 (IL-18) mRNA in BECs and secretion of activated IL-18 from BECs of BALB/c, which might have led to an up-regulation of FasL mRNA and protein expression in BECs followed by FasR/FasL interaction. These results suggest that GCDCA induces apoptosis of BECs through FasR/FasL interaction via an autocrine/paracrine effect, IL-18 is responsible for the expression of FasL in BEC, and the up-regulation of caspase-3 expression is involved in this model. This model could be useful for molecular and genetic studies of Fas-mediated apoptosis of BECs.

Role of Caspase-8 Activation in Mediating Vitamin E-Induced Apoptosis in Murine Mammary Cancer Cells

The vitamin E family of compounds is divided into two subgroups, tocopherols and tocotrienols. However, tocotrienols display more potent apoptotic activity in mammary cancer cells. Although the mechanism(s) mediating tocotrienol-induced apoptosis is presently unknown, apoptosis is carried out by activation of initiator caspases (caspase-8 or -9) that subsequently activate effector caspases (caspase-3, -6, or -7). Studies were conducted to determine whether tocotrienol-induced apoptosis is mediated by activation of the caspase-8 and/or caspase-9 pathway. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained on serum-free media. Treatment with tocotrienol-rich-fraction of palm oil (TRF) and γ-tocotrienol, but not α-tocopherol, induced a dose-dependent decrease in +SA cell viability. TRF- and γ-tocotrienol-induced cell death resulted from apoptosis, as determined by DNA fragmentation and positive TUNEL assay staining. Additional studies showed that treatment with 50 μM TRF or 20 μM γ-tocotrienol increased intracellular activity and levels of processed caspase-8 and -3 but not caspase-9. Furthermore, treatment with specific caspase-8 or -3 inhibitors, but not caspase-9 inhibitor, completely blocked the tocotrienol-induced apoptosis in +SA cells. These findings demonstrate that tocotrienol-induced apoptosis in +SA mammary cancer cells is mediated through activation of the caspase-8 signaling pathway and is independent of caspase-9 activation.

Time-course of programmed cell death during leaf senescence in Eucommia ulmoides.

Leaves of Eucommia ulmoidesOliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca(2+)-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves.

Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells

Although the influence of selective cyclooxygenase (COX)-2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX-2 inhibitors. Celecoxib strongly suppressed the proliferation of COX-2 expressing HT-29 cells at 10–40 μM. NS-398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT-29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase-3/7 activation. DNA fragmentation was also increasd in COX-2 non-expressing cell lines (SW-480 and HCT-116) by exposure to celecoxib for 6–24 h. All six COX-2 inhibitors suppressed the production of prostaglandin E 2 by HT-29 cells, suggesting that the pro-apoptotic effect of celecoxib was unrelated to inhibition of COX-2. Inactivation of Akt might explain the differential pro-apoptotic effect of these selective COX-2 inhibitors on colon adenocarcinoma cells.

Antiapoptotic effect of nicorandil mediated by mitochondrial atp-sensitive potassium channels in cultured cardiac myocytes

ObjectivesWe examined whether nicorandil, a clinically useful drug for the treatment of ischemic syndromes, inhibits myocardial apoptosis. BackgroundNicorandil has been reported to have a cardioprotective action through activation of mitochondrial ATP-sensitive potassium (mitoKATP) channels. Based on our recent observation that mitoKATP channel activation has a remarkable antiapoptotic effect in cultured cardiac cells, we hypothesized that the protective effects of nicorandil may be at least partially due to an antiapoptotic effect. MethodsCultured neonatal rat cardiac myocytes were exposed to hydrogen peroxide to induce apoptosis. Effects of nicorandil were evaluated using a number of apoptotic markers. ResultsExposure to 100 μM hydrogen peroxide resulted in apoptotic cell death as shown by TUNEL positivity, cytochrome c translocation, caspase-3 activation and dissipation of mitochondrial inner membrane potential (ΔΨm). Nicorandil (100 μM) suppressed all of these markers of apoptosis. Notably, nicorandil prevented ΔΨm depolarization in a concentration-dependent manner (EC50 ∼ 40 μM, with saturation by 100 μM), as shown by fluorescence-activated cell sorter analysis of cells stained with a fluorescent ΔΨm-indicator, tetramethylrhodamine ethyl ester (TMRE). Time-lapse confocal microscopy of individual cells loaded with TMRE shows that nicorandil suppresses ΔΨm loss. Subcellular calcein localization revealed inhibition of the mitochondrial permeability transition by nicorandil. These protective effects of nicorandil were blocked by the mitoKATP channel antagonist 5-hydroxydecanoate. ConclusionsOur findings identify nicorandil as an inhibitor of apoptosis induced by oxidative stress in cardiac myocytes, and confirm the critical role of mitoKATP channels in inhibiting apoptosis.

Hippocampal neuronal apoptosis in type 1 diabetes

Duration-related cognitive impairment is an increasingly recognized complication of type 1 diabetes. To explore potential underlying mechanisms, we examined hippocampal abnormalities in the spontaneously type 1 diabetic BB/W rat. As a functional assay of cognition, the Morris water maze test showed significantly prolonged latencies in 8-month diabetic rats not present at 2 months of diabetes. These abnormalities were associated with DNA fragmentation, positive TUNEL staining, elevated Bax/Bcl-x L ratio, increased caspase 3 activities and decreased neuronal densities in diabetic hippocampi. These changes were not caused by hypoglycemic episodes or reduced weight in diabetic animals. To explore potential mechanisms responsible for the apoptosis, we examined the expression of the IGF system. Western blotting and in situ hybridization revealed significant reductions in the expression of IGF-I, IGF-II, IGF-IR and IR preceding (2 months) and accompanying (8 months) the functional cognitive impairments and the apoptotic neuronal loss in hippocampus. These data suggest that a duration-related apoptosis-induced neuronal loss occurs in type 1 diabetes associated with cognitive impairment. The data also suggest that this is at least in part related to impaired insulin and/or IGF activities.

Postischemic reperfusion is the prerequisite for tunel positivity

Postischemic reperfusion is the prerequisite for tunel positivity

Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis.

Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.

Primary sensory neurons and satellite cells after peripheral axotomy in the adult rat: timecourse of cell death and elimination.

The timecourse of cell death in adult dorsal root ganglia after peripheral axotomy has not been fully characterised. It is not clear whether neuronal death begins within 1 week of axotomy or continues beyond 2 months after axotomy. Similarly, neither the timecourse of satellite cell death in the adult, nor the effect of nerve repair has been described. L4 and L5 dorsal root ganglia were harvested at 1-14 days, 1-6 months after sciatic nerve division in the adult rat, in accordance with the Animals (Scientific Procedures) Act 1986. In separate groups the nerve was repaired either immediately or following a 1-week delay, and the ganglia were harvested 2 weeks after the initial transection. Microwave permeabilisation and triple staining enabled combined TUNEL staining, morphological examination and neuron counting by the stereological optical dissector technique. TUNEL-positive neurons, exhibiting a range of morphologies, were seen at all timepoints (peak 25 cells/group 2 weeks after axotomy) in axotomised ganglia only. TUNEL-positive satellite cell numbers peaked 2 months after axotomy and were more numerous in axotomised than control ganglia. L4 control ganglia contained 13,983 (SD 568) neurons and L5, 16,285 (SD 1,313). Neuron loss was greater in L5 than L4 axotomised ganglia, began at 1 week (15%, P=0.045) post-axotomy, reached 35% at 2 months ( P<0.001) and was not significantly greater at 4 months or 6 months. Volume of axotomised ganglia fell to 19% of control by 6 months ( P<0.001). In animals that underwent nerve repair, both the number of TUNEL-positive neurons and neuron loss were reduced. Immediate repair was more protective than repair after a 1-week delay. Thus TUNEL positivity precedes actual neuron loss, reflecting the time taken to complete cell death and elimination. Neuronal death begins within 1 day of peripheral axotomy, the majority occurs within the first 2 months, and limited death is still occurring at 6 months. Neuronal death is modulated by peripheral nerve repair and by its timing after axotomy. Secondary satellite cell death also occurs, peaking 2 months after axotomy. These results provide a logical framework for future research into neuronal and satellite cell death within the dorsal root ganglia and provide further insight into the process of axotomy induced neuronal death.

Mutated p21/WAF/CIP transgene overexpression reduces smooth muscle cell proliferation, macrophage deposition, oxidation-sensitive mechanisms, and restenosis in hypercholesterolemic apolipoprotein E knockout mice.

We have investigated whether by introducing a mutated p21 cyclin-dependent kinase inhibitor through a standard type 5 adenovirus (Ad), it would be possible to interfere with restenosis in hypercholesterolemic apolipoprotein E knockout mice. Restenosis is a clinically relevant, undesired effect of percutaneous transluminal coronary angioplasty (PTCA). A critical event underlying restenosis is smooth muscle cell (SMC) proliferation leading to neointimal formation and vessel reocclusion. Recent data demonstrated that it is possible to reduce restenosis by introducing various genes blocking the cell cycle through Ad vectors. Nonetheless, most experiments were conducted in the healthy carotid artery of rat, which is far from the condition of human disease. Therefore, we investigated whether antiproliferative or proapoptotic genes affect restenosis in a model of atherosclerosis closer to clinical settings. Ad-mutated(m)-p21WAF/CIP1 transgene overexpression induces a significant reduction of restenosis in hypercholesterolemic apolipoprotein E knockout mice subjected to injury of common carotid artery. This was associated with reduced SMC density and proliferation, macrophage deposition, and oxidation-sensitive mechanisms. Treatment with p21/WAF also enhanced TUNEL positivity of arterial cells. We show that in an experimental model of atherosclerosis, braking the cell proliferation through increased vascular apoptosis and reduced oxidation-sensitive signal transduction and macrophage accumulation can significantly ameliorate the deleterious effects of vascular injuries similar to those that occur during PTCA and related procedures.

Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells.

Mitochondria can either enhance or suppress cell death. Cytochrome c release from mitochondria and depolarization of the mitochondrial membrane potential (DeltaPsi) are crucial events in triggering apoptosis. In contrast, activation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels prevents lethal ischemic injury in vivo, implicating these channels as key players in the process of ischemic preconditioning. We probed the relationship between mitoK(ATP) channels and apoptosis in cultured neonatal rat cardiac ventricular myocytes. Incubation with 200 micromol/L hydrogen peroxide induced TUNEL positivity, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and dissipation of DeltaPsi. Pharmacological opening of mitoK(ATP) channels by diazoxide (100 micromol/L) preserved mitochondrial integrity and suppressed all markers of apoptosis. Diazoxide prevented DeltaPsi depolarization in a concentration-dependent manner (EC(50) approximately 40 micromol/L, with saturation by 100 micromol/L), as shown by both flow cytometry and quantitative image analysis of cells stained with fluorescent DeltaPsi indicators. These cytoprotective effects of diazoxide were reproduced by pinacidil, another mitoK(ATP) agonist, and blocked by the mitoK(ATP) channel antagonist 5-hydroxydecanoate (500 micromol/L). Our findings identify a novel mitochondrial pathway that is protective against apoptosis. The results also pinpoint mitoK(ATP) channels as logical therapeutic targets in diseases of enhanced apoptosis and oxidative stress.