TdT-mediated dUTP-X Nick End Labeling Research Articles

HIF-2α regulates CD44 to promote cancer stem cell activation in triple-negative breast cancer via PI3K/AKT/mTOR signaling.

Breast cancer is a common malignant tumor that seriously threatens women's health. Breast cancer stem cell (CSC)-like cell population may be the main factor for breast cancer metastasis. Therefore, targeted therapy for CSCs has great potential significance. Hypoxia-inducible factor is a transcription factor widely expressed in tumors. Studies have shown that down-regulation of the hypoxia signaling pathway inhibits tumor stem cell self-renewal and increases the sensitivity of stem cells to radiotherapy and chemotherapy mediated by hypoxia-inducible factor-2α (HIF-2α). However, the specific mechanism remains unclear and further research is necessary. To investigate the effect of HIF-2α down-regulation on stem cell markers, microsphere formation, and apoptosis in breast cancer cell line MDA-MB-231 under hypoxia and its possible mechanism. Immunohistochemistry was used to detect the expression of HIF-2α and CD44 in triple-negative breast cancer (TNBC) and non-TNBC tissues. Double-labeling immunofluorescence was applied to detect the co-expression of HIF-2α and CD44 in MDA-MB-231 cells and MCF-7 cells. HIF-2α was silenced by RNA interference, and the expression of CD44 and transfection efficiency were detected by real-time fluorescent quantitative PCR. Further, flow cytometry, TdT-mediated X-dUTP nick end labeling, and mammosphere formation assays were used to evaluate the effect of HIF-2α on CSCs and apoptosis. The possible mechanisms were analyzed by Western blot. The results of immunohistochemistry showed that HIF-2α was highly expressed in both TNBC and non-TNBC, while the expression of CD44 in different molecular types of breast cancer cells was different. In in vitro experiments, it was found that HIF-2α and CD44 were expressed almost in the same cell. Compared with hypoxia + negative-sequence control, HIF-2α small interfering ribonucleic acid transfection can lower the expression of HIF-2α and CD44 mRNA(P < 0.05), increase the percentage of apoptotic cells (P < 0.05), and resulted in a reduction of CD44+/CD24- population (P < 0.05) and mammosphere formation (P < 0.05) in hypoxic MDA-MB-231 cells. Western blot analysis revealed that phosphorylated protein-serine-threonine kinase (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR) levels in MDA-MB-231 decreased significantly after HIF-2α silencing (P < 0.05). Down-regulation of HIF-2α expression can inhibit the stemness of human breast cancer MDA-MB-231 cells and promote apoptosis, and its mechanism may be related to the CD44/phosphoinosmde-3-kinase/AKT/mTOR signaling pathway.

JLX001 Ameliorates Ischemia/Reperfusion Injury by Reducing Neuronal Apoptosis via Down-Regulating JNK Signaling Pathway

JLX001, a novel compound with similar structure with cyclovirobuxine D (CVB-D), has been proved to exert therapeutical effects on permanent focal cerebral ischemia. However, the protective effects of JLX001 on cerebral ischemia/reperfusion (I/R) injury and its anti-apoptotic effects have not been reported. We investigated the efficacy of JLX001 in two pharmacodynamic tests (pre-treatment test and post-treatment) with rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). The pharmacodynamic tests demonstrated that JLX001 ameliorated I/R injury by reducing infarct sizes and brain edema. The results of Morris water maze, neurological scores, cylinder test and posture reflex test implied that JLX001 improved the learning, memory and motor ability after MCAO/R in the long term. Anti-apoptotic effects of JLX001 and its regulation of cytosolic c-Jun N-terminal Kinases (JNKs) signal pathway were confirmed in vivo by co-immunofluorescence staining and western immunoblotting. Furthermore, primary cortical neuron cultures were prepared and exposed to oxygen glucose deprivation/reoxygenation (OGD/R) for in vitro studies. Cytotoxicity test and mitochondrial membrane potential (MMP) test showed that JLX001 enhanced cell survival rate and maintained MMP. Flow cytometry and TdT-mediated dUTP-X nick end labeling (TUNEL) staining demonstrated the anti-apoptotic effects of JLX001 in vitro. Likewise, JLX001 regulated JNK signal pathway in vivo, which was also confirmed by western immunoblotting. Collectively, this study presents the first evidence that JLX001 exerted protective effects against I/R injury by reducing neuronal apoptosis via down-regulating JNK signaling pathway.

Lutein modulates transcription dysregulation of adhesion molecules and spermatogenesis transcription factors induced by testicular ischemia reperfusion injury: it could be SAFE.

Testicular ischemia reperfusion injury (tIRI) is considered as the underlying mechanism of testicular torsion, which can cause male infertility. tIRI-induced damage was investigated by assessing the gene expression of spermatogenesis master transcription factors (TFs) and that of major adhesion molecules of the blood-testis barrier. The effect of lutein, a hydroxyl carotenoid, in alleviating tIRI-induced damage was also studied. Male Sprague-Dawley rats were divided into three groups: sham, unilateral tIRI, and tIRI + lutein (0.2 mg/kg). tIRI was induced by occlusion of the testicular artery for 1 h, followed by 4 h of reperfusion. Lutein was injected 15 min after the start of ischemia. Histological analysis and real-time polymerase chain reaction revealed significant decreases in tissue biopsy scores, reduced seminiferous tubule diameters, and downregulated the mRNA expression of the TFs cAMP-responsive element modulator (CREM), TATA box-binding protein-related factor 2 (TRF2), and regulatory factor X 2 (RFX2) compared with the sham group. Lutein treatment reversed these effects. The mRNA expression of the adhesion molecules N-cadherin, nectin-2, claudin-11, occludin, and connexin-43 was significantly downregulated during tIRI, but this change was prevented by lutein treatment. In addition, lutein normalized the tIRI-induced increase in total antioxidant capacity, increased malondialdehyde (MDA) levels, augmented number of TdT-mediated dUTP-X nick-end labeling (TUNEL)-positive nuclei, and activated caspase-8 pathway. The components of survivor activating factor enhancement (SAFE) were also activated during tIRI. Increased tissue expression of TNF-α and its receptor, TNFR1, was accompanied by increased phosphorylation of Janus kinase (JAK) and STAT3, which was prevented by lutein treatment. Our findings suggested that tIRI-induced spermatogenic damage may involve modulation of the SAFE pathway and could benefit from lutein treatment.

Methane-rich saline attenuates ischemia/reperfusion injury of abdominal skin flaps in rats via regulating apoptosis level.

BackgroundIn plastic surgery, skin damage induced by ischemia/reperfusion (I/R) is a multifactorial process that often occurs. Methane gas has been reported to be a new therapeutic gas for attenuating I/R injury. In this study, we assessed the effects of methane-rich saline (MRS) in regulating apoptosis on skin flap I/R injury.MethodsMale Sprague–Dawley rats, 6–8 weeks old, were divided randomly into three groups: one sham surgery group (SH) and two surgery groups. After undergoing 6 h of I/R management of an abdominal skin flap, surgery groups were treated with physiological saline (I/R-P) or methane-rich saline (I/R-M). On the 3rd postoperative day, a laser Doppler flowmeter was used to measure flap blood supply, and hematoxylin and eosin (H&E) staining was used to observe morphological changes. TdT-mediated dUTP-X nick end labeling (TUNEL) staining was also used to observe early apoptosis and is presented as the percentage of TUNEL-positive cells. Moreover, pASK-1, pJNK, Bcl-2 and Bax were detected by immunohistochemical technology. Caspase-3 activity was also measured to evaluate the effects of MRS.ResultsCompared to the I/R-P group, the flaps in the I/R-M group presented a larger survival area and better blood perfusion with less inflammatory infiltration and cell apoptosis, a higher expression of Bcl-2, a lower expression of pASK-1, pJNK and Bax, and a lower caspase-3 activity.ConclusionAccording to the results, MRS attenuated I/R injury by regulating apoptosis and has the potential to be applied as a new therapy for improving skin flap survival.

Effects of Garlic Fractions Consumption on Male Reproductive Functions

Many researchers have focused on the preventive and curative effects of garlic (Allium sativum), particularly on cardiovascular diseases and cancer. However, its impacts on the male reproductive system have not been clearly defined. In this study, the effect of chronic consumption of two garlic fractions was tested: one soluble in water (aqueous solution obtained by grinding and centrifugation) and the other one precipitated by ethanol (alcoholic precipitate obtained by precipitation of the aqueous solution), on different variables of male rats' reproductive functions. These two fractions were targeted to try to identify the nature of the active garlic compounds responsible for the different modifications observed on testicular parameters. The observation of seminiferous tubules of rats treated with garlic fractions showed an increased number of tubules deprived of spermatozoa. In addition, garlic fractions induced apoptosis of testicular germ cells (TdT-mediated dUTP-X nick-end labeling [TUNEL] approach) and a decrease of serum testosterone levels and seminiferous tubule DNA concentrations. In summary, our histological and molecular results suggest that one or several substances, soluble in water and precipitated by alcohol, impaired spermatogenesis.

Characterization of a critical role for CFTR chloride channels in cardioprotection against ischemia/reperfusion injury

To further characterize the functional role of cystic fibrosis transmembrane conductance regulator (CFTR) in early and late (second window) ischemic preconditioning (IPC)- and postconditioning (POC)-mediated cardioprotection against ischemia/reperfusion (I/R) injury. CFTR knockout (CFTR(-/-)) mice and age- and gender-matched wild-type (CFTR(+/+)) and heterozygous (CFTR(+/-)) mice were used. In in vivo studies, the animals were subjected to a 30-min coronary occlusion followed by a 40-min reperfusion. In ex vivo (isolate heart) studies, a 45-min global ischemia was applied. To evaluate apoptosis, the level of activated caspase 3 and TdT-mediated dUTP-X nick end labeling (TUNEL) were examined. In the in vivo I/R models, early IPC significantly reduced the myocardial infarct size in wild-type (CFTR(+/+)) (from 40.4% ± 5.3% to 10.4% ± 2.0%, n=8, P<0.001) and heterozygous (CFTR(+/-)) littermates (from 39.4% ± 2.4% to 15.4% ± 5.1%, n=6, P<0.001) but failed to protect CFTR knockout (CFTR(-/-)) mice from I/R induced myocardial infarction (46.9% ± 6.2% vs 55.5% ± 7.8%, n=6, P>0.5). Similar results were observed in the in vivo late IPC experiments. Furthermore, in both in vivo and ex vivo I/R models, POC significantly reduced myocardial infarction in wild-type mice, but not in CFTR knockout mice. In ex vivo I/R models, targeted inactivation of CFTR gene abolished the protective effects of IPC against I/R-induced apoptosis. These results provide compelling evidence for a critical role for CFTR Cl(-) channels in IPC- and POC-mediated cardioprotection against I/R-induced myocardial injury.

Study of Neuronal Apoptosis and Changes in Expression of the Proteins Related to the Mitochondrial Pathway after Aβ1–42 Injection in the Rat Hippocampus

The objective was to study neuronal apoptosis and changes in the proteins related to the mitochondrial pathway in the hippocampus of a rat model of Alzheimer’s disease (AD) and discuss its significance in AD. Thirty-six Sprague-Dawley rats were divided into 3 groups: a normal group, a control group and an AD group. AD was established by injecting β-amyloid_1–42 (Aβ_1–42) into the hippocampus. Learning and memory ability was estimated with a Y maze at different times. The apoptotic neurons were detected by the TdT-mediated X-dUTP nick end labeling (TUNEL) method. Cytochrome C and caspase-9 protein expression in the hippocampus was detected by Western blot. The results showed that there was obvious impairment of learning and memory in rats of the AD group after Aβ_1–42injection. The apoptotic neurons in the hippocampus of the rats in the AD group were significantly increased compared with the normal group and the control group (p < 0.01), and the expression of cytochrome C and caspase-9 in hippocampus in AD group was significantly increased too (p < 0.01). The results suggest that the mitochondrial pathway is one of the important neuronic apoptotic pathways induced by Aβ_1–42in the hippocampus_,and may play an important role in the pathogenesis of AD.

Histological changes after single high-dose irradiation for squamous cell carcinoma arising from a burn scar

Single high-dose irradiation has become a safe and encouraging form of radiation therapy but variations in the histological changes after irradiation have not been fully understood in clinical settings. The aim of this study was to investigate the histological changes of tumor cells after single high-dose, direct irradiation with a case of cutaneous squamous cell carcinoma that developed on a burn scar. The patient received irradiation followed by radical resection of the tumor. Tissues before and after irradiation were stained with Hematoxylin and Eosin (H&E), TdT-mediated dUTP-X nick end labeling (TUNEL), and by immunohistochemistry for MIB-1, p53, and p21. Massive necrosis was noted after irradiation but a few viable tumor cells remained. Preradiation histology revealed MIB-1 and p53 as positive, and p21 tumor cells as partially positive. These expressions were reduced after irradiation. The TUNEL stain was consistently negative. It was established that tumor cell death after single high-dose irradiation was caused mainly by necrosis rather than apoptosis in clinical settings.

Parkin Protects Dopaminergic Neurons against Microtubule-depolymerizing Toxins by Attenuating Microtubule-associated Protein Kinase Activation

Mitogen-activated protein kinases, originally known as microtubule-associated protein (MAP) kinases, are activated in response to a variety of stimuli. Here we report that microtubule-depolymerizing agents such as colchicine or nocodazole induced strong activation of MAP kinases including JNK, ERK, and p38. This effect was markedly attenuated by parkin, whose mutations are linked to Parkinson disease (PD). Our previous study has shown that parkin stabilizes microtubules through strong interactions mediated by three independent domains. We found that each of the three microtubule-binding domains of parkin was sufficient to reduce MAP kinase activation induced by microtubule depolymerization. The ability to attenuate microtubule depolymerization and the ensuing MAP kinase activation was abrogated in B-lymphocytes and fibroblasts derived from PD patients with parkin mutations such as exon 4 deletion. Such mutations produced truncated parkin proteins lacking any microtubule binding domain and prevented parkin from protecting midbrain dopaminergic neurons against microtubule-depolymerizing toxins such as rotenone or colchicine. Consistent with these, blocking MAP kinase activation in midbrain dopaminergic neurons by knocking down MAP kinase kinases (MKK) significantly reduced the selective toxicity of rotenone or colchicine. Conversely, overexpression of MAP kinases caused marked toxicities that were significantly attenuated by parkin. Thus, the results suggest that parkin protects midbrain dopaminergic neurons against microtubule-depolymerizing PD toxins such as rotenone by stabilizing microtubules to attenuate MAP kinase activation.

BMP-7 protects mesangial cells from injury by polymeric IgA

Bone morphogenetic protein-7 (BMP-7) is a potential therapeutic agent for acute and chronic renal diseases. Here we found that addition of polymeric IgA, isolated from patients with IgA nephropathy, increased the synthesis of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and fibronectin in cultured human mesangial cells, effects blunted by BMP-7. When mesangial cells were cultured with both polymeric IgA and BMP-7 there was an increase in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The activation of NF kappaB and TNF-alpha synthesis induced by polymeric IgA or TNF-alpha were downregulated by BMP-7 or rosiglitazone. BMP-7 inhibited TNF-alpha release from polymeric IgA-stimulated mesangial cells by activation of PPAR-gamma but suppressed TGF-beta release by mechanisms independent of PPAR-gamma. The expression of inhibitory Smad6 and 7 was increased whereas the expression of active Smad2 and 3 was reduced in these mesangial cells by BMP-7. Our study shows that BMP-7 ameliorates IgA nephropathy-derived polymeric IgA-induced TNF-alpha and TGF-beta synthesis in human mesangial cells through multiple mechanisms involving inhibitory Smads and PPAR-gamma.

Novel Function of PERK as a Mediator of Force-induced Apoptosis

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response.

Acute Rejection and the Muscularis Propria After Intestinal Transplantation: The Alloresponse, Inflammation, and Smooth Muscle Function

It has been shown that in transplantation the intestinal muscularis may act as an immunologically active layer via the activation of resident macrophages and the recruitment of leukocytes. Thus we hypothesized that inflammation within the intestinal muscularis is involved in the promotion of acute rejection in intestinal allografts and that this causes smooth muscle dysfunction. Orthotopic allogenic and small bowel transplantation (Brown-Norway rats-Lewis rats) was performed without immunosuppression. Animals were sacrificed 1, 4, and 7 days after small bowel transplantation. Isogenic transplanted grafts (Brown-Norway rats-Brown-Norway rats) as well as nontransplanted bowel served as controls. Mediator mRNA expression was determined by real-time reverse-transcriptase polymerase chain reaction. Leukocyte infiltration was evaluated in muscularis whole mounts by immunohistochemistry. Apoptosis was evaluated by TdT-mediated dUTP-X nick end labeling assay. Contractility was assessed in a standard organ bath under bethanechol stimulation. Statistical analysis was performed using a Student's t test and one-way analysis of variance. Transplanted animals showed a significant early inflammatory response within the graft muscularis because of reperfusion injury. Only allogenic transplanted animals exhibited a significant second molecular inflammatory peak in the muscularis during rejection (mRNA induction for interleukin (IL)-6, intercellular adhesion molecule-1, monocyte chemoattractant protein (MCP)-1, interferon-gamma, IL-2, tumor necrosis factor-alpha, IL-10, inducible nitric oxide synthase). These findings were associated with significant leukocyte infiltration within the muscularis, increasing apoptotic cells and massive impairment of smooth muscle contractile activity by 78%. The data shows that transplantation results in an early and temporary inflammatory response within the intestinal graft muscularis, that is reactivated and intensified during acute allograft rejection. The immunoreaction within the intestinal muscularis leads to intestinal allograft smooth muscle dysfunction.

Studies on the relationship of objective response by chemotherapy and senescence induced in vitro for non-small cells lung cancer

To investigate the relationship between the objective response to combination chemotherapy of taxanes plus cisplatin in non-small cell lung cancer (NSCLC) and docetaxel plus cisplatin (DC regime) induced senescence of tumor cells in vitro. And its relation to mutant P53 protein (m-P53) was also to be evaluated. Sixty-seven specimen obtained from NSCLC patients from January 1, 2003 to June 30, 2006. The patients consisted of 48 males and 19 females, ranging in age from 54 to 82 years (mean, 67.5 years), 41 cases were diagnosed as pathological stage IIIb, 26 cases were diagnosed as stage IV. Thirty-nine tumors were confirmed to be adenocarcinomas, 28 were confirmed to be squamous cell carcinomas. All patients accepted 2–6 cycles combination chemotherapy of Taxanes (docetaxel 40 mg/m2, d1; d8, or paclitaxel 175 mg/m2, d1) plus cisplatin (CDDP, 25 mg/m2, d2–4). Patients were divided into chemoresponsive (CR + PR) and chemoresistant (SD + PD) groups according to objective response status which was evaluated by RECIST system. Tumor cells from specimens of bronchoscopic, surgical biopsy and pleural effusion cell collection had been cultured and treated with DC in vitro. The m-P53 of culture supernatant was measured by ABC-ELISA kit before DC treatment. The telomerase activity was determined by the telomeric repeat amplification protocol (TRAP) based PCR-ELISA kit and apoptosis was determined by TdT-mediated d-UTP-X nick-end labeling (TUNEL) assay. Data represent as both actual detected and positive value. The senescence of tumor cells defined as that, apoptosis rate increased more than 50% to control, and telomerase activity decreased less than 50% to control. There was no significant difference between clinical treatment response and sex, pathological type, specimen origin, or m-P53 status in cultured cell supernatant. Telomerase activity and apoptosis rate was positive in 61.1% (41/67) and 25.4%(17/67) of all samples respectively. A significant difference of senescence of tumor cells treated by DC, was existed between chemoresponsive and chemoresistant patients groups (P < 0.05). Multinomial logistic regression analyses shew that telomerase activity decreased less than 50% in vitro may be an indicator of clinical response for taxanes plus cisplatin chemotherapy. Odds ratio was 4.226, P < 0.05. For NSCLC, DC induce lung cancer tumor cells senesce in vitro may be a promising predicator for clinical response, but the relationship between objective response by chemotherapy and detectable m-P53 or DC induced apoptosis is still obscure.

Is digitalis compound-induced cardiotoxicity, mediated through guinea-pig cardiomyocytes apoptosis?

Our aim in performing this study was to analyze in vivo the cell death mechanism induced by toxic doses of digitalis compounds on guinea-pig cardiomyocytes. We analyzed three study groups of five male guinea pigs each. Guinea pigs were intoxicated under anesthesia with ouabain or digoxin (at a 50–60% lethal dose); the control group did not receive digitalis. A 5-hours period elapsed before guinea pig hearts were extracted to obtain left ventricle tissue. We carried out isolation of mitochondria and cytosol, cytochrome c and caspase-3 and -9 determination, and electrophoretic analysis of nuclear DNA. TdT-mediated DUTP-X nick end labeling (TUNEL) reaction was performed in histologic preparations to identify in situ apoptotic cell death. Ultrastructural analysis was performed by electron microscopy. Electrophoretic analysis of DNA showed degradation into fragments of 200–400 base pairs in digitalis-treated groups. TUNEL reaction demonstrated the following: in the control group, < 10 positive nuclei per field; in the digoxin-treated group, 2–14 positive nuclei per field, while in the ouabain-treated group counts ranged from 9–30 positive nuclei per field. Extracts from ouabain-treated hearts had an elevation of cytochrome c in cytosol and a corresponding decrease in mitochondria; this release of cytochrome c provoked activation of caspase-9 and -3. Electron microscopy revealed presence of autophagic vesicles in cytoplasm of treated hearts. Toxic dosages of digitalis at 50–60% of the lethal dose are capable of inducing cytochrome c release from mitochondria, processing of procaspase-9 and -3, and DNA fragmentation; these observations are mainly indicative of apoptosis, although a mixed mechanism of cell death cannot be ruled out.

Aging is Neuroprotective During Global Ischemia but Leads to Increased Caspase-3 and Apoptotic Activity in Hippocampal Neurons

Previously, we found a significantly greater number of surviving CA1 neurons to global ischemia in the aged (24-month-old) F344 rats than in young (4-month-old) rats. The present study tests the hypothesis that aging retards neuronal death in the hippocampal CA1 region following cerebral ischemia. The CA1 "living cell ratio" was significantly greater in aged than in young rats at three days (62+/-8% vs. 30+/-8%) and at eight days (36+/-6% vs. 17+/-5%), but not at 14 days (15+/-12% vs. 18+/-12%) following ischemia. The number of the CA1 cells exhibiting co-localized TdT-mediated X-dUTP nick end labeling reaction and caspase-3 active peptide (C3AP) immunoreactivity was greater in aged than young animals at three and eight days following ischemia (36+/-8/mm vs. 3+/-1/mm and 36+/-14 vs. 0+/-0, p<0.05 respectively). Also, the total number of C3AP-positive cells in the CA1 region in the aged group was significantly greater than in the young group at three and eight days post-ischemia (p<0.05). Aging appears to delay caspase-3-dependent apoptotic cell death induced by global ischemia in the CA1 region of the hippocampus, consistent with an age-induced neuroprotective process.

Adipocyte Apoptosis After Burn Injury Is Associated With Altered Fat Metabolism

Burn injury often is associated with the abnormal lipid metabolism, including hyperlipidemia, desensitization to lipolytic responses to catecholamines, and reduction in the size of the white adipose tissue. Understanding the biological mechanisms for the decrease in fat mass despite desensitization to catecholamines is important both for the study of lipid metabolism and for the study of its relationship to concomitant insulin resistance. Using epididymal adipose tissue from adult male Sprague-Dawley rats after burn injury (n = 102) or sham-burn injury (n = 102), we tested the hypothesis that a whole-body burn injury causes apoptosis in that tissue. At 1, 3, and 7 days after 40% to 50% body burn injury to the rat, epidydimal adipose tissue was harvested and studied for apoptotic changes and lipolytic properties. For apoptosis, paraformaldehyde-fixed tissue sections were analyzed by in situ TdT-mediated dUTP-X nick-end labeling (TUNEL) staining, and tissue homogenates were also analyzed for DNA fragmentation by enzyme-linked immunoassay and ligation-mediated polymerase chain reaction ladder assay. Isolated adipocytes were stimulated with isoprotenerol, and glycerol production was measured as a reflector of effectiveness of lipolysis. Epididymal adipose tissue showed increased apoptosis manifested by the positive TUNEL staining and increased DNA fragmentation by enzyme-linked immunoassay at day 3 and 7 after burn injury. The DNA fragmentation was confirmed further by the ligation-mediated polymerase chain reaction ladder assay. This elevated DNA fragmentation persisted in the burned animals from day 3 until day 7 after burn injury, the end of observation period. Increase in apoptosis was correlated with decrease in DNA content and tissue weight in the epidydimis. At the functional level, a significant decrease in isoproterenol-induced lipolytic activity (glycerol production) was observed to almost 50% of control level at day 3 and 7 but was not decreased at day 1. Apoptosis of adipocytes may play a role in the altered lipid metabolism, including hyperlipidemia observed in burned subjects.

Cytomegalovirus infection of trophoblast cells elicits an inflammatory response: A possible mechanism of placental dysfunction

We sought to determine whether cytomegalovirus infection of extravillous trophoblast cells induces inflammatory changes that lead to cell death. Extravillous trophoblast (HTR-8/SVneo) cells were infected with cytomegalovirus. Cell death assays were performed 8 to 48 hours after infection. The expression and secretion of cytokines (interleukin-6 and -8) preceding cell death was measured by polymerase chain reaction and enzyme-linked immunosorbent assay. Cell viability (lactate dehydrogenase assay) was reduced approximately 20%, and rates of apoptosis (measured by TdT-mediated dUTP-X nick end labeling [TUNEL] assay) were increased approximately 40% at 16 to 48 hours after infection. Significantly elevated levels of caspase-3 mRNA levels were observed before increased cell death. Interleukin-6 and -8 mRNA expression and protein secretion were up-regulated 8 to 16 hours after cytomegalovirus infection. Placental exposure to cytomegalovirus induces an inflammatory response that precedes invasive trophoblast cell death. Cytomegalovirus may prevent normal placental invasion, which results in adverse reproductive outcomes that are associated with placental dysfunction.

Angiogenesis Inhibitor Z24 Induces Endothelial Cell Apoptosis and Suppresses Tumor Growth and Metastasis

Z24, a small molecular compound with similar chemical structure to SU5416 designed and synthesized by our lab, has been proved to be an angiogenesis inhibitor. In this study, Z24 was shown to induce human umbilical venous endothelial cell (HUVEC) apoptosis confirmed by morphologic changes including the presence of apoptotic bodies, significant apoptotic sub-G1 peak upon flow-cytometric analysis, formation of DNA ladders upon agarose gel electrophoresis, and TUNEL (TdT mediated X-dUTP nick-end labeling) results. Systemic administration of Z24 at non-toxic dose in nude mice resulted in inhibition of subcutaneous tumor growth of human colon cancer HCT-8, while it did not inhibit this cell line in vitro, with 100-fold more potent growth-inhibition against endothelial cells. The immunohistochemical results showed that the microvessel density of tumor tissue of the Z24 group was significantly lower than that of the control groups (P<0.05), which supported its anti-angiogenic property. We further found that Z24 inhibited the pulmonary metastasis of mouse lung adenocarcinoma LA795, with fewer surface lung metastases (89.6%, P<0.0001) and decreased lung weights (38.5%, P<0.01) compared to the vehicle group. All these findings support that Z24 is a promising angiogenesis inhibitor for limiting tumor growth and metastasis.

Sonoporation with doxorubicin enhances suppression of intimal hyperplasia in a vein graft model

The purpose of the present study is to examine whether sonoporation with doxorubicin enhances suppression of intimal hyperplasia (IH) in a vein graft model. After the administration of 1.5 mg/kg doxorubicin intravenously, the right external jugular vein of six rabbits was exposed at 2 W/cm2 and 1 MHz of ultrasound for 2 min (Sonoporation group). Tissue doxorubicin concentration was measured. In 48 rabbits, the right common carotid artery was ligated after performing a vein graft bypass. The animals were divided into the following four groups: the C0 group (surgical procedure only); the C0S (sonoporation without doxorubicin); the C1 (doxorubicin administration only); the C1S (sonoporation with doxorubicin). Twenty-four grafts were subjected to Elastic van Gieson staining for morphometric analysis 4 weeks after the operation; others were subjected to TdT-mediated X-dUTP nick end-labeling for detection of apoptic cells and to staining with a monoclonal antibody against the proliferating cell nuclear antigen for assessment of cell proliferation 1 week after. The tissue doxorubicin concentration was significantly higher in the Sonoporation group than in the Control group. Compared with the C0 group, IH was not suppressed in the C1 group but was significantly suppressed in the C1S group. Sonoporation with doxorubicin administration suppressed IH significantly (C1 group versusC1S group: P < 0.05). Cell apoptosis was induced and cell proliferation was suppressed significantly in the C1S group. Sonoporation with doxorubicin suppressed IH of the vein graft. Sonoporation may be effective in coronary or peripheral revascularization using vein grafts.

Deletion of Murine Smn Exon 7 Directed to Liver Leads to Severe Defect of Liver Development Associated with Iron Overload

Spinal muscular atrophy (SMA) is characterized by degeneration of lower motor neurons caused by mutations of the survival motor neuron 1 gene (SMN1). SMN is involved in various processes including the formation of the spliceosome, pre-mRNA splicing and transcription. To know whether SMN has an essential role in all mammalian cell types or an as yet unknown specific function in the neuromuscular system, deletion of murine Smn exon 7, the most frequent mutation found among SMA patients, has been restricted to liver. Homozygous mutation results in severe impairment of liver development associated with iron overload and lack of regeneration leading to dramatic liver atrophy and late embryonic lethality of mutant mice. These data strongly suggest an ubiquitous and essential role of full-length SMN protein in various mammalian cell types. In SMA patients, the residual amount of SMN allows normal function of various organs except motor neurons. However, data from mouse and human suggest that other tissues might be involved in severe form of SMA or during prolonged disease course which reinforce the need of therapeutic approaches targeted to all tissues. In addition, liver function of patients should be carefully investigated and followed up before and during therapeutic trials.