TUNEL-positive Staining Research Articles

Vasoinhibin generation and effect on neuronal apoptosis in the hippocampus of late mouse embryos.

Morphological and behavioral evidence suggests that vasoinhibin is present in the central nervous system (CNS), triggering neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin reduces neuronal survival and differentiation of primary sensory neurons of the peripheral nervous system. To address the functional role played by vasoinhibin at the CNS, and to better understand the underlying mechanisms involved in its actions, we treated primary cultured hippocampal neurons obtained from embryonic day 16 (E16) mice with a human recombinant vasoinhibin. We examined the resulting cellular changes, focusing on neuronal cell death, and explored the local generation of vasoinhibin within the hippocampus. Our results show that vasoinhibin significantly reduced neuronal cell density and increased immunoreactive activated caspase-3 and TUNEL-positive staining at 72, 16, and 24 h, respectively. Furthermore, vasoinhibin increased the expression of proapoptotic genes BAX, BAD, BIM, and PUMA and decreased that of the antiapoptotic gene BCL-2 at 24 h, as assessed by quantitative real-time reverse transcription-polymerase chain reaction. Vasoinhibin effects were blocked by coincubation with a vasoinhibin antibody or with prolactin. Immunoreactive bands consistent with vasoinhibin were observed in hippocampal extracts by Western blot analysis, and a prolactin standard was cleaved to vasoinhibin by a hippocampal lysate in a heat- and cathepsin D inhibitor pepstatin A-dependent fashion. Taken together, these data support the notion that vasoinhibin is locally produced by cathepsin D within the embryonic mouse hippocampus, a brain region that plays a critical role in emotional regulation, resulting in decreased neuronal cell viability via the activation of the intrinsic apoptosis pathway.

Doxorubicin Induces Endotheliotoxicity and Mitochondrial Dysfunction via ROS/eNOS/NO Pathway.

Background: Doxorubicin (Dox) can induce endotheliotoxicity and damage the vascular endothelium (VE). The most principle mechanism might be excess reactive oxygen species (ROS) generation. Nevertheless, the characteristics of ROS generation, downstream mechanisms, and target organelles in Dox-induced endotheliotoxicity have yet to be elucidated. Methods and Results: In order to explore the related problems, the VE injury models were established in mice and human umbilical vein endothelial cells (HUVECs) by Dox-induced endotheliotoxicity. Results showed that the activities of lactate dehydrogenase (LDH) and creatine kinase of mice’s serum increased after injected Dox. The thoracic aortic strips’ endothelium-dependent dilation was significantly impaired, seen noticeable inflammatory changes, and brown TUNEL-positive staining in microscopy. After Dox-treated, HUVECs viability lowered, LDH and caspase-3 activities, and apoptotic cells increased. Both intracellular/mitochondrial ROS generation significantly increased, and intracellular ROS generation lagged behind mitochondria. HUVECs treated with Dox plus ciclosporin A (CsA) could basically terminate ROS burst, but plus edaravone (Eda) could only delay or inhibit, but could not completely cancel ROS burst. Meanwhile, the expression of endothelial nitric oxide synthase (eNOS) decreased, especially phosphorylation of eNOS significantly. Then nitric oxide content decreased, the mitochondrial function was impaired, mitochondrial membrane potential (MMP) impeded, mitochondrial swelled, mitochondrial permeability transition pore (mPTP) was opened, and cytochrome C was released from mitochondria into the cytosol. Conclusion: Dox produces excess ROS in the mitochondria, thereby weakens the MMP, opens mPTP, activates the ROS-induced ROS release mechanism, induces ROS burst, and leads to mitochondrial dysfunction, which in turn damages VE. Therefore, interrupting any step of the cycles, as mentioned above can end the related vicious cycle and prevent the occurrence and development of injury.

Coenzyme Q10 Regulation of Apoptosis and Oxidative Stress in H2O2 Induced BMSC Death by Modulating the Nrf-2/NQO-1 Signaling Pathway and Its Application in a Model of Spinal Cord Injury.

Spinal cord injury (SCI) has always been considered to be a devastating problem that results in catastrophic dysfunction, high disability rate, low mortality rate, and huge cost for the patient. Stem cell-based therapy, especially using bone marrow mesenchymal stem cells (BMSCs), is a promising strategy for the treatment of SCI. However, SCI results in low rates of cell survival and a poor microenvironment, which limits the therapeutic efficiency of BMSC transplantation. Coenzyme Q10 (CoQ10) is known as a powerful antioxidant, which inhibits lipid peroxidation and scavenges free radicals, and its combined effect with BMSC transplantation has been shown to have a powerful impact on protecting the vitality of cells, as well as antioxidant and antiapoptotic compounds in SCI. Therefore, we aimed to evaluate whether CoQ10 could decrease oxidative stress against the apoptosis of BMSCs in vitro and explored its molecular mechanisms. Furthermore, we investigated the protective effect of CoQ10 combined with BMSCs transplanted into a SCI model to verify its ability. Our results demonstrate that CoQ10 treatment significantly decreases the expression of the proapoptotic proteins Bax and Caspase-3, as shown through TUNEL-positive staining and the products of oxidative stress (ROS), while increasing the expression of the antiapoptotic protein Bcl-2 and the products of antioxidation, such as glutathione (GSH), against apoptosis and oxidative stress, in a H2O2-induced model. We also identified consistent results from the CoQ10 treatment of BMSCs transplanted into SCI rats in vivo. Moreover, the Nrf-2 signaling pathway was also investigated in order to detail its molecular mechanism, and the results show that it plays an important role, both in vitro and in vivo. Thus, CoQ10 exerts an antiapoptotic and antioxidant effect, as well as improves the microenvironment in vitro and in vivo. It may also protect BMSCs from oxidative stress and enhance their therapeutic efficiency when transplanted for SCI treatment.

Iron Overload Damages the Endothelial Mitochondria via the ROS/ADMA/DDAHII/eNOS/NO Pathway.

It has been recognized that iron overload may harm the body's health. Vascular endothelial cells (VECs) are one of the main targets of iron overload injury, and the mechanism involved was thought to be related to the excessive generation of reactive oxygen species (ROS). However, the subcellular and temporal characteristics of ROS generation, potential downstream mechanisms, and target organelles in VECs injured by iron overload have not been expounded yet. In this study, we elucidated the abovementioned issues through both in vivo and in vitro experiments. Mice were fed pellet diets that were supplemented with iron for 4 consecutive months. Results showed that the thoracic aortic strips' endothelium-dependent dilation was significantly impaired and associated with inflammatory changes, noticeable under brown TUNEL-positive staining in microscopy analysis. In addition, the serum content of asymmetric dimethylarginine (ADMA) increased, whereas nitric oxide (NO) levels decreased. Furthermore, the dimethylarginine dimethylaminohydrolase II (DDAHII) expression and activity, as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) in aortic tissue, were inhibited. Human umbilical vein endothelial cells were treated with 50 μM iron dextran for 48 hours, after which the cell viability, NO content, DDAHII expression and activity, and phosphorylation of eNOS decreased and lactate dehydrogenase and caspase-3 activity, ADMA content, and apoptotic cells significantly increased. After the addition of L-arginine (L-Arg) or pAD/DDAHII, the abovementioned changes were reversed. By dynamically detecting the changes of ROS generation in the cytoplasm and mitochondria and interfering with different aspects of signaling pathways, we have confirmed for the first time that excessive ROS originates from the cytoplasm and activates the ROS-induced ROS release (RIRR) mechanism, leading to mitochondrial dysfunction. Together, our data suggested that excessive free iron ions produced excess ROS in the cytoplasm. Thus, excess ROS create one vicious circle by activating the ADMA/eNOS/DDAHII/NO pathway and another vicious circle by activation of the RIRR mechanism, which, when combined, induce a ROS burst, resulting in mitochondrial dysfunction and damaged VECs.

The role of autophagy in the pathogenesis of exposure keratitis.

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure‐induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3‐methyladenine (3‐MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy‐related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy‐related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress‐related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway‐related proteins. Real‐time quantitative PCR (qRT‐PCR) determined IL‐1β, IL‐6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure‐induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress‐induced autophagy promoted cell survival. Taken together, air exposure‐induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.

Exogenous Vasohibin-2 Exacerbates Angiotensin II-Induced Ascending Aortic Dilation in Mice.

Background: Chronic angiotensin II (AngII) infusion promotes ascending aortic dilation in C57BL/6J mice. Meanwhile, vasohibin-2 (VASH2) is an angiogenesis promoter in neovascularization under various pathologic conditions. The aim of this study was to investigate whether exogenous VASH2 influences chronic AngII-induced ascending aortic dilation. Methods and Results: Eight–ten-week-old male C57BL/6J mice were injected with adenovirus (Ad) expressing either VASH2 or LacZ. One week after the injection, mice were infused with either AngII or saline s.c. for 3 weeks. Mice were divided into 4 groups: AngII+VASH2, AngII+LacZ, saline+VASH2, and saline+LacZ. Overexpression of VASH2 significantly increased AngII-induced intimal areas as well as the external diameter of the ascending aorta. In addition, VASH2 overexpression promoted ascending aortic medial elastin fragmentation in AngII-infused mice, which was associated with increased matrix metalloproteinase activity and medial smooth muscle cell (SMC) apoptosis. On western blot analysis, accumulation of apoptotic signaling proteins, p21 and p53 was increased in the AngII+VASH2 group. Furthermore, transfection of human aortic SMC with Ad VASH2 increased p21 and p53 protein abundance upon AngII stimulation. Positive TUNEL staining was also detected in the same group of the human aortic SMC. Conclusions: Exogenous VASH2 exacerbates AngII-induced ascending aortic dilation in vivo, which is associated with increased medial apoptosis and elastin fragmentation.

Nerve growth factor alleviates cerebral infarction and neurologic deficits by regulating VEGF, SDF-1 and S100A12 expression through PI3K pathway

Stroke remains the leading cause of death and disability worldwide, which destroys the quality of patients’ lives and thus is becoming a heavy burden to the society. However, the current therapeutic approaches are far from satisfaction. The objective of this study is to elucidate the impact of nerve growth factor (NGF) on the brain damage induced by cerebral ischemia and its potential molecular mechanism. Middle cerebral artery occlusion (MCAO) rats were used as animal models and neurological functions were evaluated by modified Neurological Severity Score (NSS). Brain cell apoptosis was analyzed by TUNEL-positive staining while brain infarct size was determined according to 2% 2,3,5-triphenyltetrazolium chloride (TCC) staining volume. Rats receiving NGF demonstrated significantly alleviated brain damage, reflected by a substantial improvement in the neurobehavioral outcome, a decrease in brain cell apoptosis and shrinkage of brain infarct volume. Further analysis revealed a markedly elevated circulating vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1) levels as well as a significant downregulation of SA10012 expression in NGF treated group compared with the untreated group. Strikingly, the protective effect of NGF on cerebral ischemic injury was abolished in rats treated with both NGF and PI3K inhibitors, indicating that phosphoinositide-3-kinase (PI3K) signaling is essential for NGF function. In conclusion, NGF treatment might be a potential therapeutic approach against cerebral infarction by downregulating SA10012 expression and upregulating VEGF, SDF-1 in a PI3K signaling dependent manner.

Fibroblast Growth Factor 21 Attenuates Vascular Calcification by Alleviating Endoplasmic Reticulum Stress Mediated Apoptosis in Rats.

Fibroblast growth factor 21 (FGF21), a hormone with multiple metabolic properties, has proven to be pleiotropic biological effects and may play pivotal role in numerous cardiovascular and metabolic diseases in the future. Vascular calcification (VC) is a concomitant pathological process of various cardiovascular and metabolic diseases. However, the effects of FGF21 on VC remain unclear. Therefore, in this research, we aimed to explore the roles and mechanisms of FGF21 in VC induced by vitamin D3 plus nicotine (VDN) treatment rats. After 28 days VDN treatment, the calcium overload was confirmed by blood pressure, ultrasound imaging, calcium content, ALP activity and aortic pathological characteristics. In terms of FGF21, exogenous FGF21 can ameliorate the elevation of blood pressure, aortic calcification and related injury in VC rats. To investigate the mechanisms of FGF21 on VC, the endoplasmic reticulum stress (ERS) mediated apoptosis pathways were tested. As a method to detect apoptosis, the increased positive TUNEL staining cells were alleviated by FGF21 treatment. Furthermore, exogenous FGF21 can suppress the increased ERS chaperone, GRP78, in the calcified aortas. In the three pathways of ERS mediated apoptosis, we found CHOP pathway and caspase-12 pathway were involved in the treatment of FGF21, but not p-JNK/JNK pathway. Our study proved for the first time that FGF21 can inhibit the progress of VC by alleviating ERS mediated apoptosis in rats. FGF21 might be a new target for preventing and treating VC.

C-Myc promotes tubular cell apoptosis in ischemia-reperfusion-induced renal injury by negatively regulating c-FLIP and enhancing FasL/Fas-mediated apoptosis pathway.

c-Myc plays an important role in cell proliferation, differentiation, and cell apoptosis. FasL/Fas pathway is a key regulator of cell apoptosis. This study was aimed to investigate the effects of c-Myc on the FasL/Fas pathway in ischemia-reperfusion (I/R)-induced renal injury. Rats were objected to bilateral renal ischemia for 60 min and reperfused for 24 or 48 h. NRK-52E cells were treated with hypoxia-reoxygenation (H/R) or FasL. Immunohistochemistry was used to identify the distribution of c-Myc. Cell apoptosis was assessed by TUNEL staining. Ad-c-Myc and recombinant pcDAN 3.0 were used to overexpress c-Myc and c-FLIP, respectively. ChIP assay and luciferase assay were used to detect the binding of c-Myc to c-FLIP promoter. In I/R rats, c-Myc was increased significantly and mainly located in renal tubular epithelial cells; meanwhile, c-FLIP was decreased, cleaved caspase-8, cleaved caspase-3 and TUNEL-positive staining cells were increased. Treatment of I/R rats with c-Myc inhibitor 10058-F4 significantly attenuated the decrease in c-FLIP, the increase in cleaved caspase-8, cleaved caspase-3, TUNEL-positive cells, Scr and BUN in I/R rats. In NRK-52E cells, hypoxia and reoxygen induced the increase in c-Myc and decrease in c-FLIP. ChIP and luciferase assay results indicated that c-Myc binds to the promoter region of c-FLIP gene. Overexpression of c-Myc markedly decreased c-FLIP. Overexpression of c-FLIP inhibited the increase in cleaved caspase-8 and caspase-3 induced by FasL. Data indicated that c-Myc is increased in kidneys of I/R rats and negatively regulates the expression of c-FLIP, then enhanced FasL-induced cell apoptosis in I/R stress.

Rosmarinic Acid Combined with Adriamycin Induces Apoptosis by Triggering Mitochondria-Mediated Signaling Pathway in HepG2 and Bel-7402 Cells.

BackgroundHepatic carcinoma is the third leading cause of cancer-related deaths. This study aimed to evaluate the anti-tumor effects of rosmarinic acid (RosA) combined with Adriamycin (ADM) on proliferation and apoptosis of hepatic carcinoma cell lines.Material/MethodsHuman HepG2 and Bel-7402 cells were treated with RosA and ADM and divided into HepG2 or Bel-7402, 25 μg/ml, 50 μg/m, and 100 μg/ml RosA+0.4 μg/ml ADM groups, respectively. The Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell viability. Immunohistochemistry assay was used to examine B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) expression. Cell cycle analysis was used to detect cell cycle distribution. Flow cytometry and terminal deoxynucleotidyl transferase-mediated d-UTP nick-end labeling (TUNEL) assay were utilized to evaluate apoptosis.ResultsRosA combined with ADM damaged cell morphology and decreased cell viability, and significantly decreased S-phase cell numbers compared to the HepG2 or Bel-7402 group (p<0.05). Apoptosis rates in the RosA combined with ADM group were significantly increased compared to the HepG2 or Bel-7402 group (p<0.05). TUNEL assay showed that RosA combined with ADM significantly induced DNA damage (TUNEL-positive staining) in the HepG2 and Bel-7402 groups (p<0.05). RosA combined with ADM significantly reduced Bcl-2 expression in HepG2 or Bel-7402 cells (p<0.05). RosA combined with ADM significantly increased Bax expression in HepG2 and Bel-7402 cells (p<0.05). Cell viability, apoptosis, cell cycle, and Bcl-2 and Bax expression were changed with increased concentrations of RosA.ConclusionsRosA combined with ADM damaged tumor cell morphologies, decreased cell viability, and induced apoptosis of HepG2 and Bel-7402 by triggering the mitochondria-mediated signaling pathway.

Abstract 2324: Docosahexaenoic acid-induced Nrf2 may be correlated with cell death by amplification of oxidative stress via induction of klf9 in cervical cancer cells

Abstract NF-E2-related transcription factor-2 (Nrf2) is a key transcriptional factor regulating antioxidant gene expression and protection from oxidant damage. Docosahexaenoic acid (DHA) as one of ω3-polyunsaturated fatty acids has anticancer action through several mechanisms. Here we report that DHA-induced Nrf2 is related to cell death, not survival in HeLa cell. We confirmed DHA-induced apoptotic cell death using PARP cleavage, Anexin V staing, and TUNEL positive staining assay. DHA-triggering ROS production increased the level Nrf2 and decreased keap1 in dose- and time-dependent manner. Increased Nrf2 by DHA was partially Keap1 dependent. DHA activated Nrf2 signaling by increasing its translation and the Nrf2 was translocated from cytosol to nucleus. Upregulation of DHA-mediated Nrf2 was also confirmed by increase of heme oxygenase-1 (HO-1) expression. DHA-induced Nrf2 increased the level of Kruppel-like factor 9 (klf9) and then decreased thioredoxin reductase 2 (Txnrd2) in dose- and time-dependent manner. These results suggest that DHA-induced ROS increases Nrf2 and the Nrf2 is related to cell death by increase of klf9 and decrease of Txnrd2 in HeLa cells. Thus, utilization of ω3-PUFAs may represent a promising therapeutic approach for chemoprevention and treatment of human cervical cancer [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)] Citation Format: Kaipeng Jing, Soyeon Shin, Seung-Hyeon Han, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Docosahexaenoic acid-induced Nrf2 may be correlated with cell death by amplification of oxidative stress via induction of klf9 in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2324.

Involvement of adipose tissue inflammation and dysfunction in virus-induced type 1 diabetes.

The etiopathogenesis of type 1 diabetes (T1D) remains poorly understood. We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced T1D to better understand the role of the innate immune system in the mechanism of virus-induced disease. We observed that infection with KRV results in cell influx into visceral adipose tissue soon following infection prior to insulitis and hyperglycemia. In sharp contrast, subcutaneous adipose tissue is free of cellular infiltration, whereas β cell inflammation and diabetes are observed beginning on day 14 post infection. Immunofluorescence studies further demonstrate that KRV triggers CD68+ macrophage recruitment and the expression of KRV transcripts and proinflammatory cytokines and chemokines in visceral adipose tissue. Adipocytes from naive rats cultured in the presence of KRV express virus transcripts and upregulate cytokine and chemokine gene expression. KRV induces apoptosis in visceral adipose tissue in vivo, which is reflected by positive TUNEL staining and the expression of cleaved caspase-3. Moreover, KRV leads to an oxidative stress response and downregulates the expression of adipokines and genes associated with mediating insulin signaling. Activation of innate immunity with Poly I:C in the absence of KRV leads to CD68+ macrophage recruitment to visceral adipose tissue and a decrease in adipokine expression detected 5 days following Poly (I:C) treatment. Finally, proof-of-principle studies show that brief anti-inflammatory steroid therapy suppresses visceral adipose tissue inflammation and protects from virus-induced disease. Our studies provide evidence raising the hypothesis that visceral adipose tissue inflammation and dysfunction may be involved in early mechanisms triggering β cell autoimmunity.

Towards the repair of marginal liver grafts: Mesenchymal stromal cell therapy.

Abstract Despite the recent inclusion of livers donated after cardiac death (DCD), more than 25% of people listed for a liver transplant in Canada die before obtaining a suitable graft. Over half of possible DCD livers undergo long periods of ischemia that sensitize the liver to reperfusion injury (HIRI) and render the liver grafts unsuitable for transplantation. Mesenchymal Stromal cells (MSC) show promise in protecting liver tissue against HIRI: MSC stimulated with inflammatory agents have enhanced anti-inflammatory activity. Kupffer Cells (KCs) are central to HIRI, are activated after reperfusion, and release various inflammatory mediators that promote hepatocyte death. We hypothesize that LPS-primed MSC-treatment can mitigate the tissue damage inflicted by HIRI by modulating KC activation. To test this, we injected LPS-primed MSCs(LPS) into rats undergoing warm ischemia to 70% of the liver. Rats that received MSCs(LPS) had significantly lower levels of AST (2288 ± 2121 IU/L) compared to sham controls (6614 ± 3495 IU/L). Histological analysis showed decreased TUNEL and Caspase-3 positive staining in the MSC(LPS) group: the necrotic area in the MSC- and sham-treated groups affected &amp;gt;50% of the liver parenchyma whereas, only ~20% was necrotic in the MSC(LPS) group. In this latter group, the number of infiltrating inflammatory CD68+ macrophages in the necrotic tissue and CD163+ macrophages in the viable tissue was significantly decreased. Systemic treatment of rats with activated MSCs resulted in the decreased migration of macrophages into inflamed tissue and limited parenchymal tissue damage. Primed MSCs are a viable treatment to protect marginal DCD donor liver grafts from the damaging effects of HIRI.

Chondrocyte-specific RUNX2 overexpression accelerates cartilage degeneration following traumatic injury

Purpose: Osteoarthritis (OA) is a debilitating joint disease causing irreversible loss of cartilage within the synovial joints. Studies show roughly 50% of patients with an ACL or meniscus tear will eventually develop knee OA. Despite the high prevalence of OA, exact molecular mechanisms underlying onset are unclear. RUNX2 is a transcription factor that promotes chondrocyte hypertrophy and expression of factors catabolic to the cartilage extracellular matrix (ECM). It is also known to regulate apoptosis signaling in osteoblasts. Importantly, enhanced RUNX2 expression is seen in early stages of human OA and in murine models of injury-induced OA. Here, we investigate whether chondrocyte-specific RUNX2 overexpression can accelerate OA progression following traumatic joint injury. Methods: At 2 months of age, male and female Acan-Cre+/-ERT2; ROSA-Runx2f/+ (RUNX2 GOF) and littermate Cre-negative control mice were given tamoxifen at a dosage of 100 μg/g body weight daily for 5 days (N = 6 per gender and genotype). At 10 weeks of age, mice were subjected to meniscal-ligamentous injury (MLI) in the right hindlimb, and a sham injury in the contralateral limb. Hindlimbs were harvested for histological analysis 4 and 8 weeks post MLI. Hindlimbs were fixed in 10% neutral buffered formalin for 3 days, decalcified in 14% EDTA for 7 days, and embedded into paraffin. Sagittal sections were cut from the medial knee compartment and stained with Safranin-O/Fast Green. On three sections from distinct levels of each sample, manual histomorphometry and modified OARSI scoring were performed (for OARSI scoring, scores from 4 graders were averaged, p < 0.05, N = 6, 2-way ANOVA). Histomorphometry values represent mean cartilage area ± SEM normalized to contralateral sham values (p < 0.05, n = 4, unpaired t-test). MMP13 immunohistochemistry (IHC) was performed using Abcam MMP13 antibody ab75606, and TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche). Preliminary in vitro studies used primary sternal chondrocytes from ROSA-Runx2f/f mice infected with adenovirus encoding GFP or Cre to induce RUNX2 GOF; cells were treated with 1 ng/ml IL-1β for 24 hours to simulate the inflammatory environment within an injured joint and harvested for qPCR and Western blotting. Results: Histology reveals male RUNX2 GOF mice have enhanced cartilage damage and Saf-O staining loss compared to controls; these findings were confirmed by significantly increased OARSI scores in male RUNX2 GOF mice 2 months post MLI (Figure 1A, 1B). Female RUNX2 GOF mice display a modest increase in damage histologically, but did not exhibit a significant change in OARSI score. RUNX2 GOF contralateral sham joints showed no changes relative to controls for either gender. Preliminary histomorphometry data from both genders of mice reveals significant decreases in total and unmineralized femoral cartilage area in RUNX2 GOF mice 2 months post MLI (Figure 1C). RUNX2 GOF mice also exhibit increased TUNEL-positive staining and MMP13 expression 1 month post MLI in the articular cartilage (Figure 1D). In vitro, preliminary data suggests that RUNX2 GOF chondrocytes induce enhanced expression of pro-apoptotic genes Bax and Bid relative to control cells following IL-1β treatment. Conclusions: Our results suggest chondrocyte-specific RUNX2 overexpression can accelerate OA development after traumatic injury. Upregulation of MMP13, a direct downstream target of RUNX2, implies cartilage ECM catabolism after injury is enhanced with RUNX2 GOF. Both the TUNEL staining and our preliminary in vitro data indicate RUNX2 may be exerting its effects in part via positive regulation of apoptosis. Our data highlight the contribution of genetic variability to the development of this disease and suggest that genetic alterations affecting RUNX2 expression levels may, in part, predetermine the rate of OA onset following traumatic injury.

The BET/BRD inhibitor JQ1 attenuates diabetes-induced cognitive impairment in rats by targeting Nox4-Nrf2 redox imbalance

Diabetes-induced oxidative damage is believed to play an important role in the development of cognitive dysfunction. In this study, the involvement of the Nox4-Nrf2 redox imbalance was investigated. STZ-induced diabetic rats exhibited obvious oxidative stress and apoptosis in the hippocampus assessed by augmentation of lipid peroxidation, positive TUNEL staining, elevated ratio of Bax/Bcl-2 and increased caspase 3 activity. Furthermore, hyperglycemia markedly increased Nox4 activity and reduced the activation of Nrf2 by suppressing its up-stream regulatory Akt as well as down-stream target HO-1. Significant improvement of cognitive performance was observed after treatment with the BET/BRD inhibitor JQ1, accompanied by decreased oxidative stress, neuroinflammation and apoptosis in the hippocampus. JQ1 treatment also improved changes in the neuronal cell morphology as well as increased the expression of p-AKT, Nrf2 and HO-1. Our results provide evidence indicating that JQ1 treatment could modulate Nox4-Nrf2 redox imbalance in the hippocampus and may be a promising agent for diabetes-associated cognitive dysfunction.

Alpha lipoic acid attenuates radiation-induced oral mucositis in rats.

PurposeRadiotherapy is currently one of the main treatment modalities for head and neck cancer; however, it also results in severe toxicity to the normal tissue, to the detriment of patients. This study aimed to investigate whether alpha lipoic acid (ALA) could protect against radiation-induced oral mucositis in a rat model.ResultsOn post-irradiation days 4 and 7, the epithelial layer on oral mucosa showed pronounced injury (shortening of the layer) and it is diminished by ALA pretreatment before radiation. Hif-1a expression was significantly induced in the radiation group on days 4, 7, and 28. GLUT1 expression was also induced by radiation at all time points, and the expression levels peaked on day 28. Phosphorylated p53 level was significantly higher in the radiation group on days 4 and 7, and Bax protein expression was significantly higher in the same group on day 4 than ALA-pretreated radiation group. TUNEL-positive staining was significantly lower in the ALA-pretreated radiation group.Materials and methodsRats were assigned to one of the following four groups: control, ALA only (100 mg/kg, i.p.), irradiated, and ALA administered 24 h and 30 min prior to irradiation, with the neck area including the oral mucosa evenly irradiated with 2 Gy per minute (total dose, 18 Gy) using a photon 6-MV linear accelerator. Rats were sacrificed 4, 7, 28, or 56 days after radiation.ConclusionsThe results show that ALA can be used to ameliorate radiation-induced oral mucositis with head and neck cancer.

Tumor-targeted Nanoparticle Delivery of HuR siRNA Inhibits Lung Tumor Growth In Vitro and In Vivo By Disrupting the Oncogenic Activity of the RNA-binding Protein HuR.

Selective downregulation of the human antigen R (HuR) protein by siRNA may provide a powerful approach for treating lung cancer. To this end, we investigated the efficacy of transferrin receptor-targeted liposomal nanoparticle-based HuR siRNA (HuR-TfNP) therapy and compared with control siRNA (C)-TfNP therapy both, in vitro and in vivo using lung cancer models. In vitro studies showed HuR-TfNP, but not C-TfNP, efficiently downregulated HuR and HuR-regulated proteins in A549, and HCC827 lung cancer cells, resulting in reduced cell viability, inhibition of cell migration and invasion, and induction of G1 cell-cycle arrest culminating in apoptosis. However, HuR-TfNP activity in normal MRC-9 lung fibroblasts was negligible. In vivo biodistribution study demonstrated that fluorescently labeled HuR-siRNA or ICG dye-loaded TfNP localized in tumor tissues. Efficacy studies showed intratumoral or intravenous administration of HuR-TfNP significantly inhibited A549 (>55% inhibition) and HCC827 (>45% inhibition) subcutaneous tumor growth compared with C-TfNP. Furthermore, HuR-TfNP treatment reduced HuR, Ki67, and CD31 expression and increased caspase-9 and PARP cleavage and TUNEL-positive staining indicative of apoptotic cell death in tumor tissues compared with C-TfNP treatment. The antitumor activity of HuR-TfNP was also observed in an A549-luc lung metastatic model, as significantly fewer tumor nodules (9.5 ± 3.1; P < 0.001; 88% inhibition) were observed in HuR-TfNP-treated group compared with the C-TfNP-treated group (77.7 ± 20.1). Significant reduction in HuR, Ki67, and CD31 expression was also observed in the tumor tissues of HuR-TfNP-treatment compared with C-TfNP treatment. Our findings highlight HuR-TfNP as a promising nanotherapeutic system for lung cancer treatment. Mol Cancer Ther; 16(8); 1470-86. ©2017 AACR.

Growth suppressive effect of pegylated arginase in malignant pleural mesothelioma xenografts

BackgroundMalignant pleural mesothelioma (MPM) is a difficult-to-treat global disease. Pegylated arginase (BCT-100) has recently shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and melanoma. This study aims to investigate the effects of PEG-BCT-100 in MPM.MethodsA panel of 5 mesothelioma cell lines (H28, 211H, H226, H2052 and H2452) was used to study the in vitro effects of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 were studied using 211H and H226 nude mice xenografts. Protein expression (argininosuccinate synthetase, ornithine transcarbamylase, cleaved PARP, cleaved caspase 3, cyclins (A2, D3, E1 and H), CDK4 and Ki67) and arginine concentration were evaluated by Western blot and ELISA respectively. Cellular localization of BCT-100 was detected by immunohistochemistry and immunoflorescence. TUNEL assay was used to identify cellular apoptotic events.ResultsArgininosuccinate synthetase was expressed in H28, H226, and H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 reduced in vitro cell viability (IC50 values at 13–24 mU/ml, 72 h) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth (p < 0.01) with prolonged median survival (p < 0.01) in both xenograft models. Combining BCT-100 with pemetrexed or cisplatin conferred no additional benefits over single agents. Serum and intratumoral arginine levels were effectively decreased by BCT-100, associated with cytosolic accumulation of BCT-100 within tumor cells. Apoptosis (PARP cleavage in 211H xenografts; Bcl-2 downregulation, and cleavage of PARP and caspase 3 in H226 xenografts; positive TUNEL staining in both) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenografts; suppression of cyclin A2, E1, H and CDK4 in H226 xenografts) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms.ConclusionsBCT-100 suppressed tumor growth with prolonged median survival partially mediated by intratumoral arginine depletion resulting in apoptosis and G1 arrest in mesothelioma xenograft models. The findings provide scientific evidence to support further clinical development of BCT-100 in treatment of MPM.

Abstract 640: Overexpression of Vasohibin-2 Exacerbates Development of Angiotensin II-Induced Thoracic Aortic Aneurysms Independent of VeEGF

Objective: Chronic angiotensin II (AngII) infusion promotes both thoracic (TAAs) and abdominal aortic aneurysms (AAAs) in mice. Vasohibin-2 (VASH2) is known to cause angiogenesis at the sprouting front of neovascularization. The purpose of this study was to examine whether VASH2 influenced AngII-induced TAAs. Methods: Male C57BL/6J mice (10-week-old) were injected with VASH2 or LacZ expressing adenovirus (Ad; 7.5 x 10 9 vp/100 μL) via tail vein at 2 week intervals. One week after the first injection, subcutaneous infusion of either AngII (1,000 ng/kg/min) or saline by mini osmotic pumps was started for 3 weeks. Consequently, mice were divided into 4 groups: AngII + Ad VASH2 (n=22), AngII + Ad LacZ (n=21), saline + Ad VASH2 (n=10), saline + Ad LacZ (n=8). Next, in order to examine whether VASH2 affected TAAs via VEGF regulation, bevacizumab was intraperitoneally administrated into mice; AngII + Ad VASH2 + saline (n=15), AngII + Ad VASH2 + bevacizumab (n=15). TAAs were evaluated in all mice by en face method. Third, human aortic smooth muscle cells (hSMCs) were infected with Ad VASH2 or Ad LacZ, stimulated with or without AngII to evaluate further mechanism. Result: Intima area of aortic arch was significantly larger in AngII + Ad VASH2 group than in AngII + Ad LacZ group (19.78 ± 0.40 mm 3 vs 17.74 ± 0.44 mm 3 , P &lt; 0.001). Gelatin zymography demonstrated that AngII upregulated latent MMP-2 expression, and activated MMP-2 most prominently in AngII + VASH2 group. Protein expression of p21 and p53 in thoracic aortas was enhanced in AngII + VASH2 group. Positive TUNEL staining was observed in thoracic aortic wall of AngII + VASH2 group. No significant difference in intima area of aortic arch between AngII + Ad VASH2 + saline group and AngII + Ad VASH2 + bevacizumab group. In vitro, the same results were observed regarding protein expression of p21 and p53, and TUNEL staining. In addition, Annexin-V staining was detected only in AngII + VASH2 group. Conclusion: Overexpression of VASH2 accelerated development of AngII-induced TAAs in vivo. VASH2-induced cell apoptosis may influence AngII-induced TAA formation independent of VEGF.

Nuclear Receptor PPARα Agonist Wy-14,643 Ameliorates Hepatic Cell Death in Hepatic IKKβ-Deficient Mice.

Inhibitor of nuclear factor kappa-B kinase beta (IKKβ) plays a critical role in cell proliferation and inflammation in various cells by activating NF-κB signaling. However, the interrelationship between peroxisome proliferator-activated receptor α (PPARα) and IKKβ in cell proliferation is not clear. In this study, we investigated the possible role of PPARα in the hepatic cell death in the absence of IKKβ gene using liver-specific Ikkb-null (IkkbF/F-AlbCre) mice. To examine the function of PPARα activation in hepatic cell death, wild-type (IkkbF/F) and IkkbF/F-AlbCre mice were treated with PPARα agonist Wy-14,643 (0.1% w/w chow diet) for two weeks. As a result of Wy-14,643 treatment, apoptotic markers including caspase-3 cleavage, poly (ADP-ribose) polymerase (PARP) cleavage and TUNEL-positive staining were significantly decreased in the IkkbF/F-AlbCre mice. Surprisingly, Wy-14,643 increased the phosphorylation of p65 and STAT3 in both Ikkb and IkkbF/F-AlbCre mice. Furthermore, BrdU-positive cells were significantly increased in both groups after treatment with Wy-14,643. Our results suggested that IKKβ-derived hepatic apoptosis could be altered by PPARα activation in conjunction with activation of NF-κB and STAT3 signaling.