Transferase-mediated dUTP Nick-end Labeling Staining Research Articles

The Glucagon-Like Peptide-1 Receptor Agonist Exendin 4 Has a Protective Role in Ischemic Injury of Lean and Steatotic Liver by Inhibiting Cell Death and Stimulating Lipolysis

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase - all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.

Metformin in early breast cancer: a prospective window of opportunity neoadjuvant study

Metformin may exert anti-cancer effects through indirect (insulin-mediated) or direct (insulin-independent) mechanisms. We report results of a neoadjuvant "window of opportunity" study of metformin in women with operable breast cancer. Newly diagnosed, untreated, non-diabetic breast cancer patients received metformin 500 mg tid after diagnostic core biopsy until definitive surgery. Clinical (weight, symptoms, and quality of life) and blood [fasting serum insulin, glucose, homeostasis model assessment (HOMA), C-reactive protein (CRP), and leptin] attributes were compared pre- and post-metformin as were terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Ki67 scores (our primary endpoint) in tumor tissue. Thirty-nine patients completed the study. Mean age was 51 years, and metformin was administered for a median of 18 days (range 13-40) up to the evening prior to surgery. 51 % had T1 cancers, 38 % had positive nodes, 85 % had ER and/or PgR positive tumors, and 13 % had HER2 overexpressing or amplified tumors. Mild, self-limiting nausea, diarrhea, anorexia, and abdominal bloating were present in 50, 50, 41, and 32 % of patients, respectively, but no significant decreases were seen on the EORTC30-QLQ function scales. Body mass index (BMI) (-0.5 kg/m(2), p < 0.0001), weight (-1.2 kg, p < 0.0001), and HOMA (-0.21, p = 0.047) decreased significantly while non-significant decreases were seen in insulin (-4.7 pmol/L, p = 0.07), leptin (-1.3 ng/mL, p = 0.15) and CRP (-0.2 mg/L, p = 0.35). Ki67 staining in invasive tumor tissue decreased (from 36.5 to 33.5 %, p = 0.016) and TUNEL staining increased (from 0.56 to 1.05, p = 0.004). Short-term preoperative metformin was well tolerated and resulted in clinical and cellular changes consistent with beneficial anti-cancer effects; evaluation of the clinical relevance of these findings in adequately powered clinical trials using clinical endpoints such as survival is needed.

Quantitative Determination of Apoptosis of Pancreatic β-Cells in a Murine Model of Type 1 Diabetes Mellitus

Type 1 diabetes mellitus is characterized by a significant deficit in pancreatic β-cell mass, presumably caused by β-cell apoptosis. We investigated the incidence of β-cell apoptosis in streptozotocin-treated mice and nonobese diabetic (NOD) mice with (99m)Tc-annexin A5. Vehicle-treated mice, streptozotocin-treated mice, and NOD mice at the ages of 5, 9, 16, and 20 wk (5-8 mice per group) were injected with (99m)Tc-annexin A5 and sacrificed 6 h later for autoradiography, and the regional (99m)Tc-annexin A5 level in the pancreas was evaluated. Pancreatic islets were identified by insulin immunohistochemical staining, and apoptotic cells were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The (99m)Tc-annexin A5 level in pancreatic islets was expressed as the percentage injected dose per area of pancreatic islets and normalized by animal body weight (%ID × 10(6)/mm(2)/kg). The level of apoptotic cells in pancreatic islets was expressed as the number of TUNEL-positive cells per area of pancreatic islets (cells/mm(2)). The (99m)Tc-annexin A5 accumulation level was significantly higher (2.5 ± 0.7 vs. 0.7 ± 0.1 %ID × 10(6)/mm(2)/kg, P < 0.05) and the number of TUNEL-positive cells was significantly higher (1,170 ± 535 vs. 5 ± 6 cells/mm(2), P < 0.05) in the pancreatic islets of the streptozotocin-treated mice than in those of the vehicle-treated mice. The (99m)Tc-annexin A5 accumulation level was significantly higher (1.1 ± 0.4 vs. 0.5 ± 0.1 %ID × 10(6)/mm(2)/kg, P < 0.05) and the number of TUNEL-positive cells was significantly higher (152 ± 82 vs. 4 ± 9 cells/mm(2), P < 0.05) in the pancreatic islets of 16-wk-old NOD mice than in those of 5-wk-old NOD mice. In addition, the level of (99m)Tc-annexin A5 correlated with the number of TUNEL-positive cells in the pancreatic islets of the streptozotocin-treated mice (r = 0.821, P < 0.001) and NOD mice (r = 0.721, P < 0.001). There is significant islet cell apoptosis with (99m)Tc-annexin A5 accumulation in the pancreas of both streptozotocin and NOD mice.

Interaction of diabetes and ACE2 in the pathogenesis of cardiovascular disease in experimental diabetes

Local and systemic AngII (angiotensin II) levels are regulated by ACE2 (angiotensin-converting enzyme 2), which is reduced in diabetic tissues. In the present study, we examine the effect of ACE2 deficiency on the early cardiac and vascular changes associated with experimental diabetes. Streptozotocin diabetes was induced in male C57BL6 mice and Ace2-KO (knockout) mice, and markers of RAS (renin-angiotensin system) activity, cardiac function and injury were assessed after 10weeks. In a second protocol, diabetes was induced in male ApoE (apolipoprotein E)-KO mice and ApoE/Ace2-double-KO mice, and plaque accumulation and markers of atherogenesis assessed after 20weeks. The induction of diabetes in wild-type mice led to reduced ACE2 expression and activity in the heart, elevated circulating AngII levels and reduced cardiac Ang-(1-7) [angiotensin-(1-7)] levels. This was associated structurally with thinning of the LV (left ventricular) wall and mild ventricular dilatation, and histologically with increased cardiomyocyte apoptosis on TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) staining and compensatory hypertrophy denoted by an increased cardiomyocyte cross-sectional area. By contrast Ace2-KO mice failed to increase circulating AngII concentration, experienced a paradoxical fall in cardiac AngII levels and no change in Ang-(1-7) following the onset of diabetes. At the same time the major phenotypic differences between Ace2-deficient and Ace2-replete mice with respect to BP (blood pressure) and cardiac hypertrophy were eliminated following the induction of diabetes. Consistent with findings in the heart, the accelerated atherosclerosis that was observed in diabetic ApoE-KO mice was not seen in diabetic ApoE/Ace2-KO mice, which experienced no further increase in plaque accumulation or expression in key adhesion molecules beyond that seen in ApoE/Ace2-KO mice. These results point to the potential role of ACE2 deficiency in regulating the tissue and circulating levels of AngII and their sequelae in the context of diabetes, as well as the preservation or augmentation of ACE2 expression or activity as a potential therapeutic target for the prevention of CVD (cardiovascular disease) in diabetes.

Effectiveness of Apoptotic Factors Expressed on the Wounds of Patients With Stage III Pressure Ulcers

The purpose of this study was to explore the expression of apoptotic factors in stage III pressure ulcers and explain their effects on wound healing. The sample comprised 8 patients with stage III pressure ulcers, along with 4 volunteers with normal skin who were undergoing elective surgical procedures. Eight patient tissue specimens from wound of stage III pressure ulcers were obtained from hospitalized patients. Tissue specimens from 4 volunteer patients were obtained during surgery. All 12 tissue specimens were analyzed for presence of apoptosis-related factors including bax/bcl-2 (B-cell lymphoma 2), Fas/fasL (Fas ligand), and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) as well as immunohistochemical analysis with Fas/FasL. Bax positive cells were strongly expressed from surrounding zone to center zone in the specimens obtained from stage III pressure ulcers, and Bcl-2 expression staining decreased contrarily (P < .01). Comparison, apoptotic factors bax positive cell and fas/fasL mRNA expression occurred rarely in tissue specimens from the control group. Fas/fasL mRNA-positive granules were detected in epidermal cells and fibroblasts, demonstrating a progressive increase from the margin of the pressure ulcers progressing toward the center of the wound bed. We also found an increased intensity of TUNEL staining in ulcer center as well as wound margin, compared with control skin tissue. These results suggest that molecular regulators of apoptosis are involved in pressure ulcer wound healing. Results also suggest that differences in the expression and distribution of the apoptotic related factors in stage III pressure ulcers play an important role in the delayed wound healing characteristic of these complex wounds.

PEP-1–metallothionein-III protein ameliorates the oxidative stress-induced neuronal cell death and brain ischemic insults

BackgroundOxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult. MethodsA human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1–MT-III protein. PEP-1–MT-III protein was purified using affinity chromatograph. Transduced PEP-1–MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry. ResultsPurified PEP-1–MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1–MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1–MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1–MT-III protein protects against oxidative stress induced cell death in vitro and in vivo. General significanceTransduced PEP-1–MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1–MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease.

Therapeutic administration of IL-11 exhibits the postconditioning effects against ischemia-reperfusion injury via STAT3 in the heart

Activation of cardiac STAT3 by IL-6 cytokine family contributes to cardioprotection. Previously, we demonstrated that IL-11, an IL-6 cytokine family, has the therapeutic potential to prevent adverse cardiac remodeling after myocardial infarction; however, it remains to be elucidated whether IL-11 exhibits postconditioning effects. To address the possibility that IL-11 treatment improves clinical outcome of recanalization therapy against acute myocardial infarction, we examined its postconditioning effects on ischemia/reperfusion (I/R) injury. C57BL/6 mice were exposed to ischemia (30 min) and reperfusion (24 h), and IL-11 was intravenously administered at the start of reperfusion. I/R injury mediated the activation of STAT3, which was enhanced by IL-11 administration. IL-11 treatment reduced I/R injury, analyzed by triphenyl tetrazolium chloride staining [PBS, 46.7 ± 14.4%; IL-11 (20 μg/kg), 28.6 ± 7.5% in the ratio of infarct to risk area]. Moreover, echocardiographic and hemodynamic analyses clarified that IL-11 treatment preserved cardiac function after I/R. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining revealed that IL-11 reduced the frequency of apoptotic cardiomyocytes after I/R. Interestingly, IL-11 reduced superoxide production assessed by in situ dihydroethidium fluorescence analysis, accompanied by the increased expression of metallothionein 1 and 2, reactive oxygen species (ROS) scavengers. Importantly, with the use of cardiac-specific STAT3 conditional knockout (STAT3 CKO) mice, it was revealed that cardiac-specific ablation of STAT3 abrogated IL-11-mediated attenuation of I/R injury. Finally, IL-11 failed to suppress the ROS production after I/R in STAT3 CKO mice. IL-11 administration exhibits the postconditioning effects through cardiac STAT3 activation, suggesting that IL-11 has the clinical therapeutic potential to prevent I/R injury in heart.

Effect of valsartan on the pathological progression of hepatic fibrosis in rats with type 2 diabetes

Currently there is no effective treatment for nonalcoholic fatty liver disease (NAFLD), especially hepatic fibrosis induced by type 2 diabetes. Valsartan maybe has beneficial effect on the liver disease. The aim of the present study was to investigate the effect of valsartan on the pathological progression of hepatic fibrosis in rats with type 2 diabetes. An animal model of hepatic fibrosis with type 2 diabetes was developed using a high-sucrose, high-fat diet and low-dose streptozotocin. Valsartan (15mg/kg/day, i.g.) was orally administered for four months. The livers were removed to make hematoxylin–eosin (HE) staining and Picric acid–Sirius red staining, and immunohistochemistry staining of α-smooth-muscle-actin (α-SMA), transforming growth factor β1 (TGF-β1), tumor necrosis factor (TNF-α) and monocyte chemotactic protein-1 (MCP-1). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed to detect hepatocyte apoptosis. The liver mitochondria were isolated to measure the mitochondrial respiratory function. The results showed that valsartan significantly alleviated the lesion of hepatic steatosis and hepatic fibrosis by HE staining and Picric acid–Sirius red staining. Immunohistochemical staining suggested that the expression of α-SMA, TGF-β1, TNF-α and MCP-1 in liver tissue of diabetic rats was markedly reduced by valsartan. TUNEL staining showed that there were fewer TUNEL-positive apoptotic hepatocytes in valsartan group. In addition, valsartan restored the injured hepatic mitochondrial respiratory function. The findings demonstrated that valsartan prevented the pathological progression of hepatic fibrosis in type 2 diabetic rats, correlated with reducing α-SMA, TGF-β1, TNF-α and MCP-1 expression, also anti-apoptosis and mitochondria-protective potential.

Expression of AMPA receptor subunits in hippocampus after status convulsion

The expression of 2-amino-3-(5-methyl-3-oxo-1, 2-oxazol-4-yl) propanoic acid receptor (AMPAR) subunits in the hippocampus of naive immature and adult rats (IRs, ARs) was investigated after status convulsion (SC). Seizures were induced in IRs and ARs with intraperitoneal injections of lithium and pilocarpine. Rats were killed at four time points (3h, 1day, 3days, and 7days) after SC. The proportion of apoptotic cells was quantified by Annexin V-FITC apoptosis detection. The location and type of apoptotic cells were assessed by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Immunoblotting techniques were used to demonstrate changes in AMPAR subunit expression. Severe seizures induced neuronal apoptosis in the hippocampus. The proportion of apoptotic cells in IRs was consistently lower than that in ARs after SC. The expressions of four AMPAR subunits in IRs were consistently lower than those in ARs before and after SC. SC for 1h inhibited the expression of glutamate receptors (GluR1-4) in the hippocampus of IRs and ARs and altered the subunit composition of AMPARs. GluR2 was the predominant AMPAR subunit in the hippocampus of normal ARs, while the GluR2/3 subunits were predominantly expressed 7days after SC. GluR3/4 subunits were mainly expressed in the hippocampus of normal IRs, which had the lowest levels of GluR2. Immature brain was more resistant to seizure-induced neural damage. The time course of reduction and recovery differed for each subunit and was dependent on developmental stage. The increased expression of GluR2 could confer early but transient protection in the immature brain after SC.

The neuroprotective role of TERT via an antiapoptotic mechanism in neonatal rats after hypoxia–ischemia brain injury

Telomerase reverse transcriptase (TERT) can regulate cell apoptosis and proliferation. It has been shown that TERT expression can be induced in models of adult brain ischemia. In the present study, we investigated the role of TERT in ischemic neuronal death in neonatal hypoxic–ischemic rats model. Postnatal day 10 Sprague-Dawley rats were used to establish hypoxia–ischemia (HI) model and hypoxia alone (H) model. Pups were killed at 4, 8, 12, 24, or 48h after the insult. Plasmid containing mock, TERT antisense or sense fragment mixed with Fugene HD was injected to the right lateral ventricle immediately after the insult respectively. Additional injection was performed after 24h. Pups were sacrificed 24h after the administration. TERT and cleaved caspase-3 (CC3) expression were measured by Western blot. Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. We found that H/HI treatment induced neuronal apoptosis and expression of TERT and CC3. However, TERT was higher in H than in HI pups whereas CC3 and apoptosis were opposite, TERT antisense plasmid markedly attenuated TERT expression induced by HI, upregualted CC3 expression, and increased apoptosis. Our results indicate that TERT might function as an anti-apoptotic protein by inhibiting activation of caspase-3, while further studies are needed to evaluate underlying mechanisms.

Induction of apoptosis in Eμ-myc lymphoma cells in vitro and in vivo through calpain inhibition

Calpains are cysteine proteases that have been implicated as both effectors and suppressors of apoptosis. Previously, we showed that c-myc transformation regulated calpain activity and sensitized cells to apoptosis induced by calpain inhibition. The objective of this study was to investigate the role of calpain in the Eμ-myc transgenic model of B-cell lymphoma. Calpain activity assays, apoptosis, cell cycle assays, and expression measurements were used to determine the activity and role of calpain in vitro and in vivo. We found that Eμ-myc transgenic cells have highly elevated calpain activity. Calpastatin, the negative calpain regulator, was expressed at much lower levels in Eμ-myc lymphoma cells compared to normal splenic B cells. The primary isoform in Eμ-myc lymphoma is calpain 1. Treatment of Eμ-myc lymphoma cells with the calpain inhibitors PD150606 or calpain inhibitor III induced caspase-3-dependent apoptosis in vitro. General caspase inhibitors or caspase-3/7 inhibitor protected cells from death induced by calpain inhibitor, whereas caspase-9 inhibitors failed to rescue cells. Human Burkitt's lymphoma (BL2) cells display a pattern of sensitivity and caspase-3 dependence similar to calpain inhibition. Treatment of Eμ-myc lymphoma-bearing mice with PD150606 inhibited calpain activity in vivo and induced cell death in these cells as determined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Multiple daily treatments resulted in reduced tumor load, particularly in combination with etoposide. In conclusion, calpain is highly elevated in the Eμ-myc lymphoma and calpain inhibition has therapeutic potential.

Resveratrol attenuates brain damage in a rat model of focal cerebral ischemia via up-regulation of hippocampal Bcl-2

A number of studies have demonstrated that resveratrol (Res), a natural polyphenol compound found in plants, shows potent neuroprotective, anti-inflammatory and antioxidant effects; however, its ability to prevent ischemia-induced brain damage remains unclear. Here we tested whether Res played a neuroprotective role in a rat brain ischemia model induced by middle cerebral artery occlusion (MCAO). Adult male rats were randomly assigned into four experimental groups: sham operation (sham), ischemia treatment (MCAO), Res-treated MCAO (Res+MCAO) and Res alone group (Res+sham). The brain damage size and hippocampal apoptotic neurons in each rat were evaluated by triphenyltetrazolium chloride (TTC) staining and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining, respectively. Long-term potentiation (LTP) induced by high-frequency stimulation (HFS) in the hippocampus was assessed with extracellular recording. The expression of apoptosis-related proteins, i.e., Bcl-2 and Bax, in the hippocampus was detected by western blot. Our results revealed that Res treatment significantly reduced brain infarct volume of MCAO rats as compared to MCAO rats without Res treatment. A significant increase in TUNEL-positive neurons in the hippocampal CA1 region was visualized in the MCAO rats as compared to that of the sham group, but this increase was attenuated with Res treatment. Functionally, extracellular recordings revealed that MCAO operation impaired LTP in the hippocampal CA1 region and the basal synaptic transmission between the Schaffer collaterals, whereas Res treatment rescued the impaired LTP and facilitated synaptic transmission in the CA1 region of the MCAO rats. Res treatment increased the expression of anti-apoptotic protein Bcl-2 and decreased the expression of pro-apoptotic protein Bax in the MCAO rats. The findings suggest that Res can attenuate the deleterious effects of focal cerebral ischemia/reperfusion-induced brain injury and function as a potential neuroprotective agent. The neuroprotective qualities of Res, based on our data, may be attributable to the up-regulation of Bcl-2 expression and down-regulation of Bax expression.

Testosterone improves cardiac function and alters angiotensin II receptors in isoproterenol-induced heart failure

The renin-angiotensin-aldosterone system is known to play an important role in the pathophysiology and development of heart failure. Several studies have reported the benefits of testosterone in heart failure. However, the mechanisms of testosterone-induced effects on heart failure require further study. To determine the effects of castration and testosterone administration on cardiac function and angiotensin II receptor function in rats with isoproterenol-induced heart failure. Wistar rats were divided randomly into control and heart failure groups. The heart failure groups were further divided into the following groups: castration; castration+testosterone replacement; and sham castration. Echocardiography and haemodynamic measurements were used to evaluate cardiac function. Cardiocyte apoptosis and fibrosis were determined using terminal deoxyribonucleotide transferase-mediated dUTP nick-end labelling (TUNEL) staining and Masson's Trichrome staining, respectively. Angiotensin II receptor (AT1 and AT2) messenger ribonucleic acid (mRNA) expression levels were assayed using real-time reverse transcriptase-polymerase chain reactions, while Western immunoblotting was used to estimate Bcl-2 protein expression levels. Castration significantly increased cardiomyocyte apoptosis and fibrosis that was normally induced by isoproterenol (P<0.05). AT2 receptor mRNA expression in the castration group was increased and Bcl-2 protein expression was decreased compared with the castration+testosterone replacement group (P<0.05). These data suggest that androgen therapy could play an important role in pathophysiological changes in heart failure and have beneficial effects for its treatment.

The Neuroprotective Effect of Agmatine After Focal Cerebral Ischemia in Diabetic Rats

Diabetes mellitus is a metabolic disorder associated with structural and functional alterations of various organ systems including the central nervous system. The aim of present study was to investigate the neuroprotective effect of agmatine (AGM) on cerebral ischemic damage in diabetic rats. Normoglycemic (n=30) and streptozocine-induced diabetic rats (n=82) were subjected to 30 minutes of suture-occlusion of the middle cerebral artery (MCAO) with 24 or 72 hours of reperfusion. Thirty-nine diabetic rats were treated with AGM (100 mg/kg, intraperitoneal) immediately after 30 minutes of MCAO. To evaluate the motor function, a modified neurological examination and rota-rod exercise were performed. The brain infarct volume and edema volume were assessed. Caspase-3 activity and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining were used to evaluate cellular apoptosis. Western blot and immunohistochemical analysis were performed to determine the expression of neuronal nitric oxide synthase (NOS) and inducible NOS in ischemic brain tissues. AGM posttreatment improved the neurobehavioral activity of diabetic MCAO rats at 24 and 72 hours after reperfusion. The infarct size and edema volume were reduced in AGM-treated diabetic rats compared with those in diabetic rats without AGM posttreatment (P<0.01). Immunohistochemical analysis showed that AGM treatment significantly decreased the number of caspase-3-positive and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells in diabetic MCAO rats at 24 and 72 hours after reperfusion (P<0.01). Western blotting and immunohistochemistry results indicated that AGM treatment significantly decreased neuronal NOS and inducible NOS expression in diabetic rats at 24 and 72 hours after reperfusion (all P<0.05). AGM posttreatment reduced cerebral infarct size and neurological deficit expression in diabetic rats subjected to MCAO. The reduced infarct size was associated with a decrease in apoptosis and NOS expression.

CIP-13F, a novel aminopeptidase N (APN/CD13) inhibitor, inhibits Lewis lung carcinoma growth and metastasis in mice

Aminopeptidase N (APN/CD13) is highly expressed on the surface of cancer cells and is thought to be involved in cancer growth and metastasis. The research of APN/CD13 inhibitors is considered as a strategy of cancer treatment. We aimed to evaluate the efficacy of CIP-13F, a novel APN/CD13 inhibitor, using a Lewis lung carcinoma (LLC) implantation mouse model. C57BL/6 mice were subcutaneously inoculated with LLC cells in anterior flank. Then, 0, 50 and 100 mg/kg of CIP-13F were injected via vena caudalis. Bestatin was used as the positive control. Administration of CIP-13F or bestatin was performed daily for 3 consecutive weeks. Mice were killed, and the tumors in anterior flank and metastasis nodules in lungs were examined. The assays of immunohistochemical staining, immunofluorescent flow cytometry and western blotting were performed to estimate the expression of APN/CD13 in LLC cells. We carried out the experiments of Annexin-V/PI staining, DNA fragmentation analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining to determine apoptotic cells in LLC tissues. Using immunohistochemical staining with CD34, the antiangiogenesis of CIP-13F was evaluated in LLC tissue sections. CIP-13F treatment resulted in a significant delay of LLC growth in anterior flank. Examination of lungs showed that the number of metastatic nodules of LLC was also markedly decreased. The inhibitory effect of CIP-13F on LLC growth was further evidenced by the induction of LLC apoptosis, showing the increases in Annexin-V/PI staining cells, DNA fragmentation and TUNEL staining cells. Molecular analyses of LLC tissues in CIP-13F-treated mice suggested that the decrease in APN/CD13 expression by CIP-13F might account for its actions of mechanism. Further, the inhibition of angiogenesis in LLC tissues was determined, showing the decreases in microvessel density (MVD) and angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF-α). Our results showed that CIP-13F effectively inhibited LLC growth and pulmonary metastasis in mice and suggested that CIP-13F is a potential drug for the treatment for cancers with positive APN/CD13 expression.

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.

The Central Nervous System Is a Target Organ of Acute Graft-Versus-Host Disease

Abstract 1895One of the major limitations of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is graft-versus-host disease (GVHD), which is a severe and common complication that impacts on multiple organ systems with varying degrees of severity. Neurological complications such as encephalopathy, meningoencephalitis, cerebrovascular disease, demyelination and in particular neurocognitive deficits, have been reported in patients after allo-HSCT but their etiology remains in most cases poorly understood. In this study we analyzed whether the central nervous system (CNS) is a target of acute GVHD.We used a murine MHC-disparate allo-HSCT model to study cerebral involvement in GVHD: C57BL/6 (B6) (H-2b) into lethally irradiated (850 cGy, split dose) BALB/c (H-2d). The control group received a syngeneic BMT (syn-HSCT) with BALB/c into BALB/c.CNS infiltrating lymphocytes were isolated from brains after complete perfusion of the animals. We found increasing numbers of CNS-infiltrating donor derived CD4 and CD8 alloreactive T cells on day 7, 14, and 21 (p<0.001) in allo-HSCT recipients, but almost none in syn-HSCT controls.In addition, in order to assess cytokines and homing molecules necessary for CNS infiltration in GVHD, we performed competitive BMTs by mixing control B6 (CD45.1) with either WT B6, IL17−/−, IL21R−/−, or L-selectin−/− (all CD45.2) in a 1:1 ratio. We found statistically significant and reproducibly decreased numbers of infiltrating T cells using IL21R−/− donor T cells, which we have recently reported to mediate decreased systemic GVHD and gastrointestinal infiltration as well.To confirm the cerebral involvement in situ, histological analysis and immunohistochemistry for CD3, CD4 and TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) on coronal brain sections from perfused animals were performed. We observed infiltration of T cells in Cortex, Hippocampus, Midbrain (Basal ganglia, Thalamus, Hypothalamus), Cerebellum, and Medulla oblongata of allo-HSCT recipients, which was consistent with lymphocytic multifocal meningoencephalitis. Sections from syn-HSCT recipients showed no pathology and T cells were virtually undetectable (p<0.01). Apoptotic cell death by TUNEL staining was only observed in allo-HSCT recipients.To test whether these histopathological findings were associated with cognitive deficits, we performed behavioral tests at 14 to 20 days after HSCT: Locomotor activity was evaluated in the accelerated rotorod (latency to fall) and open field (total distance moved) tests; neuromuscular function and strength were tested in the grip test (latency to fall); anxiety-related behavior and exploratory activity were tested in the open field (distance moved/time spent in center) and in the elevated plus maze (time spent in open arm); and spatial learning and memory were analyzed in the Morris water maze. Although allo-HSCT recipients had developed mild to moderate GVHD in the first three weeks after transplant as measured by GVHD score and weight loss, we found no differences between allo-HSCT versus syn-HSCT recipients or non-transplanted mice in locomotor activity, motor coordination, strength and neuromuscular function. However, allo-HSCT recipients demonstrated increased anxious behavior and decreased exploratory activity in the open field test (p<0.01). Importantly, spatial learning was impaired in allo-HSCT recipients as compared to both syn-HSCT recipients and non-transplanted controls (p<0.0001). Of note, both the decreased exploratory behaviour and the spatial learning defect were not due to a lack of mobility of allo-HSCT recipients, because the allo-HSCT recipients covered similar distances in both tests as the syn-HSCT recipients.Taken together these findings demonstrate that the CNS can be a target organ of acute GVHD in allo-HSCT recipients, which is associated with early donor T cell infiltration, histopathology consistent with lymphocytic multifocal meningoencephalitis and specific cognitive defects (increased anxious behaviour and decreased spatial learning), which are not due to lack of mobility. Further studies, particularly in the clinical setting, will reveal the extent of GVHD-related cognitive defects and may lead to new approaches to diagnose and treat a new aspect of GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Targeted Systemic Radiotherapy with scVEGF/177Lu Leads to Sustained Disruption of the Tumor Vasculature and Intratumoral Apoptosis

Tumor vessels abundantly express receptors for vascular endothelial growth factor (VEGF), despite treatment with conventional or antiangiogenic drugs. We wished to determine whether the high levels of VEGF receptor (VEGFR) within the tumor vasculature could be leveraged for intracellular delivery of therapeutically significant doses of scVEGF/(177)Lu, a novel radiopharmaceutical based on a recombinant single-chain (sc) derivative of VEGF, in orthotopic breast cancer models. scVEGF-PEG (polyethylene gycol)-DOTA conjugates containing 2.0-, 3.4-, or 5.0-kDa PEG linkers site-specifically conjugated to a cysteine-containing tag (Cys-tag) in scVEGF were radiolabeled with (177)Lu (scVEGF/(177)Lu) for in vivo studies. Human MDA231luc and mouse 4T1luc cell lines were injected orthotopically to establish breast carcinoma tumors in immunodeficient and immunocompetent hosts, respectively. The effects of scVEGF/(177)Lu were defined by analysis of changes in tumor growth and immunohistochemical staining for the endothelial markers CD31 and VEGFR-2 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining for intratumoral apoptosis. Biodistribution assays and dosimetric calculations established that scVEGF/(177)Lu with a 3.4-kDa PEG linker delivered the highest dose of radiation to tumors (69.9 cGy/MBq/g of tissue) and the lowest dose to the kidneys (33.3 cGy/MBq/organ). Total doses below 40 MBq/mouse of scVEGF/(177)Lu did not affect renal function, and 3 divided doses of 6.3 MBq/mouse or a bolus dose of 18.9 MBq/mouse induced only transient lymphopenia and weight loss (<10% baseline weight). In mice with orthotopic mammary breast carcinoma, intravenous injections of well-tolerated bolus and fractionated doses of scVEGF/(177)Lu in the range from 6.3 to 18.9 MBq/mouse (25-76 MBq/m(2)) resulted in dose-dependent tumor growth inhibition. Immunohistochemical analysis of tumors at 4-5 wk after single injections of scVEGF/(177)Lu indicated dose-dependent regression of tumor vasculature and widespread intratumoral apoptosis. A single dose of 7.4 MBq/mouse of scVEGF/(177)Lu given before a course of bevacizumab or sunitinib treatment enhanced the antiangiogenic effects of both drugs. Selective targeting of VEGFR in tumor vasculature with well-tolerated doses of scVEGF/(177)Lu is effective in orthotopic breast cancer models. As high levels of VEGFR expression in the tumor vasculature are a common feature in a variety of cancers, targeting tumor angiogenesis with scVEGF/(177)Lu warrants further exploration.

Human Retinal Development in an in situ Whole Eye Culture System

Phenotypic characterization of human retinogenesis may be facilitated by use of the tissue culture system paradigm. Traditionally, most culture protocols involve isolation of retinal tissue and/or cells, imposing various degrees of trauma, which in many cases leads to abnormal development. In this paper, we present a novel culture technique using whole embryonic eyes to investigate whether the retina in situ can develop normally in vitro. All procedures were carried out in accordance with the Declaration of Helsinki. Human embryos were obtained from elective abortions with the informed consent of the women seeking abortion. A total of 19 eyes were enucleated. The ages of the embryonic retinas were 6–7.5 weeks. Eyecups from 2 eyes were fixed immediately, to be used as controls. The remaining 17 eyes were placed on culture plates and divided into 3 groups kept for 7 (n = 4), 14 (n = 7) and 28 (n = 6) days in vitro (DIV). After fixation, the specimens were processed for hematoxylin and eosin staining, immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Antibodies against recoverin (rods and cones), protein kinase C (PKC; rod bipolar cells) and vimentin (Müller cells) were used. TUNEL was used to detect apoptotic cells. In hematoxylin- and eosin-stained sections, the control retinas displayed a neuroblast cell layer (NBL) and an inner marginal zone. Specimens kept 7–14 DIV had a similar appearance, while 28-day specimens consisted of an NBL with almost no marginal zone. Thirteen of the 17 cultured retinas displayed completely normal lamination without rosettes or double folds. Pyknotic cells were found at the inner margin of the retinas, and the proportion of these cells increased with time in vitro. TUNEL staining revealed a few scattered cells in 7-DIV specimens, and the amount of stained cells in the inner part of the retinas progressively increased in 14- and 28-DIV specimens. Vimentin labeling showed cells arranged in a vertical pattern in all retinas. Labeling with recoverin revealed photoreceptors in 4 of the retinas kept for 14 DIV, and in all retinas kept for 28 DIV. After 28 DIV, 2 of the eyes labeled with PKC contained rod bipolar cells with minimal axons. Here we showed that human embryonic retinas can be kept in culture in situ within the eye for at least 4 weeks. Abnormal lamination is not as frequent as in isolated full-thickness retinas, indicating that physical and biochemical contact with surrounding tissues is vital for proper development. Several types of the retina-specific neuronal and glial cells were seen to differentiate according to the in vivo schedule. The results are important for future studies of retinal development, and the technique can also be used for testing the effects of various drugs on the immature retina.

Vasoactive intestinal peptide protects against ischemic brain damage induced by focal cerebral ischemia in rats

Vasoactive intestinal peptide (VIP) exerts neuroprotective effects under various neurotoxic conditions in vitro. In the present study, we investigated the effects of VIP on transient ischemic brain damage. Focal cerebral ischemia was induced using middle cerebral artery occlusion (MCAO) for 120 min in the adult rat brain. Either a single intracerebroventricular injection of VIP or saline was given at the beginning of reperfusion. Forty-eight hours after MCAO, the rats were sacrificed for evaluation of the infarct volume and histological analysis. ELISA was performed to assay levels of serum S100B before being sacrificed. We also evaluated the blood–brain barrier (BBB) permeability using Evans blue dye injection method. In contrast to the cases treated with vehicle, the infarct volume was significantly ( P < 0.05) reduced, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining and immunoreactivity for S100B were also significantly ( P < 0.05) decreased in the ischemic hemisphere with VIP treatment. In addition, the elevations of serum S100B were significantly ( P < 0.01) attenuated in VIP-treated rats compared with those of control rats. Treatment with VIP did not result in a significant reduction of Evans blue leakage, although it tended to be lower than that in the control rats. Our data suggest that treatment with VIP reduces brain damage in ischemic rats, and this effect may be associated with the attenuation of apoptosis and S100B expression.