Apolipoprotein A-I mimetic peptide L-4F prevents myocardial and coronary dysfunction in diabetic mice

Diabetes is a major health problem associated with adverse cardiovascular outcomes. The apolipoprotein A-I mimetic peptide L-4F is a putative anti-diabetic drug, has antioxidant and anti-inflammatory proprieties and improves endothelial function. In obese mice L-4F increases adiponectin levels, improving insulin sensitivity, and reducing visceral adiposity. We hypothesized that the pleiotropic actions of L-4F can prevent heart and coronary dysfunction in a mouse model of genetically induced Type II diabetes. We treated db/db mice with either L-4F or vehicle for 8 weeks. Trans-thoracic echocardiography was performed; thereafter, isolated hearts were subjected to ischemia/reperfusion (IR). Glucose, insulin, adiponectin, and pro-inflammatory cytokines (IL-1β, TNF-α, MCP-1) were measured in plasma and HO-1, pAMPK, peNOS, iNOS, adiponectin, and superoxide in cardiac tissue. In db/db mice L-4F decreased accumulation of subcutaneous and total fat, and increased insulin sensitivity and adiponectin levels while lowering inflammatory cytokines (P < 0.05). L-4F normalized in vivo left ventricular (LV) function of db/db mice, increasing (P < 0.05) fractional shortening and decreasing (P < 0.05) LV dimensions. In I/R experiments, L-4F prevented coronary microvascular resistance from increasing and LV function from deteriorating in the db/db mice. These changes were associated with increased cardiac expression of HO-1, pAMPK, peNOS, and adiponectin and decreased levels of superoxide and iNOS (P < 0.01). In the present study we showed that L-4F prevented myocardial and coronary functional abnormalities in db/db mice. These effects were associated with stimulation of HO-1 resulting in increased levels of anti-inflammatory, anti-oxidative, and vasodilatatory action through a mechanism involving increased levels of adiponectin, pAMPK, and peNOS.     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications

Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposome-DNA complexing time, and the presence or absence of media components such as antibiotics and serum. The importance of these factors in Lipofectamine 2000 mediated transfection will be discussed together with some specific applications: transfection of primary neurons, high throughput transfection, and delivery of small interfering RNAs.     

Effects of Lipofectamine 2000/siRNA Complexes on Autophagy in Hepatoma Cells

Lipofectamine 2000 is commonly used for siRNA transfections. However, few studies have examined cellular responses to this delivery system. The purpose of this study is to evaluate the effect of siRNA transfection using Lipofectamine 2000 on cellular autophagy. Huh7.5 cells, stably transfected to express GFP–LC3, were treated with Lipofectamine 2000/negative control siRNA (NC siRNA) complexes. At different time points after treatment, cells were lysed and analyzed by immunoblotting and fluorescence spectroscopy. Cells were also observed using confocal microscopy. An increase of endogenous LC3 lipidation, GFP–LC3 fluorescence, and autophagosomal puncta was observed in cells treated with Lipofectamine 2000/NC siRNA complexes. The kinetics of the increase of GFP–LC3 fluorescence correlated with the concentration of NC siRNA transfected, where 50, 100, and 200 nM NC siRNA caused a significant increase at 72, 48, and 24 h, respectively, after transfection. A similar effect on the GFP–LC3 signal was also observed for cells treated with Lipofectamine 2000 complexed with two other NC siRNAs. The effects were also confirmed in another hepatoma cell line, H4IIE, by immunoblotting. Lipofectamine 2000-mediated transport of NC siRNAs led to an increase of autophagosomes in a dose- and time-dependent manner. Thus, this effect on cells should be taken into consideration when using this approach for intracellular delivery of siRNA.     

Micro-PET imaging of alphavbeta3-integrin expression with 18F-labeled dimeric RGD peptide

The alphav integrins, which act as cell adhesion molecules, are closely involved with tumor invasion and angiogenesis. In particular, alphavbeta3 integrin, which is specifically expressed on proliferating endothelial cells and tumor cells, is a logical target for development of a radiotracer method to assess angiogenesis and anti-angiogenic therapy. In this study, a dimeric cyclic RGD peptide E[c(RGDyK)]2 was labeled with 18F (t(1/2) = 109.7 min) by using a prosthetic 4-[18F]fluorobenzoyl moiety to the amino group of the glutamate. The resulting [18F]FB-E[c(RGDyK)]2, with high specific activity (200-250 GBq/micromol at the end of synthesis), was administered to subcutaneous U87MG glioblastoma xenograft models for micro-PET and autoradiographic imaging as well as direct tissue sampling to assess tumor targeting efficacy and in vivo kinetics of this PET tracer. The dimeric RGD peptide demonstrated significantly higher tumor uptake and prolonged tumor retention in comparison with a monomeric RGD peptide analog [18F]FB-c(RGDyK). The dimeric RGD peptide had predominant renal excretion, whereas the monomeric analog was excreted primarily through the biliary route. Micro-PET imaging 1 hr after injection of the dimeric RGD peptide exhibited tumor to contralateral background ratio of 9.5 +/- 0.8. The synergistic effect of polyvalency and improved pharmacokinetics may be responsible for the superior imaging characteristics of [18F]FB-E[c(RGDyK)]2.     

A novel bovine lactoferrin peptide, FKCRRWQWRM, suppresses Candida cell growth and activates neutrophils.

To identify potent new antifungal agents, the Candida cell growth inhibitory activities of six lactoferrin (Lf) peptides consisting of 6-25 amino acid residues (peptide 1, FKCRRWQWRMKKLGAPSITCVRRAF lactoferricin B; peptide 2, FKCRRWQWRM; peptide 2', FKARRWQWRM; peptide 3, GAPSITCVRRAF; peptide 4, RRWQWR; and peptide 5, RWQWRM) were examined. Of these, peptide 2 strongly suppressed the multiplication of Candida cells, but other peptides showed only weak activities. In two strains of C. albicans, the minimum inhibitory concentration 100 of peptide 2 (17.3+/-2.2 microM and 17.5+/-2.4 microM) was close to that of miconazole (13.0+/-1.7 microM and 13.1+/-1.6 microM) but markedly different from that of amphotericin B (0.52+/-0.09 microM and 0.56+/-0.11 microM). The suppression of Candida cell growth was additively increased by a combination of peptide 2 with amphotericin B and miconazole. Peptides 1, 3, 4 and 5 and Lf suppressed iron uptake by Candida cells, inversely correlated with their Candida cell growth inhibition activities. However, iron uptake was not inhibited by peptide 2. In addition, peptide 2 upregulated Candida cell killing activity of polymorphonuclear leukocytes (PMN) increasing their superoxide generation, protein kinase C activity, p38 MAPK activity and the expression of p47phox. These results indicated that the main antimicrobial activity of the Lf peptides is dependent on the N-terminal half of Lf and that the PMN upregulatory activity of peptide 2 and additive function of peptide 2 with antifungal drugs are useful for prophylaxis and control of candidiasis.     

A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids

Administered subcutaneously, D-4F or L-4F are equally efficacious, but only D-4F is orally efficacious because of digestion of L-4F by gut proteases. Orally administering niclosamide (a chlorinated salicylanilide used as a molluscicide, antihelminthic, and lampricide) in temporal proximity to oral L-4F (but not niclosamide alone) in apoE null mice resulted in significant improvement (P < 0.001) in the HDL-inflammatory index (HII), which measures the ability of HDL to inhibit LDL-induced monocyte chemotactic activity in endothelial cell cultures. Oral administration of L-[113-122]apoJ with niclosamide also resulted in significant improvement (P < 0.001) in HII. Oral administration of niclosamide and L-4F together with pravastatin to female apoE null mice at 9.5 months of age for six months significantly reduced aortic sinus lesion area (P = 0.02), en face lesion area (P = 0.033), and macrophage lesion area (P = 0.02) compared with pretreatment, indicating lesion regression. In contrast, lesions were significantly larger in mice receiving only niclosamide and pravastatin or L-4F and pravastatin (P < 0.001). In vitro niclosamide and L-4F tightly associated rendering the peptide resistant to trypsin digestion. Niclosamide itself did not inhibit trypsin activity. The combination of niclosamide with apolipoprotein mimetic peptides appears to be a promising method for oral delivery of these peptides.     

F2L, a peptide derived from heme-binding protein, inhibits LL-37-induced cell proliferation and tube formation in human umbilical vein endothelial cells

F2L, a peptide derived from heme-binding protein, was originally identified as an endogenous ligand for formyl peptide receptor-like (FPRL)2. Previously, we reported that F2L inhibits FPR and FPRL1-mediated signaling in neutrophils. Since endothelial cells express functional FPRL1, we examined the effect of F2L on LL-37 (an FPRL1 agonist)-induced signaling in human umbilical vein endothelial cells (HUVECs). F2L stimulated the chemotactic migration in HUVECs. However, F2L inhibited FPRL1 activity, resulting in the inhibition of cell proliferation and tube formation induced by LL-37 in HUVECs. We suggest that F2L will potentially be useful in the study of FPRL1 signaling and the development of drugs to treat diseases involving the FPRL1 in the vascular system.     

Relationship between muscle sympathetic nerve activity and systemic hemodynamics during nitric oxide synthase inhibition in humans

Large interindividual differences exist in resting sympathetic nerve activity (SNA) among normotensive humans with similar arterial pressure (AP). We recently showed inverse relationships of resting SNA with cardiac output (CO) and vascular adrenergic responsiveness that appear to balance the influence of differences in SNA on blood pressure. In the present study, we tested whether nitric oxide (NO)-mediated vasodilation has a role in this balance by evaluating hemodynamic responses to systemic NO synthase (NOS) inhibition in individuals with low and high resting muscle SNA (MSNA). We measured MSNA via peroneal microneurography, CO via acetylene uptake and AP directly, at baseline and during increasing systemic doses of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Baseline MSNA ranged from 9 to 38 bursts/min (13 to 68 bursts/100 heartbeats). L-NMMA caused dose-dependent increases in AP and total peripheral resistance and reflex decreases in CO and MSNA. Increases in AP with L-NMMA were greater in individuals with high baseline MSNA (PANOVA<0.05). For example, after 8.5 mg/kg of L-NMMA, in the low MSNA subgroup (n=6, 28+/-4 bursts/100 heartbeats), AP increased 9+/-1 mmHg, whereas in the high-MSNA subgroup (n=6, 58+/-3 bursts/100 heartbeats), AP increased 15+/-2 mmHg (P<0.01). The high-MSNA subgroup had lower baseline CO and smaller decreases in CO with L-NMMA, but changes in total peripheral resistance were not different between groups. We conclude that differences in CO among individuals with varying sympathetic traffic have important hemodynamic implications during disruption of NO-mediated vasodilation.     

Effects of intracerebroventricular administration of D,L-2-amino-5-phosphonovaleric acid, an N-methyl-D-aspartate receptor antagonist, on luteininizing hormone release in ovariectomized lambs.

To determine whether endogenous glutamate and aspartate control LH secretion via N-methyl-D-aspartate (NMDA) receptors in the sheep, we evaluated the effects of the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (AP5) on secretion of LH in ovariectomized lambs. Twelve lambs were ovariectomized and surgically implanted with lateral cerebroventricular cannulae. At the time of the experiment, (38 wk of age) they received intracerebrally 4 injections of either 50 (n = 4), 100 (n = 4), or 200 micrograms (n = 4) of AP5. Blood samples were collected every 10 min for 8 h with animals receiving AP5 at hours 4, 5, 6, and 7. Patterns of LH during the preinjection period were compared to those during the period encompassing AP5 injections. Mean concentrations of LH were lower during AP5 injections than during the preinjection periods, a response that was not influenced by dose (0.87 +/- 0.08 vs. 0.69 +/- .07 ng/ml; p < 0.01). LH pulse amplitude decreased during AP5 treatment relative to the preinjection periods, but this difference was not statistically significant (0.79 +/- 0.11 vs. 0.68 +/- 0.10 ng/ml; p = 0.09). There were no effects of AP5 on LH pulse frequency (1.00 +/- 0.10 vs. 0.83 +/- 0.15 pulses/h for injection and preinjection periods; p > 0.10). A second experiment was done to evaluate a higher dose of AP5. Four animals were chosen to receive 4 injections of 2 mg of AP5 in a design identical to that used in the first experiment.     

鸡原始生殖细胞转染条件优化

为获得鸡原始生殖细胞(primordial germ cells,PGCs)的最佳转染效率,本研究比较不同质粒用量和不同细胞数在3种转染试剂(Lipofectamine 2000、3000和LTX&Plus Reagent)中PGCs的转染效率,利用荧光激活细胞分选技术(fluorescence activated cell sorting technology,FACS)辅助优化Lipofectamine 3000转染试剂,经FACS进一步分选获得带绿色荧光蛋白(GFP)的PGCs,继续培养3周后,移植回注到受体鸡胚中,移植3.5 d后分离性腺拍照观察。结果显示,转染试剂Lipofectamine 3000的转染效率最高,质粒、Lipofectamine 3000转染试剂和PGCs细胞数的配比为3μg:4μL:0.5×10⁴个,转染5h转染效率最高,达到23.4%,与现有的研究结果相比提高了2倍以上。移植回注PGCs到受体鸡胚中,荧光显微镜观察到鸡胚性腺中有GFP阳性细胞。本研究综合考虑转染试剂、质粒用量和细胞数量的影响因素以优化PGCs的转染条件,为高...     

F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling

F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of (125)I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits FPR- and FPRL1-mediated signaling, and is expected to be useful in the study of FPR and FPRL1 signaling and in the development of drugs to treat diseases involving the FPR family of receptors.     

PAR-2 activation at the time of reperfusion salvages myocardium via an ERK1/2 pathway in in vivo rat hearts

Protease-activated receptor-2 (PAR-2) may have proinflammatory effects in some tissues and protective effects in other tissues. The role of PAR-2 in in vivo myocardial ischemia-reperfusion has not yet been determined. This study tested the hypothesis that PAR-2 activation with the PAR-2 agonist peptide SLIGRL (PAR-2 AP) reduces myocardial infarct size when given at reperfusion in vivo, and this cardioprotection involves the ERK1/2 pathway. Anesthetized rats were randomly assigned to the following groups with 30 min of regional ischemia and 3 h reperfusion: 1) control with saline; 2) vehicle (DMSO); 3) PAR-2 AP, 1 mg/kg given intravenously 5 min before reperfusion; 4) scrambled peptide (SP), 1 mg/kg; 5) the ERK1/2 inhibitor PD-98059 (PD), 0.3 mg/kg given 10 min before reperfusion; 6) the phosphatidylinositol 3-kinase inhibitor LY-294002 (LY), 0.3 mg/kg given 10 min before reperfusion; 7) PD + PAR-2 AP, 0.3 mg/kg PD given 5 min before PAR-2 AP; 8) LY + PAR-2 AP, 0.3 mg/kg LY given 5 min before PAR-2 AP; 9) chelerythrine (Chel) alone, 5 mg/kg given 10 min before reperfusion; and 10) Chel + PAR-2 AP, Chel was given 5 min before PAR-2 AP (10 min before reperfusion). Activation of ERK1/2, ERK5, Akt, and the downstream targets of ERK1/2 [P90 RSK and bcl-xl/bcl-2-associated death promoter (BAD)] was determined by Western blot analysis in separate experiments. PAR-2 AP significantly reduced infarct size compared with control (36 +/- 2% vs. 53 +/- 1%, P < 0.05), and SP had no effect on infarct size (53 +/- 3%). PAR-2 AP significantly increased phosphorylation of ERK1/2, p90RSK, and BAD but not Akt or ERK5. Accordingly, the infarct-size sparing effect of PAR-2 AP was abolished by PD (PAR-2 AP, 36 +/- 2% vs. PD + PAR-2 AP, 50 +/- 1%; P < 0.05) and by Chel (Chel + PAR-2 AP, 58 +/- 2%) but not by LY (PAR-2 AP, 36 +/- 2% vs. LY + PAR-2 AP, 38 +/- 3%; P > 0.05). Therefore, PAR-2 activation is cardioprotective in the in vivo rat heart ischemia-reperfusion model, and this protection involves the ERK1/2 pathway and PKC.     

C-type natriuretic peptide for reduction of restenosis: gene transfer is superior over single peptide administration

BACKGROUND: Restenosis is still a significant clinical problem limiting the long-term therapeutic success following balloon dilation or stent implantation. New approaches are necessary inhibiting neointima formation and simultaneously promoting re-endothelialization. Therefore, long-term therapeutic effects of adventitial liposome-mediated C-type natriuretic protein (CNP) gene and CNP peptide applications in a porcine model for restenosis post-angioplasty were investigated. METHODS: For in vitro applications, primary cultures of porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were used. Gene transfer was performed with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N5-carbamoylspermine)propane]. In vivo treatment of pig femoral arteries was adventitial using a needle injection catheter following balloon angioplasty. Arteries were investigated by angiography, Evan's blue staining, histomorphometry, immunohistochemistry, PCR and RT-PCR. RESULTS: Using CNP gene transfer in vitro, 29.4+/-7.2% reduction of cell proliferation in VSMCs was observed. In ECs, the CNP gene did not compromise cellular growth. For the CNP peptide the optimal concentration was 1 mM with 50.7+/-11.3% reduction of VSMC proliferation and 12.1+/-5.3% enhancement of growth of ECs. Three weeks following application in vivo complete re-endothelialization was observed in all treated groups. At 3 months significant reduction of neointima formation was observed using CNP gene vs. CNP peptide (85.9+/-7.8% vs. 63.3+/-27.6% reduction, P<0.05) compared to control treatment. CONCLUSION: Periadventitial liposome-mediated CNP gene transfer in vivo resulted in a significant long-term reduction of neointima formation without compromising endothelial repair and was superior over single CNP peptide administration. Advantages of CNP are its physiological origin and simultaneous inhibition of VSMC proliferation and promotion of EC growth.     

Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.     

Atrial natriuretic peptide prevents diabetes-induced endothelial dysfunction

Atrial natriuretic peptide (ANP) exerts beneficial effects on the cardiovascular system in part by exerting antioxidant activity. Given that oxidant stress is a key cause of endothelial dysfunction in diabetes, we investigated whether ANP improves endothelial function in rats with diabetes. Rats were injected with streptozotocin (55 mg/kg iv) to induce type 1 diabetes or the citrate vehicle as controls (n=12). After 4 weeks the diabetic rats were treated with ANP (10 pmol/kg/min sc, n=12) or the antioxidant tempol (1.5 mmol/kg/day sc, n=11), both by osmotic minipump, ramipril (1 mg/kg per day in the drinking water) or remained untreated (n=11). After a further 4 weeks, anaesthetised rats were killed by exsanguination and the thoracic aortae collected for examination of vascular activity and measurement of superoxide generation. Diabetic rats showed elevated plasma glucose concentration (45+/-3 mM) compared to controls (10+/-1 mM) and this was not affected by ANP (43+/-3 mM), ramipril (41+/-2 mM) or tempol (43+/-2 mM). Endothelium-dependent relaxation ex vivo in response to acetylcholine was impaired in diabetic rats (Rmax=66+/-4%) compared to control rats (Rmax=94+/-1%) but treatment with ANP (Rmax=80+/-4%), ramipril (Rmax=88+/-2%) or tempol (Rmax=81+/-5%) significantly improved those responses. Relaxant responses to the endothelium-independent vasodilator sodium nitroprusside were enhanced by treatment of diabetic rats with ANP or ramipril and their combination; but not by tempol. Superoxide generation was significantly elevated in aorta from untreated diabetic rats (649+/-146% of control). In diabetic rats, superoxide generation was significantly attenuated by ANP (to 229+/-78%) or tempol (to 186+/-64%). This study demonstrates that ANP improves vascular oxidant stress in concert with endothelial function, independent of any effect on plasma glucose levels. These studies may lead to new therapies, based on natriuretic peptide and/or antioxidant approaches, for ameliorating the vascular complications of diabetes.