Scramble Peptide Research Articles

New Antimicrobial Peptide with Two CRAC Motifs: Activity against Escherichia coli and Bacillus subtilis.

Due to the emergence of multiple antibiotic resistance in many pathogens, the studies on new antimicrobial peptides (AMPs) have become a priority scientific direction in fundamental and applied biology. Diverse mechanisms underlie the antibacterial action of AMPs. Among them are the effects that AMPs cause on bacterial cell membranes. In this work, we studied the antibacterial activity of a peptide named P4 with the following sequence RTKLWEMLVELGNMDKAVKLWRKLKR that was constructed from two alpha-helical fragments of the influenza virus protein M1 and containing two cholesterol-recognizing amino-acid consensus (CRAC) motifs. Previously we have shown that 50 μM of peptide P4 is toxic to cultured mouse macrophages. In the present work, we have found that peptide P4 inhibits the growth of E. coli and B. subtilis strains at concentrations that are significantly lower than the cytotoxic concentration that was found for macrophages. The half-maximal inhibitory concentration (IC50) for B. subtilis and E. coli cells were 0.07 ± 0.01 μM and 1.9 ± 0.4 μM, respectively. Scramble peptide without CRAC motifs did not inhibit the growth of E. coli cells and was not cytotoxic for macrophages but had an inhibitory effect on the growth of B. subtilis cells (IC50 0.4 ± 0.2 μM). A possible involvement of CRAC motifs and membrane sterols in the mechanism of the antimicrobial action of the P4 peptide is discussed. We assume that in the case of the Gram-negative bacterium E. coli, the mechanism of the toxic action of peptide P4 is related to the interaction of CRAC motifs with sterols that are present in the bacterial membrane, whereas in the case of the Gram-positive bacterium B. subtilis, which lacks sterols, the toxic action of peptide P4 is based on membrane permeabilization through the interaction of the peptide cationic domain and anionic lipids of the bacterial membrane. Whatever the mechanism can be, we report antimicrobial activity of the peptide P4 against the representatives of Gram-positive (B. subtilis) and Gram-negative (E. coli) bacteria.

The (pro)renin receptor antagonist, PRO20, attenuates high fat diet induced hepatic steatosis and fibrosis in non‐alcoholic fatty liver disease (NAFLD)

Non‐alcoholic fatty liver disease (NAFLD) comprises a spectrum of liver damage directly related to diabetes, metabolic syndrome, and obesity. The (Pro)renin receptor (PRR) in the liver recently has been shown to play an important role in hepatic steatosis. In this study, we test our hypothesis that PRR antagonist, PRO20, reduces hepatic steatosis and fibrosis. Wild‐type mice in C57Bl/6J background (n=9‐10/group) were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) with matching calories for 6 weeks. Methionine choline deficient (MCD) diet was used to study fibrosis development in C57BL/6J mice for 8 weeks. Two weeks following respective diet modifications, mice were implanted with a subcutaneous osmotic pump (4 or 6 week release model) containing either PRO20 (700ug/kg/d) or saline. Liver tissues were processed for Oil Red O (ORO), H&E, and Masson’s trichrome histology staining for lipid and collagen accumulation. The presence of lipids, indicative of steatosis, was quantified using ImageJ/FIJI software and presented as % of ORO staining to the total area. We found that 6 weeks of HFD (15.90 ± 1.49 %) induced a significant elevation on liver ORO staining compared with the liver from mice fed with NFD (3.60 ± 1.61 %, p<0.0001). More importantly, PRO20 treatment (9.91 ± 1.46 %; p = 0.001) significantly reduced the ORO staining in mice treated with HFD. Hepatic triglyceride concentrations were quantified with colorimetric assay and shown to be significantly lowered in PRO20 treated mice (2.110 ± 0.6175) compared with scramble peptide treated (6.470 ± 0.3553, p<0.0001) mice under HFD. Collagen area was also found to be reduced in MCD PRO20 (4.970 ± 0.9096 %) treated mice compared with the control mice (8.901 ± 0.2782, p = 0.0033). Moreover, PRO20 treatment (258 ± 53.12 U/L) decreased liver ALT levels in MCD‐fed PRO20 mice compared with the control mice (682 ± 116.4 U/L). De novo lipogenesis genes thought to contribute to the development and progression of NAFLD were evaluated by real‐time PCR and presented as fold‐change expression normalized to β‐actin. The glycerol‐3‐phosphate acetyltransferase 3 (GPAT3) (1.835 ± 0.1798, p = 0.0183) expression was found to be downregulated in the livers of HFD PRO20 treated mice. Our findings indicate that PRO20 reduces HFD‐induced hepatic steatosis and MCD‐induced liver fibrosis and liver dysfunction. We conclude that PRR antagonism using PRO20 might be an effective therapeutic approach for treatment of NAFLD.

Design of the lentivirus-driven sustained LR12 delivery system for TREM-1 inhibition for palliating atherosclerosis.

Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) has been a potential target in the management of pathophysiology and clinical sequelae of atherosclerosis. LR12 peptide effectively blocks ligand-TREM-1 interaction; however, the short half-life of LR12 is a major hurdle in its translational application in atherosclerosis management warranting new methods for sustained bioavailability in clinical applications. The present study reports a novel method of packing the coding sequence of LR12 in a lentiviral system to ensure a sustained expression and bioavailability for effective TREM-1 inhibition. Lentivirus vector systems (LV-LR12 and LV-SP) for the expression of LR12 peptide and SP (scrambled peptide) were successfully designed, constructed, and tested in vitro in smooth muscle cells (SMCs). Viral amounts obtained were 703.6 ± 145.12 and 609.3 ± 145.93ng/ml p24 for LV-LR12 and LV-SP, respectively which correspond to ~ 107IFU/ml for both vectors. Dot blot assay revealed significantly increased expression of LR12-FLAG and SP-FLAG in 125μg total protein which was doubled in 250μg protein with respect to un-transduced SMCs suggesting the sustained release of LR12/SP as confirmed by ELISA. Cellular expression of LR12-FLAG and SP-FLAG displayed 8.44-fold and 7.55-fold increase, respectively compared to the control SMCs. The findings demonstrated a promising strategy for packing the LR12 coding sequence in lentiviral vector for TREM-1 inhibition for the management of atherosclerosis and other inflammatory diseases.

Matrix metalloproteinase-sensitive poly(ethylene glycol)/peptide hydrogels as an interactive platform conducive to cell proliferation during 3D cell culture

The response of extracellular matrix (ECM) to dynamic cell signals is of great significance for the regulation of cell behavior. In the present study, we prepared a type of matrix metalloproteinase (MMP)-sensitive degradable hydrogels (MSDHs) via the catalyst-free o-phthalaldehyde (OPA)/amine cross-linking reaction between o-phthalaldehyde-grafted four-arm poly(ethylene glycol) (4aPEG-OPA) and an MMP-sensitive degradable peptide. The gelation rates and storage moduli of MSDHs and the MMP-insensitive hydrogels (MIHs) based on an MMP-insensitive scramble peptide were comparable and dependent on the concentrations of precursor polymers. MSDHs were degradable while MIHs were stable in the presence of proteinase in vitro. The degradation of MSDHs was obviously faster than that of MIHs after subcutaneous injection into rats. In addition, both types of poly(ethylene glycol)/peptide hydrogels displayed excellent cytocompatibility in vitro, and showed good histocompatibility in vivo in the subcutaneous layer of rats. Furthermore, the proliferation of several MMP-expressing cell lines including MDA-MB-231 cells within MSDHs was obviously faster than that in MIHs, indicating the influence of metabolism-mediated scaffold degradation on the cell proliferation. This study provides a new biocompatible and biodegradable 3D cell culture interactive platform for regulation of cell behavior.

GFOGER Peptide Modifies the Protein Content of Extracellular Vesicles and Inhibits Vascular Calcification.

ObjectiveVascular calcification (VC) is an active process during which vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and release extracellular vesicles (EVs). In turn, the EVs serve as calcification foci via interaction with type 1 collagen (COL1). We recently showed that a specific, six-amino-acid repeat (GFOGER) in the sequence of COL1 was involved in the latter’s interaction with integrins expressed on EVs. Our main objective was to test the GFOGER ability to inhibit VC.ApproachWe synthesized the GFOGER peptide and tested its ability to inhibit the inorganic phosphate (Pi)-induced calcification of VSMCs and aortic rings. Using mass spectrometry, we studied GFOGER’s effect on the protein composition of EVs released from Pi-treated VSMCs.ResultsCalcification of mouse VSMCs (MOVAS-1 cells), primary human VSMCs, and rat aortic rings was lower in the presence of GFOGER than with Pi alone (with relative decreases of 66, 58, and 91%, respectively; p < 0.001 for all) (no effect was observed with the scramble peptide GOERFG). A comparative proteomic analysis of EVs released from MOVAS-1 cells in the presence or absence of Pi highlighted significant differences in EVs’ protein content. Interestingly, the expression of some of the EVs’ proteins involved in the calcification process (such as osteogenic markers, TANK-binding kinase 1, and casein kinase II) was diminished in the presence of GFOGER peptide (data are available via ProteomeXchange with identifier PXD018169∗). The decrease of osteogenic marker expression observed in the presence of GFOGER was confirmed by q-RT-PCR analysis.ConclusionGFOGER peptide reduces vascular calcification by modifying the protein content of the subsequently released EVs, in particular by decreasing osteogenicswitching in VSMCs.

An engineered anti-idiotypic antibody-derived killer peptide (KP) early activates swine inflammatory monocytes, CD3+CD16+ natural killer T cells and CD4+CD8α+ double positive CD8β+ cytotoxic T lymphocytes associated with TNF-α and IFN-γ secretion

This study evaluated the early modulation of the phenotype and cytokine secretion in swine immune cells treated with an engineered killer peptide (KP) based on an anti-idiotypic antibody functionally mimicking a yeast killer toxin. The influence of KP on specific immunity was investigated using porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as ex vivo antigens. Peripheral blood mononuclear cells (PBMC) from healthy pigs were stimulated with KP and with a scramble peptide for 20 min, 1, 4 and 20 h or kept unstimulated. The cells were analyzed using flow cytometry and ELISA. The same time-periods were used for KP pre-incubation/co-incubation to determine the effect on virus-recalled interferon-gamma (IFN-γ) secreting cell (SC) frequencies and single cell IFN-γ productivity using ELISPOT.KP induced an early dose-dependent shift to pro-inflammatory CD172α+CD14+high monocytes and an increase of CD3+CD16+ natural killer (NK) T cells. KP triggered CD8α and CD8β expression on classical CD4−CD8αβ+ cytotoxic T lymphocytes (CTL) and double positive (DP) CD4+CD8α+ Th memory cells (CD4+CD8α+low CD8β+low). A fraction of DP cells also expressed high levels of CD8α. The two identified DP CD4+CD8α+high CD8β+low/+high CTL subsets were associated with tumor necrosis factor alpha (TNF-α) and IFN-γ secretion. KP markedly boosted the reactivity and cross-reactivity of PRRSV type-1- and PCV2b-specific IFN-γ SC. The results indicate the efficacy of KP in stimulating Th1-biased immunomodulation and support studies of KP as an immunomodulator or vaccine adjuvant.

(Pro)renin Receptor Antagonism Attenuates High Fat Diet Induced Non‐alcoholic Fatty Liver Disease

Non‐alcoholic fatty liver disease (NAFLD) represents a diverse array of liver damage linked to obesity, and metabolic syndrome. The mechanisms and the treatment for NAFLD remains lacking; however, recent evidence suggests a critical importance of the (pro)renin receptor (PRR) and the renin‐angiotensin system (RAS) in liver lipid metabolism. Our laboratory recently developed a novel PRR antagonistic peptide PRO20 that blocks the fibrotic signaling of the PRR and the activation of the RAS. We hypothesize that PRR antagonism reverses NAFLD development by preventing the activation of liver RAS and fibrotic signaling. To test this hypothesis, wild‐type male C57Bl/6J mice (n=9–10/group) were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) for 6 weeks. Two weeks following diet modification, mice were implanted with a subcutaneous osmotic pump containing either PRO20 or scramble peptide (700 μg/kg/d, 4 weeks). At the end of the 6‐week protocol, liver tissues were harvested and processed for Oil Red O (ORO) and Masson’s trichrome staining for assessment of lipid and collagen accumulation respectively. In scramble peptide treated groups, we found that 6 weeks of HFD induced a significant elevation on liver ORO staining (15.90 ± 1.49 %;) compared with the liver from mice fed with NFD (3.60 ± 1.61 %, p &lt; 0.0001). More importantly, PRO20 (9.91 ± 1.46 %; p = 0.001) significantly reduced the ORO staining compared with scramble peptide in mice treated with HFD. Portal vein collagen deposition was significantly lower in mice treated with PRO20 (1.93 ± 0.60%) compared to the control peptide (4.05 ± 0.60%, p &lt; 0.0211) following 6 weeks of HFD. Mechanistically, we found that PRO20 (1.99 ± 0.62) normalized the elevation of liver mRNA level (fold change) of peroxisome proliferator‐activated receptor gamma (PPARγ) induced by HFD (HFD Scramble peptide: 2.61 ± 0.47 vs. NFD Scramble peptide: 1.13 ± 0.47, p = 0.0037), a key transcription factor regulating lipogenic genes in the liver. In addition, PRO20 prevented the activation of key liver RAS components induced by HFD including the angiotensinogen (1.00 ± 0.62 vs. 6.153 ± 0.62, p &lt; 0.0001), angiotensin converting enzyme (1.03 ± 0.63 vs. 2.91 ± 0.63, p = 0.0344), and angiotensin II type 1a receptor (0.35 ± 0.46 vs. 2.20 ± 0.46, p = 0.0091 value). In summary, our data showed that subcutaneous infusion of PRO20 attenuated the development of HFD‐induced NAFLD and portal fibrosis by regulating liver PPARγ and preventing liver local RAS activation. We conclude that PRR antagonism maybe a new therapeutic target for the treatment of NALFD.Support or Funding InformationNIH/NHLBI (R01HL122770), American Heart Association (17IRG33370128), NIH/NIGMS (1P20GM130459‐5451)

Telomerase Reverse Transcriptase Mediates Restoration of Functional Vasodilation in Isolated Coronary Microvessels of Aged Female Rats

IntroductionReactive oxygen species (ROS) increase during aging, resulting in decreased NO signaling and the production of ROS damages cellular components including mitochondrial DNA. Telomerase Reverse Transcriptase (TERT) has ROS‐suppressing effects in the mitochondria and is decreased in coronary artery disease (CAD). Upregulation of Telomerase restores NO mediated vasodilation in CAD. We hypothesize that treatment of isolated coronary microvessels of aged female rats with peptide nucXTERT (shunts TERT to mitochondria) will increase functional vasodilation while treatment of isolated coronary microvessels from young female rats with mitoXTERT (shunts TERT away from mitochondria) will blunt functional vasodilation.MethodsIsolated coronary microvessels from young (4 months, n = 2) or old (24 months, n = 5) Fischer 344 female rats were assessed via pressure myography. Vessels were incubated for 12 hours with nucXTERT or mitoXTERT, AGS 499 (TERT activator), or a scramble peptide negative control. Vessel relaxation to dobutamine (10−5 – 10−10 M), isoproterenol (10−4 – 10−8 M), acetylcholine (10−4 – 10−9 M), and H2O2 (10−3 – 10−8 M) was assessed post‐incubation.ResultsnucXTERT and AGS 499 treated vessel percent relaxation in old rats for isoproterenol, acetylcholine, and dobutamine were increased compared to scramble treated vessels (Figure 1a–c). Compared to scramble, nucXTERT increase of percent relaxation was significant for isoproterenol at 10−4 – 10−6 M (p = .007, .003, and .013, respectively), dobutamine at 10−5 – 10−7 M (p = &lt; .001, .016, and .016, respectively) and near significant for acetylcholine at 10−4 and 10−6 M (p = .097 and .062, respectively). Following nucxTERT and AGS 499 treatment, the percent relaxation to H2O2 (7.3% and 3.8% at max dilation, respectively) was reduced compared to scramble‐treated vessels (34.5% at max dilation)(Figure 1d). As expected, mitoXTERT incubation of young rat microvessels abrogated vasodilation of vessels to isoproterenol, acetylcholine, and dobutamine compared to scramble and AGS 499 responses. Hydrogen peroxide percent relaxation was increased in mitoXTERT (83.9% at max dose) compared to AGS 499 and scramble (3.79% and 34.38%, respectively).ConclusionsThe data support a vasoprotective and restorative role of TERT in coronary microvessels from aged female rats as adrenergic and acetylcholine‐mediated vasodilation were increased in nucXTERT and AGS 499 relative to the scrambled peptide. With nucXTERT, TERT was being blocked from nuclear translocation, increasing extranuclear TERT available for mitochondrial protection from ROS. Shunting TERT away from the mitochondria via mitoXTERT blunted adrenergic and acetylcholine‐mediated vasodilation as less TERT was available for mitochondrial ROS protection. Considering oxidative stress is a driver of CAD and the concomitant importance of altering vessel patency to allow for adequate blood flow to match metabolic demand, the modulation of TERT may be a holistic and plausible future clinical direction.Support or Funding InformationFunding for this project was provided by NIH RO1 AG053585 and the Gheen’s Foundation to Amanda Jo LeBlanc.Old rat isolated coronary arteriole vasoreactivity with nucXTERT peptide, AGS 499, or scramble peptide incubation. Shown as percent relaxation to isoproterenol (a) dobutamine (b) acetylcholine (c) and hydrogen peroxide (d). p ≤ 0.05 for nucXTERT (*) vs scramble. Data are presented as means ± SE, analyzed with two‐way repeated measures ANOVA followed by post‐hoc Bonferroni test.Figure 1

A laminin-211-derived bioactive peptide promotes the osseointegration of a sandblasted, large-grit, acid-etched titanium implant.

Early implant loading is very important for reducing the duration of missing teeth in human patients. The laminin-derived peptide, DLTIDDSYWYRI motif (Ln2-P3), accelerates bone healing. Therefore, to investigate the hypothesis that Ln2-P3 increases the bone response to sandblasted, large-grit, acid-etched (SLA) titanium implants, the effect of the Ln2-P3 peptide on the osseointegration of SLA titanium implants was evaluated in vitro and in vivo. Human osteoblast-like cells were cultured on untreated, scrambled peptide (SP)-treated, and Ln2-P3-treated SLA titanium discs, and the cellular responses of these cells were evaluated. The Ln2-P3 treatment augmented osteoblast attachment and spreading, alkaline phosphatase activity, and the expression of osteogenic marker genes. Furthermore, the untreated and Ln2-P3-treated SLA titanium implants were inserted into the tibiae of rabbits for 9 and 11 days. Compared with the untreated implants, the Ln2-P3-treated implants showed a significantly higher bone-to-implant contact ratio at Day 9 after implantation and an increased bone area. The Ln2-P3 treatment of the SLA titanium implant surface augmented osteoblastic activity and accelerated peri-implant bone formation at the bone-implant interface. Overall, these results indicated that compared with the SLA titanium surface alone, the Ln2-P3 peptide-treated SLA titanium surface enhances initial osseointegration, thereby facilitating earlier implant loading.

Involvement of Low-Density Lipoprotein Receptor in the Pathogenesis of Pulmonary Hypertension.

BackgroundRecently, we and others have reported a causal role for oxidized lipids in the pathogenesis of pulmonary hypertension (PH). However, the role of low‐density lipoprotein receptor (LDL‐R) in PH is not known.Methods and ResultsWe examined the role of LDL‐R in the development of PH and determined the efficacy of high‐density lipoprotein mimetic peptide 4F in mitigating PH. Explanted human lungs and plasma from patients with PH and control subjects were analyzed for gene expression, histological characteristics, and lipoprotein oxidation. Male LDL‐R null (LDL‐R knockout) mice (12–15 months old) were fed chow, Western diet (WD), WD with 4F, and WD with scramble peptide for 12 weeks. Serial echocardiography, cardiac catheterization, oxidized LDL assay, real‐time quantitative reverse transcription–polymerase chain reaction, and histological analysis were performed. The effect of LDL‐R knockdown and oxidized LDL on human pulmonary artery smooth muscle cell proliferation was assessed in vitro. LDL‐R and CD36 expression levels were significantly downregulated in the lungs of patients with PH. Patients with PH also had increased lung lipid deposits, oxidized LDL, E06 immunoreactivity, and plasma oxidized LDL/LDL ratio. LDL‐R knockout mice on WD developed PH, right ventricular hypertrophy, right ventricular dysfunction, pulmonary vascular remodeling, fibrosis, and lipid deposition in lungs, aortic atherosclerosis, and left ventricular dysfunction, which were prevented by 4F. Interestingly, PH in WD group preceded left ventricular dysfunction. Oxidized LDL or LDL‐R knockdown significantly increased proliferation of human pulmonary artery smooth muscle cells in vitro.ConclusionsHuman PH is associated with decreased LDL‐R in lungs and increased oxidized LDL in lungs and plasma. WD‐fed LDL‐R knockout mice develop PH and right ventricular dysfunction, implicating a role for LDL‐R and oxidized lipids in PH.

Targeting delivery of partial VAR2CSA peptide guided N-2-Hydroxypropyl trimethyl ammonium chloride chitosan nanoparticles for multiple cancer types.

To developing a multiple cancer types targeting drug delivery carrier system, a 28 amino acids from the VAR2CSA was synthesized as the placental CSA-binding peptide (plCSA-BP). Its specific binding ability to cancer cells was tested on cancer tissue array, and the results showed that plCSA-BP could bind to multiple cancer types. Then, the plCSA-BP was used as a guiding peptide to coat nanoparticles synthesized from N-2-HACC (CSA/HACC-NPs) which were loaded with prodigiosin (CSA/HACC-PNPs) or indocyanine green (CSA/HACC-INPs). The cancer cells specific targeting and efficacy of the CSA/HACC-PNPs were tested by different cancer cells in vitro and various cancer xenograft model in vivo. A scramble peptide (SCR) was used as control and synthesized SCR/HACC-PNPs and SCR/HACC-INPs. The results showed that the CSA/HACC-INPs could specifically uptake by JEG-3, PC3 and A594 cells, and the CSA/HACC-PNPs exhibited better anti-cancer activity and lower toxic effect in subcutaneous choriocarcinoma and prostatic tumor models compared with the free prodigiosin, HACC-PNPs and SCR/HACC-PNPs. So, the CSA/HACC-NPs could be used as a specific delivery carrier for multiple cancer types, and provided an alternate treatment option of various cancers with a single recipe.

Effect of Annexin-A1 mimetic peptide Ac2-26 on activation of astrocytes in rats

Objective To evaluate the effect of Annexin-A1 mimetic peptide Ac2-26 on activation of astrocytes in rats. Methods The primarily cultured astrocytes from the cortex of fetal Sprague-Dawley rats after 4 passages were divined into 4 groups (n=24 each) using a random number table method: control group (C group), LPS group, LPS+ scramble peptide group (LPS+ Src group) and LPS+ Ac2-26 group.LPS was added to LPS group with the final concentration of 1 mg/ml.LPS at the final concentration of 1 mg/ml and scramble peptide at the final concentration of 3.3 mmol/L were added to LPS+ Src group.LPS at the final concentration of 1 mg/ml and Ac2-26 at the final concentration of 3.3 mmol/L were added to LPS+ Ac2-26 group.After 24-h incubation, the cell survival rate was measured by CCK-8 assay, the migration was determined by Transwell assay, the concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1a (MIP-1a) in the supernatant were measured (by enzyme-linked immunosorbent assay), and the expression of glial fibrillary acidic protein (GFAP), extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), p38 mitogen-activated protein kinase (p38MAPK), and phosphorylated p38MAPK (p-p38MAPK) in astrocytes was detected by Western blot. Results Compared with group C, the expression of GFAP was significantly up-regulated, and the cell mobility, concentrations of TNF-α, IL-1β, MCP-1 and MIP-1α in the supernatant, p-ERK/ERK ratio, p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were increased (P 0.05). Compared with group LPS, the expression of GFAP was significantly down-regulated, and the cell mobility, concentrations of TNF-α, IL-1β, MCP-1 and MIP-1α in the supernatant, p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were decreased in group LPS+ Ac2-26 (P 0.05). Conclusion Ac2-26 can inhibit activation of astrocytes and produces anti-inflammatory effect in rats. Key words: Astrocytes; Ac2-26

In vitro characterization of odorranalectin for peptide-based drug delivery across the blood\u2013brain barrier

BackgroundThe use of siRNA-based gene silencing has been recently underscored as a potential therapeutic strategy for the treatment of neurological disorders. However, the stability of siRNA and other small molecule therapeutics is challenged by their intrinsic instability and limited passage across the blood–brain barrier (BBB). Based on these premises, our objective was to characterize/optimize odorranalectin (OL), a small non-immunogenic lectin-like peptide, as a carrier for targeted delivery across the BBB. For this purpose, 5(6)-carboxyfluorescein-conjugated OL and scramble peptide were synthesized, and then their BBB cellular internalization/trafficking and stability were characterized versus temperature, pH and serum content in the media in hCMEC/D3 cells as a model of BBB endothelium. Specifically, integrity of the internalized peptide in cell lysates was analyzed by LC/MS while cellular distribution and intracellular trafficking of OL was examined by fluorescence microscopy with early-late endosome (pHRodo Red®) and lysosome (Lysotracker®) markers.ResultsOur data show that cellular uptake of OL increased linearly with the concentrations tested in this study at 37 °C and the uptake was two to threefolds higher when compared to scramble peptide. While there were no differences for scramble peptide, the uptake of OL decreased by 50% at 4 °C incubation (vs. 37 °C). No effects of pH were observed on endothelial uptake of OL. Immunofluorescence studies also indicated a significant cellular internalization of OL that remained intact (as evaluated by LC–MS/MS) and co-localized with endosomal, but not lysosome marker. Importantly, OL was found non-toxic to cells at all concentrations tested.ConclusionsIn summary, our data suggest the existence of a receptor-mediated transcytosis pathway for cellular uptake of OL at the BBB endothelium. However, in vivo studies will be needed to assess the siRNA loading capacity of OL and its trans-BBB transport efficiency for targeted delivery in the brain.

Spinal macrophage migration inhibitory factor and high mobility group box 1 mediate persistent bladder pain

Repeated intravesical PAR4 (protease activated receptor 4) activation elicits persistent bladder pain lasting 5 days after the last treatment. Persistent bladder pain was fully reversed by a systemic HMGB1 (high mobility group box 1) inhibitor while a MIF (macrophage migration inhibitory factor) antagonist partly reversed it. Since there is growing evidence that spinal MIF and HMGB1 mediate inflammatory and neuropathic pain we examined whether there were spinal changes occurring during persistent bladder pain that may be responsible for maintaining bladder pain. In addition, we tested whether we could modulate persistent bladder pain with spinal MIF or HMGB1 antagonists. Persistent bladder pain was elicited in female C57 mice by repeated (3x) intravesical instillation of PAR4-activating peptide while control animals received scramble peptide treatment. On day 4, spinal cord (L6-S1) changes in c-fos (non-specific marker of spinal activation) was assessed with immunofluorescence while MIF and HMGB1 were assessed with immunofluorescence, western blotting and real-time PCR. On day 7, mice received an intrathecal injection of a neutralizing MIF monoclonal antibody (15 μg in 5 μl PBS) or a HMGB1 inhibitor glycyrrhizin (25 μg in 5 μl of 5% alcohol in PBS) and abdominal mechanical threshold was tested. On day 9, mice were treated with vehicle or control and abdominal mechanical threshold was tested. Immunofluorescence showed that c-fos and MIF in the dorsal horn, dorsal grey commissure and intermediolateral areas significantly increased in PAR4-treated mice while HMGB1 was decreased. In addition, intrathecal treatment with MIF neutralizing mAb or glycyrrhizin significantly alleviated abdominal mechanical hypersensitivity at 1 and 2 h and the analgesic effect diminished at 6 h. Vehicle or control treatment had no effect. Persistent bladder pain is associated with spinal changes in MIF and HMGB1 levels. Furthermore, spinal treatment with MIF monoclonal antibody and HMGB1 inhibitor temporarily reversed bladder pain. Our findings suggest that spinal MIF and HMGB1 participate in persistent bladder pain induced by repeated intravesical PAR4 and may be potential therapeutic targets in chronic bladder pain conditions.

A laminin-derived functional peptide, PPFEGCIWN, promotes bone formation on sandblasted, large-grit, acid-etched titanium implant surfaces.

This study aimed to investigate the in vitro and in vivo bone-forming potential of a sandblasted, large-grit, acid-etched (SLA) titanium (Ti) surface treated with a laminin-derived functional peptide, PPFEGCIWN (DN3). Human osteoblast-like MG63 cells were cultured with SLA Ti discs untreated or treated with DN3 or a control scrambled peptide (SP). Cell adhesion, spreading, and viability on the discs were tested. Alkaline phosphatase gene expression and enzyme activity were also evaluated. Four DN3-coated SLA Ti implants and four untreated implants were placed into the tibiae of two rabbits (two implants/tibia). Ten days later, the bone-implant interfaces were subjected to histomorphometry to measure the bone response. The surface properties of the discs and implants were determined using scanning electron, widefield confocal, and confocal laser microscopy and x-ray photoelectron spectroscopy. The peptide-treated and untreated discs and implants were similar in terms of physical surface properties, but the peptide-treated surfaces had significantly higher nitrogen levels (P < .05). The DN3 peptide promoted cell adhesion, spreading, and alkaline phosphatase expression and enzyme activity (P < .05). Histomorphometry of the harvested implants showed rapid bone formation and affinity of the motif. This study suggests that treatment with the cell adhesion peptide DN3 promotes bone healing at the SLA Ti surface.

Abstract 055: ND-13, a DJ-1-Derived Peptide, Protects Against the Renal Fibrosis and Inflammation Associated With Unilateral Ureter Obstruction

Oxidative stress and inflammation are important players in the pathogenesis of cardiovascular and renal diseases. DJ-1 is a redox-sensitive chaperone that regulates the expression of several antioxidant genes. Activation of the DJ-1/Nrf2 pathway in the kidney inhibits the development and progression of several renal diseases. The 20 aa peptide ND-13 consists of 13 highly conserved aa from the DJ-1 sequence and a TAT- derived 7 aa sequence to help in cell penetration. ND-13 prevents neuronal degeneration in mice; however, its effects on kidney damage remain unknown. We hypothesized that treatment with ND-13 would prevent the renal damage and inflammation associated with unilateral ureter obstruction (UUO). C57Bl/6 mice and DJ1 -/- mice underwent UUO and were divided in 3 groups: control (no UUO), UUO+scrambled peptide (SP) or UUO+ND-13 (3 mg/kg, s.c. daily). After 14 days of treatment, urine and kidneys were collected for analysis of renal damage. ND-13 treatment prevented the development of fibrosis in C57Bl/6 mice (UUO+SP: 702±189% of control, UUO+ND-13: 264±8% of control, n=2-4/group, p&lt;0.05), suggesting that ND-13 is protective against connective tissue deposition in the kidney. Treatment with ND-13 decreased renal mRNA expression of TNF- α ( fold change from control: 101±46 in UUO+SP; 18±7 in UUO+ND-13, n=4-5/group, p&lt;0.05), IL-6 (6.7±2 in UUO+SP; 1.54±0.3 in UUO+ND-13, p&lt;0.05), TGF- β ( 3.3±1.12 UUO+SP; 1.4±0.03 UUO+ND-13, p&lt;0.05) and Colagen1 α 1 (79±16 UUO+SP; 33±3.8 in UUO +ND-13, p&lt;0.05) in C57Bl/6 mice. In DJ1 -/- mice, treatment with ND-13 similarly decreased expression of TNF-α, IL-6 and TGF-β, but, in contrast, failed to prevent renal fibrosis or kidney expression of col1 α 1 . UUO also led to elevated urinary NGAL, marker of proximal tubular injury, in DJ-1 -/- mice and ND-13 treatment prevented that increase (71±17% UUO vs control: -18±21% UUO+ND-13 vs control, n=5/group). Our results suggest that ND-13 has protective effects on renal injury, fibrosis and inflammation, crucial mechanisms in the pathogenesis of renal disease. Thus, ND-13 treatment may be a new therapeutic approach for the prevention of renal injury, fibrosis and inflammation in renal diseases. Funded by T32DK007545 to CDM and 5P01 HL074940-10, 7R01 DK039308-31 to PAJ.

Targeting regenerative exosomes to myocardial infarction using cardiac homing peptide.

Rationale: Cardiac stem cell-derived exosomes have been demonstrated to promote cardiac regeneration following myocardial infarction in preclinical studies. Recent studies have used intramyocardial injection in order to concentrate exosomes in the infarct. Though effective in a research setting, this method is not clinically appealing due to its invasive nature. We propose the use of a targeting peptide, cardiac homing peptide (CHP), to target intravenously-infused exosomes to the infarcted heart.Methods: Exosomes were conjugated with CHP through a DOPE-NHS linker. Ex vivo targeting was analyzed by incubating organ sections with the CHP exosomes and analyzing with fluorescence microscopy. In vitro assays were performed on neonatal rat cardiomyocytes and H9C2 cells. For the animal study, we utilized an ischemia/reperfusion rat model. Animals were treated with either saline, scramble peptide exosomes, or CHP exosomes 24 h after surgery. Echocardiography was performed 4 h after surgery and 21 d after surgery. At 21 d, animals were sacrificed, and organs were collected for analysis.Results: By conjugating the exosomes with CHP, we demonstrate increased retention of the exosomes within heart sections ex vivo and in vitro with neonatal rat cardiomyocytes. In vitro studies showed improved viability, reduced apoptosis and increased exosome uptake when using CHP-XOs. Using an animal model of ischemia/reperfusion injury, we measured the heart function, infarct size, cellular proliferation, and angiogenesis, with improved outcomes with the CHP exosomes.Conclusions: Our results demonstrate a novel method for increasing delivery of for treatment of myocardial infarction. By targeting exosomes to the infarcted heart, there was a significant improvement in outcomes with reduced fibrosis and scar size, and increased cellular proliferation and angiogenesis.

Effect of Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) Blockade in Rats with Cecal Ligation and Puncture (CLP)-Induced Sepsis.

BackgroundBlocking of TREM-1 signaling improves survival of mice with sepsis induced by Pseudomonas aeruginosa. However, whether TREM-1 blockade has beneficial effects in polymicrobial sepsis is poorly understood. Here, we aimed to investigate the effect of modulation of the TREM-1 pathway in rats with polymicrobial sepsis induced by cecal ligation and puncture (CLP).Material/MethodsNormal Sprague-Dawley (SD) rats with sepsis induced by CLP were allocated randomly to received scramble peptide or LP17 via the jugular vein. Serum level of sTREM-1, IL6, TNF-α, and IL-1β were detected by ELISA assay. The mRNA and protein levels of JAK2 and STAT3 were detected by real-time PCR and Western blot analysis.ResultsSTREM-1 concentration was greatly and progressively increased in rats with CLP-induced sepsis, and the increase was attenuated by TREM-1 inhibitory peptide LP17. More than 60% survival was observed in rats at the experiment endpoint after LP17 treatment. TREM-1 blockade also attenuated the increased level of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, and thus attenuated systematic and distant inflammatory responses. Furthermore, TREM-1 blockade significantly attenuated the increased levels of pJAK2 and pSTAT3.ConclusionsTREM-1 blockade by the use of an inhibitory peptide LP17 could prolong survival of rats with polymicrobial sepsis and attenuate systematic inflammatory responses through the JAK2/STAT3 signaling pathway. Our results suggest that modulation of TREM-1 by a synthetic peptide might be a potential therapeutic option for polymicrobial sepsis.

A New Magnetic Resonance Imaging Probe Specifically Targeting Vascular Endothelial Growth Factor Receptor 2:Synthesis, Characterization and Biological Evaluation

Vascular endothelial growth factor (VEGF) is strongly expressed in most tumours and promotes both tumour growth and vascularization. The peptide, VEGF 125-136 , consisting of 12 amino acids is encoded by exon 6 of the VEGF gene and inhibits VEGF activity by blocking the binding of VEGF to the VEGFR2 receptor. The aim of the present study was to develop a targeting probe for magnetic resonance imaging (MRI) of tumours by conjugating VEGF 125-136 to gadolinium (III) (Gd(III)) (VGd) through the formation of chelates. The targeting efficiency of VGd to human hepatocellular carcinoma (HCC) cell line, BEL-7402, was subsequently determined both in vitro and in vivo . In the in vitro studies, the MRI results of BEL-7402 cells treated with VGd showed significantly higher T1 signal-to-noise ratio than that of both the competitive group, namely, those treated with VGd and VEGF125-136 and the control group, a scramble peptide conjugated to Gd(III) (SGd). In vivo , when xenografts of BEL-7402 cells were established in mice and then VGd or SGd were injected via tail vein, MRI showed that the tumour signal from VGd initially decreased from 5 to 120 min and then it increased at 120 min post injection. The peak signal was observed at 120 min after injection. In contrast, no distinct peak was observed for SGd. These findings indicate that VGd can target VEGFR2, highly expressed by BEL-7402 cells, enabling targeting MRI with high efficacy to be achieved both in vitro and in vivo .

Ultrasmall AGuIX theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of glioblastoma.

Despite combined treatments, glioblastoma outcome remains poor with frequent local recurrences, indicating that a more efficient and local therapy is needed. In this way, vascular-targeted photodynamic therapy (VTP) could help tumor eradication by destroying its neovessels. In this study, we designed a polysiloxane-based nanoparticle (NP) combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer (PS) and a new ligand peptide motif (KDKPPR) targeting neuropilin-1 (NRP-1), a receptor overexpressed by angiogenic endothelial cells of the tumor vasculature. This structure achieves the detection of the tumor tissue and its proliferating part by MRI analysis, followed by its treatment by VTP. The photophysical properties of the PS and the peptide affinity for NRP-1 recombinant protein were preserved after the functionalization of NPs. Cellular uptake of NPs by human umbilical vein endothelial cells (HUVEC) was increased twice compared to NPs without the KDKPPR peptide moiety or conjugated with a scramble peptide. NPs induced no cytotoxicity without light exposure but conferred a photocytotoxic effect to cells after photodynamic therapy (PDT). The in vivo selectivity, evaluated using a skinfold chamber model in mice, confirms that the functionalized NPs with KDKPPR peptide moiety were localized in the tumor vessel wall.