Apolipoprotein B binding domains: evidence that they are cell-penetrating peptides that efficiently deliver antigenic peptide for cross-presentation of cytotoxic T cells

Low-density lipoproteins (LDLs) are a good source of cholesterol, which is important in cellular homeostasis and production of steroids. Apolipoprotein B-100 (ApoB-100), the sole protein component of LDL, is known to bind to cell surface LDL receptor (LDLR) or cell surface-bound proteoglycans and to be internalized into cells. We found that APCs, consisting of macrophages and dendritic cells, upregulate LDLR on culture in vitro without obvious stimulation. In contrast, T cell populations only upregulate LDLR on activation. Thus, we strategized that tagging immunogens to ApoB-100 might be a useful means to target Ag to APCs. We generated fusion proteins consisting of receptor binding sites in ApoB-100, coupled to OVA peptide (ApoB-OVA), as Ag delivery vehicles and demonstrated that this novel delivery method successfully cross-presented OVA peptides in eliciting CTL responses. Surprisingly, internalization of ApoB-OVA peptide occurred via cell surface proteoglycans rather than LDLRs, consistent with evidence that structural elements of ApoB-100 indicate it to have cell-penetrating peptide properties. Finally, we used this strategy to assess therapeutic vaccination in a tumor setting. OVA-expressing EL-4 tumors grew progressively in mice immunized with ApoB-100 alone but regressed in mice immunized with ApoB-OVA fusion protein, coinciding with development of OVA-specific CTLs. Thus, to our knowledge, this is the first article to describe the cell-penetrating properties of a conserved human origin cell penetrating peptide that may be harnessed as a novel vaccination strategy as well as a therapeutics delivery device.     

Modulation of Recombinant Antigenic Constructs Containing Multi-Epitopes towards Effective Reduction of Atherosclerotic Lesion in B6;129S-Ldlrtm1HerApobtm2Sgy/J Mice

Atherosclerosis is increasingly recognized as a complex chronic inflammatory disease. Many more studies have extended vaccination against atherosclerosis by using epitopes from self-antigens or beyond and demonstrated that vaccination with antigens or derivatives could reduce the extent of the lesions in atherosclerosis-prone mice. Our previous study has demonstrated that construct AHHC [ApoB100^688-707 + hHSP60^303-312 + hHSP60^153-163 + Cpn derived peptide (C)] significantly reduced atherosclerotic lesion. The aim of this study was to investigate whether AHHC can be modulated towards increased lesion reduction in mice by creating two other derivatives with a sequential epitope-substitution named RHHC in which A was replaced by an “R” (C5aR^1-31) and RPHC with a further “H” (hHSP60^303-312) conversion into “P” (protease-activated receptor-1^42-55) in mice. Antigenic epitopes were incorporated into a dendroaspin scaffold. Immunization of B6;129S-Ldlr^tm1HerApob^tm2Sgy/J mice with three constructs elicited production of high levels of antibodies against each epitope (apart from hHSP60^153-163 and P which induced a low antibody response). Histological analyses demonstrated that the mice immunized with either RPHC or RHHC showed significant reductions in the size of atherosclerostic lesions compared to those with AHHC (69.5±1.1% versus 55.7±3.4%, P<0.01 or 65.6±1.3% versus 55.7±3.4%, P<0.01). Reduction of plaque size in the aortic sinus and descending aorta correlated with alterations in cellular immune responses when compared with controls. We conclude that a recombinant construct RPHC may provide new antigenic and structural features which are favorable for significant reduction in atherosclerotic lesion formation. This approach offers a novel strategy for developing anti-atherosclerotic agents.     

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.     

Papaya ringspot virus coat protein gene for antigen presentation in Escherichia coli

The coat protein (CP) of Papaya ringspot virus (PRSV) was analyzed for presentation of the antigenic peptide of animal virus, Canine parvovirus (CPV), in Escherichia coli (E. coli). The 45 nucleotides fragment coding for the 15-aa peptide epitope of the CPV-VP2 protein was either inserted into the PRSV-cp gene at the 5', 3' ends, both 5' and 3' ends or substituted into the 3' end of the PRSV cp gene. Each of the chimeric PRSV cp genes was cloned into the pRSET B vector under the control of the T7 promoter and transformed into E. coli. The recombinant coat proteins expressed from different chimeric PRSV-cp genes were purified and intraperitoneally injected into mice. All of the recombinant coat proteins showed strong immunogenicity and stimulate mice immune response. The recombinant coat proteins containing the CPV epitope insertion at the C terminus and at both N and C termini elicited ten times higher specific antisera in immunized mice compared with the other two recombinant coat proteins which contain the CPV epitope insertion at the N terminus and substitution at the C terminus.     

Interacting domains of the HN and F proteins of newcastle disease virus

The activation of most paramyxovirus fusion proteins (F proteins) requires not only cleavage of F(0) to F(1) and F(2) but also coexpression of the homologous attachment protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H protein coexpression strongly suggests that an interaction between HN and F proteins is required for fusion, and studies of chimeric HN proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion proteins containing these HN protein sequences but not to GST or to GST containing HN protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant proteins. These mutations inhibited the fusion promotion activities of the proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN protein binds to the F protein HR2 domain, an interaction important for the fusion promotion activity of the HN protein.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

Butanol Extract of Acanthopanax senticosus (Rupr. et Maxim.) Harms Alleviates Atherosclerosis in Apolipoprotein E-Deficient Mice Fed a High-Fat Diet

This study investigated the effect of butanol extract of AS (ASBUE) on atherosclerosis in apolipoprotein E-deficient (ApoE^−/−) mice. The mice were administered ASBUE (390 or 130 mg/kg/day) or rosuvastatin (RSV) via oral gavage for eight weeks. In ApoE^−/− mice, ASBUE suppressed the abnormal body weight gain and improved serum and liver biochemical indicators. ASBUE remarkably reduced the aortic plaque area, improved liver pathological conditions, and lipid metabolism abnormalities, and altered the intestinal microbiota structure in ApoE^−/− mice. In the vascular tissue of ASBUE-treated mice, P-IKKβ, P-NFκB, and P-IκBα levels tended to decrease, while IκB-α increased in high fat-diet-fed atherosclerotic mice. These findings demonstrated the anti-atherosclerotic potential of ASBUE, which is mediated by the interaction between the gut microbiota and lipid metabolism and regulated via the Nuclear Factor-kappa B (NF-κB) pathway. This work paves the groundwork for subsequent studies to develop innovative drugs to treat atherosclerosis.     

Selective modification of apoB-100 in the oxidation of low density lipoproteins by myeloperoxidase in vitro

Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK..., corresponding to amino acid residues 53-66...on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M=O), cysteinyl azo (*C), RS -N= N-DNP, derivatives of EEL(*C)T(M=O)FIR and LNDLNS VLV(M=O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO2H) by HOCl.The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability.     

Immunocontraception in mice using repeated, multi-antigen peptides: immunization with purified recombinant antigens

Two immunocontraceptive antigens (AgE and AgF) were constructed that included different combinations of highly species-specific peptides from the mouse reproductive antigens SP56, ZP3, ZP2, and ZP1 in the form of multi-antigen peptides (MAPs). Both AgE and AgF contained three tandem repeats each of ZP2 and ZP3 peptide epitopes and a single copy of a ZP1 peptide sequence all of which had previously been demonstrated to individually have immunodominant or contraceptive effects. In addition, AgF contained a single contraceptive peptide derived from SP56, the putative ZP3 receptor protein on sperm. The antigens were expressed and affinity purified as recombinant repeated multi-antigen (polyepitope) peptides using an Escherichia coli maltose binding protein (MBP) expression system. Female BALB/c mice actively immunized with these antigens in Freund's adjuvants produced variable serum antibody responses to the component peptides. Fertility rates for animals immunized with AgE (40%) and AgF (20%) were significantly reduced compared to MBP immunized mice (90%), but the reduction in fertility did not correlate with peptide-specific serum antibody levels. Ovaries from all immunized mice appeared histologically normal with no evidence of oophoritis. These results demonstrate that high levels of immunocontraception can be achieved in mice, without apparent side-effects, using species-specific immunogens that include repeated peptides from proteins involved in fertilization.     

Proteomic profiling during atherosclerosis progression: Effect of nebivolol treatment

There is a great need for the identification of biomarkers for the early diagnosis of atherosclerosis and the agents to prevent its progression. The aim of this study was to explore the effect of 24 week of nebivolol (a third-generation vasodilatory beta-blocker) treatment on serum protein profiles in Apo E^?/? mice during atherosclerosis progression. Nebivolol treated and non-treated (the control group) groups consisted of 10 genetically modified homozygous Apo E^?/? mice. Proteomic analyses were performed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the serum samples from the nebivolol treated and non-treated Apo E^?/? mice. The protein profiles obtained using three different chips, CM10 (weak cation-exchange), H50 (reverse phase), and IMAC30-Cu²⁺ (immobilized metal affinity capture) were statistically analyzed using the ProteinChip data manager 3.0 program. At the end of 24 week of nebivolol-treatment period, a total of 662 protein/peptide clustering peaks were detected using 12 different conditions and reading with high and low intensity laser energy. The highest total number of protein/peptide clusters was found on H50 chip array. The peak intensities of 95 of the 662 protein/peptide clusters were significantly different in the nebivolol-treated atherosclerotic group in comparison to the non-treated control mice groups (P < 0.05). Forty-three protein/peptides were up-regulated (high signal intensity) while 52 protein/peptides had lower signal intensity (down-regulated) in the nebivolol-treated atherosclerotic group. The proteomic profiles of nebivolol-treated Apo E^?/? mice were different than the control group indicating a potential role of nebivolol in atherosclerosis. Our study contributes to understand the efficacy of nebivolol on serum protein/peptide profiles during atherosclerosis development.     

Cutting edge: a single MHC anchor residue alters the conformation of a peptide-MHC complex inducing T cells that survive negative selection

We generated transgenic mice that expressed hen egg-white lysozyme (HEL) under a class II MHC promoter. The A7 line expressed HEL with a point mutation in the Asp(52) residue, the main anchor amino acid responsible for the selection of the chemically dominant family of peptides (52-60) by I-A(k) molecules. Mice expressing HEL with Ala(52) were completely unresponsive when immunized with the same protein, i.e., HEL A52. However, the same mice immunized with wild-type HEL elicited T cells that recognized a conformation of the 52-61 core sequence uniquely different between Asp(52) and Ala(52) containing peptides. Importantly, some T cells also recognized the HEL A52 peptide given exogenously but not the same peptide processed from HEL A52 protein. Thus, a core MHC anchor residue influences markedly the specificity of the T cells. We discuss the relevance of these findings to autoimmunity and vaccination with altered peptides.     

Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.     

Immunization using an Apo B-100 related epitope reduces atherosclerosis and plaque inflammation in hypercholesterolemic apo E (-/-) mice

Immune system modulates atherosclerosis and immunization using homologous LDL reduces atherosclerosis in hyperlipidemic animals. The nature of athero-protective antigenic epitopes in LDL remains unclear. We have recently identified nearly a 100 antigenic epitopes in human apo B-100 and in this study we evaluated the effects of immunization with two such epitopes on atherosclerosis in hypercholesterolemic apo E (-/-) mice. Male apo E (-/-) mice were immunized at 6-7 weeks of age with two different apo B-100 related peptide sequences using alum as adjuvant and mice immunized with alum alone served as controls. Peptide-2 immunization reduced aortic atherosclerosis by 40% and plaque inflammation by 80% compared to controls without a reduction in circulating cholesterol levels whereas Peptide-1 immunization had no effect. Peptide-2 immunization also reduced the progression of aortic lesions when mice were immunized at 16 weeks of age, suggesting the possibility of immuno-modulation in treating established atherosclerosis. The athero-protective effect of Peptide-2 immunization was absent in splenectomized mice but could be conveyed to non-immunized mice via adoptive transfer of splenocytes from peptide-2 immunized mice. In conclusion, immunization with a specific apo B-100 related peptide sequence reduces aortic atherosclerosis and plaque inflammation. Such acquired immunity and athero-protective effect appears to be mediated by splenocytes. These data demonstrate the feasibility of peptide based immunomodulating therapy for atherosclerosis.     

A Murine Model of Obesity With Accelerated Atherosclerosis

The epidemic of obesity sweeping developed nations is accompanied by an increase in atherosclerotic cardiovascular diseases. Dyslipidemia, diabetes, hypertension, and obesity are risk factors for cardiovascular disease. However, delineating the mechanism of obesity‐accelerated atherosclerosis has been hampered by a paucity of animal models. Similar to humans, apolipoprotein E–deficient (apoE^?/?) mice spontaneously develop atherosclerosis over their lifetime. To determine whether apoE^?/? mice would develop obesity with accelerated atherosclerosis, we fed mice diets containing 10 (low fat (LF)) or 60 (high fat (HF)) kcal % from fat for 17 weeks. Mice fed the HF diet had a marked increase in body weight and atherosclerotic lesion formation compared to mice fed the LF diet. There were no significant differences between groups in serum total cholesterol, triglycerides, or leptin concentrations. Plasma concentrations of the acute‐phase reactant serum amyloid A (SAA) are elevated in both obesity and cardiovascular disease. Accordingly, plasma SAA concentrations were increased fourfold (P < 0.01) in mice fed the HF diet. SAA was associated with both pro‐ and antiatherogenic lipoproteins in mice fed the HF diet compared to those fed the LF diet, in which SAA was primarily associated with the antiatherogenic lipoprotein high‐density lipoprotein (HDL). Moreover, SAA was localized with apoB‐containing lipoproteins and biglycan in the vascular wall. Taken together, these data suggest male apoE‐deficient mice are a model of metabolic syndrome and that chronic low level inflammation associated with increased SAA concentrations may mediate atherosclerotic lesion formation.     

A mouse monoclonal antibody specific for mouse apoB48 and apoB100 produced by immunizing "apoB39-only" mice with mouse apoB48

We have generated and characterized a murine monoclonal antibody (mAb) that binds to both mouse apolipoprotein (apo) B48 and apoB100. We immunized "apoB39-only" mice (mice that synthesize a truncated form of apoB, apoB39, but no apoB48 or apoB100) with lipoproteins containing mouse apoB48 and then used splenocytes from the immunized mice to create hybridomas. We identified a hybridoma, 2G11, that secretes a mAb that binds to mouse apoB48 and apoB100 but not to apoB39. Antibody 2G11 also binds apoB48 and apoB100 from rats and hamsters but not from humans. The mAb recognizes mouse apoB equally in very low and low density lipoproteins and was used to quantify apoB in wild-type, apoE-deficient and low-density lipoprotein receptor-deficient mice and in mice treated with an antisense drug that lowers plasma apoB levels. The antibody will be an important reagent for studying mouse models of atherosclerosis. The study also underscores the utility of genetically modified mice for generating mouse mAbs against mouse proteins.     

Is it just paraoxonase 1 or are other members of the paraoxonase gene family implicated in atherosclerosis?

PURPOSE OF REVIEW: During the past decade, paraoxonase 1, a HDL-associated protein, has been demonstrated to be an important contributor to the antioxidant capacity of HDL. Studies using paraoxonase 1 null mice by gene targeting and transgenic mice corroborated the hypothesis that paraoxonase 1 protects against atherosclerosis. In contrast to paraoxonase 1, the other two members of the paraoxonase gene family, namely paraoxonase 2 and paraoxonase 3, are either undetectable (paraoxonase 2) or detected at very low levels (paraoxonase 3) on HDL, and are considered to participate in intracellular antioxidant mechanisms. In this review, we summarize studies reported in the past 2 years suggesting a protective role for paraoxonase 2 and paraoxonase 3 in the development of atherosclerosis in mice. RECENT FINDINGS: Adenovirus-mediated expression of human paraoxonase 2 or paraoxonase 3 proteins protects against the development of atherosclerosis in apolipoprotein E-deficient mice. Paraoxonase 2-deficient mice develop significantly larger atherosclerotic lesions than their wild-type and heterozygous counterparts on an atherogenic diet despite having lower levels of apolipoprotein B-containing lipoproteins. Atherosclerotic lesions were significantly lower in male hPON3Tg/LDLR null mice than in LDLR null mice on a western diet. SUMMARY: We conclude that, in addition to paraoxonase 1, both paraoxonase 2 and paraoxonase 3 proteins are protective against the development of atherosclerosis in mice. These findings underscore the utility of all members of the paraoxonase gene family as therapeutic targets for the treatment of atherosclerosis.     

Redirecting retroviral tropism by insertion of short, nondisruptive peptide ligands into envelope

A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.     

Molecular basis for the protective effects of low-density lipoprotein receptor-related protein 1 (LRP1)-derived peptides against LDL aggregation

Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly¹¹²⁷-Cys¹¹⁴⁰ exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly¹¹²⁷-Cys¹¹⁴⁰ (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA₂)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA₂-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA₂. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima.     

Mucosal Tolerance to a Combination of ApoB and HSP60 Peptides Controls Plaque Progression and Stabilizes Vulnerable Plaque in Apobtm2SgyLdlrtm1Her/J Mice

Oral tolerance to auto antigens reduces the development of atherosclerosis in mouse models. However, the effect of immune tolerance to multiple self antigenic peptides in plaque progression and stabilization is not known. We studied the protective effect of mucosal tolerance to peptides from apolipoprotein B (ApoB; 661–680) and heat shock protein 60 (HSP60; 153–163), in combination with diet, in the prevention of atherosclerotic lesion progression and plaque stabilization in ApoB^tm25gyLDLr^tm1Her mice. We found that oral administration of five doses of a combination of ApoB and HSP60 peptides (20 µg/mice/dose) induced tolerance to both the peptides and reduced early plaque development by 39.9% better than the individual peptides (ApoB = 28.7%;HSP60 = 26.8%)(P<0.001). Oral tolerance to combination of peptides along with diet modification arrested plaque progression by 37.6% which was associated with increases in T-regulatory cell and transforming growth factor-β expression in the plaque and peripheral circulation. Reduced macrophage infiltration and tumor necrosis factor-α expression in the plaque was also observed. Tolerance with continued hypercholesterolemia resulted in 60.8% reduction in necrotic core area suggesting plaque stabilization, which was supported by reduction in apoptosis and increased efferocytosis demonstrated by greater expression of receptor tyrosine kinase Mer (MerTK) in the plaque. Tolerance to the two peptides also reduced the expression of matrix metalloproteinase 9, tissue factor, calprotectin, and increased its collagen content. Our study suggests that oral tolerance to ApoB and HSP60 peptide combination induces CD4⁺ CTLA4⁺ Tregs and CD4⁺CD25⁺Foxp3⁺ Tregs secreting TGF-β, which inhibit pathogenic T cell response to both peptides thus reducing the development and progression of atherosclerosis and provides evidence for plaque stabilization in ApoB^tm25gyLDLr^tm1Her mice.     

Protective effect of Morus rubra L. leaf extract on diet-induced atherosclerosis in diabetic rats

The antiatherosclerotic effect of aqueous leaves extract of Morus rubra was studied in streptozotocin-induced diabetic rats fed with atherosclerotic (Ath) diet [1.5 ml olive oil containing 8 mg (3, 20,000 IU) vitamin D2 and 40 mg cholesterol] for 5 consecutive days. A short-term toxicity assessment was also conducted in healthy rats to examine toxic effects of the extract. Oral administration of extract to diabetic rats (100, 200 and 400 mg/kg body weight per day for a period of 30 days) produced significant (p<0.001) fall in fasting blood glucose (FBG) in a dose-dependent manner. Treatment with the extract (400 mg/kg) showed significant (p<0.001) improvement in body weight and serum lipid profile i.e., total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol, when compared with diabetic control. Endothelial dysfunction parameters (sVCAM-1, Fibrinogen, total NO levels and oxidized LDL), apolipoprotein A and apolipoprotein B were significantly (p<0.001) reversed to near normal, following treatment with the extract. Thus, our study shows that aqueous leaf extract of Morus rubra (400 mg/kg) significantly improves the homeostasis of glucose and fat and possesses significant anti-atherosclerotic activity.