Mimetic Peptide Research Articles

Biomimetic porphyrin-lipid nanoparticles - novel nanoscale theranostics for multimodal imaging and therapy in atherosclerotic cardiovascular disease

Abstract Background High-density lipoprotein (HDL) mimetic nanoagents have unrealized potential for atherosclerosis theranostics. Porphyrin-lipid HDL mimetic nanoparticles (Por-HDL-NPs) incorporate a porphyrin-lipid conjugate which permits near infrared fluorescence imaging and positron emission tomography (PET) through chelation of Copper-64 (64Cu). The outer shell contains apolipoprotein A-I mimetic peptide R4F designed to interact with scavenger receptor class B type I (SR-BI), enabling macrophage-targeting and therapeutic effects. Purpose To investigate the multimodal imaging, macrophage-targeting and therapeutic properties of Por-HDL-NPs and applications in atherosclerotic cardiovascular disease. Methods In vitro, the cholesterol efflux capacity of PBS (control), reconstituted HDL (rHDL) and Por-HDL-NPs was determined in immortalized bone marrow-derived macrophages (iBMDMs) loaded with [³H]-cholesterol. Real-time quantitative PCR (RT-qPCR) assessed mRNA levels of inflammatory mediators. ELISAs and Western blotting measured changes in protein levels. Apolipoprotein (Apo)e-/- mice were fed high-cholesterol diet (HCD) for 6 weeks, for early-stage plaque, and 13 weeks for the unstable plaque model in which two partial stenoses were made in the right carotid artery. PET and fluorescence (IVIS, flow cytometry, microscopy) imaging identified Por-HDL-NPs in plaques, circulating and aortic cells. Histological techniques assessed plaque area and composition in aortic sinus and carotid artery sections. Results Por-HDL-NPs were internalized by iBMDMs, visualized via fluorescence microscopy and flow cytometry. Por-HDL-NPs increased cholesterol efflux (49%, P<0.05), compared to rHDL. Por-HDL-NPs reduced macrophage mRNA levels of Il-1b (88%), Il-18 (54%) and Ccl5 (75%), and protein secretion of IL-1β (69%) and CCL5 (82%), P<0.05. Por-HDL-NPs suppressed inflammasome components Nlrp3 (69%) and Asc (36%), and activation of inflammatory transcription factor p65-NF-κB (53%), P<0.05. SR-BI siRNA knockdown and methyl-β-cyclodextrin, revealed that the anti-inflammatory properties of Por-HDL-NPs were independent of SR-BI and cholesterol efflux. In Apoe-/- mice, PET imaging showed 64Cu-Por-HDL-NPs localized in hearts and could detect increases in plaque size over time with HCD feeding. Por-HDL-NP fluorescence was detected within aortic sinus and unstable carotid plaques, co-localized with CD68+ macrophages. Por-HDL-NP-treated mice had smaller early-stage (22%) and unstable plaques (52%) than control PBS-treated mice, P<0.05. Por-HDL-NP mice had fewer circulating (32%) and aortic monocytes (81%), and a reduction in aortic arch Rela (26%), P<0.05. Conclusions Por-HDL-NPs present as potential nanoscale theranostics for atherosclerotic cardiovascular disease. Por-HDL-NPs can detect plaques using multiple imaging modalities and exhibit atheroprotective effects.Atherosclerosis imaging with Por-HDL-NPs

Subduing the Inflammatory Cytokine Storm

The inflammatory cytokine response is essential for protective immunity, yet bacterial and viral pathogens often elicit an exaggerated response (“cytokine storm”) harmful to the host that can cause multi-organ damage and lethality. Much has been published recently on the cytokine storm within the context of the coronavirus pandemic, yet bacterial sepsis, severe wound infections and toxic shock provide other prominent examples. The problem of the cytokine storm is compounded by the increasing incidence of multidrug-resistant bacterial strains. We created an incisive molecular tool for analyzing the role of the B7/CD28 costimulatory axis in the human inflammatory response. To attenuate the cytokine storm underlying infection pathology, yet preserve host defenses, we uniquely targeted the engagement of CD28 with its B7 co-ligands by means of short peptide mimetics of the human CD28 and B7 receptor homodimer interfaces. These peptides are not only effective tools for dissecting mechanism but also serve to attenuate the inflammatory response as a broad host-oriented therapeutic strategy against the cytokine storm. Indeed, such peptides protect mice from lethal Gram-positive bacterial superantigen-induced toxic shock even when dosed in molar amounts well below that of the superantigen and show promise in protecting humans from the severe inflammatory disease necrotizing soft tissue infections (‘flesh-eating’ bacterial sepsis) following traumatic wound injuries.

Apolipoprotein A-I Mimetic Peptide Restores VEGF-induced Angiogenesis in Hypercholesterolemic Ischemic Heart by Reducing HDL Proinflammatory Properties.

VEGF-induced angiogenesis is impaired in hypercholesterolemia. Previous studies showed that an apolipoprotein A-I(ApoA-I) mimetic peptide, D-4F, is able to reduce HDL proinflammatory index in hypercholesterolemia. Whether D-4F promotes angiogenesis in hypercholesterolemia remains unclear. Low-density lipoprotein receptor null (LDLr-/-) mice and LDLr-/-/ApoA-I-/- mice were fed with high-fat diet with or without D-4F (1mg/kg·d). C57BL/6 mice fed with normal diet served as control. The myocardial infarction was induced by ligation coronary artery, and the VEGFA-AAV 9 was injected in heart. The plasma HDL proinflammatory index, cardiac function, infarct size, and angiogenesis related signaling pathways were examined. The HDL proinflammatory index increases in hypercholesterolemic mice. VEGFA stimulates angiogenesis and improves cardiac function in ischemic heart of C57BL/6 mice, but not in hypercholesterolemic mice. D-4F reduces HDL proinflammatory index. D-4F combined with VEGFA stimulates the expression of CD31 and eNOS, activates ERK1/2, reduces infarct size, and improves cardiac function in ischemic heart in hypercholesterolemic LDLr-/- mice but not in hypercholesterolemic LDLr-/-/ApoA-I-/- mice. D-4F restores the VEGF-induced angiogenesis by reducing HDL proinflammatory properties in hypercholesterolemic ischemic heart.

Ultrasound-Responsive Hydrogel Incorporated with TGF-β Mimetic Peptides for Endometrium Recovery to Restore Fertility.

Unavoidable damage to the basal layer of the endometrium has a huge negative impact on a woman's reproductive health and menstrual cycle. However, it is difficult for medicine to penetrate a series of biological barriers toward the basal layer in the deeper area of the endometrium. To meet this challenge, we developed an ultrasound-responsive hydrogel incorporated with a transforming growth factor-beta (TGF-β) mimetic peptide to enhance pregnancy outcomes by restoring the function of a wounded endometrium due to its deep-tissue-penetration capability. In vitro studies revealed that the TGF-β-mimetic-peptide-loaded hydrogel could achieve 64.35% of cell migration under ultrasound stimulation even in phosphate-buffered saline of pH 6.0. Upon in situ sonication at the uterus, carboxymethyl chitosan can be released from degraded hydrogel to open tight junctions with reduced interstitial pressure by ultrasound to promote deep penetration. Rat studies revealed that the penetration capability of TGF-β-mimetic-peptide-loaded hydrogel with sonication was 1.6 times higher than that of the control group. Besides the rat uterine model, ex vivo human uterine tissue was also collected and imaged, demonstrating up to ∼700 μm of tissue penetration depth. In addition, compared to control groups, effective uterus recovery without intrauterine stenosis and endometrial cavity fluid was observed from rats with severe uterine injury treated by TGF-β-mimetic-peptide-loaded hydrogel. In addition, fertility restoration in the endometrial injury model was observed after treatment with such an ultrasound-responsive hydrogel incorporated with TGF-β mimetic peptides. Overall, this work provides an effective approach to treating endometrial injury for enhanced pregnancy outcomes.

High-Density Lipoprotein Mimetic Peptide 4F Reduces Toxic Amyloid-Beta Exposure to the Blood-Brain Barrier Endothelium in Alzheimer's Disease Transgenic Mice.

Aβ accumulation in the blood-brain barrier (BBB) endothelium, which lines the cerebrovascular lumen, is a significant contributor to cerebrovascular dysfunction in Alzheimer's disease (AD). Reduced high-density lipoprotein (HDL) levels are associated with increased AD risk, and the HDL mimetic peptide 4F has been developed as a promising therapeutic agent to improve cerebrovascular health in AD. In this study, we evaluated the impact of 4F on 125I-Aβ42 blood-to-brain distribution using dynamic SPECT/CT imaging in both wild-type and APP/PS1 transgenic mice. Graphical analysis of the imaging data demonstrated that 4F significantly reduced the blood-to-brain influx rate in wild-type mice and the distribution of 125I-Aβ42 in the BBB endothelium in APP/PS1 mice. To elucidate the molecular mechanisms underlying the effect of 4F, we evaluated its impact on the p38 pathway and its role in mediating Aβ42 trafficking in human BBB endothelial cell monolayers. Treatment with 4F significantly decreased Aβ42 induced p38 activation in BBB endothelial cells. Furthermore, inhibition of p38 kinase significantly reduced endothelial accumulation of fluorescence-labeled Aβ42 and luminal-to-abluminal permeability across the cell monolayer. While our previous publication has hinted at the potential of 4F to reduce Aβ accumulation in the brain parenchyma, the current findings demonstrated the protective effect of 4F in reducing Aβ42 accumulation in the BBB endothelium of AD transgenic mice. These findings revealed the impact of a clinically tested agent, the HDL mimetic peptide 4F, on Aβ exposure to the BBB endothelium and offer novel mechanistic insights into potential therapeutic strategies to treat cerebrovascular dysfunction in AD.

A new model and precious tool to study molecular mechanisms of macrophage aging.

The accumulation of senescent cells, characterized by a senescence-associated secretory phenotype (SASP), contributes to chronic inflammation and age-related diseases (ARD). During aging, macrophages can adopt a senescent-like phenotype and an altered function, which promotes senescent cell accumulation. In the context of aging and ARD, controlling the resolution of the inflammatory response and preventing chronic inflammation, especially by targeting macrophages, must be a priority. Aging being a dynamic process, we developed a model of in vitro murine peritoneal macrophage aging. Our results show that macrophages cultured for 7 or 14 days exhibit a senescence-like phenotype: proliferation decrease, the levels of cyclin-dependent kinase inhibitors p16INK4A and p21CIP1 and of pro-inflammatory SASP components (MCP-1, IL-6, IL-1β, TNF-α, and MMP-9) increase, phagocytosis capacity decline and glycolytic activity is induced. In our model, chronic treatment with CB3, a thioredoxin-1 mimetic anti-inflammatory peptide, completely prevents p21CIP1 increase and enables day 14 macrophages to maintain proliferative activity.We describe a new model of macrophage aging with a senescence-like phenotype associated with inflammatory, metabolic and functional perturbations. This model is a valuable tool for characterizing macrophage aging mechanisms and developing innovative strategies with promising therapeutical purpose in limiting inflammaging and ARD.

Polymer-Based Antimicrobial Peptide Mimetics for Treating Multi-drug Resistant Infections: Therapy and Toxicity Evaluation

Polymer-Based Antimicrobial Peptide Mimetics for Treating Multi-drug Resistant Infections: Therapy and Toxicity Evaluation

Alzheimer's Disease Immunotherapy and Mimetic Peptide Design for Drug Development: Mutation Screening, Molecular Dynamics, and a Quantum Biochemistry Approach Focusing on Aducanumab::Aβ2-7 Binding Affinity.

Seven treatments are approved for Alzheimer's disease, but five of them only relieve symptoms and do not alter the course of the disease. Aducanumab (Adu) and lecanemab are novel disease-modifying antiamyloid-β (Aβ) human monoclonal antibodies that specifically target the pathophysiology of Alzheimer's disease (AD) and were recently approved for its treatment. However, their administration is associated with serious side effects, and their use is limited to early stages of the disease. Therefore, drug discovery remains of great importance in AD research. To gain new insights into the development of novel drugs for Alzheimer's disease, a combination of techniques was employed, including mutation screening, molecular dynamics, and quantum biochemistry. These were used to outline the interfacial interactions of the Aducanumab::Aβ2-7 complex. Our analysis identified critical stabilizing contacts, revealing up to 40% variation in the affinity of the Adu chains for Aβ2-7 depending on the conformation outlined. Remarkably, two complementarity determining regions (CDRs) of the Adu heavy chain (HCDR3 and HCDR2) and one CDR of the Adu light chain (LCDR3) accounted for approximately 77% of the affinity of Adu for Aβ2-7, confirming their critical role in epitope recognition. A single mutation, originally reported to have the potential to increase the affinity of Adu for Aβ2-7, was shown to decrease its structural stability without increasing the overall binding affinity. Mimetic peptides that have the potential to inhibit Aβ aggregation were designed by using computational outcomes. Our results support the use of these peptides as promising drugs with great potential as inhibitors of Aβ aggregation.

Dysfunctional High-Density Lipoprotein Cholesterol and Coronary Artery Disease: A Narrative Review.

High-density lipoprotein (HDL) cholesterol is traditionally viewed as protective against cardiovascular disease (CVD). However, emerging evidence reveals that dysfunctional HDL, characterized by impaired reverse cholesterol transport (RCT), reduced anti-inflammatory and antioxidant activities and increased endothelial dysfunction, which can contribute to coronary artery disease (CAD). Dysfunctional HDL, resulting from oxidative modifications of Apolipoprotein A-1 (Apo A-1) and enzyme inactivation, fails to effectively remove cholesterol from peripheral tissues and may promote inflammation and atherosclerosis. Genetic mutations affecting HDL metabolism further complicate its role in cardiovascular health. Studies have shown that conventional therapies aimed at raising HDL-C levels do not necessarily reduce cardiovascular events, highlighting the need for new approaches that improve HDL functionality. Therapeutic strategies such as Apo A-1 mimetic peptides, reconstituted HDL infusions, and drugs targeting specific HDL metabolic pathways are being explored. Additionally, weight loss, statin therapy, and niacin have shown potential in enhancing HDL function. The pathophysiology of dysfunctional HDL involves complex mechanisms, including oxidative stress, inflammation, and genetic mutations, which alter its structure and function, diminishing its cardioprotective effects. New functional assays, such as the cholesterol efflux capacity (CEC) and HDL inflammatory index, provide more accurate predictions of cardiovascular risk by assessing HDL quality rather than quantity. As research progresses, the focus is shifting towards therapeutic strategies that enhance HDL function and address the root causes of its dysfunction, offering a more effective approach to reducing cardiovascular risk and preventing CAD.

Humidity-responsive pectin/AgNPs/ZnO composite films with high antimicrobial and UV-proof functions

ZnO nanoparticles and Ag nanoparticles (AgNPs) tend to agglomerate when used individually, and high Ag+concentrations can cause cytotoxicity. To address these issues, we synthesized carboxymethyl cellulose (CMC)-based antimicrobial mimetic peptides (AMPMs) by introducing amphiphilic cations on the surface of CMC using a chemical grafting method. Bis-formaldehyde-formylated AMPMs were prepared through the directional oxidation of AMPMs with periodate and used as the reducing agent to synthesize AgNPs on the surface of pectin in situ for the preparation of a pectin/AgNPs/ZnO composite film. The physical and chemical properties of the films were characterized and analyzed by SEM (EDS), TEM, XRD, and UV spectroscopy. The results showed that the film's surface was smooth and flat, with small, well-stabilized AgNPs and uniformly dispersed ZnO. The film retained the original crystalline form of pectin, but exhibited altered crystallinity, indicating that the method employed was very mild. A systematic evaluation of the film’s antimicrobial performance using the dynamic growth curve, inhibition zone, and colony counting methods showed that AgNPs and ZnO imparted excellent, long-lasting antimicrobial activity to the film (98 %). Additionally, the films exhibited good flexibility, excellent ultraviolet protection (99.9 %), biocompatibility, and biodegradability. Notably, the film was sensitive to humidity, as demonstrated by its humidity-responsive bionic application. This study provides a promising reference and strategy for advancing the development of multifunctional polysaccharide-based nanocomposites.

Synthesis, characterization, and surface modification of degradable polar hydrophobic ionic polyurethane nanoparticles for the delivery of therapeutics to vascular tissue

Degradable polar hydrophobic ionic polyurethanes (D-PHI) are an emerging class of biomaterials with particular significance for blood-contacting applications due to their immunomodulatory effects and highly customizable block chemistry. In this manuscript, D-PHI polymer was formulated as a nanoparticle excipient for the first time by inverse emulsion polymerization. The nanoparticles were optimized with consideration of diameter, surface charge, size variability, and yield as a delivery vehicle for a custom vascular therapeutic peptide. A layer-by-layer (LBL) surface modification technique using poly-L-lysine was integrated within the nanoparticle design to optimize therapeutic loading efficiency. Solvent pH played a pivotal role in emulsion micelle formation, LBL polymer secondary structure, and the polymer functional group interactions critical for high therapeutic loading. The resulting nanoparticle platform met target size (200 ± 20 nm), polydispersity (<0.07), and storage stability standards, was nontoxic, and did not affect therapeutic peptide bioactivity in vitro. Surface-modified D-PHI nanoparticles can be reproducibly manufactured at low cost, generating a highly customizable excipient platform suitable for delivery of biomolecular therapeutics. These nanoparticles have potential applications in vascular drug delivery via localized infusion, drug eluting stents, and drug-coated angioplasty balloons. Statement of significanceNanoscale excipients have become critical in the delivery of many therapeutics to enhance drug stability and targeted biodistribution through careful design of nanoparticle composition, surface chemistry, and size. This manuscript describes the development of a nanoparticle excipient derived from an immunomodulatory degradable polar hydrophobic ionic polyurethane, in combination with a layer-by-layer surface modification approach utilizing poly-L-lysine, to transport a mimetic peptide targeting smooth muscle cell migration in vascular disease. The nanoparticle platform draws on the effect of pH to maximize drug loading and tailor particle properties. The low cost and easily reproducible system presents a highly customizable platform that can be adapted for therapeutic delivery across a wide range of clinical indications.

Chemoselective Approach to Versatile Acyl Fluorides by Photoinduced Activation of p-Methoxybenzyl Esters.

A new strategy for the metal-free photoinduced activation of p-methoxybenzyl esters is developed using Selectfluor and benzil for the generation of acyl fluoride intermediates that enable various transformations. The highlight of this activation method is its high chemoselectivity in the presence of other functionalities, such as esters, amides, and ketones. A synthetic application for the preparation of peptide mimetics that possess two different amide units is also described.

Annexin A1 activates the G protein-coupled formyl peptide receptor type 2-dependent endothelial nitric oxide synthase pathway to alleviate sepsis associated acute lung injury

To investigate whether annexin A1 (ANXA1) improves sepsis-induced lung injury by activating G protein-coupled formyl peptide receptor type 2 (FPR2)-dependent endothelial nitric oxide synthase (eNOS) pathway. Twenty-four male SD rats were randomly divided into normal group (Control group), lipopolysaccharide (LPS) induced lung injury model group (LPS group), LPS+ANXA1 mimetic peptide group (LPS+Ac2-26 group) and LPS+ANXA1 mimetic peptide+FPR2 inhibitor group (LPS+Ac2-26+WRW4 group), with 6 rats in each group. On the third day before modeling, rats of the LPS+Ac2-26 group were injected with 1 mg/kg Ac2-26 by the tail vein and rats of LPS+Ac2-26+WRW4 group were injected with 1 mg/kg Ac2-26 and 2.2 mg/kg WRW4 by the tail vein. The rats of control group and LPS group were injected same volume of physiological saline. After 48 hours of modeling, the rats were anesthetized and the carotid blood was taken to detect the oxygenation index (OI). Lung tissue was taken from the euthanized rats. The wet/dry (W/D) ratio was determined. The pathological changes of lung tissue were observed under light microscope and pathological score was performed. The levels of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6, IL-10), malondialdehyde (MDA) and myeloperoxidase (MPO) were detected by enzyme-linked immunosorbent assay (ELISA). The protein expressions of eNOS, inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) were detected by Western blotting. Under light microscope, compared with LPS group, the infiltration degree of inflammatory cells in the lung tissue of LPS+Ac2-26 group was reduced, and the thickness of the alveolar septum was improved. The degree of inflammatory cell infiltration in the lung tissue of LPS+Ac2-26+WRW4 group was more severe than that of LPS+Ac2-26 group, and the thickness of the alveolar septum increased. These findings suggested that ANXA1 significantly inhibited inflammatory cell infiltration and improved alveolar septal thickness, WRW4 reversed the lung improvement effects of ANXA1. Compared with control group, OI in LPS group was significantly decreased, and W/D ratio, pathological score and TNF-α, IL-1β, IL-6, MDA and MPO levels in lung tissue were significantly increased. Compared with LPS group, OI and IL-10 levels in lung tissue were significantly increased in LPS+Ac2-26 group, while W/D ratio, pathological score, TNF-α, IL-1β, IL-6, MDA and MPO levels in lung tissue were significantly decreased. These results indicated that ANXA1 can improve the oxygenation capacity, improve lung tissue leakage, reduce edema, and inhibit lung tissue inflammation in rats with lung injury. Compared with LPS+Ac2-26 group, the LPS+Ac2-26+WRW4 group showed significant decreases in OI and lung tissue IL-10 level [OI (mmHg, 1 mmHg ≈ 0.133 kPa): 132.16±24.00 vs. 248.67±18.70, IL-10 (ng/L): 27.30±3.04 vs. 36.10±3.92, both P < 0.05], the lung tissue W/D ratio, pathological score and levels of TNF-α, IL-1β, IL-6, MDA and MPO were significantly increased [W/D ratio: 5.29±0.02 vs. 4.83±0.02, pathological score: 5.00±0.28 vs. 2.67±0.52, TNF-α (ng/L): 39.80±4.36 vs. 32.10±2.15, IL-1β (ng/L): 200.00±15.68 vs. 152.60±9.74, IL-6 (ng/L): 181.50±18.02 vs. 148.50±7.34, MDA (mmol/mg): 82.01±8.22 vs. 70.43±5.69, MPO (pg/mg): 6.50±0.32 vs. 4.60±0.56, all P < 0.05]. These results suggested that WRW4 could block the above improvement of ANXA1. Western blotting results showed that compared with control group, the expression of eNOS, iNOS and NF-κB in LPS group was significantly up-regulated. Compared with LPS group, the protein expression of eNOS in LPS+Ac2-26 group was significantly up-regulated (eNOS/β-actin: 0.25±0.01 vs. 0.14±0.01, P < 0.05), and the protein expression of iNOS and NF-κB was significantly down-regulated (iNOS/β-actin: 0.09±0.02 vs. 0.12±0.02, NF-κB/β-actin: 0.35±0.06 vs. 0.59±0.13, both P < 0.05). These findings suggested that ANXA1 might activate the eNOS pathway and down-regulate the expression of NF-κB. Compared with LPS+Ac2-26 group, the protein expression of eNOS in LPS+Ac2-26+WRW4 group was significantly down-regulated (eNOS/β-actin: 0.17±0.02 vs. 0.25±0.01, P < 0.05), while the protein expression of iNOS and NF-κB was significantly up-regulated (iNOS/β-actin: 0.12±0.02 vs. 0.09±0.02, NF-κB/β-actin: 0.52±0.10 vs. 0.35±0.06, both P < 0.05). These results suggested that WRW4 blocked the activation of the eNOS pathway by ANXA1. ANXA1 can improve lung injury associated with sepsis by activating FPR2-dependent eNOS pathway.

Investigation of the effect of apoA-I mimetic peptides on the performance of therapeutic nanodiscs

Investigation of the effect of apoA-I mimetic peptides on the performance of therapeutic nanodiscs

Apolipoprotein AI mimetic peptide CS-6253 increases the atherosclerotic lesion stability index independent of leukocyte abca1 in bone marrow-derived cells

Apolipoprotein AI mimetic peptide CS-6253 increases the atherosclerotic lesion stability index independent of leukocyte abca1 in bone marrow-derived cells

Abstract Tu005: Small Extracellular Vesicles Derived from Bovine Milk Contain an Endogenous Carboxyl Terminal Polypeptide of The Gap Junction Protein Connexin-43

Background: Connexin 43 (Cx43) is the body's most widely expressed gap junctional connexin. Researchers have shown evidence of non-canonical functions of Cx43 and the therapeutic promise of Cx43 mimetic peptides in cardiac pathologies. However, some critical barriers, including in vivo stability and potential immunotoxicity of Cx43 mimetic peptides, hinder clinical translation of these pro-drugs. To address these challenges, we are pioneering bovine milk-derived small extracellular vesicles (mEVs) as delivery vehicles for peptidic drugs. While exploring mEVs as drug delivery tools for αCT1 – a peptide mimicking Cx43 carboxyl terminus (CT) undergoing Phase III testing and known for promoting wound healing and cardioprotection - mEVs without loaded drug cargo exhibited similar bioactivity. This study aimed to test the hypothesis that Cx43, particularly CT fragments of Cx43, are naturally present in mEVs. Methods: mEVs were isolated from bovine milk using a novel protocol developed by our lab based on Tangential Flow Filtration and characterized by Western Blotting (WB), Nanoparticle Tracking Analysis, and Transmission Electron Microscopy. To determine the presence of Cx43 in mEVs, we used WB, immunoprecipitation and Single-Molecule Localization Microscopy (SMLM). Results: Characterization of mEVs confirmed relatively pure and ultrastructurally definitive mEVs isolated from milk at high density. WB for Cx43 in mEVs lysates based on two antibodies - one against the cytoplasmic loop domain and the other against CT-most 20 amino acids of Cx43, showed that full-length Cx43 was present in mEVs. Interestingly, the Cx43 CT antibody also detected a band at around 16 KDa, and this was confirmed by WB by using two more Cx43 CT antibodies. We also immunoprecipitated this Cx43 CT polypeptide from mEVs lysates using the CT antibodies. SMLM suggested that a subpopulation of mEVs contain Cx43 and demonstrated vesicle-to-vesicle heterogeneity to Cx43 immunolabeling. Conclusions: We show for the first time that an endogenous Cx43 CT polypeptide is present in mEVs. Our results provide new insight into the role of Cx43 in mEV bioactivity and potentially offer a path to safe, effective, and orally available therapeutic options based on mEVs.

Supramolecular Motion Enables Chondrogenic Bioactivity of a Cyclic Peptide Mimetic of Transforming Growth Factor-β1.

Transforming growth factor (TGF)-β1 is a multifunctional protein that is essential in many cellular processes that include fibrosis, inflammation, chondrogenesis, and cartilage repair. In particular, cartilage repair is important to avoid physical disability since this tissue does not have the inherent capacity to regenerate beyond full development. We report here on supramolecular coassemblies of two peptide amphiphile molecules, one containing a TGF-β1 mimetic peptide, and another which is one of two constitutional isomers lacking bioactivity. Using human articular chondrocytes, we investigated the bioactivity of the supramolecular copolymers of each isomer displaying either the previously reported linear form of the mimetic peptide or a novel cyclic analogue. Based on fluorescence depolarization and 1H NMR spin-lattice relaxation times, we found that coassemblies containing the cyclic compound and the most dynamic isomer exhibited the highest intracellular TGF-β1 signaling and gene expression of cartilage extracellular matrix components. We conclude that control of supramolecular motion is emerging as an important factor in the binding of synthetic molecules to receptors that can be tuned through chemical structure.

Integrating Mechanics and Bioactivity: A Detailed Assessment of Elasticity and Viscoelasticity at Different Scales in 2D Biofunctionalized PEGDA Hydrogels for Targeted Bone Regeneration.

Methods for promoting and controlling the differentiation of human mesenchymal stem cells (hMSCs) in vitro before in vivo transplantation are crucial for the advancement of tissue engineering and regenerative medicine. In this study, we developed poly(ethylene glycol) diacrylate (PEGDA) hydrogels with tunable mechanical properties, including elasticity and viscoelasticity, coupled with bioactivity achieved through the immobilization of a mixture of RGD and a mimetic peptide of the BMP-2 protein. Despite the key relevance of hydrogel mechanical properties for cell culture, a standard for its characterization has not been proposed, and comparisons between studies are challenging due to the different techniques employed. Here, a comprehensive approach was employed to characterize the elasticity and viscoelasticity of these hydrogels, integrating compression testing, rheology, and atomic force microscopy (AFM) microindentation. Distinct mechanical behaviors were observed across different PEGDA compositions, and some consistent trends across multiple techniques were identified. Using a photoactivated cross-linker, we controlled the functionalization density independently of the mechanical properties. X-ray photoelectrin spectroscopy and fluorescence microscopy were employed to evaluate the functionalization density of the materials before the culturing of hMSCs on them. The cells cultured on all functionalized hydrogels expressed an early osteoblast marker (Runx2) after 2 weeks, even in the absence of a differentiation-inducing medium compared to our controls. Additionally, after only 1 week of culture with osteogenic differentiation medium, cells showed accelerated differentiation, with clear morphological differences observed among cells in the different conditions. Notably, cells on stiff but stress-relaxing hydrogels exhibited an overexpression of the osteocyte marker E11. This suggests that the combination of the functionalization procedure with the mechanical properties of the hydrogel provides a potent approach to promoting the osteogenic differentiation of hMSCs.

Drugs targeting APOE4 that regulate beta-amyloid aggregation in the brain: Therapeutic potential for Alzheimer's disease.

Alzheimer's disease is characterized by progressive cognitive decline, and behavioural and psychological symptoms of dementia are common. The APOE ε4 allele, a genetic risk factor, significantly increases susceptibility to the disease. Despite efforts to effectively treat the disease, only seven drugs are approved for its treatment, and only two of these prevent its progression. This highlights the need to identify new pharmacological options. This review focuses on mimetic peptides, small molecule correctors and HAE-4 antibodies that target ApoE. These drugs reduce β-amyloid-induced neurodegeneration in preclinical models. In addition, loop diuretics such as bumetanide and furosemide show the potential to reduce the prevalence of Alzheimer's disease in humans, and antidepressants such as imipramine improve cognitive function in individuals diagnosed with Alzheimer's disease. Consistent with this, both classes of drugs have been shown to exert neuroprotective effects by inhibiting ApoE4-catalysed Aβ aggregation in preclinical models. Moreover, peroxisome proliferator-activated receptor ligands, particularly pioglitazone and rosiglitazone, reduce ApoE4-induced neurodegeneration in animal models. However, they do not prevent the cognitive decline in APOE ε4 allele carriers. Finally, ApoE4 impairs the integrity of the blood-brain barrier and haemostasis. On this basis, ApoE4 modulation is a promising avenue for the treatment of late-onset Alzheimer's disease.

A mimetic peptide of ACE2 protects against SARS-CoV-2 infection and decreases pulmonary inflammation related to COVID-19

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22–46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.