Endothelial cell-derived extracellular vesicles induce pro-angiogenic responses in mesenchymal stem cells.

Angiogenesis is a central component of vital biological processes such as wound healing, tissue nourishment, and development. Therefore, angiogenic activities are precisely maintained with secreted factors such as angiopoietin-1 (Ang1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). As an element of intracellular communication, extracellular vesicles (EVs)-particularly EVs of vascular origin-could have key functions in maintaining angiogenesis. However, the functions of EVs in the control of angiogenesis have not been fully studied. In this study, human umbilical vein endothelial cell line (HUVEC)-derived small EVs (<200 nm; HU-sEVs) were investigated as a potential pro-angiogenic agent. Treating mesenchymal stem cells (MSCs) and mature HUVEC cells with HU-sEVs induced their tube formation under in vitro conditions and significantly increased the expression of angiogenesis-related genes, such as Ang1, VEGF, Flk-1 (VEGF receptor 2), Flt-1 (VEGF receptor 1), and vWF (von Willebrand Factor), in a dose-dependent manner. These results indicate that HU-sEVs take part in angiogenesis activities in physiological systems, and suggest endothelial EVs as a potential therapeutic candidate for the treatment of angiogenesis-related diseases.

A pilot study for treatment of severe COVID-19 pneumonia by aerosolized formulation of convalescent human immune plasma exosomes (ChipEXO™).

This is a single-center prospective, open-label, single arm interventional study to test the safety and efficacy of recently described ChipEXO™ for severe COVID-19 pneumonia. The ChipEXO™ is a natural product derived from convalescent human immune plasma of patients recovered from moderate COVID-19 infection. In September 2021, 13 patients with pending respiratory failure were treated with ChipEXO™ adapted for aerosolized formulation delivered via jet nebulizer. Patients received 1-5x1010 nano vesicle/5 mL in distilled water twice daily for five days as an add-on to ongoing conventional COVID-19 treatment. The primary endpoint was patient safety and survival over a 28-day follow-up. The secondary endpoint was longitudinal assessment of clinical parameters following ChipEXO™ to evaluate treatment response and gain insights into the pharmacodynamics. ChipEXO™ was tolerated well without any allergic reaction or acute toxicity. The survival rate was 84.6% and 11 out of 13 recovered without any sequel to lungs or other organs. ChipEXO™ treatment was effective immediately as shown in arterial blood gas analyses before and two hours after exosome inhalation. During the 5 days of treatment, there was a sustainable and gradual improvement on oxygenation parameters: i.e. respiratory rate (RR) [20.8% (P < 0.05)], oxygen saturation (SpO2) [6,7% (P < 0.05)] and partial pressure of oxygen to the fraction of inspired oxygen (PaO2/FiO2) [127.9% (P < 0.05)] that correlated with steep decrease in the disease activity scores and inflammatory markers, i.e. the sequential organ failure assessment (SOFA) score (75%, p < 0.05), C-reactive protein (46% p < 0.05), ferritin (58% p = 0.53), D-dimer (28% p=0.46). In conclusion, aerosolized ChipEXO™ showed promising safety and efficacy for life-threatening COVID-19 pneumonia. Further studies on larger patient populations are required to confirm our findings and understand the pathophysiology of improvement toward a new therapeutic agent for the treatment of severe COVID-19 pneumonia.

Preclinical Studies on Convalescent Human Immune Plasma-Derived Exosome: Omics and Antiviral Properties to SARS-CoV-2.

The scale of the COVID-19 pandemic forced urgent measures for the development of new therapeutics. One of these strategies is the use of convalescent plasma (CP) as a conventional source for passive immunity. Recently, there has been interest in CP-derived exosomes. In this report, we present a structural, biochemical, and biological characterization of our proprietary product, convalescent human immune plasma-derived exosome (ChipEXO), following the guidelines set forth by the Turkish Ministry of Health and the Turkish Red Crescent, the Good Manufacturing Practice, the International Society for Extracellular Vesicles, and the Gene Ontology Consortium. The data support the safety and efficacy of this product against SARS-CoV-2 infections in preclinical models.

Lead Borate Nanoparticles Induce Apoptotic Gene Activity in P53 Mutant Cancer Cells.

Cancer is a complex and multistage disease that causes suffering worldwide. Several mutations in tumor suppressor proteins are mostly responsible for tumorigenic development. Thus, determination of the mutations and developing a mutation targeted therapy are crucial in order to cure cancer. Moreover, since healthy cells do not have mutations in their tumor suppressor genes, mutation-specific treatment is responsible for selective treatment without harming a healthy tissue in the body. In this current study, lead borate nanoparticles (LB-Np) have been synthesized, and their effects on P53 mutant cancer cells were investigated. The synthesis method includes steps of mixing a borate buffer solution with the lead nitrate solution, washing the resulting precipitate with distilled water and eventually preparing stable LB-Np solutions. Cell viability analysis was conducted to identify the toxicity of LB-Np in HaCaT, A549, MCF7, and T47D cell lines. The changes in morphologies of breast cancer cell lines were demonstrated by using microscopical analysis. Additionally, alterations in gene expressions were determined in breast cancer cell lines after LB-Np treatment. This multidisciplinary study also identified the selective effect of LB-Np in cancer cell lines, in vitro. MTS and quantitative polymerase chain reaction assays demonstrated the effect of LB-Np were specific for p53 mutation cell line, T47D. Breast cancer cell line T47D has 580 C/T mutation which affects the activation of p53 tumor suppressor protein. However, LB-Np treatment effectively killed T47D cell lines and did not affect any other cell lines that have no p53 mutations such as MCF7, A549, and healthy HaCaT. Overall, synthesized LB-Np were found to be effective in p53-mutated cell lines and showed a remarkable selective anti-cancer activity.

Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing.

Due to the prevalence of individuals suffering from chronic wounds, developing safe and effective wound care agents are one of the more prominent fields of research in biology. However, wound healing is a complex, multi-stage biological process, involving multiple sequences of biological responses from different types of cells, secreted mediators, and extracellular matrix elements. Plants have a long history of use in the treatment of wounds. Plant-derived extracellular vesicles, which are secreted nano vesicle messengers responsible for intercellular communications, show promise as a new, biotechnological wound-care agent. In this study, we assessed the wound healing potential of extracellular vesicles isolated from grapefruits - a plant with well-known anti-inflammatory and wound healing properties. Grapefruit extracellular vesicles (GEVs) increased cell viability and cell migration while reducing intracellular ROS production in a dose-dependent manner in HaCaT cells. Expression of proliferation and migration-related genes were raised by GEV treatment in a dose dependent manner. Additionally, GEV treatment increased the tube formation capabilities of treated HUVEC cells. These findings suggest that GEVs can be used as plant-derived wound healing agents, and have shown potential as a biotechnological agent for wound healing. Further development and study of plant-derived extracellular vesicles may lead to the realization of their full potential.

Effective Scarless Wound Healing Mediated by Erbium Borate Nanoparticles.

The developments of nanoparticle-based treatments that benefit from novel discoveries have an essential place in the regeneration of acute and chronic wounds. Furthermore, research about the treatment methods which attempt to swiftly and scarless wound recovery has increased over time. In recent years, it has been shown that metallic-based nanoparticles, especially silver and gold derived, have an accelerating effect on chronic and contaminated wound healing. The crucial factors of inducing and completion of regeneration of wound are enhanced epithelialization rate and neovascularization in the tissue. In our study, the main purpose is the investigation of the boosting effects of erbium borate nanoparticles on the wound healing process, especially scarless ones. Newly syntesized erbium borate nanoparticles (ErB-Nps) were characterized by their concentration and particle size using nanoparticle tracking analysis (NTA). In order to examine the effect of ErB-Np on wound closure, scratch assay for dermal epithelial cells and tube formation assay for endothelial cells were performed. In addition, in order to examine the effect of the ErB-Np at a molecular level, the levels of genes related to both wound healing, inflammation, and scarless wound closure were determined with the RT-PCR experiment. Consequently, it has been shown that erbium borate nanoparticles have increased the melioration speed of scar tissue and have given clues about scarless healing potential. The investigation of the regeneration potential of erbium borate nanoparticles was done via MTS assay, quantitative PCR analysis, reactive oxygen species assay, and scratch assay. Our results show that ErB-Np is a proper agent that can be used for scarless wound healing.

In vitro tooth-shaped scaffold construction by mimicking late bell stage.

Neogenesis of osseous and ligamentous interfacial structures is essential for the regeneration of large oral or craniofacial defects. However, current treatment strategies are inadequate in renewing supporting tissues of teeth after trauma, chronic infections or surgical resection. Combined use of 3D scaffolds with stem cells became a promising treatment option for these injuries. Matching different scaffolding materials with different tissues can induce the correct cytokines and the differentiation of cells corresponding to that particular tissue. In this study, a hydroxyapatite (HA) based scaffold was used together with human adipose stem cells (hASCs), human bone marrow stem cells (hBMSCs) and gingival epithelial cells to mimic human tooth dentin-pulp-enamel tissue complexes and model an immature tooth at the late bell stage in vitro. Characteristics of the scaffold were determined via SEM, FTIR, pore size and density measurements. Changes in gene expression, protein secretions and tissue histology resulting from cross-interactions of different dental tissues grown in the system were shown. Classical tooth tissues such as cementum, pulp and bone like tissues were formed within the scaffold. Our study suggests that a HA-based scaffold with different cell lineages can successfully mimic early stages of tooth development and can be a valuable tool for hard tissue engineering.

Optimized Isolation of Extracellular Vesicles From Various Organic Sources Using Aqueous Two-Phase System.

From biomarkers to drug carriers, Extracellular Vesicles (EVs) are being used successfully in numerous applications. However, while the subject has been steadily rising in popularity, current methods of isolating EVs are lagging behind, incapable of isolating EVs at a high enough quantity or quality while also requiring expensive, specialized equipment. The "isolation problem" is one of the major obstacles in the field of EV research - and even more so for their potential, widespread use for clinical diagnosis and therapeutic applications. Aqueous Two-Phase Systems (ATPS) has been reported previously as a promising method for isolating EVs quickly and efficiently, and with little contaminants - however, this method has not seen widespread use. In this study, an ATPS-based isolation protocol is used to isolate small EVs from plant, cell culture, and parasite culture sources. Isolated EVs were characterized in surface markers, size, and morphological manner. Additionally, the capacity of ATPS-based EV isolation in removing different contaminants was shown by measuring protein, fatty acid, acid, and phenol red levels of the final isolate. In conclusion, we have shown that EVs originating from different biological sources can be isolated successfully in a cost-effective and user-friendly manner with the use of aqueous two-phase systems.

Immunomodulatory Behavior of Mesenchymal Stem Cells.

The use of Mesenchymal Stem Cells (MSCs) in the treatment of diseases where immunomodulation impacts therapy is increasing steadily. Recent studies aim to achieve effective use of MSCs in treatment of Graft versus Host Disease (GvHD), Crohn's disease and organ transplantations. The molecular mechanisms governing immunomodulatory properties of MSCs have not been fully understood, although current studies are indicating progress. Especially, in vitro studies and animal models provide a major contribution to our knowledge in clinical use of MSCs. The immunosuppressive and immune-enhancer properties of MSCs are -typically- determined with respect to type and concentrations of soluble molecules found in their physiological environment. In mammals the immune system protects the organism -not only- from certain microorganisms, but also from any entity that it recognizes as foreign, including its own cells when it is received as a threat. This protection can sometimes occur by increasing the number of immune cells and sometimes by suppressing a pathologically hyper-induced immunological response. In particular, realization of the bi-directional effect of MSCs on immune cells has placed substantial emphasis on this area of research. This chapter focuses on the interaction of MSCs with the immune cells, the bilateral role of these interactions, and whether studies that aim to understand these interactions can yield promising results in terms of developing improved use of MSCs in treatment.