Circ_0011129 Encapsulated by the Small Extracellular Vesicles Derived from Human Stem Cells Ameliorate Skin Photoaging

Photoaging is not only the main cause of skin aging caused by exogenous factors, it is also related to a variety of skin diseases and even malignant tumors. Excessive and repeated exposure to ultraviolet radiation, especially UVA induces oxidative stress, DNA damage, inflammation, and collagen and elastin degeneration, ultimately leads to skin photoaging, manifested by skin redness, coarse wrinkles, and pigmentation even skin cancer. There has been a large demand of effective prevention and medications but approaches in the current management of photoaging are very limited. In the previous study, we found that a non-coding circular RNA circ_0011129 acts as a miR-6732-5p adsorption sponge to inhibit the reduction of type I collagen and the denaturation and accumulation of elastin in UVA-induced HDF cells photoaging model. However, in vivo instability and efficient delivery to the target cell of circRNA is a major challenge for its clinical application. Therefore, improving its stability and delivery efficiency are desired. In this study, we proposed a strategy of delivering circ_0011129 with small extracellular vesicles (sEVs) from human adipose-derived stem cells (hADSCs) to intervene in the photoaging process. The results showed that sEVs from hADSCs in 3D bioreactor culture (3D-sEVs) can prevent photoaging. Consequently, by overexpressing circ_0011129 in hADSCs, we successfully loaded it into 3D-sEVs (3D-circ-sEVs) and its protective effect was better. Our studies provide a novel approach to preventing skin photoaging, which has important clinical significance and application value for the development of non-coding RNA drugs to treat skin photoaging. We first screened out hADSCs-derived sEVs with excellent anti-oxidant effects. We then compared the sEVs collected from traditional 2D culture with 3D bioreactor culture. By miRNA-seq and GEO data analysis, we found that miRNAs in 3D-sEVs were enriched in cell activities related to apoptosis, cellular senescence, and inflammation. Subsequently, we prepared circ_0011129-loaded 3D-sEVs (3D-circ-sEVs) by overexpressing it in hADSCs for the treatment of photoaging in vitro. We proved that 3D-circ-sEVs can interfere with the process of cell photoaging and protect cells from UVA radiation damage, as well as in a H2O2-induced oxidative stress model.     

Evaluation of Circulating Platelet Extracellular Vesicles and Hypertension Mediated Organ Damage

Elevated circulating platelet-derived extracellular vesicles (pEVs) have been associated with arterial hypertension. The role of hypertension-mediated organ damage (HMOD) to induce EV release is still unknown. We studied the micro- and macro-vascular changes (retinal vascular density and pulse wave velocity), endothelial function (flow-mediated vasodilation of brachial artery and finger plethysmography), and assessed the psychosocial status (anxiety and depression) in hypertensive patients to determine their relationship with EV release. Pulse wave velocity showed a significant positive correlation with pEVs (r = 0.33; p = 0.01). Systolic blood pressure (SBP) negatively correlated with retinal vascularity. The superficial retinal vascular plexus density in the whole image showed a significant negative correlation with 24 h SBP (r = −0.38, p < 0.01), day-SBP (r = −0.35, p = 0.01), and night-SBP (r = −0.27, p = 0.04). pEVs did not show significant associations with microvascular damage (retinal vascular density), endothelial function (flow-mediated vasodilation of brachial artery and finger plethysmography), or psychosocial status (anxiety and depression). Our results indicate that the pEV levels were associated with macrovascular damage measured by PWV, whereas no significant association between pEVs and microvascular damage, endothelial function, or emotional status could be detected. The potential utility of pEV in clinical practice in the context of HMOD may be limited to macrovascular changes.     

What Are the Reasons for Continuing Failures in Cancer Therapy? Are Misleading/Inappropriate Preclinical Assays to Be Blamed? Might Some Modern Therapies Cause More Harm than Benefit?

Over 50 years of cancer research has resulted in the generation of massive amounts of information, but relatively little progress has been made in the treatment of patients with solid tumors, except for extending their survival for a few months at best. Here, we will briefly discuss some of the reasons for this failure, focusing on the limitations and sometimes misunderstanding of the clinical relevance of preclinical assays that are widely used to identify novel anticancer drugs and treatment strategies (e.g., “synthetic lethality”). These include colony formation, apoptosis (e.g., caspase-3 activation), immunoblotting, and high-content multiwell plate cell-based assays, as well as tumor growth studies in animal models. A major limitation is that such assays are rarely designed to recapitulate the tumor repopulating properties associated with therapy-induced cancer cell dormancy (durable proliferation arrest) reflecting, for example, premature senescence, polyploidy and/or multinucleation. Furthermore, pro-survival properties of apoptotic cancer cells through phoenix rising, failed apoptosis, and/or anastasis (return from the brink of death), as well as cancer immunoediting and the impact of therapeutic agents on interactions between cancer and immune cells are often overlooked in preclinical studies. A brief review of the history of cancer research makes one wonder if modern strategies for treating patients with solid tumors may sometimes cause more harm than benefit.     

Microbiota-Derived Extracellular Vesicles Detected in Human Blood from Healthy Donors

The microbiota constitutes an important part of the holobiont in which extracellular vesicles (EVs) are key players in health, especially regarding inter- and intra-kingdom communications. Analysis of EVs from the red blood cell concentrates of healthy donors revealed variable amounts of OmpA and LPS in 12 of the 14 analyzed samples, providing indirect experimental evidence of the presence of microbiota EVs in human circulating blood in the absence of barrier disruption. To investigate the role of these microbiota EVs, we tracked the fusion of fluorescent Escherichia coli EVs with blood mononuclear cells and showed that, in the circulating blood, these EVs interacted almost exclusively with monocytes. This study demonstrates that bacterial EVs constitute critical elements of the host–microbiota cellular communication. The analysis of bacterial EVs should thus be systematically included in any characterization of human EVs.     

Brown Adipose Tissue Sheds Extracellular Vesicles That Carry Potential Biomarkers of Metabolic and Thermogenesis Activity Which Are Affected by High Fat Diet Intervention

Brown adipose tissue (BAT) is a key target for the development of new therapies against obesity due to its role in promoting energy expenditure; BAT secretory capacity is emerging as an important contributor to systemic effects, in which BAT extracellular vesicles (EVs) (i.e., batosomes) might be protagonists. EVs have emerged as a relevant cellular communication system and carriers of disease biomarkers. Therefore, characterization of the protein cargo of batosomes might reveal their potential as biomarkers of the metabolic activity of BAT. In this study, we are the first to isolate batosomes from lean and obese Sprague–Dawley rats, and to establish reference proteome maps. An LC-SWATH/MS analysis was also performed for comparisons with EVs secreted by white adipose tissue (subcutaneous and visceral WAT), and it showed that 60% of proteins were exclusive to BAT EVs. Precisely, batosomes of lean animals contain proteins associated with mitochondria, lipid metabolism, the electron transport chain, and the beta-oxidation pathway, and their protein cargo profile is dramatically affected by high fat diet (HFD) intervention. Thus, in obesity, batosomes are enriched with proteins involved in signal transduction, cell communication, the immune response, inflammation, thermogenesis, and potential obesity biomarkers including UCP1, Glut1, MIF, and ceruloplasmin. In conclusion, the protein cargo of BAT EVs is affected by the metabolic status and contains potential biomarkers of thermogenesis activity.     

Therapeutic Potential of Extracellular Vesicles in Aging and Age-Related Diseases

Aging is associated with an alteration of intercellular communication. These changes in the extracellular environment contribute to the aging phenotype and have been linked to different aging-related diseases. Extracellular vesicles (EVs) are factors that mediate the transmission of signaling molecules between cells. In the aging field, these EVs have been shown to regulate important aging processes, such as oxidative stress or senescence, both in vivo and in vitro. EVs from healthy cells, particularly those coming from stem cells (SCs), have been described as potential effectors of the regenerative potential of SCs. Many studies with different animal models have shown promising results in the field of regenerative medicine. EVs are now viewed as a potential cell-free therapy for tissue damage and several diseases. Here we propose EVs as regulators of the aging process, with an important role in tissue regeneration and a raising therapy for age-related diseases.     

Rhodococcus equi-Derived Extracellular Vesicles Promoting Inflammatory Response in Macrophage through TLR2-NF-κB/MAPK Pathways

Rhodococcus equi (R. equi) is a Gram-positive coccobacillus that causes pneumonia in foals of less than 3 months, which have the ability of replication in macrophages. The ability of R. equi persist in macrophages is dependent on the virulence plasmid pVAPA. Gram-positive extracellular vesicles (EVs) carry a variety of virulence factors and play an important role in pathogenic infection. There are few studies on R. equi-derived EVs (R. equi-EVs), and little knowledge regarding the mechanisms of how R. equi-EVs communicate with the host cell. In this study, we examine the properties of EVs produced by the virulence strain R. equi 103⁺ (103⁺-EVs) and avirulenct strain R. equi 103⁻ (103⁻-EVs). We observed that 103⁺-EVs and 103⁻-EVs are similar to other Gram-positive extracellular vesicles, which range from 40 to 260 nm in diameter. The 103⁺-EVs or 103⁻-EVs could be taken up by mouse macrophage J774A.1 and cause macrophage cytotoxicity. Incubation of 103⁺-EVs or 103⁻-EVs with J774A.1 cells would result in increased expression levels of IL-1β, IL-6, and TNF-α. Moreover, the expression of TLR2, p-NF-κB, p-p38, and p-ERK were significantly increased in J774A.1 cells stimulated with R. equi-EVs. In addition, we presented that the level of inflammatory factors and expression of TLR2, p-NF-κB, p-p38, and p-ERK in J774A.1 cells showed a significant decreased when incubation with proteinase K pretreated-R. equi-EVs. Overall, our data indicate that R. equi-derived EVs are capable of mediating inflammatory responses in macrophages via TLR2-NF-κB/MAPK pathways, and R. equi-EVs proteins were responsible for TLR2-NF-κB/MAPK mediated inflammatory responses in macrophage. Our study is the first to reveal potential roles for R. equi-EVs in immune response in R. equi-host interactions and to compare the differences in macrophage inflammatory responses mediated by EVs derived from virulent strain R. equi and avirulent strain R. equi. The results of this study have improved our knowledge of the pathogenicity of R. equi.     

Brownian Motion Influence on AFM Exosomes’ Size Measurements

Extracellular vesicles are evaluated by nanoparticle tracking analysis (NTA), providing information on their hydrodynamic diameters, and by atomic force microscopy (AFM) to calculate their geometric diameters. The aim of this study is to explore the influence of Brownian movements in a sample drop and preparation time on imaging-based measurements and to determine the relationship between the geometric and hydrodynamic sizes of the extracellular vesicles measured by the AFM and the NTA, respectively. Exosomes derived from the human prostate cancer cell line PC3 were evaluated by NTA and AFM, and those results were compared with Monte Carlo simulations. The mean size, evaluated by AFM shortly after application on the mica substrate, is less than its real value. It obtains the correct value faster for a thinner sample drop. Fitting the log-normal distribution to the geometric and hydrodynamic diameters leads to the conclusion that the latter could arise from the former by linear scaling by a factor that could be used to characterize the analyzed extracellular vesicles. The size of the vesicles attached to the mica substrate depends on time. The effect of Brownian motion and stretch of the lipid bilayer should be considered in the context of exosome AFM studies.     

CD73-Adenosinergic Axis Mediates the Protective Effect of Extracellular Vesicles Derived from Mesenchymal Stromal Cells on Ischemic Renal Damage in a Rat Model of Donation after Circulatory Death

We propose a new organ-conditioning strategy based on mesenchymal stromal cell (MSCs)/extracellular vesicle (EVs) delivery during hypothermic perfusion. MSCs/EVs marker CD73 is present on renal proximal tubular cells, and it protects against renal ischemia-reperfusion injury by converting adenosine monophosphate into adenosine (ADO). In this study, after checking if CD73-silenced EVs (EVsi) would impact in vitro tubular-cell proliferation, we perfused kidneys of a rat model of donation after circulatory death, with Belzer solution (BS) alone, BS supplemented with MSCs, EVs, or EVsi. The ADO and ATP levels were measured in the effluents and tissues. Global renal ischemic damage score (GRS), and tubular cell proliferation index (IPT) were evaluated in the tissue. EVsi did not induce cell proliferation in vitro. Ex vivo kidneys perfused with BS or BS + EVsi showed the worst GRS and higher effluent ADO levels than the MSC- and EV-perfused kidneys. In the EV-perfused kidneys, the tissue and effluent ATP levels and IPT were the highest, but not if CD73 was silenced. Tissue ATP content was positively correlated with tissue ADO content and negatively correlated with effluent ADO level in all groups. In conclusion, kidney conditioning with EVs protects against ischemic damage by activating the CD73/ADO system.