SARS-CoV-2 Accessory Protein Orf7b Induces Lung Injury via c-Myc Mediated Apoptosis and Ferroptosis.

The pandemic of coronavirus disease 2019 (COVID-19) has been the foremost modern global public health challenge. The airway is the primary target in severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) infection, with substantial cell death and lung injury being signature hallmarks of exposure. The viral factors that contribute to cell death and lung injury remain incompletely understood. Thus, this study investigated the role of open reading frame 7b (Orf7b), an accessory protein of the virus, in causing lung injury. In screening viral proteins, we identified Orf7b as one of the major viral factors that mediates lung epithelial cell death. Overexpression of Orf7b leads to apoptosis and ferroptosis in lung epithelial cells, and inhibitors of apoptosis and ferroptosis ablate Orf7b-induced cell death. Orf7b upregulates the transcription regulator, c-Myc, which is integral in the activation of lung cell death pathways. Depletion of c-Myc alleviates both apoptotic and ferroptotic cell deaths and lung injury in mouse models. Our study suggests a major role of Orf7b in the cell death and lung injury attributable to COVID-19 exposure, supporting it as a potential therapeutic target.

Nanotopographical Cues Tune the Therapeutic Potential of Extracellular Vesicles for the Treatment of Aged Skeletal Muscle Injuries.

Skeletal muscle regeneration relies on the tightly temporally regulated lineage progression of muscle stem/progenitor cells (MPCs) from activation to proliferation and, finally, differentiation. However, with aging, MPC lineage progression is disrupted and delayed, ultimately causing impaired muscle regeneration. Extracellular vesicles (EVs) have attracted broad attention as next-generation therapeutics for promoting tissue regeneration. As a next step toward clinical translation, strategies to manipulate EV effects on downstream cellular targets are needed. Here, we developed an engineering strategy to tune the therapeutic potential of EVs using nanotopographical cues. We found that EVs released by young MPCs cultured on flat substrates (fEVs) promoted the proliferation of aged MPCs while EVs released by MPCs cultured on nanogratings (nEVs) promoted myogenic differentiation. We then employed a bioengineered 3D muscle aging model to optimize the administration protocol and test the therapeutic potential of fEVs and nEVs in a high-throughput manner. We found that the sequential administration first of fEVs during the phase of MPC proliferative expansion (i.e., 1 day after injury) followed by nEV administration at the stage of MPC differentiation (i.e., 3 days after injury) enhanced aged muscle regeneration to a significantly greater extent than fEVs and nEVs delivered either in isolation or mixed. The beneficial effects of the sequential EV treatment strategy were further validated in vivo, as evidenced by increased myofiber size and improved functional recovery. Collectively, our study demonstrates the ability of topographical cues to tune EV therapeutic potential and highlights the importance of optimizing the EV administration strategy to accelerate aged skeletal muscle regeneration.

Arsenic disrupts extracellular vesicle-mediated signaling in regenerating myofibers.

Chronic exposure to environmental arsenic is a public health crisis affecting hundreds of millions of individuals worldwide. Though arsenic is known to contribute to many pathologies and diseases, including cancers, cardiovascular and pulmonary diseases, and neurological impairment, the mechanisms for arsenic-promoted disease remain unresolved. This is especially true for arsenic impacts on skeletal muscle function and metabolism, despite the crucial role that skeletal muscle health plays in maintaining cardiovascular health, systemic homeostasis, and cognition. A barrier to researching this area is the challenge of interrogating muscle cell-specific effects in biologically relevant models. Ex vivo studies investigating mechanisms for muscle-specific responses to arsenic or other environmental contaminants primarily utilize traditional 2-dimensional culture models that cannot elucidate effects on muscle physiology or function. Therefore, we developed a contractile 3-dimensional muscle construct model-composed of primary mouse muscle progenitor cells differentiated in a hydrogel matrix-to study arsenic exposure impacts on skeletal muscle regeneration. Muscle constructs exposed to low-dose (50 nM) arsenic exhibited reduced strength and myofiber diameter following recovery from muscle injury. These effects were attributable to dysfunctional paracrine signaling mediated by extracellular vesicles (EVs) released from muscle cells. Specifically, we found that EVs collected from arsenic-exposed muscle constructs recapitulated the inhibitory effects of direct arsenic exposure on myofiber regeneration. In addition, muscle constructs treated with EVs isolated from muscles of arsenic-exposed mice displayed significantly decreased strength. Our findings highlight a novel model for muscle toxicity research and uncover a mechanism of arsenic-induced muscle dysfunction by the disruption of EV-mediated intercellular communication.

Utility of extracellular vesicles as a potential biological indicator of physiological resilience during military operational stress.

Extracellular vesicles (EVs) transport biological content between cells to mediate physiological processes. The association between EVs and resilience, the ability to cope with stress, is unknown. Using unbiased machine learning approaches, we aimed to identify a biological profile of resilience. Twenty servicemen (27.8 ± 5.9 years) completed the Connor Davidson Resilience (CD-RISC) questionnaire and were exposed to daily physical and cognitive exertion with 48-hr sleep and caloric restriction. Blood samples from baseline and the second day of stress were analyzed for neuroendocrine biomarkers impacted by military stress. EVs were isolated from plasma and stained with antibodies associated with exosomes (CD63), microvesicles (VAMP3), and apoptotic bodies (THSD1). Individuals were separated into high (n = 10, CD-RISC > 90) and low (n = 10, CD-RISC < 79) resilience. EV features were stratified by size, then down-selected using regression trees and compared between groups. Diagnostic accuracy was assessed using receiver operating characteristic curves. Compared to low resilience, high resilience demonstrated a greater increase in variability of THSD1 local bright spot intensities among large-sized EVs in response to stress (p = 0.002, Hedges' g = 1.59). Among medium-sized EVs, high resilience exhibited a greater decrease in side scatter intensity (p = 0.014, Hedges' g = 1.17). Both features demonstrated high to moderate diagnostic accuracy for high resilience (AUC = 0.90 and 0.79). In contrast, neuroendocrine biomarker concentrations were similar between groups. The increase in variability among THSD1 + EVs in high, but not low, resilient individuals following stress may suggest high resilience is accompanied by stress-triggered apoptotic adaptations to the environment that are not detected in neuroendocrine biomarkers.

Men and women display distinct extracellular vesicle biomarker signatures in response to military operational stress.

Extracellular vesicles (EVs) are mediators of physiological changes that occur during physical exertion. This study examined the effects of physical exertion with and without sleep and caloric restriction on EV size, concentration, and surface proteins in men and women. Twenty participants (10 men) completed a 5-day simulated military operational stress protocol with daily physical exertion. Blood was drawn before and immediately after exertion at baseline (D1) and following 48-h of sleep and caloric restriction (D3). EV size and concentration were assessed using nanoparticle tracking analysis. EVs were identified with markers associated with exosomes (CD63), microvesicles (VAMP3), apoptotic bodies (THSD1), and skeletal muscle-derived EVs (SGCA) and quantified using imaging flow cytometry. Interactive and main effects of sex, day, and time on EVs were assessed using three-way ANOVAs. EV concentration declined pre to postexertion in women on D1 and D3 but was stable in men. EV size increased from pre to postexertion and from D1 to D3 in men and women. Physical exertion following sleep and caloric restriction increased CD63+ EV concentration, proportion of total EVs, and CD63 surface protein expression regardless of sex. The proportion of SGCA+ EVs increased in men and women following exertion and from D1 to D3 but was higher in women than in men. No differences were observed in VAMP3+ and THSD1+ EVs. This study identified sexually dimorphic EV profiles in response to various stressors. Further investigations are necessary to determine if dimorphic EV responses affect health and performance outcomes during stress.NEW & NOTEWORTHY Sex is understudied in EV research, and most studies limit EV analysis to single stress conditions such as exercise. Multistress conditions consisting of physical exertion and sleep and caloric restriction are common in real-world settings. We demonstrate that physical exertion results in sex-specific EV signatures and that EV profiles vary according to single versus multistress conditions. Our data highlight important biological and ecological characteristics that should be considered in EV research.

The biphasic and age-dependent impact of klotho on hallmarks of aging and skeletal muscle function.

Aging is accompanied by disrupted information flow, resulting from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders including skeletal muscle wasting, or sarcopenia. To derive a global metric of growing 'disorderliness' of aging muscle, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Escalating network entropy reached an inflection point at old age, while structural and functional alterations progressed into oldest-old age. To probe the potential for restoration of molecular 'order' and reversal of the sarcopenic phenotype, we systemically overexpressed the longevity protein, Klotho, via AAV. Klotho overexpression modulated genes representing all hallmarks of aging in old and oldest-old mice, but pathway enrichment revealed directions of changes were, for many genes, age-dependent. Functional improvements were also age-dependent. Klotho improved strength in old mice, but failed to induce benefits beyond the entropic tipping point.

Regulation of aged skeletal muscle regeneration by circulating extracellular vesicles.

Heterochronic blood exchange (HBE) has demonstrated that circulating factors restore youthful features to aged tissues. However, the systemic mediators of those rejuvenating effects remain poorly defined. We show here that the beneficial effect of young blood on aged muscle regeneration was diminished when serum was depleted of extracellular vesicles (EVs). Whereas EVs from young animals rejuvenate aged cell bioenergetics and skeletal muscle regeneration, aging shifts EV subpopulation heterogeneity and compromises downstream benefits on recipient cells. Machine learning classifiers revealed that aging shifts the nucleic acid, but not protein, fingerprint of circulating EVs. Alterations in sub-population heterogeneity were accompanied by declines in transcript levels of the pro-longevity protein, α-Klotho, and injection of EVs improved muscle regeneration in a Klotho mRNA-dependent manner. These studies demonstrate that EVs play a key role in the rejuvenating effects of HBE and that Klotho transcripts within EVs phenocopy the effects of young serum on aged skeletal muscle.

Klotho: An Elephant in Aging Research.

The year 2017 marked the 20th anniversary of the first publication describing Klotho. This single protein was and is remarkable in that its absence in mice conferred an accelerated aging, or progeroid, phenotype with a dramatically shortened life span. On the other hand, genetic overexpression extended both health span and life span by an impressive 30%. Not only has Klotho deficiency been linked to a number of debilitating age-related illnesses but many subsequent reports have lent credence to the idea that Klotho can compress the period of morbidity and extend the life span of both model organisms and humans. This suggests that Klotho functions as an integrator of organ systems, making it both a promising tool for advancing our understanding of the biology of aging and an intriguing target for interventional studies. In this review, we highlight advances in our understanding of Klotho as well as key challenges that have somewhat limited our view, and thus translational potential, of this potent protein.