Comparison of aggregation enhancement and inhibition as strategies for reducing the cytotoxicity of the aortic amyloid polypeptide medin

Aortic medial amyloid (AMA) occurs as localised non-atheromatous plaques in virtually all individuals over the age of 50. The major protein component of AMA is the 50-residue polypeptide medin. Here we propose two methods of manipulating medin aggregation to reduce the cytotoxic species of medin: either by promoting formation of larger benign species or retaining small non-cytotoxic species. Medin co-localises with a variety of factors including glycosaminoglycans (GAGs). The first approach shows that the GAG heparin enhances the rate of medin aggregation and alters the morphology of the amyloid fibrils. Cellular viability measurements suggest that heparin eliminates small cytotoxic species of medin, promoting formation of benign fibrils. The second approach applies a previously successful approach of designing small peptide moieties that are complementary to the key amyloidogenic sequence but which contain modified amino acids known to disrupt hydrogen bonding and therefore prevent aggregation of the target protein. This approach also reduces cellular toxicity of medin at all stages of the aggregation process examined exhibiting a different mode of action to heparin. These results raise the question of whether enhancement of medin aggregation by GAGs is beneficial, by eliminating toxic oligomers, or has deleterious effects by reducing arterial plasticity associated with increased fibril load and whether small peptide inhibitors can be applied as drug candidates for amyloid diseases.     

Oxidative Stress Alters the Morphology and Toxicity of Aortic Medial Amyloid

The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity.     

Emerging roles of extracellular vesicles in cellular senescence and aging

Cellular senescence is a cellular program that prevents the proliferation of cells at risk of neoplastic transformation. On the other hand, age‐related accumulation of senescent cells promotes aging at least partially due to the senescence‐associated secretory phenotype, whereby cells secrete high levels of inflammatory cytokines, chemokines, and matrix metalloproteinases. Emerging evidence, however, indicates that extracellular vesicles (EVs) are important mediators of the effects of senescent cells on their microenvironment. Senescent cells secrete more EphA2 and DNA via EVs, which can promote cancer cell proliferation and inflammation, respectively. Extracellular vesicles secreted from DNA‐damaged cells can also affect telomere regulation. Furthermore, it has now become clear that EVs actually play important roles in many aspects of aging. This review is intended to summarize these recent progresses, with emphasis on relationships between cellular senescence and EVs.     

Increased production of functional small extracellular vesicles in senescent endothelial cells

Small extracellular vesicles (EVs) are novel players in vascular biology. However, a thorough understanding of their production and function remains elusive. Endothelial senescence is a key feature of vascular ageing and thus, is an attractive therapeutic target for the treatment of vascular disease. In this study, we sought to characterize the EV production of senescent endothelial cells. To achieve this, Human Umbilical Vascular Endothelial Cells (HUVECs) were replicated until they reached senescence, as determined by measurement of Senescence‐Associated β‐Galactosidase activity via microscopy and flow cytometry. Expression of the endosomal marker Rab7 and the EV marker CD63 was determined by immunofluorescence. Small EVs were isolated by ultracentrifugation and characterized using electron microscopy, nanoparticle tracking analysis and immunoassays to assess morphology, size, concentration and expression of exosome markers CD9 and CD81. Migration of HUVECs in response to EVs was studied using a transwell assay. The results showed that senescent endothelial cells express higher levels of Rab7 and CD63. Moreover, senescent endothelial cells produced higher levels of CD9‐ and CD81‐positive EVs. Additionally, small EVs from both young and senescent endothelial cells promoted HUVEC migration. Overall, senescent endothelial cells produce an increased number of functional small EVs, which may have a role in vascular physiology and disease.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C assignment of the amyloidogenic protein medin using fast-pulsing NMR techniques

Thirty-one proteins are known to form extracellular fibrillar amyloid in humans. Molecular information about many of these proteins in their monomeric, intermediate or fibrillar form and how they aggregate and interact to form the insoluble fibrils is sparse. This is because amyloid proteins are notoriously difficult to study in their soluble forms, due to their inherent propensity to aggregate. Using recent developments in fast NMR techniques, band-selective excitation short transient and band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence we have been able to assign a 5 kDa full-length amyloidogenic protein called medin. Medin is the key protein component of the most common form of localised amyloid with a proposed role in aortic aneurysm and dissection. This assignment will now enable the study of the early interactions that could influence initiation and progression of medin aggregation. The chemical shifts have been deposited in the BioMagRes-Bank accession Nos. 25399 and 26576.     

Medin Oligomer Membrane Pore Formation: A Potential Mechanism of Vascular Dysfunction

Medin, a 50-amino-acid cleavage product of the milk fat globule-EGF factor 8 protein, is one of the most common forms of localized amyloid found in the vasculature of individuals older than 50 years. Medin induces endothelial dysfunction and vascular inflammation, yet despite its prevalence in the human aorta and multiple arterial beds, little is known about the nature of its pathology. Medin oligomers have been implicated in the pathology of aortic aneurysm, aortic dissection, and more recently, vascular dementia. Recent in vitro biomechanical measurements found increased oligomer levels in aneurysm patients with altered aortic wall integrity. Our results suggest an oligomer-mediated toxicity mechanism for medin pathology. Using lipid bilayer electrophysiology, we show that medin oligomers induce ionic membrane permeability by pore formation. Pore activity was primarily observed for preaggregated medin species from the growth-phase and rarely for lag-phase species. Atomic force microscopy (AFM) imaging of medin aggregates at different stages of aggregation revealed the gradual formation of flat domains resembling the morphology of supported lipid bilayers. Transmission electron microscopy images showed the coexistence of compact oligomers, largely consistent with the AFM data, and larger protofibrillar structures. Circular dichroism spectroscopy revealed the presence of largely disordered species and suggested the presence of β-sheets. This observation and the significantly lower thioflavin T fluorescence emitted by medin aggregates compared to amyloid-β fibrils, along with the absence of amyloid fibers in the AFM and transmission electron microscopy images, suggest that medin aggregation into pores follows a nonamyloidogenic pathway. In silico modeling by molecular dynamics simulations provides atomic-level structural detail of medin pores with the CNpNC barrel topology and diameters comparable to values estimated from experimental pore conductances.     

Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell-derived extracellular vesicles in stirred-tank bioreactors

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) act as signaling mediators of cellular responses. However, despite representing a promising alternative to cell-based therapies, clinical translation of EVs is currently limited by their lack of scalability and standardized bioprocessing. Herein, we integrated scalable downstream processing protocols with standardized expansion of large numbers of viable cells in stirred-tank bioreactors to improve EV production. Higher EV yields were linked to EV isolation by tangential flow filtration followed by size exclusion chromatography, rendering 5 times higher number of EVs comparatively to density gradient ultracentrifugation protocols. Additionally, when compared to static culture, EV manufacture in bioreactors resulted in 2.2 higher yields. Highlighting the role of operating under optimal cell culture conditions to maximize the number of EVs secreted per cell, MSCs cultured at lower glucose concentration favored EV secretion. While offline measurements of metabolites concentration can be performed, in this work, Raman spectroscopy was also applied to continuously track glucose levels in stirred-tank bioreactors, contributing to streamline the selection of optimal EV collection timepoints. Importantly, MSC-derived EVs retained their quality attributes and were able to stimulate angiogenesis in vitro, therefore highlighting their promising therapeutic potential.     

Localized amyloids important in diseases outside the brain--lessons from the islets of Langerhans and the thoracic aorta

It has long been understood that amyloids can be lethal in systemic diseases. More recently, it has been accepted that local cerebral aggregation of the small peptide Aβ is involved in the pathogenesis of Alzheimer's disease. Protein aggregation, with the generation of small amyloid deposits in specific organs, also occurs outside the central nervous system and often is associated with increased cell death. In this review, we discuss two lesser known but common localized amyloid fibril-forming proteins: the polypeptide hormone islet amyloid polypeptide (IAPP) and the lactadherin-derived peptide medin. IAPP aggregates and induces the depletion of islet β-cells in type 2 diabetes and in islets transplanted into type 1 diabetic subjects. Initial amyloid deposition occurs intracellularly and parts of this amyloid consist of proIAPP. Medin derived from lactadherin expressed by smooth muscle cells aggregates into amyloid in certain arteries, particularly the thoracic aortic media layer, and may have a role in the generation of the potentially lethal conditions of thoracic aortic aneurysm and dissection.     

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype

The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct “autophagosome-exosome-like” profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.     

Production cell analysis and compound-based boosting of small extracellular vesicle secretion using a generic and scalable production platform

Extracellular vesicles (EVs) are a novel format of advanced therapeutical medicinal products (ATMPs). They can act regenerative or immune-modulatory as cell therapy substitutes or as a platform for designer exosomes. The biotechnological production of therapeutic EVs is still very much uncharted territory so standardized host cells, production setups, and isolation methods are not yet implemented. In this work, we present a tangential flow filtration (TFF) and fast-performance liquid chromatography (FPLC)-based size exclusion chromatography (SEC) purification setup that is compatible for industry applications. Moreover, we evaluated a series of potential host cell lines regarding their EV productivity, characteristics, and biological functionality. It was found that telomerase-immortalized Wharton's jelly mesenchymal stromal cells (WJ-MSC/TERT273) secrete high amounts of EVs per cell with regenerative capabilities. On the other hand, Cevec's amniocyte producer cells® (CAP®) and human embryonic kidney (HEK293) suspension cells are suitable platforms for designer EVs with high yields. Finally, we aimed to boost the EV secretion of HEK293 cells via chemical adjuvants and verified four compounds that heighten cellular EV secretion in a presumably cAMP-dependent manner. A combination of fenoterol, iodoacetamide, and dinitrophenol increased the EV yield in HEK293 cells threefold and cellular secretion rate fivefold.     

One night of sleep deprivation induces release of small extracellular vesicles into circulation and promotes platelet activation by small EVs

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. Few studies have focused on EV levels in subjects with sleep disorders. Here, we aimed to explore the role of acute sleep deprivation on the quantity and functionality of circulating EVs, and their tissue distribution. EVs were isolated by ultracentrifugation from the plasma of volunteers and animals undergoing one night of sleep deprivation. Arterio‐venous shunt, FeCl₃ thrombus test and thrombin‐induced platelet aggregation assay were conducted to evaluate the in vivo and in vitro bioactivity of small EVs. Western blotting was performed to measure the expression of EV proteins. The fate and distribution of circulating small EVs were determined by intravital imaging. We found that one night of sleep deprivation triggers release of small EVs into the circulation in both healthy individuals and animals. Injection of sleep deprivation‐liberated small EVs into animals increased thrombus formation and weight in thrombosis models. Also, sleep deprivation‐liberated small EVs promoted platelet aggregation induced by thrombin. Mechanistically, sleep deprivation increased the levels of HMGB1 protein in small EVs, which play important roles in platelet activation. Furthermore, we found sleep deprivation‐liberated small EVs are more readily localize in the liver. These data suggested that one night of sleep deprivation is a stress for small EV release, and small EVs released here may increase the risk of thrombosis. Further, small EVs may be implicated in long distance signalling during sleep deprivation‐mediated adaptation processes.     

The role of extracellular vesicles in iron homeostasis and ferroptosis: Focus on musculoskeletal diseases

Iron homeostasis is crucial for maintaining proper cellular function, and its disruption is considered one of the pathogenic mechanisms underlying musculoskeletal diseases. Under conditions of oxidative stress, the accumulation of cellular iron overload and lipid peroxidation can lead to ferroptosis. Extracellular vesicles (EVs), serving as mediators in the cell-to-cell communication, play an important role in regulating the outcome of cell ferroptosis. Growing evidence has proven that EV biogenesis and secretion are tightly associated with cellular iron export. Furthermore, different sources of EVs deliver diverse cargoes to bring about phenotypic changes in the recipient cells, either activating or inhibiting ferroptosis. Thus, delivering therapies targeting ferroptosis through EVs may hold significant potential for treating musculoskeletal diseases. This review aims to summarize current knowledge on the role of EVs in iron homeostasis and ferroptosis, as well as their therapeutic applications in musculoskeletal diseases, and thereby provide valuable insights for both research and clinical practice.     

The Role of Extracellular Vesicles in Senescence

Cells can communicate in a variety of ways, such as by contacting each other or by secreting certain factors. Recently, extracellular vesicles (EVs) have been proposed to be mediators of cell communication. EVs are small vesicles with a lipid bilayer membrane that are secreted by cells and contain DNA, RNAs, lipids, and proteins. These EVs are secreted from various cell types and can migrate and be internalized by recipient cells that are the same or different than those that secrete them. EVs harboring various components are involved in regulating gene expression in recipient cells. These EVs may also play important roles in the senescence of cells and the accumulation of senescent cells in the body. Studies on the function of EVs in senescent cells and the mechanisms through which nonsenescent and senescent cells communicate through EVs are being actively conducted. Here, we summarize studies suggesting that EVs secreted from senescent cells can promote the senescence of other cells and that EVs secreted from nonsenescent cells can rejuvenate senescent cells. In addition, we discuss the functional components (proteins, RNAs, and other molecules) enclosed in EVs that enter recipient cells.     

Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus

Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system that mediates the release of macromolecule‐degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non‐conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.     

Cellular senescence contributes to age‐dependent changes in circulating extracellular vesicle cargo and function

Extracellular vesicles (EVs) have emerged as important regulators of inter‐cellular and inter‐organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63⁺ particles mediate these effects. Treatment of macrophages with EV‐like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV‐like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging‐associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.     

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.     

Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture

Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na(+)/H(+) exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV-like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection.     

Unwinding fibril formation of medin, the peptide of the most common form of human amyloid

Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.