Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

Antisense oligonucleotide activity in tumour cells is influenced by intracellular LBPA distribution and extracellular vesicle recycling.

Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.

Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles.

OBJECTIVE: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. CONCLUSIONS: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

Using macropinocytosis for intracellular delivery of therapeutic nucleic acids to tumour cells.

Nucleic acids are a rapidly emerging therapeutic modality with the potential to become the third major drug modality alongside antibodies and small molecules. Owing to the unfavourable physico-chemical characteristics of nucleic acids, such as large size and negative charge, intracellular delivery remains a fundamental challenge to realizing this potential. Delivery technologies such as lipids, polymers and peptides have been used to facilitate delivery, with many of the most successful technologies using macropinocytosis to gain cellular entry; mostly by default rather than design. Fundamental knowledge of macropinocytosis is rapidly growing, presenting opportunities to better tailor design strategies to target this pathway. Furthermore, certain types of tumour cells have been observed to have high levels of macropinocytic activity and traffic cargo to favourable destinations within the cell for endosomal release, providing unique opportunities to further use this entry route for drug delivery. In this article, we review the delivery systems reported to be taken up by macropinocytosis and what is known about the mechanisms for regulating macropinocytosis in tumour cells. From this analysis, we identify new opportunities for exploiting this pathway for the intracellular delivery of nucleic acids to tumour cells. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

Publisher Correction: Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.

In the version of this Article originally published, the affiliations for Roland A. Fleck and José Antonio Del Río were incorrect due to a technical error that resulted in affiliations 8 and 9 being switched. The correct affiliations are: Roland A. Fleck: 8Centre for Ultrastructural Imaging, Kings College London, London, UK. José Antonio Del Río: 2Cellular and Molecular Neurobiotechnology, Institute for Bioengineering of Catalonia, Barcelona, Spain; 9Department of Cell Biology, Physiology and Immunology, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; 10Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain. This has now been amended in all online versions of the Article.

Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-ß1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.

Calcium phosphate particles stimulate interleukin-1ß release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release.

AIMS: Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1ß (IL-1ß). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs. METHODS AND RESULTS: Using ELISA to measure IL-1ß release from VSMCs, we demonstrated that CaP particles stimulated IL-1ß release from proliferating and senescent human VSMCs, but with substantially greater IL-1ß release from senescent cells; this required caspase-1 activity but not LPS-priming of cells. Potential inflammasome agonists including ATP, nigericin and monosodium urate crystals did not stimulate IL-1ß release from VSMCs. Western blot analysis demonstrated that CaP particles induced rapid activation of spleen tyrosine kinase (SYK) (increased phospho-Y525/526). The SYK inhibitor R406 reduced IL-1ß release and caspase-1 activation in CaP particle-treated VSMCs, indicating that SYK activation occurs upstream of and is required for caspase-1 activation. In addition, IL-1ß and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1ß in exosomes isolated from VSMC media. Furthermore, CaP particle treatment stimulated exosome secretion by VSMCs in a SYK-dependent manner, while the exosome-release inhibitor spiroepoxide reduced IL-1ß release. CONCLUSIONS: CaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1ß, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification.

Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques.

BACKGROUND: The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the "vulnerable plaque," necessitating an improved approach for predicting onset of symptoms. METHODS: We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. RESULTS: Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. CONCLUSION: The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. FUNDING: UK: British Heart Foundation (BHF); King's BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London in partnership with King's College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.

Prothrombin Loading of Vascular Smooth Muscle Cell-Derived Exosomes Regulates Coagulation and Calcification.

OBJECTIVE: The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. APPROACH AND RESULTS: Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleation site formation on the exosomal surface. PT was deposited in the calcified vasculature, and there was a negative correlation between vascular calcification and the levels of circulating PT. In addition, we found that VSMC exosomes induced thrombogenesis in a tissue factor-dependent and PS-dependent manner. CONCLUSIONS: Gamma-carboxylated coagulation proteins are potent inhibitors of vascular calcification suggesting warfarin action on these factors also contributes to accelerated calcification in patients receiving this drug. VSMC exosomes link calcification and coagulation acting as novel activators of the extrinsic coagulation pathway and inducers of calcification in the absence of Gla-containing inhibitors.

Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.

RATIONALE: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Neointimal hyperplasia and calcification in medium sized arteries in adult patients with chronic kidney disease.

The nature of arterial changes resulting in cardiovascular events and dialysis vascular access failures in adult predialysis patients is not well known. This study examined intimal changes, calcium deposition, and consequent stiffness in brachial and radial arteries of adult CKD patients. Ten brachial-artery and seven radial-artery specimens were obtained during fistula creation from nine predialysis and eight dialysis-dependent, nondiabetic patients; and age-gender matched controls undergoing coronary bypass grafts (6 radial) or kidney donation (6 renal). Arterial stiffness was measured at baseline. Vessel histology, morphometric analysis of intima-media, and direct quantification of calcium load was performed using standard techniques. Both predialysis and dialysis patients demonstrated significant arterial intimal hyperplasia with intima:media ratio higher than controls (0.13 ± 0.12 vs. 0.02 ± 0.05, p = 0.01). Calcium deposition was demonstrated on histology and the calcium content in patients was higher than controls (34.68 ± 26.86 vs. 10.95 ± 9.18 µg/µg, p = 0.003). The blood vessel calcium content correlated with arterial stiffness (r = 0.64, p = 0.018). This study for the first time describes, and suggests mechanistic linkage between, intimal hyperplasia, pathological calcium deposition, and increased functional arterial stiffness in dialysis and predialysis patients. Our research could serve as a unique window into the in vivo status of the uremic vasculature impacting fistula maturation and cardiovascular disease.

Calcium regulation of vascular smooth muscle cell-derived matrix vesicles.

Vascular calcification is a pathological process common in patients with disorders of mineral metabolism and mediated by vascular smooth muscle cells (VSMCs). A key event in the initiation of VSMC calcification is the release of mineralization-competent matrix vesicles (MVs), small membrane-bound bodies with structural features enabling them to efficiently nucleate hydroxyapatite. These bodies are similar to MVs secreted by chondrocytes during bone development and their properties include the absence of calcification inhibitors, formation of nucleation sites, and accumulation of matrix metalloproteinases such as MMP-2. The mechanisms of MV biogenesis and loading remain poorly understood; however, emerging data have demonstrated that alterations in cytosolic calcium homeostasis can trigger multiple changes in MV composition that promote their mineralization.

Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated ß1-integrin-dependent cell migration.

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human ß1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of ß1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from ß1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

Arterial calcification in chronic kidney disease: key roles for calcium and phosphate.

Vascular calcification contributes to the high risk of cardiovascular mortality in chronic kidney disease (CKD) patients. Dysregulation of calcium (Ca) and phosphate (P) metabolism is common in CKD patients and drives vascular calcification. In this article, we review the physiological regulatory mechanisms for Ca and P homeostasis and the basis for their dysregulation in CKD. In addition, we highlight recent findings indicating that elevated Ca and P have direct effects on vascular smooth muscle cells (VSMCs) that promote vascular calcification, including stimulation of osteogenic/chondrogenic differentiation, vesicle release, apoptosis, loss of inhibitors, and extracellular matrix degradation. These studies suggest a major role for elevated P in promoting osteogenic/chondrogenic differentiation of VSMC, whereas elevated Ca has a predominant role in promoting VSMC apoptosis and vesicle release. Furthermore, the effects of elevated Ca and P are synergistic, providing a major stimulus for vascular calcification in CKD. Unraveling the complex regulatory pathways that mediate the effects of both Ca and P on VSMCs will ultimately provide novel targets and therapies to limit the destructive effects of vascular calcification in CKD patients.

Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization.

RATIONALE: Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)-derived MVs. OBJECTIVES: To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress. METHODS AND RESULTS: Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites. CONCLUSIONS: In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.

Targeting vascular calcification: softening-up a hard target.

Widespread vascular calcification is a ubiquitous feature of aging and is prevalent in association with a number of common pathologies including atherosclerosis, renal failure, and diabetes. Once thought of as innocuous, emerging evidence suggests that calcification is causal in precipitating vascular events and mediating chronic cardiovascular damage, independent of disease context. Importantly, a large body of data has shed light on the factors that favor the formation of calcification in vivo, as well as on the complex mechanisms that initiate and promote it. This has identified some novel targets and allowed for the possibility that calcification can potentially be blocked and ultimately regressed. Targets include local and circulating inhibitors of calcification as well as factors that may ameliorate vascular smooth muscle cell (VSMC) apoptosis. Despite this, the vasculature remains a difficult tissue to target and currently there are no effective treatments in general use. More crucially, any potential treatments will need to be carefully evaluated as they may impinge on bone metabolism. Our best hope for the near future is to normalize factors associated with accelerated calcification in pathologies such as renal failure where, aberrant mineral metabolism, as well as treatment regimes, may contribute to the initiation and progression of calcification.

Urokinase gene transfer augments angiogenesis in ischemic skeletal and myocardial muscle.

Urokinase plasminogen activator (uPA) is required for both endogenous and vascular endothelial growth factor (VEGF)-augmented angiogenesis in normal tissues, leading us to hypothesize that uPA augmentation by gene transfer might promote angiogenesis in ischemic tissues. Overexpression of uPA was studied in rat myocardial infarction (MI) and mouse hind limb ischemia models and compared with VEGF overexpression effects. Animals were divided into control and three experimental groups (n = 6), receiving intramuscular injections of plasmids as follows: (i) control (empty vector or expressing beta-galactosidase); (ii) uPA; (iii) VEGF(165); (iv) a 1:1 mixture of uPA and VEGF(165). The capillary densities in both ischemic models were greater (P < 0.05) in tissues treated with uPA, VEGF, or a combination of both than in controls. Infarct size was reduced in hearts from uPA and VEGF experimental groups compared with controls (P < 0.05). Local overexpression of uPA induced a marked increase in the number of macrophages and myofibroblasts present within infarcts. Hind limb blood flow was greater in all experimental groups by day 10 (P < 0.05). Overall, the effects of uPA and VEGF were uniformly comparable. Additional analysis revealed association of local edema with VEGF but not with uPA treatment. This study established that uPA gene therapy effectively induces functionally significant angiogenesis in models of acute MI and hind limb ischemia.

Proteasomal dysfunction in sporadic Parkinson's disease.

The cause and mechanism of neuronal death in sporadic Parkinson's disease (PD) continue to elude investigators. Recently, alterations in proteasomal function have been detected in the brain of patients with the illness. The biochemical basis of the defect and its relevance to the disease process are now being studied. The available results suggest that proteasomal dysfunction could underlie protein accumulation, Lewy body formation, and neuron death in PD. The cause of proteasomal dysfunction is unknown at present, but this could relate to gene mutations, oxidative damage, ATP depletion, or the actions of environmental toxins. It remains to be established if proteasomal dysfunction plays a primary or a secondary role in the initiation or progression of the neurodegenerative process in PD.