Daniel Soong

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.

The first World Cell Race

Summary Motility is a common property of animal cells. Cell motility is required for embryogenesis [1], tissue morphogenesis [2] and the immune response [3] but is also involved in disease processes, such as metastasis of cancer cells [4]. Analysis of cell migration in native tissue in vivo has yet to be fully explored, but motility can be relatively easily studied in vitro in isolated cells. Recent evidence suggests that cells plated in vitro on thin lines of adhesive proteins printed onto culture dishes can recapitulate many features of in vivo migration on collagen fibers [5,6]. However, even with controlled in vitro measurements, the characteristics of motility are diverse and are dependent on the cell type, origin and external cues. One objective of the first World Cell Race was to perform a large-scale comparison of motility across many different adherent cell types under standardized conditions. To achieve a diverse selection, we enlisted the help of many international laboratories, who submitted cells for analysis. The large-scale analysis, made feasible by this competition-oriented collaboration, demonstrated that higher cell speed correlates with the persistence of movement in the same direction irrespective of cell origin.

Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo

Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.

Emergence of embryonic pattern through contact inhibition of locomotion

The pioneering cell biologist Michael Abercrombie first described the process of contact inhibition of locomotion more than 50 years ago when migrating fibroblasts were observed to rapidly change direction and migrate away upon collision. Since then, we have gleaned little understanding of how contact inhibition is regulated and only lately observed its occurrence in vivo. We recently revealed that Drosophila macrophages (haemocytes) require contact inhibition for their uniform embryonic dispersal. Here, to investigate the role that contact inhibition plays in the patterning of haemocyte movements, we have mathematically analysed and simulated their contact repulsion dynamics. Our data reveal that the final pattern of haemocyte distribution, and the details and timing of its formation, can be explained by contact inhibition dynamics within the geometry of the Drosophila embryo. This has implications for morphogenesis in general as it suggests that patterns can emerge, irrespective of external cues, when cells interact through simple rules of contact repulsion.

Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis

The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin‐mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F‐actin‐dependent manner, suggesting that F‐actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin‐coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.

Mammalian microtubule P-body dynamics are mediated by nesprin-1

Nesprin-1 is a multi-functional processing body component and microtubule scaffold that is necessary for RNA granule dynamics.

The importance of serine 776 in Ataxin-1 partner selection: A FRET Analysis

Anomalous expansion of a polymorphic tract in Ataxin-1 causes the autosomal dominant spinocerebellar ataxia type 1. In addition to polyglutamine expansion, requirements for development of pathology are phosphorylation of serine 776 in Ataxin-1 and nuclear localization of the protein. The phosphorylation state of serine 776 is also crucial for selection of the Ataxin-1 multiple partners. Here, we have used FRET for an in cell study of the interaction of Ataxin-1 with the spliceosome-associated U2AF65 and the adaptor 14-3-3 proteins. Using wild-type Ataxin-1 and Ser776 mutants to a phosphomimetic aspartate and to alanine, we show that U2AF65 binds Ataxin-1 in a Ser776 phosphorylation independent manner whereas 14-3-3 interacts with phosphorylated wild-type Ataxin-1 but not with the mutants. These results indicate that Ser776 acts as the molecular switch that discriminates between normal and aberrant function and that phosphomimetics is not a generally valid approach whose applicability should be carefully validated.

Prelamin A Accumulation Attenuates Rac1 Activity and Increases the Intrinsic Migrational Persistence of Aged Vascular Smooth Muscle Cells

Vascular smooth muscle cell (VSMC) motility is essential during both physiological and pathological vessel remodeling. Although ageing has emerged as a major risk factor in the development of cardiovascular disease, our understanding of the impact of ageing on VSMC motility remains limited. Prelamin A accumulation is known to drive VSMC ageing and we show that presenescent VSMCs, that have accumulated prelamin A, display increased focal adhesion dynamics, augmented migrational velocity/persistence and attenuated Rac1 activity. Importantly, prelamin A accumulation in proliferative VSMCs, induced by depletion of the prelamin A processing enzyme FACE1, recapitulated the focal adhesion, migrational persistence and Rac1 phenotypes observed in presenescent VSMCs. Moreover, lamin A/C-depleted VSMCs also display reduced Rac1 activity, suggesting that prelamin A influences Rac1 activity by interfering with lamin A/C function at the nuclear envelope. Taken together, these data demonstrate that lamin A/C maintains Rac1 activity in VSMCs and prelamin A disrupts lamin A/C function to reduce Rac1 activity and induce migrational persistence during VSMC ageing.

162 Regulated Exosome Secretion by Vascular Smooth Muscle Cells Mediates Vascular Calcification

Arterial stiffening caused by deposition of calcium phosphate salts in the vessel wall is common in patients with atherosclerosis, renal failure and diabetes and contributes to high cardiovascular morbidity and mortality in these groups. Medial calcification is mediated by vascular smooth muscle cells (VSMCs) which undergo phenotypic transitions and secrete matrix vesicles (MVs) that form the first nidus for mineralization. In this study we studied the mechanisms regulating MV secretion and compared them with circulating calcifying calciprotein complexes and exosomes. Phenotypic modulation of human aortic VSMCs was induced by TGF-β1 or PDGF-BB. MVs and extracellular vesicles were isolated from cell media or platelet-free plasma, by differential ultracentrifugation and compared using Nanosight LM-10, flow cytometry, immunogold labelling, transmission electron microscopy and western blotting. We found that MVs are secreted from multivesicular bodies and enriched with the exosomal markers, CD63, CD81, CD9 and MHC I. Exosome secretion was regulated by the exosome biogenesis regulator, sphingomyelin phosphodiesterase 3 (SMPD3), and inhibition of SMPD3 prevented VSMC calcification. Treatment of VSMCs in calcifying conditions and loss of the contractile phenotype were both associated with elevated exosome secretion and increased calcification. In agreement with our in vitro data, MVB-like structures were observed in VSMCs ex vivo as shown by EM analysis of human vessel rings. Immunohistochemical staining revealed that the exosome markers, CD63 and annexin A6 were present in the extracellular (ECM) at sites of vascular calcification in vivo . Importantly, only exosomes secreted by VSMCs in calcifying conditions contained amorphous calcium phosphate crystals as detected by EDX analysis. Nanosite analysis revealed that calcifying VSMC-derived exosomes are similar in size to non-calcifying exosomes (mode 136 +/- 3.6 nm and 147+/- 5.9 nm, correspondingly) and were similar to exosomes, isolated from the blood (Mode 146+/-14 nm). Calcifying exosomes were also distinct from calcium phosphate crystals (mode 107 +/- 7 nm) and fetuin-A containing calciprotein particles (mode 182 +/- 9.3 nm). Our data indicate that vascular calcification is mediated by VSMC-derived exosomes and that loss of the contractile VSMC phenotype and mineral imbalance promote calcification by enhanced exosome secretion. Calcifying exosomes are distinct from calcium phosphate crystals or calciprotein particles, and may be presented in the circulation where their levels may be a novel indicator of vascular calcification.

192 MODULATED EXOSOME SECRETION BY VASCULAR SMOOTH MUSCLE CELLS IS A NOVEL REGULATORY MECHANISM OF VASCULAR CALCIFICATION

Vascular calcification is a regulated pathological process similar to bone formation which is mediated by vascular smooth muscle cells (VSMCs) undergoing osteogenic transdifferentiation. Initiation of vascular calcification occurs in small membrane-bound matrix vesicles (MVs), secreted by VSMCs into the extracellular matrix however the mechanisms regulating MV biogenesis and secretion are unclear. Fetuin-A, a circulating protein abundant in MVs was used to trace the origin of VSMC-derived MVs. Alexa488-labelled fetuin-A was rapidly uptaken by human VSMCs and appeared in the late endosomal system and multivesicular bodies (MVBs) indicating that MVs are secreted from the endosomal compartment similar to exosomes; extracellular vesicles originating from MVBs and secreted by the range of cells. Biochemical analysis of MVs showed that they were enriched with exosomal markers, CD9 and CD63. Furthermore, inhibition of sphingomyelin phosphodiesterase 3 (SMPD3), which in involved in exosome biogenesis abrogated MV secretion by VSMCs confirming that MVs represent exosomes. Calcifying conditions induced exosome secretion by VSMCs and this was accompanied by elevated SMPD3 expression. Importantly, an inhibition of SMPD3 prevented VSMC calcification. Phenotypic modulation of VSMCs and loss of the contractile phenotype resulted in elevated exosome secretion while contractile VSMCs secreted significantly less exosomes. Notably, elevated extracellular calcium rapidly induced calcification of synthetic VSMCs whilst contractile VSMCs did not calcify. In agreement with this data, abundant immunohistochemical staining for CD63 was observed only in atherosclerotic human aorta in close association with calcified areas with little staining detected in the healthy vessel wall. Taken together this study demonstrates that MVs originate from MVBs and are secreted via the exosomal pathway. Loss of the contractile phenotype and mineral imbalance promote VSMC calcification by enhanced exosome secretion. Targeting the mechanisms of VSMC exosome secretion and/or their loading with calcification inhibitors may provide novel therapeutic interventions aimed for the prevention of vascular calcification.