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Nesprins (nuclear envelope spectrin repeat proteins) are multi-isomeric scaffolding proteins. Giant nesprin-1 and -2 localise to the outer nuclear membrane, interact with SUN (Sad1p/UNC-84) domain-containing proteins at the inner nuclear membrane to form the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, which, in association with lamin A/C and emerin, mechanically couples the nucleus to the cytoskeleton. Despite ubiquitous expression of nesprin giant isoforms, pathogenic mutations in nesprin-1 and -2 are associated with tissue-specific disorders, particularly related to striated muscle such as dilated cardiomyopathy and Emery–Dreifuss muscular dystrophy. Recent evidence suggests this muscle-specificity might be attributable in part, to the small muscle specific isoform, nesprin-1α2, which has a novel role in striated muscle function. Our current understanding of muscle-specific functions of nesprin-1 and its isoforms will be summarised in this review to provide insight into potential pathological mechanisms of nesprin-related muscle disease and may inform potential targets of therapeutic modulation.

ObjectiveThe differentiation of vascular smooth muscle cells (VSMCs) into an osteo-chondrogenic phenotype and consequent vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular ageing. Sox9 (SRY-Box Transcription Factor 9) is a major regulator of chondrogenesis that is also expressed in the vasculature during development and disease. Here, we investigated the relationship between VSMC ageing and Sox9-driven ECM regulation in VSMCs in vitro and in vivo.Approach and ResultsWe found that Sox9 expression in the aortic media was not associated with vascular calcification but strongly correlated with the senescence marker p16. Analysis of Sox9 expression in vitro showed that it was not a direct regulator of VSMC senescence but was highly mechanosensitive exclusively in senescent VSMCs, showing increased expression on stiff matrices. Manipulation of Sox9 via overexpression and depletion in VSMCs revealed that Sox9 regulates both ECM stiffness and organisation by orchestrating changes in collagen expression and reducing VSMC contractility. Importantly these ECM changes promoted VSMC phenotypic modulation such that senescent cells plated onto ECM synthesized from senescent cells depleted of Sox9 returned to a proliferative state and reduced p16 and p21 expression, as well as decreased the senescence associated secretory phenotype (SASP) marker IL6. We identified procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) as a novel Sox9 target, and key regulator of Sox9-driven ECM stiffness during VSMC senescence. LH3 is packaged into extracellular vesicles (EVs) and secreted into the ECM and we found that Sox9 also increases EV secretion, leading to increased LH3 deposition within the ECM.ConclusionsThese findings identify Sox9 as a key driver of increased ECM stiffness during VSMC ageing through upregulation of LH3 deposition via increased EV release. They also highlight a crucial role for ECM structure and composition in regulating VSMC phenotype and identify a novel cycle where cellular senescence and increased ECM stiffening promote Sox9 expression which acts to further enhance ECM modifications thus accelerating vascular stiffening, ageing and senescence.

Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid peptide. Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid accumulation in the extracellular matrix (ECM). To determine the mechanisms of AMA formation with age, we explored the impact of vascular smooth muscle cell (VSMC) senescence, EV secretion, and ECM remodeling on medin accumulation. Medin was detected in EVs secreted from primary VSMCs. Small, round medin aggregates colocalized with EV markers in decellularized ECM in vitro and medin was shown on the surface of EVs deposited in the ECM. Decreasing EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age and VSMC senescence increased EV secretion, increased EV medin loading and triggered deposition of fibril-like medin. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition, which led to enhanced EV-ECM binding and accelerated medin aggregation. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalized with EVs and medin. Isolated EVs selectively bound to HSPG2 in the ECM and its knock-down decreased formation of fibril-like medin structures. These data identify VSMC-derived EVs and HSPG2 in the ECM as key mediators of medin accumulation, contributing to age-associated AMA development.

Cardiovascular disease is the most common cause of death worldwide, especially beyond the age of 65 years, with the vast majority of morbidity and mortality due to myocardial infarction and stroke. Vascular pathology stems from a combination of genetic risk, environmental factors, and the biologic changes associated with aging. The pathogenesis underlying the development of vascular aging, and vascular calcification with aging, in particular, is still not fully understood. Accumulating data suggests that genetic risk, likely compounded by epigenetic modifications, environmental factors, including diabetes and chronic kidney disease, and the plasticity of vascular smooth muscle cells to acquire an osteogenic phenotype are major determinants of age-associated vascular calcification. Understanding the molecular mechanisms underlying genetic and modifiable risk factors in regulating age-associated vascular pathology may inspire strategies to promote healthy vascular aging. This article summarizes current knowledge of concepts and mechanisms of age-associated vascular disease, with an emphasis on vascular calcification.

The lamin A precursor, prelamin A, requires extensive processing to yield mature lamin A and effect its primary function as a structural filament of the nucleoskeleton. When processing is perturbed, nuclear accumulation of prelamin A is toxic and causes laminopathic diseases such as Hutchinson–Gilford progeria syndrome and cardiomyopathy. However, the physiological role of prelamin A is largely unknown and we sought to identify novel insights about this. Using rodent heart tissue, primary cells and the C2C12 model of myofibrillogenesis, we investigated the expression and localization patterns of prelamin A in heart and skeletal muscle cells. We found that endogenous prelamin A was detectable in mouse heart localized to the sarcomere in both adult mouse heart and isolated neonatal rat cardiomyocytes. We investigated the regulation of prelamin A in C2C12 myofibrillogenesis and found it was dynamically regulated and organized into striations upon myofibril formation, colocalizing with the Z-disc protein α-actinin. These data provide evidence that prelamin A is a component of the sarcomere, underpinning a physiological purpose for unprocessed prelamin A. This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Vascular smooth muscle biology is increasingly exploited as an interventional target in vascular disease. Vascular smooth muscle Notch3–Rho kinase–cGMP interaction has been implicated in brain and peripheral arteriopathy in CADASIL. In the present commentary, we discuss the potential implications for other, more common non-atherosclerotic microvascular diseases: INOCA and HFpEF. The relation to mechanotransduction, to cellular senescence and to sGC activators as potential intervention agents are described.

Purpose of review This review examines the current knowledge and recent developments in the field of vascular calcification focusing on the emerging role of senescence and inflammation in driving this disorder and exploring the overlap and relevance of these pathways to calcinosis in rheumatic disease. Recent findings Vascular calcification is an age-associated disorder. Recent studies have identified DNA damage, cellular senescence and consequent inflammation as key drivers of vascular smooth muscle cell osteogenic change and mineralization. Similar ageing and inflammatory factors are associated with calcinosis in rheumatic disease and some are targets of experimental drugs currently undergoing clinical trials. Summary Calcinosis in the vascular system and in rheumatic disease share similarities in terms of biomineralization and cardiovascular outcomes. Although research into the role of senescence and inflammation has recently been advanced in vascular calcification, little is known about the mechanistic role of inflammation in calcinosis in rheumatic disease. This review explores whether lessons from one calcinosis can be transferred and applied to the other to provide further insights and inform treatment strategies.

Supplemental Digital Content is available in the text., Rationale: BCL11B (B-cell leukemia 11b) is a transcription factor known as an essential regulator of T lymphocytes and neuronal development during embryogenesis. A genome-wide association study showed that a gene desert region downstream of BCL11B, known to function as a BCL11B enhancer, harbors single nucleotide polymorphisms associated with increased arterial stiffness. However, a role for BCL11B in the adult cardiovascular system is unknown. Objective: Based on these human findings, we sought to examine the relation between BCL11B and arterial function. Methods and Results: Here we report that BCL11B is expressed in the vascular smooth muscle where it regulates vascular stiffness. RNA sequencing of aortas from wild-type and Bcl11b null mice (BSMKO) identified the cGMP (cyclic guanosine monophosphate)-cGMP-dependent protein kinase G (PKG) as the most significant differentially regulated signaling pathway in BSMKO compared with wild-type mice. BSMKO aortas showed decreased levels of PKG1, increased levels of Ca++-calmodulin-dependent serine/threonine phosphatase calcineurin (PP2B) and decreased levels of their common phosphorylation target, phosphorylated vasodilator-stimulated phosphoprotein (pVASPS239), a regulator of cytoskelatal actin rearrangements. Decreased pVASPS239 in BSMKO aortas was associated with increased actin polymerization (filamentous/globular actin ratio). Functionally, aortic force, stress, wall tension, and stiffness, measured ex vivo in organ baths, were increased in BSMKO aortas, and BSMKO mice had increased pulse wave velocity, the in vivo index of arterial stiffness. Despite having no effect on blood pressure or microalbuminuria, increased arterial stiffness in BSMKO mice was associated with increased incidence of cerebral microbleeds compared with age-matched wild-type littermates. Conclusions: We have identified vascular smooth muscle BCL11B as a crucial regulator of aortic smooth muscle function and a potential therapeutic target for vascular stiffness.

Physiological calcification plays an essential part in the development of the skeleton and teeth; however, the occurrence of calcification in soft tissues such as the brain, heart, and kidneys associates with health impacts, creating a massive social and economic burden. The current paradigm for pathological calcification focuses on the biological factors responsible for bone‐like mineralization, including osteoblast‐like cells and proteins inducing nucleation and crystal growth. However, the exact mechanism responsible for calcification remains unknown. Toward this goal, this review dissects the current understanding of structure–function relationships and physico‐chemical properties of pathologic calcification from a materials science point of view. We will discuss a range of potential mechanisms of pathological calcification, with the purpose of identifying universal mechanistic pathways that occur across multiple organs/tissues at multiple length scales. The possible effect of extracellular components in signaling and templating mineralization, as well as the role of intrinsically disordered proteins in calcification, is reviewed. The state‐of‐the‐art in vitro models and strategies that can recreate the highly dynamic environment of calcification are identified.

This project was supported by grants from the British Heart Foundation (PG/11/71/29091 and PG/16/68/31991) and the Orthopaedic Institute Ltd., (RJAH Orthopaedic Hospital, Oswestry, UK). Nesprins, nuclear envelope spectrin-repeat proteins encoded by the SYNE1 and SYNE2 genes, are involved in localization of nuclei. The short isoform, nesprin-1-alpha2, is required for relocation of the microtubule organizer function from centromeres to the nuclear rim during myogenesis. Using specific antibodies, we now show that both nesprin-1-alpha2 and nesprin-1-giant co-localize with kinesin at the junctions of concatenated nuclei and at the outer poles of nuclear chains in human skeletal myotubes. In adult muscle, nesprin-1-alpha2 was found, together with kinesin, only on nuclei associated with neuromuscular junctions, whereas all adult cardiomyocyte nuclei expressed nesprin-1-alpha2. In a proteomics study, kinesin heavy and light chains were the only significant proteins in myotube extracts pulled down by nesprin-1-alpha2, but not by a mutant lacking the highly-conserved STAR domain (18 amino-acids, including the LEWD motif). The results support a function for nesprin-1-alpha2 in the specific localization of skeletal muscle nuclei mediated by kinesins and suggest that its primary role is at the outer nuclear membrane. Publisher PDF

Highlights •CKD is associated with very high risk of CAD. CAD management is complicated in CKD patients, due to comorbid conditions and potential side effects during interventions. •There are few trials related to CAD with focus on CKD patients, particularly in those with advanced CKD. •Additional prospective studies focusing on diagnosis, prevention, and treatment of CAD are needed in CKD.

Supplemental Digital Content is available in the text., 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.

Objective: The development of ectopic vascular calcification is strongly linked with organismal aging, which is primarily caused by the accumulation of DNA damage over time. As Runx2 (Runt-related transcription factor 2) has been identified as a regulator of vascular smooth muscle cell osteogenic transition, a key component of vascular calcification, we examined the relationship between DNA damage and Runx2 activation. Approach and Results: We found genotoxic stress-stimulated Runx2 accumulation and transactivation of its osteogenic target genes, leading to enhanced calcification. Inhibition of DNA damage signaling attenuated this response. Runx2 localized to sites of DNA damage and participated in DNA repair by regulating phosphorylation events on histone H2AX, with exogenous expression of Runx2 resulting in unrepaired DNA damage and increased apoptosis. Mechanistically, Runx2 was PARylated in response to genotoxic stress, and inhibition of this modification disrupted its localization at DNA lesions and reduced its binding to osteogenic gene promoters. Conclusions: These data identify Runx2 as a novel component of the DNA damage response, coupling DNA damage signaling to both osteogenic gene transcription and apoptosis and providing a mechanism for accelerated mineralization in aging and chronic disease.

The pathobiology of atherosclerotic disease requires further elucidation to discover new approaches to address its high morbidity and mortality. To date, over 17 million cardiovascular-related deaths have been reported annually, despite a multitude of surgical and nonsurgical interventions and advances in medical therapy. Existing strategies to prevent disease progression mainly focus on management of risk factors, such as hypercholesterolemia. Even with optimum current medical therapy, recurrent cardiovascular events are not uncommon in patients with atherosclerosis, and their incidence can reach 10–15% per year. Although treatments targeting inflammation are under investigation and continue to evolve, clinical breakthroughs are possible only if we deepen our understanding of vessel wall pathobiology. Vascular smooth muscle cells (VSMCs) are one of the most abundant cells in vessel walls and have emerged as key players in disease progression. New technologies, includingin situhybridization proximity ligation assays,in vivocell fate tracing with the CreERT2-loxP system and single-cell sequencing technology with spatial resolution, broaden our understanding of the complex biology of these intriguing cells. Our knowledge of contractile and synthetic VSMC phenotype switching has expanded to include macrophage-like and even osteoblast-like VSMC phenotypes. An increasing body of data suggests that VSMCs have remarkable plasticity and play a key role in cell-to-cell crosstalk with endothelial cells and immune cells during the complex process of inflammation. These are cells that sense, interact with and influence the behavior of other cellular components of the vessel wall. It is now more obvious that VSMC plasticity and the ability to perform nonprofessional phagocytic functions are key phenomena maintaining the inflammatory state and senescent condition and actively interacting with different immune competent cells.

Many cardiovascular diseases (CVD) are driven by pathological remodelling of blood vessels, which can lead to aneurysms, myocardial infarction, ischaemia and strokes. Aberrant remodelling is driven by changes in vascular cell behaviours combined with degradation, modification, or abnormal deposition of extracellular matrix (ECM) proteins. The underlying mechanisms that drive the pathological remodelling of blood vessels are multifaceted and disease specific; however, unravelling them may be key to developing therapies. Reductionist models of blood vessels created in vitro that combine cells with biomaterial scaffolds may serve as useful analogues to study vascular disease progression in a controlled environment. This review presents the main considerations for developing such in vitro models. We discuss how the design of blood vessel models impacts experimental readouts, with a particular focus on the maintenance of normal cellular phenotypes, strategies that mimic normal cell-ECM interactions, and approaches that foster intercellular communication between vascular cell types. We also highlight how choice of biomaterials, cellular arrangements and the inclusion of mechanical stimulation using fluidic devices together impact the ability of blood vessel models to mimic in vivo conditions. In the future, by combining advances in materials science, cell biology, fluidics and modelling, it may be possible to create blood vessel models that are patient-specific and can be used to develop and test therapies. Statement of significance Simplified models of blood vessels created in vitro are powerful tools for studying cardiovascular diseases and understanding the mechanisms driving their progression. Here, we highlight the key structural and cellular components of effective models and discuss how including mechanical stimuli allows researchers to mimic native vessel behaviour in health and disease. We discuss the primary methods used to form blood vessel models and their limitations and conclude with an outlook on how blood vessel models that incorporate patient-specific cells and flows can be used in the future for personalised disease modelling., Graphical abstract Image, graphical abstract

Rationale: Vascular calcification, the formation of calcium phosphate crystals in the vessel wall, is mediated by vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms remain elusive, precluding mechanism-based therapies. Objective: Phenotypic switching denotes a loss of contractile proteins and an increase in migration and proliferation, whereby VSMCs are termed synthetic. We examined how VSMC phenotypic switching influences vascular calcification and the possible role of the uniquely calcium-dependent reactive oxygen species (ROS)-forming Nox5 (NADPH oxidase 5). Methods and Results: In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN-1 (calponin 1), α-SMA (α-smooth muscle actin), and SM22-α (smooth muscle protein 22α) and an increase in synthetic marker S100A4 (S100 calcium binding protein A4) compared with contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca 2+ . Phenotypic switching was accompanied by increased levels of ROS and Ca 2+ -dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knockdown of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca 2+ -dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca 2+ loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca 2+ . Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca 2+ and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs. Conclusions: We show here that contractile VSMCs are resistant to calcification and identify Nox5 as a key regulator of VSMC phenotypic switching. Additionally, we describe a new mechanism of Ca 2+ uptake via EVs and show that Ca 2+ induces ROS production in VSMCs via Nox5. ROS production is required for release of EVs, which promote calcification. Identifying molecular pathways that control Nox5 and VSMC-derived EVs provides potential targets to modulate vascular remodeling and calcification in the context of mineral imbalance. Graphic Abstract: A graphic abstract is available for this article.

Cardiomyopathies are progressive diseases of heart muscle often caused by mutations in genes encoding sarcomeric, cytoskeletal and nucleoskeletal proteins though in many cases the cause of disease is not identified. Whilst nucleus hypertrophy has been described, it is not known whether nucleus shape changes are a general feature of cardiomyopathy. Due to the rod-shaped nature of cardiomyocytes and their elliptical nuclei we hypothesised that orientation of analysis would be an important determinant of any changes observed between patients exhibiting primarily unexplained cardiomyopathy and control samples from non-failing donors. To investigate this we performed image analysis of cardiomyocyte nuclei in myocardial cryosections from a cohort of cardiomyopathy patients. We discovered that circularity, solidity and aspect ratio were sensitive to orientation of the myocardium and that in the transverse plane only circularity was reduced in cardiomyocyte nuclei of cardiomyopathy patients. These findings show that orientation dependent changes in nucleus shape may be a property of cardiomyopathy and with appropriate follow up studies, may prove to have mechanistic and diagnostic value.

Vascular calcification is a ubiquitous pathology of aging. Oxidative stress, persistent DNA damage, and senescence are major pathways driving both cellular and tissue aging, and emerging evidence suggests that these pathways are activated, and even accelerated, in patients with vascular calcification. The DNA damage response—a complex signaling platform that maintains genomic integrity—is induced by oxidative stress and is intimately involved in regulating cell death and osteogenic differentiation in both bone and the vasculature. Unexpectedly, a posttranslational modification, PAR (poly[ADP-ribose]), which is a byproduct of the DNA damage response, initiates biomineralization by acting to concentrate calcium into spheroidal structures that can nucleate apatitic mineral on the ECM (extracellular matrix). As we start to dissect the molecular mechanisms driving aging-associated vascular calcification, novel treatment strategies to promote healthy aging and delay pathological change are being unmasked. Drugs targeting the DNA damage response and senolytics may provide new avenues to tackle this detrimental and intractable pathology.

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils – potentially important in the microenvironment of actively dividing cells, such as cancer cells – disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

Dysregulated mineral metabolism and factors in uraemic serum place chronic kidney disease (CKD) patients at high risk of ectopic calcification, particularly vascular calcification. CKD patients have impaired phosphate excretion accompanied by klotho deficiency and vitamin D deficiency. The parathyroid hormone and fibroblast growth factor 23 negative feedback loops are ineffective and over activated leading to secondary hyperparathyroidism, CKD bone mineral disorder and a chronic increase in calcium (Ca) and phosphate (P). High serum Ca and P drive several mechanisms of calcification including apoptosis, osteogenic differentiation and the accumulation of both extracellular vesicles and calcium phosphate-containing particles (CPPs). Furthermore, the accumulation of uraemic toxins in serum has been shown to increase both oxidative and endoplasmic reticulum stress, which activate the inflammatory and DNA damage response pathways and promote calcification. There is no cure for vascular calcification, and treatment strategies involve preventing mineral dysregulation or directly targeting individual mechanisms of calcification.

Prevention of medial calcification in patients with chronic kidney disease requires the maintenance of vascular smooth muscle cell fitness. To preserve viability under chronic kidney disease–induced stress, vascular smooth muscle cells increase exosome formation and release, but the result is aggravated pathological calcification. Now Chen et al. report that microvesicles from calcifying vascular smooth muscle cells may propagate procalcifying signals to normal vascular smooth muscle cells. To help design effective strategies to impair procalcifying cell-to-cell communication, this commentary updates the current understanding of the main regulators of microvesicle/exosome biogenesis and secretion. Fil: Dusso, Adriana. Instituto de Investigación Sanitaria del Principado de Asturias; España Fil: Colombo, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Shanahan, Catherine M.. King’s College London; Reino Unido

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-1β (IL-1β). 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-1β release from VSMCs, we demonstrated that CaP particles stimulated IL-1β release from proliferating and senescent human VSMCs, but with substantially greater IL-1β 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-1β 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-1β 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-1β and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1β 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-1β release. Conclusions CaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1β, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification., Graphical abstract Image 2, Highlights • CaP particles induce IL-1β release from human VSMCs. • Senescent cells display higher basal and CaP-stimulated IL-1β release. • Inflammasome agonists ATP, nigericin and MSU crystals do not induce IL-1β release from human VSMCs. • CaP particle-induced IL-1β release is dependent on SYK, caspase-1 and exosome release.

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.

Pathological glycation of extracellular matrix modelled with 13C-labelled sugars yields unique novel atomic level NMR structural and chemical insights non-destructively., Non-enzymatic glycation of extracellular matrix with (U-13C5)-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH3–CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes.

Brayson et al. preview work from Wong et al. that provides evidence supporting the hypothesis that cell and tissue mechanics play a crucial role in the regulation of key nuclear functions., In this issue, Wang et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201708137) show that disruption to different mechanical domains of muscle cells converge at the linker of nucleoskeleton to cytoskeleton complex to affect DNA endoreplication potentially via barrier to autointegration factor–mediated epigenetic mechanisms.

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues, yet its impact upon the heart is unknown. Here, we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy, and we show that a potentially novel mouse model of cardiac-specific prelamin A accumulation exhibited a phenotype consistent with inflammatory cardiomyopathy, which we observed to be similar to HIV-associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings (a) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (b) have implications for the management of HIV patients with cardiac disease, suggesting protease inhibitors should be replaced with alternative therapies (i.e., nonnucleoside reverse transcriptase inhibitors); and (c) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy., Accumulation of prelamin A occurs in dilated and HIV associated cardiomyopathy causing inflammation, which could be ameliorated by switching therapies in HIV patients.

Chronic kidney disease (CKD) is a major risk factor for valvular heart disease (VHD). Mitral annular and aortic valve calcifications are highly prevalent in CKD patients and commonly lead to valvular stenosis and regurgitation, as well as complications including conduction system abnormalities and endocarditis. VHD, especially mitral regurgitation and aortic stenosis, is associated with significantly reduced survival among CKD patients. Knowledge related to VHD in the general population is not always applicable to CKD patients because the pathophysiology may be different, and CKD patients have a high prevalence of comorbid conditions and elevated risk for periprocedural complications and mortality. This Kidney Disease: Improving Global Outcomes (KDIGO) review of CKD and VHD seeks to improve understanding of the epidemiology, pathophysiology, diagnosis, and treatment of VHD in CKD by summarizing knowledge gaps, areas of controversy, and priorities for research.

A cell’s ability to react to mechanical stimuli is known to be affected by the transport of transcription factors, the proteins responsible for regulating transcription of DNA into RNA, across the membrane enveloping its nucleus. Yet the molecular mechanisms by which mechanical cues control this process remain unclear. Here we show that one such protein, myocardin-related transcription factor A (MRTFA), is imported into the nucleus at a rate that is inversely correlated with its nanomechanical stability, but independent of its thermodynamic stability. Attaching mechanically stable proteins to MRTFA results in reduced gene expression and the subsequent slowing down of cell migration. We conclude that the mechanical unfolding of proteins regulates their nuclear translocation rate, and highlight the role of the nuclear pore complex as a selective mechanosensor that is capable of detecting forces as low as ∼10 pN. The modulation of the mechanical stability of transcription factors may represent a general strategy for the control of gene expression. The rate at which proteins are imported into the nucleus of a cell is shown to be regulated by their mechanical unfolding, a mechanism that identifies the nuclear pore machinery as a highly sensitive force detector.

Summary Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification., Graphical Abstract, Highlights • Poly(ADP-ribose) is found close to ECM calcification in developing bone and arteries • Poly(ADP-ribose) is produced in response to oxidative stress and delivered to the ECM • Poly(ADP-ribose) forms dense liquid droplets with calcium ions • Inhibiting PARP enzyme activity blocks calcification in vitro and in vivo, Müller et al. investigate the physicochemical process of extracellular matrix calcification in both physiological (bone) and pathological (vascular calcification) contexts. They find that oxidative stress-induced poly(ADP-ribose) nucleates calcium phosphate mineral crystals on extracellular matrix substrates and that calcification is inhibited by poly(ADP-ribose) polymerase (PARP) enzyme inhibitors.

Vascular smooth muscle cell (VSMC) phenotypic conversion from a contractile to 'synthetic' state contributes to vascular pathologies including restenosis, atherosclerosis and vascular calcification. We have recently found that the secretion of exosomes is a feature of 'synthetic' VSMCs and that exosomes are novel players in vascular repair processes as well as pathological vascular thrombosis and calcification. Pro-inflammatory cytokines and growth factors as well as mineral imbalance stimulate exosome secretion by VSMCs, most likely by the activation of sphingomyelin phosphodiesterase 3 (SMPD3) and cytoskeletal remodelling. Calcium stress induces dramatic changes in VSMC exosome composition and accumulation of phosphatidylserine (PS), annexin A6 and matrix metalloproteinase-2, which converts exosomes into a nidus for calcification. In addition, by presenting PS, VSMC exosomes can also provide the catalytic surface for the activation of coagulation factors. Recent data showing that VSMC exosomes are loaded with proteins and miRNA regulating cell adhesion and migration highlight VSMC exosomes as potentially important communication messengers in vascular repair. Thus, the identification of signalling pathways regulating VSMC exosome secretion, including activation of SMPD3 and cytoskeletal rearrangements, opens up novel avenues for a deeper understanding of vascular remodelling processes.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall. Changes in VSMC actomyosin activity and morphology are prevalent in cardiovascular disease. The actin cytoskeleton actively defines cellular shape and the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, comprised of nesprin and the Sad1p, UNC-84 (SUN)-domain family members SUN1/2, has emerged as a key regulator of actin cytoskeletal organisation. Although SUN1 and SUN2 function is partially redundant, they possess specific functions and LINC complex composition is tailored for cell-type-specific functions. We investigated the importance of SUN1 and SUN2 in regulating actomyosin activity and cell morphology in VSMCs. We demonstrate that siRNA-mediated depletion of either SUN1 or SUN2 altered VSMC spreading and impaired actomyosin activity and RhoA activity. Importantly, these findings were recapitulated using aortic VSMCs isolated from wild-type and SUN2 knockout (SUN2 KO) mice. Inhibition of actomyosin activity, using the rho-associated, coiled-coil-containing protein kinase1/2 (ROCK1/2) inhibitor Y27632 or blebbistatin, reduced SUN2 mobility in the nuclear envelope and decreased the association between SUN2 and lamin A, confirming that SUN2 dynamics and interactions are influenced by actomyosin activity. We propose that the LINC complex exists in a mechanical feedback circuit with RhoA to regulate VSMC actomyosin activity and morphology.

The sparse but functionally essential post-translational collagen modification 5-hydroxylysine can undergo further transformations, including crosslinking, O-glycosylation, and glycation. Dynamic nuclear polarization (DNP) and stable isotope enriched lysine incorporation provide sufficient solid-state NMR sensitivity to identify these adducts directly in skin and vascular smooth muscle cell extracellular matrix (ECM), without extraction procedures, by comparison with chemical shifts of model compounds. Thus, DNP provides access to the elucidation of structural consequences of collagen modifications in intact tissue.

Cardiomyopathies are complex heart muscle diseases that can be inherited e.g. dilated cardiomyopathy resulting from LMNA gene mutations, or acquired, e.g. cardiomyopathy associated with HIV. In both cases the lamin A precursor, prelamin A, may play a central role: mutations in LMNA and certain HIV protease inhibitors acting via the enzyme ZMPSTE24 both inhibit prelamin A processing. Firstly, we show that myocardial prelamin A accumulation occurs in both these cardiomyopathies in patients. Secondly, we developed a novel mouse model of cardiac specific prelamin A accumulation which mimicked tissue and molecular features of HIV associated cardiomyopathy, including inflammation. These findings: (1) confirm a central pathological role of prelamin A common to genetic and acquired cardiomyopathies; (2) have implications for the management of HIV patients with cardiac disease in whom protease inhibitors with low/no binding to ZMPSTE24 may be preferred; and (3) suggest that targeting inflammation may be a useful treatment strategy for some forms of inherited cardiomyopathy.

Fundamentals of Bone Formation In vitro models can help guide research for tissue engineering or drug delivery, but the extent to which results from in vitro experiments may mimic in vivo ones will depend on the robustness of the model. For complex tissues like the extracellular matrix or bone, this means matching the chemical organization of the tissue at both the atomic scale and the structural level. Chow et al. (p. 742 ) used nuclear magnetic resonance (NMR) spectroscopy to analyze a sample on both these length scales. First an isotope-enriched mouse was produced to enhance the NMR signal. Samples from these mice were then used to study the extracellular matrix of developing bone and the calcification front during fetal bone growth.

Nuclear and mitochondrial DNA damage contribute to aging and related cardiovascular disease. To explore if the aging features are cell autonomous we compared cardiovascular function of mice with a specific DNA repair system knockout in vascular endothelial cells (EC-KO) to that of mice with specific knockout in smooth muscle cells (SMC-KO). We evaluated cardiac function by echocardiography, blood pressure by the tail cuff method and ex vivo vascular function in aorta, coronary and carotid arteries using wire myographs organ baths setups. EC-KO showed macrovascular and microvascular vasodilator dysfunction due to specific loss of endothelium-dependent nitric oxide (NO) signaling (maximal vasorelaxation response to acetylcholine in aorta: 59.0% in EC-KO vs 75.0% in WT mice; p-value lt 0.0001). The reduction of vasodilator response was associated with a temporary systolic blood pressure increase at 3 months (138 mm Hg in EC-KO vs 125 mm Hg in WT mice; p-value= 0.03). In addition, EC-KO mice showed a severely compromised microvascular barrier function in the kidney, leading to papillary necrosis. Cardiac output was slightly affected at 5 months of age (16 ml/min in EC-KO vs 18 ml/min in WT mice; p-value= 0.13) and aortic distensibility was reduced (0.2 mm in EC-KO vs 0.3 mm in WT mice; p-value =0.05), suggesting decreased cardiac contractility and increased vascular stiffness. In sharp contrast, SMC-KO mice showed a specific decrease of endothelium-independent NO-mediated relaxation (maximal vasorelaxation response to the NO donor sodium nitroprusside: 83.4% in SMC-KO vs 98.6% in WT mice; p-value lt 0.001). Furthermore, SMC-KO showed increased carotid artery stiffness (Mediastress at intraluminal pressure of 120 mm Hg: 1.44*10 6 in SMC-KO vs 1.82*10 6 dyness/cm 2 in WT mice; p-value lt 0.001), displayed aortic root dilation and regurgitation. We conclude that DNA damage in EC and VSMC each lead to specific pathological changes that are found also in human cardiovascular aging. In complement, the changes reproduce the vascular phenotype of whole body DNA repair knockout mice ( Ercc1 d/- ) previously found by us. Therefore, the cardiovascular aging effects of DNA damage are at least partly cell autonomic, and represent an important treatment target.