Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores.

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation.

Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

BACKGROUND: Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. METHODS: Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. RESULTS: In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. CONCLUSIONS: These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere.

Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study, we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localize to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52 to 53, localized to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four-and-half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin, and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in the maintenance and/or regulation of sarcomeric organization and function.

Molecular Mechanisms of Vascular Health: Insights From Vascular Aging and Calcification.

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.

Nesprin-1: novel regulator of striated muscle nuclear positioning and mechanotransduction.

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.

Runx2 (Runt-Related Transcription Factor 2) Links the DNA Damage Response to Osteogenic Reprogramming and Apoptosis of Vascular Smooth Muscle Cells.

[Figure: see text].

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.

Notch-Rho-cGMP interaction: common point of convergence in microvascular aging-related 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.

DNA Damage Response: A Molecular Lynchpin in the Pathobiology of Arteriosclerotic Calcification.

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.

Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling.

Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine-threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species-generating NADPH oxidase-4 (Nox4) is induced downstream of ATF4, binds to a PP1-targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4-regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia-reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4-GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.

Calcinosis: insights from other calcinoses.

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.

Extracellular Matrix Proteomics Reveals Interplay of Aggrecan and Aggrecanases in Vascular Remodeling of Stented Coronary Arteries.

BACKGROUND: Extracellular matrix (ECM) remodeling contributes to in-stent restenosis and thrombosis. Despite its important clinical implications, little is known about ECM changes post-stent implantation. METHODS: Bare-metal and drug-eluting stents were implanted in pig coronary arteries with an overstretch under optical coherence tomography guidance. Stented segments were harvested 1, 3, 7, 14, and 28 days post-stenting for proteomics analysis of the media and neointima. RESULTS: A total of 151 ECM and ECM-associated proteins were identified by mass spectrometry. After stent implantation, proteins involved in regulating calcification were upregulated in the neointima of drug-eluting stents. The earliest changes in the media were proteins involved in inflammation and thrombosis, followed by changes in regulatory ECM proteins. By day 28, basement membrane proteins were reduced in drug-eluting stents in comparison with bare-metal stents. In contrast, the large aggregating proteoglycan aggrecan was increased. Aggrecanases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family contribute to the catabolism of vascular proteoglycans. An increase in ADAMTS-specific aggrecan fragments was accompanied by a notable shift from ADAMTS1 and ADAMTS5 to ADAMTS4 gene expression after stent implantation. Immunostaining in human stented coronary arteries confirmed the presence of aggrecan and aggrecan fragments, in particular, at the contacts of the stent struts with the artery. Further investigation of aggrecan presence in the human vasculature revealed that aggrecan and aggrecan cleavage were more abundant in human arteries than in human veins. In addition, aggrecan synthesis was induced on grafting a vein into the arterial circulation, suggesting an important role for aggrecan in vascular plasticity. Finally, lack of ADAMTS-5 activity in mice resulted in an accumulation of aggrecan and a dilation of the thoracic aorta, confirming that aggrecanase activity regulates aggrecan abundance in the arterial wall and contributes to vascular remodeling. CONCLUSIONS: Significant differences were identified by proteomics in the ECM of coronary arteries after bare-metal and drug-eluting stent implantation, most notably an upregulation of aggrecan, a major ECM component of cartilaginous tissues that confers resistance to compression. The accumulation of aggrecan coincided with a shift in ADAMTS gene expression. This study provides the first evidence implicating aggrecan and aggrecanases in the vascular injury response after stenting.

Arterial "inflammaging" drives vascular calcification in children on dialysis.

Children on dialysis have a cardiovascular mortality risk equivalent to older adults in the general population, and rapidly develop medial vascular calcification, an age-associated pathology. We hypothesized that premature vascular ageing contributes to calcification in children with advanced chronic kidney disease (CKD). Vessels from children with Stage 5 CKD with and without dialysis had evidence of increased oxidative DNA damage. The senescence markers p16 and p21 were also increased in vessels from children on dialysis. Treatment of vessel rings ex vivo with calcifying media increased oxidative DNA damage in vessels from children with Stage 5 CKD, but not in those from healthy controls. Vascular smooth muscle cells cultured from children on dialysis exhibited persistent DNA damage, impaired DNA damage repair, and accelerated senescence. Under calcifying conditions vascular smooth muscle cells from children on dialysis showed increased osteogenic differentiation and calcification. These changes correlated with activation of the senescence-associated secretory phenotype (SASP), an inflammatory phenotype characterized by the secretion of proinflammatory cytokines and growth factors. Blockade of ataxia-telangiectasia mutated (ATM)-mediated DNA damage signaling reduced both inflammation and calcification. Clinically, children on dialysis had elevated circulating levels of osteogenic SASP factors that correlated with increased vascular stiffness and coronary artery calcification. These data imply that dysregulated mineral metabolism drives vascular "inflammaging" by promoting oxidative DNA damage, premature senescence, and activation of a pro-inflammatory SASP. Drugs that target DNA damage signaling or eliminate senescent cells may have the potential to prevent vascular calcification in patients with advanced CKD.

Novel nesprin-1 mutations associated with dilated cardiomyopathy cause nuclear envelope disruption and defects in myogenesis.

Nesprins-1 and -2 are highly expressed in skeletal and cardiac muscle and together with SUN (Sad1p/UNC84)-domain containing proteins and lamin A/C form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) bridging complex at the nuclear envelope (NE). Mutations in nesprin-1/2 have previously been found in patients with autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). In this study, three novel rare variants (R8272Q, S8381C and N8406K) in the C-terminus of the SYNE1 gene (nesprin-1) were identified in seven DCM patients by mutation screening. Expression of these mutants caused nuclear morphology defects and reduced lamin A/C and SUN2 staining at the NE. GST pull-down indicated that nesprin-1/lamin/SUN interactions were disrupted. Nesprin-1 mutations were also associated with augmented activation of the ERK pathway in vitro and in hearts in vivo. During C2C12 muscle cell differentiation, nesprin-1 levels are increased concomitantly with kinesin light chain (KLC-1/2) and immunoprecipitation and GST pull-down showed that these proteins interacted via a recently identified LEWD domain in the C-terminus of nesprin-1. Expression of nesprin-1 mutants in C2C12 cells caused defects in myoblast differentiation and fusion associated with dysregulation of myogenic transcription factors and disruption of the nesprin-1 and KLC-1/2 interaction at the outer nuclear membrane. Expression of nesprin-1α2 WT and mutants in zebrafish embryos caused heart developmental defects that varied in severity. These findings support a role for nesprin-1 in myogenesis and muscle disease, and uncover a novel mechanism whereby disruption of the LINC complex may contribute to the pathogenesis of DCM.

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.

Not all vascular smooth muscle cell exosomes calcify equally in chronic kidney disease.

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.

Nesprin-1/2: roles in nuclear envelope organisation, myogenesis and muscle disease.

Nesprins (nuclear envelope spectrin repeat proteins) are multi-isomeric scaffolding proteins. Nesprin-1 and -2 are highly expressed in skeletal and cardiac muscles and together with SUN (Sad1p/UNC84) domain-containing proteins form the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex at the nuclear envelope in association with lamin A/C and emerin. Mutations in nesprin-1/2 have been found in patients with autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). Several lines of evidence indicate that compromised LINC complex function is the critical step leading to muscle disease. Here, we review recent advances in our understanding of the functions of nesprin-1/2 in the LINC complex and mechanistic insights into how mutations in nesprin-1/2 lead to nesprin-related muscle diseases, in particular DCM and EDMD.

Mouse models of nesprin-related diseases.

Nesprins (nuclear envelope spectrin repeat proteins) are a family of multi-isomeric scaffolding proteins. Nesprins form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) complex with SUN (Sad1p/UNC84) domain-containing proteins at the nuclear envelope, in association with lamin A/C and emerin, linking the nucleoskeleton to the cytoskeleton. The LINC complex serves as both a physical linker between the nuclear lamina and the cytoskeleton and a mechanosensor. The LINC complex has a broad range of functions and is involved in maintaining nuclear architecture, nuclear positioning and migration, and also modulating gene expression. Over 80 disease-related variants have been identified in SYNE-1/2 (nesprin-1/2) genes, which result in muscular or central nervous system disorders including autosomal dominant Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy and autosomal recessive cerebellar ataxia type 1. To date, 17 different nesprin mouse lines have been established to mimic these nesprin-related human diseases, which have provided valuable insights into the roles of nesprin and its scaffold LINC complex in a tissue-specific manner. In this review, we summarise the existing nesprin mouse models, compare their phenotypes and discuss the potential mechanisms underlying nesprin-associated diseases.

Magnesium Counteracts Vascular Calcification: Passive Interference or Active Modulation?

Over the last decade, an increasing number of studies report a close relationship between serum magnesium concentration and cardiovascular disease risk in the general population. In end-stage renal disease, an association was found between serum magnesium and survival. Hypomagnesemia was identified as a strong predictor for cardiovascular disease in these patients. A substantial body of in vitro and in vivo studies has identified a protective role for magnesium in vascular calcification. However, the precise mechanisms and its contribution to cardiovascular protection remain unclear. There are currently 2 leading hypotheses: first, magnesium may bind phosphate and delay calcium phosphate crystal growth in the circulation, thereby passively interfering with calcium phosphate deposition in the vessel wall. Second, magnesium may regulate vascular smooth muscle cell transdifferentiation toward an osteogenic phenotype by active cellular modulation of factors associated with calcification. Here, the data supporting these major hypotheses are reviewed. The literature supports both a passive inorganic phosphate-buffering role reducing hydroxyapatite formation and an active cell-mediated role, directly targeting vascular smooth muscle transdifferentiation. However, current evidence relies on basic experimental designs that are often insufficient to delineate the underlying mechanisms. The field requires more advanced experimental design, including determination of intracellular magnesium concentrations and the identification of the molecular players that regulate magnesium concentrations in vascular smooth muscle cells.