The WD Domain of Atg16l1 Crucial for LC3-Associated Phagocytosis Is Not Required for Preserving Skin Barrier Function in Mice.

The skin is a multifunctional organ, forming a barrier between the external and internal environment, thereby functioning as a safeguard against extrinsic factors. Autophagy has been implicated in epidermal differentiation and in preserving skin homeostasis. LC3-associated phagocytosis (LAP) uses some but not all components of autophagy. The Atg16l1 (Δ WD) mouse model lacks the WD40 domain required for LAP and has been widely used to study the effects of LAP deficiency and autophagy on tissue homeostasis and response to infection. In this study, the Δ WD model was used to study the relationship between LAP and skin homeostasis by determining whether LAP-deficient mice display a cutaneous phenotype. Skin histology of wild-type and Δ WD mice aged 1 year revealed minor morphological differences in the tail skin dermal layer. RT-qPCR and western blot analysis showed no differences in key keratin expression between genotypes. Skin barrier formation, assessed by dye permeation assays, demonstrated full and proper formation of the skin barrier at embryonic day 18.5 in both genotypes. Biomechanical analysis of the skin showed decreased skin elasticity in aged Δ WD but not wild-type mice. In summary, the LAP-deficient Δ WD mice displayed subtle alterations in dermal histology and age-related biomechanical changes.

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.

Piezo1-mediated regulation of smooth muscle cell volume in response to enhanced extracellular matrix rigidity.

BACKGROUND AND PURPOSE: Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined. EXPERIMENTAL APPROACH: Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation. KEY RESULTS: On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic-like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin-1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin-1 gene expression were up-regulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation. CONCLUSIONS AND IMPLICATIONS: In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin-1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin-1 gene expression is observed in disease relevant VSMC phenotypes.

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.

HGF/c-Met/ß1-integrin signalling axis induces tunneling nanotubes in A549 lung adenocarcinoma cells.

Tunneling nanotubes (TNTs) are thin cytoplasmic extensions involved in long-distance intercellular communication and can transport intracellular organelles and signalling molecules. In cancer cells, TNT formation contributes to cell survival, chemoresistance, and malignancy. However, the molecular mechanisms underlying TNT formation are not well defined, especially in different cancers. TNTs are present in non-small cell lung cancer (NSCLC) patients with adenocarcinoma. In NSCLC, hepatocyte growth factor (HGF) and its receptor, c-Met, are mutationally upregulated, causing increased cancer cell growth, survival, and invasion. This study identifies c-Met, ß1-integrin, and paxillin as novel components of TNTs in A549 lung adenocarcinoma cells, with paxillin localised at the protrusion site of TNTs. The HGF-induced TNTs in our study demonstrate the ability to transport lipid vesicles and mitochondria. HGF-induced TNT formation is mediated by c-Met and ß1-integrin in conjunction with paxillin, followed by downstream activation of MAPK and PI3K pathways and the Arp2/3 complex. These findings demonstrate a potential novel approach to inhibit TNT formation through targeting HGF/c-Met receptor and ß1-integrin signalling interactions, which has implications for multi-drug targeting in NSCLC.

Low-Protein Diets Composed of Protein Recovered from Food Processing Supported Growth, but Induced Mild Hepatic Steatosis Compared with a No-Protein Diet in Young Female Rats.

BACKGROUND: Living in low-income countries often restricts the consumption of adequate protein and animal protein. OBJECTIVES: This study aimed to investigate the effects of feeding low-protein diets on growth and liver health using proteins recovered from animal processing. METHODS: Female Sprague-Dawley rats (aged 28 d) were randomly assigned (n = 8 rats/group) to be fed standard purified diets with 0% or 10% kcal protein that was comprised of either carp, whey, or casein. RESULTS: Rats that were fed low-protein diets showed higher growth but developed mild hepatic steatosis compared to rats that were fed a no-protein diet, regardless of the protein source. Real-time quantitative polymerase chain reactions targeting the expression of genes involved in liver lipid homeostasis were not significantly different among groups. Global RNA-sequencing technology identified 9 differentially expressed genes linked to folate-mediated 1-carbon metabolism, endoplasmic reticulum (ER) stress, and metabolic diseases. Canonical pathway analysis revealed that mechanisms differed depending on the protein source. ER stress and dysregulated energy metabolism were implicated in hepatic steatosis in carp- and whey-fed rats. In contrast, impaired liver one-carbon methylations, lipoprotein assembly, and lipid export were implicated in casein-fed rats. CONCLUSIONS: Carp sarcoplasmic protein showed comparable results to commercially available casein and whey protein. A better understanding of the molecular mechanisms in hepatic steatosis development can assist formulation of proteins recovered from food processing into a sustainable source of high-quality protein.

RNA-sequencing revealed apple pomace ameliorates expression of genes in the hypothalamus associated with neurodegeneration in female rats fed a Western diet during adolescence to adulthood.

OBJECTIVES: Apple pomace, a waste byproduct of apple processing, is rich in nutrients (e.g. polyphenols and soluble fiber) with the potential to be neuroprotective. The aim of this study was to employ RNA-sequencing (RNASeq) technology to investigate diet-gene interactions in the hypothalamus of rats after feeding a Western diet calorically substituted with apple pomace. METHODS: Adolescent (age 21-29 days) female Sprague-Dawley rats were randomly assigned (n = 8 rats/group) to consume either a purified standard diet, Western (WE) diet, or Western diet calorically substituted with 10% apple pomace (WE/AP) for 8 weeks. RNA-seq was performed (n = 5 rats/group) to determine global differentially expressed genes in the hypothalamus. RESULTS: RNA-seq results comparing rats fed WE to WE/AP revealed 15 differentially expressed genes in the hypothalamus. Caloric substitution of WE diet with 10% apple pomace downregulated (q < 0.06) five genes implicated in brain aging and neurodegenerative disorders: synuclein alpha, phospholipase D family member 5, NADH dehydrogenase Fe-S protein 6, choline O-acetyltransferase, and frizzled class receptor 6. DISCUSSION: Altered gene expression of these five genes suggests that apple pomace ameliorated synthesis of the neurotransmitter, acetylcholine, in rats fed a WE diet. Apple pomace, a rich source of antioxidant polyphenols and soluble fiber, has been shown to reverse nonalcoholic fatty liver disease (NAFLD). Diet-induced NAFLD decreases hepatic de novo synthesis of choline, a precursor to acetylcholine. Based on preclinical evidence, apple pomace has the potential to be a sustainable functional food for maintaining brain function and for reducing the risk of neurodegeneration.

A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells.

Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8 nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO- macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.

Enabling genomic island prediction and comparison in multiple genomes to investigate bacterial evolution and outbreaks.

Outbreaks of virulent and/or drug-resistant bacteria have a significant impact on human health and major economic consequences. Genomic islands (GIs; defined as clusters of genes of probable horizontal origin) are of high interest because they disproportionately encode virulence factors, some antimicrobial-resistance (AMR) genes, and other adaptations of medical or environmental interest. While microbial genome sequencing has become rapid and inexpensive, current computational methods for GI analysis are not amenable for rapid, accurate, user-friendly and scalable comparative analysis of sets of related genomes. To help fill this gap, we have developed IslandCompare, an open-source computational pipeline for GI prediction and comparison across several to hundreds of bacterial genomes. A dynamic and interactive visualization strategy displays a bacterial core-genome phylogeny, with bacterial genomes linearly displayed at the phylogenetic tree leaves. Genomes are overlaid with GI predictions and AMR determinants from the Comprehensive Antibiotic Resistance Database (CARD), and regions of similarity between the genomes are also displayed. GI predictions are performed using Sigi-HMM and IslandPath-DIMOB, the two most precise GI prediction tools based on nucleotide composition biases, as well as a novel blast-based consistency step to improve cross-genome prediction consistency. GIs across genomes sharing sequence similarity are grouped into clusters, further aiding comparative analysis and visualization of acquisition and loss of mobile GIs in specific sub-clades. IslandCompare is an open-source software that is containerized for local use, plus available via a user-friendly, web-based interface to allow direct use by bioinformaticians, biologists and clinicians (at https://islandcompare.ca).

Using Polyacrylamide Hydrogels to Model Physiological Aortic Stiffness Reveals that Microtubules Are Critical Regulators of Isolated Smooth Muscle Cell Morphology and Contractility.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aortic wall and normally exist in a quiescent, contractile phenotype where actomyosin-derived contractile forces maintain vascular tone. However, VSMCs are not terminally differentiated and can dedifferentiate into a proliferative, synthetic phenotype. Actomyosin force generation is essential for the function of both phenotypes. Whilst much is already known about the mechanisms of VSMC actomyosin force generation, existing assays are either low throughput and time consuming, or qualitative and inconsistent. In this study, we use polyacrylamide hydrogels, tuned to mimic the physiological stiffness of the aortic wall, in a VSMC contractility assay. Isolated VSMC area decreases following stimulation with the contractile agonists angiotensin II or carbachol. Importantly, the angiotensin II induced reduction in cell area correlated with increased traction stress generation. Inhibition of actomyosin activity using blebbistatin or Y-27632 prevented angiotensin II mediated changes in VSMC morphology, suggesting that changes in VSMC morphology and actomyosin activity are core components of the contractile response. Furthermore, we show that microtubule stability is an essential regulator of isolated VSMC contractility. Treatment with either colchicine or paclitaxel uncoupled the morphological and/or traction stress responses of angiotensin II stimulated VSMCs. Our findings support the tensegrity model of cellular mechanics and we demonstrate that microtubules act to balance actomyosin-derived traction stress generation and regulate the morphological responses of VSMCs.

Mechanical programming of arterial smooth muscle cells in health and ageing.

Arterial smooth muscle cells (ASMCs), the predominant cell type within the arterial wall, detect and respond to external mechanical forces. These forces can be derived from blood flow (i.e. pressure and stretch) or from the supporting extracellular matrix (i.e. stiffness and topography). The healthy arterial wall is elastic, allowing the artery to change shape in response to changes in blood pressure, a property known as arterial compliance. As we age, the mechanical forces applied to ASMCs change; blood pressure and arterial wall rigidity increase and result in a reduction in arterial compliance. These changes in mechanical environment enhance ASMC contractility and promote disease-associated changes in ASMC phenotype. For mechanical stimuli to programme ASMCs, forces must influence the cell's load-bearing apparatus, the cytoskeleton. Comprised of an interconnected network of actin filaments, microtubules and intermediate filaments, each cytoskeletal component has distinct mechanical properties that enable ASMCs to respond to changes within the mechanical environment whilst maintaining cell integrity. In this review, we discuss how mechanically driven cytoskeletal reorganisation programmes ASMC function and phenotypic switching.

Protein quality and safety evaluation of sarcoplasmic protein derived from silver carp (Hypophthalmichthys molitrix) using a rat model.

Consisting of 25 to 30% of protein in carp, water-soluble sarcoplasmic proteins lost in wash water, have been recovered and freeze-dried into a protein-rich powder. Study objectives were to evaluate protein quality and safety of a silver carp sarcoplasm derived protein powder (CSP) compared to commercial protein supplements, casein, and whey. In vivo protein quality assessment of CSP showed a lower (P < 0.05) protein digestibility corrected amino acid score compared to the commercial protein sources. Despite greater (P < 0.05) fecal amino acid excretion in casein-fed rats, there were no significant differences in liver and muscle amino acid profiles. All low (10% kcal) protein diets supported growth with the normal range. However, whey protein supplementation resulted in greater (P < 0.05) adiposity. CSP, casein, or whey-fed rats showed no differences in major organ weights, renal damage biomarkers, or bone indices. Collectively, results indicated CSP was safe with protein quality comparable to casein. PRACTICAL APPLICATION: As much as 40 percent of protein in fish can be lost due to sarcoplasmic protein solubilization in processing wash water. Silver carp sarcoplasm protein powder may have similar commercial potential as a sustainable and nutritious alternative to whey and casein proteins. This project aimed to verify the protein quality and safety of this economical protein source.

SUN1/2 Are Essential for RhoA/ROCK-Regulated Actomyosin Activity in Isolated Vascular Smooth Muscle Cells.

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.

Emerging regulators of vascular smooth muscle cell migration.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall and normally adopt a quiescent, contractile phenotype. VSMC migration is tightly controlled, however, disease associated changes in the soluble and insoluble environment promote VSMC migration. Classically, studies investigating VSMC migration have described the influence of soluble factors. Emerging data has highlighted the importance of insoluble factors, including extracellular matrix stiffness and porosity. In this review, we will recap on the important signalling pathways that regulate VSMC migration and reflect on the potential importance of emerging regulators of VSMC function.

Apple Pomace Consumption Favorably Alters Hepatic Lipid Metabolism in Young Female Sprague-Dawley Rats Fed a Western Diet.

Apple pomace, which is a waste byproduct of processing, is rich in several nutrients, particularly dietary fiber, indicating potential benefits for diseases that are attributed to poor diets, such as non-alcoholic fatty liver disease (NAFLD). NAFLD affects over 25% of United States population and is increasing in children. Increasing fruit consumption can influence NAFLD. The study objective was to replace calories in standard or Western diets with apple pomace to determine the effects on genes regulating hepatic lipid metabolism and on risk of NAFLD. Female Sprague-Dawley rats were randomly assigned (n = 8 rats/group) to isocaloric diets of AIN-93G and AIN-93G/10% w/w apple pomace (AIN/AP) or isocaloric diets of Western (45% fat, 33% sucrose) and Western/10% w/w apple pomace (Western/AP) diets for eight weeks. There were no significant effects on hepatic lipid metabolism in rats fed AIN/AP. Western/AP diet containing fiber-rich apple pomace attenuated fat vacuole infiltration, elevated monounsaturated fatty acid content, and triglyceride storage in the liver due to higher circulating bile and upregulated hepatic DGAT2 gene expression induced by feeding a Western diet. The study results showed the replacement of calories in Western diet with apple pomace attenuated NAFLD risk. Therefore, apple pomace has the potential to be developed into a sustainable functional food for human consumption.

Nutritional Quality and Physical Characteristics of Soluble Proteins Recovered from Silver Carp.

The objective of this study was to evaluate the nutritional quality and physical characteristics of soluble proteins separated from silver carp at 4, 20, and 40 °C. Ground silver carp was diluted, and soluble proteins were separated by centrifugation and dried. The proximate composition (dry wt) of the protein powders averaged 82.42% protein, 3.25% lipid, and 14.50% ash. Average protein recovery yield was 11.78% with the better yields occurring at 20 °C (P < 0.05). Mineral profile revealed greater concentrations of Fe, Mg, P, and Na when compared to the initial homogenate. More saturated and monounsaturated fatty acids were recovered in the 4 °C powder and the least in the 40 °C powder (P < 0.05). Polyunsaturated fatty acids displayed a reverse trend, with the greatest concentration in the 40 °C powder and the least in the 4 °C powder (P < 0.05). The amino acid profile revealed that the protein powder met all FAO/WHO/UNO amino acid requirements for adults. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed high amounts of low and medium molecular weight (MW) proteins (10-15 and 25-50 kDa, respectively). Two-dimensional (2-D) electrophoresis indicated that the low MW proteins possessed a neutral isoelectric point relative to that of the medium MW proteins. The protein powder was significantly less soluble (P < 0.05) than whey protein concentrate 80 at every pH tested (pH 3.0 to 11.0). Similar tendencies were seen when ionic strength was shifted (0.0 to 1.1 I; P < 0.05). Soluble protein powders derived from silver carp are nutrient rich and have physical characteristics resembling whey protein concentrate. Changes in process temperature had limited effects on protein powder composition. PRACTICAL APPLICATION: Soluble proteins contribute to 20 to 40% of fish protein and are soluble in neutral salt solutions. Much of the sarcoplasmic proteins are lost when they solubilize in processing water and are discarded similarly to how whey protein was once discarded during dairy processing. When government regulations on whey disposal were implemented, the dairy industry responded by repurposing the high-quality protein for human use and it is now a billion dollar industry. The aim of this research project was to verify the composition of an otherwise overlooked protein source.

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.

Adventitial SCA-1+ Progenitor Cell Gene Sequencing Reveals the Mechanisms of Cell Migration in Response to Hyperlipidemia.

Adventitial progenitor cells, including SCA-1+ and mesenchymal stem cells, are believed to be important in vascular remodeling. It has been shown that SCA-1+ progenitor cells are involved in neointimal hyperplasia of vein grafts, but little is known concerning their involvement in hyperlipidemia-induced atherosclerosis. We employed single-cell sequencing technology on primary adventitial mouse SCA-1+ cells from wild-type and atherosclerotic-prone (ApoE-deficient) mice and found that a group of genes controlling cell migration and matrix protein degradation was highly altered. Adventitial progenitors from ApoE-deficient mice displayed an augmented migratory potential both in vitro and in vivo. This increased migratory ability was mimicked by lipid loading to SCA-1+ cells. Furthermore, we show that lipid loading increased miRNA-29b expression and induced sirtuin-1 and matrix metalloproteinase-9 levels to promote cell migration. These results provide direct evidence that blood cholesterol levels influence vascular progenitor cell function, which could be a potential target cell for treatment of vascular disease.

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.

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.