Aged-vascular niche hinders osteogenesis of mesenchymal stem cells through paracrine repression of Wnt-axis.

Age-induced decline in osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) potentiates osteoporosis and increases the risk for bone fractures. Despite epidemiology studies reporting concurrent development of vascular and bone diseases in the elderly, the underlying mechanisms for the vascular-bone cross-talk in aging are largely unknown. In this study, we show that accelerated endothelial aging deteriorates bone tissue through paracrine repression of Wnt-driven-axis in BMSCs. Here, we utilize physiologically aged mice in conjunction with our transgenic endothelial progeria mouse model (Hutchinson-Gilford progeria syndrome; HGPS) that displays hallmarks of an aged bone marrow vascular niche. We find bone defects associated with diminished BMSC osteogenic differentiation that implicate the existence of angiocrine factors with long-term inhibitory effects. microRNA-transcriptomics of HGPS patient plasma combined with aged-vascular niche analyses in progeria mice reveal abundant secretion of Wnt-repressive microRNA-31-5p. Moreover, we show that inhibition of microRNA-31-5p as well as selective Wnt-activator CHIR99021 boosts the osteogenic potential of BMSCs through de-repression and activation of the Wnt-signaling, respectively. Our results demonstrate that the vascular niche significantly contributes to osteogenesis defects in aging and pave the ground for microRNA-based therapies of bone loss in elderly.

Aging impairs the neurovascular interface in the heart.

Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.

Premature aging in genetic diseases: what conclusions can be drawn for physiological aging.

According to current views the major hallmarks of physiological aging may be subdivided into three categories, primary causes of cellular damage (genomic instability, telomere attrition, loss of proteostasis, epigenetic alterations and compromised macroautophagy), antagonistic hallmarks that represent response to damage (deregulated nutrient sensing, cellular senescence, mitochondrial dysfunction) and integrative hallmarks that represent culprits of the phenotype (stem cell exhaustion, altered intercellular communication, chronic inflammation, dysbiosis). In contrast to physiological aging, premature aging diseases are driven by one or two distinct primary causes of aging, such as genomic instability in the case of Werner syndrome (WS), each displaying other hallmarks of aging to a variable extent. In this review we will focus on primary causes of well-investigated premature aging diseases Hutchinson-Gilford progeria syndrome (HGPS), WS, and Cockayne syndrome (CS) and for each provide an overview of reported aging hallmarks to elucidate resemblance to physiological aging on the mechanistic level and in the context of characteristic age-related diseases. Ubiquitous and tissue specific animal models of premature aging diseases will be discussed as useful tools to decipher fundamental aging-related mechanisms and develop intervention strategies to combat premature aging and age-related diseases.

Endothelial and systemic upregulation of miR-34a-5p fine-tunes senescence in progeria.

Endothelial defects significantly contribute to cardiovascular pathology in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Using an endothelium-specific progeria mouse model, we identify a novel, endothelium-specific microRNA (miR) signature linked to the p53-senescence pathway and a senescence-associated secretory phenotype (SASP). Progerin-expressing endothelial cells exert profound cell-non-autonomous effects initiating senescence in non-endothelial cell populations and causing immune cell infiltrates around blood vessels. Comparative miR expression analyses revealed unique upregulation of senescence-associated miR34a-5p in endothelial cells with strong accumulation at atheroprone aortic arch regions but also, in whole cardiac- and lung tissues as well as in the circulation of progeria mice. Mechanistically, miR34a-5p knockdown reduced not only p53 levels but also late-stage senescence regulator p16 with no effect on p21 levels, while p53 knockdown reduced miR34a-5p and partially rescued p21-mediated cell cycle inhibition with a moderate effect on SASP. These data demonstrate that miR34a-5p reinforces two separate senescence regulating branches in progerin-expressing endothelial cells, the p53- and p16-associated pathways, which synergistically maintain a senescence phenotype that contributes to cardiovascular pathology. Thus, the key function of circulatory miR34a-5p in endothelial dysfunction-linked cardiovascular pathology offers novel routes for diagnosis, prognosis and treatment for cardiovascular aging in HGPS and potentially geriatric patients.

The structural and gene expression hypotheses in laminopathic diseases-not so different after all.

Laminopathies are a diverse group of rare diseases with various pathologies in different tissues, which are linked to mutations in the LMNA gene. Historically, the structural disease model proposed mechanical defects of the lamina and nuclear fragility, the gene expression model impairment of spatial chromatin organization and signaling pathways as underlying mechanisms leading to the pathologies. Exciting findings in the past few years showing that mechanical forces are directly transmitted into the nucleus, where they affect chromatin organization and mechanoresponsive signaling molecules, have led to a revised concept of an integrative unified disease model, in which lamin-mediated pathways in mechanotransduction and chromatin regulation are highly interconnected and mutually dependent. In this Perspective we highlight breakthrough findings providing new insight into lamin-linked mechanisms of mechanotransduction and chromatin regulation and discuss how a combined and interrelated impairment of these functions by LMNA mutations may impair the complex mechanosignaling network and cause tissue-specific pathologies in laminopathies.

Endothelial progerin expression causes cardiovascular pathology through an impaired mechanoresponse.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder characterized by accelerated cardiovascular disease with extensive fibrosis. It is caused by a mutation in LMNA leading to expression of truncated prelamin A (progerin) in the nucleus. To investigate the contribution of the endothelium to cardiovascular HGPS pathology, we generated an endothelium-specific HGPS mouse model with selective endothelial progerin expression. Transgenic mice develop interstitial myocardial and perivascular fibrosis and left ventricular hypertrophy associated with diastolic dysfunction and premature death. Endothelial cells show impaired shear stress response and reduced levels of endothelial nitric oxide synthase (eNOS) and NO. On the molecular level, progerin impairs nucleocytoskeletal coupling in endothelial cells through changes in mechanoresponsive components at the nuclear envelope, increased F-actin/G-actin ratios, and deregulation of mechanoresponsive myocardin-related transcription factor-A (MRTFA). MRTFA binds to the Nos3 promoter and reduces eNOS expression, thereby mediating a profibrotic paracrine response in fibroblasts. MRTFA inhibition rescues eNOS levels and ameliorates the profibrotic effect of endothelial cells in vitro. Although this murine model lacks the key anatomical feature of vascular smooth muscle cell loss seen in HGPS patients, our data show that progerin-induced impairment of mechanosignaling in endothelial cells contributes to excessive fibrosis and cardiovascular disease in HGPS patients.

Arteriovenous specification: BMPER and TWSG1 determine endothelial cell fate via activation of synergistic BMP and Notch signaling.

Two extracellular BMP modulators, BMPER and TWSG1, act in a pro-BMP fashion to activate endothelial-specific members of the TGF-ß/BMP receptor family. Through cross-talk with the Notch signaling pathways, they are key regulators of downstream Notch targets, including ephrin B2. This adds to our understanding of BMP and Notch signaling, how these pathways converge, and thereby control arteriovenous specification.

Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks.

Mutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility 'in-check' and maintains AJ homeostasis.

Lamins at the crossroads of mechanosignaling.

The intermediate filament proteins, A- and B-type lamins, form the nuclear lamina scaffold adjacent to the inner nuclear membrane. B-type lamins confer elasticity, while A-type lamins lend viscosity and stiffness to nuclei. Lamins also contribute to chromatin regulation and various signaling pathways affecting gene expression. The mechanical roles of lamins and their functions in gene regulation are often viewed as independent activities, but recent findings suggest a highly cross-linked and interdependent regulation of these different functions, particularly in mechanosignaling. In this newly emerging concept, lamins act as a "mechanostat" that senses forces from outside and responds to tension by reinforcing the cytoskeleton and the extracellular matrix. A-type lamins, emerin, and the linker of the nucleoskeleton and cytoskeleton (LINC) complex directly transmit forces from the extracellular matrix into the nucleus. These mechanical forces lead to changes in the molecular structure, modification, and assembly state of A-type lamins. This in turn activates a tension-induced "inside-out signaling" through which the nucleus feeds back to the cytoskeleton and the extracellular matrix to balance outside and inside forces. These functions regulate differentiation and may be impaired in lamin-linked diseases, leading to cellular phenotypes, particularly in mechanical load-bearing tissues.

Networking and anchoring through plectin: a key to IF functionality and mechanotransduction.

Intermediate filaments (IFs) are involved in multiple cellular processes that are essential for the maintenance of cell and tissue integrity as well as response and adaption to stress. Mainly through pathological manifestations in patients and the analysis of genetic mouse models, it became evident that cytolinker proteins of the plakin protein family are essential for many of the functions ascribed to IFs. As discussed in this review, one of them, plectin, affects the assembly properties, interaction potential, compartmentalization, and linkage properties of IFs, making it to a key player for IF functionality. The far reaching consequences of IFs not being well-connected for skin and muscular integrity, migration, and mechanotransduction are highlighted.

Mechanosensing through focal adhesion-anchored intermediate filaments.

Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up-regulation of "stretched" high-affinity integrin complexes. By exploiting tension-independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton-regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.

Plectin-controlled keratin cytoarchitecture affects MAP kinases involved in cellular stress response and migration.

Plectin is a major intermediate filament (IF)-based cytolinker protein that stabilizes cells and tissues mechanically, regulates actin filament dynamics, and serves as a scaffolding platform for signaling molecules. In this study, we show that plectin deficiency is a cause of aberrant keratin cytoskeleton organization caused by a lack of orthogonal IF cross-linking. Keratin networks in plectin-deficient cells were more susceptible to osmotic shock-induced retraction from peripheral areas, and their okadaic acid-induced disruption (paralleled by stress-activated MAP kinase p38 activation) proceeded faster. Basal activities of the MAP kinase Erk1/2 and of the membrane-associated upstream protein kinases c-Src and PKCdelta were significantly elevated, and increased migration rates, as assessed by in vitro wound-closure assays and time-lapse microscopy, were observed. Forced expression of RACK1, which is the plectin-binding receptor protein for activated PKCdelta, in wild-type keratinocytes elevated their migration potential close to that of plectin-null cells. These data establish a link between cytolinker-controlled cytoarchitecture/scaffolding functions of keratin IFs and specific MAP kinase cascades mediating distinct cellular responses.

Targeted ablation of plectin isoform 1 uncovers role of cytolinker proteins in leukocyte recruitment.

Plectin, a typical cytolinker protein, is essential for skin and skeletal muscle integrity. It stabilizes cells mechanically, regulates cytoskeleton dynamics, and serves as a scaffolding platform for signaling molecules. A variety of isoforms expressed in different tissues and cell types account for this versatility. To uncover the role of plectin 1, the major isoform expressed in tissues of mesenchymal origin, against the background of all other variants, we raised plectin isoform 1-specific antibodies and generated isoform-deficient mice. In contrast to plectin-null mice (lacking all plectin isoforms), which die shortly after birth because of severe skin blistering, plectin isoform 1-deficient mice were viable at birth, had a normal lifespan, and did not display the skin blistering phenotype. However, dermal fibroblasts isolated from plectin 1-deficient mice exhibited abnormalities in their actin cytoskeleton and impaired migration potential. Similarly, plectin 1-deficient T cells isolated from nymph nodes showed diminished chemotactic migration in vitro. Most strikingly, in vivo we found that leukocyte infiltration during wound healing was reduced in the mutant mice. These data show a specific role of a cytolinker protein in immune cell motility. Single isoform-deficient mice thus represent a powerful tool to unravel highly specific functions of plectin variants.

Plectin-RACK1 (receptor for activated C kinase 1) scaffolding: a novel mechanism to regulate protein kinase C activity.

Agonist-induced translocation of protein kinase C (PKC) isozymes is mediated by receptors for the activated form of the kinase, shuttling it from one intracellular site to another and enhancing its catalytic activity. It is however unknown whether the receptors themselves are anchored to certain intracellular structures prior to their engagement with PKC. We show here sequestering of receptor for activated C kinase 1 (RACK1) to the cytoskeleton through the cytoskeletal linker protein plectin during the initial stages of cell adhesion. We found that upon PKC activation, RACK1 was released from the cytoskeleton and transferred to the detergent-soluble cell compartment, where it formed an inducible triple complex with one of the PKC isozymes, PKCdelta, and with plectin. In plectin-deficient cells the cytoskeleton-associated RACK1 fraction was reduced, and the protein was found predominantly at sites to which it normally translocated upon PKC activation. Concomitantly, dislocation of PKCdelta and elevated enzymatic activity were observed in these cells. PKCdelta was also more rapidly degraded, likely due to its overactivation. We propose a previously unrecognized function of plectin as cytoskeletal regulator of PKC signaling, and possibly other signaling events, through sequestration of the scaffolding protein RACK1.