Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening.

Vascular stiffening and its sequelae are major causes of morbidity and mortality in the elderly. The increasingly accepted concept of "smooth muscle cell (SMC) stiffness syndrome" along with matrix deposition has emerged in vascular biology to account for the mechanical phenotype of arterial aging, but the molecular targets remain elusive. In this study, using an unbiased proteomic analysis, we identified lysyl oxidase-like 2 (LOXL2) as a critical SMC mediator for age-associated vascular stiffening. We tested the hypothesis that loss of LOXL2 function is protective in aging-associated vascular stiffening. We determined that exogenous and endogenous nitric oxide markedly decreased LOXL2 abundance and activity in the extracellular matrix of isolated SMCs and LOXL2 endothelial cells suppress LOXL2 abundance in the aorta. In a longitudinal study, LOXL2+/- mice were protected from age-associated increase in pulse-wave velocity, an index of vascular stiffening, as occurred in littermate wild-type mice. Using isolated aortic segments, we found that LOXL2 mediates vascular stiffening in aging by promoting SMC stiffness, augmented SMC contractility, and vascular matrix deposition. Together, these studies establish LOXL2 as a nodal point for a new therapeutic approach to treat age-associated vascular stiffening. NEW & NOTEWORTHY Increased central vascular stiffness augments risk of major adverse cardiovascular events. Despite significant advances in understanding the genetic and molecular underpinnings of vascular stiffening, targeted therapy has remained elusive. Here, we show that lysyl oxidase-like 2 (LOXL2) drives vascular stiffening during aging by promoting matrix remodeling and vascular smooth muscle cell stiffening. Reduced LOXL2 expression protects mice from age-associated vascular stiffening and delays the onset of isolated systolic hypertension, a major consequence of stiffening.

TGF-ß1 Evokes Human Airway Smooth Muscle Cell Shortening and Hyperresponsiveness via Smad3.

Transforming growth factor ß1 (TGF-ß1), a cytokine whose levels are elevated in the airways of patients with asthma, perpetuates airway inflammation and modulates airway structural cell remodeling. However, the role of TGF-ß1 in excessive airway narrowing in asthma, or airway hyperresponsiveness (AHR), remains unclear. In this study, we set out to investigate the direct effects of TGF-ß1 on human airway smooth muscle (HASM) cell shortening and hyperresponsiveness. The dynamics of AHR and single-cell excitation-contraction coupling were measured in human precision-cut lung slices and in isolated HASM cells using supravital microscopy and magnetic twisting cytometry, respectively. In human precision-cut lung slices, overnight treatment with TGF-ß1 significantly augmented basal and carbachol-induced bronchoconstriction. In isolated HASM cells, TGF-ß1 increased basal and methacholine-induced cytoskeletal stiffness in a dose- and time-dependent manner. TGF-ß1-induced single-cell contraction was corroborated by concomitant increases in myosin light chain and myosin phosphatase target subunit 1 phosphorylation levels, which were attenuated by small interfering RNA-mediated knockdown of Smad3 and pharmacological inhibition of Rho kinase. Strikingly, these physiological effects of TGF-ß1 occurred through a RhoA-independent mechanism, with little effect on HASM cell [Ca2+]i levels. Together, our data suggest that TGF-ß1 enhances HASM excitation-contraction coupling pathways to induce HASM cell shortening and hyperresponsiveness. These findings reveal a potential link between airway injury-repair responses and bronchial hyperreactivity in asthma, and define TGF-ß1 signaling as a potential target to reduce AHR in asthma.

Mediator Med23 deficiency enhances neural differentiation of murine embryonic stem cells through modulating BMP signaling.

Unraveling the mechanisms underlying early neural differentiation of embryonic stem cells (ESCs) is crucial to developing cell-based therapies of neurodegenerative diseases. Neural fate acquisition is proposed to be controlled by a 'default' mechanism, for which the molecular regulation is not well understood. In this study, we investigated the functional roles of Mediator Med23 in pluripotency and lineage commitment of murine ESCs. Unexpectedly, we found that, despite the largely unchanged pluripotency and self-renewal of ESCs, Med23 depletion rendered the cells prone to neural differentiation in different differentiation assays. Knockdown of two other Mediator subunits, Med1 and Med15, did not alter the neural differentiation of ESCs. Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23 depletion attenuated BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1, which is involved in Bmp4 promoter-enhancer communication. Interestingly, med23 knockdown in zebrafish embryos also enhanced neural development at early embryogenesis, which could be reversed by co-injection of bmp4 mRNA. Taken together, our study reveals an intrinsic, restrictive role of MED23 in early neural development, thus providing new molecular insights for neural fate determination.

Inflammatory Role of Milk Fat Globule-Epidermal Growth Factor VIII in Age-Associated Arterial Remodeling.

Background Age-associated aortic remodeling includes a marked increase in intimal medial thickness (IMT), associated with signs of inflammation. Although aortic wall milk fat globule-epidermal growth factor VIII (MFG-E8) increases with age, and is associated with aortic inflammation, it is not known whether MFG-E8 is required for the age-associated increase in aortic IMT. Here, we tested whether MFG-E8 is required for the age-associated increase in aortic IMT. Methods and Results To determine the role of MFG-E8 in the age-associated increase of IMT, we compared aortic remodeling in adult (20-week) and aged (96-week) MFG-E8 (-/-) knockout and age matched wild-type (WT) littermate mice. The average aortic IMT increased with age in the WT from 50±10 to 70±20 µm (P<0.0001) but did not significantly increase with age in MFG-E8 knockout mice. Because angiotensin II signaling is implicated as a driver of age-associated increase in IMT, we infused 30-week-old MFG-E8 knockout and age-matched littermate WT mice with angiotensin II or saline via osmotic mini-pumps to determine whether MFG-E8 is required for angiotensin II-induced aortic remodeling. (1) In WT mice, angiotensin II infusion substantially increased IMT, elastic lamina degradation, collagen deposition, and the proliferation of vascular smooth muscle cells; in contrast, these effects were significantly reduced in MFG-E8 KO mice; (2) On a molecular level, angiotensin II treatment significantly increased the activation and expression of matrix metalloproteinase type 2, transforming growth factor beta 1, and its downstream signaling molecule phosphorylated mother against decapentaplegic homolog 2, and collagen type I production in WT mice; however, in the MFG-E8 knockout mice, these molecular effects were significantly reduced; and (3) in WT mice, angiotensin II increased levels of aortic inflammatory markers phosphorylated nuclear factor-kappa beta p65, monocyte chemoattractant protein 1, tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 molecular expression, while in contrast, these inflammatory markers did not change in knockout mice. Conclusions Thus, MFG-E8 is required for both age-associated proinflammatory aortic remodeling and also for the angiotensin II-dependent induction in younger mice of an aortic inflammatory phenotype observed in advanced age. Targeting MFG-E8 would be a novel molecular approach to curb adverse arterial remodeling.

Calorie Restriction Curbs Proinflammation That Accompanies Arterial Aging, Preserving a Youthful Phenotype.

Background Aging exponentially increases the incidence of morbidity and mortality of quintessential cardiovascular disease mainly due to arterial proinflammatory shifts at the molecular, cellular, and tissue levels within the arterial wall. Calorie restriction ( CR ) in rats improves arterial function and extends both health span and life span. How CR affects the proinflammatory landscape of molecular, cellular, and tissue phenotypic shifts within the arterial wall in rats, however, remains to be elucidated. Methods and Results Aortae were harvested from young (6-month-old) and old (24-month-old) Fischer 344 rats, fed ad libitum and a second group maintained on a 40% CR beginning at 1 month of age. Histopathologic and morphometric analysis of the arterial wall demonstrated that CR markedly reduced age-associated intimal medial thickening, collagen deposition, and elastin fractionation/degradation within the arterial walls. Immunostaining/blotting showed that CR effectively prevented an age-associated increase in the density of platelet-derived growth factor, matrix metalloproteinase type II activity, and transforming growth factor beta 1 and its downstream signaling molecules, phospho-mothers against decapentaplegic homolog-2/3 (p- SMAD -2/3) in the arterial wall. In early passage cultured vascular smooth muscle cells isolated from AL and CR rat aortae, CR alleviated the age-associated vascular smooth muscle cell phenotypic shifts, profibrogenic signaling, and migration/proliferation in response to platelet-derived growth factor. Conclusions CR reduces matrix and cellular proinflammation associated with aging that occurs within the aortic wall and that are attributable to platelet-derived growth factor signaling. Thus, CR reduces the platelet-derived growth factor-associated signaling cascade, contributing to the postponement of biological aging and preservation of a more youthful aortic wall phenotype.

TGFß1 reinforces arterial aging in the vascular smooth muscle cell through a long-range regulation of the cytoskeletal stiffness.

Here we report exquisitely distinct material properties of primary vascular smooth muscle (VSM) cells isolated from the thoracic aorta of adult (8 months) vs. aged (30 months) F344XBN rats. Individual VSM cells derived from the aged animals showed a tense internal network of the actin cytoskeleton (CSK), exhibiting increased stiffness (elastic) and frictional (loss) moduli than those derived from the adult animals over a wide frequency range of the imposed oscillatory deformation. This discrete mechanical response was long-lived in culture and persistent across a physiological range of matrix rigidity. Strikingly, the pro-fibrotic transforming growth factor ß1 (TGFß1) emerged as a specific modifier of age-associated VSM stiffening in vitro. TGFß1 reinforced the mechanical phenotype of arterial aging in VSM cells on multiple time and length scales through clustering of mechanosensitive α5ß1 and αvß3 integrins. Taken together, these studies identify a novel nodal point for the long-range regulation of VSM stiffness and serve as a proof-of-concept that the broad-based inhibition of TGFß1 expression, or TGFß1 signal transduction in VSM, may be a useful therapeutic approach to mitigate the pathologic progression of central arterial wall stiffening associated with aging.

Reduced vasorin enhances angiotensin II signaling within the aging arterial wall.

The glycosylated protein vasorin physically interacts with the transforming growth factor-beta1 (TGF-ß1) and functionally attenuates its fibrogenic signaling in the vascular smooth muscle cells (VSMCs) of the arterial wall. Angiotensin II (Ang II) amplifies TGF-ß1 activation in the VSMCs of the arterial wall with aging. In this study, we hypothesized that a reduced expression of the protein vasorin plays a contributory role in magnifying Ang II-associated fibrogenic signaling in the VSMCs of the arterial wall with aging. The current study shows that vasorin mRNA and protein expression were significantly decreased both in aortic wall and VSMCs from old (30 mo) vs. young (8 mo) FXBN rats. Exposing young VSMCs to Ang II reduced vasorin protein expression to the levels of old untreated cells while treating old VSMCs with the Ang II type AT1 receptor antagonist Losartan upregulated vasorin protein expression up to the levels of young. The physical interaction between vasorin and TGF-ß1 was significantly decreased in old vs. young VSMCs. Further, exposing young VSMCs to Ang II increased the levels of matrix metalloproteinase type II (MMP-2) activation and TGF-ß1 downstream molecules p-SMAD-2/3 and collagen type I production up to the levels of old untreated VSMCs, and these effects were substantially inhibited by overexpressing vasorin. Administration of Ang II to young rats (8 mo) for 28 days via an osmotic minipump markedly reduced the expression of vasorin. Importantly, vasorin protein was effectively cleaved by activated MMP-2 both in vitro and in vivo. Administration of the MMP inhibitor, PD 166793, for 6 mo to young adult (18 mo) via a daily gavage markedly increased levels of vasorin in the aortic wall. Thus, reduced vasorin amplifies Ang II profibrotic signaling via an activation of MMP-2 in VSMCs within the aging arterial wall.

Defining an olfactory receptor function in airway smooth muscle cells.

Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma.