Cortical actin regulation modulates vascular contractility and compliance in veins.

Most cardiovascular research focuses on arterial mechanisms of disease, largely ignoring venous mechanisms. Here we examine ex vivo venous stiffness, spanning tissue to molecular levels, using biomechanics and magnetic microneedle technology, and show for the first time that venous stiffness is regulated by a molecular actin switch within the vascular smooth muscle cell in the wall of the vein. This switch connects the contractile apparatus within the cell to adhesion structures and facilitates stiffening of the vessel wall, regulating blood flow return to the heart. These studies also demonstrate that passive stiffness, the component of total stiffness not attributable to vascular smooth muscle activation, is severalfold lower in venous tissue than in arterial tissue. We show here that the activity of the smooth muscle cells plays a dominant role in determining total venous stiffness and regulating venous return. The literature on arterial mechanics is extensive, but far less is known about mechanisms controlling mechanical properties of veins. We use here a multi-scale approach to identify subcellular sources of venous stiffness. Portal vein tissue displays a severalfold decrease in passive stiffness compared to aortic tissues. The α-adrenergic agonist phenylephrine (PE) increased tissue stress and stiffness, both attenuated by cytochalasin D (CytoD) and PP2, inhibitors of actin polymerization and Src activity, respectively. We quantify, for the first time, cortical cellular stiffness in freshly isolated contractile vascular smooth muscle cells using magnetic microneedle technology. Cortical stiffness is significantly increased by PE and CytoD inhibits this increase but, surprisingly, PP2 does not. No detectable change in focal adhesion size, measured by immunofluorescence of FAK and zyxin, accompanies the PE-induced changes in cortical stiffness. Probing with phospho-specific antibodies confirmed activation of FAK/Src and ERK pathways and caldesmon phosphorylation. Thus, venous tissue stiffness is regulated both at the level of the smooth muscle cell cortex, via cortical actin polymerization, and by downstream smooth muscle effectors of Src/ERK signalling pathways. These findings identify novel potential molecular targets for the modulation of venous capacitance and venous return in health and disease.

Aging impairs smooth muscle-mediated regulation of aortic stiffness: a defect in shock absorption function?

Increased aortic stiffness is an early and independent biomarker of cardiovascular disease. Here we tested the hypothesis that vascular smooth muscle cells (VSMCs) contribute significantly to aortic stiffness and investigated the mechanisms involved. The relative contributions of VSMCs, focal adhesions (FAs), and matrix to stiffness in mouse aorta preparations at optimal length and with confirmed VSMC viability were separated by the use of small-molecule inhibitors and activators. Using biomechanical methods designed for minimal perturbation of cellular function, we directly quantified changes with aging in aortic material stiffness. An alpha adrenoceptor agonist, in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME) to remove interference of endothelial nitric oxide, increases stiffness by 90-200% from baseline in both young and old mice. Interestingly, increases are robustly suppressed by the Src kinase inhibitor PP2 in young but not old mice. Phosphotyrosine screening revealed, with aging, a biochemical signature of markedly impaired agonist-induced FA remodeling previously associated with Src signaling. Protein expression measurement confirmed a decrease in Src expression with aging. Thus we report here an additive model for the in vitro biomechanical components of the mouse aortic wall in which 1) VSMCs are a surprisingly large component of aortic stiffness at physiological lengths and 2) regulation of the VSMC component through FA signaling and hence plasticity is impaired with aging, diminishing the aorta's normal shock absorption function in response to stressors.

The contribution of vascular smooth muscle to aortic stiffness across length scales.

The operation of the cardiovascular system in health and disease is inherently mechanical. Clinically, aortic stiffness has proven to be of critical importance as an early biomarker for subsequent cardiovascular disease; however, the mechanisms involved in aortic stiffening are still unclear. The etiology of aortic stiffening with age has been thought to primarily involve changes in extracellular matrix protein composition and quantity, but recent studies suggest a significant involvement of the differentiated contractile vascular smooth muscle cells in the vessel wall. Here, we provide an overview of vascular physiology and biomechanics at different spatial scales. The processes involved in aortic stiffening are examined with particular attention given to recent discoveries regarding the role of vascular smooth muscle.

Reversal of Aging-Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein-Protein Interfaces.

BACKGROUND: The proximal aorta normally functions as a critical shock absorber that protects small downstream vessels from damage by pressure and flow pulsatility generated by the heart during systole. This shock absorber function is impaired with age because of aortic stiffening. METHODS AND RESULTS: We examined the contribution of common genetic variation to aortic stiffness in humans by interrogating results from the AortaGen Consortium genome-wide association study of carotid-femoral pulse wave velocity. Common genetic variation in the N-WASP (WASL) locus is associated with carotid-femoral pulse wave velocity (rs600420, P=0.0051). Thus, we tested the hypothesis that decoy proteins designed to disrupt the interaction of cytoskeletal proteins such as N-WASP with its binding partners in the vascular smooth muscle cytoskeleton could decrease ex vivo stiffness of aortas from a mouse model of aging. A synthetic decoy peptide construct of N-WASP significantly reduced activated stiffness in ex vivo aortas of aged mice. Two other cytoskeletal constructs targeted to VASP and talin-vinculin interfaces similarly decreased aging-induced ex vivo active stiffness by on-target specific actions. Furthermore, packaging these decoy peptides into microbubbles enables the peptides to be ultrasound-targeted to the wall of the proximal aorta to attenuate ex vivo active stiffness. CONCLUSIONS: We conclude that decoy peptides targeted to vascular smooth muscle cytoskeletal protein-protein interfaces and microbubble packaged can decrease aortic stiffness ex vivo. Our results provide proof of concept at the ex vivo level that decoy peptides targeted to cytoskeletal protein-protein interfaces may lead to substantive dynamic modulation of aortic stiffness.

Live-cell phenotypic-biomarker microfluidic assay for the risk stratification of cancer patients via machine learning.

The risk stratification of prostate cancer and breast cancer tumours from patients relies on histopathology, selective genomic testing, or on other methods employing fixed formalin tissue samples. However, static biomarker measurements from bulk fixed-tissue samples provide limited accuracy and actionability. Here, we report the development of a live-primary-cell phenotypic-biomarker assay with single-cell resolution, and its validation with prostate cancer and breast cancer tissue samples for the prediction of post-surgical adverse pathology. The assay includes a collagen-I/fibronectin extracellular-matrix formulation, dynamic live-cell biomarkers, a microfluidic device, machine-vision analysis and machine-learning algorithms, and generates predictive scores of adverse pathology at the time of surgery. Predictive scores for the risk stratification of 59 prostate cancer patients and 47 breast cancer patients, with values for area under the curve in receiver-operating-characteristic curves surpassing 80%, support the validation of the assay and its potential clinical applicability for the risk stratification of cancer patients.

The focal adhesion: a regulated component of aortic stiffness.

Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress.

Stimulus-specific activation and actin dependency of distinct, spatially separated ERK1/2 fractions in A7r5 smooth muscle cells.

A proliferative response of smooth muscle cells to activation of extracellular signal regulated kinases 1 and 2 (ERK1/2) has been linked to cardiovascular disease. In fully differentiated smooth muscle, however, ERK1/2 activation can also regulate contraction. Here, we use A7r5 smooth muscle cells, stimulated with 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA) to induce cytoskeletal remodeling or fetal calf serum (FCS) to induce proliferation, to identify factors that determine the outcomes of ERK1/2 activation in smooth muscle. Knock down experiments, immunoprecipitation and proximity ligation assays show that the ERK1/2 scaffold caveolin-1 mediates ERK1/2 activation in response to DPBA, but not FCS, and that ERK1/2 is released from caveolin-1 upon DPBA, but not FCS, stimulation. Conversely, ERK1/2 associated with the actin cytoskeleton is significantly reduced after FCS, but not DPBA stimulation, as determined by Triton X fractionation. Furthermore, cytochalasin treatment inhibits DPBA, but not FCS-induced ERK1/2 phosphorylation, indicating that the actin cytoskeleton is not only a target but also is required for ERK1/2 activation. Our results show that (1) at least two ERK1/2 fractions are regulated separately by specific stimuli, and that (2) the association of ERK1/2 with the actin cytoskeleton regulates the outcome of ERK1/2 signaling.