LIMK (LIM Kinase) Inhibition Prevents Vasoconstriction- and Hypertension-Induced Arterial Stiffening and Remodeling

Increased arterial stiffness and vascular remodeling precede and are consequences of hypertension. They also contribute to the development and progression of life-threatening cardiovascular diseases. Yet, there are currently no agents specifically aimed at preventing or treating arterial stiffening and remodeling. Previous research indicates that vascular smooth muscle actin polymerization participates in the initial stages of arterial stiffening and remodeling and that LIMK (LIM kinase) promotes F-actin formation and stabilization via cofilin phosphorylation and consequent inactivation. Herein, we hypothesize that LIMK inhibition is able to prevent vasoconstriction- and hypertension-associated arterial stiffening and inward remodeling. We found that small visceral arteries isolated from hypertensive subjects are stiffer and have greater cofilin phosphorylation than those from nonhypertensives. We also show that LIMK inhibition prevents arterial stiffening and inward remodeling in isolated human small visceral arteries exposed to prolonged vasoconstriction. Using cultured vascular smooth muscle cells, we determined that LIMK inhibition prevents vasoconstrictor agonists from increasing cofilin phosphorylation, F-actin volume, and cell cortex stiffness. We further show that localized LIMK inhibition prevents arteriolar inward remodeling in hypertensive mice. This indicates that hypertension is associated with increased vascular smooth muscle cofilin phosphorylation, cytoskeletal stress fiber formation, and heightened arterial stiffness. Our data further suggest that pharmacological inhibition of LIMK prevents vasoconstriction-induced arterial stiffening, in part, via reductions in vascular smooth muscle F-actin content and cellular stiffness. Accordingly, LIMK inhibition should represent a promising therapeutic means to stop the progression of arterial stiffening and remodeling in hypertension.

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Actin polymerization in differentiated vascular smooth muscle cells requires vasodilator-stimulated phosphoprotein

AJP Cell Physiology ◽

10.1152/ajpcell.00431.2009 ◽

2010 ◽

Vol 298 (3) ◽

pp. C559-C571 ◽

Cited By ~ 40

Author(s):

Hak Rim Kim ◽

Philip Graceffa ◽

François Ferron ◽

Cynthia Gallant ◽

Malgorzata Boczkowska ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Actin Filament ◽

Hot Spots ◽

Actin Polymerization ◽

Muscle Cells ◽

Cell Cortex ◽

Sites Of Action

Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768–778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with α-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with α-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC.

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Vascular Smooth Muscle Cells and Arterial Stiffening: Relevance in Development, Aging, and Disease

Physiological Reviews ◽

10.1152/physrev.00003.2017 ◽

2017 ◽

Vol 97 (4) ◽

pp. 1555-1617 ◽

Cited By ~ 167

Author(s):

Patrick Lacolley ◽

Véronique Regnault ◽

Patrick Segers ◽

Stéphane Laurent

Keyword(s):

Smooth Muscle ◽

Arterial Stiffness ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Cytoskeletal Proteins ◽

Arterial Tree ◽

Arterial Stiffening ◽

Wall Stiffness

The cushioning function of large arteries encompasses distension during systole and recoil during diastole which transforms pulsatile flow into a steady flow in the microcirculation. Arterial stiffness, the inverse of distensibility, has been implicated in various etiologies of chronic common and monogenic cardiovascular diseases and is a major cause of morbidity and mortality globally. The first components that contribute to arterial stiffening are extracellular matrix (ECM) proteins that support the mechanical load, while the second important components are vascular smooth muscle cells (VSMCs), which not only regulate actomyosin interactions for contraction but mediate also mechanotransduction in cell-ECM homeostasis. Eventually, VSMC plasticity and signaling in both conductance and resistance arteries are highly relevant to the physiology of normal and early vascular aging. This review summarizes current concepts of central pressure and tensile pulsatile circumferential stress as key mechanical determinants of arterial wall remodeling, cell-ECM interactions depending mainly on the architecture of cytoskeletal proteins and focal adhesion, the large/small arteries cross-talk that gives rise to target organ damage, and inflammatory pathways leading to calcification or atherosclerosis. We further speculate on the contribution of cellular stiffness along the arterial tree to vascular wall stiffness. In addition, this review provides the latest advances in the identification of gene variants affecting arterial stiffening. Now that important hemodynamic and molecular mechanisms of arterial stiffness have been elucidated, and the complex interplay between ECM, cells, and sensors identified, further research should study their potential to halt or to reverse the development of arterial stiffness.

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Bcl11b is a Newly Identified Regulator of Vascular Smooth Muscle Phenotype and Arterial Stiffness

10.1101/193110 ◽

2017 ◽

Cited By ~ 2

Author(s):

Jeff Arni C. Valisno ◽

Pavania Elavalakanar ◽

Christopher Nicholson ◽

Kuldeep Singh ◽

Dorina Avram ◽

...

Keyword(s):

Smooth Muscle ◽

Arterial Stiffness ◽

Vascular Smooth Muscle ◽

Actin Polymerization ◽

Genome Wide Association Study ◽

Ex Vivo ◽

Aortic Aneurysms ◽

Cell Phenotype ◽

Gene Desert ◽

A Genome

ABSTRACTB-cell leukemia 11b (Bcl11b) is a zinc-finger transcription factor known as master regulator of T lymphocytes and neuronal development during embryogenesis. Bcl11b-interacting protein COUP-TFII is required for atrial development and vasculogenesis, however a role of Bcl11b in the adult cardiovascular system is unknown. A genome-wide association study (GWAS) recently showed that a gene desert region downstream ofBCL11Band known to function asBCL11Benhancer harbors single nucleotide polymorphisms (SNPs) associated with increased arterial stiffness. Based on these human findings, we sought to examine relations between Bcl11b and arterial function using mice with Bcl11b deletion. We report for the first time that Bcl11b is expressed in vascular smooth muscle (VSM) and transcriptionally regulates the expression of VSM contractile proteins smooth muscle myosin and smooth muscle α-actin. Lack of Bcl11b in VSM-specific Bcl11b null mice (BSMKO) resulted in increased expression of Ca++-calmodulin-dependent serine/threonine phosphatase calcineurin in BSMKO VSM cells, cultured in serum-free condition, and in BSMKO aortas, which showed an inverse correlation with levels of phosphorylated VASPS239, a regulator of cytoskeletal actin rearrangements. Moreover, decreased pVASPS239in BSMKO aortas was associated with increased actin polymerization (F/G actin ratio). Functionally, aortic force, stress and wall tension, measured ex vivo in organ baths, were increased in BSMKO aortas and BSMKO mice had increased pulse wave velocity, thein vivoindex of arterial stiffness, compared to WT littermates. Despite having no effect on baseline blood pressure or angiotensin II-induced hypertension, Bcl11b deletion in VSM increased the incidence of aortic aneurysms in BSMKO mice. Aneurysmal aortas from angII-treated BSMKO mice had increased number of apoptotic VSM cells. In conclusion, we identified VSM Bcl11b as a novel and crucial regulator of VSM cell phenotype and vascular structural and functional integrity.

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Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.3.l653 ◽

1999 ◽

Vol 277 (3) ◽

pp. L653-L661 ◽

Cited By ~ 44

Author(s):

Carol A. Hirshman ◽

Charles W. Emala

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Airway Smooth Muscle ◽

Actin Polymerization ◽

Muscle Cells ◽

Fiber Formation ◽

Airway Smooth Muscle Cells ◽

Actin Reorganization ◽

Endothelin 1 ◽

In Cells

Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells and Ca2+ sensitization in intact smooth muscle preparations by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. In airway smooth muscle, the agonists responsible for cytoskeletal reorganization via actin polymerization are poorly understood. Carbachol-, lysophosphatidic acid (LPA)-, and endothelin-1-induced increases in filamentous actin staining are indicative of actin reorganization (filamentous-to-globular actin ratios of 2.4 ± 0.3 in control cells, 6.7 ± 0.8 with carbachol, 7.2 ± 0.8 with LPA, and 7.4 ± 0.9 with endothelin-1; P < 0.001; n = 14 experiments). Although the effect of all agonists was blocked by C3 exoenzyme (inactivator of Rho), only carbachol was blocked by pertussis toxin. Although carbachol-induced actin reorganization was blocked in cells pretreated with antisense oligonucleotides directed against Gαi-2 alone, LPA- and endothelin-1-induced actin reorganization were only blocked when both Gαi-2 and Gqα were depleted. These data indicate that in human airway smooth muscle cells, carbachol induces actin reorganization via a Gαi-2pathway, whereas LPA or endothelin-1 induce actin reorganization via either a Gαi-2 or a Gqα pathway.

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PKC induces uterine artery contraction through vascular smooth muscle actin polymerization

The FASEB Journal ◽

10.1096/fasebj.20.4.a303-c ◽

2006 ◽

Vol 20 (4) ◽

Author(s):

DaLiao Xiao ◽

Lubo Zhang

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Uterine Artery ◽

Actin Polymerization ◽

Smooth Muscle Actin ◽

Muscle Actin

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Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

AJP Cell Physiology ◽

10.1152/ajpcell.00467.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. C463-C475 ◽

Cited By ~ 45

Author(s):

Ana C. P. Thirone ◽

Pam Speight ◽

Matthew Zulys ◽

Ori D. Rotstein ◽

Katalin Szászi ◽

...

Keyword(s):

Actin Polymerization ◽

De Novo ◽

Rho Kinase ◽

Small Gtpases ◽

Hyperosmotic Stress ◽

Dominant Negative ◽

Lim Kinase ◽

Tubular Cells ◽

Cofilin Phosphorylation ◽

Underlying Mechanisms

Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation.We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.

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A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00210.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. C988-C993 ◽

Cited By ~ 8

Author(s):

Hak Rim Kim ◽

Paul C. Leavis ◽

Philip Graceffa ◽

Cynthia Gallant ◽

Kathleen G. Morgan

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Actin Polymerization ◽

Direct Detection ◽

New Method ◽

Actin Dynamics ◽

Tat Peptide ◽

Isolated Cells ◽

Intact Cells ◽

Permeabilized Cells

Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH2-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled actin. When TAT-Cys-S-STNB-tagged rhodamine actin (TSSAR) was added to live, freshly enzymatically isolated cells, we observed punctae of incorporated actin at the cortex of the cell. The punctae are indistinguishable from those we have previously reported to occur in the same cell type when rhodamine G-actin is added to permeabilized cells. Thus this new method allows the delivery of labeled G-actin into intact cells without disrupting the native state and will allow its further use to study the effect of physiological intracellular Ca2+ concentration transients and signal transduction on actin dynamics in intact cells.

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