scholarly journals Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

2008 ◽  
Vol 295 (1) ◽  
pp. C268-C278 ◽  
Author(s):  
Zhe Sun ◽  
Luis A. Martinez-Lemus ◽  
Michael A. Hill ◽  
Gerald A. Meininger
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Mechanical Activity ◽  
Tissue Blood Flow ◽  
Cell Contact ◽  
Type I ◽  
Adhesion Sites

Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2∼5 μm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of α5- and β3-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward ( z-axis) pulling force (800∼1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to α5β1- and αvβ3-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces.

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

2007 ◽  
Vol 293 (3) ◽  
pp. H1978-H1986 ◽  
Author(s):  
Charles S. Wallace ◽  
Sophie A. Strike ◽  
George A. Truskey
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Tissue Engineered Blood Vessels ◽  
Focal Adhesion Formation ◽  
Fibrillar Adhesion

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the α5β1-integrin complex, whereas ECs used either α5β1- or αvβ3-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC α5β1-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

scholarly journals Degraded Collagen Fragments Promote Rapid Disassembly of Smooth Muscle Focal Adhesions That Correlates with Cleavage of Pp125FAK, Paxillin, and Talin

1999 ◽  
Vol 147 (3) ◽  
pp. 619-630 ◽  
Author(s):  
Neil O. Carragher ◽  
Bodo Levkau ◽  
Russell Ross ◽  
Elaine W. Raines
Keyword(s):  
Smooth Muscle ◽  
Focal Adhesion ◽  
Type I Collagen ◽  
Focal Adhesions ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Type I ◽  
Focal Adhesion Complex ◽  
Adhesion Complex

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125FAK), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125FAK is a substrate for both calpain I– and II–mediated processing. Mapping of the proteolytic cleavage fragments of pp125FAK predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125FAK to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125FAK with the cytoskeletal fraction, while pp125FAK cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125FAK cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125FAK, paxillin, and talin and dissolution of the focal adhesion complex.

scholarly journals Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization

2014 ◽  
Vol 307 (7) ◽  
pp. H945-H957 ◽  
Author(s):  
Srinivasa Raju Datla ◽  
Daniel J. McGrail ◽  
Sasa Vukelic ◽  
Lauren P. Huff ◽  
Alicia N. Lyle ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Traction Force ◽  
Force Generation ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Nadph Oxidase 4 ◽  
Focal Adhesion Turnover

Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner.

scholarly journals Type I Phosphatidylinositol Phosphate Kinase Beta Regulates Focal Adhesion Disassembly by Promoting β1 Integrin Endocytosis

2010 ◽  
Vol 30 (18) ◽  
pp. 4463-4479 ◽  
Author(s):  
Wei-Ting Chao ◽  
Felicity Ashcroft ◽  
Alexes C. Daquinag ◽  
Tegy Vadakkan ◽  
Zhubo Wei ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Type I ◽  
Adhesion Sites ◽  
Phosphatidylinositol Phosphate ◽  
Β1 Integrins ◽  
Focal Adhesion Turnover ◽  
Dynamin 2 ◽  
Phosphatidylinositol Phosphate Kinase

ABSTRACT Cell migration requires the regulated disassembly of focal adhesions, but the underlying mechanisms remain poorly defined. We have previously shown that focal adhesion disassembly requires the dynamin 2- and clathrin-dependent endocytosis of ligand-activated β1 integrins. Here, we identify type I phosphatidylinositol phosphate kinase beta (PIPKIβ), an enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI4,5P2), as a key regulator of this process. We found that knockdown of PIPKIβ by RNA interference blocks the internalization of active β1 integrins and impairs focal adhesion turnover and cell migration. These defects are caused by the failure to target the endocytic machinery, including clathrin adaptors and dynamin 2, to focal adhesion sites. As a consequence, depletion of PIPKIβ blocks clathrin assembly at adhesion plaques and prevents complex formation between dynamin 2 and focal adhesion kinase (FAK), a critical step in focal adhesion turnover. Together, our findings identify PIPKIβ as a novel regulator of focal adhesion disassembly and suggest that PIPKIβ spatially regulates integrin endocytosis at adhesion sites to control cell migration.

scholarly journals Mechanotransduction through fibronectin-integrin focal adhesion in microvascular smooth muscle cells: is calcium essential?

2012 ◽  
Vol 302 (10) ◽  
pp. H1965-H1973 ◽  
Author(s):  
Zhe Sun ◽  
Zhaohui Li ◽  
Gerald A. Meininger
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Focal Adhesion ◽  
Kinase Inhibitor ◽  
Contractile Response ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Mechanical Force ◽  
Type I ◽  
Low Passage

It is believed that increased transmural pressure exerts force on vascular smooth muscle cells (VSMCs) and triggers Ca2+ signaling as an initiating event responsible for the arteriolar myogenic response. However, the mechanisms linking the pressure increase to Ca2+ signaling are unclear. We have shown previously using atomic force microscopy (AFM) that mechanical force induces a VSMC contractile response when applied to single fibronectin (FN; Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA. Am J Physiol Cell Physiol 295; C268–C278, 2008) focal adhesion sites. This current study seeks to determine whether application of force to single focal adhesions can cause a change in VSMC Ca2+. Experiments were performed in low passage (p3∼10) as well as in freshly isolated skeletal muscle arteriole VSMCs. AFM-attached microbeads (5 μm) were coated with FN or collagen type I (CN-I) or type IV (CN-IV) and placed on a VSMC for 20 min, resulting in formation of a focal adhesion between the cell and the microbead. In low passage VSMCs, mechanically pulling on the FN-coated beads (800∼3000 pN) did not induce a Ca2+ increase but did cause a contractile response. In freshly isolated VSMCs, application of an FN or CN-I-coated bead onto the cell surface induced global Ca2+ increases. However, these Ca2+ increases were not correlated with the application of AFM pulling force to the bead or with the VSMC contractile responses to FN-coupled pulling. Chelating cytosolic Ca2+ using BAPTA loading had no negative effect on the focal adhesion-related contractile response in both freshly isolated and low passage VSMCs, while the Rho-kinase inhibitor Y27632 abolished the micromyogenic response in both cases. These observations suggest that, in freshly isolated and cultured VSMCs, application of mechanical force to a focal adhesion does not invoke an acute global Ca2+ increase. On the other hand, our data support a role for Rho-linked signaling mechanism involved in mechanotransduction leading to focal contraction that is independent of the need for a global increase in VSMC Ca2+.

scholarly journals Caspase-mediated Cleavage of Focal Adhesion Kinase pp125FAK and Disassembly of Focal Adhesions in Human Endothelial Cell Apoptosis

1998 ◽  
Vol 187 (4) ◽  
pp. 579-586 ◽  
Author(s):  
Bodo Levkau ◽  
Barbara Herren ◽  
Hidenori Koyama ◽  
Russell Ross ◽  
Elaine W. Raines
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Caspase 3 ◽  
Umbilical Vein ◽  
Focal Adhesions ◽  
Focal Adhesion Complex ◽  
Adhesion Sites ◽  
Induced Apoptosis ◽  
Adhesion Complex

Normal endothelial and epithelial cells undergo apoptosis when cell adhesion and spreading are prevented, implying a requirement for antiapoptotic signals from the extracellular matrix for cell survival. We investigated some of the molecular changes occurring in focal adhesions during growth factor deprivation–induced apoptosis in confluent monolayers of human umbilical vein endothelial cells. Among the first morphologic changes after initiation of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the matrix followed by detachment. We observe a specific proteolytic cleavage of focal adhesion kinase (pp125FAK), an important component of the focal adhesion complex, and identify pp125FAK as a novel substrate for caspase-3 and caspase-3–like apoptotic caspases. The initial cleavage precedes detachment, and coincides with loss of pp125FAK and paxillin from focal adhesion sites and their redistribution into the characteristic membrane blebs of apoptotically dying cells. Cleavage of pp125FAK differentially affects its association with signaling and cytoskeletal components of the focal adhesion complex; binding of paxillin, but not pp130Cas (Cas, Crk-associated substrate) and vinculin, to the COOH terminally truncated pp125FAK is abolished. Therefore, caspase-mediated cleavage of pp125FAK may be participating in the disassembly of the focal adhesion complex and actively interrupting survival signals from the extracellular matrix, thus propagating the cell death program.

scholarly journals Chronic Mineral Dysregulation Promotes Vascular Smooth Muscle Cell Adaptation and Extracellular Matrix Calcification

2009 ◽  
Vol 21 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Rukshana C. Shroff ◽  
Rosamund McNair ◽  
Jeremy N. Skepper ◽  
Nichola Figg ◽  
Leon J. Schurgers ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Smooth Muscle Cell ◽  
Cell Adaptation

scholarly journals Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells

2000 ◽  
Vol 11 (3) ◽  
pp. 929-939 ◽  
Author(s):  
Seunghyi Kook ◽  
Sang Ryeol Shim ◽  
Soo Jeon Choi ◽  
Joohong Ahnn ◽  
Jae Il Kim ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Caspase 3 ◽  
Morphological Changes ◽  
Point Mutations ◽  
Membrane Blebbing ◽  
Adhesion Sites ◽  
Focal Adhesion Proteins ◽  
Induced Apoptosis

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell–cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD416G and DSPD748G) in vitro, and point mutations substituting the Asp of DVPD416G and DSPD748G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD416G generated a 74-kDa fragment, which was in turn cleaved at DSPD748G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas–paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.

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