Non-muscle myosin II regulates aortic stiffness through effects on specific focal adhesion proteins and the non-muscle cortical cytoskeleton

Non-muscle myosin II (NMII) plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in mammalian vascular function is not well understood. Here, we investigated the function of NMII in the biomechanical and signalling properties of mouse aorta. We found that blebbistatin, an inhibitor of NMII, decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate that in freshly isolated, contractile, aortic smooth muscle cells, the non-muscle myosin IIA (NMIIA) isoform is associated with contractile filaments in the core of the cell as well as those in the non-muscle cell cortex. However, the non-muscle myosin IIB (NMIIB) isoform is excluded from the cell cortex and colocalizes only with contractile filaments. Furthermore, both siRNA knockdown of NMIIA and NMIIB isoforms in the differentiated A7r5 smooth muscle cell line and blebbistatin-mediated inhibition of NM myosin II suppress agonist-activated increases in phosphorylation of the focal adhesion proteins FAK Y925 and paxillin Y118. Thus, we show in the present study, for the first time that NMII regulates aortic stiffness and stress and that this regulation is mediated through the tension-dependent phosphorylation of the focal adhesion proteins FAK and paxillin.     

Src modulates contractile vascular smooth muscle function via regulation of focal adhesions

Src is a known regulator of focal adhesion turnover in migrating cells; but, in contrast, Src is generally assumed to play little role in differentiated, contractile vascular smooth muscle (dVSM). The goal of the present study was to determine if Src-family kinases regulate focal adhesion proteins and how this might affect contractility of non-proliferative vascular smooth muscle. We demonstrate here, through the use of phosphotyrosine screening, deconvolution microscopy imaging, and differential centrifugation, that the activity of Src family kinases in aorta is regulated by the alpha agonist and vasoconstrictor phenylephrine, and leads to focal adhesion protein phosphorylation and remodeling in dVSM. Furthermore, Src inhibition via morpholino knockdown of Src or by the small molecule inhibitor PP2 prevents phenylephrine-induced adhesion protein phosphorylation, markedly slows the tissue's ability to contract, and decreases steady state contractile force amplitude. Significant vasoconstrictor-induced and Src-dependent phosphorylation of Cas pY-165, FAK pY-925, paxillin pY-118, and Erk1/2 were observed. However, increases in FAK 397 phosphorylation were not seen, demonstrating differences between cells in tissue versus migrating, proliferating cells. We show here that Src, in a cause and effect manner, regulates focal adhesion protein function and, consequently, modulates contractility during the action of a vasoconstrictor. These data point to the possibility that vascular focal adhesion proteins may be useful drug discovery targets for novel therapeutic approaches to cardiovascular disease.     

Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II–mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II–dependent manner and to examine the mechanism for their myosin II–sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II–mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation.     

An aortic spontaneously active phosphatase dephosphorylates myosin and inhibits actin-myosin interaction

Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase $\text{a}$ and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.     

Synergistic effect of angiotensin II and nitric oxide synthase inhibitor in increasing aortic stiffness in mice

Although they are implicated on their own as risk factors for cardiovascular disease, the potential link between nitric oxide (NO) deficiency, ANG II, and vascular stiffening has not been tested before. We evaluated the role of chronic ANG II treatment and NO deficiency, alone and in combination, on aortic stiffness in mice and tested parameters contributing to increases in active or passive components of vascular stiffness, including blood pressure, vascular smooth muscle contractility, and extracellular matrix components. Untreated (control) mice and mice treated with a NO synthase (NOS) inhibitor [N(omega)-nitro-L-arginine methyl ester (L-NAME), 0.5 g/l] were implanted with osmotic minipumps delivering ANG II (500 ng.kg(-1).min(-1)) for 28 days. Aortic stiffness was then measured in vivo by pulse wave velocity (PWV) and ex vivo by load-strain analysis to obtain values of maximal passive stiffness (MPS). Blood pressure and aortic contractility ex vivo were measured. ANG II treatment or NOS inhibition with L-NAME did not independently increase vascular stiffness; however, the combined treatments worked synergistically to increase PWV and MPS. The combined treatments of ANG II + L-NAME also significantly increased aortic wall collagen content while decreasing elastin. These novel results suggest that NO deficiency and ANG II act synergistically to increase aortic stiffness in mice predominantly via changes in aortic wall collagen/elastin ratio.     

Regulatable stiffness in relaxed airway smooth muscle: a target for asthma treatment?

The airway smooth muscle (ASM) layer within the airway wall modulates airway diameter and distensibility. Even in the relaxed state, the ASM layer possesses finite stiffness and limits the extent of airway distension by the radial force generated by parenchymal tethers and transmural pressure. Airway stiffness has often been attributed to passive elements, such as the extracellular matrix in the lamina reticularis, adventitia, and the smooth muscle layer that cannot be rapidly modulated by drug intervention such as ASM relaxation by β-agonists. In this study, we describe a calcium-sensitive component of ASM stiffness mediated through the Rho-kinase signaling pathway. The stiffness of ovine tracheal smooth muscle was assessed in the relaxed state under the following conditions: 1) in physiological saline solution (Krebs solution) with normal calcium concentration; 2) in calcium-free Krebs with 2 mM EGTA; 3) in Krebs with calcium entry blocker (SKF-96365); 4) in Krebs with myosin light chain kinase inhibitor (ML-7); and 5) in Krebs with Rho-kinase inhibitor (Y-27632). It was found that a substantial portion of the passive stiffness could be abolished when intracellular calcium was removed; this calcium-sensitive stiffness appeared to stem from intracellular source and was not sensitive to ML-7 inhibition of myosin light chain phosphorylation, but was sensitive to Y-27632 inhibition of Rho kinase. The results suggest that airway stiffness can be readily modulated by targeting the calcium-sensitive component of the passive stiffness within the muscle layer.     

Caveolin-1 regulates contractility in differentiated vascular smooth muscle

Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an alpha agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. alpha-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the alpha-agonist were obtained with the cholesterol-depleting agent methyl-beta-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.     

Myosin phosphatase-Rho interacting protein. A new member of the myosin phosphatase complex that directly binds RhoA

Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments. M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins. An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner. M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells. M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.     

The pro-apoptotic protein Par-4 facilitates vascular contractility by cytoskeletal targeting of ZIPK

Par-4 (prostate apoptosis response 4) is a pro-apoptotic protein and tumour suppressor that was originally identified as a gene product up-regulated during apoptosis in prostate cancer cells. Here, we show, for the first time, that Par-4 is expressed and co-localizes with the actin filament bundles in vascular smooth muscle. Furthermore, we demonstrate that targeting of ZIPK to the actin filaments, as observed upon PGF-2α stimulation, is inhibited by the presence of a cell permeant Par-4 decoy peptide. The same decoy peptide also significantly inhibits PGF-2α induced contractions of smooth muscle tissue. Moreover, knockdown of Par-4 using antisense morpholino nucleotides results in significantly reduced contractility, and myosin light chain and myosin phosphatase target subunit phosphorylation. These results indicate that Par-4 facilitates contraction by targeting ZIPK to the vicinity of its substrates, myosin light chain and MYPT, which are located on the actin filaments. These results identify Par-4 as a novel regulator of myosin light chain phosphorylation in differentiated, contractile vascular smooth muscle.     

Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin

Rho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin-targetting subunit (MYPT1) of the myosin-associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited.     

M-RIP targets myosin phosphatase to stress fibers to regulate myosin light chain phosphorylation in vascular smooth muscle cells

Vascular smooth muscle cell contraction and relaxation are directly related to the phosphorylation state of the regulatory myosin light chain. Myosin light chains are dephosphorylated by myosin phosphatase, leading to vascular smooth muscle relaxation. Myosin phosphatase is localized not only at actin-myosin stress fibers where it dephosphorylates myosin light chains, but also in the cytoplasm and at the cell membrane. The mechanisms by which myosin phosphatase is targeted to these loci are incompletely understood. We recently identified myosin phosphatase-Rho interacting protein as a member of the myosin phosphatase complex that directly binds both the myosin binding subunit of myosin phosphatase and RhoA and is localized to actin-myosin stress fibers. We hypothesized that myosin phosphatase-Rho interacting protein targets myosin phosphatase to the contractile apparatus to dephosphorylate myosin light chains. We used RNA interference to silence the expression of myosin phosphatase-Rho interacting protein in human vascular smooth muscle cells. Myosin phosphatase-Rho interacting protein silencing reduced the localization of the myosin binding subunit to stress fibers. This reduction in stress fiber myosin phosphatase-Rho interacting protein and myosin binding subunit increased basal and lysophosphatidic acid-stimulated myosin light chain phosphorylation. Neither cellular myosin phosphatase, myosin light chain kinase, nor RhoA activities were changed by myosin phosphatase-Rho interacting protein silencing. Furthermore, myosin phosphatase-Rho interacting protein silencing resulted in marked phenotypic changes in vascular smooth muscle cells, including increased numbers of stress fibers, increased cell area, and reduced stress fiber inhibition in response to a Rho-kinase inhibitor. These data support the importance of myosin phosphatase-Rho interacting protein-dependent targeting of myosin phosphatase to stress fibers for regulating myosin light chain phosphorylation state and morphology in human vascular smooth muscle cells.     

A shear stress responsive gene product PP1201 protects against Fas-mediated apoptosis by reducing Fas expression on the cell surface

Cells that form vascular system employ different mechanisms to offset deleterious consequences of exposure to cytokines and cells present in blood. Vascular homeostasis is sustained in part by genes, whose expression increases in response to hemodynamic forces in these cells. PP1201 (also known as RECS1) is one such gene whose expression level increases in response to laminar shear stress. Aged mice deficient in PP1201 are prone to develop cystic medial degeneration (CMD), a form of aortic aneurism manifested with loss of smooth muscle cells and accumulation of basophilic substances. Here we found that higher levels of PP1201 can protect against Fas ligand (FasL)-induced apoptosis. PP1201 interacted with the Fas receptor (CD95/Apo1) and colocalized with it in the Golgi compartment. Unlike its homolog lifeguard (LFG), PP1201 overexpression in several types of cells including primary human aortic smooth muscle cells (AoSMC) decreased the expression of Fas on the plasma membrane without changing the total Fas levels. Only high but not constitutive level of PP1201 controls Fas signaling. Our data suggest that PP1201 functions as an anti-apoptotic protein and its increased expression in vascular cells can contribute to homeostasis by reducing Fas trafficking to the cell membrane.     

Myosin phosphatase: Structure,regulation and function

Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PPlc; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a smaller subunit, M20, of unknown function. Most of the properties of MP are due to MYPT and include binding of PP1c and substrate. Other interactions are discussed. A recent discovery is the existence of an MYPT family and members include, MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. Characteristics of each are outlined. An important discovery was that the activity of MP could be regulated and both activation and inhibition were reported. Activation occurs in response to elevated cyclic nucleotide levels and various mechanisms are presented. Inhibition of MP is a major component of Ca2+-sensitization in smooth muscle and various molecular mechanisms are discussed. Two mechanisms are cited frequently: (1) Phosphorylation of an inhibitory site on MYPT1, Thr696 (human isoform) and resulting inhibition of PP1c activity. Several kinases can phosphorylate Thr696, including Rho-kinase that serves an important role in smooth muscle function; and (2) Inhibition of MP by the protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17). Examples where these mechanisms are implicated in smooth muscle function are presented. The critical role of RhoA/Rho-kinase signaling in various systems is discussed, in particular those vascular smooth muscle disorders involving hypercontractility.     

Ageing causes an aortic contractile dysfunction phenotype by targeting the expression of members of the extracellular signal‐regulated kinase pathway

The extracellular signal‐regulated kinase (ERK) pathway is a well‐known regulator of vascular smooth muscle cell proliferation, but it also serves as a regulator of caldesmon, which negatively regulates vascular contractility. This study examined whether aortic contractile function requires ERK activation and if this activation is regulated by ageing. Biomechanical experiments revealed that contractile responses to the alpha1‐adrenergic agonist phenylephrine are attenuated specifically in aged mice, which is associated with downregulation of ERK phosphorylation. ERK inhibition attenuates phenylephrine‐induced contractility, indicating that the contractile tone is at least partially ERK‐dependent. To explore the mechanisms of this age‐related downregulation of ERK phosphorylation, we transfected microRNAs, miR‐34a and miR‐137 we have previously shown to increase with ageing and demonstrated that in A7r5 cells, both miRs downregulate the expression of Src and paxillin, known regulators of ERK signalling, as well as ERK phosphorylation. Further studies in aortic tissues transfected with miRs show that miR‐34a but not miR‐137 has a negative effect on mRNA levels of Src and paxillin. Furthermore, ERK phosphorylation is decreased in aortic tissue treated with the Src inhibitor PP2. Increases in miR‐34a and miR‐137 with ageing downregulate the expression of Src and paxillin, leading to impaired ERK signalling and aortic contractile dysfunction.     

Focal adhesion kinase is abundant in developing blood vessels and elevation of its phosphotyrosine content in vascular smooth muscle cells is a rapid response to angiotensin II

Focal adhesion kinase (FAK) is a structurally unique nonreceptor protein-tyrosine kinase that localizes to focal adhesion plaques. Regulation of its activity has been implicated in diverse signaling pathways, including those mediated by extracellular matrix/integrin interactions, G-protein coupled receptors for mitogenic neuropeptides, and certain oncogene products. To gain evidence for specific processes in which FAK may be involved in vivo, a study was initiated to determine its expression pattern during mouse development. FAK expression was detected in early embryos and appeared to be distributed throughout all cell types at about the time of neurulation. Subsequent to neural tube closure, expression became particularly abundant in the developing vasculature. This included expression in the medial layer of arteries populated by smooth muscle cells. In vitro studies using cultured rat aortic vascular smooth muscle cells demonstrate that FAK phosphotyrosine content is dramatically elevated in response to plating cells onto the adhesive glycoprotein, fibronectin. Also, enhanced tyrosine phosphorylation of FAK is observed in these cells upon stimulation with the vasoconstrictor angiotensin II. Thus, in vascular smooth muscle cells, like fibroblasts, FAK appears to play a role in signaling mechanisms induced by extracellular matrix components as well as G-protein coupled receptor agonists. The combined results of this study suggest that signaling through FAK may play an important role in blood vessel morphogenesis and function. © 1994 Wiley-Liss, Inc.     

Periostin Links Mechanical Strain to Inflammation in Abdominal Aortic Aneurysm

Aims

Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammation, which contributes to the pathological remodeling of the extracellular matrix. Although mechanical stress has been suggested to promote inflammation in AAA, the molecular mechanism remains uncertain. Periostin is a matricellular protein known to respond to mechanical strain. The aim of this study was to elucidate the role of periostin in mechanotransduction in the pathogenesis of AAA.

Methods and Results

We found significant increases in periostin protein levels in the walls of human AAA specimens. Tissue localization of periostin was associated with inflammatory cell infiltration and destruction of elastic fibers. We examined whether mechanical strain could stimulate periostin expression in cultured rat vascular smooth muscle cells. Cells subjected to 20% uniaxial cyclic strains showed significant increases in periostin protein expression, focal adhesion kinase (FAK) activation, and secretions of monocyte chemoattractant protein-1 (MCP-1) and the active form of matrix metalloproteinase (MMP)-2. These changes were largely abolished by a periostin-neutralizing antibody and by the FAK inhibitor, PF573228. Interestingly, inhibition of either periostin or FAK caused suppression of the other, indicating a positive feedback loop. In human AAA tissues in ex vivo culture, MCP-1 secretion was dramatically suppressed by PF573228. Moreover, in vivo, periaortic application of recombinant periostin in mice led to FAK activation and MCP-1 upregulation in the aortic walls, which resulted in marked cellular infiltration.

Conclusion

Our findings indicated that periostin plays an important role in mechanotransduction that maintains inflammation via FAK activation in AAA.     

MicroRNA‐203 mimics age‐related aortic smooth muscle dysfunction of cytoskeletal pathways

Increased aortic stiffness is a biomarker for subsequent adverse cardiovascular events. We have previously reported that vascular smooth muscle Src‐dependent cytoskeletal remodelling, which contributes to aortic plasticity, is impaired with ageing. Here, we use a multi‐scale approach to determine the molecular mechanisms behind defective Src‐dependent signalling in an aged C57BL/6 male mouse model. Increased aortic stiffness, as measured in vivo by pulse wave velocity, was found to have a comparable time course to that in humans. Bioinformatic analyses predicted several miRs to regulate Src‐dependent cytoskeletal remodelling. qRT‐PCR was used to determine the relative levels of predicted miRs in aortas and, notably, the expression of miR‐203 increased almost twofold in aged aorta. Increased miR‐203 expression was associated with a decrease in both mRNA and protein expression of Src, caveolin‐1 and paxillin in aged aorta. Probing with phospho‐specific antibodies confirmed that overexpression of miR‐203 significantly attenuated Src and extracellular signal regulated kinase (ERK) signalling, which we have previously found to regulate vascular smooth muscle stiffness. In addition, transfection of miR‐203 into aortic tissue from young mice increased phenylephrine‐induced aortic stiffness ex vivo, mimicking the aged phenotype. Upstream of miR‐203, we found that DNA methyltransferases (DNMT) 1, 3a, and 3b are also significantly decreased in the aged mouse aorta and that DNMT inhibition significantly increases miR‐203 expression. Thus, the age‐induced increase in miR‐203 may be caused by epigenetic promoter hypomethylation in the aorta. These findings indicate that miR‐203 promotes a re‐programming of Src/ERK signalling pathways in vascular smooth muscle, impairing the regulation of stiffness in aged aorta.     

Increased arterial load alters aortic structural and functional properties during embryogenesis

As in the adult dorsal aorta, the embryonic dorsal aorta is an important determinant of cardiovascular function, and increased stiffness may have secondary effects on cardiac and microcirculatory development. We previously showed that acutely and chronically increased arterial load via vitelline artery ligation (VAL) increases systemic arterial stiffness. To test the hypothesis that local dorsal aortic stiffness also increases, we measured aortic pulse-wave velocity (PWV) and assessed the active and passive properties (stress and strain) of isolated aortic segments. PWV along the dorsal aorta increased acutely and chronically after VAL. Analysis of isolated aortic active properties suggests that load-exposed aortas experienced higher stress, but not strain, at similar intraluminal pressures. When smooth muscle tone was relaxed, strain decreased in VAL vessels, whereas stress became similar to control vessels. Immunohistochemical analysis revealed that although aortic smooth muscle alpha-actin content was similar between groups, more cell layers expressed smooth muscle alpha-actin, and myocyte cell shape was markedly rounder in VAL embryos. Additionally, aortic and perivascular collagen type I and III content significantly increased in load-exposed VAL vessels. Increased production of these proteins is consistent with the observed increase in aortic PWV and decreased strain in VAL passive aortic segments. Thus the embryonic dorsal aorta is sensitive to increased arterial load and adapts by altering its material properties via changes in collagen content.     

XIAP reverses various functional activities of FRNK in endothelial cells

In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.