Expression of cathepsin K is regulated by shear stress in cultured endothelial cells and is increased in endothelium in human atherosclerosis

2007 ◽  
Vol 292 (3) ◽  
pp. H1479-H1486 ◽  
Author(s):  
Manu O. Platt ◽  
Randall F. Ankeny ◽  
Guo-Ping Shi ◽  
Daiana Weiss ◽  
J. D. Vega ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Molecular Mechanisms ◽  
Cathepsin K ◽  
Immunohistochemical Analysis ◽  
Cysteine Proteases ◽  
Laminar Shear Stress ◽  
Gelatinase Activity ◽  
Aortic Endothelial Cells ◽  
Expression And Activity

Cathepsins, the lysosomal cysteine proteases, are involved in vascular remodeling and atherosclerosis. Genetic knockout of cathepsins S and K in mice has shown to reduce atherosclerosis, although the molecular mechanisms remain unclear. Because atherosclerosis preferentially occurs in arteries exposed to disturbed flow conditions, we hypothesized that shear stress would regulate cathepsin K expression and activity in endothelial cells. Mouse aortic endothelial cells (MAEC) exposed to proatherogenic oscillatory shear (OS, ± 5 dyn/cm2 for 1 day) showed significantly higher cathepsin K expression and activity than that of atheroprotective, unidirectional laminar shear stress (LS, 15 dyn/cm2 for 1 day). Western blot and active-site labeling studies showed an active, mature form of cathepsin K in the conditioned medium of MAEC exposed to OS but not in that of LS. Functionally, MAEC exposed to OS significantly increased elastase and gelatinase activity above that of LS. The OS-dependent elastase and gelatinase activities were significantly reduced by knocking down cathepsin K with small-interfering (si) RNA, but not by a nonsilencing siRNA control, suggesting that cathepsin K is a shear-sensitive protease. In addition, immunohistochemical analysis of atherosclerotic human coronary arteries showed a positive correlation between the cathepsin K expression levels in endothelium and elastic lamina integrity. These findings suggest that cathepsin K is a mechanosensitive, extracellular matrix protease that, in turn, may be involved in arterial wall remodeling and atherosclerosis.

Abstract 927: The Role Of Peroxiredoxins As Mechanosensitive Antioxidants In Endothelial Cells

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Amy Mowbray ◽  
Sang Won Kang ◽  
Sue Goo Rhee ◽  
Hanjoong Jo
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Subcellular Localization ◽  
Molecular Mechanisms ◽  
Immunofluorescent Staining ◽  
Dependent Manner ◽  
Laminar Shear Stress ◽  
Arterial Tree ◽  
Aortic Endothelial Cells ◽  
Stress Dependent

Atherosclerosis is an inflammatory disease occurring primarily in curved or branching regions of the arterial tree where disturbed flow patterns, such as oscillation, exist. We have previously shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, while laminar shear stress (LS) reduces ROS compared to static controls. OS stimulation of ROS has been shown to occur in an NADPH oxidase-dependent manner. However, the mechanism by which LS removes ROS remains unclear. Peroxiredoxins (PRX) are a family of antioxidant proteins that have been linked to the prevention of oxidative stress and inflammation, but their role in atherosclerosis is unknown. Here, we hypothesize that shear stress regulates ROS levels in endothelial cells by controlling antioxidant peroxiredoxins. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to static, laminar, and oscillatory fluid flow conditions via cone-and-plate viscometer. Western blot analysis and immunofluorescent staining were used to evaluate the expression and subcellular localization of six known mammalian peroxiredoxins (PRX I-VI). Immunoblots indicated that BAEC express all six isoforms of peroxiredoxin proteins and that LS upregulated PRX I levels significantly compared to static controls and OS. Immunofluorescence also showed a distinct subcellular localization of each PRX: PRX I, II, IV, V and VI in the cytoplasm, PRX I, IV and V in the Golgi, PRX III in the mitochondria, and PRX I in the nucleus. These results indicate that peroxiredoxins are mechanosensitive antioxidants, removing ROS in a subcellular-specific manner. Based on these data, we suggest that peroxiredoxin antioxidants are likely involved in the molecular mechanisms that control shear stress-dependent atherosclerotic plaque development.

Sustained JNK activation induces endothelial apoptosis: studies with colchicine and shear stress

1999 ◽  
Vol 277 (4) ◽  
pp. H1593-H1599 ◽  
Author(s):  
Ying-Li Hu ◽  
Song Li ◽  
John Y.-J. Shyy ◽  
Shu Chien
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Colchicine Treatment ◽  
Laminar Shear Stress ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Jnk Activation ◽  
Laminar Shear ◽  
Sustained Activation ◽  
Aortic Endothelial

The disruption of microtubules by treating bovine aortic endothelial cells with 10−7–10−5M colchicine caused apoptosis, as evidenced by DNA laddering and TdT-mediated dUTP nick end labeling fluorescence staining. Colchicine treatment also induced a sustained activation of c-Jun NH2-terminal kinase (JNK) that lasted for ≥12 h. The blockade of JNK activity by using the negative interfering mutant JNK(K-R) markedly decreased the apoptosis induced by colchicine. Exposure of bovine aortic endothelial cells to laminar shear stress (12 dyn/cm2) caused a transient (<2 h) activation of JNK, and there was no induction of apoptosis. The sustained activation of JNK may play a significant role in the apoptosis induced by colchicine.

scholarly journals Laminar Shear Stress Enhanced Glycosaminoglycan Synthesis in Cultured Porcine Aortic Endothelial Cells

1996 ◽  
Vol 24 (4-5) ◽  
pp. 197-203
Author(s):  
Tomoyuki ARISAKA ◽  
Masako MITSUMATA ◽  
Masahiko KAWASUMI ◽  
Toshio TOHJIMA ◽  
Shunichi HIROSE ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Laminar Shear Stress ◽  
Glycosaminoglycan Synthesis ◽  
Aortic Endothelial Cells ◽  
Laminar Shear ◽  
Porcine Aortic Endothelial Cells ◽  
Aortic Endothelial

Biomechanical and biochemical regulation of cathepsin K expression in endothelial cells converge at AP-1 and NF-κB

2016 ◽  
Vol 397 (5) ◽  
pp. 459-468 ◽  
Author(s):  
Philip M. Keegan ◽  
Suhaas Anbazhakan ◽  
Baolin Kang ◽  
Betty S. Pace ◽  
Manu O. Platt
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Shear Stress ◽  
Cathepsin K ◽  
Mrna Levels ◽  
Arterial Remodeling ◽  
Aortic Endothelial Cells ◽  
Protein Levels ◽  
Biomechanical Stimuli ◽  
Factor Α

Abstract Cathepsins K and V are powerful elastases elevated in endothelial cells by tumor necrosis factor-α (TNFα) stimulation and disturbed blood flow both of which contribute to inflammation-mediated arterial remodeling. However, mechanisms behind endothelial cell integration of biochemical and biomechanical cues to regulate cathepsin production are not known. To distinguish these mechanisms, human aortic endothelial cells (HAECs) were stimulated with TNFα and exposed to pro-remodeling or vasoprotective shear stress profiles. TNFα upregulated cathepsin K via JNK/c-jun activation, but vasoprotective shear stress inhibited TNFα-stimulated cathepsin K expression. JNK/c-jun were still phosphorylated, but cathepsin K mRNA levels were significantly reduced to almost null indicating separate biomechanical regulation of cathepsin K by shear stress separate from biochemical stimulation. Treatment with Bay 11-7082, an inhibitor of IκBα phosphorylation, was sufficient to block induction of cathepsin K by both pro-remodeling shear stress and TNFα, implicating NF-κB as the biomechanical regulator, and its protein levels were reduced in HAECs by vasoprotective shear stress. In conclusion, NF-κB and AP-1 activation were necessary to activate cathepsin K expression in endothelial cells, highlighting integration of biochemical and biomechanical stimuli to control cathepsins K and V, powerful elastases implicated for arterial remodeling due to chronic inflammation and disturbed blood flow.

Endothelial albumin permeability is shear dependent, time dependent, and reversible

1991 ◽  
Vol 260 (6) ◽  
pp. H1992-H1996 ◽  
Author(s):  
H. Jo ◽  
R. O. Dull ◽  
T. M. Hollis ◽  
J. M. Tarbell
Keyword(s):  
Shear Stress ◽  
Endothelial Cell ◽  
Fluorescein Isothiocyanate ◽  
Vascular Wall ◽  
Laminar Shear Stress ◽  
Aortic Endothelial Cells ◽  
Transendothelial Permeability ◽  
Steady Laminar Shear Stress ◽  
Laminar Shear

Altered permeability of vascular endothelium to macromolecules may play a role in vascular disease as well as vascular homeostasis. Because the shear stress of flowing blood on the vascular wall is known to influence many endothelial cell properties, an in vitro system to measure transendothelial permeability (Pe) to fluorescein isothiocyanate conjugated bovine serum albumin under defined physiological levels of steady laminar shear stress was developed. Bovine aortic endothelial cells grown on polycarbonate filters pretreated with gelatin and fibronectin constituted the model system. Onset of 1 dyn/cm2 shear stress resulted in a Pe rise from 5.1 +/- 1.3 x 10(-6) cm/s to 21.9 +/- 4.6 X 10(-6) cm/s at 60 min (n = 6); while 10 dyn/cm2 shear stress increased Pe from 4.8 +/- 1.5 X 10(-6) cm/s to 50.2 +/- 6.8 X 10(-6) cm/s at 30 min and 49.6 +/- 8.9 X 10(-6) cm/s at 60 (n = 9). Pe returned to preshear values within 120 and 60 min after removal of 1 and 10 dyn/cm2 shear stress, respectively. The data show that endothelial cell Pe in vitro is acutely sensitive to shear stress.

scholarly journals Primary cilia of human endothelial cells disassemble under laminar shear stress

2004 ◽  
Vol 164 (6) ◽  
pp. 811-817 ◽  
Author(s):  
Carlo Iomini ◽  
Karla Tejada ◽  
Wenjun Mo ◽  
Heikki Vaananen ◽  
Gianni Piperno
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Primary Cilia ◽  
Umbilical Vein ◽  
Intraflagellar Transport ◽  
Mechanical Stimuli ◽  
Laminar Shear Stress ◽  
Human Endothelial Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Laminar Shear

We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-α-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.

Matrix metalloproteinase-9 : surrogate marker and therapeutic target against neurovascular impairment after cerebral ischemia

2015 ◽  
Author(s):  
◽  
Shanyan Chen
Keyword(s):  
Endothelial Cells ◽  
Ischemic Stroke ◽  
Cerebral Ischemia ◽  
Molecular Mechanisms ◽  
Combined Treatment ◽  
Cell Penetrating Peptides ◽  
Therapeutic Window ◽  
Gelatinase Activity

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Stroke ranks fourth among all causes of death, and acute ischemic stroke is the most common form. The neurovascular unit (NVU) describes a basic functional structure in the brain and is primarily composed of endothelial cells, pericytes, astrocytes, microglia and neurons. The dynamic structure of the NVU is highly regulated due to interactions between different cells and extracellular matrix (ECM) components. Proteolysis of the ECM by matrix metalloproteinases (MMPs), especially MMP-9, plays an important role in the pathophysiology of cerebral ischemia and administration of tissue plasminogen activator (tPA). The activation of gelatinases (MMP-2/9) is considered a key mechanism involved in the impairment of NVU. The overall goal of this research project is to examine the role of MMP-9 in the neurovascular impairment after ischemic stroke in mice. In this project, we implemented a new strategy using gelatinase-activatable cell-penetrating peptides (ACPPs) tagged with fluorescence and/or gadolinium-based contrast agents to investigate proteolysis of gelatinases as surrogate markers of neurovascular integrity. We presented evidence that the combination of a sensitive fluorescent chromatophore and MRI contrast enhancement agent can be used to monitor gelatinase activity and its distribution in cultured neurons as well as in mice after focal cerebral ischemia. Detection of the activity of gelatinases in vivo using ACPPs could provide insights into the underlying mechanism for gelatinase proteolysis that mediate ischemia-related neurovascular impairment. We also applied a two-dimensional (2D) gelatin zymography technique that combines isoelectric focusing (IEF) with zymographic electrophoresis. We demonstrated that the 2D zymography approach can improve separation of different isoforms of gelatinases in both in vitro and in vivo conditions. 2D zymography is an effective method to separate posttranslational modification isoforms of gelatinases and to identify modifications that regulate their enzymatic activity in acute brain injuries. In work that follows, we used a fibrin-rich blood clot to occlude the middle cerebral artery (MCA) in mice as a model to represent the critical thromboembolic features of ischemic stroke in humans. In this study, we evaluated effects of SB-3CT, a mechanism-based inhibitor selective for gelatinases. We demonstrated MMP-9 activation and neurovasculature impairment in this stroke model, and showed the ability of SB-3CT to inhibit MMP-9 activity in vivo, which in turn resulted in maintenance of laminin, antagonism of pericyte contraction and loss, preservation of laminin-positive pericytes and endothelial cells, and thus rescuing neurons from apoptosis and preventing intracerebral hemorrhage. We further demonstrated that SB-3CT/tPA combined treatment could attenuate MMP-9 -- mediated degradation of endothelial laminin, impairment of endothelial cells, and decrease of caveolae -- mediated transcytosis. Early inhibition of MMP-9 proteolysis by SB-3CT decreased brain damage, reduced BBB disruption, and prevented hemorrhagic transformation after delayed tPA treatment. Therefore usage of SB-3CT will be helpful in accessing combination therapy with tPA in ischemic stroke. Results from these studies indicate the important role of MMP-9 in cerebral ischemia and thus the need for further studies to explore the molecular mechanisms underlying its activation and regulation. Results further demonstrated that the combined use of MMP-9 inhibitor with tPA may extend tPA therapeutic window for mitigating stroke damage.

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