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To study the roles of anisotropic cell morphology and directionality of mechanical force in apoptosis, the spreading of human umbilical vein endothelial cells (HUVECs) was constrained by growing on micropatterned (MP) strips of fibronectin (FN, 20 g/cm 2 ) with widths of 15, 30, and 60 m on silicone membrane. Cells on 30-and 60-m strips, like cells on a nonpatterned (NP) surface coated with FN, showed clear actin stress fibers with anchoring spots of phosphorylated focal adhesion kinase (p-FAK) and no significant apoptosis. On 15-m strips, cells had few stress fibers, no p-FAK, and significant apoptosis. After seeding for 12 h, the cells were subjected to pulsatile shear stress (12 ؎ 4 dyn/cm 2 ) parallel or perpendicular to MP strips, or kept under static condition. Parallel flow caused cell elongation with enhanced stress fibers and p-FAK, and a reduction in apoptosis, but perpendicular flow did not. The Rho inhibitory C3 exoenzyme abolished the effects of parallel flow. RhoV14, the constitutively active Rho, enhanced stress fibers and p-FAK, and prevented apoptosis of HUVECs on 15-m strips under static condition. RhoV14 also reduced cell apoptosis under both parallel and perpendicular flows. Our results indicate that cell apoptosis can be modulated by changes in ECM micropatterning, anisotropic cell morphology, and mechanical forces. These extracellular microenvironment factors affect cell survival through alterations in Rho GTPase activity, stress fiber organization, and FAK phosphorylation.actin stress fibers ͉ focal adhesion kinase ͉ micropatterning ͉ Rho GTPase ͉ shear stress T he regulation of survival and death of endothelial cells (ECs) is critical to vascular homeostasis. Perturbations of this balance contribute to vascular diseases (1). Apoptosis is a process for orderly disposal of unwanted cells and is necessary for homeostasis. It has been shown that modulation of the geometry of extracellular matrix (ECM) affects cell spreading and adhesion under static condition, thus modifying EC growth, differentiation, migration, and/or apoptosis (2). Microfabrication techniques have been used to investigate and control cell functions. Chen et al. (2) found that cell fate can be switched from proliferation to apoptosis by decreasing the size of the microfabricated ECM islands with a symmetric geometry.Vascular ECs at the blood-vascular interface are constantly exposed to hemodynamic forces. It has been shown that the shear stress due to flow in straight parts of the vascular tree upregulates the genes involved in anti-apoptosis, cell cycle arrest, and morphological remodeling, thus contributing to atheroprotective effects (for review, see ref.3). Such cell remodeling processes require both biochemical signaling and structural reorganization in response to the flow direction. There have been many reports on the role of ECM microenvironment in regulating cell morphogenesis and mechanotransduction in relation to vascular development and cardiovascular physiology (4), but the effects of changes in cell morpholog...

Sudden coronary artery occlusion is one of the leading causes of death. Several in vitro models have been used to study the relationship between hemodynamic forces and platelet function. However, very few in vivo studies exist that fully explore this relationship due to the lack of rheologic data for the platelet. For this purpose, micropipette aspiration techniques were used in the present study to determine the mechanical properties of platelets. The data were analyzed by two mathematical models: (1) an erythrocyte-type membrane model which yielded a platelet shear modulus of 0.03+/-0.01 dyn cm[-1] (mean+/-SD) and a viscous modulus of 0.12+/-0.04 dyn s cm[-1]. (2) An endothelial-type cell model which approximated the platelet Young's modulus to be 1.7+/-0.6 x 10(3) dyn cm(-2) with a viscous modulus of 1.0+/-0.5 x 10(4) dyn s cm(-2). The endothelial-type cell model more accurately describes the mechanics occurring at the micropipette tip and permits more appropriate assumptions to be made in quantifying the rheologic properties of a platelet. Results from this study can be integrated into numerical models of blood flow in stenosed coronary arteries to elucidate the impact of local hemodynamics on platelets and thrombus formation in coronary artery disease.

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Landeen LK, Dederko DA, Kondo CS, Hu BS, Aroonsakool N, Haga JH, Giles WR. Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 294: H736-H749, 2008. First published November 16, 2007 doi:10.1152/ajpheart.00316.2007.-Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K ϩ current (IK ACh ) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by ϳ25% (vs. untreated controls) in fieldstimulated myocytes. S1P 1 was shown to be involved by using the S1P 1-selective agonist SEW2871 on myocytes isolated from S1P3-null mice. However, in these myocytes, S1P 3 can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P 1 antagonist VPC23019. Since S1P1 activates Gi exclusively, whereas S1P 3 activates both Gi and Gq, these results strongly implicate the involvement of mainly G i. Additional experiments using the IK ACh blocker tertiapin demonstrated that IK ACh can contribute to the negative inotropy following S1P activation of S1P 1 (perhaps through Gi␤␥ subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by I K ACh ) can reduce L-type calcium current and thus can decrease the intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via G i to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca 2ϩ ]i; and 2) a second pathway that acts via Gi to activate IK ACh and reduce APD. This decrease in APD is expected to decrease Ca 2ϩ influx and reduce [Ca 2ϩ ]i and myocyte contractility.calcium; contraction; cell shortening; inhibitory G protein; acetylcholine-sensitive potassium; myocyte SPHINGOSINE-1-PHOSPHATE (S1P) is a biologically active, cell membrane-associated sphingolipid that binds with high affinity to five distinct G-coupled protein receptor isoforms (S1P 1-5 ). S1P 1 has been detected in abundance in neonatal rat cardiomyocytes (39). In these cells, exposure to S1P (500 nM) results in an initial negative inotropic effect (reduction of systolic calcium). However, this may be followed by calcium overload (increased diastolic calcium) and then a cessation of contractility. In isolated atrial myocytes, S1P has been shown to activate a weakly inwardly rectifying potassium (K ϩ ) current. This K ϩ conductance is very similar to the K ϩ current activated by ACh (I K ACh ). Activation of this current can shorte...

We investigated the effects of oscillatory flow in regulating the gene expressions of type I collagen (COL1, the main component of human bone tissues) and osteopontin (OPN, the key gene for calcium deposition) in human osteoblast-like (MG-63) cells, and the roles of mitogen-activated protein kinases (MAPKs) in this regulation. The cells were subjected to oscillatory flow (0.5 +/- 4 dyn/cm(2)) or kept under static condition for various time periods (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h). Oscillatory flow caused significant up-regulations of both COL1 and OPN gene expressions over the 16 h of study, and a transient activation of MAPKs was starting at 15 min and declining to basal level in 2 h. The flow-induction of COL1 was blocked by an ERK inhibitor (PD98059) and reduced by a JNK inhibitor (SP600125), whereas that of OPN was abolished by PD98059. Analysis of the cis-elements in the COL1 and OPN promoters suggests the involvement of transacting factors Elk-1 and AP-1 in the transcription regulation. The ERK inhibitor (PD98059) blocked Elk-1 phosphorylation, as well as COL1 and OPN gene expression. The JNK inhibitor (SP600125) abolished c-jun phosphorylation and COL1 expression. These results suggest that the flow-induction of OPN was mediated through the ERK-Elk1-OPN pathway, and that COL1 was regulated by both the ERK-Elk1-COL1 and JNK-c-JUN-COL1 pathway.