Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.
Insulin-like growth factor (IGF)-1 inhibits apoptosis, but its mechanism is unknown. Myocyte stretching activates p53 and p53-dependent genes, leading to the formation of angiotensin II (Ang II) and apoptosis. Therefore, this in vitro system was used to determine whether IGF-1 interfered with p53 function and the local renin-angiotensin system (RAS), decreasing stretch-induced cell death. A single dose of 200 ng/ml IGF-1 at the time of stretching decreased myocyte apoptosis 43% and 61% at 6 and 20 hours. Ang II concentration was reduced 52% at 20 hours. Additionally, p53 DNA binding to angiotensinogen (Aogen), AT1 receptor, and Bax was markedly down-regulated by IGF-1 via the induction of Mdm2 and the formation of Mdm2-p53 complexes. Concurrently, the quantity of p53, Aogen, renin, AT1 receptor, and Bax was reduced in stretched myocytes exposed to IGF-1. Conversely, Bcl-2 and the Bcl-2-to-Bax protein ratio increased. The effects of IGF-1 on cell death, Ang II synthesis, and Bax protein were the consequence of Mdm2-induced down-regulation of p53 function. In conclusion, the anti-apoptotic impact of IGF-1 on stretched myocytes was mediated by its capacity to depress p53 transcriptional activity, which limited Ang II formation and attenuated the susceptibility of myocytes to trigger their endogenous cell death pathway. (+info)
Fibrocartilage in tendons and ligaments--an adaptation to compressive load.
Where tendons and ligaments are subject to compression, they are frequently fibrocartilaginous. This occurs at 2 principal sites: where tendons (and sometimes ligaments) wrap around bony or fibrous pulleys, and in the region where they attach to bone, i.e. at their entheses. Wrap-around tendons are most characteristic of the limbs and are commonly wider at their point of bony contact so that the pressure is reduced. The most fibrocartilaginous tendons are heavily loaded and permanently bent around their pulleys. There is often pronounced interweaving of collagen fibres that prevents the tendons from splaying apart under compression. The fibrocartilage can be located within fascicles, or in endo- or epitenon (where it may protect blood vessels from compression or allow fascicles to slide). Fibrocartilage cells are commonly packed with intermediate filaments which could be involved in transducing mechanical load. The ECM often contains aggrecan which allows the tendon to imbibe water and withstand compression. Type II collagen may also be present, particularly in tendons that are heavily loaded. Fibrocartilage is a dynamic tissue that disappears when the tendons are rerouted surgically and can be maintained in vitro when discs of tendon are compressed. Finite element analyses provide a good correlation between its distribution and levels of compressive stress, but at some locations fibrocartilage is a sign of pathology. Enthesis fibrocartilage is most typical of tendons or ligaments that attach to the epiphyses of long bones where it may also be accompanied by sesamoid and periosteal fibrocartilages. It is characteristic of sites where the angle of attachment changes throughout the range of joint movement and it reduces wear and tear by dissipating stress concentration at the bony interface. There is a good correlation between the distribution of fibrocartilage within an enthesis and the levels of compressive stress. The complex interlocking between calcified fibrocartilage and bone contributes to the mechanical strength of the enthesis and cartilage-like molecules (e.g. aggrecan and type II collagen) in the ECM contribute to its ability to withstand compression. Pathological changes are common and are known as enthesopathies. (+info)
Receptor mechanisms underlying heterogenic reflexes among the triceps surae muscles of the cat.
The soleus (S), medial gastrocnemius (MG), and lateral gastrocnemius (LG) muscles of the cat are interlinked by rapid spinal reflex pathways. In the decerebrate state, these heterogenic reflexes are either excitatory and length dependent or inhibitory and force dependent. Mechanographic analysis was used to obtain additional evidence that the muscle spindle primary ending and the Golgi tendon organ provide the major contributions to these reflexes, respectively. The tendons of the triceps surae muscles were separated and connected to independent force transducers and servo-controlled torque motors in unanesthetized, decerebrate cats. The muscles were activated as a group using crossed-extension reflexes. Electrical stimulation of the caudal cutaneous sural nerve was used to provide a particularly strong activation of MG and decouple the forces of the triceps surae muscles. During either form of activation, the muscles were stretched either individually or in various combinations to determine the strength and characteristics of autogenic and heterogenic feedback. The corresponding force responses, including both active and passive components, were measured during the changing background tension. During activation of the entire group, the excitatory, heterogenic feedback linking the three muscles was found to be strongest onto LG and weakest onto MG, in agreement with previous results concerning the strengths of heteronymous Ia excitatory postsynaptic potentials among the triceps surae muscles. The inhibition, which is known to affect only the soleus muscle, was dependent on active contractile force and was detected essentially as rapidly as length dependent excitation. The inhibition outlasted the excitation and was blocked by intravenous strychnine. These results indicate that the excitatory and inhibitory effects are dominated by feedback from primary spindle receptors and Golgi tendon organs. The interactions between these two feedback pathways potentially can influence both the mechanical coupling between ankle and knee. (+info)
Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear.
Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress. (+info)
Cloning of a stretch-inhibitable nonselective cation channel.
A homologue of the capsaicin receptor-nonselective cation channel was cloned from the rat kidney to investigate a mechanosensitive channel. We found this channel to be inactivated by membrane stretch and have designated it stretch-inactivated channel (SIC). SIC encodes a 563-amino acid protein with putative six transmembrane segments. The cDNA was expressed in mammalian cells, and electophysiological studies were performed. SIC-induced large cation currents were found to be regulated by cell volume, with currents being stimulated by cell shrinkage and inhibited by cell swelling. Single channel analysis showed a conductance of 250 pS with cation permeability (PCl/PNa < 0.1), and the channel possessed some of the characteristics of a stretch-inactivated channel in that it was permeable to calcium, sensitive to membrane stretch, and blocked by Gd3+. Therefore, we cloned one of the mechanosensitive cation channels of mammals, which is considered to regulate Ca2+ influx in response to mechanical stress on the cell membrane. (+info)
Distinct structural attributes regulating von Willebrand factor A1 domain interaction with platelet glycoprotein Ibalpha under flow.
We have used recombinant von Willebrand factor (vWF) fragments to investigate the properties regulating A1 domain interaction with platelet glycoprotein (GP) Ibalpha. One fragment, rvWF508-704, represented the main portion of domain A1 (mature subunit residues 497-716) within the Cys509-Cys695 disulfide loop. The other, rvWF445-733, included the carboxyl-terminal region of domain D3, preceding A1, and corresponded to the proteolytic fragment originally identified as the GP Ibalpha-binding site (residues 449-728). Conformational changes were induced by reduction and alkylation of the Cys509-Cys695 bond and/or exposure to acidic pH. The cyclic rvWF445-733 fragment exhibited the function of native vWF A1 domain. When immobilized onto a surface, it tethered platelets at shear rates up to 6,300 s-1 mediating low velocity translocation but not stable attachment; in solution, it exhibited limited interaction with GP Ibalpha. In contrast, fragments with perturbed conformation could not tether platelets at high shear rates but promoted stable adhesion at lower shear and bound tightly to GP Ibalpha. Only in the presence of the exogenous modulator, botrocetin, did cyclic rvWF445-733 mediate irreversible adhesion. Thus, conformational transitions in the vWF A1 domain may influence differentially the efficiency of bond formation with GP Ibalpha and the stability of binding. (+info)
Calculation of a Gap restoration in the membrane skeleton of the red blood cell: possible role for myosin II in local repair.
Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network. (+info)
The forward rate of binding of surface-tethered reactants: effect of relative motion between two surfaces.
The reaction of molecules confined to two dimensions is of interest in cell adhesion, specifically for the reaction between cell surface receptors and substrate-bound ligand. We have developed a model to describe the overall rate of reaction of species that are bound to surfaces under relative motion, such that the Peclet number is order one or greater. The encounter rate between reactive species is calculated from solution of the two-dimensional convection-diffusion equation. The probability that each encounter will lead to binding depends on the intrinsic rate of reaction and the encounter duration. The encounter duration is obtained from the theory of first passage times. We find that the binding rate increases with relative velocity between the two surfaces, then reaches a plateau. This plateau indicates that the increase in the encounter rate is counterbalanced by the decrease in the encounter duration as the relative velocity increases. The binding rate is fully described by two dimensionless parameters, the Peclet number and the Damkohler number. We use this model to explain data from the cell adhesion literature by incorporating these rate laws into "adhesive dynamics" simulations to model the binding of a cell to a surface under flow. Leukocytes are known to display a "shear threshold effect" when binding selectin-coated surfaces under shear flow, defined as an increase in bind rate with shear; this effect, as calculated here, is due to an increase in collisions between receptor and ligand with increasing shear. The model can be used to explain other published data on the effect of wall shear rate on the binding of cells to surfaces, specifically the mild decrease in binding within a fixed area with increasing shear rate. (+info)