Track 15. Microcirculation 15.6. Microvascular Mechanotransduction $337

In 42 guinea pigs, PL pressure measurements were carried out under ke- tamine anesthesia with spontaneous respiration. The extracochlear pressure transducer was connected with the PL via a glass capillary in the scala tympani. Via this connection, test signals were applied, e.g. PL was aspirated or artificial PL was infused stationary or as a microbolus [1]. Substantial volume displacements of the cochlear PL were observed as a result of autonomously induced contractions of the tensor tympani muscle, artificially generated eardrum movements by air pressure pulses in the external auditory canal and influences of physiological functions of the body. The model is implemented as a mechano-electrical analogy. It is an RC network with concentrated parameters that also contains non-linear resistances. The number of elements varies with the viewed frequency range from mHz and into the lower audible range. The non-linearities are originated by the pressure- dependent and directionally dependent shape of the perilymphatic connecting canals. Artificial obliterations of individual connecting canals impair pressure homeostasis and increase the exchange time of the entire PL volume, which is relevant for the long-term development of progressive hearing disorders in humans.

References [1] Neumann H J, Haberland E J: Biophysikalische Untersuchungen der Perilymph-

bewegung und Sauerstoffdiffusion im cochle~ren Perilymphraum unter patho- physiologischen Bedingungen. Eine tierexperimentelle Studie. Tectum Verlag, Marburg 1998.

4604 Tu, 17:00-17:15 (P25) Compression and hypoxia in an engineered skeletal muscle model D. Gawlitta, C.W.J. Oomens, F.ET. Baaijens, C.V.C. Bouten. Technical University Eindhoven, Department of Biomedical Engineering, Eindhoven, The Netherlands

The most adhered hypothesis for the development of (deep) pressure ulcers is that local ischemia initiates tissue breakdown. Another, more recent hypothesis is that cellular deformation due to tissue compression causes tissue degen- eration. The aim of the present study was to monitor in real time the relative contributions of ischemia and compression in the development of cell damage in engineered muscle tissue. Engineered skeletal muscle tissue was molded from C2C12 mouse myoblasts in a collagen I gel. After gelling in growth medium, the medium was changed to differentiation medium to initiate maturation of the cells into muscle fibers. For imposing compression and ischemia on the engineered muscle a compres- sion device was assembled consisting of an indenter for tissue compression in an incubator chamber. To model ischemia, the oxygen level in the incubation chamber was adjustable between hypoxic and normoxic conditions. The tissue was located on a glass window for monitoring cell death with a confocal microscope. A protocol was developed for real-time monitoring of cell death development in the muscle construct. A green-fluorescent probe was applied for marking apoptotic nuclei, whereas necrotic nuclei were indicated by red fluorescence. Engineered muscle tissue was subjected to 0, 20 or 40% compression under normal oxygen tension or hypoxic conditions (6 groups). Development of cell death was monitored every hour on several locations for 22 hours in each sample. Compression resulted in more necrotic cell death compared to the control or hypoxic experiments. Hypoxia induced a strong green fluorescent signal, which was not observed in the other experiments. Staining of cytoplasm besides staining of the nucleus contributed to this signal. It was concluded that hypoxia per se did not lead to cell death in time, whereas compression resulted in cell death immediately after initiation. However, when compression and hypoxia were combined, the mere effect of compression on cell death appeared to be decreased by the additional hypoxic conditions.

4781 Tu, 17:15-17:30 (P25) Microstructural analysis of deformation-induced hypoxic damage in skeletal muscle K.K. Ceelen, C.W.J. Oomens, F.P.T. Baaijens. Eindheven University of Technology, Department of Biomedical Engineering, The Netherlands

Deep pressure ulcers are caused by sustained mechanical loading and involve skeletal muscle tissue injury. The prevalence is high, partly due to a lack of understanding of the exact underlying mechanisms. Our hypothesis is that the aetiology is dominated by damage due to cellular deformation [1,2,3] and deformation-induced ischaemia. The experimental observation that mechanical compression induced a pattern of interspersed healthy and dead muscle cells [3] strongly suggests taking into account the muscle microstructure when studying damage development. A computational model on the microstructural level for deformation-induced hypoxic damage in skeletal muscle tissue was developed. Dead cells stop consuming oxygen and their stiffness is assumed to decrease due to loss of structure. The questions addressed are if these two consequences of cell death

influence the development of cell injury in the remaining cells. The results show that weakening of dead cells indeed affects the damage accumulation in other cells. Further, the fact that cells stop consuming oxygen after they have died is beneficial for tissue viability. Although the shape of the predicted damage development in time at different compression levels was quite similar to experimentally obtained curves, the time axis was shifted. This is because hypoxic damage will not occur within 4 hours, and therefore, the model is currently being extended to include damage due to cellular deformation, which is hypothesized to be the result of stretch-induced increased membrane permeability for Ca 2+.

References [1] Bouten et al. Ann Biomed Eng. 2001; 29: 153-163. [2] Breuls et al. Ann Biomed Eng. 2003; 31: 1357-1364. [3] Stekelenburg et al. J App Physiol. 2005; submitted.

15.6. Microvascular Mechanotransduction

6236 Th, 08:15-08:30 (P39) Unraveling hypertensive transduction cascades in the vasculature S. Lehoux. Inserm U689, Cardiovascular Research Center Inserm Lariboisiere, Paris, France

Blood vessels are permanently subjected to mechanical forces in the form of stretch, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow, and shear stress. Alterations in blood pressure or flow invariably produce transformations in the vessel wall that will aim to accommodate the new conditions and to ultimately restore basal levels of tensile stress and shear stress. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by mechanical stress, initiating complex signal transduction cascades leading to functional changes within the cell. One such cascade is that of the transcription factor NF-kB, now known to be activated in vessels exposed to hypertensive conditions. We have recently uncovered the pathway whereby stretch activates NF-kB in isolated arteries: strain induces the release of reactive oxygen species (ROS), which in turn cleave and activate the metalloproteinase TACE, liberating an epidermal growth factor receptor (EGFR) ligand that stimulates the EGFR. Using vessels from EGFR ligand-deficient mice, we found that transforming growth factor-a (TGF-a), but not epiregulin or HB-EGF, is the key mediator of this process. Activation of the EGFR then triggers the NF-kB pathway. The importance of this signalling cascade is substantiated in vivo. In wild-type mice rendered hypertensive by angiotensin II (Angll) administration, activation of NF-kB is associated with vascular remodelling characterized by gelatinase activation, cell apoptosis and proliferation, and vascular wall thickening. However, Angll- induced hypertension fails to activate NF-kB in TGF-a-deficient mice, and vascular remodelling is much reduced in these animals. Our data therefore identify a new pathway whereby hypertensive conditions activate NF-kB, and identify TGF-a as a potential target to modulate mechanosensitive vascular remodelling.

6977 Th, 08:30-08:45 (P39) Shear stress dependence of leukocyte rolling interactions on nanopatterned substrates of P-selectin that mimic activated endothelial surfaces X. Lin, A.S.W. Ham, M.L. Reed, M.B. Lawrence, B.P. Helmke. University ef Virginia, Charlottesville, Virginia, USA

Nano-fabricated surfaces patterned with P-selectin represent a novel set of tools that enable detailed investigation of leukocyte rolling mechanisms on substrates that mimic the surface of activated endothelium. In this study, leukocyte rolling behavior on nanopatterned substrates (dot size 30-45 nm and dot pitch 55-85nm) or on a uniform surface concentration of P-selectin was measured with wall shear stress values of 0.5-10dyn/cm 2. For low shear stress values comparable to those in postcapillary venules, the spatial distribution of P-selectin did not affect rolling velocity, pause time between displacement steps, or variance of velocity. However, when the shear stress was increased greater than 2dyne/cm 2, the rolling velocity was significantly increased as a function of nanodot size and spacing relative to that on a uniform spatial distribution of P-selectin. The influence of the nanodot size and spacing on pause time, which was shown by previous studies to be related to the number of stressed bonds at the peeling edge, was also significant at higher shear stress values. Variance of rolling velocity was significant for shear stress greater than 6 dyne/cm 2, indicating that the size and spacing of patterns also affected the randomness of rolling behavior at higher shear stress values. These results suggest that the spatial distribution of P-selectin on the surface of activated endothelium is not critical to leukocyte rolling behavior in postcapillary venules but may serve to modulate leukocyte-endothelial interactions in larger vessels with high levels of hemodynamic shear stress. Supported by NSF MRSEC Grant DMR-0080016.