The long term goal of this grant application Is to understand the mechanism by which cells respond to mechanical signals which are a part of their immediate in vivo environment. We have developed an apparatus which permits us to apply precise levels of biaxial deformation to cells in culture and to assess the effects of this mechanical strain morphologically, biochemically and physiologically. We propose that mechanical strain modifies cell function and that the cell's ability to acclimate to its mechanical environment may compromise cell function and ultimately cell survival. Using this apparatus, we propose to: 1) determine If matrix protein synthesis and matrix gene expression are altered as a result of applied biaxial strain and 2), to determine a mechanism of mechanochemical transduction in endothelial cells In response to biaxial elongation. To accomplish aim 1, we will analyze matrix synthesis at the protein and RNA levels with particular emphasis on the fibronectin and the multiple forms which arise from splicing of the primary transcript. To accomplish the second aim, we will measure the Intracellular transients associated with mechanical stimulation and develop an analytical model for the calcium kinetics with emphasis on calcium accumulation as a result of injury. We will also determine if phosphorylation events are involved with the mechano-transduction process in endothelial cells. Changes in matrix protein synthesis will be assessed by radioisotope incorporation studies, polyacrylamide gel electrophoresis of isotopically labeled proteins, protease digestion of labeled proteins, Western blotting and ELISAs. Effects on matrix gene expression will be evaluated using specific matrix protein cDNA probes for collagens and fibronectin isoforms. These probes will be used in Northern hybridization and slot blot analysis of RNAs extracted from cell cultures which have been biaxially strained for different periods of time. In addition, RNAs from biaxially strained cultures will amplified and quantitated using polymerase chain reaction technology. Phosphorylation events which result from biaxial deformation of cells in culture will be characterized with a view toward understanding the mechanism of cellular response to applied mechanical forces. We will also attempt to measure the Intracellular ion transients associated with mechanical stimulation using specific fluorescent dyes which bind intracellular free Call stoichiometrically. The goal of these studies is to define the mechanotransduction system present in vascular cells and to define the molecular mechanism of cellular response to applied mechanical forces.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL034005-08
Application #
3346488
Study Section
Pathology A Study Section (PTHA)
Project Start
1985-04-01
Project End
1995-06-30
Budget Start
1993-08-01
Budget End
1994-06-30
Support Year
8
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Dentistry
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Chaqour, B; Howard, P S; Macarak, E J (1999) Identification of stretch-responsive genes in pulmonary artery smooth muscle cells by a two arbitrary primer-based mRNA differential display approach. Mol Cell Biochem 197:87-96
Chaqour, B; Howard, P S; Richards, C F et al. (1999) Mechanical stretch induces platelet-activating factor receptor gene expression through the NF-kappaB transcription factor. J Mol Cell Cardiol 31:1345-55
Cargill 2nd, R S; Thibault, L E (1996) Acute alterations in [Ca2+]i in NG108-15 cells subjected to high strain rate deformation and chemical hypoxia: an in vitro model for neural trauma. J Neurotrauma 13:395-407
Baskin, L S; Constantinescu, S; Duckett, J W et al. (1994) Type III collagen decreases in normal fetal bovine bladder development. J Urol 152:688-91
Baskin, L; Meaney, D; Landsman, A et al. (1994) Bovine bladder compliance increases with normal fetal development. J Urol 152:692-5;discussion 696-7
Winston, F K; Thibault, L E; Macarak, E J (1993) An analysis of the time-dependent changes in intracellular calcium concentration in endothelial cells in culture induced by mechanical stimulation. J Biomech Eng 115:160-8
Baskin, L S; Constantinescu, S C; Howard, P S et al. (1993) Biochemical characterization and quantitation of the collagenous components of urethral stricture tissue. J Urol 150:642-7
Baskin, L; Howard, P S; Macarak, E (1993) Effect of physical forces on bladder smooth muscle and urothelium. J Urol 150:601-7
Baskin, L S; Howard, P S; Duckett, J W et al. (1993) Bladder smooth muscle cells in culture: I. Identification and characterization. J Urol 149:190-7
Baskin, L; Howard, P S; Macarak, E (1993) Effect of mechanical forces on extracellular matrix synthesis by bovine urethral fibroblasts in vitro. J Urol 150:637-41

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