|Dr Barry S. Gow||Associate Professor (in-charge)||University||1973-94|
|Carolyn Murtagh||Research Assistant, 0.6||CRC||1993-94|
This Laboratory studied the physiology of arteries and vascular cells. The major focus has been on vascular hyperplasia, particularly the role of haemodynamic factors such as shear stress on cultured endothelial cells and cyclic stretching of vascular smooth muscle cells (VSM). This is the final report from this Laboratory.
The overall aim of the CRC-funded project is to be able ultimately to grow endothelial and vascular smooth muscle cells in co-culture while subjecting them to the normal haemodynamic stresses that arterial cells are subjected to in vivo. Virtually all cell culture research that has been done to elucidate the mechanisms of restenosis following balloon angioplasty has so far been done in an abnormal static cell culture environment. Because the dynamic cell culture module (DCCM) is difficult to manufacture and is primarily designed for both combined flow and stretch studies it was expedient to develop a stretch module that would allow cyclic stretch on smooth muscle cells to be evaluated independently of the endothelial cells. Two projects were undertaken in 1994: further development of the DCCM and its evaluation using bovine endothelial cells, and development of a cyclic stretch system (CSS) to evaluate the effects of cyclic stretch on cultured smooth muscle cells.
Further development and evaluation of dynamic cell culture module (DCCM)
The module has been refined to allow simultaneous stretching of the silicone rubber membrane which forms the floor of the flow pathway. The flow rig was completed to deliver shear stresses of 40 ± 15 dynes to cultured cells while maintaining a mean and steady pressure within the normal range. The basic rig was described in the 1993 Report. Adjustment of pulsatile flow and pressure is possible by setting stroke of a computer-controlled piston pump, and compliance within the rig. Progress was hampered by major technical difficulties, particularly those leading to premature cell death within the module but the Lab was ultimately successful in observing changes in orientation, area and shape index of ovine endothelial cells subject to both steady and pulsatile flow. Cells elongated and orientated parallel with the axis of flow within 12 h of applying the stress. Moreover, F-actin fibres were seen to form, also aligning with the direction of shear stress. Preliminary data suggested increased prostacyclin production by the shear stressed cells. This work is being continued at the Univ. of N.S.W.
Development of a dynamic stretch system
The system consists of a specially fabricated CO2 incubator in which six stretch modules are clamped. Each module has within it a slab of silicone rubber which was cyclically stretched at 1 Hz from without at an amplitude of 2.5 or 5% assisted by a servomotor. Six silicone rubber slabs within culture dishes served as static controls. The silicone rubber is first hydrophilized and cell attachment factor adsorbed on prior to seeding of the smooth muscle cells (SMC). Initially, the efficacy of attachment factors, fibronectin, laminin, vitronectin and collagens I and IV were evaluated, with the latter two being substantially inferior to the remainder. Seeding efficiency was similarly inferior on the collagens. The cyclic stretching produced early orientation of human SMC with alignment of their long axes approaching a direction normal to the line of stretch. The orientation of F-actin stress fibres also parallelled the axes of cells. This project is also being continued at UNSW.
The Laboratory was located in rooms 265-267 of the Anderson Stuart Building. Dr Gow's office was room 268. The Lab had a surgical operating and experimental facility for large animals. It contained an X-ray image intensifier, cardiac and arterial catheterization equipment, apparatus for measuring pressure, flow and arterial diameter, multichannel data aquisition equipment, equipment for data processing, analysis, plotting and stepper motor control using microcomputers, equipment for automated measurement of arterial smooth muscle mechanics in vitro and of microrheological properties of the arterial intima, and a special instrument to measure the effect of fluid shear on the arterial intima. There were two research microscopes, one inverted, with phase contrast. Most recently, apparatus had been built to measure the response of vascular cells to haemodynamic conditions in culture.
|Co-operative Research Centre||Cardiac Technology, Project 1.3||Gow BS||1993|
|(*committed to June 1994, with $35,400 requested for July 1994 to June 1995)|
Total for 1994: $15,000
|Practical classes (no.)||24||(8)||-||8||32|
|(*Lectures to Dent 2, Sc 2 and Sc Aux were given concurrently)|
Total formal contact teaching time = 89 h
In addition, time was spent in preparation of lectures, consultations by students, setting and marking exams and essays and course supervisor duties.