In-vitro Experiments

 

Abstract

Cerebral aneurysms tend to develop at bifurcations where the blood vessel wall experiences complex hemodynamics characterized by impinging flow, high wall shear stress (WSS) and high wall shear gradient (WSSG). Endothelial cells (ECs) directly contact blood flow and act as sensors of WSS. To better understand aneurysm initiation, we wish to know how ECs respond to such complex hemodynamics. Therefore, a T-shaped chamber was used to study effects of impinging flow on cultured bovine aortic ECs. Cultures exposed to impinging flow for 24-72 hrs exhibited a decrease in cell density in the stagnation area and a sharp increase in density slightly downstream in the region of flow acceleration. To determine whether this effect was due to local hemodynamic stimulation of cell proliferation or to hemodynamically directed cell migration, proliferation was inhibited with mitomycin-C. In the absence of proliferation a peak in EC density still occurred. Furthermore, between 24-72 hrs, the density peak gradually moved away from the impingement, but remained within the region of maximal WSS; i.e., cells eventually accumulated at the downstream end of the region of flow acceleration. Thus, impinging flow induced localized EC migration. The depletion of cells from the stagnation area and accumulation downstream of flow acceleration suggest that high WSSG is the critical hemodynamic factor driving this migration. In vivo, high motility and turnover of ECs adjacent to flow impingements may make bifurcation apices particularly susceptible to disruption and subsequent aneurysm formation. In order to expose endothelial cells to impinging flow, a confluent monolayer of bovine aortic ECs was subjected to flow impingement in an in vitro flow loop consisting of a reservoir, a peristaltic pump, a dampener, and an impingement flow chamber. The chamber was designed to direct flow through a T-junction resulting in a 2-D jet which impinged at the center of a cover slip of ECs and then split in opposite directions along the cover slip.

 

    Background

● Intracranial aneurysms commonly form at bifurcations, regions that are exposed to high Wall Shear Stress (WSS >40dynes/cm2) and high Wall Shear Stress Gradients (WSSG >300dynes/cm3) [1]

● ECs are the mechanotransducers of WSS and WSSG [2]

● Dysfunction of ECs correlates with aneurysm initiation

● Exposure to flow results in the migration and redistribution of ECs, critical events in wound healing, angiogenesis and vessel wall remodeling

In this study we examined the effect of bifurcation hemodynamics on the    motile behavior of endothelial cells.

 
      Method

 

Impinging flow culture chamber

In order to expose endothelial cells to impinging flow, a confluent monolayer of bovine aortic ECs was subjected to flow impingement in an in vitro flow loop consisting of a reservoir, a peristaltic pump, a dampener, and an impingement flow chamber. The chamber was designed to direct flow through a T-junction resulting in a 2-D jet which impinged at the center of a cover slip of ECs and then split in opposite directions along the cover slip.

 
 

Exposing cells to Hemodynamics Envirenment

The hemodynamics of this bifurcation are described by three distinct regions. Region I, a stagnation region characterized by a stagnation point, and low to normal WSS, and high WSSG. Region II, an accelerating flow region characterized by high positive WSSG and high WSS, and Region III, a recovery region characterized by negative to zero WSSG and high WSS.

 

Cells are depleted from impingement zone and accumulate in region of accelerating flow

After exposing cells to impinging flow for 48hrs the cell density was measured across the monolayer. Cell density decreased at the impingement (trough) but increased downstream with a discrete peak in the acceleration zone. Further downstream density returned to a relatively constant value that was lower than that at the peak but higher than that of Region I.

 

Flow-induced changes in cell density are due to cell movement, not local proliferation

To determine if the cell density rearrangement shown above is independent of cell proliferation, cells were treated with an irreversible inhibitor of proliferation, mitomycin-C (MMC). In the absence of proliferation, cells are still depleted from the impingement and accumulate in the acceleration zone. Furthermore, the cell density peak occurred before the peak in WSS suggesting WSS alone is not responsible for the migration.

 
   WSSG drives the redistribution of cells from the impingement zone to the acceleration region

The location of accumulating cells was tracked over different flow exposure times. Even after 72  hours of flow exposure, the peak in cell density still remains in the acceleration zone (region II). Because cells “pile up” here, movement out of the impingement region must be faster than the downstream movement in the recover region (III). Since cells at the beginning of region III experience higher WSS than the cells in region I and II, it suggests that a gradient in WSS causes a much greater stimulation of downstream movement than WSS alone.  

   

 Significance

 If the impingement environment elicits similar behavior in vivo, it would be expected that ECs at the apex would increase proliferation in order to replace endothelium lost to the continuous downstream migration.  High motility and turnover of ECs adjacent to flow impingements may make bifurcation apices particularly susceptible to disruption and subsequent aneurysm formation.

 

 References

1. Meng H, D. Swartz, Z. Wang, Y. Hoi, J. Kolega, E. Metaxa, M.P. Szymanski, J. Yamamoto, E. Sauvageau, E. I. Levy. A Model System for Mapping Vascular Responses to Complex Hemodynamics at Arterial Bifurcations In Vivo. Neurosurgery. 2006;59(5).

2. Chien S. Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. Am J Physiol Heart Circ Physiol.  2007; 292:1209-1224.

 

Acknowlegements

This work was funded by NIH (Grant NS047242) and the Cummings Foundation.

 

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