Natacha DePaola, Ph.D.

  Associate Professor, Biomedical Engineering

Research Area

Atherosclerosis Research

In humans, the localization of atherosclerotic lesions coincides with the presence of altered hemodynamics ("disturbed blood flows") where endothelial cells exhibit an altered phenotype. The cell mechanical environment at these atherogenesis-prone regions is a complex one in which the shear stress force on the endothelial surface changes its magnitude and direction in space and time at the macroscopic (monolayer level) and microscopic (individual cell) level.

Experimental models, in which endothelial cell morphology, function, and gene regulation are studied in a well-controlled fluid mechanical environment, are essential to the investigation of the cellular and molecular mechanisms by which complex hemodymanics may alter endothelial cell function leading to lesion development. However, the effect of fluid forces in vascular endothelial biology has been traditionally investigated in vitro using flow models in which endothelial monolayers are exposed to unidirectional flows of uniform shear stress magnitudes characteristic of normal segments of the vasculature. I developed the first in vitro model to study the effect of complex flow characteristics associated with arterial disease in endothelial cell function. These in vitro studies recreated the spatial and temporal variations of fluid shear stresses experienced in vivo by the endothelium in areas prone to atherosclerosis (i.e. carotid sinus). The cell culture environment was engineered so that the principal hemodynamic characteristics of the circulation at arterial bifurcations (secondary flows, flow separation, reattachment and recirculation) were reproduced in a well-controlled fashion. Therefore, advancing the state of the art in engineering the cell environment for cell culture studies.

Using this in vitro model we demonstrated for the first time that shear stress gradients (rather than shear stress magnitude) is the key element of disturbed flow fields responsible for local manifestations of "endothelial dysfunction" in vitro that are consistent with cellular alterations seen in vivo in areas of altered hemodynamics. Specifically, localized increased cell DNA synthesis and evidence of flow-induced cell migration and cell loss were reported for the first time (DePaola, et al Arteriosclerosis and Thrombosis 12, 1992).

We have further investigated the role of steady and unsteady disturbed flows in lesion development by evaluating various spatial and temporal alterations in endothelial cell function associated with early atherosclerosis. Results have demonstrated that cell proliferation, cell migration, cell density, monolayer permeability to macromolecules, endothelial-monocyte adhesion, monolayer remodeling, and gap junctional intercellular communication are dynamically regulated by macroscopic shear stress gradients in vitro. The current data strongly support the existence of a mechanistic link between gap junction gene expression and cell proliferation and show evidence of cell signaling processes initiated by flow. These studies (currently ongoing) are providing fundamental information expected to contribute to the identification of physical mechanisms involved in cell pathobiology and to the understanding of the role of hemodynamics in early arterial disease.

My research work on endothelial intercellular communication has been conducted in close collaboration with Dr. Polacek and Dr. Davies from the Institute for Medicine and Engineering at Penn. We have reported the first in vitro study on gap junctional regulation by fluid forces and demonstrated that spatial gradients in fluid shear stress (associated with disturbed flow regions) induce local changes in endothelial gap junction-mediated intercellular communication. The results demonstrate that gene expression, protein organization, and function of the gap junctional protein connexin 43 is regionally mapped to hemodynamic forces.

Differences between hemodynamically defined regions of the endothelial layer demonstrated compartmentalization of cell-cell communication in vitro. This finding is highly relevant since it could aid the understanding of the focal nature of atherosclerotic lesions. The altered communication rate observed in areas of disturbed flow in vitro may constitute localized areas of altered cell function.

Motivated by the above findings we are continuing our collaborative work with the IME under the hypothesis that gradients in fluid shear stress induce regional changes in endothelial gap junctional intercellular communication (GJIC) which in turn, alter cell phenotype, contributing to "functional compartmentalization" (adjacent regions with altered cell function) of the endothelial monolayer in vivo and in vitro. This project, funded by NIH, will be one of the main research activities in my laboratory for the next 5 years. The long-term objective of this research is to investigate the role of hemodynamic forces in endothelial cell communication and to identify the cellular and molecular mechanisms by which flow regulates endothelial GJIC, contributing to localized vessel wall remodeling and the development of atherosclerotic lesions.

More recently in collaboration with the IME at Penn (Dr. D. Polacek and Dr. P.F. Davies) we are investigating the hypothesis that within any defined region of the vessel wall there is significant heterogeneity of endothelial signaling and gene expression regulated by differential shear stress from cell to cell. This study involves the analysis of gene expression in single cells, and group of cells, removed from precise hemodynamic locations in vitro and in vivo. The expression of known shear stress-responsive genes is being evaluated and new hemodynamic genes are expected to be identified. Our in vitro flow studies consider the complex spatial and temporal flow characteristics found in regions prone to atherosclerosis (flow disturbance). Hemodynamic gene expression is being studied based on spatial and temporal variations in shear stress defined at the local cell surface. Well-characterized and precise experimental models of spatially defined flow are combined with regional and single-cell gene-expression profiling to investigate the relationships linking hemodynamics to vessel-wall pathobiology.•