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Signaling Mechanisms Involved in Endothelial Cell Activation by Perturbed Flow

Contact Person: Peter I Lelkes (pilelkes@facstaff.wisc.edu)

Discussion and Conclusion

The main outcome of this study is that perturbed flow of low shear stress upregulates VCAM-1 expression in cultured human aortic endothelial cells. This confirms recent observations by us and by others that non-steady, non-laminar flow patterns, similar to those found at sites of atherosclerotic predilections result in the upregulation of endothelial cell adhesion molecules, notably of VCAM-1 (Kettlun et al., 1996, Chappell et al., 1998). The findings by others describing down-regulation of VCAM-1 expression by laminar flow may be physiologically relevant in those areas where laminar shear stress prevails, e.g. in the venous circulation. However, in those areas that are prone to atherosclerotic lesions, such as in the arterial circulation (coronaries, carotid), in particular in the aortic arch or in the vicinity of bifurcations, the flow pattern of the pulsatile blood flow is realistically described as "perturbed" (Asakura and Karino, 1990). And it is exactly in these regions that in vivo VCAM-1 expression is upregulated. Therefore, we postulated that perturbed flow activates unique signaling mechanisms which are distinct from those induced by laminar flow. Our data, which to the best of our knowledge are the first to show an upregulation of VCAM-1 by non-laminar flow, support our hypothesis.

Our findings are novel, since previous in vitro studies consistently showed a decrease in VCAM-1 expression by laminar shear stress(Ando et al., 1994). Recently Korenaga et al. (1997) identified a cis-acting negative flow response element located between -0.7 and -0.3 kb of the VCAM-1 promoter. This shear inhibitory element, which is activated by laminar shear stress, consists of a tandem sequence of two AP-1 consensus binding sites with an oligonucleotide sequence of TGACTCA.

A cornerstone of the current concept of EC activation by cytokines is the generation an ROS and activation of ROS dependent signaling pathways, e.g. via NF-kappaB (Collins, 1993; Collins et al ., 1993, 1995; Gimbrone et al., 1997). Certain similarities in VCAM-1 upregulation by cytokines and by PF, e.g., in terms of time course, extent of VCAM-1 induction and its inhibition by PDTC, might imply that the regulation of VCAM-1 expression by these two dissimilar agonists involve similar mechanisms. Indeed, this argumentation holds in the case of ICAM-1 upregulation by laminar flow: EC activation by laminar shear stress and cytokines results in the formation of ROS and upregulation of NF-kappaB, (Collins, 1993; Marui et al., 1993; Lan et al., 1994; Read et al., 1994; Collins et al., 1995; Gimbrone et al., 1997), which in turn binds to a specific nuclear binding site which entails the shear stress response element (SSRE) (Khachigian et al., 1995).

Our results seem to disprove this simple correlation between mechanical forces, ROS generation and NF-kappaB induction and endothelial cell activation. In our system, PF fails to activate the nuclear translocat ion of NF-kappaB, and, yet, VCAM-1 induction by PF is inhibited by the ROS scavenger PDTC. Thus, in terms of the possible mechanisms by which VCAM-1 is upregulated by PF, this apparent dichotomy is the most striking result of our study and calls for a fresh interpretation of possible PF-sensitive regulatory mechanism which may be quite distinct from the cytokine-induced signaling pathways.

Since the discovery of the "prototypic' SSRE, several new candidate shear activated response elements have been described which seem to be activated by known transcription factors. For example, activator protein-1 (AP-1), is a, ROS-sensitive transcription factor, which is involved in the flow-mediated regulation of several genes, such a monocyte chemotactic protein 1 (MCP-1) and, probably, also of endothelin and eNOS (Shyy et al., 1995; Malek and Izumo, 1996; Wung et al., 1997). In our system, in contrast to NF-kappaB, the level of nuclear translocation of AP-1is increased, in a manner which is independent o n the local flow patterns (Lelkes et al., manuscript in preparation). Interestingly, Korenaga et al. (1997) demonstrated that the downregulation of VCAM-1 requires an activation of a tandem AP-1 site. Clearly, the upregulation of VCAM-1 by PF requires more complex regulation than solely via AP-1.

In preliminary studies we established that PF also upregulates ICAM-1 expression (not shown). However, in contrast to ICAM-1 induction by laminar stress, PF-stimulated ICAM-1 activation occurs in the absence of NF-kappaB induction. The induction of egr-1 and of SP1 by PF in a manner that seems to depend on the local flow patterns (Lelkes et al., manuscript in preparation) might suggest that this transcription factor is also important in the flow-mediated upregulation of VCAM-1. Hence, we hypothesize that egr-1, SP1 and perhaps other, not yet identified transcription factors might be part of the unique sensing/signaling machinery which is involved in PF-induced VCAM-1 upregulation.

The present work proves the feasibility and usefulness of our flow chamber as a unique model system for testing the effects of realistic hemodynamic flow patterns (e.g. non-laminar, perturbed flow) on the expression of genes which are of clinical relevance as early warning signs/ predictors/hallmarks vascular diseases, such as atherosclerosis. Specifically, the present work suggests that the upregulation of VCAM-1 expression by perturbed flow is distinct from the down-regulation of VCAM-1 expression by laminar shear stress. Details of the molecular mechanisms remain to be elucidated, but it appears from our data that PF induces transcription factors (such as egr-1 and SP1), which are different from those responsive to laminar shear stress. We anticipate that promoter deletion assays in conjunction with our model system will yield detailed information on the molecular mechanisms of PF- sensitive gene regulation. Specifically we anticipate that such studies might yield (one or more) unique perturbed-flow res ponse elements (PFRE). Such a PFRE might become the target of therapeutic interventions for preventing or reverting damage to the vascular wall, e.g during the onset and progression of atherosclerotic lesions.

Acknowledgement: This study was supported, in part, by grants (to PIL) from the American Heart Association (National Center), the Milwaukee Heart Research Foundation, and from Berlex Biosciences. We thank Dr. Mary E. Gerritsen (formerly with Bayer Corporation, West Haven, CT) for her generous gift of the -VCAM-1 antibody 313.4B. We are indebted to Dr. Mark M. Samet for his invaluable help in the realization and operation of the flow system discussed in this communication.

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Lelkes, P.I.; Kettlun, C.S.; Wigboldus, J.; Waters, C.R.; Sukovich, D.A.; Rubanyi, G.M.; (1998). Signaling Mechanisms Involved in Endothelial Cell Activation by Perturbed Flow. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/
© 1998 Author(s) Hold Copyright