Endothelial Mechanisms of Flow-Mediated Athero-Protection and Susceptibility
- flow characteristics
- TNF-α, caspase
- endothelial apoptosis
See related article, pages 97–105
The arterial endothelium survives remarkably well as the interface between blood and vessel wall in an environment of constantly changing biomechanical stresses as well as acute and chronic exposure to inflammatory stimulants (eg, cytokines and hypercholesterolemia respectively).1 Cell turnover, which tends to occur in regional clusters,2 is otherwise very low in this monolayer. The endothelium also plays an important regulatory role in the pathogenesis of vascular disease. The cells readily respond to diverse stimuli through a repertoire of mechanisms to enhance their own survival even as they facilitate inflammatory, proatherogenic responses in the subendothelial tissue. The necessity to be a responsive cellular interface probably accounts for much of the endothelial phenotype heterogeneity that exists between vascular beds as well as within discrete regions of the arterial circulation.3,4
Hemodynamic characteristics that vary with blood vessel geometry predict the location of arterial sites that are susceptible to atherosclerosis.5 Curved and branching vessel geometries create sites of flow separation that contain transient flow reversals, lower average shear stresses, and occasional turbulence, (collectively, disturbed flow) and that are predictive of lesion formation. In contrast, pulsatile unidirectional laminar flow (and higher average shear stresses) is associated with regions where atherosclerosis rarely occurs, despite there being equivalent exposure to plasma risk factors such as hypercholesterolemia throughout the circulation. Although the signatures of endothelial phenotype in such regions in vivo are varied and complex, data are emerging from genomic5–7 and protein8 analyses of endothelium at such sites that identify molecular differences. Some of these are accessible for study in vitro to investigate detailed mechanisms under more controlled conditions. An interesting example that addresses mechanisms of flow-related differential regulation of endothelial cell phenotype leading to cell survival is reported in this issue of Circulation Research.
Garin et al9 demonstrate that the introduction of undisturbed laminar flow in vitro protects endothelial cells from tumor necrosis factor (TNF)-α–induced apoptosis through inhibition of a pathway that cleaves protein kinase C zeta (PKCζ), one of several PKC isoforms expressed by the endothelium. Flow promoted cell survival instead of programmed cell death when compared with control cells not exposed to flow. The study extends previous demonstrations of flow inhibition of TNF-α signaling, apoptosis, and adhesion protein expression in endothelial cells10–12 by clarifying the role of PKCζ cleavage in TNF-α–induced caspase 3 activation.
The PKC families of enzymes are serine/threonine kinases that phosphorylate effector proteins leading to multiple outcomes. PKCζ, a member of the atypical family of PKC enzymes, has acquired particular significance in endothelial biology. In earlier studies of cytokine-mediated apoptosis, Rahman et al (1999)13 had demonstrated that TNF-α–induced activation of NF-κB leading to proinflammatory ICAM-1 expression is mediated through the generation of reactive oxidants controlled by an atypical PKC family isoform, (subsequently identified as PKCζ).14 Other studies have since shown that adhesion molecule expression results from prolonged exposure to disturbed flow in vitro and is associated with disturbed flow in vivo.15 Recently, Magid, and Davies8 identified differential post-translational modification of PKCζ in endothelial lysates isolated from a disturbed flow region of swine aorta when compared with undisturbed flow regions in the same animals. PKCζ was shown to account entirely for the increased endothelial PKC enzyme activity in the athero-susceptible, disturbed flow region. In exploring a central role for PKCζ in flow-regulation of cytokine-activated endothelial signaling under controlled conditions in the present study, Garin et al first showed that PKCζ is necessary for JNK activation by TNF-α and that JNK in turn mediates caspase-3 activation (see Figure, panel A). In HeLa cells, TNF-α induces PKCζ processing to a shorter highly active catalytic domain, CATζ, by removal of an auto-inhibitory sequence.16 In endothelium, CATζ, generated by caspase-3 cleavage of PKCζ, potentiates a feedback loop that activates JNK to amplify caspase-3 activation and the cleavage of more PKCζ. The same pathway was shown to be present in endothelial cell fractions from rabbit aorta. Thus PKCζ with its internal amplification loop operates at multiple levels directed to apoptosis. The experiments revealed, however, that exposure to unidirectional laminar flow (athero-protective hemodynamics) inhibited the JNK-caspase-3-CATζ generation (box in Figure, panel A) reducing or preventing apoptosis and pro-inflammatory endothelial adhesion protein expression. The implication is that regions of undisturbed laminar flow in vivo protect the endothelium via these mechanisms. The interpretation is consistent with elevated PKCζ activity noted in vivo in regions susceptible to atherogenesis when compared with protected regions where undisturbed flow dominates.8 Increased phosphorylation of the activation loop at Thr410, which confers increased catalytic activity17 and which was differentially measured at the arterial sites (see Figure, panel B) is consistent with a proinflammatory signature of flow-mediated PKCζ involvement in vivo when the flow is disturbed but not when the flow is unidirectional and undisturbed. The report is an interesting contribution to the interplay between cytokine-mediated and flow-mediated endothelial signaling and phenotype, and it raises several additional considerations.
Cell culture studies can be extended to create a stronger link between flow in vitro and in vivo. The flow comparison in this study uses no-flow as a reference control. In vivo in humans, arterial flow is zero only for a brief moment when the flow reverses in a region of flow separation (disturbed flow); otherwise flow is accelerating or decelerating with approx 2Hz frequency at these sites and in most other locations (where the flow is undisturbed) it is unidirectional and pulsatile. The addition of cells subjected to disturbed flow would be an important reference group for in vitro studies and may provide further insights into athero-susceptible flow. The unidirectional steady laminar flow shown to inhibit PKCζ cleavage could also readily be replaced by a pulsatile waveform of unidirectional flow that more closely simulates flow in athero-protected regions in vivo. However, even in present form, the experiments remain a convincing proof-of-principle demonstration of flow-mediation of an important amplification pathway.
It is unclear how flow inhibits this signaling cascade. Flow effects generally fall into two broad mechanisms; deformation forces that modify the mechanotransduction responses of the cell, and changes of convective transport within the cell and/or at the cell surface. Either or both may be relevant to PKC signaling. In the study by Rahman et al13 atypical PKC activation alone was insufficient to activate NF-κB and induce adhesion proteins; the downstream generation of reactive oxygen species (ROS) was required. In the present study, ROS may also be an intermediate (not measured). Could the flow wash out high oxidant concentrations in the cells and thereby inhibit CATζ generation by removing a required cofactor(s)? The role of ROS in tbe pathway can be investigated by antioxidant pretreatment (N-acetylcysteine, glutathione) or by local generation of peroxides or other oxidants. If ROS are also important regulators of the pathway, extrapolation to in vivo dynamics (where flow is always present) would require more sophisticated in vitro flow experiments than simple laminar flow versus no flow because the transport properties at the arterial endothelium in vivo are complex in both undisturbed and disturbed flow.
In summary, mechanisms for the transduction and amplification of apoptotic signals involving PKC enzymes in tandem with caspases play an important role in a variety of cells. In the endothelium, flow-inhibition of PKCζ processing is an important, although not exclusive, prosurvival mechanism. In vivo measurements of endothelial PKCζ in the arterial circulation are consistent with differential protective mechanisms linked to hemodynamic characteristics.
Sources of Funding
The author’s research is supported by grants HL64388 and HL62250 from the National Heart Lung and Blood Institute of the NIH.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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