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(Circulation Research. 2006;98:1117.)
© 2006 American Heart Association, Inc.

Editorials

A Farewell Kiss Triggers a Broken Heart?

Nicholas A. Flavahan

From the Heart and Lung Research Institute, Ohio State University, Columbus.

Correspondence to Nicholas A. Flavahan, The Ohio State University, 473 West 12th Ave, R 110E, Columbus, OH 43210. E-mail nicholas.flavahan{at}osumc.edu

See related article, pages 1168–1176

Key Words: vascular smooth muscle • apoptosis • atherosclerosis • T cells • CD4

The evolving atherosclerotic lesion reflects a coordinated interaction between vascular and immune/inflammatory cells.^1,2 Foam cells and extracellular lipid droplets form the central core of the atheroma, which is covered by vascular smooth muscle cells (VSMCs) and a collagen-rich matrix. T cells and macrophages infiltrate the lesion and are particularly abundant in the shoulder region. VSMCs are responsible for producing extracellular matrix proteins that provide strength and stability to the lesion. Indeed, the atherosclerotic plaque can remain relatively stable in which a cap of VSMCs and matrix proteins covers the lipid core. Alternatively, the plaque can develop into a chronic active inflammatory lesion.^1,2 These unstable plaques are characterized by increased numbers of activated immune/inflammatory cells and increased expression and release of numerous inflammatory mediators and proteolytic enzymes. Thinning of the fibrous cap reflecting reductions in VSMCs and matrix proteins is a key feature of unstable plaques.^1,2 The subsequent rupture of weakened unstable plaques, which exposes prothrombotic material from the core of the plaque, is the principal mechanism underlying acute coronary syndromes (ACS) including unstable angina, myocardial infarction, and sudden cardiac death.^1,2

Activated macrophages have been considered a primary culprit in destabilizing atherosclerotic plaques. They are a major source of matrix metalloproteinases (MMPs), which can degrade all components of the extracellular matrix and represent a principal mechanism for collagen breakdown and cap weakness.^1,2 Macrophages are also highly effective at inducing apoptosis of VSMCs, a characteristic feature of unstable plaques.^3,4 Although CD4⁺ T cells are prominent within unstable plaques, they have generally been considered to regulate the lesional activity of other cell types, including macrophages. In this issue of Circulation Research, Pryshchep et al⁵ highlight the direct role of CD4⁺ T cells in causing apoptosis of VSMCs mediated by the cytotoxic T cell "Kiss of Death", which may play an important pathological role in plaque destabilization.

CD4⁺ T cells from ACS patients (from blood or cloned from endarterectomy samples) were highly effective inducers of apoptosis in autologous VSMCs (from the patient’s endarterectomy samples) or allogeneic VSMCs (mismatched endarterectomy samples or from commercial sources).⁵ In contrast, CD4⁺ T cells from healthy controls or from patients with stable angina (SA) were much less effective. Although the CD4⁺ T cells used in the study were not fully characterized,⁵ they presumably represent CD4⁺CD28^null cells. These T cells are selectively expanded in the peripheral blood of ACS but not SA patients, and have increased prominence within unstable compared with stable plaques.⁶ Indeed, 10% of circulating ACS CD4⁺ cells were effective at killing VSMCs,⁵ consistent with the circulating CD4⁺CD28^null cell population in ACS patients.⁶ CD4⁺CD28^null T cells appear to be pseudo-senescent lymphocytes generated by repeated antigenic stimulation of CD28⁺ precursors.⁷ The loss of CD28 is accompanied by acquisition of new aberrant functions. CD4⁺CD28^null cells are resistant to apoptosis and produce high levels of cytokines, in particular interferon (IFN)- and tumor necrosis factor (TNF)-.^6,7 CD28^null T cells mediate the increased production of IFN- in ACS patients. They are tissue damaging and highly autoreactive T cells. Indeed, CD4⁺CD28^null cells have several cytotoxic mechanisms normally associated with natural killer (NK) cells, including granzyme B (GrB) and perforin-containing granules, and a variety of killer-cell immunoglobulin-like receptors (KIRs).^6–9 Expression of KIRs and the adapter molecule DAP12 in CD4⁺CD28^null cells enables killing of target cells via release of cytotoxic granules in a T cell receptor (TCR)-independent manner.

Pryshchep et al⁵ use elegant imaging techniques to probe the kinetics and mechanisms underlying T cell–induced VSMC apoptosis, including visualization of immunological synapses between these cells. To induce apoptosis, T cells had to interact with VSMCs for 60 minutes; removing the T cells after 10 or 30 minutes prevented VSMC death. Consistent with their effects on apoptosis, only CD4⁺ cells from ACS patients were able to maintain a sustained (>40 minutes) contact zone with VSMCs. The initial interaction between T cells and VSMCs was similar for control and ACS T cells, with early localization of T cell signaling molecules (CD3/TCR, pZAP-70) and formation of an immunological synapse: a central core composed of CD3/TCR molecules (central supramolecular activation complex, c-SMAC), surrounded by a ring of LFA-1 (peripheral p-SMAC). This outer adhesive ring stabilizes cell interactions, forming a protected inner zone in which secreted cytokines and enzymes attain maximum effect at minimal cost to adjacent cells.¹⁰ With control T cells, the signaling molecules and synapse quickly dissipated, whereas with ACS T cells the immunological synapse was maintained and associated with sustained signaling in T cells and VSMCs.⁵ The immunological synapse between ACS T cells and VSMCs underwent significant remodeling, with decreased intensities of the c-SMAC and p-SMAC.⁵ The authors interpreted the partial loss of c-SMAC to represent formation of a secretory synapse, enabling the directed release of cytotoxic mediators toward the VSMCs. Indeed, T cell–induced apoptosis of VSMCs was inhibited by strontium, which causes degranulation of T cells, suggesting that VSMC apoptosis might be mediated by the perforin–GrB pathway.⁵ GrB has been localized with apoptotic VSMCs in atherosclerotic lesions.¹¹ The classical view of perforin–GrB-induced cell death is that perforin enables access of GrB inside the target cell to initiate apoptosis by caspase-dependent and independent mechanisms. However, GrB can also function as a matrix protease and can induce VSMC apoptosis by an extracellular action.¹¹ Indeed, the dismantling of p-SMAC during sustained CD4⁺CD28^null:VSMC interactions⁵ might result in a less effective seal with overflow of GrB and apoptosis of surrounding cells.

T cell–induced apoptosis of VSMCs was reduced by blocking HLA-DR, indicating involvement of MHC Class II molecules and suggesting that VSMC apoptosis was dependent on TCR activation.⁵ ACS T cells were also more effective at forming sustained synapses and killing superantigen-enriched THP-1 cells suggesting that the ACS cells are hyperresponsive to TCR stimulation. A surprising finding of the present study was that ACS T cells did not distinguish between VSMCs derived from carotid endarterectomy samples and those from normal coronary arteries (commercial source of VSMCs).⁵ Although MHC class II molecules are expressed on VSMCs of atherosclerotic lesions, especially at sites prone to rupture, they are not expressed by normal VSMCs either in the arterial wall or during cell culture.^12,13 IFN- causes a dramatic upregulation of MHC class II molecules in normal VSMCs.¹³ Indeed, the increased expression of MHC class II in lesional VSMCs might reflect the elevated production of IFN- by CD4⁺CD28^null cells, which are also present in unstable lesions. Indeed, this activity could prime VSMCs for subsequent apoptosis mediated by these T cells. However, the IFN-–induction of MHC class II occurs slowly and would not have occurred during the short time-course of the present experiments. The authors did not provide any details on whether VSMC apoptosis was dependent on the phenotype (including lesional versus medial cells) or activity (including cell cycle) of the VSMCs, which regulate the sensitivity of VSMCs to apoptosis and the biological activity of GrB.^3,14

Unfortunately, the authors did not discuss the present results in light of their other recently published report on CD4⁺-induced apoptosis of VSMCs.¹⁵ In that report, the authors adopted a similar approach using CD4⁺ cells/cell lines and autologous/allogeneic VSMCs. Although a slightly longer incubation time and a variety of T cell:target cell ratios were analyzed, the observed rates of apoptosis were consistent with those reported in the present study. Apoptosis was also prevented by inhibition of MHC class II (HLA-DR) molecules.¹⁵ However, the increased VSMC apoptosis caused by ACS CD4⁺ T cells was largely prevented by inhibition of TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family of cytokines. VSMCs expressed receptors for TRAIL and could be killed after exposure to TRAIL.¹⁵ Although the CD4⁺ cells expressed TRAIL, the protein was present on the cell surface only after TCR activation. Therefore, the initial interaction between ACS T cells and VSMCs apparently stimulated translocation of TRAIL to the cell surface, triggering apoptosis of VSMCs. The pathological relevance of this mechanism was elegantly demonstrated using adoptive transfer of CD4⁺ cells into immunodeficient mice implanted with human carotid plaque tissue. The ACS CD4⁺ cells caused impressive apoptosis of VSMCs in the plaque, which was prevented by inhibiting TRAIL.¹⁵

Assuming that a similar population of CD4⁺ T cells was used in the two studies,^5,15 the combined results suggest that TRAIL may have translocated to the immunological synapse to engage death receptors on the VSMC surface. Presumably this translocation was prevented by strontium, although strontium may also have impaired the function of the immunological synapse. Alternatively, apoptosis of VSMCs may have been mediated by synergistic interactions between multiple mediators (eg, GrB and TRAIL). Regardless of the mechanism, the results demonstrate that CD4⁺ T from ACS patients are highly effective at killing VSMCs and this mechanism can function in vivo in human atherosclerotic plaques.^5,15 However, a drawback to the elegant adoptive transfer and transplant model is that it emphasizes the role of T cells, while reducing the contribution of macrophages.

Although CD4⁺CD28^null cells can be enticed to kill endothelial cells,⁸ they appear to have a more deadly attraction to VSMCs.^5,15 Given this predilection and the highly-reactive nature of these abnormal cells, their increased presence in ACS patients and in unstable lesions suggests that they contribute significantly to vascular pathology. By directly stimulating apoptosis of VSMCs (eg, through TRAIL, KIR, FasL, GrB/perforin) or by coordinating and activating macrophages to kill these cells, CD4⁺CD28^null could weaken the cap and destabilize angiogenic vessels, precipitating atherosclerotic plaque rupture and the clinical features of ACS. Increased understanding of the mechanisms regulating the emergence and activity of these pathological T cells and their role in human atherosclerotic lesions could provide new insight into therapeutic options for this devastating disease.

	Acknowledgments

 
Research in the author’s laboratory is supported by National Institutes of Health grants HL067331, HL080119, HL056091, and OH008531.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

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T Cell Recognition and Killing of Vascular Smooth Muscle Cells in Acute Coronary Syndrome

Sergey Pryshchep, Kayoko Sato, Jörg J. Goronzy, and Cornelia M. Weyand
Circ. Res. 2006 98: 1168-1176. [Abstract] [Full Text] [PDF]

This Article Extract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Flavahan, N. A. Search for Related Content PubMed PubMed Citation Articles by Flavahan, N. A. Related Collections Related Article