The document discusses the role of apoptosis in atherosclerosis and the effects of heat on apoptosis of different cell types in atherosclerotic plaques. It finds that heating human carotid plaques to 42°C for 15 minutes significantly increases macrophage apoptosis from 8% to 46% while only slightly increasing smooth muscle cell apoptosis from 4% to 10%. Heat treatment also decreases the expression of pro-inflammatory cytokines TNF-α and IL-6 and inactivates the NFκB transcription factor. The conclusion is that gentle, short-term heating may help treat advanced atherosclerotic plaques by reducing inflammation and triggering macrophage apoptosis.

1. Abstract
Macrophage apoptosis: a double
edge sword?
Apoptosis, a form of genetically programmed cell death, plays an essential role in different
physiologic and pathologic processes including atherosclerosis, in which it affects all cell
types including endothelial cells, vascular smooth muscle cells (VSMCs), and macrophages.
Over the course of the plaque progression, pro- and anti-apoptotic signals abound. In other
organ systems, apoptosis limits the number of a particular cell type that accumulates in the
lesion. The issue in atherosclerosis, however, is clearly more complex. The loss of VSMCs
can be detrimental for plaque stability since most of the fibrous cap collagen required for the
tensile strength of the cap is produced by VSMCs. Apoptosis of macrophages, on the other
hand, could be beneficial for plaque stability if apoptotic bodies were removed. Several
investigators have reported, however, that apoptotic bodies in the advanced atherosclerotic
plaque are often not scavenged, can activate the coagulation cascade, potentially leading to
plaque rupture and luminal thrombosis. Many of the apoptotic bodies are of macrophage
origin. Moreover, interventions like statin therapy have shown that beneficial effects on the
plaque, namely shrinkage of the lipid core, decrease of the inflammatory burden and
thickening of the fibrous cap, are accompanied by a decrease in apoptotic activity. It is
therefore not surprising that most investigators believe that apoptosis is detrimental to
plaque stability.

2. Abstract (con’t)
Macrophage apoptosis: a double
edge sword?
Our group has long been interested in the thermal heterogeneity of the
atherosclerotic plaque and on the effect of plaque heating on the processes of
inflammation and apoptosis. In a recent study by Dr. Birendra Lal in Dr. Yong-Jian
Geng’s laboratory at the University of Texas Houston, eleven freshly living human
carotid endarterectomy specimens were heated in DMEM medium at 42°C for 15
minutes followed by incubation at 37°C for 6 hours. In unheated controls, 4% of
the VSMCs and 8% of macrophages were TUNEL positive. In the specimens with
the short term heating, 46% of the macrophages and 10% of the SMCs were
TUNEL positive. Immunostaining for tumor necrosis factor-α (TNF-α) and
interleukin-6 (IL-6) demonstrated lower levels of both cytokines in the heated
group. Moreover, thermal stimulation also inactivated NF-κB (a transcription
factor involved in cytokine expression, cell proliferation, etc) in macrophages
derived from THP-1 cells by phorbol esters as demonstrated by gel shift assays.

3. Abstract (con’t)
Macrophage apoptosis: a double
edge sword?
In another set of experiments performed by Dr. Mitra Rajabi in Dr. Yong-Jian
Geng’s laboratory at the University of Texas Houston, mouse VSMCs were
divided in two groups, half heated at 42°C for 15 minutes before returning to
37°C. Two hours after heating, both heated and non-heated dishes were divided in
3 groups: a) TNF-α 10ng/ml, b) TNF-α 10ng/ml and IFN-γ 10ng/ml, and c) no
cytokines. After 12, 36 and 48 hours, the nitrite production, a marker of iNOS
expression, was statistically significant lower in the heated as compared to the non-
heated groups.

4. Abstract (con’t)
Macrophage apoptosis: a double
edge sword?
We therefore believe that specific therapies like local gentle heating have a
potential therapeutic effect by decreasing markers of inflammation coupled to
their pro-apoptotic effects on macrophages. In addition, the operator in the
catheterization laboratory could add adjuvant therapy like balloon dilation,
stenting and anticoagulation, thereby preventing the potential complications of
plaque rupture and thrombosis from happening in vivo.
In summary, although large body of evidence considers apoptosis in the plaque
to be risky and detrimental, we believe that under certain controlled conditions,
gentle heating could decrease the plaque vulnerability.

5. APOPTOSIS &
ATHEROSCLEROSIS
∀• While apoptosis is a key negative regulator of the cell density in
oncogenesis, organ development, and immune response, the role of apoptosis in
atherosclerosis is more complex.
• Variation in the rate of apoptosis of different cell types promotes differences
in growth rates, structure and stability of the plaques.
• Several cytokines known to be pro-apoptotic, such as tumor necrosis-α
(TNF-α), interleukin-1β, and interferon γ (IFN- γ) and products of genes involved
in the cell cycle regulation (Fas/Fas ligand, caspase, p53 and c-Myc) have been
found in vascular cells and atherosclerotic plaques.
• Apoptotic rate is higher in advanced plaques

6. APOPTOSIS OF DIFFERENT
CELL TYPES IN PLAQUE TISSUE
ENDOTHELIAL CELLS
∀ • Lesion-prone regions show increased endothelial cell (EC) turnover ratio.
∀ • ECs undergo apoptosis when coming in contact with circulating or local
factors like angiotensin II, oxidized LDL, reactive oxygen species (ROS) and
inflammatory cytokines.
• Apoptotic ECs assume pro-coagulant characteristics due to increased
exposure to phosphatidylserine and loss of normal anticoagulant membrane
properties.
• Apoptotic ECs increase migration of monocytes and T-lymphocytes.

7. APOPTOSIS OF DIFFERENT
CELL TYPES IN PLAQUE TISSUE
VASCULAR SMOOTH MUSCLE CELLS
 Apoptosis of vascular smooth muscle cells (VSMC) reduces the rate of
plaque growth. At the same time, since VSMCs are the source of interstitial
collagen fibers type I, plaque stability might be affected.
 Migration of macrophages to areas of VSMC apoptosis has been described.
 Overall effects of VSMCs apoptosis are complex and difficult to predict but
generally felt to be deleterious for plaque stability.

8. APOPTOSIS OF DIFFERENT
CELL TYPES IN PLAQUE TISSUE
MACROPHAGES
•• Macrophages may activate several matrix metalloproteinases which degrade
interstitial collagen, thus weakening the fibrous cap.
• Macrophages produce cytokines that may induce VSMCs apoptosis.
• Loss of macrophages results in decreased scavenging products of cell degradation,
leading to accumulation of necrotic debris and coagulation activation.
• Therefore, apoptosis of macrophages, may have both pro- and anti-destabilizing
effects.

9. APOPTOSIS OF DIFFERENT
CELL TYPES IN PLAQUE TISSUE
T-LYMPHOCYTES
∀• Lymphocytes produce molecules with important regulatory
functions on the plaque cell death (cytokines, perforin, Fas).
∀• Apoptosis of lymphocytes is not well understood in the context of
atherosclerosis.

10. Effect of heat on apoptosis of
macrophages and smooth muscle
cells
At 37 °C, the proportion of apoptotic SMC and macrophage were 4% and 8%
respectively. At 42 °C, these proportions increased to 10% and 46%
respectively.

11. Effect of heat on macrophage
apoptosis
∀
TUNEL and HAM-56 double staining. There is significant increase
in the number of TUNEL positive macrophages after heating (8% to 46%)

12. Effect of heat on SMC apoptosis
∀
TUNEL and α actin double staining. There is insignificant increase in the
number of TUNEL positive SMCs after heating (4% to 10%).

13. Effect of heat on macrophage
ultrastructure
∀
Human carotid atherectomy specimen. A: Normal macrophage in unheated
plaque. B: Two apoptotic macrophages inheated plaque condensed chromatin is
same in both cells.

14. Effect of heat on macrophage
ultrastructure (con’t)
∀
C: Enlarged view of B. D: Foam cell at the end stage of apoptotic process. Extra
cellular debris is also present.

15. Effect of heat on TNF -α
immunoreactivity
∀
TNF- α immunoreactivity decreases markedly with heating

16. Conclusion
∀
 Gentle short-term thermal treatment induces apoptosis in human
atherosclerotic lesions, reduces expression of pro-inflammatory cytokines
TNFα and IL-6, and inactivates NFκB (as demonstrated by electrophoretic
mobility shift assay, data not shown).
 These data suggest that thermal treatment may have potential for
treating advanced atherosclerotic lesions by reducing inflammation and
triggering apoptosis in macrophages.