205 macrophage apoptosis

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.

Abstract (con’t)


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 42C for 15 minutes followed by incubation at 37C 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- (TNF-) and interleukin-6 (IL-6) demonstrated lower levels of both cytokines in the heated group. Moreover, thermal stimulation also inactivated NF-B (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.

Abstract (con’t)


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 42C for 15 minutes before returning to 37C. Two hours after heating, both heated and non-heated dishes were divided in 3 groups: a) TNF- 10ng/ml, b) TNF- 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.

Abstract (con’t)


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.

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

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.


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.


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.


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.

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.

Effect of heat on macrophage apoptosis

TUNEL and HAM-56 double staining. There is significant increasein the number of TUNEL positive macrophages after heating (8% to 46%)

Effect of heat on SMC apoptosis

TUNEL and actin double staining. There is insignificant increase in the number of TUNEL positive SMCs after heating (4% to 10%).

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.

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.

Effect of heat on TNF - immunoreactivity

TNF- immunoreactivity decreases markedly with heating

Conclusion

Gentle short-term thermal treatment induces apoptosis in human atherosclerotic lesions, reduces expression of pro-inflammatory cytokines TNF and IL-6, and inactivates NFB (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.

Health & Medicine

205 macrophage apoptosis