Increased apoptosis and cysteinyl aspartate specific protease-3 gene expression in human intracranial aneurysm

doi:10.1016/j.jocn.2005.11.018

Journal of Clinical Neuroscience

Volume 14, Issue 6, June 2007, Pages 550-555

https://doi.org/10.1016/j.jocn.2005.11.018 Get rights and content

Abstract

Objective

To investigate apoptosis in vascular smooth muscle cells (VSMCs) and caspase-3 expression in ruptured intracranial aneurysm.

Methods

Tissue samples of 15 ruptured intracranial aneurysms, 6 abdominal aortic aneurysms (AAA) and 6 normal vessels were evaluated. Apoptosis in VSMCs was determined on transmission electron microscopy. Immunohistochemistry for alpha-SMC actin and direct cell counts (medial VSMCs per high-power field (HPF)) were employed to determine medial VSMC density. Additionally, gene expression of caspase-3 was determined using real-time RT-PCR.

Results

We demonstrated medial VSMCs exhibiting morphological apoptotic changes in cerebral aneurysm and AAA. Medial VSMC density was significantly decreased in intracranial aneurysm (43.9 ± 4.3 SMCs/HPF) and AAA (53.2 ± 9.4 SMCs/HPF) compared with the normal arteries (222.8 ± 12.1 SMCs/HPF; p < 0.01). An 8.94-fold and 6.73-fold increase in expression of caspase-3mRNA in intracranial aneurysm and AAA, respectively, were obtained relative to the normal vessels.

Conclusions

These results suggest that real time RT-PCR provides a useful tool to test gene expression in small samples, and may contribute to a better understanding of the role of apoptosis in ruptured intracranial aneurysm.

Introduction

Subarachnoid hemorrhage due to ruptured intracranial aneurysm is a devastating event associated with high morbidity and mortality.[1], [2] Apoptosis of vascular smooth muscle cells (VSMCs) is an important acquired degenerative feature strongly associated with rupture of human intracranial aneurysms.[3], [4], [5], [6] Experimental evidence confirms that apoptosis of medial VSMCs is involved in the formation and development of saccular cerebral aneurysms.⁷ In addition, excessive apoptosis is a feature of abdominal aortic aneurysm (AAA).[8], [9], [10]

Apoptosis, programmed cell death, plays an important role in a wide range of physiological conditions. In brief, it functions as a homeostatic regulator, balancing cell proliferation and loss. Activation of cysteine proteases, called caspases, plays a major role in the execution of apoptosis. Caspase-3 is the most critical determiner of cell apoptosis. Activated caspase cascades selectively cleave cellular proteins, which results in apoptotic cell morphology and cell death.[11], [12], [13], [14] However, the relationship between caspase-3 and intracranial aneurysm has not been fully elucidated. Apoptosis in cerebral aneurysm tissue has been detected by means of a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP biotin nick end labelling technique (TUNEL).[3], [4], [6] However, quantitative assessment of caspase-3 gene expression using real time RT-PCR in intracranial aneurysm has not been reported.

In this study, we show increased apoptosis of VSMCs and decreased medical VSMC density as well as elevated caspase-3mRNA expression with real time RT-PCR in aneurysmal tissue. These findings raise the possibility that excessive apoptosis of VSMC may contribute to the pathogenesis of intracranial aneurysms.

Section snippets

Materials and methods

The protocol of this study was approved by the national ethical committee. Between September 2003 and January 2005, 15 specimens from ruptured intracranial aneurysm were obtained from patients during surgical clipping in West China Hospital, Sichuan University. There were 12 female and 3 male patients aged between 34 and 68 years (mean, 55 years). Ten aneurysms were located on the posterior communicating artery, 3 on the middle cerebral artery and 2 on the anterior communicating artery.

Results

Histological sections of the intracranial aneurysm with hematoxylin-eosin staining showed obvious thinning of the arterial wall and regional arterial wall dilatation as well as disruption of the vascular wall integrity (Fig. 1A). Intimal damage and loss of the elastic lamellae as well as focal hemorrhage in the arterial wall were observed (Fig. 1B). Under the electron microscope, variable degrees of chromatin condensation were visible. In addition, ultrastructural changes including cell body

Discussions

The balance between proliferation and apoptosis of VSMCs is believed to contribute to the vascular remodeling process. Ranganna et al.[17], [18] point out that excessive proliferation of VSMCs is involved in vascular proliferative disorders, such as atherosclerosis, hypertension and stenosis. They have shown that treatment with butyrate produces inhibition of VSMC proliferation via the down-regulation of proliferating genes in animal models. Inversely, excessive apoptosis of VSMCs is associated

Conclusions

Our results suggest that real time RT-PCR is reliable for the detection of mRNA in human intracranial aneurysm, and this method provides useful tool for studying gene expression in small samples. We have simultaneously demonstrated excessive apoptosis of VSMCs and augmented caspase-3m RNA expression. Upregulation of the caspase-3 gene is closely implicated in the pathological process of the ruptured intracranial aneurysm. Further study is required that may lead to the development of new

Acknowledgements

We thank Dr. Xia QJ, Wang L, Yan ShH and Cao GQ for their skilful technical assistance.

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^☆: This research were Supported by The National Natural Scientific Foundation of China. No: 30300363.

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Laboratory studyIncreased apoptosis and cysteinyl aspartate specific protease-3 gene expression in human intracranial aneurysm☆

Abstract

Objective

Methods

Results

Conclusions

Introduction

Section snippets

Materials and methods

Results

Discussions

Conclusions

Acknowledgements

J Vasc Surg

J Clin Neurosci

Neurosci Lett

Neurosci Lett

Methods

Atherosclerosis

Eur J Vasc Endovasc Surg

Brain Res

Molecular anatomy of an intracranial aneurysm: coordinated expression of genes involved in wound healing and tissue remodeling

Stroke

Intracranial aneurysms

N Engl J Med

Loss and apoptosis of smooth muscle cells in intracranial aneurysms. Studies with in situ DNA end labeling and antibody against single-stranded DNA

Acta Neurochir

Evidence for apoptosis in human intracranial aneurysms

Neurol Res

Laboratory study
Increased apoptosis and cysteinyl aspartate specific protease-3 gene expression in human intracranial aneurysm☆