Kotaro Ogihara

Apoptosis of endothelial cells in vessels affected by cerebral vasospasm

BACKGROUND Cerebral vasospasm after subarachnoid hemorrhage is a prolonged contraction that leads to cerebral ischemia or infarction. Morphological studies of cerebral arteries during vasospasm have shown extensive necrosis of smooth-muscle cells and desquamation and dystrophy of endothelial cells. The mechanism of cellular death is unknown. METHODS We report an observation of apoptotic changes in the cerebral arteries of a patient who died after suffering severe cerebral vasospasm caused by aneurysmal rupture. Subarachnoid hemorrhage and cerebral vasospasm were confirmed by computed tomography scanning and angiogram. Histological and immunohistological examinations for apoptosis were performed in cerebral arteries. For control, the arteries from another patient, who died of trauma without head injury, were used. RESULTS Corrugation of the internal elastic lamina and increased amounts of connective tissue was demonstrated by light microscopy. Apoptotic changes, characterized by condensation of chromatin of the nucleus and detachment from the basal membrane, were found on transmission electron microscopy in endothelial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling reaction revealed positive staining of the nuclei of the endothelial cells. CONCLUSIONS This study demonstrates that apoptosis occurred in the cerebral arteries in a patient who died of cerebral vasospasm. The possible role of apoptosis in cerebral vasospasm is discussed.

Oxyhemoglobin produces apoptosis and necrosis in cultured smooth muscle cells

Confluent rat aortic smooth muscle cells were treated with OxyHb in a concentration- and time-dependent manner. A high concentration of OxyHb (100 microM) within 24 h decreased cell density. DNA analysis showed a smear pattern characteristic of cell necrosis. Transmission electron microscopy demonstrated disintegration of the cell membrane and destruction of cell organelles. Western blotting using PARP antibody revealed that 116 kDa PARP was not cleaved to 85 kDa, an apoptosis-related fragment. On the contrary, a low concentration of OxyHb (10 microM) produced apoptotic cell death at 72 h that was supported by DNA analysis and TUNEL staining. These results demonstrated that a high level of OxyHb induced necrosis within 24 h and a low concentration of OxyHb produced apoptosis after 72 h in cultured smooth muscle cells. Morphological alterations induced by OxyHb might contribute to the vascular wall changes in the cerebral arteries following subarachnoid hemorrhage (SAH).

Oxyhemoglobin produces necrosis in cultured smooth muscle cells.

OBJECT Myonecrosis in the tunica media, which is defined morphologically, is one of the most striking alterations in the cerebral arterial wall following subarachnoid hemorrhage (SAH). In this study, oxyhemoglobin (OxyHb) was added to cultured rat aortic smooth muscle cells to determine the pattern of cell death by morphological and biochemical techniques. METHODS Confluent rat aortic smooth muscle cells were treated with OxyHb in a concentration- and time-dependent manner. Cell density was assayed by counting the number of cells that attached to the culture dishes after exposed to OxyHb. To identify cell death pattern, DNA analysis, electron microscopy, and Western blotting using poly (ADP-ribose) polymerase (PARP) antibody were performed. CONCLUSIONS OxyHb decreased cell density in a concentration- and time-dependent manner. DNA analysis showed a smear pattern characteristic of cell necrosis. Transmission electron microscopy demonstrated disintegration of cell membrane and destruction of cell organelles. No apoptotic changes, such as condensation of chromatin or apoptotic bodies were observed. Western blotting using PARP antibody revealed that 116 kDa PARP was not cleaved to 85 kDa, an apoptosis-related fragment. These results demonstrated morphologically and biochemically that OxyHb induced necrosis, not apoptosis, in cultured smooth muscle cells.

Apoptosis in Basilar Endothelial Cells in a Canine Double Hemorrhage Model

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a combination of prolonged contraction and vessel remodeling that may lead to cerebral ischemia or infarction [2]. Morphological studies of cerebral arteries affected by vasospasm often show extensive necrosis of smooth-muscle cells [1] as well as desquamation and dystrophy of endothelial cells [7, 11]. The actual mechanisms of cellular death following cerebral vasospasm are not known. Recent studies of post-SAH vasospasm in this laboratory strongly suggest that endothelial cells may be dying through apoptotic pathways [8, 10].

Effect of endothelin receptor antagonists on non-muscle matrix compaction in a cell culture vasospasm model

Abstract Endothelin-1 (ET-1), a potent vascular smooth muscle constrictor, is one of the possible spasmogens in cerebral vasospasm. However, the role of ET-I in non-muscle compaction (another aspect of the pathogenesis of cerebral vasospasm) has not been reported. This study was undertaken to demonstrate the effect of ET-I, as well as erythrocyte lysate and bloody cerebrospinal fluid (CSF), on fibroblast populated collagen lattice (FPCL) compaction. Human dermal fibroblasts were used to form FPCL. The concentration- dependent effect of ET-I was examined in the absence and presence of an ETA receptor antagonist (BQ- 485), or an ETB receptor antagonist (BQ-788), or both. FPCL compaction was determined by measuring reduction of areas over five days following treatment. To compare the effect of ET-1 on lattice compaction, erythrocyte lysate and bloody CSF obtained from a cerebral vasospasm patient were also tested. We found that ET-1 increased FPCL compaction in a concentration-dependent (but not time-dependent) manner. Erythrocyte lysate produced the strongest compaction, however, without time-dependence. Bloody CSF promoted FPCL compaction in a time-dependent fashion. Compaction induced by ET-1 was inhibited by BQ-485 but not by BQ-788. We concluded that ET-1 promotes FPCL compaction by activation of ETA receptors. Other components in bloody CSF or erythrocytes may also contribute to FPCL compaction. [Neurol Res 2000; 22: 209-214]

Bloody cerebrospinal fluid alters contractility of cultured arteries.

The pathogenesis of cerebral vasospasm that follows aneurysmal subarachnoid hemorrhage (SAH) is poorly understood. Multiple methods have been used to clarify the mechanism of spasmogen-induced vasospasm, however, each method has its own limitations. Cultured cells lose their phenotype and inter-cellular interactions, and animal models are expensive and can be used only in some established centers. Isolated cerebral arteries have been used extensively to study the contractility by transient exposure to spasmogens that, however, can hardly represent cerebral vasospasm that occurs 2-4 days after SAH. In this study, we cultured arteries with bloody cerebrospinal fluid (CSF) from patients of cerebral vasospasm and studied the contractility of the arteries 1, 3 and 4 days later. This method preserves artery wall structure, prolongs exposure of artery to bloody CSF, and is simple and inexpensive. Cultured rat aorta showed enhanced contractile response to 5-HT (p < 0.001) but reduced response to KCl (p < 0.05) 4 days after culturing with bloody CSF. We concluded that the contractility of arteries was modified by prolonged incubation with bloody CSF. Our observations in this study could be important and may explain some aspects of pathogenesis of cerebral vasospasm.

Mitogen-activated protein kinase plays an important role in hemolysate-induced contraction in rabbit basilar artery.

OBJECT Mitogen-activated protein kinase (MAPK) is an important signaling factor in the vascular proliferation and contraction, the two features of cerebral vasospasm following subarachnoid hemorrhage. We studied the possible involvement of MAPK in hemolysate-induced signal transduction and contraction in rabbit basilar artery. METHODS Isometric tension was used to record the contractile response of rabbit basilar artery to hemolysate. Western blots using antibodies for MAPK were conducted. 1) Hemolysate produced a concentration-dependent contraction of rabbit basilar artery. Pre-incubation of arteries with MAPK kinase inhibitor PD-98059 markedly reduced the contraction induced by hemolysate. PD-98059 also relaxed, in a concentration-dependent fashion, the sustained contraction induced by hemolysate (10%). 2) Hemolysate produced a time-dependent elevation of MAPK immunoreactivity in Western blot in rabbit basilar artery. MAPK was enhanced 3 min after hemolysate exposure and the effect reached maximum at 5 min. The immunoreactivity of MAPK decayed slowly with time, but the level of MAPK was still higher than the basal level even at two hours after exposure to hemolysate. 3) Pre-incubation of arteries with MAPK kinase inhibitor PD-98059 abolished the effect of hemolysate on MAPK immunoreactivity. CONCLUSION Hemolysate produced contraction of rabbit basilar artery possibly by activation of MAPK. MAPK inhibitors may be useful in the treatment of cerebral vasospasm.

Role of Tyrosine Kinase in Fibroblast Compaction and Cerebral Vasospasm

Hemolysate, a proposed causative agent for cerebral vasospasm following subarachnoid hemorrhage, produces contraction of cerebral arteries by activation of tyrosine kinases. In addition, hemolysate accelerates fibroblast collagen compaction that could play a role in cerebral vasospasm. We studied the effect of hemolysate on tyrosine phosphorylation and fibroblast collagen compaction in cultured dog cerebral and human dermal fibroblasts using tyrosine kinase inhibitors and tyrosine antibodies (Western blot). 1) Hemolysate was found to enhance tyrosine phosphorylation of two proteins approximately 64 and 120 kDa. The effect of hemolysate was time- and concentration-dependent. 2) Two main components in hemolysate, oxyhemoglobin and adenosine triphosphate (ATP), produced similar results to that of hemolysate. 3) Tyrosine kinase inhibitor genistein and tyrphostin A51 (30 microM) markedly reduced the effect of hemolysate on tyrosine phosphorylation. 4) In another study, hemolysate increased fibroblast collagen compaction and the effect of hemolysate was reduced by genistein and tyrphostin A51. We conclude that hemolysate activates tyrosine kinase that may lead to acceleration of fibroblast compaction. This effect of hemolysate may contribute to cerebral vasospasm.

Apoptosis in Cerebral Vasospasm After Subarachnoid Hemorrhage

We hypothesize that the mechanism of cerebral vasospasm after subarachnoid hemorrhage may not be a delayed and prolonged vascular response to certain spasmogens but a more complicated cascade, including contraction, cell death, and proliferation. This study was undertaken to demonstrate the apoptotic cell death induced by oxyhemoglobin in cultured endothelial cells in vitro, apoptosis in endothelium in vivo in a canine double hemorrhage model of experimental subarachnoid hemorrhage, and apoptosis in cerebral endothelial cells in a patient who died of cerebral vasospasm. (1) Oxyhemoglobin-produced endothelial apoptotic cell was demonstrated by apoptotic bodies [transmission electron microscopy (TEM)] and DNA ladders. (2) Endothelial cell damage and detachment were observed in dog middle cerebral arteries 5 days after blood injection. (3) In a patient who died 20 days after aneurysmal subarachnoid hemorrahge, endothelial detachment and apoptotic endothelial cells were demonstrated by TEM and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) reaction. These studies demonstrated that apoptosis may be an important step in vasospasm development. They opened a new avenue for treatment of vasospasm.