Toshinari Meguro

Cytotoxicity of cytokines in cerebral microvascular endothelial cell.

OBJECTIVEnSeveral studies reported that the levels of proinflammatory cytokines such as TNF-alpha, IL-1beta, IL-6, and IL-8 are elevated in the cerebrospinal fluid (CSF) of patients after subarachnoid hemorrhage (SAH). Cytokines in CSF may contribute to the development of vasospasm and cerebral ischemia. In the present study, we investigated the possible cytotoxic effects of these cytokines on cultured cerebral microvascular endothelial cells.nnnMETHODnThe effects of TNF-alpha, IL-1beta, IL-6, and IL-8 were tested using cell viability assay, DNA fragmentation analysis (DNA laddering), Western blot analysis (Anti-poly-(ADP-ribose) polymerase [PARP] antibody), and caspase-3 activity.nnnRESULTSnTNF-alpha and IL-1beta, but not IL-6 or IL-8, caused cell detachment in a dose-dependent manner (p<0.05). TNF-alpha (200 pg/ml) and IL-1beta (150 pg/ml) produced DNA ladders at 24-72 h. TNF-alpha but not IL-1beta cleaved the PARP from 116- to 85-kDa fragments and enhanced caspase-3 activity at 24-72 h after incubation with endothelial cells. Caspase-3 inhibitor at 10 micromol/l significantly prevented TNF-alpha-induced cell detachment (p<0.05).nnnDISCUSSIONnTNF-alpha induces apoptosis in cultured cerebral endothelial cells through the cleavage of caspase-3. IL-1beta decreases the adherent cells, produces DNA ladders, but fails to cleave PARP or increase caspase-3 activity. IL-1beta may induce apoptosis in cerebral endothelial cells through different pathway from that of TNF-alpha.

Oxyhemoglobin induces caspase‐mediated cell death in cerebral endothelial cells

Damaged endothelium is one of the pathological changes of the cerebral vasospastic vessels following subarachnoid hemorrhage. Our recent study shows that oxyhemoglobin (OxyHb) induces apoptosis in vascular endothelial cells. Apoptosis generally requires the action of various classes of proteases, including a family of cysteine proteases, known collectively as the caspases. This study was undertaken to investigate the activation of caspases and the efficacy of caspase inhibitors, z‐IETD‐fmk and z‐LEHD‐fmk, for oxyhemoglobin‐induced apoptosis in vascular endothelial cells. Cultured bovine brain microvascular endothelial cells (passages 5–9) were used for this study. OxyHb (10u2003µmol/L) was added during the 24–72u2003h incubation with and without caspase‐8 or −u200a9 inhibitors (z‐IETD‐fmk and z‐LEHD‐fmk). Counting surviving cells, DNA laddering, western blotting of poly(ADP‐ribose) polymerase, and measurement of caspase activities were employed to confirm the cytotoxic effects of OxyHb and the protective effects of the caspase inhibitors. OxyHb produced cell detachment in a time‐dependent manner and increased caspase‐8 and ‐9 activities in the cells. z‐IETD‐fmk and z‐LEHD‐fmk (100u2003µmol/L) attenuated OxyHb‐induced cell loss, DNA laddering, and proteolytic cleavage of PARP, although a lower concentration (10u2003µmol/L) of caspase inhibitors showed partial effects. OxyHb activates caspase‐8 and ‐9 in cultured vascular endothelial cells, and blocking the action of the caspases with the inhibitors efficiently prevents loss of vascular endothelial cells from OxyHb‐induced apoptosis in vitro. These results suggest that the caspase cascade participates in OxyHb‐induced apoptosis.

Improved rat model for cerebral vasospasm studies

Abstract While the rat has been used extensively in subarachnoid hemorrhage (SAH)-cerebral vasospasm studies, concerns exist whether this animal represents a usable model because its time course and pattern of cerebral vasospasm following SAH is not comparable to that observed in man. At present, our knowledge of the rat model is based almost exclusively on studies using a single hemorrhage method. Since there is a positive correlation between severity of cerebral vasospasm, and volume of subarachnoid blood, an obvious question is whether the rat will show modifications in vascular responses when insulted by a second SAH. Here, an SAH was produced in rats using a double hemorrhage method. Following SAH, cerebral arteries showed pathological alterations, significant decreases in luminal perimeter, and increases in arterial wall thickness, over a 7-day post-SAH period. The above vascular features are considered to be indicative of cerebral vasospasm and their presence over a 7-day post-SAH period represents a significant time extension when compared to a single hemorrhage. These modified vascular responses made the double hemorrhaged rat a much-improved animal model. [Neurol Res 2001; 23: 761-766]

Caspase Inhibitors Attenuate Oxyhemoglobin-Induced Apoptosis in Endothelial Cells

Background and Purpose — Our recent study showed that oxyhemoglobin (OxyHb) induces apoptosis in cultured endothelial cells. Apoptosis requires the action of various classes of proteases, including a family of cysteine proteases known collectively as the caspases. This study was undertaken to investigate the effect of 2 caspase inhibitors, Z-VDVAD-FMK and Z-DEVD-FMK, in the protection of endothelial cells from OxyHb-induced apoptosis. Methods — Cultured bovine brain microvascular endothelial cells (passages 5 to 9) were exposed to OxyHb (10 &mgr;mol/L) for 24 to 72 hours with and without caspase inhibitors. Cell attachment, DNA ladder, Western blotting of poly(ADP-ribose) polymerase (PARP), and caspase activities were measured to confirm the cytotoxic effect of OxyHb and the protective effect of the caspase inhibitors. Results — (1) OxyHb produced cell detachment in a time-dependent manner. (2) OxyHb increased caspase-2 and -3 activities, produced DNA ladders, and cleaved PARP in endothelial cells. (3) Z-VDVAD-FMK and Z-DEVD-FMK (100 &mgr;mol/L) attenuated OxyHb-induced cell detachment, reduced caspase-2 and -3 activities, abolished OxyHb-induced DNA ladders, and prevented OxyHb-induced cleavage of PARP. Conclusions — OxyHb activates caspase-2 and -3 in cultured brain microvessel endothelial cells. Caspase inhibitors attenuated the cytotoxic effect of OxyHb.

Efficacy of intrathecal liposomal fasudil for experimental cerebral vasospasm after subarachnoid hemorrhage.

OBJECTIVETo investigate the safety and efficacy of liposomal fasudil in a sustained-release form for the prevention of cerebral vasospasm after subarachnoid hemorrhage (SAH). METHODSEighteen rats were divided into three groups, each of which received 2.5 mg/kg of liposomal fasudil, 5 mg/kg of liposomal fasudil, or drug-free liposomes after SAH. Next, experimental SAH was induced in 15 dogs by injection of autologous arterial blood into the cisterna magna twice after baseline vertebral angiography. In six dogs, 0.94 mg/kg of liposomal fasudil was injected into the cisterna magna (treatment group). In four dogs, drug-free liposomes were similarly injected (placebo group), and the remaining five dogs were not treated with liposomal injection after SAH (control group). Angiography was repeated on Day 7, and cerebrospinal fluid was collected before the dogs were killed. RESULTSA high dose of liposomal fasudil caused no significant changes in mean arterial blood pressure and did not induce seizures during the observation period. Gross and microscopic examination of the brains revealed no abnormalities, but severe vasospasm was noted in the rat basilar artery, mainly in the group treated with drug-free liposomes. Likewise, in the canine placebo and control groups, significant vasospasm occurred in the basilar artery on Day 7. In the treatment group, vasospasm in the basilar artery was significantly ameliorated (P < 0.01). In vivo, 90% of fasudil was released from liposomes in the cerebrospinal fluid. CONCLUSIONA single injection of intrathecal liposomal fasudil is safe and effective for the prevention of vasospasm in experimental SAH.

Intrathecal application with liposome-entrapped Fasudil for cerebral vasospasm followingsubarachnoid hemorrhage in rats

To date, the pharmacological approach to cerebral vasospasm following subarachnoid hemorrhage has been hampered in part by an inability to attain sufficiently high concentrations of vasodilator drugs in the cerebrospinal fluid (CSF). To overcome this limitation of current drug therapy, we have developed a sustained-release preparation of protein kinase inhibitor Fasudil. Cerebral vasospasm in rats was induced by double-injection method. Treated rats received 0.417 mg liposome-entrapped Fasudil via the cisterna magna and control rats received drug-free liposomes in the same manner. The diameter of the basilar artery was assessed at 7 days after the initial blood injection. Vasoconstriction of the rat basilar artery was significantly reduced in group treated with liposomal Fasudil compared to the control group (treated group: 87.7 +/- 6.18%, n= 10; control group: 66.3 +/- 9.82%, n = 10; ***P< 0.001). This new approach for cerebral vasospasm may have significant potential for use in the clinical setting.

Role of MAPK in chronic cerebral vasospasm.

Many extracellular stimuli (including growth factors, hormones, stress, elevated temperature and G-protein coupled receptor agonists) initialize the phosphorylation cascades leading to the activation of mitogen-activated protein kinase (p44/42 MAPK) [2]. p44/42 MAPK activation may lead to an expression of the early genes, such as c-jun or c-fos, thus leading to the cellular proliferation [3]. p44/42 MAPK can cause the phosphorylation of caldesmon, thus promoting prolonged contraction [1]. It has been reported in several in vitro studies that p44/42 MAPK is involved in the prolonged contraction of the cerebral arteries, caused by hemolysate [8] and endothelin-1 [9], the most important causative agents for vasospasm. The levels of c-fos, and c-jun were increased in the presence of hemolysate, reaching maximum expression between 30 and 60 minutes in rat aorta smooth muscle cells [7].

A Novel Drug Delivery System as Prophylaxis for Cerebral Vasospasm

Experimental work has extensively demonstrated that calcium antagonists can reverse established angiographically identified vasospasm when administered by the intrathecal route, despite being ineffective when administered by the peripheral route [3, 5]. However, the effective time in the therapeutic window may be temporary even if bolus application by intrathecal route was feasible. Therefore, externalized ventricular catheter or repeated lumbar puncture is needed to keep a therapeutic drug concentration in the cerebrospinal fluid (CSF), which is troublesome in a clinical setting.