Betty Chen

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 (10 µmol/L) was added during the 24–72 h incubation with and without caspase‐8 or − 9 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 (100 µmol/L) attenuated OxyHb‐induced cell loss, DNA laddering, and proteolytic cleavage of PARP, although a lower concentration (10 µmol/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.

Calcium entry via ORAI1 regulates glioblastoma cell proliferation and apoptosis.

INTRODUCTION Calcium entry plays a critical role in the proliferation and survival of certain tumors. Ca(2+) release activated Ca(2+) (CRAC) channels constitute one of the most important pathways for calcium entry especially that of store-operated calcium entry (SOCE). ORAI1 and stromal interaction molecule1 (STIM1) are essential protein components of CRAC channels. In this study we tested the effect of inhibiting CRAC through ORAI1 and STIM1 on glioblastoma multiforme (GBM) tumor cell proliferation and survival. METHODS Two glioblastoma cell lines, C6 (rat) and U251 (human), were used in the study. ORAI1 and STIM1 expressions were examined using Western blot and immunohistochemistry. CRAC channel activity and its components were inhibited with ion channel blockers and using siRNA knockdown. Changes in intracellular calcium concentration were recorded using Fura-2 fluorescent calcium imaging. Cell proliferation and apoptosis were examined using MTS and TUNEL assays, respectively. RESULTS CRAC blockers, such as SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole), 2-aminoethoxydiphenyl borate (2-APB) and Diethylstilbestrol (DES), inhibited cell proliferations and SOCE in GBM cells. Knockdown of ORAI1 and STIM1 proteins using siRNA significantly inhibited C6 cell proliferation and SOCE compared with those in control cells, and a more significant effect was observed in cells with ORAI1 siRNA knockdown than that of STIM1-treated cells. Both CRAC blockers and siRNA treatments increased apoptosis in C-6 cells compared with control. CONCLUSION Calcium entry via ORAI1 and CRAC channels are important for GBM proliferation and survival.

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.

20-Hydroxyeicosatetraenoic Acid Inhibition Attenuates Balloon Injury-Induced Neointima Formation and Vascular Remodeling in Rat Carotid Arteries

20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to the migration and proliferation of vascular smooth muscle cells (VSMC) in vitro, but there are few studies that address its effects on vascular remodeling in vivo. The present study determined whether inhibition of 20-HETE production attenuates intimal hyperplasia (IH) and vascular remodeling after balloon injury (BI). Sprague Dawley rats underwent BI of the common carotid artery and were treated with vehicle, 1-aminobenzotriazole (ABT, 50 mg/kg i.p. once daily), or HET0016 (N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine) (2 mg/kg s.c. twice daily) for 14 days. Fourteen days after BI and treatment, the animals underwent carotid angiography, and the arteries were harvested for morphometric, enzymatic and immunohistochemical analysis. There was a 96% reduction of angiographic stenosis in the rats treated with 1-ABT. There was a 61 and 66% reduction of the intima/media area ratios in the 1-ABT and HET0016 treated rats compared with the vehicle-treated group. 20-HETE levels were elevated in BI carotid arteries, and the levels were markedly suppressed in the groups treated with 1-ABT and HET0016 (P < 0.001). Immunostaining revealed that the expression of CYP4A enzyme was markedly increased in the neointima of BI arteries, and it colocalized with the expression of smooth muscle-specific actin, indicating increased proliferation of VSMC. An increase in the expression of CYP4A and the production of 20-HETE contributes to neointimal growth in BI rat carotid arteries. Systemic administration 1-ABT or HET0016 prevents the increase in 20-HETE levels and attenuates VSMC migration and proliferation, resulting in a marked reduction in IH and vascular remodeling after endothelial injury.

Angiographic evaluation of the rat carotid balloon injury model.

RATIONALE The rat carotid balloon-injury (BI) model is a widely used model of intimal hyperplasia (IH) and vascular remodeling. A variable degree of IH after BI has been previously reported, and we have encountered technical challenges and suboptimal results with the original method. OBJECTIVE To evaluate the original rat carotid artery BI method with the use of micro-angiography. We tested the hypothesis that in order to obtain an optimal arterial response, BI should be limited to the common carotid artery with preservation of blood flow. METHODS AND RESULTS The left common carotid artery (CCA) was injured by one of three different methods. Carotid angiograms and pathology were examined 14 days after BI. A 2F Fogarty balloon catheter inflated to 2 atm inside the aortic arch would not slide back into the common carotid artery until deflation to 0.5 to 0.7 atm. Four out of five (80%) vessels injured with this method developed excessive inflammation without discernible IH. Six out of nine (66%) arteries that underwent BI limited to the CCA at 2 atm developed the largest angiographic stenosis (p=0.003) and IH (0.20±0.03 mm(2), p=0.028). Ten out of eleven (91%) arteries injured with a variable pressure of 1.5 to 2.2 atm, based on the operators feedback, developed considerable IH (0.12±0.02 mm(2)). All injured carotid arteries with preserved blood flow on angiography developed IH with intact histological boundaries. CONCLUSIONS Optimal IH with preservation of histological boundaries is achieved by graded BI limited to the CCA that preserves carotid blood flow.

Aortic response to balloon-injury in the obese Zucker rat: a translational animal model for endovascular interventions

Background: The small diameter of the carotid balloon-injury (BI) model impedes the evaluation of available endovascular devices. We developed an endovascular BI model in the rat’s descending aorta that is compatible with available endovascular instruments. This study also tested the hypothesis that neointimal formation is enhanced in the aorta of obese Zucker (OZ) rats. Methods: Left external carotid arteriotomies and BI of the thoracic and abdominal aorta were performed using a Gateway balloon catheter. Aortograms and aortic pathology were examined at 2, 4 and 10 weeks. Results: Ten weeks after BI the OZ abdominal aorta narrowed 8.34 ± 1.10 % vs. an expansion of 2.36 ± 2.24 % in the lean rat (LZ) (p < 0.001). Simultaneously, the LZ thoracic aorta expanded 9.50 ± 4.27 % vs. a stenosis of 2.78 ± 1.65 % in the OZ (p = 0.003). A significant increase in neointimal formation, as measured by the intima to media (I:M) ratio, was observed in the OZ descending aorta (p <0.001). Conclusions: This is a minimally invasive BI model to the rat’s descending aorta compatible with available endovascular instruments. Following BI the OZ descending aorta demonstrates enhanced neointimal formation and constrictive vascular remodeling.