Ken Fujise

Physical and functional interaction between myeloid cell leukemia 1 protein (MCL1) and Fortilin. The potential role of MCL1 as a fortilin chaperone.

Myeloid cell leukemia 1 protein (MCL1) is an anti-apoptotic protein that is structurally related to Bcl-2. Unlike other Bcl-2 family proteins that are constitutively expressed, MCL1 is inducibly expressed in cells that are recently exposed to growth and differentiation stimuli. Here, we report the identification of fortilin as a novel MCL1-interacting protein by screening of a yeast two-hybrid library with MCL1 as bait. Fortilin specifically interacted with MCL1 both in vitro andin vivo. The intracellular localization of fortilin was predominantly nuclear and identical to that of MCL1, as shown by immunostaining and confocal microscopy analysis. Fortilin, like MCL1, was rapidly inducible in serum-stimulated human aortic vascular smooth muscle cells. Although the depletion of intracellular fortilin by small interfering RNA (siRNA) against fortilin (siRNA-fortilin) did not affect intracellular MCL1 level, the depletion of intracellular MCL1 by siRNA-MCL1 was associated with the significant reduction of the fortilin protein level, without affecting the fortilin transcript numbers. In addition, a pulse-chase experiment showed that the depletion of MCL1 by siRNA-MCL1 was associated with the rapid degradation of fortilin protein, which was found quite stable in the presence of MCL1. Furthermore, the half-life of fortilinR21A, a point mutant of fortilin lacking the binding to MCL1, was significantly shorter than that of wild-type fortilin as shown by a pulse-chase experiment. These data suggest that MCL1, in addition to being an anti-apoptotic molecule, serves as a chaperone of fortilin, binding and stabilizing fortilin in vivo. Taken together with our previous observation that fortilin overexpression prevents cells from undergoing apoptosis (Li, F., Zhang, D., and Fujise, K. (2001) J. Biol. Chem. 276, 47542–47549), it is likely that MCL1, an anti-apoptotic protein inducible by growth and differentiation stimuli, stabilizes another anti-apoptotic protein fortilin maximizing the prosurvival environment in cells.

Intracoronary adenosine administered during percutaneous intervention in acute myocardial infarction and reduction in the incidence of 'no reflow' phenomenon

Percutaneous intervention in acute myocardial infarction has been associated with a high incidence of “no reflow,” ranging from 11% to 30%, with an increased risk of complications. The role of intracoronary adenosine for the prevention of this phenomenon has not been evaluated fully. We studied the procedural outcomes of 79 patients who underwent percutaneous intervention in the context of acute myocardial infarction. Twenty‐eight patients received no intracoronary adenosine, and 51 received intracoronary adenosine boluses (24–48 μg before and after each balloon inflation). Eight patients who were not given adenosine experienced no reflow (28.6%) and higher rates of in‐hospital death, while only three of 51 patients (5.9%; P =0.014) in the adenosine group experienced no reflow. No untoward complications were noted during adenosine infusion. Intracoronary adenosine bolus administration during percutaneous intervention in the context of acute myocardial infarction is easy and safe and may significantly lessen the incidence of no reflow, which may improve the outcome of this procedure. Cathet. Cardiovasc. Intervent. 51:27–31, 2000.

Morelloflavone, a Biflavonoid, Inhibits Tumor Angiogenesis by Targeting Rho GTPases and Extracellular Signal-Regulated Kinase Signaling Pathways

Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action.

Infrapopliteal transcatheter interventions for limb salvage in diabetic patients: Importance of aggressive interventional approach and role of transcutaneous oximetry

OBJECTIVES This study sought to determine whether infrapopliteal transcatheter interventions can salvage ischemic limbs in diabetic patients referred for below the knee amputation at our institution. BACKGROUND The value of transcatheter interventions in diabetic crural arteries is controversial. Tissue oxygen partial pressure (TCO2) levels < 40 mm Hg predict poor wound healing. METHODS Percutaneous interventions were performed in 29 consecutive diabetic patients in need of limb salvage. Technical success was defined as < 20% residual vessel stenosis. Clinical success was defined as the avoidance of amputation and achievement of wound healing. At hospital discharge, patients were treated with Coumadin and aspirin. Ankle-brachial index (ABI) and TCO2 measurements were obtained before and after the intervention. RESULTS After 12-month follow-up, six patients had presistent wounds, whereas 23 experienced wound healing. Forty of the 50 infrapopliteal arteries successfully dilated were occluded, with a mean (+/-SD) lesion length of 18.0 +/- 3.5 cm. After the procedure, TCO2 improved from 27.82 +/- 9.97 mm Hg (95% confidence interval [CI] 23.95 to 31.69) to 54.5 +/- 14.73 mm Hg (95% CI 48.79 to 60.21, p < 0.0001), whereas the ABI did not (p > 0.2). TCO2 predicted procedural and clinical success (p < 0.0182). CONCLUSIONS Infrapopliteal transcatheter interventions in diabetic patients may salvage the majority of limbs doomed to amputation. Although TCO2 measurements are valuable in predicting wound healing and success after interventions, ABI measurements are not.

Adenosine use during aortocoronary vein graft interventions reverses but does not prevent the slow-no reflow phenomenon.

Slow or no reflow (SNR) complicates 10–15% of cases of percutaneous intervention (PI) in saphenous vein bypass graft (SVG). To date there have been limited options for the prevention and treatment of this common and potentially serious complication. We evaluated the procedural outcome of 143 consecutive SVG interventions. We compared patients who received pre‐intervention intra‐graft adenosine boluses with those who did not. In addition we examined the efficacy of adenosine boluses to reverse slow‐no reflow events. Angiograms were reviewed and flow graded (TIMI grade) by film readers blinded to the use of any intraprocedural drug or clinical history. Seventy patients received intragraft adenosine boluses before percutaneous intervention (APPI), 73 received no preintervention adenosine (NoAPPI). There were no significant angiographic differences between the two groups at baseline. A total of 20 patients experienced SNR. The incidence of SNR was similar in the two groups (APPI = 14.2% vs. NoAPPI = 13.6%, P = 0.9). SNR was treated with repeated, rapid boluses (24 μg each) of intra‐graft adenosine. Reversal of SNR was observed in 10 of 11 patients (91%) who received high doses of adenosine (≥5 boluses, mean 7.7 ± 2.6) and in 3 of 9 (33%) of those who received low doses (<5 boluses, mean 1.5 ± 1.2). Final TIMI flow was significantly better in the high dose than in the low dose group (final TIMI 2.7 ± 0.6 vs. 2 ± 0.8, P = 0.04). No significant untoward complications were observed during adenosine infusion. These findings suggest that SNR after PI in SVG is not prevented by pre‐intervention adenosine, but it can be safely and effectively reversed by delivery of multiple, rapid and repeated boluses of 24 μg of intra‐graft adenosine. Cathet. Cardiovasc. Intervent. 51:394–399, 2000.

Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

Outcome of access site in patients treated with platelet glycoprotein IIb/IIIa inhibitors in the era of closure devices

The most consistent procedural predictor of vascular access site complications thus far has been the intensity and duration of anticoagulant therapy during and after percutaneous coronary interventions (PCI). Several devices have been developed to aid in the closure of the femoral arteriotomy. This report describes the clinical outcome of unsuccessful deployment of femoral closure devices in a cohort of 285 consecutive patients who underwent PCI and were treated with platelet glycoprotein (GP) IIb/IIIa inhibitors. Manual femoral artery compression was used in 123 patients, Perclose in 123 patients, and AngioSeal in 39 patients. Successful homeostasis was achieved in 98.4% of patients who received manual compression, in 91.9% of the Perclose‐sealed arteriotomy, and in 84.6% of patients who received the AngioSeal closure device (P = 0.004). The incidence of vascular complications after successful deployment was 9%. Patients not achieving hemostasis with closure device or 1° manual compression developed complications in the majority of cases (> 80%; P < 0.05). By multivariate analysis (with adjustment for baseline differences), the use of AngioSeal closure device was found to be an independent risk factors leading to primary deployment failure and all access site complications (OR 2.97; 95% CI 1.5–6.0; P = 0.006). In summary, failed hemostasis by artery closure devices in patients undergoing PCI who are treated with GP IIb/IIIa inhibitors is associated with significant vascular complications. AngioSeal may be associated with a higher failure rate, while manual compression and Perclose seem to be more effective with a lower complication rate. Cathet Cardiovasc Intervent 2003;58:1–5.

Effects of clopidogrel pretreatment before percutaneous coronary intervention in patients treated with glycoprotein IIb/IIIa inhibitors (abciximab or tirofiban)

T clinical efficacy and safety of clopidogrel pretreatment in addition to glycoprotein (GP) IIb/IIIa antagonists during percutaneous coronary intervention (PCI) is unknown. This study compares the in-hospital clinical outcome of patients who received clopidogrel pretreatment before PCI with that in patients who did not receive it as adjunctive antiplatelet therapy to GP IIb/IIIa antagonists. • • • Data were collected from the Memorial Hermann Heart Center Interventional Cardiology database. We examined a consecutive series of 299 patients undergoing PCI. All patients received GP IIb/IIIa inhibitor therapy in the form of abciximab or tirofiban. Use and type of GP IIb/IIIb antagonists was at the discretion of the operator. Abciximab was given as a bolus dose of 0.25 mg/kg body weight followed by continuous infusion of 10 g/min for 12 hours. Tirofiban was given as a loading dose of 0.4 g/kg/min for 30 minutes followed by a maintenance dose of 0.1 g/kg/min for 12 to 24 hours after the procedure. All patients were taking aspirin and received a heparin bolus to achieve an activated clotting time between 250 and 300 seconds. Patients presenting to the catheterization laboratory with a developing myocardial infarction (MI) for a primary or rescue angioplasty were excluded. Patients were divided into 2 groups: those who received clopidogrel pretreatment before PCI (starting within 5 days before PCI or a loading dose of 300 mg the morning of the day of the procedure at the discretion of the primary operator, group I), and those who received aspirin alone (group II) with clopidogrel (300 mg loading dose and 75 mg/day for 1 month) given after stent deployment. Therapy with clopidogrel (75 mg/day) was continued for 1 month in both groups if the stent was deployed during the procedure. Coronary angioplasty and intracoronary stent implantation were performed using standard percutaneous techniques. Balloon size was selected to match the reference vessel diameter obtained from on-line angiographic analysis (1.1:1 balloon/artery ratio). Different types of stents were used (excluding open coil type). After stent implantation, high-pressure balloon dilatation was performed for angiographic optimization. Intravascular ultrasonography–guided coronary stenting was not performed in most patients. Each operator relied on his own judgment or on other objective measurements, such as online quantitative coronary angiography, to assess PCI results. On completion of the procedure, patients were moved to a monitored unit and the arterial sheath was removed. Successful PCI was defined as final residual stenosis within the treated lesion of 20%, with achievement of Thrombolysis In Myocardial Infarction grade 3 flow, without in-lab occurrence of death, MI, or a complication requiring immediate coronary revascularization. A major adverse cardiac event was defined as any 1 of the following: (1) Q-wave or non–Q-wave MI, (2) need for urgent repeat target vessel revascularization, or (3) cardiovascular death that occurred during the period of hospitalization after the index coronary procedure. Postprocedural MI was defined as the occurrence of typical ischemic chest pain of 30 minutes duration with a creatine kinase elevation of 3 times the upper limit of normal with an associated increase in the MB fraction. There was no routine protocol for acquisition of postprocedure creatine kinase. All postprocedural cardiac enzymes were obtained for suspected recurrent myocardial ischemia, manifested by recurrent postprocedural chest pain, hemodynamic instability, or new electrocardiographic changes of ischemia. Urgent target vessel revascularization was defined as a repeat PCI or coronary artery bypass grafting of the index artery due to presumed recurrent ischemia manifested by recurrent angina, arrhythmias, or hemodynamic compromise. Adverse effects of therapy were recorded and compared between the 2 groups. These consist of major bleeding and thrombocytopenia. Major bleeding was defined as bleeding requiring transfusion of blood products or precipitating hemodynamic compromise. Intracerebral hemorrhage of any extent was considered a major adverse effect of therapy. Thrombocytopenia was defined as platelet count 100,000/mm. Chi-square and Fischer exact tests were used for analysis of categorical variables when appropriate, and Student t testing was used for analysis of continuous variables. Multivariate logistic regression analysis was performed to determine significance of variables related to an in-hospital major adverse cardiac From the Cardiology Division, University of Texas Medical School and Hermann Heart Center, Memorial Hermann Hospital, Houston, Texas. Dr. Rosales’ address is: Division of Cardiology, University of Texas Health Science Center-Houston, PO Box 20708, Houston, Texas 77225-0708. Manuscript received March 29, 2001; revised manuscript received and accepted May 30, 2001.

Human fortilin is a molecular target of dihydroartemisinin

Dehydroartemisinin (DHA) is an effective anti‐malaria agent. Fortilin is an anti‐apoptotic molecule overexpressed in many human cancers. Here, we show that DHA binds human fortilin, increases the ubiquitination of fortilin, shortens fortilins half‐life in a proteasome‐dependent fashion, and reduces cellular levels of fortilin in varieties of cells. DHA induced DNA fragmentation in U2OS cells in a fortilin‐dependent manner. The fortilin‐knocked‐down cells were less susceptible—and fortilin‐overexpressing cells more susceptible—to DHA than were wild‐type cells, suggesting that apoptotic effects of DHA are—at least partly—conferred through fortilin. Together, these data suggest that fortilin is a molecular target of DHA. DHA and its derivative may prove to be viable anti‐cancer agents in fortilin‐overexpressing cancers.

Adenoviral Gene Transfer of Fortilin Attenuates Neointima Formation Through Suppression of Vascular Smooth Muscle Cell Proliferation and Migration

Background—Fortilin, a recently characterized nuclear antiapoptotic factor structurally distinct from inhibitor of apoptosis proteins (IAPs) and Bcl-2 family member proteins, has been suggested to be involved in cell survival and regulation of apoptosis within the cardiovascular system. In this continued investigation, we characterized the influence of adenovirus-mediated fortilin (Ad-fortilin) gene delivery on vascular remodeling after experimental angioplasty. Methods and Results—Vessel wall expression of Ad-fortilin or adenoviral luciferase (Ad-luc) was demonstrated 72 hours and 14 days after rat carotid artery (CA) balloon angioplasty. Morphometric analyses 14 days after injury revealed significantly diminished neointima development in the Ad-fortilin–treated CAs compared with Ad-luc or PBS controls, with no changes in medial wall morphometry observed between the 3 groups. The Ad-fortilin–treated CAs demonstrated a 50% reduction in medial wall proliferating cell nuclear antigen (PCNA) labeling after 72 hours, with significantly reduced neointimal and medial wall PCNA labeling and cell counts after 14 days. Terminal dUTP nick-end labeling results and morphological changes characteristic of programmed cell death suggest a trend toward reduced apoptosis in the fortilin-transfected balloon-injured vessels compared with Ad-luc injured controls. Temporal analysis of human aorta smooth muscle cell (SMC) proliferation demonstrated a marked time-dependent inhibition in Ad-fortilin treated SMCs without the influence of elevated apoptosis. Thymidine incorporation was significantly inhibited in the Ad-fortilin–treated cells compared with Ad-luc controls. Ad-fortilin transfected SMCs also demonstrated significantly decreased migration compared with Ad-luc controls. Conclusions—These cumulative results suggest that the novel antiapoptotic protein fortilin may play important redundant pathophysiological roles in modulating the vascular response to experimental angioplasty through suppression of SMC proliferation and migration concomitant with reduction of vessel wall apoptosis.