Citations

Objective—The ubiquitin-proteasome system is the principal degradation route of intracellular and oxidized proteins, thus regulating many cellular processes conceivably important for atherosclerosis. The aim of this study was to evaluate the activity of ubiquitin-proteasome system in human carotid artery plaques in relation to oxidative stress and clinical manifestation.Methods and Results—In carotid endarterectomy specimens from 83 asymptomatic and 94 symptomatic patients, content of ubiquitin, ubiquitin conjugates, matrix metalloproteases, and NADPH-oxidase-p67 was evaluated by immunoblotting; proteolytic proteasome activity by fluorometric assay; single and double immunostaining for ubiquitin conjugates, 3-nitrotyrosine, apoptosis, smooth muscle α-actin, and macrophage CD-68, as well as Sirius Red staining for collagen were performed. Compared with asymptomatic patients, symptomatic patients showed a more unstable plaque phenotype, an increased degree of apoptosis, a significantly higher ubiquitin conjugates content (17.72±1.36 versus 10.99±1.04;P<0.001), and lower proteasome activity (5.01±0.70 versus 9.41±1.19 nmol AMC/mg protein/min;P<0.01). Ubiquitin conjugates content was directly correlated to NADPH-p67 and degree of apoptosis. Immunostaining revealed colocalization of ubiquitin conjugates and 3-nitrotyrosine, and accumulation of ubiquitin conjugates in smooth muscle cells and macrophages.Conclusions—In human carotid plaques increased oxidative stress is associated with inhibition of the proteasome activity and accumulation of ubiquitin conjugates, particularly in symptomatic patients. These results suggest a possible role of the ubiquitin-proteasome system in influencing plaque stability.

Abstract We previously identified HSulf-1 as a down-regulated gene in several tumor types including ovarian, breast, and hepatocellular carcinomas. Loss of HSulf-1, which selectively removes 6-O-sulfate from heparan sulfate, up-regulates heparin-binding growth factor signaling and confers resistance to chemotherapy-induced apoptosis. Here we report that HSulf-1 expression in MDA-MB-468 breast carcinoma clonal lines leads to reduced proliferation in vitro and reduced tumor burden in athymic nude mice in vivo. Additionally, xenografts derived from HSulf-1–expressing stable clones of carcinoma cells showed reduced vessel density, marked necrosis, and apoptosis, indicative of inhibition of angiogenesis. Consistent with this observation, HSulf-1–expressing clonal lines showed reduced staining with the endothelial marker CD31 in Matrigel plug assay, indicating that HSulf-1 expression inhibits angiogenesis. More importantly, HSulf-1 expression in the xenografts was associated with a reduced ability of vascular endothelial cell heparan sulfate to participate in a complex with fibroblast growth factor 2 (FGF-2) and its receptor tyrosine kinase FGF receptor 1c. In vitro, short hairpin RNA–mediated down-regulation of HSulf-1 in human umbilical vein endothelial cells (HUVEC) resulted in an increased proliferation mediated by heparan sulfate–dependent FGF-2, hepatocyte growth factor, and vascular endothelial growth factor 165 (VEGF165) but not by heparan sulfate–independent VEGF121. HSulf-1 down-regulation also enhanced downstream signaling through the extracellular signal–regulated kinase pathway compared with untreated cells. Consistent with the role of heparan sulfate glycosaminoglycan sulfation in VEGF-mediated signaling, treatment of HUVEC cells with chlorate, which inhibits heparan sulfate glycosaminoglycan sulfation and therefore mimics HSulf-1 overexpression, led to an attenuated VEGF-mediated signaling. Collectively, these observations provide the first evidence of a novel mechanism by which HSulf-1 modulates the function of heparan sulfate binding VEGF165 in proliferation and angiogenesis. (Cancer Res 2006; 66(12): 6025-32)

Abstract Objective: Spontaneous intracranial hypotension is typically caused by a spontaneous spinal cerebrospinal fluid (CSF) leak. The configuration of the related dural defects can be complex. We describe our experience with the surgical anatomy of these defects. Methods: Thirteen consecutive patients with spontaneous spinal CSF leaks who underwent surgical exploration at Mayo Clinic between 1994 and 2003 were studied. All patients' records, imaging studies, detailed intraoperative findings, and outcomes were reviewed. Results: There were four men and nine women with an average age of 40 years (range, 12–62 yr). Preoperative imaging studies revealed a single site of CSF leak in eight patients, two sites in three patients, and multiple sites in two patients. Intraoperatively, the exact site of leakage could not be found in four patients. Among the other nine patients, primary closure of a meningeal diverticulum was achieved in one patient. Significant regional attenuation of the dura prevented primary repair of the leak site in eight patients. Muscle, fibrin glue, and Gelfoam (Upjohn Co., Kalamazoo, MI) soaked in patient's own blood were commonly used to pack the epidural space in an attempt to seal the site of the leak. Ligation of two nonappendicular nerve roots allowed closure of the leak in one of these patients. Postoperatively, resolution of symptoms occurred in eight patients, significant improvement was noted in three patients, and only transient resolution in two. The mean duration of follow-up was 20.5 months. Conclusion: Surgery for closure of spontaneous spinal CSF leaks may not be straightforward. Even when extradural CSF leakage is discovered preoperatively by imaging studies, it may not always be possible to identify the exact site of the leakage intraoperatively. Furthermore, the anatomy of the dural defects may be complex and not amenable to primary closure. In such cases, the use of adjuvant techniques during surgical exploration may be effective.

AbstractMetastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options. (Cancer Res 2006; 66(4): 2328-37)

Purpose This phase I study was undertaken to define the toxicity, pharmacokinetics, pharmacodynamics, maximum tolerated dose (MTD), and clinical activity of CI-1040, a small-molecule inhibitor of the dual-specificity kinases MEK(mitogen-activated protein kinase kinase) -1 and MEK2 , in patients with advanced malignancy. Patients and Methods CI-1040 was tested in multiple daily dosing frequencies administered for 21 days repeated every 28 days leading ultimately to continuous administration, and effect of food on absorption was tested. Single dose and steady-state pharmacokinetics were assessed during cycle 1 and phosphorylated extracellular receptor kinase (pERK) levels were assessed in WBCs and also in tumor tissue from selected patients. Results Seventy-seven patients received CI-1040 at dose levels ranging from 100 mg QD to 800 mg tid. Grade 3 asthenia was dose limiting at the highest dose level tested, 800 mg tid administered with food. Ninety-eight percent of all drug-related adverse events were grade 1 or 2 in severity; most common toxicities included diarrhea, asthenia, rash, nausea, and vomiting. Plasma concentrations of CI-1040 and its active metabolite, PD 0184264, increased in a less than dose proportional manner from 100 to 800 mg QD. Administration with a high-fat meal resulted in an increase in drug exposure. The MTD and recommended phase II dose was 800 mg BID administered with food. Sixty-six patients were assessable for response. One partial response was achieved in a patient with pancreatic cancer and 19 patients (28%) achieved stable disease lasting a median of 5.5 months (range, 4 to 17 months). Inhibition of tumor pERK (median, 73%; range, 46% to 100%) was demonstrated in 10 patients. Conclusion CI-1040 was well tolerated at 800 mg BID administered with food. Both target suppression and antitumor activity were demonstrated in this phase I study.

Background— Heart failure (HF) with normal ejection fraction (diastolic HF [DHF]) usually occurs in elderly patients with hypertension. The presence and significance of altered systolic and diastolic ventricular function in DHF is increasingly controversial. Our objective was to develop a clinically relevant large-animal model to better understand the pathophysiology of DHF. Methods and Results— Ventricular structure and function were characterized in young control (YC group; n=6), old control (OC group; n=7), and old dogs made hypertensive by renal wrapping (experimental DHF [ExDHF] group; n=8). The ExDHF group was associated with normal left ventricular (LV) volume, increased LV mass, and myocardial fibrosis. LV relaxation was impaired in ExDHF (τ=53±6 ms) compared with OC (τ=35±3 ms; P <0.05) and YC (τ=33±6 ms; P <0.05) dogs. The percent diastole at which relaxation is complete was increased in ExDHF (116±30%) compared with OC (69±8%; P <0.05) and YC (35±5%; P <0.05) dogs. The coefficient of LV diastolic stiffness was similar in OC, YC, and ExDHF dogs. Diastolic pressures increased dramatically in response to increases in blood pressure. End-systolic LV stiffness was enhanced in ExDHF dogs and after load enhancement of myocardial performance was maintained. Arterial stiffness was increased in ExDHF dogs. Conclusions— Aged dogs with chronic hypertension exhibit LV hypertrophy and fibrosis with impaired LV relaxation but no increase in the coefficient of LV diastolic stiffness. LV systolic and arterial stiffness are increased, which may exacerbate load-dependent impairment of relaxation and contribute to increased filling pressures with hypertensive episodes. This model mimics many of the structural and functional characteristics described in the limited studies of human DHF and provides insight into the pathogenesis of DHF.

Background and Purpose— The local renin-angiotensin system (RAS) and cyclooxygenase-2 contribute to the activation of nuclear factor κB (NFκB) and C-reactive protein (CRP). We hypothesized that the combination of RAS blockers (RASb) and ASA reduces NFκB and CRP within atherosclerotic plaques. Methods— Patients undergoing carotid endarterectomy were divided into groups according to treatment (RASb–acetylsalicylic acid [ASA], ASA, RASb, and control). The expression of NFκB, CRP, and CD40L was analyzed through Western blots in the obtained plaques. Results— Plaques from patients treated with the combination of RASb and ASA showed lower expression of NFκB (25.4±9.8 densitometric units [DU]) than those of the control group (57.6±13.2 DU, P =0.03) as well as lower expression of CRP (20.9±9.6 DU) than those of the other treatment groups (ASA 86.1±13 DU, RASb 88.4±31 DU, controls 67.8±18.6, P =0.004). A negative expression of NFκB was associated with a reduced incidence of symptoms compared with a positive expression (5/33 [15.1%] versus 14/35 [40%], P =0.031). Conclusions— The combined treatment with RASb and ASA decreases the expression of inflammatory markers in atherosclerosis in humans. This study supports the role of the local RAS and cyclooxygenase-2 in the progression of atherosclerosis.