Nivolumab in sorafenib-naive and sorafenib-experienced patients with advanced hepatocellular carcinoma: 5-year follow-up from CheckMate 040.

BACKGROUND: Patients with advanced hepatocellular carcinoma (aHCC) have a poor prognosis and high mortality. Nivolumab monotherapy demonstrated clinical benefit with an acceptable safety profile in patients with aHCC in the CheckMate 040 study. Five-year follow-up of the sorafenib-naive and sorafenib-experienced groups of CheckMate 040 is presented here. PATIENTS AND METHODS: Patients received nivolumab monotherapy at dose levels of 0.1-10.0 mg/kg (dose-escalation phase) or 3 mg/kg (dose-expansion phase) every 2 weeks until disease progression or unacceptable toxicity. Primary endpoints were safety and tolerability (dose escalation), and objective response rate (ORR) by blinded independent central review (BICR) and by investigator as per RECIST version 1.1 (dose expansion). RESULTS: Eighty sorafenib-naive and 154 sorafenib-experienced patients were treated. Minimum follow-up in both groups was 60 months. ORR as per BICR was 20% [95% confidence interval (CI) 12% to 30%] and 14% (95% CI 9% to 21%) in the sorafenib-naive and sorafenib-experienced groups, respectively. Responses occurred regardless of HCC etiology or baseline tumor cell programmed death-ligand 1 (PD-L1) expression levels. Median overall survival (OS) was 26.6 months (95% CI 16.6-30.6 months) and 15.1 months (95% CI 13.0-18.2 months) in sorafenib-naive and sorafenib-experienced patients, respectively. The 3-year OS rates were 28% in the sorafenib-naive and 20% in the sorafenib-experienced groups; 5-year OS rates were 14% and 12%, respectively. No new safety signals were identified; grade 3/4 treatment-related adverse events were observed in 33% and 21% of patients in the sorafenib-naive and sorafenib-experienced groups, respectively. Biomarker analyses showed that baseline PD-L1 expression ≥1% was associated with higher ORR and longer OS compared with PD-L1 <1%. In the sorafenib-naive group, patients with OS ≥3 years exhibited higher baseline CD8 T-cell density compared with those with OS <1 year. CONCLUSION: With 5 years of follow-up, nivolumab monotherapy continued to provide durable clinical benefit with manageable safety in sorafenib-naive and sorafenib-experienced patients with aHCC.

Systemic delivery of the interleukin-1 receptor antagonist protein using a new strategy of direct adenoviral-mediated gene transfer to skeletal muscle capillary endothelium in the isolated rat hindlimb.

Current gene therapy strategies using adenoviral vectors to target the lung or liver have been complicated by an acute inflammatory response that can result in loss of transgene expression as well as tissue injury and necrosis. Skeletal muscle comprises 40% of total body weight; it possesses a high density, accessible capillary network that is resistant to injury and thus may be a logical target for adenoviral vectors. We hypothesized that adenoviral transduction of the rat skeletal muscle capillary bed during vascular isolation would achieve efficient gene transfer sufficient to achieve systemic serum levels of a recombinant protein without significant tissue injury. During vascular isolation of the hindleg, a replication-incompetent adenovirus (Ad) encoding for either the marker gene, human placental alkaline phosphatase (hpAP), or interleukin-1 receptor antagonist (IL-1ra) was infused and subsequently flushed from the circulation after a 30-min dwell period. Gene transfer over a 10(9)-10(12) particle/ml range to the gastrocnemius capillary endothelium and muscle fibers was highly efficient and titer-dependent, reaching maximum transduction rates of 71 +/- 7% and 25 +/- 5%, respectively, 5 days after gene transfer (n = 3-8 rats/group, p < 0.05). hpAP transgene expression was barely detectable at 14 days. No significant tissue injury or necrosis of the skeletal muscle was observed at 5 and 14 days, and distant organ gene transfer was minimal or absent. Gastrocnemius muscle from rats (n = 4) given Ad-IL-1ra had 241 +/- 66 pg IL-1ra/mg protein at 5 days, while those given Ad-hpAP, negative control (n = 3) had 35 +/- 14 pg IL-1ra/mg protein (p < 0.05). Ad-IL-1ra rats (n = 4) had serum levels of 185 +/- 20 pg/ml IL-1ra at 5 days whereas Ad-hpAP control rats (n = 5) had no IL-1ra detectable (p < 0.0001). Athymic rats given Ad-IL-1ra (n = 6) had serum levels of 493 +/- 62 pg/ml IL-1ra 14 days after transduction, and IL-1ra was detected for up to 98 days. Sera from Ad-IL-1ra athymic rats significantly inhibited IL-1 beta-induced (1 ng/ml) prostaglandin E2 (PGE2) production from cultured endothelial cells by 82 +/- 2% (p < 0.001). Thus, this gene transfer strategy is the first to result in substantial transduction of both skeletal muscle capillary endothelium and fibers, sufficient to achieve pharmacologic levels of IL-1ra. Although no acute tissue injury or necrosis was observed, persistence of transgene expression in athymic rats suggests that loss of expression in normal rats was by an immune-mediated mechanism.

Gastrin-releasing peptide stimulation of amylase release from rat pancreatic lobules involves intrapancreatic neurons.

Gastrin releasing peptide (GRP) immunoreactivity has been localized to nerve fibers innervating pancreatic acini and identified in nerve cell bodies within intrapancreatic ganglia. The role of intrapancreatic neurotransmission in GRP- and neuromedin C (NmC)-stimulated amylase release was investigated using rat pancreatic lobules in vitro. Lobule responsiveness to neuronal depolarization was demonstrated by amylase release upon exposure to 55 mM potassium (207 +/- 7% of control) or veratridine (294 +/- 12%). Both GRP and NmC produced dose-dependent increases in lobular amylase release, with ED50 values of 1.1 nM and 0.13 nM, respectively. Amylase release in response to submaximal concentrations of GRP were significantly inhibited by tetrodotoxin (78 +/- 5% of control) or hexamethonium (71 +/- 5% of control). GRP-stimulated amylase release was decreased to 71 +/- 5% of control by atropine coincubation. NmC-stimulated amylase release was not affected by tetrodotoxin, hexamethonium, or atropine. GRP (10(-10) to 10(-6) M) produced dose-dependent increments in [3H]acetylcholine release from pancreatic lobules. GRP stimulates amylase release from rat pancreatic lobules by a neurally mediated mechanism in addition to direct action on acinar membrane receptors.

The clinical presentation and prognostic factors for intrahepatic and extrahepatic cholangiocarcinoma in a tertiary care centre.

BACKGROUND: The incidence of cholangiocarcinoma is rising. Accurate predictors of survival at diagnosis are not well defined. AIM: To clarify the clinical presentation and prognostic factors of intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma in a contemporary cohort of patients. METHODS: Records for consecutive patients at the University of Michigan hospital diagnosed with cholangiocarcinoma between January 2003 and April 2008 were reviewed. RESULTS: In all, 136 patients had cholangiocarcinoma (79 intra- and 57 extrahepatic cholangiocarcinoma). Median survival was 27.3 months-25.8 months for intrahepatic cholangiocarcinoma and 30.3 months for extrahepatic cholangiocarcinoma. Independent predictors of mortality at presentation on multivariate analysis were elevated bilirubin level (HR 1.04, 95%CI 1.01-1.07), CA 19-9 levels >100 U/mL (HR 1.90, 95%CI 1.17-3.08) and stage of disease (HR 1.51, 95%CI 1.16-1.96). After adjusting for baseline prognostic factors, surgical therapy was associated with improved survival (HR 0.48; 95% CI 0.26-0.88). There were no significant differences regarding clinical presentation, disease stage (P = 0.98), and survival (P = 0.51) between intra- and extrahepatic cholangiocarcinoma. CONCLUSIONS: Survival for cholangiocarcinoma remains poor with no significant difference in outcomes between intra- and extrahepatic cholangiocarcinoma. Stage of disease, bilirubin level and CA 19-9 level are important prognostic factors at presentation. Surgical therapy provides similar efficacy for both tumours when adjusted for other prognostic variables.

Financial implications of pancreas transplant complications: a business case for quality improvement.

We quantified the financial implications of surgical complications following pancreas transplantation. We reviewed medical and financial records of 49 pancreas transplant recipients at the University of Michigan Health System (UMHS) between 1/6/2002 and 11/22/2004. The association of donor, transplant recipient and financial variables was assessed. The median costs to UMHS of procedures and follow-up were $92,917 for recipients without surgical complications versus $108,431 when a surgical complication occurred, a difference of $15,514 (p = 0.03). Median reimbursement by the payer was $17,363 higher in patients with a surgical complication (p = 0.001). Similar trends (higher insurer costs) were noted when stratifying by payer (public and private) and specific procedure (SPK and PAK). All parties (patient, physician, payer and medical center) should benefit from quality improvement, with payers having a financial interest in pancreas transplant surgical quality initiatives.

Who pays for biliary complications following liver transplant? A business case for quality improvement.

We use biliary complication following liver transplantation to quantify the financial implications of surgical complications and make a case for surgical improvement initiatives as a sound financial investment. We reviewed the medical and financial records of all liver transplant patients at the UMHS between July 1, 2002 and June 30, 2005 (N = 256). The association of donor, transplant, recipient and financial data points was assessed using both univariable (Student's t-test, a chi-square and logistic regression) and multivariable (logistic regression) methods. UMHS made a profit of $6822 +/- 39087 on patients without a biliary complication while taking a loss of $5742 +/- 58242 on patients with a biliary complication (p = 0.04). Reimbursement by the payer was $5562 higher in patients with a biliary complication compared to patients without a biliary complication (p = 0.001). Using multivariable logistic regression analysis, the two independent risk factors for a negative margin included private insurance (compared to public) (OR 1.88, CI 1.10-3.24, p = 0.022) and biliary leak (OR = 2.09, CI 1.06-4.13, p = 0.034). These findings underscore the important impact of surgical complications on transplant finances. Medical centers have a financial interest in transplant surgical quality improvement, but payers have the most to gain with improved surgical outcomes.

Excessive oral amphetamine use as a possible cause of renal and splanchnic arterial aneurysms: a report of two cases.

INTRODUCTION: Multiple visceral aneurysms are uncommon and usually result from connective tissue diseases, systemic arteritis, or mycotic lesions. An association between multiple visceral aneurysms and excessive oral amphetamine use has not been reported. METHODS: The clinical features of 2 patients at the University of Michigan Medical Center for treatment of multiple visceral aneurysms and amphetamine use were reviewed. RESULTS: The patients had histories of excessive oral amphetamine use that ranged from 50 mg daily for 22 years to 200 mg daily for 2 years. No evidence was seen of systemic arteritis, connective tissue disorder, or an infectious process that may have caused the aneurysms. The arteriograms documented multiple splanchnic and renal artery aneurysms that involved both the large and the small arteries. The aneurysms of 1 patient were managed conservatively, and the patient has not had any clinical sequelae of the aneurysms during 14 years of follow-up. The second patient had hematobilia from a ruptured hepatic artery aneurysm that was treated with transcatheter embolic occlusion of the bleeding vessel. The patient had no recurrent gastrointestinal problems and continued to use amphetamines until his death from a cerebrovascular accident 6 years later. CONCLUSION: A possible association between excessive oral amphetamine use and multiple visceral aneurysms is reported for 2 patients in whom other risk factors were absent. The potential for chronic oral amphetamine use to cause multiple visceral aneurysms is an ill-defined but not unexpected complication of this substance that is known to contribute to arterial hypertension and to produce a form of necrotizing arteritis.

Effects of retroviral-mediated tissue plasminogen activator gene transfer and expression on adherence and proliferation of canine endothelial cells seeded onto expanded polytetrafluoroethylene.

PURPOSE: Seeding prosthetic arterial grafts with genetically modified endothelial cells (ECs) has the potential to substantially improve graft function. However, preliminary applications suggest that grafts seeded with retrovirally transduced ECs yield a significantly lower percent surface coverage than those seeded with nontransduced ECs. The objective of this study was to test the hypothesis that canine ECs transduced with the human tissue plasminogen activator (tPA) gene would have a lower rate of adherence to pretreated expanded polytetrafluoroethylene (ePTFE) both in vitro and in vivo and that they would proliferate at a slower rate on pretreated ePTFE in vitro. METHODS: Early passage ECs derived from canine external jugular vein were transduced with the retroviral MFG vector containing the gene for human tPA. ECs exposed to media alone served as controls. Iodine 125-labeled ECs were seeded in vitro onto ePTFE graft segments pretreated with canine whole blood, fibronectin (50 micrograms/ml), or media alone, and the percent of ECs adherent at 1 hour were determined (n = 3). Additional tPA-transduced and -nontransduced ECs were grown for 10 days on either fibronectin (50 micrograms/ml)-pretreated ePTFE wafers or tissue culture plastic pretreated with gelatin (1%) or fibronectin (50 micrograms/ml), and the EC proliferation rates were determined (n = 3). Furthermore, 125I-labeled ECs were seeded onto fibronectin (50 micrograms/ml)-pretreated ePTFE graft segments implanted as carotid and femoral artery interposition grafts (n = 3). The grafts were harvested after 1 hour, and the percent of ECs adherent was determined. RESULTS: Human tPA was detected by immunohistochemical staining in 61% +/- 5% of the transduced ECs and was expressed at 35.4 +/- 12.9 ng/hr/10(6) cells. Fibronectin and whole blood pretreatment of the ePTFE grafts led to greater EC adherence in vitro than did media alone (90.9% +/- 5.3% vs 77.8% +/- 5.8% vs 4.7% +/- 1.1%, p < or = 0.05). No significant difference in the rates of adherence or proliferation was seen in vitro between the transduced and nontransduced ECs. No significant difference in proliferation was found for the transduced ECs on the three matrices tested in vitro. In contrast, adherence of the transduced ECs in vivo was significantly lower than that of nontransduced ECs (64.7% +/- 2.1% vs 73.7% +/- 4.1%, p < or = 0.05) 1 hour after implantation. CONCLUSIONS: Lower rates of surface endothelialization by genetically modified ECs in vivo do not appear to be due to an impaired capacity to initially adhere or proliferate on the synthetic graft but may result from decreased adherence after exposure to in vivo hemodynamic forces.

Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers.

Vascular cells are an important target for gene transfer because of their potential to deliver gene products both locally and systemically. Direct retroviral gene transfer to vascular cells in vivo has been limited by inefficient rates of transduction. We hypothesized that vascular cell transduction efficiency (TE), during short retroviral incubation periods, is significantly improved in vitro and in vivo using centrifugation to increase viral titer. Furthermore, we hypothesized a linear relationship between concentration of viable viral particles (measured as colony-forming units (CFUs)/cell) and retroviral TE during short incubation periods. Cultured rat pulmonary artery endothelial cells (RPAECs), rat aortic smooth muscle cells (RSMCs), and human iliac artery endothelial cells (HIAECs) demonstrated a strong correlation between TE and high concentrations of virus (> 100 CFU/cell) during retroviral incubation periods of 10 to 60 minutes. High titers, and thereby high concentrations, were achieved by centrifugation and resuspension in a fraction of the original volume. Titers was consistently increased tenfold, for a twentyfold increase in concentration by volume. A 20-minute incubation with a Moloney murine leukemia-derived retroviral vector coding for human placental alkaline phosphatase, pLJhpAP, at a concentration of 1150 CFU/cell yielded TEs of 10.6% +/- 0.7%, 40.4% +/- 1.6%, and 15.1% +/- 2.0% for RPAECs, RSMCs, and HIAECs, respectively. A similar effect was shown using the Moloney murine leukemia-derived MFGlacZ retroviral vector, coding for Escherichia coli beta-galactosidase. Increased titer and concentration had no effect on target cell viability, as shown by trypan blue exclusion. Although RSMCs had the most cells transduced in a given incubation period (p < 0.05), RPAECs had the highest replication rate (p < 0.05), suggesting the importance of factors other than cell cycle on retroviral TEs during short, clinically relevant incubation periods. In subsequent in vivo experiments, gene transfer was achieved in the rat carotid artery during a 20-minute incubation period infusing the concentrated pLJhpAP retrovirus after carotid balloon injury. Rats infused with virus 2 days after balloon injury exhibited hpAP activity (0 to 10 cells/section/rat) in the neointima of five out of six rats. Rats infused 4 days after balloon injury exhibited hpAP activity (0 to 25 cells/section/rat) in the media and adventitia of five out of five rats. Control rats that received the balloon injury alone or the balloon injury and unconcentrated retrovirus exhibited zero hpAP activity. We conclude that the TE of retroviral-mediated gene transfer to vascular cells in vitro and in vivo can be improved during short, clinically relevant incubation periods using centrifugation to increase retroviral titer, and thereby concentration of viable viral particles.

Tissue plasminogen activator increases canine endothelial cell proliferation rate through a plasmin-independent, receptor-mediated mechanism.

BACKGROUND: Tissue plasminogen activator (tPA) is elevated in cancer patients and is thought to promote tumor angiogenesis by facilitating endothelial cell migration through plasmin-mediated degradation of extracellular matrix. Due to the presence of an epidermal growth factor (EGF)-finger domain in the tPA A-chain and the existence of an endothelial cell (EC) receptor that binds this domain, it was hypothesized that tPA has a direct receptor-mediated effect on EC proliferation, independent of plasmin. METHODS AND RESULTS: Using cultured canine ECs, tPA (7.25 microg/ml, approximately 107 nM) increased proliferation as much as 50 and 170% in the absence and presence of growth factors, respectively. tPA-induced increases in EC proliferation occurred independent of plasmin generation, as the plasmin inhibitor, aprotinin (10 microg/ml) did not inhibit tPA-induced proliferation. However, tPA-induced proliferation was inhibited dose-dependently to a maximum of 78% using a monoclonal antibody against the tPA EGF-finger domain. This antibody, known to inhibit tPA binding to its receptor, did not inhibit tPA-induced plasmin generation. To investigate the role of potential signal transduction pathways, ECs were exposed to lavendustin A, a tyrosine kinase inhibitor, at 33.5 microM (IC50 for basic fibroblast growth factor). Lavendustin A did not inhibit tPA-induced EC proliferation. However, Rp-cAMP, an inhibitor of cAMP-dependent kinases, specifically inhibited tPA-induced EC proliferation in a dose-dependent manner (IC50 = 50.5 microM). Pertussis toxin at maximal concentrations for this system (0.5 ng/ml) did not inhibit tPA-induced EC proliferation. CONCLUSION: These results lend support to the hypothesis that tPA may have a direct receptor-mediated effect on EC proliferation and that this effect occurs independent of plasmin and may be dependent upon protein kinase A activity.

PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking.

PKH26, a fluorescent cell label, and PKH95, a 125 I-radioactive cell label, are both potentially valuable endothelial cell labels because they bind irreversibly within cell membranes. These labels would be particularly well suited to tract transplanted endothelial cells in vivo. However, no previous studies documenting lack of transfer of the label to unlabeled endothelial cells, as well as the effect of the label on endothelial cell function, have been undertaken. The purpose of this study was to determine the optimal method of endothelial cell (EC) labeling with PKH26 and PKH95, whether significant to EC-to-EC transfer of the label occurs, the effects of these labels on EC proliferation and membrane function, and the feasibility of using these labels for long-term quantitative EC tracking in vivo. Canine ECs in confluent monolayers or in cell suspension were labeled by exposure to 1.0 or 5.0 microM PKH26 for 1, 3, or 5 min. Cell viability was determined by trypan blue exclusion. The percentage of cells labeled and their fluorescence intensity were determined in a fluorescent-activated cell sorter (FACS). Effect of the label on cell function was assessed by measuring EC proliferation rates as well as intercellular adhesion molecule (ICAM) expression before and after induction with tumor necrosis factor (TNF). To determine if transfer of the label occurs, both labeled and nonlabeled ECs were grown in coculture and subjected to FACS analysis. For in vivo cell tracking, doubly labeled ECs were injected into the femoral artery of rat hind-limbs, and whole-body tissue analysis was undertaken to determine labeled-EC distribution at 60 days. Endothelial cells were labeled with equal efficacy as monolayers or in suspension. Labeling had no effect on EC proliferation rates nor on TNF-induced upregulation of ICAM expression. Coculture experiments revealed no significant label transfer to nonlabeled ECs. In vivo cell tracking studies documented that 8% of label remained within the skeletal muscle capillaries at 60 days after injection. PKH26 and PKH95 labels incorporate stably into EC membranes, do not alter endothelial cell function, and provide a precise means for quantitative EC tracking and histologic localization both in vitro and in vivo. These labels should prove to be very useful for studies of endothelial cell biology and transplantation.

Inhibition of aortic aneurysm development in blotchy mice by beta adrenergic blockade independent of altered lysyl oxidase activity.

PURPOSE: This study was designed to define the effects of beta-adrenergic blockade on aortic lysyl oxidase (LO), an enzyme responsible for elastin and collagen cross-linking, and aneurysm formation in the blotchy mouse. It was hypothesized that beta-blockade would inhibit the development of aneurysms because of its hemodynamic effect rather than a direct effect on LO activity. METHODS: Three groups of mice were studied: group I--normal littermates of blotchy mice; group II--untreated blotchy mice; group III--blotchy mice given either propranolol, atenolol, or nadolol. Data from the three different beta blocker-treated animals, group III, were statistically identical and were combined for analysis. The study was concluded when the mice were 4 months of age. At that time systolic blood pressure, heart rate, and aortic diameters were measured, and the entire aorta from each mouse was subjected to a bioassay for LO activity. RESULTS: Group I normal mice had an aortic arch diameter of 0.10 +/- 0.02 cm. Group II blotchy mice developed aortic arch aneurysms with a diameter of 0.21 +/- 0.03 cm. In Group III, beta blockade reduced the aortic arch diameter in blotchy mice to 0.11 +/- 0.03 cm. Mean heart rate in group III beta-blocked mice was reduced 25% compared with group I normal mice, and 18% compared with group II untreated blotchy mice. Blood pressures were similar in all three groups. Group II blotchy mice exhibited approximately half of the aortic LO activity (2.43 +/- 0.57 cpm/micrograms protein) noted in group I normal mice (5.82 +/- 1.06 cpm/micrograms protein). Aortic LO activity in group III blotchy mice remained low (2.09 +/- 0.85 cpm/micrograms protein) despite administration of beta-blockers. CONCLUSIONS: This is the first study to document an actual decrease in the level of aortic LO activity in blotchy mouse. beta-Blockade inhibits development of aortic aneurysms in blotchy mice. This is associated with a reduction in heart rate, but not by alterations in LO activity.

Pulmonary endothelial cell ATP depletion following intestinal ischemia.

Multiple organ failure (MOF) is known to follow systemic inflammatory mediator activation associated with intestinal ischemia-reperfusion injury. In particular, the pulmonary microvasculature appears to be susceptible to MOF-related injury. This study was designed to evaluate the hypothesis that non-cellular plasma factors associated with intestinal ischemia without reperfusion also mediate pulmonary endothelial cell injury. Male Sprague-Dawley rats had intestinal ischemia induced by microvascular clip occlusion of the superior mesenteric artery for 30, 60, 90, or 120 min. Following each period of ischemia, plasma samples were obtained from the protal vein. Time-matched sham-operated animals served as controls. Monolayers of cultured rat pulmonary artery endothelial cells were then incubated with the plasma samples and ATP levels determined using a luciferin-luciferase assay. A 51Cr-release assay using labeled endothelial cells was performed under identical conditions to assess cytotoxicity. Endothelial cell ATP levels were 1.99 +/- 0.23 x 10(-11) mole/micrograms DNA in sham preparations. After a 4-hr incubation in plasma from the 90 and 120 min ischemia groups, cellular ATP levels fell significantly to 1.07 +/- 0.23 x 10(-11) mole/micrograms DNA, respectively (P less than 0.005). No significant cytotoxic injury resulted from incubation with plasma from the 120 min group (1.0 +/- 0.4% versus 0.8 +/- 0.4% in sham group, P = NS). All animals survived 24 hr in the sham, 30, and 60 min groups. However, survival was 40 and 0% in the 90 and 120 min groups, respectively (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviral-mediated gene transfer of ICP47 inhibits major histocompatibility complex class I expression on vascular cells in vitro.

PURPOSE: Many viruses have evolved mechanisms to evade detection by the host immune system. The herpes simplex gene ICP47 encodes a protein that binds to the host antigen-processing transporter, inhibiting the formation of major histocompatibility complex class I (MHC-I) antigens in infected cells. MHC-I antigen expression is also important in acute allograft rejection. This study was designed to quantitate the effect of adenoviral-mediated gene transfer of ICP47 on MHC-I cell surface expression of human vascular cells. We hypothesized that the transduction of vascular cells with a replication-incompetent adenoviral vector that was expressing ICP47 (AdICP47) would inhibit constitutive and inducible MHC-I expression and thereby reduce the rate of cytolysis of ICP47-transduced vascular cells by sensitized cytotoxic T lymphocytes (CTL). METHODS: A replication-incompetent adenoviral vector, AdICP47, was created to express ICP47 driven by the cytomegalovirus immediate early promoter. Cultured human vascular endothelial and smooth muscle cells and human dermal fibroblasts were transduced with either AdICP47 or the control empty vector AddlE1. Cell surface constitutive and gamma-interferon-induced MHC-I expression were quantitated by flow cytometry. A standard 4-hour chromium release cytotoxicity assay was used to determine the percent cytolysis of transduced and nontransduced endothelial cells by sensitized CTL. Finally, to quantitate the specificity of the effect of ICP47 on MHC-I expression, adhesion molecule expression was quantitated in both transduced and nontransduced cells. RESULTS: Constitutive MHC-I expression in AdICP47-transduced endothelial cells was inhibited by a mean of 84% +/- 5% (SEM) in five experiments. After 48 hours of exposure to gamma-interferon, AdICP47-transduced cells exhibited a mean of 66% +/- 8% lower MHC-I expression than nontransduced cells. Similar inhibition in MHC-I expression was achieved in AdICP47-transduced vascular smooth muscle cells and dermal fibroblasts. Percent cytolysis of AdICP47-transduced endothelial cells by CTL was reduced by 72%. Finally, the specificity of the effect of transduction of ICP47 on vascular cell MHC-I expression was confirmed by a lack of significant change in either constitutive or tumor necrosis factor-induced vascular cell adhesion molecule/intercellular adhesion molecule expression. CONCLUSION: Transduction of vascular cells with AdICP47 strongly inhibits both constitutive and inducible MHC-I expression in human vascular cells. AdICP47-transduced cells exhibited a substantial reduction in cytolysis by CTL. Thus AdICP47 transduction holds promise as a technique to characterize the role of MHC-I expression in acute vascular allograft rejection in vivo and as a potential therapeutic intervention.

Transforming growth factor-beta 1 increases lysyl oxidase enzyme activity and mRNA in rat aortic smooth muscle cells.

PURPOSE: This investigation was designed to test the hypothesis that transforming growth factor-beta 1 (TGF-beta 1) regulates lysyl oxidase secretion from vascular smooth muscle cells. Lysyl oxidase is an enzyme that catalyzes an essential step in collagen and elastin cross-linking in the extracellular matrix, and TGF-beta 1 has been implicated in the pathogenesis of restenosis after vascular injury. The effect of TGF-beta 1 on lysyl oxidase in vascular smooth muscle cells has not been previously defined. METHODS: Rat aortic smooth muscle cells were grown in culture to confluence. Cells in passage 2 to 6 were incubated for 24 hours in media containing 0.1, 0.5, 1.0, or 10.0 ng/ml of TGF-beta 1. Lysyl oxidase activity in the media was quantitated with a tritium-release bioassay against an insoluble 3H-labeled aortic clastin substrate. Northern blot analyses were performed to determine steady-state levels of lysyl oxidase mRNA in the smooth muscle cells. RESULTS: Lysyl oxidase activity in the media increased 1.5-fold above control levels after exposure to 10 ng/ml of TGF-beta 1 (p < 0.01). This increase in lysyl oxidase activity was associated with a concentration-dependent increase in steady-state levels of lysyl oxidase mRNA, being 4.3- and 6.2-fold above control levels after exposure to 1 and 10 ng/ml TGF-beta 1, respectively (p < 0.01). The observed increase in steady-state lysyl oxidase mRNA after exposure to TGF-beta 1 was also time-dependent over the 24-hour experimental period. CONCLUSIONS: TGF-beta 1 appears to regulate lysyl oxidase in cultured rat aortic smooth muscle cells. Increases in lysyl oxidase activity may be one of the mechanisms by which TGF-beta 1 contributes to arterial restenosis after vascular injury.

Induction of angiogenesis by lidocaine and basic fibroblast growth factor: a model for in vivo retroviral-mediated gene therapy.

A strategy of direct, in vivo retroviral-mediated gene therapy targeting capillary endothelial cells must provide an environment of active angiogenesis. Both lidocaine and basic fibroblast growth factor (bFGF) promote angiogenesis, but the angiogenic response invoked by these substances in normal skeletal muscle has not been fully characterized. We sought to characterize these agents' angiogenic effects in anterior tibialis muscles of male Sprague-Dawley rats. An injection of either 1% lidocaine with 1:100,000 epinephrine or alternate-day injections of bFGF (0.025 or 0.25 microgram) with or without heparin were tested (n = 6 muscles/condition). Rats were sacrificed 4, 7, 10, or 12 days later and muscles were evaluated histologically to determine the number of proliferating cells using 5-bromo-2'-deoxycytidine (BrdC) and evaluated for capillary density using Griffonia simplicifolia I (GSI) lectin. At all time points, lidocaine produced at least 20-fold greater capillary density and cellular proliferation than PBS control (P < 0.0001). Injections of high-dosage bFGF produced more than fivefold greater capillary density than control injections at 7 and 10 days (P < 0.001), and more than twofold greater proliferation at 4, 7, and 12 days (P < 0.001). Capillary density returned to control levels 12 days following bFGF administration, whereas it remained well above control levels for 12 days after lidocaine administration. To confirm that lidocaine can be utilized in gene therapy strategies targeting vascular endothelium and skeletal muscle fibers, concentrated pLJ retrovirus containing cDNA for the heat-stable human placental alkaline phosphatase (hpAP) marker gene was infused into the rat hindlimb vasculature 4 days post-lidocaine administration. Rats receiving pLJhpAP retrovirus demonstrated significant hpAP transgene expression in endothelial cells and myocytes 21 days after the lidocaine injection (n = 6 muscles). In contrast, controls receiving pLJhpAP infusion without prior lidocaine administration failed to demonstrate any hpAP transgene expression. Lidocaine treatment evokes a substantially higher proliferative response than bFGF and, importantly, a durable angiogenic response in skeletal muscle. Thus, lidocaine is an ideal agent to induce angiogenesis in preparation for direct in vivo retroviral-mediated gene therapy targeting vascular endothelium.

Renal artery aneurysms: a 35-year clinical experience with 252 aneurysms in 168 patients.

OBJECTIVE: To define the relevance of treating renal artery aneurysms (RAAs) surgically. SUMMARY BACKGROUND DATA: Most prior definitions of the clinical, pathologic, and management features of RAAs have evolved from anecdotal reports. Controversy surrounding this clinical entity continues. METHODS: A retrospective review was undertaken of 168 patients (107 women, 61 men) with 252 RAAs encountered over 35 years at the University of Michigan Hospital. Aneurysms were solitary in 115 patients and multiple in 53 patients. Bilateral RAAs occurred in 32 patients. Associated diseases included hypertension (73%), renal artery fibrodysplasia (34%), systemic atherosclerosis (25%), and extrarenal aneurysms (6.5%). Most RAAs were saccular (79%) and noncalcified (63%). The main renal artery bifurcation was the most common site of aneurysms (60%). RAAs were often asymptomatic (55%), with a diagnosis made most often during arteriographic study for suspected renovascular hypertension (42%). RESULTS: Surgery was performed in 121 patients (average RAA size 1.5 cm), including 14 patients undergoing unilateral repair with contralateral RAA observation. The remaining 47 patients (average RAA size 1.3 cm) were not treated surgically. Operations included aneurysmectomy and angioplastic renal artery closure or segmental renal artery reimplantation, aneurysmectomy and renal artery bypass, and planned nephrectomy for unreconstructable renal arteries or advanced parenchymal disease. Eight patients underwent unplanned nephrectomy, being considered a technical failure of surgical therapy. Dialysis-dependent renal failure occurred in one patient. There were no perioperative deaths. Late follow-up (average 91 months) was available in 145 patients (86%). All but two arterial reconstructions remained clinically patent. Secondary renal artery procedures included percutaneous angioplasty, branch embolization, graft thrombectomy, and repeat bypass for late aneurysmal change of a vein conduit. Among 40 patients with clearly documented preoperative and postoperative blood pressure measurements, 60% had a significant decline in blood pressure after surgery while taking fewer antihypertensive medications. Late RAA rupture did not occur in the nonoperative patients, but no lessening of this group's hypertension was noted. CONCLUSION: Surgical therapy of RAAs in properly selected patients provides excellent long-term clinical outcomes and is often associated with decreased blood pressure.