Heterogeneity of treatment effects by risk in pulmonary arterial hypertension.

BACKGROUND: It is currently unknown if disease severity modifies response to therapy in pulmonary arterial hypertension (PAH). We aimed to explore if disease severity, as defined by established risk-prediction algorithms, modified response to therapy in randomised clinical trials in PAH. METHODS: We performed a meta-analysis using individual participant data from 18 randomised clinical trials of therapy for PAH submitted to the United States Food and Drug Administration to determine if predicted risk of 1-year mortality at randomisation modified the treatment effect on three outcomes: change in 6-min walk distance (6MWD), clinical worsening at 12 weeks and time to clinical worsening. RESULTS: Of 6561 patients with a baseline US Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL 2.0) score, we found that individuals with higher baseline risk had higher probabilities of clinical worsening but no difference in change in 6MWD. We detected a significant interaction of REVEAL 2.0 risk and treatment assignment on change in 6MWD. For every 3-point increase in REVEAL 2.0 score, there was a 12.49 m (95% CI 5.86-19.12 m; p=0.001) greater treatment effect in change in 6MWD. We did not detect a significant risk by treatment interaction on clinical worsening with most of the risk-prediction algorithms. CONCLUSIONS: We found that predicted risk of 1-year mortality in PAH modified treatment effect as measured by 6MWD, but not clinical worsening. Our findings highlight the importance of identifying sources of treatment heterogeneity by predicted risk to tailor studies to patients most likely to have the greatest treatment response.

Lower DHEA-S levels predict disease and worse outcomes in post-menopausal women with idiopathic, connective tissue disease- and congenital heart disease-associated pulmonary arterial hypertension.

High oestradiol (E2) and low dehydroepiandrosterone-sulfate (DHEA-S) levels are risk factors for pulmonary arterial hypertension (PAH) in men, but whether sex hormones are related to PAH in women is unknown.Post-menopausal women aged ≥55 years with PAH were matched by age and body mass index to women without cardiovascular disease. Plasma sex hormone levels were measured by immunoassay.Lower levels of DHEA-S (p<0.001) and higher levels of E2 (p=0.02) were associated with PAH. In PAH cases (n=112), lower DHEA-S levels were associated with worse haemodynamics (all p<0.01) and more right ventricular dilatation and dysfunction (both p=0.001). Lower DHEA-S levels were associated with shorter 6-min walking distance (6MWD) (p=0.01) and worse functional class (p=0.004). Each Ln(1 µg·dL-1) decrease in DHEA-S was associated with a doubling in the risk of death (hazard ratio 2.0, 95% CI 1.5-2.7; p<0.001). Higher levels of E2 were associated with shorter 6MWD (p=0.03) and worse functional class (p=0.01).High E2 and low DHEA-S levels are associated with the risk and severity of PAH in post-menopausal women. Hormonal modulation should be studied as a treatment strategy in PAH.

Anastrozole in Pulmonary Arterial Hypertension. A Randomized, Double-Blind, Placebo-controlled Trial.

RATIONALE: The aromatase inhibitor anastrozole blocks the conversion of androgens to estrogen and blunts pulmonary hypertension in animals, but its efficacy in treating patients with pulmonary arterial hypertension (PAH) is unknown. OBJECTIVES: We aimed to determine the safety and efficacy of anastrozole in PAH. METHODS: We performed a randomized, double-blind, placebo-controlled trial of anastrozole in patients with PAH who received background therapy at two centers. MEASUREMENTS AND MAIN RESULTS: A total of 18 patients with PAH were randomized to anastrozole 1 mg or matching placebo in a 2:1 ratio. The two co-primary outcomes were percent change from baseline in 17ß-estradiol levels (E2) and tricuspid annular plane systolic excursion (TAPSE) at 3 months. Anastrozole significantly reduced E2 levels compared with placebo (percent change: -40%; interquartile range [IQR], -61 to -26% vs. -4%; IQR, -14 to +4%; P = 0.003), but there was no difference in TAPSE. Anastrozole significantly increased the 6-minute-walk distance (median change = +26 m) compared with placebo (median change = -12 m) (median percent change: anastrozole group, 8%; IQR, 2 to 17% vs. placebo -2%; IQR, -7 to +1%; P = 0.042). Anastrozole had no effect on circulating biomarkers, functional class, or health-related quality of life. There was no difference in adverse events. CONCLUSIONS: Anastrozole significantly reduced E2 levels in patients with PAH but had no effect on TAPSE. Anastrozole was safe, well tolerated, and improved 6-minute-walk distance in this small "proof-of-principle" study. Larger and longer phase II clinical trials of anastrozole may be warranted in patients with PAH. Clinical trial registered with www.clinicaltrials.gov (NCT 1545336).

Carcinogenicity of ethylene oxide: key findings and scientific issues.

In support of the Integrated Risk Information System (IRIS), the U.S. Environmental Protection Agency (EPA) completed an evaluation of the inhalation carcinogenicity of ethylene oxide (EtO) in December 2016. This article reviews key findings and scientific issues regarding the carcinogenicity of EtO in EPA's Carcinogenicity Assessment. EPA's assessment critically reviewed and characterized epidemiologic, laboratory animal, and mechanistic studies pertaining to the human carcinogenicity of EtO, and addressed some key scientific issues such as the analysis of mechanistic data as part of the cancer hazard evaluation and to inform the quantitative risk assessment. The weight of evidence from the epidemiologic, laboratory animal, and mechanistic studies supports a conclusion that EtO is carcinogenic in humans, with the strongest human evidence linking EtO exposure to lymphoid and breast cancers. Analyses of the mechanistic data establish a key role for genotoxicity and mutagenicity in EtO-induced carcinogenicity and reveal little evidence supporting other mode-of-action hypotheses. In conclusion, EtO was found to be carcinogenic to humans by inhalation, posing a potential human health hazard for lymphoid and breast cancers.

Higher Estradiol and Lower Dehydroepiandrosterone-Sulfate Levels Are Associated with Pulmonary Arterial Hypertension in Men.

RATIONALE: Recent studies have focused on the role of female sex and estradiol (E2) in pulmonary arterial hypertension (PAH), but it is not known whether sex hormones are risk factors for PAH in men. OBJECTIVES: We performed a case-control study to determine whether hormone levels (E2, dehydroepiandrosterone-sulfate [DHEA-S], and testosterone) are associated with PAH in men. METHODS: Plasma sex hormone levels in men with idiopathic, heritable, or connective tissue disease-associated PAH were compared with those from age- and body mass index-matched men without clinical cardiovascular disease. MEASUREMENTS AND MAIN RESULTS: There were 23 cases with PAH (70% had idiopathic PAH, 65% were functional class III/IV) and 67 control subjects. Higher E2 and E2/testosterone levels were associated with the risk of PAH (odds ratio per 1 ln[E2:testosterone], 6.0; 95% confidence interval, 2.2-16.4; P = 0.001), whereas higher levels of DHEA-S were associated with a reduced risk (odds ratio per 1 ln[DHEA-S], 0.1; 95% confidence interval, 0.0-0.3; P = 0.001). E2 and DHEA-S levels were strong predictors of case status (C statistic for both, 0.82) but testosterone was not (C statistic, 0.53). Higher levels of E2 were associated with shorter 6-minute-walk distances (P = 0.03), whereas higher levels of DHEA-S were associated with lower right atrial pressure (P = 0.02) and pulmonary vascular resistance (P = 0.01) in men with PAH. CONCLUSIONS: Higher levels of E2 and lower levels of DHEA-S were associated with PAH in men. Sex-based differences in sex hormone processing and signaling may contribute to unique phenotypes in pulmonary vascular disease.

Emetine Di-HCl Attenuates Type 1 Diabetes Mellitus in Mice.

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterized by beta cell destruction, insulin deficiency and hyperglycemia. Activated macrophages and autoimmune T cells play a crucial role in the pathogenesis of hyperglycemia in NOD murine diabetes models, but the molecular mechanisms of macrophage activation are unknown. We recently identified pigment epithelium-derived factor (PEDF) as an adipocyte-derived factor that activates macrophages and mediates insulin resistance. Reasoning that PEDF might participate as a proinflammatory mediator in murine diabetes, we measured PEDF levels in NOD mice. PEDF levels are significantly elevated in pancreas, in correlation with pancreatic TNF levels in NOD mice. To identify experimental therapeutics, we screened 2,327 compounds in two chemical libraries (the NIH Clinical Collection and Pharmakon-1600a) for leads that inhibit PEDF mediated TNF release in macrophage cultures. The lead molecule selected, "emetine" is a widely used emetic. It inhibited PEDF-mediated macrophage activation with an EC50 or 146 nM. Administration of emetine to NOD mice and to C57Bl6 mice subjected to streptozotocin significantly attenuated hyperglycemia, reduced TNF levels in pancreas, and attenuated insulitis. Together, these results suggest that targeting PEDF with emetine may attenuate TNF release and hyperglycemia in murine diabetes models. This suggests that further investigation of PEDF and emetine in the pathogenesis of human diabetes is warranted.

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.

Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of ß-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.

Are hemodynamics surrogate end points in pulmonary arterial hypertension?

BACKGROUND: Although frequently assessed in trials and clinical practice, hemodynamic response to therapy has never been validated as a surrogate end point for clinical events in pulmonary arterial hypertension (PAH). METHODS AND RESULTS: We performed a patient-level pooled analysis of 4 randomized, placebo-controlled trials to determine whether treatment-induced changes in hemodynamic values at 12 weeks accounted for the relationship between treatment assignment and the probability of early clinical events (death, lung transplantation, atrial septostomy, PAH hospitalization, withdrawal for clinical worsening, or escalation in PAH therapy). We included 1119 subjects with PAH. The median (interquartile range) age was 48 years (37-59 years), and 23% were men. A total of 656 patients (59%) received active therapy (101 [15%] iloprost, 118 [18%] sitaxsentan, 204 [31%] sildenafil, and 233 [36%] subcutaneous treprostinil). Active treatment significantly lowered right atrial pressure, mean pulmonary artery pressure, and pulmonary vascular resistance and increased cardiac output and index (P<0.01 for all). Changes in hemodynamic values (except for right atrial pressure and mean pulmonary artery pressure) were significantly associated with the risk of a clinical event (P<0.02 for all). Although active treatment approximately halved the odds of a clinical event compared with placebo (P<0.001), changes in hemodynamics accounted for only 1.2% to 13.9% of the overall treatment effect. CONCLUSIONS: Treatment-induced changes in hemodynamics at 12 weeks only partially explain the impact of therapy on the probability of early clinical events in PAH. These findings suggest that resting hemodynamics are not valid surrogate end points for short-term events in PAH clinical trials.

Endothelial Krüppel-like factor 4 modulates pulmonary arterial hypertension.

Krüppel-like factor 4 (KLF4) is a transcription factor expressed in the vascular endothelium, where it promotes anti-inflammatory and anticoagulant states, and increases endothelial nitric oxide synthase expression. We examined the role of endothelial KLF4 in pulmonary arterial (PA) hypertension (PAH). Mice with endothelial KLF4 knockdown were exposed to hypoxia for 3 weeks, followed by measurement of right ventricular and PA pressures, pulmonary vascular muscularization, and right ventricular hypertrophy. The effect of KLF4 on target gene expression was assessed in lungs from these mice, verified in vitro by small interfering RNA (siRNA) knockdown of KLF4, and further studied at the promoter level with cotransfection experiments. KLF4 expression was measured in lung tissue from patients with PAH and normal control subjects. We found that, after hypoxia, right ventricular and PA pressures were significantly higher in KLF4 knockdown animals than controls. Knockdown animals also had more severe pulmonary vascular muscularization and right ventricular hypertrophy. KLF4 knockdown resulted in increased pulmonary expression of endothelin-1 and decreased expression of endothelial nitric oxide synthase, endothelin receptor subtype B, and prostacyclin synthase. Concordant findings were observed in vitro, both with siRNA knockdown of KLF4 and promoter activity assays. Finally, KLF4 expression was reduced in lungs from patients with PAH. In conclusion, endothelial KLF4 regulates the transcription of genes involved in key pathways implicated in PAH, and its loss exacerbates pulmonary hypertension in response to chronic hypoxia in mice. These results introduce a novel transcriptional modulator of PAH, with the potential of becoming a new therapeutic target.

Pulmonary vascular complications of liver disease.

Hepatopulmonary syndrome and portopulmonary hypertension are two pulmonary vascular complications of liver disease. The pathophysiology underlying each disorder is distinct, but patients with either condition may be limited by dyspnea. A careful evaluation of concomitant symptoms, the physical examination, pulmonary function testing and arterial blood gas analysis, and echocardiographic, imaging, and hemodynamic studies is crucial to establishing (and distinguishing) these diagnoses. Our understanding of the pathobiology, natural history, and treatment of these disorders has advanced considerably over the past decade; however, the presence of either still increases the risk of morbidity and mortality in patients with underlying liver disease. There is no effective medical treatment for hepatopulmonary syndrome. Although liver transplantation can resolve hepatopulmonary syndrome, there appears to be worse survival even with transplantation. Liver transplantation poses a very high risk of death in those with significant portopulmonary hypertension, where targeted medical therapies may improve functional status and allow successful transplantation in a small number of select patients.

Laser-assisted indocyanine green dye angiography accurately predicts the split-thickness graft timing of integra artificial dermis.

BACKGROUND: The use of an artificial dermal substitute such as Integra-a bilaminate combination of thin silicone and cross-linked bovine tendon collagen and chondroitin-6-sulfate-has become a popular method to address large surface area wounds or smaller, complex wounds devoid of a vascular bed. The incorporation of Integra depends on a vascular wound bed or periphery and can take 4 weeks or longer to occur. If the Integra has not fully incorporated at the time of placement of the split-thickness graft, complete graft loss may result. The availability of a minimally invasive method to assess the incorporation of Integra would be of great value. METHODS: Two 5 × 10-cm paraspinal full-thickness wounds were created on 3 female swine. Wounds were randomly assigned full-thickness skin graft or Integra (Plainsboro, NJ) treatment. Both types of grafts were placed after the application of fibrin glue (Tisseel, Deerfield, Ill) to the wound bed. Laser Doppler imaging (LDI) (Moor), indocyanine green dye (ICG) angiography (LifeCell SPY), and clinical scoring were performed weekly for a period of 8 weeks after grafting. At 4 weeks, the silicone layer of the Integra was removed, and a culture of autologous keratinocytes was applied. A 4-mm punch biopsy sample of each graft was taken 1, 2, 4, 6, 7, and 8 weeks postoperatively for histologic analysis. RESULTS: Both ICG angiography and LDI perfusion measurements noted an increase in perfusion at the Integra graft site that peaked 3 weeks after grafting, corresponding with the start of neovascularization and the optimal time for the application of a split-thickness skin graft. indocyanine green dye angiography measurements exhibit greater reproducibility between animals at late time points as compared with LDI. This decrease in LDI precision is directly related to increases in scar tissue thickness of greater than 5 mm as determined via histologic analysis and corresponds with the accepted maximum penetration depth of the LDI laser. CONCLUSIONS: Indocyanine green dye angiography may provide valuable information as to graft integrity and split-thickness skin graft timing at late time points. Range of LDI seems to be insufficient for split-thickness graft timing or late time point accuracy. Future exploration of ICG angiography potential will involve tracking Integra graft delay in porcine models.

Electrocardiographic Abnormalities and Their Association with Outcomes in Randomized Clinical Trials of Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is a progressive disease with manifestations including right atrial enlargement, right ventricular dysfunction, dilation, and hypertrophy. Electrocardiography (ECG) is a noninvasive, inexpensive test that is routinely performed in clinical settings. Prior studies have described separate abnormal findings in the electrocardiograms of patients with PAH. However, the role of composite ECG findings reflective of right heart disease (RHD) for risk stratification, clinical trial enrichment, and management of patients with PAH has not been explored. Objectives: To describe a pattern of RHD on ECG in patients with PAH and to investigate the association of this pattern with clinical measures of disease severity and outcomes. Methods: We harmonized individual participant data from 18 phase III randomized clinical trials of therapies for PAH (1998-2013) submitted to the U.S. Food and Drug Administration. RHD was defined as the presence of right ventricular hypertrophy, right axis deviation, right atrial enlargement, or right bundle branch block on ECG. Random effects linear regression, multilevel ordinal regression (cumulative link model), and Cox proportional hazards models were used to assess the association of RHD by ECG with 6-minute walk distance (6MWD), World Health Organization (WHO) functional class, and clinical worsening after a priori adjustment for age, sex, body mass index, and PAH etiology. Effect modification of treatment and ECG abnormalities was assessed by including an interaction term. Results: A total of 4,439 patients had baseline ECG, and 68% of patients had evidence of RHD. RHD on ECG was associated with higher pulmonary vascular resistance (P < 0.001) and higher mean pulmonary artery pressures (P < 0.001). Patients with RHD on ECG had 10 meters shorter 6MWD (P = 0.005) and worse WHO functional class (P < 0.001) at baseline. RHD on baseline ECG was associated with increased risk of clinical worsening (hazard ratio, 1.42; 95% confidence interval; 1.21, 1.67; P < 0.001). Patients with RHD had greater treatment effect in terms of 6MWD, WHO functional class, and time to clinical worsening than those without (P for interaction = 0.03, 0.001, and 0.03, respectively). Conclusions: RHD by ECG may be associated with worse outcomes and potentially greater treatment effect. Electrocardiograms could be an inexpensive, widely available noninvasive method to enrich clinical trial populations in PAH.

Is low-risk status a surrogate outcome in pulmonary arterial hypertension? An analysis of three randomised trials.

BACKGROUND: Targeting short-term improvements in multicomponent risk scores for mortality in patients with pulmonary arterial hypertension (PAH) could result in improved long-term outcomes. We aimed to determine whether PAH risk scores were adequate surrogates for clinical worsening or mortality outcomes in PAH randomised clinical trials (RCTs). METHODS: We performed an individual participant data meta-analysis of RCTs selected from PAH trials provided by the US Food and Drug Administration (FDA). We calculated predicted risk using the COMPERA, COMPERA 2.0, non-invasive FPHR, REVEAL 2.0, and REVEAL Lite 2 risk scores. The primary outcome of interest was time to clinical worsening, a composite endpoint composed of any of the following events: all-cause death, hospitalisation for worsening PAH, lung transplantation, atrial septostomy, discontinuation of study treatment (or study withdrawal) for worsening PAH, initiation of parenteral prostacyclin analogue therapy, or decrease of at least 15% in 6-min walk distance from baseline, combined with either worsening of WHO functional class from baseline or the addition of an approved PAH treatment. The secondary outcome of interest was time to all-cause mortality. We assessed the surrogacy of these risk scores, parameterised as attainment of low-risk status by 16 weeks, for improvement in long-term clinical worsening and survival using mediation and meta-analysis frameworks. FINDINGS: Of 28 trials received from the FDA, three RCTs (AMBITION, GRIPHON, and SERAPHIN; n=2508) had the data necessary to assess long-term surrogacy. The mean age was 49 years (SD 16), 1956 (78%) participants were women, 1704 (68%) were classified as White, and 280 (11%) were Hispanic or Latino. 1388 (55%) of 2503 participants with available data had idiopathic PAH and 776 (31%) of 2503 had PAH associated with connective tissue disease. In a mediation analysis, the proportions of treatment effects explained by attainment of low-risk status ranged only from 7% to 13%. In a meta-analysis of trial-regions, the treatment effects on low-risk status were not predictive of the treatment effects on time to clinical worsening (R2 values 0·01-0·19) nor the treatment effects on time to all-cause mortality (R2 values 0-0·2). A leave-one-out analysis suggested that the use of these risk scores as surrogates might lead to biased inferences regarding the effect of therapies on clinical outcomes in PAH RCTs. Results were similar when using absolute risk scores at 16 weeks as the potential surrogates. INTERPRETATION: Multicomponent risk scores have utility for the prediction of outcomes in patients with PAH. Clinical surrogacy for long-term outcomes cannot be inferred from observational studies of outcomes. Our analyses of three PAH trials with long-term follow-up suggest that further study is necessary before using these or other scores as surrogate outcomes in PAH RCTs or clinical care. FUNDING: Cardiovascular Medical Research and Education Fund, US National Institutes of Health.

Vagus nerve stimulation primes platelets and reduces bleeding in hemophilia A male mice.

Deficiency of coagulation factor VIII in hemophilia A disrupts clotting and prolongs bleeding. While the current mainstay of therapy is infusion of factor VIII concentrates, inhibitor antibodies often render these ineffective. Because preclinical evidence shows electrical vagus nerve stimulation accelerates clotting to reduce hemorrhage without precipitating systemic thrombosis, we reasoned it might reduce bleeding in hemophilia A. Using two different male murine hemorrhage and thrombosis models, we show vagus nerve stimulation bypasses the factor VIII deficiency of hemophilia A to decrease bleeding and accelerate clotting. Vagus nerve stimulation targets acetylcholine-producing T lymphocytes in spleen and α7 nicotinic acetylcholine receptors (α7nAChR) on platelets to increase calcium uptake and enhance alpha granule release. Splenectomy or genetic deletion of T cells or α7nAChR abolishes vagal control of platelet activation, thrombus formation, and bleeding in male mice. Vagus nerve stimulation warrants clinical study as a therapy for coagulation disorders and surgical or traumatic bleeding.

Exogenous seeding of cerebral ß-amyloid deposition in ßAPP-transgenic rats.

Deposition of the amyloid-ß (Aß) peptide in senile plaques and cerebral Aß angiopathy (CAA) can be stimulated in Aß-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aß seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aß itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aß seeding have employed animal models that, as they age, eventually will generate Aß lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aß within the course of its median lifespan (30 months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aß. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aß-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aß.

Neuronal RING finger protein 11 (RNF11) regulates canonical NF-κB signaling.

BACKGROUND: The RING domain-containing protein RING finger protein 11 (RNF11) is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases. METHODS AND RESULTS: Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA) knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11's association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and TNF-α mRNA and proteins, suggesting that NF-κB signaling and associated inflammatory responses are aberrantly regulated in the absence of RNF11. CONCLUSIONS: Our findings support the hypothesis that, in the nervous system, RNF11 negatively regulates canonical NF-κB signaling. Reduced or functionally compromised RNF11 could influence NF-κB-associated neuronal functions, including exaggerated inflammatory responses that may have implications for neurodegenerative disease pathogenesis and progression.

Comparison of native porcine skin and a dermal substitute using tensiometry and digital image speckle correlation.

Dermal substitutes are currently used in plastic surgery to cover various soft tissue defects caused by trauma, burns, or ablative cancer surgery. Little information is available on the biomechanical properties of these dermal substitutes after adequate incorporation as compared to normal skin. Determining parameters such as tensile strength in these skin substitutes will help us further understand their wound healing properties and potential in developing artificial tissue constructs. We hypothesize that a dermal substitute has a lower stress-strain curve and altered stress-induced deformation quantified with tensiometry and digital image speckle correlation (DISC) analysis. Two separate 5×10-cm full-thickness wounds were created on the dorsum of 3 female swine. Fibrin glue was applied before either a full-thickness skin graft (FTSG) or application of artificial dermal matrix. On day 42, cultured autologous keratinocytes were applied as a cell sheet to the wound covered with Integra. On day 56, the wounds were fully excised and fresh tissue specimens, including normal skin, were stored in a physiological solution and prepared for analysis. Rectangular samples were excised from the center of each specimen measuring 4×4×30 mm. Using a tensiometer and DISC analysis, we evaluated the tensile strength of 3 different groups of skin, namely, normal, FTSG, and Integra. There is a significant difference between the Integra specimen when compared to normal skin and FTSG. We found a minimal difference in the stress-strain curves of the latter two. Integra alone shows plastic deformation with continued stretching before ultimate midline fracture. There is significant change between the Young's moduli of the normal skin and the Integra, whereas there is little difference between the FTSG and the normal skin; DISC confirms this analysis. The normal skin and FTSG show a convergence of vectors to a linear plane, whereas Integra shows very little organization. Using 2 different methods of analysis, we have shown a dermal substitute does not display similar biomechanical properties after adequate incorporation. These major tensile strength differences are shown between normal, grafted, and Integra constructs under physiological conditions. These properties will lead to further understanding of artificial tissue and engineered constructs in laboratory and clinical applications.