Non-invasive candidate protein signature predicts hepatic venous pressure gradient reduction in cirrhotic patients after sustained virologic response.

BACKGROUND AND AIMS: A reduction in hepatic venous pressure gradient (HVPG) is the most accurate marker for assessing the severity of portal hypertension and the effectiveness of intervention treatments. This study aimed to evaluate the prognostic potential of blood-based proteomic biomarkers in predicting HVPG response amongst cirrhotic patients with portal hypertension due to Hepatitis C virus (HCV) and had achieved sustained virologic response (SVR). METHODS: The study comprised 59 patients from two cohorts. Patients underwent paired HVPG (pretreatment and after SVR), liver stiffness (LSM), and enhanced liver fibrosis scores (ELF) measurements, as well as proteomics-based profiling on serum samples using SomaScan® at baseline (BL) and after SVR (EOS). Machine learning with feature selection (Caret, Random Forest and RPART) methods were performed to determine the proteins capable of classifying HVPG responders. Model performance was evaluated using AUROC (pROC R package). RESULTS: Patients were stratified by a change in HVPG (EOS vs. BL) into responders (greater than 20% decline in HVPG from BL, or <10 mmHg at EOS with >10 mmHg at BL) and non-responders. LSM and ELF decreased markedly after SVR but did not correlate with HVPG response. SomaScan (SomaLogic, Inc., Boulder, CO) analysis revealed a substantial shift in the peripheral proteome composition, reflected by 82 significantly differentially abundant proteins. Twelve proteins accurately distinguished responders from non-responders, with an AUROC of .86, sensitivity of 83%, specificity of 83%, accuracy of 83%, PPV of 83%, and NPV of 83%. CONCLUSIONS: A combined non-invasive soluble protein signature was identified, capable of accurately predicting HVPG response in HCV liver cirrhosis patients after achieving SVR.

Efficacy and safety of MAS825 (anti-IL-1ß/IL-18) in COVID-19 patients with pneumonia and impaired respiratory function.

MAS825, a bispecific IL-1ß/IL-18 monoclonal antibody, could improve clinical outcomes in COVID-19 pneumonia by reducing inflammasome-mediated inflammation. Hospitalized non-ventilated patients with COVID-19 pneumonia (n = 138) were randomized (1:1) to receive MAS825 (10 mg/kg single i.v.) or placebo in addition to standard of care (SoC). The primary endpoint was the composite Acute Physiology and Chronic Health Evaluation II (APACHE II) score on Day 15 or on the day of discharge (whichever was earlier) with worst-case imputation for death. Other study endpoints included safety, C-reactive protein (CRP), SARS-CoV-2 presence, and inflammatory markers. On Day 15, the APACHE II score was 14.5 ± 1.87 and 13.5 ± 1.8 in the MAS825 and placebo groups, respectively (P = 0.33). MAS825 + SoC led to 33% relative reduction in intensive care unit (ICU) admissions, ~1 day reduction in ICU stay, reduction in mean duration of oxygen support (13.5 versus 14.3 days), and earlier clearance of virus on Day 15 versus placebo + SoC group. On Day 15, compared with placebo group, patients treated with MAS825 + SoC showed a 51% decrease in CRP levels, 42% lower IL-6 levels, 19% decrease in neutrophil levels, and 16% lower interferon-γ levels, indicative of IL-1ß and IL-18 pathway engagement. MAS825 + SoC did not improve APACHE II score in hospitalized patients with severe COVID-19 pneumonia; however, it inhibited relevant clinical and inflammatory pathway biomarkers and resulted in faster virus clearance versus placebo + SoC. MAS825 used in conjunction with SoC was well tolerated. None of the adverse events (AEs) or serious AEs were treatment-related.

Licogliflozin for nonalcoholic steatohepatitis: a randomized, double-blind, placebo-controlled, phase 2a study.

Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease that may advance to fibrosis and lead to mortality; however, no pharmacotherapy is currently available. We tested the hypothesis that inhibition of both the sodium-glucose cotransporters 1 and 2 with licogliflozin would lead to improvement in NASH. A total of 107 patients with phenotypic or histologic NASH were randomized (1:2:2) to receive oral administration of either placebo (n = 21), licogliflozin 30 mg (n = 43) or 150 mg (n = 43) once daily for 12 weeks. Licogliflozin 150 mg showed a significant 32% (80% confidence interval (CI): 21-43%; P = 0.002) placebo-adjusted reduction in serum alanine aminotransferase after 12 weeks of treatment, the primary endpoint of the study. However, the 30 mg dose of licogliflozin did not meet the primary endpoint (placebo-adjusted reduction 21% (80% CI: 7-32%; P = 0.061)). Diarrhea occurred in 77% (33 of 43), 49% (21 of 43) and 43% (9 of 21) of patients treated with licogliflozin 150 mg, 30 mg and placebo, respectively, which was mostly mild in severity. No other major safety concerns were identified. Treatment with 150 mg licogliflozin led to reductions in serum alanine aminotransferase in patients with NASH. Studies of longer duration and in combination with drugs that have different mechanisms of action are needed to validate these findings and to define a role of licogliflozin as a therapeutic option for NASH. ClinicalTrials.gov identifier: NCT03205150.

Metabolic adaptation characterizes short-term resistance to weight loss induced by a low-calorie diet in overweight/obese individuals.

BACKGROUND: Low-calorie diet (LCD)-induced weight loss demonstrates response heterogeneity. Physiologically, a decrease in energy expenditure lower than what is predicted based on body composition (metabolic adaptation) and/or an impaired capacity to increase fat oxidation may hinder weight loss. Understanding the metabolic components that characterize weight loss success is important for optimizing weight loss strategies. OBJECTIVES: We tested the hypothesis that overweight/obese individuals who had lower than expected weight loss in response to a 28-d LCD would be characterized by 1) impaired fat oxidation and 2) whole-body metabolic adaptation. We also characterized the molecular mechanisms associated with weight loss success/failure. METHODS: This was a retrospective comparison of participants who met their predicted weight loss targets [overweight/obese diet sensitive (ODS), n = 23, females = 21, males = 2] and those that did not [overweight/obese diet resistant (ODR), n = 14, females = 12, males = 2] after a 28-d LCD (900-1000 kcal/d). We used whole-body (energy expenditure and fat oxidation) and tissue-specific measurements (metabolic proteins in skeletal muscle, gene expression in adipose tissue, and metabolites in serum) to detect metabolic properties and biomarkers associated with weight loss success. RESULTS: The ODR group had greater mean ± SD metabolic adaptation (-175 ± 149 kcal/d; +119%) than the ODS group (-80 ± 108 kcal/d) after the LCD (P = 0.030). Mean ± SD fat oxidation increased similarly for both groups from baseline (0.0701 ± 0.0206 g/min) to day 28 (0.0869 ± 0.0269 g/min; P < 0.001). A principal component analysis factor comprised of serum 3-hydroxybutyric acid, citrate, leucine/isoleucine, acetyl-carnitine, and 3-hydroxylbutyrlcarnitine was associated with weight loss success at day 28 (std. ß = 0.674, R2 = 0.479, P < 0.001). CONCLUSIONS: Individuals who achieved predicted weight loss targets after a 28-d LCD were characterized by reduced metabolic adaptation. Accumulation of metabolites associated with acetyl-CoA excess and enhanced ketogenesis was identified in the ODS group.This trial was registered at clinicaltrials.gov as NCT01616082.

Glucosuric, renal and haemodynamic effects of licogliflozin, a dual inhibitor of sodium-glucose co-transporter-1 and sodium-glucose co-transporter-2, in patients with chronic kidney disease: A randomized trial.

AIM: To investigate the glucosuric, renal and haemodynamic effects of licogliflozin, a dual sodium-glucose co-transporter-1 and sodium-glucose co-transporter-2 inhibitor, in patients with chronic kidney disease (CKD). METHODS: This multiple-dose, parallel-group, phase II mechanistic study randomized 53 participants (aged 18-78 years, body mass index ≤ 50 kg/m2 ) with varying degrees of CKD or normal renal function to treatment with licogliflozin (50 mg once daily) or placebo for 7 days. The effects of licogliflozin on 24-h urinary glucose excretion (UGE24 ), renal function, haemodynamics, pharmacokinetics and safety were assessed. RESULTS: Licogliflozin treatment for 7 days significantly (p < .01) increased UGE24 from baseline in participants with normal renal function (adjusted mean change: 41.8 [33.6, 49.9] g) or with mild (32.6 [24.1, 41.0] g), moderate A (35.7 [28.6, 42.9] g) or moderate B (20.3 [13.1, 27.5] g) CKD, but not in severe (6.2 [-0.71, 13.18] g) CKD. Licogliflozin reduced urinary electrolytes (sodium, potassium and chloride), blood pressure and urinary volume to varying extents among different groups. Significant increases in renin (p < .05), angiotensin II (p < .05) and aldosterone (p < .01) levels were observed. Adverse events were generally mild, and most commonly included diarrhoea (94%), flatulence (68%) and abdominal pain (21%). CONCLUSION: Licogliflozin treatment results in significantly increased UGE and favourable changes in urinary electrolytes and haemodynamics in patients with varying degrees of CKD (estimated glomerular filtration rate ≥ 45 mL/min/1.73 m2 ).

Using Target Engagement Biomarkers to Predict Clinical Efficacy of MetAP2 Inhibitors.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

Basal insulin peglispro increases lipid oxidation, metabolic flexibility, thermogenesis and ketone bodies compared to insulin glargine in subjects with type 1 diabetes mellitus.

AIMS: When treated with basal insulin peglispro (BIL), patients with type 1 diabetes mellitus (T1DM) exhibit weight loss and lower prandial insulin requirements versus insulin glargine (GL), while total insulin requirements remain similar. One possible explanation is enhanced lipid oxidation and improved ability to switch between glucose and lipid metabolism with BIL. This study compared the effects of BIL and GL on glucose and lipid metabolism in subjects with T1DM. MATERIALS AND METHODS: Fifteen subjects with T1DM were enrolled into this open-label, randomised, crossover study, and received once-daily stable, individualised, subcutaneous doses of BIL and GL for 4 weeks each. Respiratory quotient (RQ) was measured using whole-room calorimetry, and energy expenditure (EE) and concentrations of ketone bodies (3-hydroxybutyrate) and acylcarnitines were assessed. RESULTS: Mean sleep RQ was lower during the BIL (0.822) than the GL (0.846) treatment period, indicating greater lipid metabolism during the post-absorptive period with BIL. Increases in carbohydrate oxidation following breakfast were greater during BIL than GL treatment (mean change in RQ following breakfast 0.111 for BIL, 0.063 for GL). Furthermore, BIL treatment increased total daily EE versus GL (2215.9 kcal/d for BIL, 2135.5 kcal/d for GL). Concentrations of ketone bodies and acylcarnitines appeared to be higher following BIL than GL treatment. CONCLUSIONS: BIL increased sleeping fat oxidation, EE, ketone bodies, acylcarnitines and post-prandial glucose metabolism when switching from conventional insulin, thus, restoring metabolic flexibility and increasing thermogenesis. These changes may explain the previously observed weight loss with BIL versus GL.

Reduced skeletal muscle secreted frizzled-related protein 3 is associated with inflammation and insulin resistance.

OBJECTIVE: To investigate the role of secreted frizzled-related protein 3 (Sfrp3) in insulin sensitivity (ISi) and ß-cell function in humans across a spectrum of glucose homeostasis. METHODS: Subjects included those with normal glucose homeostasis (NGT; n = 18), prediabetes (PD; n = 11), or type 2 diabetes (T2D; n=12). Serum and skeletal muscle (SkM) Sfrp3 levels were measured by ELISA and qPCR, respectively, and insulin signaling pathway was assessed by Western blot. IS and ß-cell function were assessed by indices derived from frequently sampled intravenous glucose tolerance test. RESULTS: SkM Sfrp3 mRNA levels were significantly reduced in PD and T2D versus NGT. Similarly, serum Sfrp3 levels tended to be decreased in PD and T2D versus NGT. SkM Sfrp3 mRNA levels correlated negatively with circulating proinflammatory cytokines (IL-6, IFN-γ) and positively with IS. In vitro-differentiated myotubes from lean insulin-sensitive subjects treated with either lipopolysaccharide (LPS) or recombinant IL-6 demonstrated a dose-dependent reduction in Sfrp3 gene expression. Treatment of myotubes with recombinant Sfrp3 restored LPS- and IL-6-induced attenuation of insulin-stimulated Akt phosphorylation. CONCLUSIONS: Inflammation-induced reduction in SkM Sfrp3 expression may contribute to insulin resistance, and this effect may be prevented by addition of exogenous Sfrp3. Thus, Sfrp3 may be a novel target for insulin sensitization.

Inhibition of JNK mitochondrial localization and signaling is protective against ischemia/reperfusion injury in rats.

To build upon recent findings that mitochondrial JNK signaling is inhibited by selectively blocking the interaction between JNK and Sab, we utilized a cell-permeable peptide to demonstrate that ischemia/reperfusion (I/R) injury could be protected in vivo and that JNK mitochondrial signaling was the mechanism by which reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cardiomyocyte cell death occur. We also demonstrated that 5 mg/kg SR-3306 (a selective JNK inhibitor) was able to protect against I/R injury, reducing infarct volume by 34% (p < 0.05) while also decreasing I/R-induced increases in the activity of creatine phosphokinase and creatine kinase-MB. TUNEL staining showed that the percent TUNEL positive nuclei in rat hearts increased 10-fold after I/R injury and that this was reduced 4-fold (p < 0.01) by SR-3306. These data suggest that blocking JNK mitochondrial translocation or JNK inhibition prevents ROS increases and mitochondrial dysfunction and may be an effective treatment for I/R-induced cardiomyocyte death.

Small Molecule c-jun-N-terminal Kinase (JNK) Inhibitors Protect Dopaminergic Neurons in a Model of Parkinson's Disease.

There are currently no drugs to treat neurodegeneration in Parkinson's disease (PD) and all existing medications only treat symptoms, lose efficacy over time, and produce untoward side effects. In the current work, we report the first highly selective, orally bioavailable, c-jun-N-terminal kinase (JNK) inhibitor for protection of dopaminergic neurons in vitro and in vivo. At 300 nM this compound showed statistically significant protection of primary dopaminergic neurons exposed to 1-methyl-4-phenylpyridinium (MPP(+)), had pharmacokinetic properties in rodents consistent with twice daily (b.i.d.) dosing, and was orally efficacious at 30 mg/kg in a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Moreover, a dose-dependent target modulation of c-jun phosphorylation served as a biomarker for demonstrating on-target inhibition of JNK as the mechanism of action for this compound. Collectively these results suggest that this JNK inhibitor could be a promising therapeutic neuroprotective agent in the treatment of Parkinson's disease.

Tetrahydroisoquinoline derivatives as highly selective and potent Rho kinase inhibitors.

Rho kinase (ROCK) is a promising drug target for the treatment of many diseases including hypertension, multiple sclerosis, cancer, and glaucoma. The structure-activity relationships (SAR) around a series of tetrahydroisoquinolines were evaluated utilizing biochemical and cell-based assays to measure ROCK inhibition. These novel ROCK inhibitors possess high potency, high selectivity, and appropriate pharmacokinetic properties for glaucoma applications. The lead compound, 35, had subnanomolar potency in enzyme ROCK-II assays as well as excellent cell-based potency (IC(50) = 51 nM). In a kinase panel profiling, 35 had an off-target hit rate of only 1.6% against 442 kinases. Pharmacology studies showed that compound 35 was efficacious in reducing intraocular pressure (IOP) in rats with reasonably long duration of action. These results suggest that compound 35 may serve as a promising agent for further development in the treatment of glaucoma.

Bone morphogenetic protein 4 mediates myocardial ischemic injury through JNK-dependent signaling pathway.

Bone morphogenetic protein (BMP) signaling regulates embryonic development of many organ systems and defective BMP signaling has been implicated in adult disorders of many of these systems. However, its relevance in cardiac disease has not been reported. Here we demonstrate for the first time that Bmp4 activity promotes cellular apoptosis following ischemia-reperfusion (I/R) injury induced myocardial infarction (MI). Bmp4 heterozygous null mice (Bmp4(+/)(-)) demonstrated reduced infarct size, less myocardial apoptosis and down-regulation of pro-apoptotic proteins relative to wild-type mice following I/R injury. This was associated with reduction in I/R induced BMP4 levels in the left ventricular infarcted region. Furthermore, treatment of neonatal cardiomyocytes with BMP4 resulted in time and dose-dependent increase in cellular apoptosis and activation of the JNK MAP kinase pathway. In contrast, while JNK activation was significantly attenuated in Bmp4(+/)(-) mice and following Smad1 inhibition in myocytes, inhibition of JNK with a specific inhibitory peptide, TAT-JBD(20,) blocked BMP4 induced apoptosis. In vivo treatment of mice with Noggin, an endogenous extracellular BMP antagonist, or dorsomorphin, a small molecule inhibitor of BMP signaling, reduced infarct size, and inhibited pro-apoptotic signaling accompanied by an inhibition of Smad1 phosphorylation and JNK activation. These studies identify a novel role for Bmp4 in the pathogenesis of myocardial infarction and illustrate the use of a small molecule inhibitor of BMP signaling for treatment of acute I/R injury.

Discovery of Potent and Selective Urea-Based ROCK Inhibitors and Their Effects on Intraocular Pressure in Rats.

A series of urea-based Rho kinase (ROCK) inhibitors were designed and evaluated. The discovered compounds had excellent enzyme and cellular potency, high kinase selectivity, high aqueous solubility, good porcine corneal penetration, and appropriate DMPK profiles for topical applications as antiglaucoma therapeutics.

Nasal vaccination with troponin reduces troponin specific T-cell responses and improves heart function in myocardial ischemia-reperfusion injury.

Myocardial ischemia with subsequent reperfusion (MI/R) can lead to significant myocardial damage. Ischemia initiates inflammation at the blood-microvascular endothelial cell interface and contributes significantly to both acute injury and repair of the damaged tissue. We have found that MI/R injury in mice is associated with a cellular immune response to troponin. Myocardial cells exclusively synthesize troponin and release the troponin into the bloodstream following injury. Mucosally administered proteins induce T cells that secrete anti-inflammatory cytokines such as IL-10 and transforming growth factor beta at the anatomical site where the protein localizes. We found that nasal administration of the three subunits of troponin (C, I and T isoforms), given prior to or 1 h following MI/R, decreased infarct size by 40% measured 24 h later. At 1.5 months following MI/R, there was a 50% reduction in infarct size and improvement in cardiac function as measured by echocardiography. Protection was associated with a reduction of cellular immunity to troponin. Immunohistochemistry demonstrated increased IL-10 and reduced IFN-gamma in the area surrounding the ischemic infarct following nasal troponin. Adoptive transfer of CD4+ T cells to mice from nasally troponin-treated mice 1 h after the MI/R decreased infarct size by 72%, whereas CD4+ T cells from IL-10-/- mice or nasally BSA-treated mice had no effect. Our results demonstrate that IL-10-secreting CD4+ T cells induced by nasal troponin reduce injury following MI/R. Modulation of cardiac inflammation by nasal troponin provides a novel treatment to decrease myocardial damage and enhance recovery after myocardial ischemia.

Secreted frizzled related protein 2 protects cells from apoptosis by blocking the effect of canonical Wnt3a.

We have demonstrated that mesenchymal stem cells overexpressing the survival gene Akt can confer paracrine protection to ischemic myocytes both in vivo and in vitro through the release of secreted frizzled related protein 2 (Sfrp2). However, the mechanisms mediating these effects of Sfrp2 have not been fully elucidated. In this study, we studied rat cardiomyoblasts subjected to hypoxia reoxygenation (HR) injury to test the hypothesis that Sfrp2 exerts anti-apoptotic effect by antagonizing pro-apoptotic properties of specific Wnt ligands. We examined the effect of Wnt3a and Sfrp2 on HR-induced apoptosis. Wnt3a significantly increased cellular caspase activities and TUNEL staining in response to HR. Sfrp2 attenuated significantly Wnt3a-induced caspase activities in a concentration dependent fashion. Using a solid phase binding assay, our data demonstrates that Sfrp2 physically binds to Wnt3a. In addition, we observed that Sfrp2 dramatically inhibits the beta-catenin/TCF transcriptional activities induced by Wnt3a. Impressively, Dickkopf-1, a protein that binds to the Wnt coreceptor LRP, significantly inhibited the Wnt3a-activated caspase and transcriptional activities. Similarly, siRNA against beta-catenin markedly inhibited the Wnt3a-activated caspase activities. Consistent with this, significantly fewer TUNEL positive cells were observed in siRNA transfected cells than in control cells. Together, our data provide strong evidence to support the notion that Wnt3a is a canonical Wnt with pro-apoptotic action whose cellular activity is prevented by Sfrp2 through, at least in part, the direct binding of these molecules. These results can explain the in vivo protective effect of Sfrp2 and highlight its therapeutic potential for the ischemic heart.

Heme-oxygenase-1-induced protection against hypoxia/reoxygenation is dependent on biliverdin reductase and its interaction with PI3K/Akt pathway.

Heme-oxygenase-1 (HO-1), a stress-inducible protein, is an important cytoprotective agent against ischemia/reperfusion (I/R) injury. However, the role of downstream mediators involved in HO-1-induced cytoprotection is not clear. In the current study we investigated the role of biliverdin reductase, an enzyme involved in the conversion of HO-1-derived biliverdin into bilirubin and the PI3K/Akt pathway in mediating the cytoprotective effects of HO-1 against hypoxia and reoxygenation (H/R) injury in vitro and in vivo. H9c2 cardiomyocytes were transfected with a plasmid expressing HO-1 or LacZ and exposed to 24 h of hypoxia followed by 12 h of reoxygenation. At the end of reoxygenation, reactive oxygen species generation was determined using CM-H(2)DCFDA dye and apoptosis was assessed by TUNEL, caspase activity and Bad phosphorylation. p85 and Akt phosphorylation were determined using cell-based ELISA and phospho-specific antibodies, respectively. HO-1 overexpression increased phosphorylation of the regulatory subunit of the PI3K (p85alpha) and downstream effector Akt in H9c2 cells, leading to decreased ROS and apoptosis. Furthermore, cardiac expression of HO-1 increased basal phosphorylated Akt levels and decreased infarct size in response to LAD ligation and release induced I/R injury. Conversely, PI3K inhibition reversed the effects of HO-1 on Akt phosphorylation, cell death and infarct size. In addition, knockdown of biliverdin reductase (BVR) expression with siRNA attenuated HO-1-induced Akt phosphorylation and increased H/R-induced apoptosis of H9c2 cells. Co-immunoprecipitation revealed protein-protein interaction between BVR and the phosphorylated p85 subunit of the PI3 kinase. Taken together, these results suggest that the enzyme biliverdin reductase plays an important role in mediating cytoprotective effects of HO-1. This effect is mediated, at least in part, via interaction with and activation of the PI3K/Akt pathway.

Preemptive heme oxygenase-1 gene delivery reveals reduced mortality and preservation of left ventricular function 1 yr after acute myocardial infarction.

We reported previously that predelivery of heme oxygenase-1 (HO-1) gene to the heart by adeno-associated virus-2 (AAV-2) markedly reduces ischemia and reperfusion (I/R)-induced myocardial injury. However, the effect of preemptive HO-1 gene delivery on long-term survival and prevention of postinfarction heart failure has not been determined. We assessed the effect of HO-1 gene delivery on long-term survival, myocardial function, and left ventricular (LV) remodeling 1 yr after myocardial infarction (MI) using echocardiographic imaging, pressure-volume (PV) analysis, and histomorphometric approaches. Two groups of Lewis rats were injected with 2 x 10(11) particles of AAV-LacZ (control) or AAV-human HO-1 (hHO-1) in the anterior-posterior apical region of the LV wall. Six weeks after gene transfer, animals were subjected to 30 min of ischemia by ligation of the left anterior descending artery followed by reperfusion. Echocardiographic measurements and PV analysis of LV function were obtained at 2 wk and 12 mo after I/R. One year after acute MI, mortality was markedly reduced in the HO-1-treated animals compared with the LacZ-treated animals. PV analysis demonstrated significantly enhanced LV developed pressure, elevated maximal dP/dt, and lower end-diastolic volume in the HO-1 animals compared with the LacZ animals. Echocardiography showed a larger apical anterior-to-posterior wall ratio in HO-1 animals compared with LacZ animals. Morphometric analysis revealed extensive myocardial scarring and fibrosis in the infarcted LV area of LacZ animals, which was reduced by 62% in HO-1 animals. These results suggest that preemptive HO-1 gene delivery may be useful as a therapeutic strategy to reduce post-MI LV remodeling and heart failure.

Secreted frizzled related protein 2 (Sfrp2) is the key Akt-mesenchymal stem cell-released paracrine factor mediating myocardial survival and repair.

Stem cell therapy has emerged as a promising tool for the treatment of a variety of diseases. Previously, we have shown that Akt-modified mesenchymal stem cells mediate tissue repair through paracrine mechanisms. Using a comprehensive functional genomic strategy, we show that secreted frizzled related protein 2 (Sfrp2) is the key stem cell paracrine factor that mediates myocardial survival and repair after ischemic injury. Sfrp2 is known to modulate Wnt signaling, and we demonstrate that cardiomyocytes treated with secreted frizzled related protein increase cellular beta-catenin and up-regulate expression of antiapoptotic genes. These findings reveal the key role played by Sfrp2 in mediating the paracrine effects of Akt-mesenchymal stem cells on tissue repair and identify modulation of Wnt signaling as a therapeutic target for heart disease.

Heme oxygenase-1 (HO-1) inhibits postmyocardial infarct remodeling and restores ventricular function.

We reported previously that predelivery of the anti-oxidant gene heme oxygenase-1 (HO-1) to the heart by adeno associated virus (AAV) markedly reduces injury after acute myocardial infarction (MI). However, the effect of HO-1 gene delivery on postinfarction recovery has not been investigated. In the current study, we assessed the effect of HO-1 gene delivery on post-MI left ventricle (LV) remodeling and function using echocardiographic imaging and histomorphometric approaches. Two groups of Sprague-Dawley rats were injected with 4 x 10(11) particles of AAV-LacZ (control) or AAV-hHO-1 in the LV wall. Eight wk after gene transfer, the animals were subjected to 30 min of ischemia by ligation of left anterior descending artery (LAD) followed by reperfusion. Echocardiographic measurements were obtained in a blinded fashion prior and at 1.5 and 3 months after I/R. Ejection fraction (EF) was reduced by 13% and 40% in the HO-1 and LacZ groups, respectively at 1.5 months after MI. Three months after MI, EF recovered fully in the HO-1, but only partially in the LacZ-treated animals. Post-MI LV dimensions were markedly increased and the anterior wall was markedly thinned in the LacZ-treated animals compared with the HO-1-treated animals. Significant myocardial scarring and fibrosis were observed in the LacZ-group in association with elevated levels of interstitial collagen I and III and MMP-2 activity. Post-MI myofibroblast accumulation was reduced in the HO-1-treated animals, and retroviral overexpression of HO-1 reduced proliferation of isolated cardiac fibroblasts. Our data indicate that rAAV-HO-1 gene transfer markedly reduces fibrosis and ventricular remodeling and restores LV function and chamber dimensions after myocardial infarction.

Chronic recurrent myocardial ischemic injury is significantly attenuated by pre-emptive adeno-associated virus heme oxygenase-1 gene delivery.

OBJECTIVES: We assessed the hypothesis that overexpression of the antioxidant enzyme heme oxygenase (HO)-1 may protect against chronic recurrent ischemia/reperfusion injury. BACKGROUND: Multiple and recurring episodes of myocardial ischemia can result in significant myocardial damage, including myocyte death, fibrosis, and wall thinning, leading to impaired ventricular function and cardiac failure. METHODS: In this study we used a closed-chest rodent model of chronic recurring myocardial ischemia and reperfusion to investigate the efficacy of pre-emptive gene therapy in overexpressing the antioxidant enzyme HO-1, using adeno-associated virus (AAV)-2 as the delivery vector. RESULTS: We show that constitutive overexpression of HO-1 can prevent myocardial wall thinning, inflammation, fibrosis, and deterioration of cardiac function (as measured by echocardiography, histology, and immunohistochemistry) induced by repeated transient myocardial ischemia and reperfusion injury. With HO-1 therapy, there was a significant reduction in apoptosis as determined by levels of markers of survival proteins and terminal deoxynucleotidyltransferase dUTP nick end-labeling staining. This prevention of tissue damage was also associated with reduction in superoxide generation. CONCLUSIONS: Taken together we provide the first evidence of the therapeutic efficacy of pre-emptive AAV-HO-1 delivery for prevention against multiple ischemic injury. This approach protects myocytes by simultaneously activating protective response and inhibiting pathological left ventricular remodeling and, therefore, may be a useful cardio-protective strategy for patients with coronary artery disease at a high risk for recurrent myocardial ischemia.