Functional characterization of 3D contractile smooth muscle tissues generated using a unique microfluidic 3D bioprinting technology.

Conditions such as asthma and inflammatory bowel disease are characterized by aberrant smooth muscle contraction. It has proven difficult to develop human cell-based models that mimic acute muscle contraction in 2D in vitro cultures due to the nonphysiological chemical and mechanical properties of lab plastics that do not allow for muscle cell contraction. To enhance the relevance of in vitro models for human disease, we describe how functional 3D smooth muscle tissue that exhibits physiological and pharmacologically relevant acute contraction and relaxation responses can be reproducibly fabricated using a unique microfluidic 3D bioprinting technology. Primary human airway and intestinal smooth muscle cells were printed into rings of muscle tissue at high density and viability. Printed tissues contracted to physiological concentrations of histamine (0.01-100 µM) and relaxed to salbutamol, a pharmacological compound used to relieve asthmatic exacerbations. The addition of TGFß to airway muscle rings induced an increase in unstimulated muscle shortening and a decreased response to salbutamol, a phenomenon which also occurs in chronic lung diseases. Results indicate that the 3D bioprinted smooth muscle is a physiologically relevant in vitro model that can be utilized to study disease pathways and the effects of novel therapeutics on acute contraction and chronic tissue stenosis.

PCSK9 is a critical regulator of the innate immune response and septic shock outcome.

A decrease in the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9) increases the amount of low-density lipoprotein (LDL) receptors on liver cells and, therefore, LDL clearance. The clearance of lipids from pathogens is related to endogenous lipid clearance; thus, PCSK9 may also regulate removal of pathogen lipids such as lipopolysaccharide (LPS). Compared to controls, Pcsk9 knockout mice displayed decreases in inflammatory cytokine production and in other physiological responses to LPS. In human liver cells, PCSK9 inhibited LPS uptake, a necessary step in systemic clearance and detoxification. Pharmacological inhibition of PCSK9 improved survival and inflammation in murine polymicrobial peritonitis. Human PCSK9 loss-of-function genetic variants were associated with improved survival in septic shock patients and a decrease in inflammatory cytokine response both in septic shock patients and in healthy volunteers after LPS administration. The PCSK9 effect was abrogated in LDL receptor (LDLR) knockout mice and in humans who are homozygous for an LDLR variant that is resistant to PCSK9. Together, our results show that reduced PCSK9 function is associated with increased pathogen lipid clearance via the LDLR, a decreased inflammatory response, and improved septic shock outcome.

A common polymorphism in the 5' region of the human protein c gene binds USF1.

INTRODUCTION: Genetic variation in the Protein C gene (PROC) is associated with altered risk of adverse outcome for a number of diseases. Common single nucleotide polymorphisms (SNPs) in the promoter region and the adjacent 5' region of PROC are associated with Protein C expression. We tested the hypothesis that common SNPs (minor allele frequency >10%) between the frequently studied promoter SNPs -1654 (rs1799808) and -1641 (rs1799809), and the end of PROC intron 2 alter nuclear transcription factor binding. MATERIALS AND METHODS: We used electrophoretic mobility shift assays with 25-mer oligonucleotides centered on each of the 10 SNPs assessed in this potential regulatory region of the Protein C gene to test for differential binding to nuclear factors isolated from Hep-G2 cells. RESULTS: We found that the G-allele oligo of the intron 2 SNP rs2069915[G/A] bound nuclear factors more avidly than the A-allele (p=1.9 × 10(-9), n=24). Similarly, we found that the C-allele oligo of the intron 2 SNP rs2069916[C/T] bound nuclear factors more avidly than the T-allele, (p=3.7 × 10(-6), n=19). Cold competition and supershift assays suggested that the protein differentially binding to the C-allele of rs2069916 was USF1. Notably, we observed minimal nuclear factor binding to oligos containing haplotypes of the previously reported -1654 and -1641 SNPs. Luciferase reporter assays that showed the A-T haplotype of rs2069915 and rs2069916 drives transcription significantly more than the C-G haplotype (t-test, P=0.015, n=12). CONCLUSION: Differential transcription factor binding occurs for common SNPs in the 5' intronic regions of PROC which may contribute to PROC regulation and reported PROC SNP - phenotype associations.

Late intervention with a myeloperoxidase inhibitor stops progression of experimental chronic obstructive pulmonary disease.

RATIONALE: Inflammation and oxidative stress are linked to the deleterious effects of cigarette smoke in producing chronic obstructive pulmonary disease (COPD). Myeloperoxidase (MPO), a neutrophil and macrophage product, is important in bacterial killing, but also drives inflammatory reactions and tissue oxidation. OBJECTIVES: To determine the role of MPO in COPD. METHODS: We treated guinea pigs with a 2-thioxanthine MPO inhibitor, AZ1, in a 6-month cigarette smoke exposure model, with one group receiving compound from Smoking Day 1 and another group treated after 3 months of smoke exposure. RESULTS: At 6 months both treatments abolished smoke-induced increases in lavage inflammatory cells, largely ameliorated physiological changes, and prevented or stopped progression of morphologic emphysema and small airway remodeling. Cigarette smoke caused a marked increase in immunohistochemical staining for the myeloperoxidase-generated protein oxidation marker dityrosine, and this effect was considerably decreased with both treatment arms. Serum 8-isoprostane, another marker of oxidative stress, showed similar trends. Both treatments also prevented muscularization of the small intrapulmonary arteries, but only partially ameliorated smoke-induced pulmonary hypertension. Acutely, AZ1 prevented smoke-induced increases in expression of cytokine mediators and nuclear factor-κB binding. CONCLUSIONS: We conclude that an MPO inhibitor is able to stop progression of emphysema and small airway remodeling and to partially protect against pulmonary hypertension, even when treatment starts relatively late in the course of long-term smoke exposure, suggesting that inhibition of MPO may be a novel and useful therapeutic treatment for COPD. Protection appears to relate to inhibition of oxidative damage and down-regulation of the smoke-induced inflammatory response.

Leucyl/cystinyl aminopeptidase gene variants in septic shock.

BACKGROUND: Vasopressin is an essential peptide hormone regulating cardiovascular homeostasis and an adjunctive vasopressor therapy for septic shock. METHODS: We tested for association between single nucleotide polymorphisms (SNPs) in vasopressin pathway genes and altered outcome in derivation (n = 589) and replication (n = 616) cohorts of patients with septic shock. The primary outcome was 28-day mortality and the secondary outcome was vasopressin clearance. In a third cardiac surgical cohort (n = 977), we tested for locus-specific heritability of serum sodium concentrations. RESULTS: Of 17 tested tag SNPs in five vasopressin pathway genes (arginine vasopressin [AVP], arginine vasopressin receptor 1A and 1B [AVPR1A, AVPR1B], leucyl/cystinyl aminopeptidase [LNPEP], and oxytocin receptor [OXTR]), rs18059 in LNPEP (also known as vasopressinase) was associated with 28-day mortality in the derivation cohort (P = .037). Therefore, we resequenced the 160-kb haplotype block encompassing the LNPEP gene, including rs18059, and genotyped the 230 identified SNPs in the derivation cohort. The strongest signal was found for LNPEP rs4869317 (adjusted P = .044). The rs4869317 TT genotype was associated with increased 28-day mortality in the derivation cohort (51.0% [TT] vs 34.5% [AA/AT]; adjusted hazard ratio [HR], 1.58; 95% CI, 1.21-2.06; P = .00073) and the replication cohort (38.6% vs 29.6%; HR, 1.36; 95% CI, 1.03-1.80; P = .030). We found that the TT genotype was associated with increased plasma vasopressin clearance (P = .028), and the rs4869317 genotype accounted for 80% of the variance of serum sodium concentrations (locus-specific heritability) in cardiac surgical patients. CONCLUSIONS: The genetic variation in LNPEP (vasopressinase) is associated with 28-day mortality in septic shock and is associated with biologic effects on vasopressin clearance and serum sodium regulation. Further confirmation in additional cohorts is required.

beta2-Adrenergic receptor gene polymorphism is associated with mortality in septic shock.

RATIONALE: The CysGlyGln haplotype of the beta(2)-adrenergic receptor gene (ADRB2) is functional and associated with altered responses to adrenergic agonists in patients with asthma. Whether this functional haplotype alters outcome in patients receiving adrenergic agonists in septic shock is unknown. OBJECTIVES: To determine whether genetic variation of ADRB2 influences outcome in septic shock. METHODS: Two cohorts of patients with septic shock were studied: a single center (St. Paul's Hospital [SPH]) cohort (n = 589) and the Vasopressin and Septic Shock Trial (VASST) cohort (n = 616). The A allele of the rs1042717 G/A polymorphism is in complete linkage disequilibrium with the CysGlyGln haplotype of ADRB2; therefore, rs1042717 was genotyped. Modulation by norepinephrine and salbutamol of IL-6 production by stimulated in vitro lymphoblastoid cells was measured by genotype. MEASUREMENTS AND MAIN RESULTS: Patients who had the AA genotype of rs1042717 displayed increased 28-day mortality in SPH (adjusted hazard ratio, 2.23; 95% confidence interval, 1.33-3.72; P = 0.0022), and this result was replicated in VASST (adjusted hazard ratio 2.82; 95% confidence interval, 1.56-5.09; P = 0.0006). This genotypic effect was eliminated in patients treated with acute low-dose corticosteroids. In all patients, the AA genotype was associated with more organ dysfunction. Patients with the AA genotype had a higher heart rate (SPH; P < 0.05; VASST; P < 0.05) and required a higher norepinephrine dose over Days 1 through 3 (VASST; P < 0.05). The AA genotype was associated with decreased norepinephrine and salbutamol inhibition of IL-6 production by stimulated lymphoblastoid cells in vitro (P < 0.05). CONCLUSIONS: The AA genotype of ADRB2 rs1042717, identifying homozygotes for the CysGlyGln haplotype, was associated with increased mortality and more organ dysfunction in septic shock.