Bioactive Suture with Added Innate Defense Functionality for the Reduction of Bacterial Infection and Inflammation.

Surgical site infections (SSI) are a clinical and economic burden. Suture-associated SSI may develop when bacteria colonize the suture surface and form biofilms that are resistant to antibiotics. Thrombin-derived C-terminal peptide (TCP)-25 is a host defense peptide with a unique dual mode of action that can target both bacteria and the excessive inflammation induced by bacterial products. The peptide demonstrates therapeutic potential in preclinical in vivo wound infection models. In this study, the authors set out to explore whether TCP-25 can provide a new bioactive innate immune feature to hydrophilic polyglactin sutures (Vicryl). Using a combination of biochemical, biophysical, antibacterial, biofilm, and anti-inflammatory assays in vitro, in silico molecular modeling studies, along with experimental infection and inflammation models in mice, a proof-of-concept that TCP-25 can provide Vicryl sutures with a previously undisclosed host defense capacity, that enables targeting of bacteria, biofilms, and the accompanying inflammatory response, is shown.

Peptide-coated polyurethane material reduces wound infection and inflammation.

There is an urgent need for treatments that not only reduce bacterial infection that occurs during wounding but that also target the accompanying excessive inflammatory response. TCP-25, a thrombin-derived antibacterial peptide, scavenges toll-like receptor agonists such as endotoxins and lipoteichoic acid and prevents toll-like receptor-4 dimerization to reduce infection-related inflammation in vivo. Using a combination of biophysical, cellular, and microbiological assays followed by experimental studies in mouse and pig models, we show that TCP-25, when delivered from a polyurethane (PU) material, exerts anti-infective and anti-inflammatory effects in vitro and in vivo. Specifically, TCP-25 killed the common wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, in both in vitro and in vivo assays. Furthermore, after its release from the PU material, the peptide retained its capacity to induce its helical conformation upon endotoxin interaction, yielding reduced activation of NF-κB in THP-1 reporter cells, and diminished accumulation of inflammatory cells and subsequent release of IL-6 and TNF-α in subcutaneous implant models in vivo. Moreover, in a porcine partial thickness wound infection model, TCP-25 treated infection with S. aureus, and reduced the concomitant inflammatory response. Taken together, these findings demonstrate a combined antibacterial and anti-inflammatory effect of TCP-25 delivered from PU in vitro, and in mouse and porcine in vivo models of localized infection-inflammation. STATEMENT OF SIGNIFICANCE: Local wound infections may result in systemic complications and can be difficult to treat due to increasing antimicrobial resistance. Surgical site infections and biomaterial-related infections present a major challenge for hospitals. In recent years, various antimicrobial coatings have been developed for infection prevention and current concepts focus on various matrices with added anti-infective components, including various antibiotics and antiseptics. We have developed a dual action wound dressing concept where the host defense peptide TCP-25, when delivered from a PU material, targets both bacterial infection and the accompanying inflammation. TCP-25 PU showed efficacy in in vitro and experimental wound models in mouse and minipigs.

Crosstalk between Mast Cells and Lung Fibroblasts Is Modified by Alveolar Extracellular Matrix and Influences Epithelial Migration.

Mast cells play an important role in asthma, however, the interactions between mast cells, fibroblasts and epithelial cells in idiopathic pulmonary fibrosis (IPF) are less known. The objectives were to investigate the effect of mast cells on fibroblast activity and migration of epithelial cells. Lung fibroblasts from IPF patients and healthy individuals were co-cultured with LAD2 mast cells or stimulated with the proteases tryptase and chymase. Human lung fibroblasts and mast cells were cultured on cell culture plastic plates or decellularized human lung tissue (scaffolds) to create a more physiological milieu by providing an alveolar extracellular matrix. Released mediators were analyzed and evaluated for effects on epithelial cell migration. Tryptase increased vascular endothelial growth factor (VEGF) release from fibroblasts, whereas co-culture with mast cells increased IL-6 and hepatocyte growth factor (HGF). Culture in scaffolds increased the release of VEGF compared to culture on plastic. Migration of epithelial cells was reduced by IL-6, while HGF and conditioned media from scaffold cultures promoted migration. In conclusion, mast cells and tryptase increased fibroblast release of mediators that influenced epithelial migration. These data indicate a role of mast cells and tryptase in the interplay between fibroblasts, epithelial cells and the alveolar extracellular matrix in health and lung disease.

PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes.

The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3-II and PNPLA3 on lipid droplets. We show that the M148-PNPLA3 variant is associated with lipid droplets that are resistant to starvation-mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148-PNPLA3 and M148-PNPLA3 colocalize and interact with LC3-II, but the M148-PNPLA3 variant has lower ability to bind LC3-II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148-PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.

Rosiglitazone drives cavin-2/SDPR expression in adipocytes in a CEBPα-dependent manner.

Caveolae are abundant adipocyte surface domains involved in insulin signaling, membrane trafficking and lipid homeostasis. Transcriptional control mechanisms for caveolins and cavins, the building blocks of caveolae, are thus arguably important for adipocyte biology and studies in this area may give insight into insulin resistance and diabetes. Here we addressed the hypothesis that one of the less characterized caveolar components, cavin-2 (SDPR), is controlled by CCAAT/Enhancer Binding Protein (CEBPα) and Peroxisome Proliferator-Activated Receptor Gamma (PPARG). Using human mRNA expression data we found that SDPR correlated with PPARG in several tissues. This was also observed during differentiation of 3T3-L1 fibroblasts into adipocytes. Treatment of 3T3-L1-derived adipocytes with the PPARγ-activator Rosiglitazone increased SDPR and CEBPα expression at both the mRNA and protein levels. Silencing of CEBPα antagonized these effects. Further, adenoviral expression of PPARγ/CEBPα or Rosiglitazone-treatment increased SDPR expression in primary rat adipocytes. The myocardin family coactivator MKL1 was recently shown to regulate SDPR expression in human coronary artery smooth muscle cells. However, we found that actin depolymerization, known to inhibit MKL1 and MKL2, was without effect on SDPR mRNA levels in adipocytes, even though overexpression of MKL1 and MKL2 had the capacity to increase caveolins and cavins and to repress PPARγ/CEBPα. Altogether, this work demonstrates that CEBPα expression and PPARγ-activity promote SDPR transcription and further supports the emerging notion that PPARγ/CEBPα and MKL1/MKL2 are antagonistic in adipocytes.

Salt-inducible kinase 2 and -3 are downregulated in adipose tissue from obese or insulin-resistant individuals: implications for insulin signalling and glucose uptake in human adipocytes.

AIMS/HYPOTHESIS: Salt-inducible kinases (SIKs) are related to the metabolic regulator AMP-activated protein kinase (AMPK). SIK2 is abundant in adipose tissue. The aims of this study were to investigate the expression of SIKs in relation to human obesity and insulin resistance, and to evaluate whether changes in the expression of SIKs might play a causal role in the development of disturbed glucose uptake in human adipocytes. METHODS: SIK mRNA and protein was determined in human adipose tissue or adipocytes, and correlated to clinical variables. SIK2 and SIK3 expression and phosphorylation were analysed in adipocytes treated with TNF-α. Glucose uptake, GLUT protein levels and localisation, phosphorylation of protein kinase B (PKB/Akt) and the SIK substrate histone deacetylase 4 (HDAC4) were analysed after the SIKs had been silenced using small interfering RNA (siRNA) or inhibited using a pan-SIK-inhibitor (HG-9-91-01). RESULTS: We demonstrate that SIK2 and SIK3 mRNA are downregulated in adipose tissue from obese individuals and that the expression is regulated by weight change. SIK2 is also negatively associated with in vivo insulin resistance (HOMA-IR), independently of BMI and age. Moreover, SIK2 protein levels and specific kinase activity display a negative correlation to BMI in human adipocytes. Furthermore, SIK2 and SIK3 are downregulated by TNF-α in adipocytes. Silencing or inhibiting SIK1-3 in adipocytes results in reduced phosphorylation of HDAC4 and PKB/Akt, less GLUT4 at the plasma membrane, and lower basal and insulin-stimulated glucose uptake in adipocytes. CONCLUSION/INTERPRETATION: This is the first study to describe the expression and function of SIKs in human adipocytes. Our data suggest that SIKs might be protective in the development of obesity-induced insulin resistance, with implications for future treatment strategies.

Perilipin 1 binds to aquaporin 7 in human adipocytes and controls its mobility via protein kinase A mediated phosphorylation.

Accumulating evidence suggests that dysregulated glycerol metabolism contributes to the pathophysiology of obesity and type 2 diabetes. Glycerol efflux from adipocytes is regulated by the aquaglyceroporin AQP7, which is translocated upon hormone stimulation. Here, we propose a molecular mechanism where the AQP7 mobility in adipocytes is dependent on perilipin 1 and protein kinase A. Biochemical analyses combined with ex vivo studies in human primary adipocytes, demonstrate that perilipin 1 binds to AQP7, and that catecholamine activated protein kinase A phosphorylates the N-terminus of AQP7, thereby reducing complex formation. Together, these findings are indicative of how glycerol release is controlled in adipocytes, and may pave the way for the future design of drugs against human metabolic pathologies.

ApoA-I Milano stimulates lipolysis in adipose cells independently of cAMP/PKA activation.

ApoA-I, the main protein component of HDL, is suggested to be involved in metabolic homeostasis. We examined the effects of Milano, a naturally occurring ApoA-I variant, about which little mechanistic information is available. Remarkably, high-fat-fed mice treated with Milano displayed a rapid weight loss greater than ApoA-I WT treated mice, and a significantly reduced adipose tissue mass, without an inflammatory response. Further, lipolysis in adipose cells isolated from mice treated with either WT or Milano was increased. In primary rat adipose cells, Milano stimulated cholesterol efflux and increased glycerol release, independently of ß-adrenergic stimulation and phosphorylation of hormone sensitive lipase (Ser563) and perilipin (Ser522). Stimulation with Milano had a significantly greater effect on glycerol release compared with WT but similar effect on cholesterol efflux. Pharmacological inhibition or siRNA silencing of ABCA1 did not diminish Milano-stimulated lipolysis, although binding to the cell surface was decreased, as analyzed by fluorescence microscopy. Interestingly, methyl-ß-cyclodextrin, a well-described cholesterol acceptor, dose-dependently stimulated lipolysis. Together, these results suggest that decreased fat mass and increased lipolysis following Milano treatment in vivo is partly explained by a novel mechanism at the adipose cell level comprising stimulation of lipolysis independently of the canonical cAMP/protein kinase A signaling pathway.

SIK2 regulates CRTCs, HDAC4 and glucose uptake in adipocytes.

Salt-inducible kinase 2 (SIK2) is an AMP-activated protein kinase (AMPK) related kinase abundantly expressed in adipose tissue. Our aim was to identify molecular targets and functions of SIK2 in adipocytes, and to address the role of PKA-mediated phosphorylation of SIK2 on Ser358. Modulation of SIK2 in adipocytes resulted in altered phosphorylation of CREB-regulated transcription co-activator 2 (CRTC2), CRTC3 and class IIa histone deacetylase 4 (HDAC4). Furthermore, CRTC2, CRTC3, HDAC4 and protein phosphatase 2A (PP2A) interacted with SIK2, and the binding of CRTCs and PP2A to wild-type but not Ser358Ala SIK2, was reduced by cAMP elevation. Silencing of SIK2 resulted in reduced GLUT4 (also known as SLC2A4) protein levels, whereas cells treated with CRTC2 or HDAC4 siRNA displayed increased levels of GLUT4. Overexpression or pharmacological inhibition of SIK2 resulted in increased and decreased glucose uptake, respectively. We also describe a SIK2­CRTC2­HDAC4 pathway and its regulation in human adipocytes, strengthening the physiological relevance of our findings. Collectively, we demonstrate that SIK2 acts directly on CRTC2, CRTC3 and HDAC4, and that the cAMP­PKA pathway reduces the interaction of SIK2 with CRTCs and PP2A. Downstream, SIK2 increases GLUT4 levels and glucose uptake in adipocytes.