Gut microbiome of a porcine model of metabolic syndrome and HF-pEF.

Metabolic syndrome (MetS) is a composite of cardiometabolic risk factors, including obesity, dyslipidemia, hypertension, and insulin resistance, with a range of secondary sequelae such as nonalcoholic fatty liver disease and diastolic heart failure. This syndrome has been identified as one of the greatest global health challenges of the 21st century. Herein, we examine whether a porcine model of diet- and mineralocorticoid-induced MetS closely mimics the cardiovascular, metabolic, gut microbiota, and functional metataxonomic phenotype observed in human studies. Landrace pigs with deoxycorticosterone acetate-induced hypertension fed a diet high in fat, salt, and sugar over 12 wk were assessed for hyperlipidemia, hyperinsulinemia, and immunohistologic, echocardiographic, and hemodynamic parameters, as well as assessed for microbiome phenotype and function through 16S rRNA metataxonomic and metabolomic analysis, respectively. All MetS animals developed obesity, hyperlipidemia, insulin resistance, hypertension, fatty liver, structural cardiovascular changes including left ventricular hypertrophy and left atrial enlargement, and increased circulating saturated fatty acid levels, all in keeping with the human phenotype. A reduction in α-diversity and specific microbiota changes at phylum, family, and genus levels were also observed in this model. Specifically, this porcine model of MetS displayed increased abundances of proinflammatory bacteria coupled with increased circulating tumor necrosis factor-α and increased secondary bile acid-producing bacteria, which substantially impacted fibroblast growth factor-19 expression. Finally, a significant decrease in enteroprotective bacteria and a reduction in short-chain fatty acid-producing bacteria were also noted. Together, these data suggest that diet and mineralocorticoid-mediated development of biochemical and cardiovascular stigmata of metabolic syndrome in pigs leads to temporal gut microbiome changes that mimic key gut microbial population signatures in human cardiometabolic disease.NEW & NOTEWORTHY This study extends a prior porcine model of cardiometabolic syndrome to include systemic inflammation, fatty liver, and insulin sensitivity. Gut microbiome changes during evolution of porcine cardiometabolic disease recapitulate those in human subjects with alterations in gut taxa associated with proinflammatory bacteria, bile acid, and fatty acid pathways. This clinical scale model may facilitate design of future interventional trials to test causal relationships between gut dysbiosis and cardiometabolic syndrome at a systemic and organ level.

Bio-inspired microneedle design for efficient drug/vaccine coating.

Biomimetics is the interdisciplinary scientific field focused on the study and imitation of biological systems, with the aim of solving complex technological problems. In this paper, we present a new bio-inspired design for microneedles (MNs) and MN arrays, intended for rapidly coating the MNs with drug/vaccine. The biomimetic approach consists in ornamenting the lateral sides of pyramidal MNs with structures inspired by the external scent efferent systems of some European true bugs, which facilitate a directional liquid transport. To realize these MNs, two-photon polymerization (TPP) technique was used. Liquid coating capabilities of structured and non-structured MNs were compared. Moreover, both in-vivo and ex-vivo skin tests were performed to prove that MNs pierce the skin. We show that the arrays of MNs can be accurately replicated using a micro-moulding technique. We believe this design will be beneficial for the process of drug/vaccine loading onto the needles' surfaces, by making it more efficient and by reducing the drug/vaccine wastage during MN coating process.

A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.

Selective M2 Macrophage Depletion Leads to Prolonged Inflammation in Surgical Wounds.

BACKGROUND: A prolonged inflammatory phase is seen in aberrant wound healing and in chronic wounds. Macrophages are central to wound healing. Distinct macrophage subtypes have differing roles both in initial inflammation and in later tissue repair. Broadly, these cells can be divided into M1 and M2 macrophages. M2 macrophage proliferation and differentiation is regulated by colony-stimulating factor 1 (CSF-1) signalling and can be blocked by GW2580, a competitive cFMS kinase inhibitor, thereby allowing for analysis of the effect of M2 blockade on progression of surgical wounds. MATERIALS AND METHODS: Macrophage Fas-induced apoptosis (MaFIA) transgenic mice with a macrophage-specific promoter used to express green fluorescent protein (GFP) were used to allow for cell tracking. The animals were treated by oral gavage with GW2580. Surgical wounds were created and harvested after 2 weeks for analysis. RESULTS: GW2580-treated mice had significantly more GFP+ cells in the surgical scar than vehicle-treated animals (GW2580, 68.0 ± 3.1%; vehicle, 42.8 ± 1.7%; p < 0.001), and GW2580 treatment depleted CD206+ M2 macrophages in the scar (GW2580, 1.4%; vehicle, 19.3%; p < 0.001). Treated animals showed significantly higher numbers of neutrophils (vehicle, 18.0%; GW2580, 51.3%; p < 0.01) and M1 macrophages (vehicle, 3.8%; GW2580, 12.8%; p < 0.01) in the scar compared to vehicle-treated animals. The total collagen content in the area of the scar was decreased in animals treated with GW2580 as compared to those treated with vehicle alone (GW2580, 67.1%; vehicle, 79.9%; p < 0.005). CONCLUSIONS: Depletion of M2 macrophages in surgical wounds via CSF-1 signalling blockade leads to persistent inflammation, with an increase in neutrophils and M1 macrophages and attenuated collagen deposition.

Bone Marrow-Derived Mesenchymal Stem Cells Have Innate Procoagulant Activity and Cause Microvascular Obstruction Following Intracoronary Delivery: Amelioration by Antithrombin Therapy.

Mesenchymal stem cells (MSCs) are currently under investigation as tools to preserve cardiac structure and function following acute myocardial infarction (AMI). However, concerns have emerged regarding safety of acute intracoronary (IC) MSC delivery. This study aimed to characterize innate prothrombotic activity of MSC and identify means of its mitigation toward safe and efficacious therapeutic IC MSC delivery post-AMI. Expression of the initiator of the coagulation cascade tissue factor (TF) on MSC was detected and quantified by immunofluorescence, FACS, and immunoblotting. MSC-derived TF antigen was catalytically active and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet thrombus deposition on collagen at arterial shear, an effect abolished by heparin coadministration. In a porcine AMI model, IC infusion of 25 × 10(6) MSC during reperfusion was associated with a decrease in coronary flow reserve but not when coadministered with an antithrombin agent (heparin). Heparin reduced MSC-associated thrombosis incorporating platelets and VWF within the microvasculature. Heparin-assisted therapeutic MSC delivery also reduced apoptosis in the infarct border zone at 24 hours, significantly improved infarct size, left ventricular (LV) ejection fraction, LV volumes, wall motion, and attenuated histologic evidence of scar formation at 6 weeks post-AMI. Heparin alone or heparin-assisted fibroblast control cell delivery had no such effect. Procoagulant TF activity of therapeutic MSCs is associated with reductions in myocardial perfusion when delivered IC may be successfully managed by heparin coadministration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic IC MSC delivery for AMI.

Synthetic chemically modified mrna-based delivery of cytoprotective factor promotes early cardiomyocyte survival post-acute myocardial infarction.

To extend the temporal window for cytoprotection in cardiomyocytes undergoing apoptosis after hypoxia and myocardial infarction (MI), a synthetic chemically modified mRNA (modRNA) was used to drive delivery of insulin-like growth factor-1 (IGF1) within the area at risk in an in vivo murine model of MI. Delivery of IGF1 modRNA, with a polyethylenimine-based nanoparticle, augmented secreted and cell-associated IGF1, promoting cardiomyocyte survival and abrogating cell apoptosis under hypoxia-induced apoptosis conditions. Translation of modRNA-IGF1 was sufficient to induce downstream increases in the levels of Akt and Erk phosphorylation. Downregulation of IGF1 specific miRNA-1 and -133 but not miR-145 expression was also confirmed. As a proof of concept, intramyocardial delivery of modRNA-IGF1 but not control modRNA-GFP significantly decreased the level of TUNEL positive cells, augmented Akt phosphorylation, and decreased caspase-9 activity within the infarct border zone 24 h post-MI. These findings demonstrate the potential for an extended cytoprotective effect of transient IGF1 driven by synthetic modRNA delivery.

Hollow silicon microneedles, fabricated using combined wet and dry etching techniques, for transdermal delivery and diagnostics.

Microneedle-based technologies are the subject of intense research and commercial interest for applications in transdermal delivery and diagnostics, primarily because of their minimally invasive and painless nature, which in turn could lead to increased patient compliance and self-administration. In this paper, a process for the fabrication of arrays of hollow silicon microneedles is described. This method uses just two bulk silicon etches - a front-side wet etch to define the 500 µm tall octagonal needle structure itself, and a rear-side dry etch to create a 50 µm diameter bore through the needle. This reduces the number of etches and process complexity over the approaches described elsewhere. Ex-vivo human skin and a customised applicator were used to demonstrate biomechanical reliability and the feasibility of using these microneedles for both transdermal delivery and diagnostics. Microneedle arrays show no damage even when applied to skin up to 40 times, are capable of delivering several mL of fluid at flowrates of 30 µL/min, and of withdrawing 1 µL of interstitial fluid using capillary action.

Development and clinical translation of tubular constructs for tracheal tissue engineering: a review.

Effective restoration of extensive tracheal damage arising from cancer, stenosis, infection or congenital abnormalities remains an unmet clinical need in respiratory medicine. The trachea is a 10-11 cm long fibrocartilaginous tube of the lower respiratory tract, with 16-20 tracheal cartilages anterolaterally and a dynamic trachealis muscle posteriorly. Tracheal resection is commonly offered to patients suffering from short-length tracheal defects, but replacement is required when the trauma exceeds 50% of total length of the trachea in adults and 30% in children. Recently, tissue engineering (TE) has shown promise to fabricate biocompatible tissue-engineered tracheal implants for tracheal replacement and regeneration. However, its widespread use is hampered by inadequate re-epithelialisation, poor mechanical properties, insufficient revascularisation and unsatisfactory durability, leading to little success in the clinical use of tissue-engineered tracheal implants to date. Here, we describe in detail the historical attempts and the lessons learned for tracheal TE approaches by contextualising the clinical needs and essential requirements for a functional tracheal graft. TE manufacturing approaches explored to date and the clinical translation of both TE and non-TE strategies for tracheal regeneration are summarised to fully understand the big picture of tracheal TE and its impact on clinical treatment of extensive tracheal defects.

Mesenchymal stromal cell derived CCL2 is required for accelerated wound healing.

Mesenchymal stromal cells (MSC) have immunomodulatory effects impacting macrophages, promoting polarisation towards a reparative phenotype. CCL2 is a potent cytokine involved in the recruitment of macrophages. We hypothesised that MSC derived CCL2 may be involved in the MSC therapeutic effect by facilitating macrophage repolarisation. To further delineate this mechanism, MSC isolated from CCL2 deficient mice (MSC-KO) were applied to excisional wounds in wild-type (WT) mice. CCL2 deficiency in MSC completely abrogated the therapeutic response compared to MSC-WT. MSC-KO were unable to repolarise macrophages to the same extent as WT and this was accompanied by a reduced angiogenesis and re-epithelialisation of the wounds at day 10. This study demonstrates that MSC derived CCL2 is required for MSC induced accelerated wound healing. The role of CCL2 in the interaction between MSC and Macrophages has not been previously demonstrated in accelerated wound healing. CCL2 has a potent effect on the ability to reduce the inflammatory response through local recruitment of macrophages. This research highlights CCL2 as a possible target for augmentation of MSC therapy to enhance therapeutic potential.

The Use of Thromboelastography to Measure the Influence Inclusion of a Local Anesthetic Agent has on the Mechanical and Kinetic Properties of Fibrin.

CONTEXT: Delivery of slow-release local anesthesia has considerable potential for postoperative analgesia. Fibrin gel has shown huge potential for drug delivery, but has not been fully investigated for the delivery of local anesthetics nor has whether incorporation of anesthetic drugs into fibrin alters its mechanical properties. AIMS: This study aimed to evaluate the effects of bupivacaine inclusion on the mechanical and kinetic properties of fibrin as measured by thromboelastography (TEG). MATERIALS AND METHODS: Serial dilutions of fibrinogen with thrombin were tested with TEG to identify the optimal concentrations to give reproducible results. Following this, fibrinogen samples diluted with bupivacaine 0.5% in place of normal saline (also 1:20 dilution) were added to thrombin to assess what influence this had on clot strength and kinetics as measured by TEG values (with R, K, and α angle relating to clot kinetics and MA and G (or shear elastic modulus strength) relating to clot strength). RESULTS: The mean values yielded for R were higher and lower for α angle, suggesting that the inclusion of bupivacaine produced a fibrin clot at a slower rate. The values for MA and G were both lower when bupivacaine was included, suggesting inclusion of the local anaesthetic also resulted in a fibrin clot of inferior strength. These results were not statistically significant. CONCLUSION: Although TEG failed to consistently measure these properties, the results suggest that inclusion of local anesthetic affects the clotting process of fibrin, potentially interfering with its ability to function as a sealant, adhesive, or hemostat.

Novel methods of local anesthetic delivery in the perioperative and postoperative setting-potential for fibrin hydrogel delivery.

The benefits of high-quality postoperative analgesia are well documented and include earlier mobilization, fewer respiratory and cardiovascular complications, and shorter hospital stay. Local anesthesia-based acute pain regimens are at worst equal to and at best superior to opiate-based regimens from the perspective of analgesia. A multimodal approach limiting opioids by combining with local anesthetics has additional beneficial effect on outcomes such as nausea and vomiting, pruritus, gastrointestinal function, respiratory complications, and neutrophil function. Wound catheters providing continuous infiltration of local anesthetics offer a rational approach to effective perioperative analgesia, but their use is limited by a short duration of action. There is an identified need for further methods to optimize longer-acting delivery of these agents. This article reviews current and evolving longer-acting techniques and their limitations with particular focus on the potential advantages of a fibrin hydrogel-based system.

Allogeneic mesenchymal stem cells, but not culture modified monocytes, improve burn wound healing.

The use of cell therapy to improve burn wound healing is limited as a validated cell source is not rapidly available after injury. Progenitor cells have shown potential to drive the intrinsic wound regeneration. Two sources of cells, allogeneic mesenchymal stem cells (MSC) and autologous culture modified monocytes (CMM), were assessed for their ability to influence burn wound healing. Both could be widely available shortly after injury. Cells were delivered in a fibrin matrix following contact burns in a porcine burns model. Application of MSC significantly decreased the area of unhealed burn compared to CMM or delivery matrix alone (6% MSC, 27% CMM, 24% Matrix, p<0.001). MSC treated wounds showed histological evidence of improved wound healing with increased collagen content (MSC 49%, CMM 42%, p<0.01), increased epidermal area (MSC 8.8%, CMM 6.1%, p<0.01) and dermal thickness (MSC 1108 µm, CMM 1007 µm, p<0.01) compared to CMM treated wounds. Labelled MSC and CMM were identified in the wounds after 2 weeks by immunohistochemistry and FACS. A single application of allogeneic MSC improves the rate of burn wound healing and improves the histological appearance of the burn wound. These cells show potential as a cell therapy that is rapidly available following burn.