Genome-wide, integrative analysis of microRNA-mediated gene regulation in human wounds reveals cooperating microRNAs as therapeutic targets

MicroRNAs (miR), as important epigenetic control factors, reportedly regulate wound repair. However, our insufficient knowledge of clinically relevant miRs hinders their potential therapeutic use. For this, we performed miR and mRNA paired expression profiling in human acute wounds and chronic non-healing venous ulcers (VU) and presented our findings on a browsable web portal (http://130.229.28.87/shiny/miRNA_Xulab/). Integrative miR and mRNA-omics analysis unraveled miR-mediated gene regulatory networks in each repair phase of the wound repair process. Importantly, we identified 17 pathologically relevant miRs exhibiting abnormal expression in VU and displaying their targetome enriched in the VU gene signature. Study of the targetome and functions of eight clinically relevant miRs in skin cells revealed that the miRs upregulated in VU (i.e., miR-34a/c-5p, miR-424-5p, miR-450-5p, miR-7704, and miR-516-5p) promoted inflammation but inhibited proliferation. In contrast, the miRs downregulated in VU (i.e., miR-218-5p and miR-96-5p) were required for cell growth and activation. Moreover, we demonstrated miR-34a, miR-424, and miR-516 cooperativity in regulating keratinocyte growth and inflammatory response. Collectively, our study suggests that VU-dysregulated miRs cooperatively contribute to stalled wound healing characterized by failed transition from inflammatory-to-proliferative phase. Targeting cooperating miRNAs provide mew opportunity for development of clinically-relevant targeted therapy to attain higher therapeutic efficacy and specificity.

Properdin Deficiency Impairs Phagocytosis and Enhances Injury at Kidney Repair Phase Post Ischemia–Reperfusion

Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia–reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (PKO) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by PKO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H2O2-increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H2O2 but inhibited by PKO. These results were confirmed by counting phagocytosed H2O2-induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, PKO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.

The Bone Cartilage Interface and Osteoarthritis

This review describes results obtained with tissue from prior studies of equine and human osteoarthritis (OA). The main methods considered are scanning electron microscopy, novel methods in light microscopy and X-ray Micro-tomography. The same samples have been re-utilised in several ways. The tissues described are hyaline articular cartilage (HAC; or substitutes), with its deep layer, articular calcified cartilage (ACC), whose deep surface is resorbed in cutting cone events to allow the deposition of subchondral bone (SCB). Multiple tidemarks are normal. Turnover at the osteochondral (ACC-HAC-SCB) junction is downregulated by overload exercise, conversely, during rest periods. Consequent lack of support predisposes to microfracture of the ACC-SCB plate, in the resorption-related repair phase of which the plate is further undermined to form sink holes. The following characteristics contribute to the OA scenario: penetrating resorption canals and local loss of ACC; cracking of ACC and SCB; sealing of cracks with High-Density Mineral Infill (HDMI); extrusion of HDMI into HAC to form High-Density Mineral Protrusions (HDMP) in HAC which may fragment and contribute to its destruction; SCB marrow space infilling and densification with (at first) woven bone; disruption, fibrillation and loss of HAC; eburnation; repair with abnormal tissues including fibrocartilage and woven bone; attachment of Sharpey fibres to SCB trabeculae and adipocyte-moulded extensions to trabeculae (excrescences).

Digital Slickline: Case Studies Highlighting Impact of Real-Time Slickline and Efficiency Improvements Compared to Traditional Slickline & E-Line Interventions

Digital Slickline (DSL) using radio frequency (RF) communications has been deployed in the field since late 2016 and has completed more than 600 jobs, and 2000 runs globally. Several papers have been published outlining how DSL has been deployed for eline replacement services such as perforating, explosive and non-explosive plug setting, production logging, and various other services. What has been less discussed are the efficiencies with surface readout (SRO) downhole data during typical slickline (SL) interventions where jarring is the prominent feature. RF DSL was introduced to the market in late 2016, and since this time, the split between SL and eline replacement services has been relatively consistent at 60/40. The separation isn't unreasonable as most interventions start as SL to prepare the well, move to a diagnostic or well repair phase, and close-out with SL to bring the well back onto production. Case histories presented will outline how SRO in-situ data give operations confidence tasks were completed as planned on gas lift change-outs and non-typical functions like a smart hole finder for leak detection. Today, we have an adequate sample size to validate the efficiency improvements deploying RF DSL compared to the traditional SL/eline intervention model. The one rig up setup off a small footprint slickline unit has proven to save multiple hours depending on the intervention complexity, and the number of eline rig up & rig down sequences eliminated. As the technology gains acceptance, the tool portfolio has continuously expanded, and we have started to leverage opportunities on traditional slickline services to minimize deferred production. Efficiency savings are well documented, but the paper will also detail the polymer-coated cable performance, with focus on breaking strength, corrosive parameters, wellbore fluid compatibility and new critical performance indicators completed before each job. We will close out by summarizing some of the newer technologies that will continue the improved efficiency theme.

TSC1 Affects the Process of Renal Ischemia-Reperfusion Injury by Controlling Macrophage Polarization

Renal ischemia-reperfusion injury (IRI) contributes to acute kidney injury (AKI), increases morbidity and mortality, and is a significant risk factor for chronic kidney disease (CKD). Macrophage infiltration is a common feature after renal IRI, and infiltrating macrophages can be polarized into the following two distinct types: M1 macrophages, i.e., classically activated macrophages, which can not only inhibit infection but also accelerate renal injury, and M2 macrophages, i.e., alternatively activated macrophages, which have a repair phenotype that can promote wound healing and subsequent fibrosis. The role of TSC1, which is a negative regulator of mTOR signaling that regulates macrophage polarization in inflammation-linked diseases, has been well documented, but whether TSC1 contributes to macrophage polarization in the process of IRI is still unknown. Here, by using a mouse model of renal ischemia-reperfusion, we found that myeloid cell-specific TSC1 knockout mice (termed Lyz-TSC1 cKO mice) had higher serum creatinine levels, more severe histological damage, and greater proinflammatory cytokine production than wild-type (WT) mice during the early phase after renal ischemia-reperfusion. Furthermore, the Lyz-TSC1 cKO mice showed attenuated renal fibrosis during the repair phase of IRI with decreased levels of M2 markers on macrophages in the operated kidneys, which was further confirmed in a cell model of hypoxia-reoxygenation (H/R) in vitro. Mechanistically, by using RNA sequencing of sorted renal macrophages, we found that the expression of most M1-related genes was upregulated in the Lyz-TSC1 cKO group (Supplemental Table 1) during the early phase. However, C/EBPβ and CD206 expression was decreased during the repair phase compared to in the WT group. Overall, our findings demonstrate that the expression of TSC1 in macrophages contributes to the whole process of IRI but serves as an inflammation suppressor during the early phase and a fibrosis promoter during the repair phase.

Repeated social defeat exaggerates CaCl2-induced abdominal aortic aneurysm expansion by eliminating periaortic fibrosis in tissue repair phase: possible involvement of specific subtypes of macrophages

Background and objective Depression is an independent risk factor of cardiovascular disease (CVD) and significantly associated with the prevalence of abdominal aortic aneurysm (AAA). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviors in apoE−/− mice and exaggerates atherosclerosis development by enhancing leukocyte activation. Here, we investigated the impact of RSD on AAA formation. Methods and results Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (28 of 48) and control mice (31 of 36) underwent application of 0.5 M calcium chloride (CaCl2) on the infrarenal aorta to induce AAA. At 1 week after application, maximum diameter and circumference of external elastic membrane were comparable between the two groups. The number of F-4/80, MMP-9, and TNF-α-positive cells in immunofluorescent images were also comparable. Further, in vitro bone marrow derived macrophages stimulation by LPS did not show any difference in mRNA expression levels of inflammatory cytokines, suggesting no discernable difference in acute inflammatory response between the two groups. In contrast, at 2 weeks after application, at the time point when MMP-9 and TNF-α-positive cells were scarcely observed, maximum diameter and circumference of external elastic membrane were significantly increased in defeated mice (0.72 mm vs. 0.90 mm, 1.59 mm vs. 2.00 mm, respectively, Control vs. Defeat, p<0.01). Intriguingly, periaortic fibrotic area in aneurysmal portion was markedly decreased in defeated mice (12.5×103 μm2 vs. 3.7×103 μm2, Control vs. Defeat, p<0.01). Consistently, accumulation of α-SMA-positive cells in adventitia of aneurysmal portion was much less in defeated mice than control mice (876 cells/mm2 vs. 319 cells/mm2, Control vs. Defeat, p<0.05), whereas those in tunica media of non-aneurysmal portion did not show any difference between the two groups. We next focused on the segregated nucleus-containing atypical monocyte (SatM), specific subtypes of monocytes/macrophages that are involved in fibrosis in injured tissues during the healing phase. We could observe SatM fraction in AAA tissue of control mice using flow cytometry. We also found that mRNA expression level of C/EBPβ, an essential regulator for SatM differentiation, was markedly decreased by 76% in BM cells of defeated mice compared with control mice (p<0.05). Conclusions Our findings demonstrate for the first time that RSD enhances AAA expansion by eliminating periaortic fibrosis in tissue repair phase, suggesting that the impaired resolution of acute inflammation after CaCl2 application contributes, at least in part, to the augmented expansion of AAA in defeated mice. Funding Acknowledgement Type of funding source: None

ÇELİK FİBERLİ ASFALT BETONU KARIŞIMLARIN MARSHALL STABİLİTESİ VE İNDİREKT ÇEKME MUKAVEMETİNİN İNCELENMESİ

One of the conductive fiber types used in the mixtures in order to provide the desired performance by reducing the deterioration of asphalt concrete coatings during the project is steel fibers. In recent years, studies on the use of steel fiber to provide self-healing property by heating the layers during the repair phase have attracted attention. In order to evaluate the economy of steel fiber layers, it is necessary to know the mechanical properties of these layers before they come into repair. In this study, the abrasion made Marshall design to binder mixtures, according to the design result, at the rate of 0.1%, 0.15% and 0.2% of the total weight of aggregate and bitumen, a steel fiber of 10 mm length and 1 mm diameter was added to the mixtures. After the first Marshall stability, conditioned indirect tensile strength tests on the mixtures, the same tests were applied again without removing the samples from the device. As a result, the highest Marshall stability and conditioned indirect tensile strength were obtained in binder mixes with 0.10% steel fiber compared to the unadulterated, while in the additive wear mixture tests, close to the additive-free or lower results were obtained.

MO067PHASE-SPECIFIC IRF4 AND IRF8 REGULATION/EXPRESSION OF MONONUCLEAR PHAGOCYTES DURING ISCHEMIC ACUTE KIDNEY INJURY/DISEASE

Background Ischemic acute tubular necrosis is a common cause of acute kidney disease (AKD) and subsequent chronic kidney disease (CKD). The different phases of AKD involve a greater functional diversity of mononuclear phagocytes (MPCs) including resident and infiltrating macrophages and conventional dendritic cells (cDCs). In addition, hematopoietic transcription factors such as IRF4 and IRF8 play an important role in immune cell maturation and polarization that contribute to tissue inflammation and remodeling. However, their role on MPCs polarization and function during AKD are not well understood. Hence, we hypothesized that the dynamically altered MPC subsets contribute to the progression of AKD, accompanied with varied expression of IRF4 and IRF8. Methods AKD was induced by transient unilateral renal pedicle clamping in C57BL/6N mice. After 1, 3, and 7 days kidneys, renal lymph nodes and spleens were collected. Flow cytometry was performed to identify different MPC subsets. GFR was measured and mRNA expression of inflammatory, anti-inflammatory transcription factors determined via RT-PCR. For in vitro experiments, bone marrow-derived macrophages and cDCs, tubular epithelial cells (TECs), renal and splenic resident CD11b+ immune cells isolated from naïve mice were stimulated with LPS or cultured under hypoxic condition for 3 and 10 hours. Bone marrow-derived monocytes were differentiated into macrophages or cDCs, and stimulated with LPS for 3 and 10 hours. After stimulation, cells were harvested for mRNA analysis via RT-PCR. Results We identified four renal phenotypically distinct MPC subsets with diverse expression patterns of CD11b/CD11c during the different phases of post-ischemic AKD. During the early (day 1) and late injury phase (day 3), the number of infiltrating CD11bhiCD11clow R2 and CD11bhiCD11chi R3 macrophage-like subsets increased, along with a significant GFR decline compared with sham-operated mice (see Figure). During the repair phase (day 7), the number of CD11blowCD11clow R1 (resident macrophage-like MPCs) and CD11blowCD11chi R4 (infiltrating cDC-like MPCs) subsets significantly increased (see Figure). Both resident macrophage-like (R1) and cDC-like (R4) MPCs significantly upregulated the expression of IRF8, whereas cDCs-like (R4) MPCs were also positive for IRF4 during the repair phase. This pattern of MPCs was consistent in spleen and renal lymph node. In vitro stimulation of renal and splenic CD11b+ cells from naïve mice with LPS or under hypoxic condition induced a significant upregulation of IRF4 and IRF8 compared to untreated cells. This was also observed in bone marrow-derived macrophages and cDCs but not in TECs. Conclusions Our data indicate that infiltrating macrophage-like and cDC-like MPCs appear in high numbers during the early and late injury phase. Furthermore, both resident macrophage-like and cDC-like MPCs are predominately present during the late injury and recovery phase in AKD with altered IRF4 and IRF8 expression pattern. Further studies are needed to unravel a potential role of IRF4 and IRF8 during the progression of AKD and CKD by e.g. using fate mapping approaches.