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Acute myocardial infarction (MI) provokes an inflammatory response in the heart that removes damaged tissues to facilitate tissue repair/regeneration. However, overactive and prolonged inflammation compromises healing, which may be counteracted by antiinflammatory mechanisms. A key regulatory factor in an inflammatory response is the antiinflammatory cytokine IL-10, which can be produced by a number of immune cells, including subsets of B lymphocytes. Here, we investigated IL-10-producing B cells in pericardial adipose tissues (PATs) and their role in the healing process following acute MI in mice. We found that IL-10-producing B cells were enriched in PATs compared to other adipose depots throughout the body, with the majority of them bearing a surface phenotype consistent with CD5 B-1a cells (CD5 B cells). These cells were detected early in life, maintained a steady presence during adulthood, and resided in fat-associated lymphoid clusters. The cytokine IL-33 and the chemokine CXCL13 were preferentially expressed in PATs and contributed to the enrichment of IL-10-producing CD5 B cells. Following acute MI, the pool of CD5 B cells was expanded in PATs. These cells accumulated in the infarcted heart during the resolution of MI-induced inflammation. B cell-specific deletion of IL-10 worsened cardiac function, exacerbated myocardial injury, and delayed resolution of inflammation following acute MI. These results revealed enrichment of IL-10-producing B cells in PATs and a significant contribution of these cells to the antiinflammatory processes that terminate MI-induced inflammation. Together, these findings have identified IL-10-producing B cells as therapeutic targets to improve the outcome of MI.
BACKGROUND & AIMS - Pancreatitis is a major cause of morbidity and mortality and is a risk factor for pancreatic tumorigenesis. Upon tissue damage, an inflammatory response, made up largely of macrophages, provides multiple growth factors that promote repair. Here, we examine the molecular pathways initiated by macrophages to promote pancreas recovery from pancreatitis.
METHODS - To induce organ damage, mice were subjected to cerulein-induced experimental pancreatitis and analyzed at various times of recovery. CD11b-DTR mice were used to deplete myeloid cells. Hbegf;LysM-Cre mice were used to ablate myeloid cell-derived heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). To ablate EGFR specifically during recovery, pancreatitis was induced in Egfr;Ptf1a;FSF-Rosa26 mice followed by tamoxifen treatment.
RESULTS - Macrophages infiltrating the pancreas in experimental pancreatitis make high levels of HB-EGF. Both depletion of myeloid cells and ablation of myeloid cell HB-EGF delayed recovery from experimental pancreatitis, resulting from a decrease in cell proliferation and an increase in apoptosis. Mechanistically, ablation of myeloid cell HB-EGF impaired epithelial cell DNA repair, ultimately leading to cell death. Soluble HB-EGF induced EGFR nuclear translocation and methylation of histone H4, facilitating resolution of DNA damage in pancreatic acinar cells in vitro. Consistent with its role as the primary receptor of HB-EGF, in vivo ablation of EGFR from pancreatic epithelium during recovery from pancreatitis resulted in accumulation of DNA damage.
CONCLUSIONS - By using novel conditional knockout mouse models, we determined that HB-EGF derived exclusively from myeloid cells induces epithelial cell proliferation and EGFR-dependent DNA repair, facilitating pancreas healing after injury.
Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.
Sepsis-associated acute kidney injury (S-AKI) significantly worsens patient prognosis, and recent evidence suggests that the injury process begins early and may be sustained by therapies used to treat the sepsis (e.g., fluids resuscitation, antibiotics). While efforts to develop less-injurious treatments are making progress, some degree of secondary injury is to be expected. So too is the inevitable nature of organ injury, which is often present at the time the patient seeks medical attention. We recently found that most patients presenting with septic shock and developing AKI had evidence of kidney damage at the time of, or within 24 h of their admission. In such patients, prevention is not a viable option, as injury has already occurred by the time of presentation. Since S-AKI patients are at increased risk of developing chronic kidney disease, a fundamental target for interventions in S-AKI is to prevent fibrosis (maladaptive repair) while stimulating regeneration (proliferation of viable epithelial cells). Using a pathway-agnostic, proliferation-based phenotypic assay, we discovered phenylthiobutanoic acid, a small molecule histone deacetylase inhibitor, that enhances renal recovery and reduces fibrosis in both zebrafish and mouse models of AKI.
© 2019 S. Karger AG, Basel.
All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state in vitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied in vivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for in vivo use and allows for simultaneous delivery of multiple genes. Using an inducible piggyBac transposon system, we optimized a protocol for the direct reprogramming of HK2 cells to induced nephron progenitor-like cells with expression of only 3 transcription factors (SNAI2, EYA1, and SIX1). Culture in conditions supportive of the nephron progenitor state further increased the expression of nephron progenitor genes. The refined protocol was then applied to primary human renal epithelial cells, which integrated into developing nephron structures in vitro and in vivo. Such inducible reprogramming to nephron progenitor-like cells could facilitate direct cellular reprogramming for kidney regeneration.
Copyright © 2019 International Society of Nephrology. All rights reserved.
BACKGROUND - Diffusion tensor tractography (DTT) has recently been shown to accurately detect nerve injury and regeneration. This study assesses whether 7-tesla (7T) DTT imaging is a viable modality to observe axonal outgrowth in a 4 cm rabbit sciatic nerve injury model fixed by a reverse autograft (RA) surgical technique.
METHODS - Transection injury of unilateral sciatic nerve (4 cm long) was performed in 25 rabbits and repaired using a RA surgical technique. Analysis of the nerve autograft was performed at 3, 6, and 11 weeks postoperatively and compared to normal contralateral sciatic nerve, used as control group. High-resolution DTT from ex vivo sciatic nerves were obtained using 3D diffusion-weighted spin-echo acquisitions at 7-T. Total axons and motor and sensory axons were counted at defined lengths along the graft.
RESULTS - At 11 weeks, histologically, the total axon count of the RA group was equivalent to the contralateral uninjured nerve control group. Similarly, by qualitative DTT visualization, the 11-week RA group showed increased fiber tracts compared to the 3 and 6 weeks counterparts. Upon immunohistochemical evaluation, 11-week motor axon counts did not significantly differ between RA and control; but significantly decreased sensory axon counts remained. Nerves explanted at 3 weeks and 6 weeks showed decreased motor and sensory axon counts.
DISCUSSION - 7-T DTT is an effective imaging modality that may be used qualitatively to visualize axonal outgrowth and regeneration. This has implications for the development of technology that non-invasively monitors peripheral nerve regeneration in a variety of clinical settings.
At least 26% of recent battlefield injuries are to the craniomaxillofacial (CMF) region. Recombinant human bone morphogenetic protein 2 (rhBMP-2) is used to treat CMF open fractures, but several complications have been associated with its use. This study tested the efficacy and safety of a lower (30% recommended) dose of rhBMP-2 to treat mandibular fractures. rhBMP-2 delivered via a polyurethane (PUR) and hydroxyapatite/β-tricalcium phosphate (Mastergraft®) scaffold was evaluated in a 2 cm segmental mandibular defect in minipigs. Bone regeneration was analyzed at 4, 8, and 12 weeks postsurgery using clinical computed tomography (CT) and rhBMP-2, and inflammatory marker concentrations were analyzed in serum and surgery-site drain effluent. CT scans revealed that pigs treated with PUR-Mastergraft® + rhBMP-2 had complete bone bridging, while the negative control group showed incomplete bone-bridging (n = 6). Volumetric analysis of regenerated bone showed that the PUR-Mastergraft® + rhBMP-2 treatment generated significantly more bone than control by 4 weeks, a trend that continued through 12 weeks. Variations in inflammatory analytes were detected in drain effluent samples and saliva but not in serum, suggesting a localized healing response. Importantly, the rhBMP-2 group did not exhibit an excessive increase in inflammatory analytes compared to control. Treatment with low-dose rhBMP-2 increases bone regeneration capacity in pigs with mandibular continuity defects and restores bone quality. Negative complications from rhBMP-2, such as excessive inflammatory analyte levels, were not observed. Together, these results suggest that treatment with low-dose rhBMP-2 is efficacious and may improve safety when treating CMF open fractures. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1491-1503, 2019.
© 2018 Wiley Periodicals, Inc.
Reconstructive treatment after trauma and tumor resection would greatly benefit from an effective soft tissue regeneration. The use of cell-free scaffolds for adipose tissue regeneration in vivo is emerging as an attractive alternative to tissue-engineered constructs, since this approach avoids complications due to cell manipulation and lack of synchronous vascularization. In this study, we developed a biodegradable polyurethane-based scaffold for soft tissue regeneration, characterized by an exceptional combination between softness and resilience. Exploring the potential as a cell-free scaffold required profound understanding of the impact of its intrinsic physico-chemical properties on the biological performance in vivo. We investigated the effect of the scaffold's hydrophilic character, degradation kinetics, and internal morphology on (i) the local inflammatory response and activation of MGCs (foreign body response); (ii) its ability to promote rapid vascularisation, cell infiltration and migration through the scaffold over time; and (iii) the grade of maturation of the newly formed tissue into vascularized soft tissue in a murine model. The study revealed that soft tissue regeneration in vivo proceeded by gradual infiltration of undifferentiated mesenchymal cells though the periphery toward the center of the scaffold, where the rapid formation of a functional and well-formed vascular network supported cell viability overtime.
STATEMENT OF SIGNIFICANCE - Exploring the potential of polyurethane-based soft foam as cell-free scaffold for soft tissue regeneration. In this work, we address the unmet need for synthetic functional soft tissue substitutes that provide adequate biological and mechanical support to soft tissue. We developed a series of flexible cross-linked polyurethane copolymer scaffolds with remarkable fatigue-resistance and tunable physico-chemical properties for soft tissue regeneration in vivo. Accordingly, we could extend the potential of this class of biomaterials, which was so far confined for bone and osteochondral tissue regeneration, to other types of connective tissue.
Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
PURPOSE - Given no definite consensus on the accepted autograft orientation during peripheral nerve injury repair, we compare outcomes between reverse and normally oriented autografts using an advanced magnetic resonance imaging technique, diffusion tensor imaging.
METHODS - Thirty-six female Sprague-Dawley rats were divided into 3 groups: sham-left sciatic nerve isolation without injury, reverse autograft-10-mm cut left sciatic nerve segment reoriented 180° and used to coapt the proximal and distal stumps, or normally oriented autograft-10-mm cut nerve segment kept in its normal orientation for coaptation. Animals underwent sciatic functional index and foot fault behavior studies at 72 hours, and then weekly. At 6 weeks, axons proximal, within, and distal to the autograft were evaluated using diffusion tensor imaging and choline acetyltransferase motor staining for immunohistochemistry. Toluidine blue staining of 1-μm sections was used to assess axon count, density, and diameter. Bilateral gastrocnemius/soleus muscle weights were compared to obtain a net wet weight. Comparison of the groups was performed using Mann-Whiney U or Kruskal-Wallis H tests to determine significance.
RESULTS - Diffusion tensor imaging findings including fractional anisotropy, radial diffusivity, and axial diffusivity were similar between reverse and normally oriented autografts. Diffusion tensor imaging tractography demonstrated proximodistal nerve regeneration in both autograft groups. Motor axon counts proximal, within, and distal to the autografts were similar. Likewise, axon count, density, and diameter were similar between the autograft groups. Muscle net weight at 6 weeks and behavioral outcomes (sciatic functional index and foot fault) at any tested time point were also similar between reverse and normally oriented autografts.
CONCLUSIONS - Diffusion tensor imaging may be a useful assessment tool for peripheral nerve regeneration. Reversing nerve autograft polarity did not demonstrate to have an influence on functional or regenerative outcomes.
Failure to properly repair damaged due to myocardial infarction is a major cause of heart failure. In contrast with adult mammals, zebrafish hearts show remarkable regenerative capabilities after substantial damage. To characterize protein dynamics during heart regeneration, we employed an HPLC-ESI-MS/MS (mass spectrometry) approach. Myocardium tissues were taken from sham-operated fish and ventricle-resected sample at three different time points (2, 7, and 14 days); dynamics of protein expression were analyzed by an ion-current-based quantitative platform. More than 2000 protein groups were quantified in all 16 experiments. Two hundred and nine heart-regeneration-related protein groups were quantified and clustered into six time-course patterns. Functional analysis indicated that multiple molecular function and metabolic pathways were involved in heart regeneration. Interestingly, Ingenuity Pathway Analysis revealed that P53 signaling was inhibited during the heart regeneration, which was further verified by real-time quantitative polymerase chain reaction (Q-PCR). In summary, we applied systematic proteomics analysis on regenerating zebrafish heart, uncovered the dynamics of regenerative genes expression and regulatory pathways, and provided invaluable insight into design regenerative-based strategies in human hearts.
Purpose - Mammalian central nervous system axons fail to regenerate after injury. Contributing factors include limited intrinsic growth capacity and an inhibitory glial environment. Inflammation-induced optic nerve regeneration (IIR) is thought to boost retinal ganglion cell (RGC) intrinsic growth capacity through progrowth gene expression, but effects on the inhibitory glial environment of the optic nerve are unexplored. To investigate progrowth molecular changes associated with reactive gliosis during IIR, we developed an imaging mass spectrometry (IMS)-based approach that identifies discriminant molecular signals in and around optic nerve crush (ONC) sites.
Methods - ONC was performed in rats, and IIR was established by intravitreal injection of a yeast cell wall preparation. Optic nerves were collected at various postcrush intervals, and longitudinal sections were analyzed with matrix-assisted laser desorption/ionization (MALDI) IMS and data mining. Immunohistochemistry and confocal microscopy were used to compare discriminant molecular features with cellular features of reactive gliosis.
Results - IIR increased the area of the crush site that was occupied by a dense cellular infiltrate and mass spectral features consistent with lysosome-specific lipids. IIR also increased immunohistochemical labeling for microglia and macrophages. IIR enhanced clearance of lipid sulfatide myelin-associated inhibitors of axon growth and accumulation of simple GM3 gangliosides in a spatial distribution consistent with degradation of plasma membrane from degenerated axons.
Conclusions - IIR promotes a robust phagocytic response that improves clearance of myelin and axon debris. This growth-permissive molecular remodeling of the crush injury site extends our current understanding of IIR to include mechanisms extrinsic to the RGC.