Production Of Extracellular Proteins Research Articles

Enhanced extracellular Bacillus stearothermophilus α-amylase production in Bacillus subtilis by balancing the entire secretion process in an optimal strain

A novel strategy that combines host strain optimization with balancing of the entire secretion process has been used to enhance the extracellular production of Bacillus stearothermophilus α-amylase (AmySA) in Bacillus subtilis. B. subtilis strain WS9, which lacks six extracellular proteases, was selected as the most suitable host for recombinant AmySA expression. This strain was further modified by inactivating the hrcA gene, which increases the expression of intracellular molecular chaperones. Then, with co-expressing the transport channel component SecYEG complex, the entire secretion process of α-amylase was balanced through the signal peptide SPRpmG. The resulting recombinant strain (WHS9GSAB) efficiently produced AmySA, showing an extracellular AmySA activity of 2835.1 U/mL during shake-flask cultivation. This was 2.9-fold greater than that of the original strain WS11YSA (976.9 U/mL). Cultivation of WHS9GSAB in a 3-L fermenter produced an extracellular AmySA activity of 35779.5 U/mL at 93 h, with a high productivity of 384.7 U/mL·h. This represents the highest level of recombinant α-amylase production in B. subtilis reported to date. The expression strategy developed in this study may be useful for both the industrial production of α-amylases and improving the extracellular production of other recombinant proteins in B. subtilis.

Umbilical Cord Pericytes Provide a Viable Alternative to Mesenchymal Stem Cells for Neonatal Vascular Engineering.

Reconstructive surgery of congenital heart disease (CHD) remains inadequate due to the inability of prosthetic grafts to match the somatic growth of pediatric patients. Functionalization of grafts with mesenchymal stem cells (MSCs) may provide a solution. However, MSCs represent a heterogeneous population characterized by wide diversity across different tissue sources. Here we investigated the suitability of umbilical cord pericytes (UCPs) in neonatal vascular engineering. Explant outgrowth followed by immunomagnetic sorting was used to isolate neural/glial antigen 2 (NG2)+/CD31− UCPs. Expanded NG2 UCPs showed consistent antigenic phenotype, including expression of mesenchymal and stemness markers, and high proliferation rate. They could be induced to a vascular smooth muscle cell-like phenotype after exposure to differentiation medium, as evidenced by the expression of transgelin and smooth muscle myosin heavy chain. Analysis of cell monolayers and conditioned medium revealed production of extracellular matrix proteins and the secretion of major angiocrine factors, which conferred UCPs with ability to promote endothelial cell migration and tube formation. Decellularized swine-derived grafts were functionalized using UCPs and cultured under static and dynamic flow conditions. UCPs were observed to integrate into the outer layer of the graft and modify the extracellular environment, resulting in improved elasticity and rupture strain in comparison with acellular grafts. These findings demonstrate that a homogeneous pericyte-like population can be efficiently isolated and expanded from human cords and integrated in acellular grafts currently used for repair of CHD. Functional assays suggest that NG2 UCPs may represent a viable option for neonatal tissue engineering applications.

Role of core protein mutations in the development of occult HBV infection

Occult HBV infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis have concentrated on mutations in the S region and the regulatory elements. Herein, we aimed to determine the role of mutations in the core region on OBIs. An OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production invitro and invivo. A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (WT) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of WT-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg, and HBsAg and viral RNA was quantified from individual SZA and WT Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An analysis of the effect of Cp mutants on intracellular or extracellular viral protein production indicated that the W62R mutation in Cp had a critical impact on the reduction of HBcAg and HBeAg production during HBV replication, whereas P50H and/or S74G mutations played a limited role in influencing viral protein production invivo. W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI. Occult hepatitis B virus infections (OBIs) have been found to be associated with amino acid mutations in the S region of the HBV, but the role of mutations in the core protein (Cp) remains unclear. In this study, an OBI strain (SZA) carrying 9 amino acid substitutions in Cp has been examined comprehensively invitro and invivo. The W62R mutation in Cp majorly reduces HBcAg and HBeAg production during HBV replication, potentially contributing to the occurrence of OBI.

Role of c-di-GMP in improving stress resistance of alginate-chitosan microencapsulated Bacillus subtilis cells in simulated digestive fluids

Probiotics (Bacillus subtilis 04178) were entrapped in alginate-chitosan microcapsules by high-voltage electrostatic process. The encapsulation pattern was established as entrapped low density cells with culture (ELDCwc). The performance of ELDCwc cells was investigated against stress environments of simulated digestive fluids. After incubation in simulated gastric (pH 2.5) and intestinal fluids (4% bile salt) for 2h, the survival rate of ELDCwc cells (18.19% and 27.54%) was significantly higher than that of the free cells (0.0000009% and 0.0005%). The reason why B. subtilis embedded in microcapsules can resist the stress environments was that the mass production of extracellular proteins and polysaccharides prompted B. subtilis to form cell aggregates. The production of extracellular proteins and polysaccharides were regulated by the concentration of c-di-GMP and the expression of ydaJKLMN operon, abbA, sinI, slrA, slrB, abrR and sinR. c-di-GMP is important for the production of extracellular polymer substance to enhance probiotic viability in stress environments.

Development of improved method to identify and analyze lung fibrocytes with flow cytometry in a reporter mouse strain.

IntroductionFibrocytes are emerging myeloid‐derived circulating cells that can migrate into damaged tissues and usually contribute to their repair. Key features of fibrocytes include the expression myeloid markers, production of extracellular matrix proteins, and secretion of various humoral factors that activate resident fibroblasts; they also have the potential to differentiate into fibroblasts. However, no specific surface markers have been identified to identify fibrocytes in vivo. One reason could be that the method used to detect fibrocytes requires intracellular collagen staining.MethodsIn the present study, to establish an improved method for the detection of lung fibrocytes and to analyze viable fibrocytes, we used collagen I(α)2‐green fluorescent protein (Col‐GFP) reporter mice, which had undergone the intratracheal instillation of bleomycin (BLM).ResultsUsing flow cytometry to gate out cells with autofluorescence, we clearly found that CD45+ GFP+ cells resided in the lungs of Col‐GFP mice at a steady state and these cells increased after BLM injury, peaking at Day 14. These cells expressed not only known cell surface markers of fibrocytes, but also some novel markers, in addition to a low level of collagen I in comparison to CD45− GFP+ cells.ConclusionOur findings suggest that the improved method can be a useful for the detection of pure lung fibrocytes and allows us to further analyze the characteristics of viable fibrocytes.

Reducing the cell lysis to enhance yield of acid-stable alpha amylase by deletion of multiple peptidoglycan hydrolase-related genes in Bacillus amyloliquefaciens

Bacillus amyloliquefaciens is a major industrial host for extracellular protein production, with great potential in the enzyme industry. However, the strain has accelerated the autolysis drawback in the process of secreting extracellular enzymes, which can significantly lower the density of cells and decrease the product yield. To identify target genes, we employed comparative transcriptome sequencing and KEGG analysis to indicate the increased expression of peptidoglycan hydrolase-regulated genes from the exponential phase to the apoptotic phase of growth; this was further confirmed by quantitative RT-PCR. By deleting lytD, lytE, and sigD genes, cell lysis was reduced and the production of acid-stable Bacillus licheniformis alpha-amylase was enhanced. After 36 h of culture, multiple deletion mutant BA ΔSDE had significantly more viable cells compared to the control strain BA Δupp, and flow cytometry analysis indicated that 48.43% and 64.03% of the cells were lysed in cultures of BA ΔSDE and BA Δupp, respectively. In a 2-L fed-batch fermenter, viable cell number of the triple deletion mutant BA ΔSDE increased by 2.79 Log/cfu/mL, and the activity of acid-stable alpha-amylase increased by 48.4%, compared to BA Δupp. Systematic multiple peptidoglycan hydrolases deletion relieved the autolysis and increased the production of industrial enzymes, and provided a useful strategy for guiding efforts to manipulate the genomes of other B. amyloliquefaciens used for chassis host.

Extracellular Matrix Proteins and Transcription of Matrix-Associated Genes in Mesenchymal Stromal Cells during Modeling of the Effects of Microgravity.

We studied the effects of simulated microgravity (10 days) on the production of extracellular matrix proteins and expression of extracellular matrix-associated genes in human mesenchymal stem cells. A decrease in collagen production, reduced expression of TIMP-1, TIMP-3, and MMP-11 genes, and enhanced expression of tenascin and laminin subunit were revealed. The results attest to activation of proteolytic processes in the matrix of mesenchymal stromal cells and weakening of cell adhesion to extracellular matrix under conditions of simulated microgravity.

Validation of an implantable bioink using mechanical extraction of human skin cells: First steps to a 3D bioprinting treatment of deep second degree burn.

Clinical grade cultured epithelial autograft (CEA) are routinely used to treat burns covering more than 60% of the total body surface area. However, although the epidermis may be efficiently repaired by CEA, the dermal layer, which is not spared in deep burns, requires additional treatment strategies. Our aim is to develop an innovative method of skin regeneration based on in situ 3D bioprinting of freshly isolated autologous skin cells. We describe herein bioink formulation and cell preparation steps together with experimental data validating a straightforward enzyme-free protocol of skin cell extraction. This procedure complies with both the specific needs of 3D bioprinting process and the stringent rules of good manufacturing practices. This mechanical extraction protocol, starting from human skin biopsies, allows harvesting a sufficient amount of both viable and growing keratinocytes and fibroblasts. We demonstrated that a dermis may be reconstituted in vitro starting from a medical grade bioink and mechanically extracted skin cells. In these experiments, proliferation of the extracted cells can be observed over the first 21 days period after 3D bioprinting and the analysis of type I collagen exhibited a de novo production of extracellular matrix proteins. Finally, in vivo experiments in a murine model of severe burn provided evidences that a topical application of our medical grade bioink was feasible and well-tolerated. Overall, these results represent a valuable groundwork for the design of future 3D bioprinting tissue engineering strategies aimed at treating, in a single intraoperative step, patients suffering from extended severe burns.

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness.

With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS ) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6mg g-1 , respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

5-Hydroxymethylfurfural inhibits Acinetobacter baumannii biofilms: an in vitro study.

The present study was aimed to investigate the antibiofilm activity of 5-hydroxymethylfurfural against Acinetobacter baumanni and Vellar estuary isolates v3 (Acinetobacter nosocomialis). The biofilm inhibitory concentration (BIC) of 5HMF against A. baumannii and v3 (A. nosocomialis) was found to be 100µg/ml) exhibited non-bactericidal concentration-dependent antibiofilm activities against Acinetobacter species. The present study found that 5HMF treatment is very effective in the initial stage of A. baumannii biofilms and it significantly disrupted the mature biofilms. Moreover, 5HMF treatment inhibited the extracellular polymeric substances (EPS), including polysaccharides and proteins production. Results from gene expression and in vitro assays further demonstrated the 5HMF treatment downregulated the expression of bfmR, bap, csuA/B, ompA and katE virulence genes, which consistently affects biofilm formation and its mediated virulence property. The present study suggests that 5HMF unveil its antibiofilm activity by interfering initial biofilm formation and suppressing the virulence regulator genes in A. baumannii. Further studies are required to explore the 5HMF mode of action responsible for the antibiofilm activity.

The Role of Cell Wall Degrading Enzymes in Antagonistic Traits of Trichoderma virens Against Rhizoctonia solani.

Background: High antagonistic ability of different Trichoderma species against a diverse range of plant pathogenic fungi has led them to be used as a biological fungicide in agriculture. They can also promote plant growth, fertility, resistance to stress, and absorption of nutrients. They are also opportunistic and symbiotic pathogens, which can lead to the activation of plant defense mechanisms.Objectives: The aim of this present study was to investigate possible enhancement of lytic enzymes production and biocontrol activity of T. virens against Rhizoctonia solani through gamma radiation and to find the relationship between changes in lytic enzyme production and antagonistic activity of T. virens.Material and Methods: Dual culture conditions were used to evaluate the antagonistic effect of T. virens and its gamma mutants against R. solani. Then, their chitinase and cellulase activities were measured. For more detailed investigation of enzymes, densitometry pattern of the proteins was extracted from the T. virens wild-type and its mutants were obtained via SDS-polyacrylamide gel electrophoresis.Results: The mutant T.vi M8, T. virens wild-type and mutant T.vi M20 strains showed the maximum antagonistic effects against the pathogen, respectively. Data showed that the mutant T. vi M8 reduced the growth of R. solani by 58 %. The mutants revealed significantly different (p<0.05) protein contents, chitinase and cellulase production (mg.mL-1) and activity (U.mL-1) compared to the wild-type strain. The highest extracellular protein production in the supernatant of chitinase and cellulase TFM was observed for the T.vi M11 and T.vi M17 strains, respectively. The T.vi M12 and wild-type strains secreted chitinase and cellulase significantly more than other strains did. Densitometry of SDS-PAGE gel bands indicated that both the amount and diversity of chitinase related proteins in the selected mutant (T. vi M8) were far higher than those of the wild-type. The diversity of molecular weight of proteins extracted from the T. virens M8 (20 proteins or bands) was very high compared to the wild-type (10 proteins) and mutant T.vi M15 (2 proteins).Conclusions: Overall, there was a strong link between the diversity of various chitinase proteins and the antagonistic properties of the mutant M8.

The effect of microplastics pollution in microalgal biomass production: A biochemical study

Microplastics (MPs) are widely spread throughout aquatic systems and water bodies. Given that water quality is one of the most important parameters in the microalgal-based industry, it is critical to assess the biochemical impact of short- and long-term exposure to MPs pollution. Here, the microalga Phaeodactylum tricornutum was exposed to water contaminated with 0.5 and 50 mg L−1 of polystyrene (PS) and/or polymethyl methacrylate (PMMA). Results show that the microalgal cultures exposed to lower concentrations of PS displayed a growth enhancement of up to 73% in the first stage (days 3-9) of the exponential growth phase. Surprisingly, and despite the fact that long-term exposure to MPs contamination did not impair microalgal growth, a steep decrease in biomass production (of up to 82%) was observed. The production of photosynthetic pigments was shown to be pH-correlated during the full growth cycle, but cell density-independent in later stages of culturing. The extracellular carbohydrates production exhibited a major decrease during long-term exposure. Still, the production of extracellular proteins was not affected by the presence of MPs. This pilot laboratory-scale study shows that the microalgal exposure to water contaminated with MPs disturbs its biochemical equilibrium in a time-dependent manner, decreasing biomass production. Thus, microalgal industry-related consequences derived from the use of MPs-contaminated water are a plausible possibility.

Liraglutide suppresses production of extracellular matrix proteins and ameliorates renal injury of diabetic nephropathy by enhancing Wnt/β-catenin signaling.

The Wnt/β-catenin signaling pathway is involved in production of the extracellular matrix (ECM) by mesangial cells (MCs). Recent studies by us and others have demonstrated that glucagon-like peptide-1 receptor agonists (GLP-1RAs) have protective effects against diabetic nephropathy. The purpose of the present study was to investigate whether the Wnt/β-catenin signaling in MCs contributes to GLP-1RA-induced inhibition of ECM accumulation and mitigation of glomerular injury in diabetic nephropathy. In cultured human mesangial cells, liraglutide (a GLP-1RA) treatment significantly reduced high glucose (HG)-stimulated production of fibronectin, collagen type IV, and α-smooth muscle actin, and the liraglutide effects were significantly attenuated by XAV-939, a selective inhibitor of Wnt/β-catenin signaling. Furthermore, HG treatment significantly decreased protein abundance of Wnt4, Wnt5a, phospho-glycogen synthase kinase-3β, and β-catenin. These HG effects on Wnt/β-catenin signaling proteins were significantly blunted by liraglutide treatment. For in vivo experiments, we administered liraglutide (200 μg·kg-1·12 h-1) by subcutaneous injection to streptozocin-induced type 1 diabetic rats for 8 wk. Administration of liraglutide significantly improved elevated blood urine nitrogen, serum creatinine, and urinary albumin excretion rate and alleviated renal hypertrophy, mesangial expansion, and glomerular fibrosis in type 1 diabetic rats, whereas blood glucose level and body weight did not have significant changes. Consistent with the in vitro experiments, liraglutide treatment significantly reduced the diabetes-induced increases in glomerular fibronectin, collagen type IV, and α-smooth muscle actin and decreases in glomerular Wnt/β-catenin signaling proteins. These results suggest that liraglutide alleviated glomerular ECM accumulation and renal injury in diabetic nephropathy by enhancing Wnt/β-catenin signaling.

Scleraxis is required for the growth of adult tendons in response to mechanical loading.

Scleraxis is a basic helix-loop-helix transcription factor that plays a central role in promoting tenocyte proliferation and matrix synthesis during embryonic tendon development. However, the role of scleraxis in the growth and adaptation of adult tendons is not known. We hypothesized that scleraxis is required for tendon growth in response to mechanical loading and that scleraxis promotes the specification of progenitor cells into tenocytes. We conditionally deleted scleraxis in adult mice using a tamoxifen-inducible Cre-recombinase expressed from the Rosa26 locus (ScxΔ) and then induced tendon growth in Scx+ and ScxΔ adult mice via plantaris tendon mechanical overload. Compared with the WT Scx+ group, ScxΔ mice demonstrated blunted tendon growth. Transcriptional and proteomic analyses revealed significant reductions in cell proliferation, protein synthesis, and extracellular matrix genes and proteins. Our results indicate that scleraxis is required for mechanically stimulated adult tendon growth by causing the commitment of CD146+ pericytes into the tenogenic lineage and by promoting the initial expansion of newly committed tenocytes and the production of extracellular matrix proteins.

Identification of Locally Isolated Cellulolytic Stenotrophomonas maltophilia from Rice Straw and Optimization of its Cellulase Activity

A highly cellulolytic bacterium was locally isolated from rice straw and identified as Stenotrophomonas maltophilia. Identification of the isolate based on the morphological, cultural and biochemical characteristics was confirmed with 16S rDNA analysis. The bacterium showed the highest level of reducing sugar and extracellular protein production when incubated for 3 days (348.75 μg/ml and 288.5 μg/ml respectively) at 40°C temperature (463.0 μg/ ml and 333.0 μg/ml respectively) and pH 6.5 (360.0 μg/ml and 349.0 μg/ml respectively) in Winstead’s broth having 1.5% CMC and 0.2% Yeast Extract as carbon and nitrogen source respectively. Crude cellulase enzymes produced by the bacterium showed the highest CMCase activity rather than FPase, Avicelase and ²-Glucosidase activities. Cellulase activity of the crude enzyme was also determined using the same parameters. The crude cellulase enzyme showed the highest CMCase activity when incubated for 60 minutes (232.5 U/ml), at pH 6.5 (105.0 U/ml), 35°C temperature (69.75 U/ml) using CMC and Peptone as carbon and nitrogen source respectively. Crude cellulase showed the highest activity in presence of mercury and SDS as metal and detergent respectively. Substrate specificity and SDS-PAGE analysis reveals that the cellulase may be an endo-1,4-glucanase.&#x0D; Bangladesh J Microbiol, Volume 37 Number 1 June 2020, pp 15-22

Quantitative Hybrid Cardiac [18F]FDG-PET-MRI Images for Assessment of Cardiac Repair by Preconditioned Cardiosphere-Derived Cells

Cardiosphere-derived cells (CDCs) are progenitor cells derived from heart tissue and have shown promising results in preclinical models. APOSEC, the secretome of irradiated peripheral blood mononuclear cells, has decreased infarct size in acute and chronic experimental myocardial infarction (MI). We enhanced the effect of CDCs with APOSEC preconditioning (apoCDC) and investigated the reparative effect in a translational pig model of reperfused MI. Supernatants of CDCs, assessed by proteomic analysis, revealed reduced production of extracellular matrix proteins after in vitro APOSEC preconditioning. In a porcine model of catheter-based reperfused anterior acute MI (AMI), CDCs with (apoCDC, n = 8) or without APOSEC preconditioning (CDC, n = 6) were infused intracoronary, 15 min after the start of reperfusion. Untreated AMI animals (n = 7) and sham procedures (n = 5) functioned as controls. 2-deoxy-2-(18 F)-fluoro-D-glucose-positron emission tomography-magnetic resonance imaging ([18F]FDG-PET-MRI), with late enhancement after 1 month, showed reduced scar volume and lower transmurality of the infarcted area in CDC and apoCDC compared to AMI controls. Segmental quantitative PET images displayed indicated more residual viability in apoCDC. The left-ventricle (LV) ejection fraction was improved nonsignificantly to 45.8% ± 8.6% for apoCDC and 43.5% ± 7.1% for CDCs compared to 38.5% ± 4.4% for untreated AMI. Quantitative hybrid [18F]FDG-PET-MRI demonstrated improved metabolic and functional recovery after CDC administration, whereas apoCDCs induced preservation of viability of the infarcted area.

TNFα and Reactive Oxygen Signaling in Vascular Smooth Muscle Cells in Hypertension and Atherosclerosis.

Hypertension and atherosclerosis, the predecessors of stroke and myocardial infarction, are chronic vascular inflammatory reactions. Tumor necrosis factor alpha (TNFα), the "master" proinflammatory cytokine, contributes to both the initiation and maintenance of vascular inflammation. TNFα induces reactive oxygen species (ROS) production which drives the redox reactions that constitute "ROS signaling." However, these ROS may also cause oxidative stress which contributes to vascular dysfunction. Mice lacking TNFα or its receptors are protected against both acute and chronic cardiovascular injury. Humans suffering from TNFα-driven inflammatory conditions such as rheumatoid arthritis and psoriasis are at increased cardiovascular risk. When treated with highly specific biologic agents that target TNFα signaling (Etanercept, etc.) they display marked reductions in that risk. The ability of TNFα to induce endothelial dysfunction, often the first step in a progression toward serious vasculopathy, is well recognized and has been reviewed elsewhere. However, TNFα also has profound effects on vascular smooth muscle cells (VSMCs) including a fundamental change from a contractile to a secretory phenotype. This "phenotypic switching" promotes proliferation and production of extracellular matrix proteins which are associated with medial hypertrophy. Additionally, it promotes lipid storage and enhanced motility, changes that support the contribution of VSMCs to neointima and atherosclerotic plaque formation. This review focuses on the role of TNFα in driving the inflammatory changes in VSMC biology that contribute to cardiovascular disease. Special attention is given to the mechanisms by which TNFα promotes ROS production at specific subcellular locations, and the contribution of these ROS to TNFα signaling.

Phosphoinositide 3-kinase γ deficiency attenuates kidney injury and fibrosis in angiotensin II-induced hypertension.

We have shown that the CXCL16/CXCR6 axis plays a critical role in recruiting inflammatory cells and bone marrow-derived fibroblasts into the kidney leading to renal injury and fibrosis. However, the underlying signaling mechanisms are not known. In the present study, we examined the role of phosphoinositide-3 kinase γ (PI3Kγ) signaling in the recruitment of inflammatory cells and bone marrow-derived fibroblasts into the kidney and development of renal injury and fibrosis in an experimental model of hypertension induced by angiotensin II. Blood pressure was comparable between wild-type (WT) and PI3Kγ knockout (KO) mice at baseline. Angiotensin II treatment led to an increase in blood pressure that was similar between WT and PI3Kγ KO mice. Compared with WT mice, PI3Kγ KO mice were protected from angiotensin II-induced renal dysfunction and injury and developed less proteinuria. PI3Kγ deficiency suppressed bone marrow-derived fibroblast accumulation and myofibroblast formation in the kidney and inhibited total collagen deposition and extracellular matrix protein production in the kidney in response to angiotensin II. PI3Kγ deficiency inhibited the infiltration of F4/80+ macrophages and CD3+ T cells into the kidney and reduced gene expression levels of pro-inflammatory cytokines in the kidney following angiotensin II treatment. Finally, inhibition of PI3Kγ suppressed CXCL16-induced monocyte migration in vitro. These results indicate that PI3Kγ mediates the influx of macrophages, T cells and bone marrow-derived fibroblasts into the kidney resulting in kidney injury and fibrosis.

WJ-39, an Aldose Reductase Inhibitor, Ameliorates Renal Lesions in Diabetic Nephropathy by Activating Nrf2 Signaling.

Diabetic nephropathy (DN) is a chronic diabetic microvascular complication. Hyperactivity of the polyol pathway is involved in the pathogenesis of DN. Aldose reductase (AR), the rate-limiting enzyme of the polyol pathway, is expected to be an effective target in the treatment of DN. WJ-39 is a novel inhibitor of AR. The present study aimed at exploring the effects of WJ-39 in DN. DN was induced in rats by injecting 30 mg/kg streptozotocin (STZ). After 14 weeks, WJ-39 (10, 20, and 40 mg/kg) was intragastrically administered to the rats for 12 weeks. Treatment with WJ-39 significantly inhibited AR activation and ameliorated renal dysfunction and fibrosis in DN rats. WJ-39 reduced oxidative stress in the kidneys of DN rats by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. WJ-39 suppressed the activation of the nuclear factor-kappa B (NF-κB) pathway and the nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome to reduce the secretion of inflammatory factors. Rat mesangial cells (RMCs) were cultured under hyperglycemic conditions. WJ-39 abrogated the high glucose- (HG-) induced, excessive production of reactive oxygen species (ROS) and inflammatory factors. However, transfection with Nrf2 small interfering RNA abolished the effects of WJ-39. WJ-39 also blocked the transforming growth factor-β1/Smad pathway to reduce the production of glomerular extracellular matrix proteins, ultimately reducing fibrogenesis in DN. Our results show that WJ-39 ameliorated renal injury in DN rats, and its effects on oxidative stress and inflammation were associated with the activation of Nrf2 signaling. Thus, WJ-39 and its mechanism of amelioration of renal lesions in DN rats by reducing renal inflammation, oxidative stress, and fibrosis injury could be an effective strategy for the treatment of DN.

Neutrophils Modulate Fibroblast Function and Promote Healing and Scar Formation after Murine Myocardial Infarction.

Aim: Recruitment of neutrophils to the heart following acute myocardial infarction (MI) initiates inflammation and contributes to adverse post-infarct left ventricular (LV) remodeling. However, therapeutic inhibition of neutrophil recruitment into the infarct zone has not been beneficial in MI patients, suggesting a possible dual role for neutrophils in inflammation and repair following MI. Here, we investigate the effect of neutrophils on cardiac fibroblast function following MI. Methods and Results: We found that co-incubating neutrophils with isolated cardiac fibroblasts enhanced the production of provisional extracellular matrix proteins and reduced collagen synthesis when compared to control or co-incubation with mononuclear cells. Furthermore, we showed that neutrophils are required to induce the transient up-regulation of transforming growth factor (TGF)-ß1 expression in fibroblasts, a key requirement for terminating the pro-inflammatory phase and allowing the reparatory phase to form a mature scar after MI. Conclusion: Neutrophils are essential for both initiation and termination of inflammatory events that control and modulate the healing process after MI. Therefore, one should exercise caution when testing therapeutic strategies to inhibit neutrophil recruitment into the infarct zone in MI patients.