Role of Transglutaminase 2 in Cell Death, Survival, and Fibrosis.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme catalyzing the crosslinking between Gln and Lys residues and involved in various pathophysiological events. Besides this crosslinking activity, TG2 functions as a deamidase, GTPase, isopeptidase, adapter/scaffold, protein disulfide isomerase, and kinase. It also plays a role in the regulation of hypusination and serotonylation. Through these activities, TG2 is involved in cell growth, differentiation, cell death, inflammation, tissue repair, and fibrosis. Depending on the cell type and stimulus, TG2 changes its subcellular localization and biological activity, leading to cell death or survival. In normal unstressed cells, intracellular TG2 exhibits a GTP-bound closed conformation, exerting prosurvival functions. However, upon cell stimulation with Ca2+ or other factors, TG2 adopts a Ca2+-bound open conformation, demonstrating a transamidase activity involved in cell death or survival. These functional discrepancies of TG2 open form might be caused by its multifunctional nature, the existence of splicing variants, the cell type and stimulus, and the genetic backgrounds and variations of the mouse models used. TG2 is also involved in the phagocytosis of dead cells by macrophages and in fibrosis during tissue repair. Here, we summarize and discuss the multifunctional and controversial roles of TG2, focusing on cell death/survival and fibrosis.

Higher intestinal and circulatory lactate associated NOX2 activation leads to an ectopic fibrotic pathology following microcystin co-exposure in murine fatty liver disease.

Clinical studies implicated an increased risk of intestinal fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Our previous studies have shown that microcystin-LR (MC-LR) exposure led to altered gut microbiome and increased abundance of lactate producing bacteria and intestinal inflammation in underlying NAFLD. This led us to further investigate the effects of the MC-LR, a PP2A inhibitor in activating the TGF-ß fibrotic pathway in the intestines that might be mediated by increased lactate induced redox enzyme NOX2. Exposure to MC-LR led to higher lactate levels in circulation and in the intestinal content. The higher lactate levels were associated with NOX2 activation in vivo that led to increased Smad2/3-Smad4 co-localization and high alpha-smooth muscle actin (α-SMA) immunoreactivity in the intestines. Mechanistically, primary mouse intestinal epithelial cells treated with lactate and MC-LR separately led to higher NOX2 activation, phosphorylation of TGFßR1 receptor and subsequent Smad 2/3-Smad4 co-localization inhibitable by apocynin (NOX2 inhibitor), FBA (a peroxynitrite scavenger) and DMPO (a nitrone spin trap), catalase and superoxide dismutase. Inhibition of NOX2-induced redox signaling also showed a significant decrease in collagen protein thus suggesting a strong redox signaling induced activation of an ectopic fibrotic manifestation in the intestines. In conclusion, the present study provides mechanistic insight into the role of microcystin in dysbiosis-linked lactate production and subsequently advances our knowledge in lactate-induced NOX2 exacerbation of the cell differentiation and fibrosis in the NAFLD intestines.

Inhibition of glutaminase to reverse fibrosis in iatrogenic laryngotracheal stenosis.

OBJECTIVES/HYPOTHESIS: Glutamine metabolism is a critical energy source for iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts, and glutaminase (GLS) is an essential enzyme converting glutamine to glutamate. We hypothesize that the GLS-specific inhibitor BPTES will block glutaminolysis and reduce iLTS scar fibroblast proliferation, collagen deposition, and fibroblast metabolism in vitro. STUDY DESIGN: Test-tube Lab Research. METHODS: Immunohistochemistry of a cricotracheal resection (n = 1) and a normal airway specimen (n = 1) were assessed for GLS expression. GLS expression was assessed in brush biopsies of subglottic/tracheal fibrosis and normal airway from patients with iLTS (n = 6). Fibroblasts were isolated and cultured from biopsies of subglottic/tracheal fibrosis (n = 6). Fibroblast were treated with BPTES and BPTES + dimethyl α-ketoglutarate (DMK), an analogue of the downstream product of GLS. Fibroblast proliferation, gene expression, protein production, and metabolism were assessed in all treatment conditions and compared to control. RESULTS: GLS was overexpressed in brush biopsies of iLTS scar specimens (P = .029) compared to normal controls. In vitro, BPTES inhibited iLTS scar fibroblast proliferation (P = .007), collagen I (Col I) (P < .0001), collagen III (P = .004), and α-smooth muscle actin (P = .0025) gene expression and protein production (P = .031). Metabolic analysis demonstrated that BPTES reduced glycolytic reserve (P = .007) but had no effects on mitochondrial oxidative phosphorylation. DMK rescued BPTES inhibition of Col I gene expression (P = .0018) and protein production (P = .021). CONCLUSIONS: GLS is overexpressed in iLTS scar. Blockage of GLS with BPTES significantly inhibits iLTS scar fibroblasts proliferation and function, demonstrating a critical role for GLS in iLTS. Targeting GLS to inhibit glutaminolysis may be a successful strategy to reverse scar formation in the airway. LEVEL OF EVIDENCE: NA Laryngoscope, 2020.

The lysyl oxidase like 2/3 enzymatic inhibitor, PXS-5153A, reduces crosslinks and ameliorates fibrosis.

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.

Quercetin Shows the Pharmacological Activity to Simultaneously Downregulate the Inflammatory and Fibrotic Responses to Tissue Injury in Association with its Ability to Target Multi-Kinases.

AIM: Previous studies have suggested that quercetin is effective for treating diverse chronic disorders including organ fibrosis and airway and cardiovascular disorders. To access the pharmacological background for its broad efficacy, we examined the ability of quercetin to modulate the inflammatory and fibrotic responses associated with organ injury that commonly underlie the pathogenesis of those disorders. METHODS: A cutaneous wound model on rabbit ear was used for in vivo study. Quercetin was topically applied to the wounds, and the number of macrophages and myofibroblasts and the size of the hypertrophic scar formed were estimated. An in vitro study examined the ability of quercetin to inhibit cell-signaling pathways that activate RAW264.7 macrophages and primary dermal fibroblasts and the tyrosine kinase activity of discoidin domain receptor 2. RESULTS: Quercetin reduced the population of macrophages and myofibroblasts and the scar formation in cutaneous wound healing. Quercetin suppressed the signaling pathways activating RAW264.7 macrophages and dermal fibroblasts, which is associated with its inhibition of multiple tyrosine kinases to regulate the pathways. This pharmacological activity of quercetin to simultaneously inhibit the inflammatory and fibrotic responses upon tissue damage by targeting multi-kinases could be the action mechanism to support its broad efficacy for various chronic disorders.

C1QTNF1 attenuates angiotensin II-induced cardiac hypertrophy via activation of the AMPKa pathway.

RATIONALE: Complement C1q tumor necrosis factor related proteins (C1QTNFs) have been reported to have diverse biological influence on the cardiovascular system. C1QTNF1 is a member of the CTRP superfamily. C1QTNF1 is expressed in the myocardium; however, its function in myocytes has not yet been investigated. OBJECTIVE: To systematically investigate the roles of C1QTNF1 in angiotensin II (Ang II)-induced cardiac hypertrophy. METHODS AND RESULTS: C1QTNF1 knock-out mice were used with the aim of determining the role of C1QTNF1 in cardiac hypertrophy in the adult heart. Data from experiments showed that C1QTNF1 was up-regulated during cardiac hypertrophic processes, which were triggered by increased reactive oxygen species. C1QTNF1 deficiency accelerated cardiac hypertrophy, fibrosis, inflammation responses, and oxidative stress with deteriorating cardiac dysfunction in the Ang II-induced cardiac hypertrophy mouse model. We identified C1QTNF1 as a negative regulator of cardiomyocyte hypertrophy in Ang II-stimulated neonatal rat cardiomyocytes using the recombinant human globular domain of C1QTNF1 and C1QTNF1 siRNA. Injection of the recombinant human globular domain of C1QTNF1 also suppressed the Ang II-induced cardiac hypertrophic response in vivo. The anti-hypertrophic effects of C1QTNF1 rely on AMPKa activation, which inhibits mTOR P70S6K phosphorylation. An AMPKa inhibitor abrogated the anti-hypertrophic effects of the recombinant human globular domain of C1QTNF1 both in vivo and vitro. Moreover, C1QTNF1-mediated AMPKa activation was triggered by the inhibition of PDE1-4, which subsequently activated the cAMP/PKA/LKB1 pathway. CONCLUSION: Our results demonstrated that C1QTNF1 improves cardiac function and inhibits cardiac hypertrophy and fibrosis by increasing and activating AMPKa, suggesting that C1QTNF1 could be a therapeutic target for cardiac hypertrophy and heart failure.

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP).

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

BMP7-induced-Pten inhibits Akt and prevents renal fibrosis.

Bone morphogenetic protein-7 (BMP-7) counteracts pro-fibrotic effects of TGFß1 in cultured renal cells and protects from fibrosis in acute and chronic renal injury models. Using the unilateral ureteral obstruction (UUO) model of chronic renal fibrosis, we investigated the effect of exogenous-rhBMP-7 on pro-fibrotic signaling pathways mediated by TGFß1 and hypoxia. Mice undergoing UUO were treated with vehicle or rhBMP-7 (300µg/kg i.p.) every other day for eight days and kidneys analysed for markers of fibrosis and SMAD, MAPK, and PI3K signaling. In the kidney, collecting duct and tubular epithelial cells respond to BMP-7 via activation of SMAD1/5/8. Phosphorylation of SMAD1/5/8 was reduced in UUO kidneys from vehicle-treated animals yet maintained in UUO kidneys from BMP-7-treated animals, confirming renal bioactivity of exogenous rhBMP-7. BMP-7 inhibited Collagen Iα1 and Collagen IIIα1 gene expression and Collagen I protein accumulation, while increasing expression of Collagen IVα1 in UUO kidneys. Activation of SMAD2, SMAD3, ERK, p38 and PI3K/Akt signaling occurred during fibrogenesis and BMP-7 significantly attenuated SMAD3 and Akt signaling in vivo. Analysis of renal collecting duct (mIMCD) and tubular epithelial (HK-2) cells stimulated with TGFß1 or hypoxia (1% oxygen) to activate Akt provided further evidence that BMP-7 specifically inhibited PI3K/Akt signaling. PTEN is a negative regulator of PI3K and BMP-7 increased PTEN expression in vivo and in vitro. These data demonstrate an important mechanism by which BMP-7 orchestrates renal protection through Akt inhibition and highlights Akt inhibitors as anti-fibrotic therapeutics.

Loss of PHD3 in myeloid cells dampens the inflammatory response and fibrosis after hind-limb ischemia.

Macrophages are essential for the inflammatory response after an ischemic insult and thereby influence tissue recovery. For the oxygen sensing prolyl-4-hydroxylase domain enzyme (PHD) 2 a clear impact on the macrophage-mediated arteriogenic response after hind-limb ischemia has been demonstrated previously, which involves fine tuning a M2-like macrophage population. To analyze the role of PHD3 in macrophages, we performed hind-limb ischemia (ligation and excision of the femoral artery) in myeloid-specific PHD3 knockout mice (PHD3-/-) and analyzed the inflammatory cell invasion, reperfusion recovery and fibrosis in the ischemic muscle post-surgery. In contrast to PHD2, reperfusion recovery and angiogenesis was unaltered in PHD3-/- compared to WT mice. Macrophages from PHD3-/- mice showed, however, a dampened inflammatory reaction in the affected skeletal muscle tissues compared to WT controls. This was associated with a decrease in fibrosis and an anti-inflammatory phenotype of the PHD3-/- macrophages, as well as decreased expression of Cyp2s1 and increased PGE2-secretion, which could be mimicked by PHD3-/- bone marrow-derived macrophages in serum starvation.

Adenosine Formed by CD73 on T Cells Inhibits Cardiac Inflammation and Fibrosis and Preserves Contractile Function in Transverse Aortic Constriction-Induced Heart Failure.

BACKGROUND: Structural damage during heart failure development leads to increased infiltration of leukocytes. Because purinergic signaling on immune cells may impact on the inflammatory response, we evaluated the role of ecto-5'-nucleotidase (CD73) on the development of heart failure after transverse aortic constriction (TAC) using global and T-cell-specific CD73-/- mice. METHODS AND RESULTS: Leukocytes infiltrating the failing heart were analyzed by a multistep enzymatic procedure over a period of 16 weeks using fluorescence-activated cell sorting. TAC significantly enhanced the infiltration of leukocytes, especially T cells. The fraction of CD73 expressing cells increased over time exclusively on cytotoxic T cells, T-helper cells, and regulatory T cells. Cardiac function significantly declined in T-cell-specific CD4-Cre+/-CD73flox/flox mice identical to that observed in global CD73 mutants and was associated with enhanced fibrosis (collagen, laminin, vimentin, periostin). Expression analysis by quantitative reverse transcription polymerase chain reaction of extracellular purine degrading enzymes and P1 and P2 receptors on T cells isolated from the injured heart revealed profound upregulation of the enzymatic machinery for hydrolysis of extracellular adenosine triphosphate and nicotinamide adenine dinucleotide, both pathways converging in the formation of AMP and adenosine via CD73. Among the P1 receptors, only the A2a receptor was significantly upregulated after TAC. T cells isolated from TAC-treated hearts show enhanced production of proinflammatory cytokines (interleukin-3, interleukin-6, interleukin-13, interleukin-17, macrophage inflammatory proteins-1α, and macrophage inflammatory proteins-1ß) when CD73 was lacking. CONCLUSIONS: Our data provide first evidence that CD73 on T cells plays an important anti-inflammatory role in TAC-induced heart failure, which is associated with antifibrotic activity and reduced production of proinflammatory cytokines most likely by activation of the adenosine A2a receptor.

Dipeptidyl Peptidase-4, Wound Healing, Scarring, and Fibrosis.

Scarring and fibrosis are an enormous public health concern, resulting in excessive morbidity and mortality in addition to countless lost health care dollars. Recent advances in cell and developmental biology promise a better understanding of scarring and fibrosis and may translate to new clinical therapies.

Role and Association of Inflammatory and Apoptotic Caspases in Renal Tubulointerstitial Fibrosis.

BACKGROUND/AIMS: Caspases, an evolutionary conserved family of aspartate-specific cystein proteases, play pivotal roles in apoptotic and inflammatory signaling. Thus far, 14 mammalian caspases are identified and categorized into 3 distinct sub-types: inflammatory caspases, apoptotic initiator and apoptotic executioner. Caspase-1 is an inflammatory caspase, while caspase-7 belongs to apoptotic executioner. The roles and association of these two distinct types of caspases in renal tubulointerstitial fibrosis (TIF) have not been well recognized. METHODS: Caspase-1 inhibitor Z-YVAD-FMK and caspase-7 siRNA were used in tubular epithelial cell line NRK-52E (TECs) to test their effects on transforming growth factor-beta1 (TGF-ß1) stimulation. In vivo, Unilateral ureteral obstruction (UUO) animal model was employed in wild-type (WT) and caspase-1 knock out (KO) (caspase-1-/-) mice. RESULTS: In current study, we found that caspase-7 was obviously activated in cultured TECs stimulated by TGF-ß1 and in UUO model of WT mice. While in UUO model of caspase-1 KO mice, the increased caspase-7 activation was suppressed significantly along with reduced trans-differentiation and minimized extracellular matrix (ECM) accumulation, as demonstrated by western blot, Masson trichrome staining and immunohistochemistry. In addition, pharmacological inhibition of caspase-1 dampened caspase-7 activation and TECs' transdifferentiation induced by TGF-ß1 exposure, which was consistent with in vivo study. Notably, caspase-7 gene knock down by specific siRNA abrogated TGF-ß1 driven TECs' trans-differentiation and reduced ECM accumulation. CONCLUSIONS: Our study associated inflammatory and apoptotic caspases in TIF for the first time and we further confirmed that caspase-1 activation is an upstream event of apoptotic caspase-7 induction in TIF triggered by UUO and in TECs mediated by TGF-ß1 induced transdifferentiation.

Inhibition of spleen tyrosine kinase (syk) suppresses renal fibrosis through anti-inflammatory effects and down regulation of the MAPK-p38 pathway.

Renal fibrosis results from an excessive accumulation of extracellular matrix that occurs in most types of chronic kidney disease. Among the many fibrogenic factors that regulate renal fibrotic processes, transforming growth factor-ß1 (TGF-ß1) and inflammation after injury play critical roles. Spleen tyrosine kinase (Syk) is important for signaling processes implicated in autoimmune, inflammatory, and allergic diseases. We examined the effects of Syk inhibition on renal fibrosis in vivo and on TGF-ß1-induced renal fibroblast activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male B6 mice. Mice with UUO were administered a Syk inhibitor or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro experiments, NRK-49F rat fibroblasts were pre-incubated with a Syk inhibitor before TGF-ß1 stimulation. The inhibitory effects of Syk inhibition on signaling pathways down-stream of TGF-ß1 were analyzed. In the UUO mouse model, administration of a Syk inhibitor attenuated extracellular matrix protein deposition and expression of α-smooth muscle actin, type I collagen, and fibronectin in a dose-dependent manner. In addition, macrophage infiltration in UUO kidney was reduced by Syk inhibition. Pre-incubation of NRK-49F cells with a Syk inhibitor suppressed TGF-ß1-induced myofibroblast activation. Furthermore, inhibitory effects of Syk inhibition on TGF-ß1-mediated myofibroblast activation were associated with down-regulation of MAPK-p38. These results suggest that Syk inhibition reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-ß1-induced kidney myofibroblast activation. Syk inhibition could have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.

Lysyl Oxidase Isoforms and Potential Therapeutic Opportunities for Fibrosis and Cancer.

INTRODUCTION: The lysyl oxidase family of enzymes is classically known as being required for connective tissue maturation by oxidizing lysine residues in elastin and lysine and hydroxylysine residues in collagen precursors. The resulting aldehydes then participate in cross-link formation, which is required for normal connective tissue integrity. These enzymes have biological functions that extend beyond this fundamental biosynthetic role, with contributions to angiogenesis, cell proliferation, and cell differentiation. Dysregulation of lysyl oxidases occurs in multiple pathologies including fibrosis, primary and metastatic cancers, and complications of diabetes in a variety of tissues. AREAS COVERED: This review summarizes the major findings of novel roles for lysyl oxidases in pathologies, and highlights some of the potential therapeutic approaches that are in development and which stem from these new findings. EXPERT OPINION: Fundamental questions remain regarding the mechanisms of novel biological functions of this family of proteins, and regarding functions that are independent of their catalytic enzyme activity. However, progress is underway in the development of isoform-specific pharmacologic inhibitors, potential therapeutic antibodies and gaining an increased understanding of both tumor suppressor and metastasis promotion activities. Ultimately, this is likely to lead to novel therapeutic agents.

[Research progress of NADPH oxidases and their inhibitors].

NADPH oxidases (NOXs) are the key enzymes of redox signaling in vivo and also the main source of reactive oxygen species (ROS) in the body. ROS plays a role of double-edged sword. On the one hand, ROS, at the level of physiological amount, has the effect of immune defense and also acts as a second messenger involved in the regulation of cellular signaling pathways. On the other hand, excessive ROS can cause oxidative stress, leading to the disorder of cellular functions. Recently, studies showed that ROS plays an important role in acceleration of some pathological reactions such as inflammation, fibrosis and tumor formation. As a major source of ROS, NOX has become a popular target in treating oxidative stress, inflammation, fibrosis and tumor. Herein, the role of NOX in these pathological processes and the research progress of NOX inhibitors are reviewed.

Recent data concerning heparanase: focus on fibrosis, inflammation and cancer.

Heparanase (HPSE) is a multitasking protein characterized by enzymatic and non-enzymatic activities. By means of its enzymatic activity, HPSE catalyzes the cutting of the side chains of heparan sulfate (HS) proteoglycans, thereby inducing the remodeling of the extracellular matrix and basement membranes. Thanks to the cleavage of HS, HPSE also promotes the release and diffusion of several HS-linked molecules such as growth factors, cytokines and enzymes. In addition to degrading HS chains, HPSE has non-enzymatic functions that trigger several signaling pathways. This signaling activity is achieved by interacting with transmembrane proteins, activating kinases such as Akt and Src, or modulating the activity of factors such as FGF-2 and TGF-ß. Several studies have recently highlighted a possible intracellular activity for HPSE, particularly at nuclear level. While HPSE activity is quite limited in physiological conditions, its demonstrated increasing involvement in various pathological conditions, such as in tumor progression and renal disease, have attracted the attention of a growing number of researchers. The fact that no other molecule is capable of performing the same function as HPSE makes this enzyme an attractive potential target of medical treatment. With this short conceptual overview, we aim to provide an update on current knowledge concerning the HPSE protein in the experimental and clinical settings, paying particular attention to its role in fibrosis, inflammation and cancer.

Macrophage epoxygenase determines a profibrotic transcriptome signature.

Epoxygenases belong to the cytochrome P450 family. They generate epoxyeicosatrienoic acids, which are known to have anti-inflammatory effects, but little is known about their role in macrophage function. By high-throughput sequencing of RNA in primary macrophages derived from rodents and humans, we establish the relative expression of epoxygenases in these cells. Zinc-finger nuclease-mediated targeted gene deletion of the major rat macrophage epoxygenase Cyp2j4 (ortholog of human CYP2J2) resulted in reduced epoxyeicosatrienoic acid synthesis. Cyp2j4(-/-) macrophages have relatively increased peroxisome proliferator-activated receptor-γ levels and show a profibrotic transcriptome, displaying overexpression of a specific subset of genes (260 transcripts) primarily involved in extracellular matrix, with fibronectin being the most abundantly expressed transcript. Fibronectin expression is under the control of epoxygenase activity in human and rat primary macrophages. In keeping with the in vitro findings, Cyp2j4(-/-) rats show upregulation of type I collagen following unilateral ureter obstruction of the kidney, and quantitative proteomics analysis (liquid chromatography-tandem mass spectrometry) showed increased renal type I collagen and fibronectin protein abundance resulting from experimentally induced crescentic glomerulonephritis in these rats. Taken together, these results identify the rat epoxygenase Cyp2j4 as a determinant of a profibrotic macrophage transcriptome that could have implications in various inflammatory conditions, depending on macrophage function.

Involvement of Rho-Kinase/LIM Kinase/Cofilin Signaling Pathway in Corporal Fibrosis after Cavernous Nerve Injury in Male Rats.

INTRODUCTION: The molecular mechanism of corporal fibrosis leading to erectile dysfunction (ED) following cavernous nerve (CN) injury is poorly understood. AIM: To determine whether the LIMK2/cofilin pathway, the downstream effectors of ROCK1, was involved in ED and corporal fibrosis following bilateral CN injury in male rats. METHODS: Forty-eight 10-week-old male Sprague-Dawley rats were equally divided into three groups: sham surgery (S); bilateral CN crush injury (I); and bilateral CN resection (R). Within each groups, two subgroups were analyzed at 1 and 4 weeks postoperatively. MAIN OUTCOME MEASURES: Electrostimulation was performed to assess erectile function by the ratio of maximal intracavernous pressure to mean arterial pressure (ICP/MAP) and areas under the ICP curve to MAP (AUC/MAP). Penile tissue was processed for Masson's trichrome staining, Western blot (ROCK1, total LIMK2, phospho-LIMK2, total cofilin, phospho-cofilin), immunohistochemistry (alpha-SM actin [α-SMA]), and double immunofluorescent staining (ROCK1, phospho-LIMK2, vimentin). RESULTS: At each time point, both I and R groups showed a significantly lower percent of ICP/MAP and AUC, and decreased SM cell/collagen ratio and expression of α-SMA than S group. Densitometry revealed a significantly higher expression of ROCK1 in I and R groups compared with S group at all time points. The LIMK2 phosphorylation in I and R groups significantly increased at 1 week, but not at 4 weeks. The cofilin phosphorylation in R group significantly increased to that in S group starting at 1 week, while that in I group was increased significantly at 4 weeks. The double immunofluorescent staining noted that coexpression of vimentin with ROCK1 or phospho-LIMK2 in I and R groups was significantly increased mainly in the subtunical area at 1 week but not at 4 weeks. CONCLUSIONS: The ROCK1/LIMK2/cofilin pathway may be involved in ED related to corporal fibrosis, and it appears to be functional particularly in the early period after CN injury.

Genetic deletion of soluble epoxide hydrolase attenuates inflammation and fibrosis in experimental obstructive nephropathy.

Soluble epoxide hydrolase (sEH) is abundantly expressed in kidney and plays a potent role in regulating inflammatory response in inflammatory diseases. However, the role of sEH in progression of chronic kidney diseases such as obstructive nephropathy is still elusive. In current study, wild-type (WT) and sEH deficient (sEH (-/-)) mice were subjected to the unilateral ureteral obstruction (UUO) surgery and the kidney injury was evaluated by histological examination, western blotting, and ELISA. The protein level of sEH in kidney was increased in UUO-treated mice group compared to nonobstructed group. Additionally, UUO-induced hydronephrosis, renal tubular injury, inflammation, and fibrosis were ameliorated in sEH (-/-) mice with the exception of glomerulosclerosis. Moreover, sEH (-/-) mice with UUO showed lower levels of inflammation-related and fibrosis-related protein such as monocyte chemoattractant protein-1, macrophage inflammatory protein-2, interleukin-1ß (IL-1ß), IL-6, inducible nitric oxide synthase, collagen 1A1, and α-actin. The levels of superoxide anion radical and hydrogen peroxide as well as NADPH oxidase activity were also decreased in UUO kidneys of sEH (-/-) mice compared to that observed in WT mice. Collectively, our findings suggest that sEH plays an important role in the pathogenesis of experimental obstructive nephropathy and may be a therapeutic target for the treatment of obstructive nephropathy-related diseases.

Review of the Multiscale Effects of Female Sex Hormones on Matrix Metalloproteinase-Mediated Collagen Degradation.

Collagenases and gelatinases regulate many physiological processes and are involved in the pathogenesis and progression of various disease states, such as osteoarthritis, renal fibrosis, and atherosclerosis. These enzymes belong to the matrix metalloproteinase (MMP) family and are regulated by a number of factors, including sex hormones. Estrogen, relaxin, and progesterone can alter the balance between tissue degradation and repair by modulating MMPs, leading to gender disparities in many MMP-related disease states. In these diseases, MMPs initiate collagen degradation at the nanoscale when they cleave and denature collagen molecules. However, the net effect on tissue is generally observed at the macroscale. To understand how nanoscale events lead to macroscale changes, we must examine the intermediate scales. In this article, we review the literature that examines the effects of estrogen, relaxin, and progesterone on MMP production and activity, connecting the nanoscale, microscale, and macroscale details to relevant disease states.