Protective Effect of CD137 Deficiency Against Postinfarction Cardiac Fibrosis and Adverse Cardiac Remodeling by ERK1/2 Signaling Pathways.

ABSTRACT: Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart after MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared with wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings because CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. In addition, the administration of CD137L recombinant protein further promoted alpha-smooth muscle actin expression and collagen I synthesis, suggesting a profibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the profibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function.

Transient Receptor Potential Melastatin 7 Promotes Vascular Adventitial Fibroblasts Phenotypic Transformation and Inflammatory Reaction Induced by Mechanical Stretching Stress via p38 MAPK/JNK Pathway.

Remodeling of the arteries is one of the pathological bases of hypertension. We have previously shown that transient receptor potential melastatin 7 (TRPM7) aggravates the vascular adventitial remodeling caused by pressure overload in the transverse aortic constriction (TAC) model. In this study, we sought to explore the functional expression and downstream signaling of TRPM7 in vascular adventitial fibroblasts (AFs) stimulated by mechanical stretching stress (MSS). The expression of TRPM7 was upregulated with a concomitant translocation to the cytoplasm in the AFs stimulated with 20% MSS. Meanwhile, the expression of α-smooth muscle actin (α-SMA), a marker of transformation from AFs to myofibroblasts (MFs) was also increased. Moreover, AF-conditioned medium caused a significant migration of macrophages after treatment with MSS and contained high levels of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Pharmacological and RNA interference approaches using the TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB) and specific anti-TRPM7 small interfering RNA (si-RNA-TRPM7) abrogated these changes significantly. Further exploration uncloaked that inhibition of TRPM7 reduced the phosphorylation of p38 MAP kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) in the AFs stimulated with MSS. Furthermore, inhibition of the phosphorylation of p38MAPK or JNK could also alleviate the MSS-induced expression of α-SMA and secretion of inflammatory factors. These observations indicate that activated TRPM7 participates in the phenotypic transformation and inflammatory action of AFs in response to MSS through the p38MAPK/JNK pathway and suggest that TRPM7 may be a potential therapeutic target for vascular remodeling caused by hemodynamic changes in hypertension.

Nanostring-based screening for tyrosine kinase fusions in inflammatory myofibroblastic tumors.

Gene expression imbalances were measured for tyrosine kinase (TK) genes using Nanostring in 19 samples of inflammatory myofibroblastic tumor (IMT). All cases were immunohistochemically stained with anaplastic lymphoma kinase (ALK) and pan-tropomyosin-related-kinase (pan-Trk) antibodies. Five cases with imbalanced ALK expression, reported with Nanostring, were tested using fluorescence in situ hybridization (FISH); two cases with imbalanced neurotrophic tyrosine receptor kinase 3 (NTRK3) expression were tested using reverse transcription-polymerase chain reaction (RT-PCR). One case with imbalanced expression for ROS proto-oncogene 1 (ROS1) was tested using RNA sequencing and RT-PCR. TK fusions were detected in all cases with imbalanced TK expression. RNA sequencing detected a FN1-ROS1 fusion gene in an adult IMT case. IMT with ALK rearrangement showed myofibroblast-dominant features. IMT with ETV6-NTRK3 fusion showed prominent lymphoplasmacytic infiltration with scattered myofibroblasts. Pan-Trk IHC revealed only scattered positively stained cells in IMT with ETV6-NTRK3 fusion gene. ROS1-positive IMT showed myofibroblast-dominant features.

ADAMTS16 activates latent TGF-ß, accentuating fibrosis and dysfunction of the pressure-overloaded heart.

AIMS: Cardiac fibrosis is a major cause of heart failure (HF), and mediated by the differentiation of cardiac fibroblasts into myofibroblasts. However, limited tools are available to block cardiac fibrosis. ADAMTS16 is a member of the ADAMTS superfamily of extracellular protease enzymes involved in extracellular matrix (ECM) degradation and remodelling. In this study, we aimed to establish ADAMTS16 as a key regulator of cardiac fibrosis. METHODS AND RESULTS: Western blot and qRT-PCR analyses demonstrated that ADAMTS16 was significantly up-regulated in mice with transverse aortic constriction (TAC) associated with left ventricular hypertrophy and HF, which was correlated with increased expression of Mmp2, Mmp9, Col1a1, and Col3a1. Overexpression of ADAMTS16 accelerated the AngII-induced activation of cardiac fibroblasts into myofibroblasts. Protein structural analysis and co-immunoprecipitation revealed that ADAMTS16 interacted with the latency-associated peptide (LAP)-transforming growth factor (TGF)-ß via a RRFR motif. Overexpression of ADAMTS16 induced the activation of TGF-ß in cardiac fibroblasts; however, the effects were blocked by a mutation of the RRFR motif to IIFI, knockdown of Adamts16 expression, or a TGF-ß-neutralizing antibody (ΝAb). The RRFR tetrapeptide, but not control IIFI peptide, blocked the interaction between ADAMTS16 and LAP-TGF-ß, and accelerated the activation of TGF-ß in cardiac fibroblasts. In TAC mice, the RRFR tetrapeptide aggravated cardiac fibrosis and hypertrophy by up-regulation of ECM proteins, activation of TGF-ß, and increased SMAD2/SMAD3 signalling, however, the effects were blocked by TGF-ß-NAb. CONCLUSION: ADAMTS16 promotes cardiac fibrosis, cardiac hypertrophy, and HF by facilitating cardiac fibroblasts activation via interacting with and activating LAP-TGF-ß signalling. The RRFR motif of ADAMTS16 disrupts the interaction between ADAMTS16 and LAP-TGF-ß, activates TGF-ß, and aggravated cardiac fibrosis and hypertrophy. This study identifies a novel regulator of TGF-ß signalling and cardiac fibrosis, and provides a new target for the development of therapeutic treatment of cardiac fibrosis and HF.

Infantile inflammatory myofibroblastic tumors: clinicopathological and molecular characterization of 12 cases.

Inflammatory myofibroblastic tumors arising in infants are rare, poorly investigated and mostly reported as isolated cases or as a part of larger series thus, their clinicopathological and molecular features are essentially unknown. Archival files from two large pediatric institutions and a tumor registry were queried for pediatric inflammatory myofibroblastic tumors. Available material from patients ≤12 months of age was reviewed. Additional immunostains (ALK-1, D240, WT1) and ALK-FISH studies were performed as needed. Targeted anchored multiplex PCR with next-generation sequencing was done in all cases. A total of 12 of 131 infantile cases (mean 5.5 months) were identified (M:F of 2:1). Anatomic locations included intestinal/mesenteric (n = 6), head/neck (n = 3), and viscera (n = 3). Half of tumors showed a hypocellular myxoid pattern, perivascular condensation, and prominent vasculature with vague glomeruloid structures present in four of them. The remaining cases exhibited a more cellular pattern with minimal myxoid component. ALK-1 immunohistochemistry was positive in most cases (11/12) with cytoplasmic-diffuse (n = 6), cytoplasmic-granular (n = 2), and dot-like (n = 3) staining patterns. ALK fusion partners identified in five cases included EML4, TPM4, RANBP2, and a novel KLC1. Three inflammatory myofibroblastic tumors showed fusions with other kinases including TFG-ROS1 and novel FN1-ROS1 and RBPMS-NTRK3 rearrangements. Favorable outcome was documented in most cases (10/11) with available follow-up (median 17 months) while three patients were successfully treated with crizotinib. In summary, infantile inflammatory myofibroblastic tumors are rare and can exhibit paucicellular, extensively myxoid/vascular morphology with peculiar immunophenotype mimicking other mesenchymal or vascular lesions. All tumors harbored kinase fusions involving ALK, ROS1, and NTRK3 including three novel fusion partners (KLC1, FN1, and RBPMS, respectively). A favorable response to crizotinib seen in three cases supports its potential use in infants as seen in older patients. Awareness of these unusual morphologic, immunophenotypic, and molecular features is critical for appropriate diagnosis and optimized targeted therapy.

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction.

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (αSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfß), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.

Abrogation of myofibroblast activities in metastasis and fibrosis by methyltransferase inhibition.

Myofibroblasts are a population of highly contractile fibroblasts that express and require the activity of the transcription factor Snail1. Cancer-associated fibroblasts (CAFs) correlate with low survival of cancer patients when present in the stroma of primary tumors. Remarkably, the presence of myofibroblastic CAFs (which express Snail1) creates mechanical properties in the tumor microenvironment that support metastasis. However, therapeutic blockage of fibroblast activity in patients with cancer is a double-edged sword, as normal fibroblast activities often restrict tumor cell invasion. We used fibroblasts depleted of Snail1 or protein arginine methyltransferases 1 and 4 (PRMT1/-4) to identify specific epigenetic modifications induced by TGFß/Snail1. Furthermore, we analyzed the in vivo efficiency of methyltransferase inhibitors using mouse models of wound healing and metastasis, as well as fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF). Mechanistically, TGFß-induced Snail1 promotes the epigenetic mark of asymmetrically dimethylated arginine. Critically, we found that inhibitors of methyltransferases prevent myofibroblast activity (but not regular fibroblast activity) in the extracellular matrix, both in cell culture and in vivo. In a mouse breast cancer model, the inhibitor sinefungin reduces both the myofibroblast activity in the tumor stroma and the metastatic burden in the lung. Two distinct inhibitors effectively blocked the exacerbated myofibroblast activity of patient-derived IPF fibroblasts. Our data reveal epigenetic regulation of myofibroblast transdifferentiation in both wound healing and in disease (fibrosis and breast cancer). Thus, methyltransferase inhibitors are good candidates as therapeutic reagents for these diseases.

Neutral endopeptidase inhibitors blunt kidney fibrosis by reducing myofibroblast formation.

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor ß (TGF-ß) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.

Antifibrotic Synergy Between Phosphodiesterase Type 5 Inhibitors and Selective Oestrogen Receptor Modulators in Peyronie's Disease Models.

BACKGROUND: Peyronie's disease (PD) is a fibrotic disorder of the penile tunica albuginea, characterised by the formation of a localised fibrous plaque that can lead to deformity and erectile dysfunction. Nonsurgical therapeutic options for PD are limited in efficacy and safety. Myofibroblasts are key cells in the pathogenesis of PD, and inhibition of myofibroblast transformation has been suggested as a therapeutic option. OBJECTIVE: To identify potential drugs using a novel phenotypic assay and then to test them using in vitro and in vivo models of PD. DESIGN, SETTING, AND PARTICIPANTS: We have developed and validated a phenotypic screening assay that measures myofibroblast transformation, by which we tested 21 compounds that were suggested to be efficacious in treating PD. The successful hits from this assay were further tested using in vitro and in vivo models of PD. RESULTS AND LIMITATIONS: The new assay was able to detect transforming growth factor-ß1-induced myofibroblast transformation. Using this assay, phosphodiesterase type 5 inhibitors (PDE5i) and selective oestrogen receptor modulators (SERMs) were identified to significantly inhibit myofibroblast transformation. A PDE5i (vardenafil) and an SERM (tamoxifen) inhibited myofibroblast transformation, collagen gel contraction, and extracellular matrix production in a synergistic fashion. In a rat model of PD, the antifibrotic effect of the combination of vardenafil and tamoxifen was greater than that of each drug alone. This study is limited by not providing a molecular mechanism for the proposed synergy. CONCLUSIONS: This is the first demonstration of a synergistic activity between a PDE5i and an SERM discovered through a phenotypic screening approach. Future clinical trials using a combination of these drugs should be considered during the active phase of PD, given the early evidence of benefit in both in vitro and in vivo models. PATIENT SUMMARY: This report suggests that the combination of a phosphodiesterase type 5 inhibitor and a selective oestrogen receptor modulator may be efficacious in treating Peyronie's disease in its active phase.

The distribution and time-dependent expression of HIPK2 during the repair of contused skeletal muscle in mice.

HIPK2 is an evolutionarily conserved serine/threonine kinase and is considered a co-regulator of an increasing number of transcription factors modulating a variety of cellular processes, including inflammation, proliferation and fibrosis. Skeletal muscle injuries repair is an overlapping event between inflammation and tissue repair. There are no reports about HIPK2 expression in skeletal muscles after trauma. A foundational study on distribution and time-dependent expression of HIPK2 was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR, which is expected to obtain a preliminary insight into the functions of HIPK2 during the repair of contused skeletal muscle in mice. An animal model of skeletal muscle contusion was established in 50 C57B6/L male mice. Samples were taken at 1, 3, 5, 7, 9, 14, 17, 21 and 28 days after contusion, respectively (5 mice at each posttraumatic interval). 5 mice were employed as control. No HIPK2-positive staining was detected in uninjured skeletal muscle. Intensive immunoreactivties of HIPK2 were observed in polymorphonuclear cells, round-shaped mononuclear cells, regenerated multinucleated myotubes and spindle-shaped fibroblastic cells in the contused tissue. The HIPK2-positive cells were identified as neutrophils, macrophages and myofibroblasts by double immunofluorescent procedure. HIPK2 protein and mRNA expression were remarkably up-regulated after contusion by Western blotting and qPCR analysis. The results demonstrated that the expression of HIPK2 is distributed in certain cell types and is time-dependently expressed in skeletal muscle after contusion, which suggested that HIPK2 may participate in the whole process of skeletal muscle wound healing, including inflammatory response, muscle regeneration and fibrogenesis.

HDAC8 inhibition ameliorates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative lung disease, and fibroblast-myofibroblast differentiation (FMD) is thought to be a key event in the pathogenesis of IPF. Histone deacetylase-8 (HDAC8) has been shown to associate with α-smooth muscle actin (α-SMA; a marker of FMD) and regulates cell contractility in vascular smooth muscle cells. However, the role of HDAC8 in FMD or pulmonary fibrosis has never been reported. This study investigated the role of HDAC8 in pulmonary fibrosis with a focus on FMD. We observed that HDAC8 expression was increased in IPF lung tissue as well as transforming growth factor (TGF)ß1-treated normal human lung fibroblasts (NHLFs). Immunoprecipitation experiments revealed that HDAC8 was associated with α-SMA in TGFß1-treated NHLFs. HDAC8 inhibition with NCC170 (HDAC8-selective inhibitor) repressed TGFß1-induced fibroblast contraction and α-SMA protein expression in NHLFs cultured in collagen gels. HDAC8 inhibition with HDAC8 siRNA also repressed TGFß1-induced expression of profibrotic molecules such as fibronectin and increased expression of antifibrotic molecules such as peroxisome proliferator-activated receptor-γ (PPARγ). Chromatin immunoprecipitation quantitative PCR using an antibody against H3K27ac (histone H3 acetylated at lysine 27; a known HDAC8 substrate and a marker for active enhancers) suggested that HDAC8 inhibition with NCC170 ameliorated TGFß1-induced loss of H3K27ac at the PPARγ gene enhancer. Furthermore, NCC170 treatment significantly decreased fibrosis measured by Ashcroft score as well as expression of type 1 collagen and fibronectin in bleomycin-treated mouse lungs. These data suggest that HDAC8 contributes to pulmonary fibrosis and that there is a therapeutic potential for HDAC8 inhibitors to treat IPF as well as other fibrotic lung diseases.

Hirudin Protects Ang II-Induced Myocardial Fibroblasts Fibrosis by Inhibiting the Extracellular Signal-Regulated Kinase1/2 (ERK1/2) Pathway.

BACKGROUND Myocardial fibrosis is closely related to all types of cardiovascular diseases. Hirudin is widely used in the prevention and treatment of cardiovascular diseases and cancers. In this study, we examined the potential role(s) and mechanism of hirudin in angiotensin II (Ang II)-induced myocardial fibrosis. MATERIAL AND METHODS The viability of myocardial fibroblasts, and reactive oxygen species (ROS) rates were measured respectively using cell counting kit-8 (CCK-8) and flow cytometry. Malondialdehyde (MDA) content, the activities of lactate dehydrogenase (LDH), and superoxide dismutase (SOD) were detected by the respective kits. The mRNA and protein levels of fibrosis-related factors were separately assessed by qRT-PCR and western blot. RESULTS Our data revealed that hirudin suppressed the viability of myocardial fibroblasts, and that it relieved the proliferation induced by Ang II in a dose-dependent manner. We also found that hirudin reduced ROS production, LDH activity, and MDA content; however, it enhanced SOD activity. Moreover, while hirudin significantly downregulated the levels of matrix metalloproteinase-2 (MMP-2), MMP-9, fibronectin (FN), transforming growth factor beta 1 (TGF-ß1), collagen-I (COL-I), and COL-III, it upregulated the expression level of tissue inhibitor of metalloproteinases-2 (TIMP-2). Furthermore, phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2) was decreased by hirudin, compared to the Ang-II group. CONCLUSIONS Hirudin depressed Ang II-induced myocardial fibroblasts via inhibiting oxidative stress, regulating fibrosis-related factors, and repressing the ERK1/2 pathway.

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.

A77 1726 (leflunomide) blocks and reverses cardiac hypertrophy and fibrosis in mice.

T-cell infiltration and the subsequent increased intracardial chronic inflammation play crucial roles in the development of cardiac hypertrophy and heart failure (HF). A77 1726, the active metabolite of leflunomide, has been reported to have powerful anti-inflammatory and T cell-inhibiting properties. However, the effect of A77 1726 on cardiac hypertrophy remains completely unknown. Herein, we found that A77 1726 treatment attenuated pressure overload or angiotensin II (Ang II)-induced cardiac hypertrophy in vivo, as well as agonist-induced hypertrophic response of cardiomyocytes in vitro In addition, we showed that A77 1726 administration prevented induction of cardiac fibrosis by inhibiting cardiac fibroblast (CF) transformation into myofibroblast. Surprisingly, we found that the protective effect of A77 1726 was not dependent on its T lymphocyte-inhibiting property. A77 1726 suppressed the activation of protein kinase B (AKT) signaling pathway, and overexpression of constitutively active AKT completely abolished A77 1726-mediated cardioprotective effects in vivo and in vitro Pretreatment with siRNA targetting Fyn (si Fyn) blunted the protective effect elicited by A77 1726 in vitro More importantly, A77 1726 was capable of blocking pre-established cardiac hypertrophy in mice. In conclusion, A77 1726 attenuated cardiac hypertrophy and cardiac fibrosis via inhibiting FYN/AKT signaling pathway.

Osteoglycin attenuates cardiac fibrosis by suppressing cardiac myofibroblast proliferation and migration through antagonizing lysophosphatidic acid 3/matrix metalloproteinase 2/epidermal growth factor receptor signalling.

Aims: Cardiac myofibroblasts (CMFs) play a crucial role in the progression of pathological fibrotic cardiac remodelling. The expression of osteoglycin (OGN) is increased in diseased hearts; however, the role of OGN in pathological cardiac remodelling is not understood. Here, we sought to determine the effect of OGN on cardiac interstitial fibrosis and investigate the molecular mechanisms of OGN in CMF activation and matrix production. Methods and results: We found that OGN expression was significantly upregulated in mouse hearts in response to chronic 14-day angiotensin II (Ang II) infusion. Mice lacking OGN (OGN-/-) exhibited enhanced cardiac interstitial fibrosis and significantly more severe cardiac dysfunction following Ang II infusion compared to wild-type mice. OGN deficiency did not alter blood pressure, nor had effect on transforming growth factor-beta signalling activation, but presented with increased proliferative activity in hearts. In vitro studies with isolated CMFs revealed that OGN deficiency significantly increased proliferation and migration and enhanced the transactivation of epidermal growth factor receptor (EGFR) signalling by Ang II. On the other hand, OGN overexpression in CMFs decreased their proliferation and migration via reducing EGFR activation. Overexpression of OGN also suppressed the shedding of membrane anchored EGFR ligand. Moreover, OGN was found to interact with a lysophosphatidic acid (LPA) receptor isoform 3 and thus to attenuate EGFR transactivation through blocking cell surface translocation of membrane type 1 matrix metalloproteinase (MT1-MMP) and subsequent pro-MMP-2 activation in a Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase (ROCK)-dependent manner. Conclusion: These findings suggest that OGN negatively regulates cardiac fibrotic remodelling by attenuating CMF proliferation and migration through LPA3-mediated and Rho/ROCK-dependent inhibition of MT1-MMP translocation, MMP2 activation and EGFR transactivation.

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling.

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast-like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF-ß, fibronectin (FN), α-SMA, and NG2. LPS also increased protein and gene expression levels of anti-inflammatory COX-2 and pro-inflammatory IL-6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS-treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen-stimulated proliferation of CD4+ and the ratio of CD4+ CD25high /CD4+ CD25low lymphocytes. LPS-treated PDLSCs did not change the frequency of CD34+ and CD45+ cells, but decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU-GM number. The results indicated that LPS-activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.

The proto-oncogene tyrosine protein kinase Src is essential for macrophage-myofibroblast transition during renal scarring.

Src activation has been associated with fibrogenesis after kidney injury. Macrophage-myofibroblast transition is a newly identified process to generate collagen-producing myofibroblasts locally in the kidney undergoing fibrosis in a TGF-ß/Smad3-dependent manner. The potential role of the macrophage-myofibroblast transition in Src-mediated renal fibrosis is unknown. In studying this by RNA sequencing at single-cell resolution, we uncovered a unique Src-centric regulatory gene network as a key underlying mechanism of macrophage-myofibroblast transition. A total of 501 differentially expressed genes associated with macrophage-myofibroblast transition were identified. However, Smad3-knockout largely reduced the transcriptome diversity. More importantly, inhibition of Src largely suppresses ureteral obstruction-induced macrophage-myofibroblast transition in the injured kidney in vivo along with transforming growth factor-ß1-induced elongated fibroblast-like morphology, α-smooth muscle actin expression and collagen production in bone marrow derived macrophages in vitro. Unexpectedly, we further uncovered that Src serves as a direct Smad3 target gene and also specifically up-regulated in macrophages during macrophage-myofibroblast transition. Thus, macrophage-myofibroblast transition contributes to Src-mediated tissue fibrosis. Hence, targeting Src may represent as a precision therapeutic strategy for macrophage-myofibroblast transition-driven fibrotic diseases.

The Rho Kinase Isoforms ROCK1 and ROCK2 Each Contribute to the Development of Experimental Pulmonary Fibrosis.

Pulmonary fibrosis is thought to result from dysregulated wound repair after repetitive lung injury. Many cellular responses to injury involve rearrangements of the actin cytoskeleton mediated by the two isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), ROCK1 and ROCK2. In addition, profibrotic mediators such as transforming growth factor-ß, thrombin, and lysophosphatidic acid act through receptors that activate ROCK. Inhibition of ROCK activation may be a potent therapeutic strategy for human pulmonary fibrosis. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models; however, the specific roles of each ROCK isoform are poorly understood. Furthermore, the pleiotropic effects of this kinase have raised concerns about on-target adverse effects of ROCK inhibition such as hypotension. Selective inhibition of one isoform might be a better-tolerated strategy. In the present study, we used a genetic approach to determine the roles of ROCK1 and ROCK2 in a mouse model of bleomycin-induced pulmonary fibrosis. Using ROCK1- or ROCK2-haploinsufficient mice, we found that reduced expression of either ROCK1 or ROCK2 was sufficient to protect them from bleomycin-induced pulmonary fibrosis. In addition, we found that both isoforms contribute to the profibrotic responses of epithelial cells, endothelial cells, and fibroblasts. Interestingly, ROCK1- and ROCK2-haploinsufficient mice exhibited similar protection from bleomycin-induced vascular leak, myofibroblast differentiation, and fibrosis; however, ROCK1-haploinsufficient mice demonstrated greater attenuation of epithelial cell apoptosis. These findings suggest that selective inhibition of either ROCK isoform has the potential to be an effective therapeutic strategy for pulmonary fibrosis.

Inflammatory myofibroblastic tumors of the lung carrying a chimeric A2M-ALK gene: report of 2 infantile cases and review of the differential diagnosis of infantile pulmonary lesions.

We report 2 infantile cases of pulmonary tumor carrying a chimeric A2M-ALK gene. A2M-ALK is a newly identified anaplastic lymphoma kinase (ALK)-related chimeric gene from a tumor diagnosed as fetal lung interstitial tumor (FLIT). FLIT is a recently recognized infantile pulmonary lesion defined as a mass-like lesion that morphologically resembles the fetal lung. Grossly, FLIT characteristically appears as a well-circumscribed spongy mass, whereas the tumors in these patients were solid and firm. Histologically, the tumors showed intrapulmonary lesions composed of densely proliferating polygonal or spindle-shaped mesenchymal cells with diffuse and dense infiltrations of inflammatory cells forming microcystic or micropapillary structures lined by thyroid transcription factor 1-positive pneumocytes, favoring inflammatory myofibroblastic tumor rather than FLIT. The proliferating cells were immunoreactive for ALK, and A2M-ALK was identified in both tumors with reverse-transcription polymerase chain reaction. The dense infiltration of inflammatory cells, immunoreactivity for ALK, and identification of an ALK-related chimeric gene suggested a diagnosis of inflammatory myofibroblastic tumor. Histologically, most reported FLITs show sparse inflammatory infiltrates and a relatively low density of interstitial cells in the septa, although prominent infiltration of inflammatory cells and high cellularity of interstitial cells are seen in some FLITs. The present cases suggest that ALK rearrangements, including the chimeric A2M-ALK gene, may be present in these infantile pulmonary lesions, especially those with inflammatory cell infiltration. We propose that these infantile pulmonary lesions containing a chimeric A2M-ALK gene be categorized as a specific type of inflammatory myofibroblastic tumor that develops exclusively in neonates and infants.

Haplodeficiency of Ataxia Telangiectasia Mutated Accelerates Heart Failure After Myocardial Infarction.

BACKGROUND: Cell senescence is involved in the process of organ damage and repair; however, the underlying molecular mechanism needs to be further explored. METHODS AND RESULTS: Senescence-related genes (ie, p21, p53, and ataxia telangiectasia mutated [ATM]) were shown to be elevated after myocardial infarction (MI) in both mouse and human hearts. Ten- to 12-week-old male wild-type littermates (ATM+/+) and ATM heterozygous mice (ATM+/-) were subjected to MI. Cardiac echography showed that ATM haplodeficiency did not affect the survival rate but aggravated heart failure at day 28 post MI. Histologic analysis showed increased fibrosis in the noninfarct area of ATM+/- mice compared with that in ATM+/+ mice. Senescence-associated ß-galactosidase staining showed that the number of senescent fibroblasts was decreased when ATM was haplodeficient both in vivo and in vitro. Costaining of α-smooth muscle actin with p53 or p19 showed fewer senescent myofibroblasts in ATM+/- mouse hearts. Moreover, angiogenesis was also examined using the endothelial markers CD31 both at early (day 7) and late stages (day 28) after MI, and ATM haplodeficiency reduced angiogenesis after MI. Finally, cardiac fibroblasts were isolated from infarcted mouse heart and the medium were tested for its capacity of endothelial tubing formation, revealing that ATM haplodeficiency led to lower vascular endothelial growth factor production from cardiac fibroblast and reduced capacity of endothelial tube formation in vitro. CONCLUSIONS: The present study shows that ATM haplodeficiency decreases fibroblast senescence and vascular endothelial growth factor production and impaired angiogenesis in response to MI, leading to accelerated heart failure.