Chitinase 1 (CHIT1) is secreted by activated macrophages. Chitinase activity is raised in atherosclerotic patient sera and is present in atherosclerotic plaque. However, the role of CHIT1 in atherosclerosis is unknown. Preliminary studies of atherosclerosis in cynomolgous monkeys revealed CHIT1 to be closely correlated with areas of macrophage infiltration. Thus, we investigated the effects of a chitinase inhibitor, allosamidin, on macrophage function in vitro and on atherosclerotic development in vivo. In RAW264.7 cells, allosamidin elevated monocyte chemoattractant protein 1 and tumor necrosis factor alpha expression, and increased activator protein 1 and nuclear factor-κB transcriptional activity. Although inducible nitric oxide synthase, IL-6, and IL-1β expression were increased, Arg1 expression was decreased by chitinase inhibition, suggesting that suppression of CHIT1 activity polarizes macrophages into a M1 phenotype. Allosamidin decreased scavenger receptor AI, CD36, ABCA1, and ABCG1 expression presumably through peroxisome proliferator-activated receptor γ and liver X receptor α inhibition. Changes in these lipid-related proteins led to suppression of cholesterol uptake and apolipoprotein AI-mediated cholesterol efflux in macrophages. These effects were confirmed with CHIT1 siRNA transfection and CHIT1 plasmid transfection experiments in primary macrophages. Apolipoprotein E-deficient hyperlipidemic mice treated for 6 weeks with constant administration of allosamidin and fed an atherogenic diet showed aggravated atherosclerotic lesions in the aortic sinus and aortic arch. These data suggest that CHIT1 exerts protective effects against atherosclerosis by suppressing inflammatory responses and polarizing macrophages toward an M2 phenotype, and promoting lipid uptake and cholesterol efflux in macrophages.

Decorin, a small leucine-rich proteoglycan (SLRP), is involved in the pathophysiology of human congenital stromal corneal dystrophy (CSCD). This disease is characterized by corneal opacities and vision impairment. In reported cases, the human gene encoding decorin contains point mutations in exon 10, generating a truncated form of decorin lacking the C-terminal 33 amino acid residues. We have previously described a transgenic mouse model carrying a similar mutation in the decorin gene that leads to an ocular phenotype characterized by corneal opacities identical to CSCD in humans. We have also identified abnormal synthesis and secretion of various SLRPs in mutant mouse corneas. In the present study, we found that mutant C-terminal truncated decorin was retained in the cytoplasm of mouse keratocytes in vivo and of transfected human embryonic kidney cells. This resulted in endoplasmic reticulum stress and an unfolded protein response. Thus, we propose a novel cell-based mechanism underlying CSCD in which a truncated SLRP protein core is retained intracellularly, its accumulation triggering endoplasmic reticulum stress that results in abnormal SLRP synthesis and secretion, which ultimately affects stromal structure and corneal transparency.

Severe acute kidney injury (AKI) is frequently accompanied by maladaptive repair and renal fibrogenesis; however, the molecular mechanisms that mediate these acute and chronic consequences of AKI remain poorly understood. In this study, we examined the role of epidermal growth factor receptor (EGFR) in these processes using waved-2 (Wa-2) mice, which have reduced EGFR activity, and their wild-type (WT) littermates after renal ischemia. Renal EGFR phosphorylation was induced within 2 days after ischemia, increased over time, and remained elevated at 28 days in WT mice, but this was diminished in Wa-2 mice. At the early stage of postischemia (2 days), Wa-2 mice developed more severe acute renal tubular damage with less reparative responses as indicated by enhanced tubular cell apoptosis, and reduced dedifferentiation and proliferation as compared to WT animals. At the late stage of postischemia (28 days), Wa-2 mice exhibited a less severe renal interstitial fibrosis as shown by reduced activation/proliferation of renal myofibroblasts and decreased deposition of extracellular matrix proteins. EGFR activation also contributed to cell cycle arrest at the G2/M phase, a cellular event associated with production of profibrogenetic factors, in the injured kidney. Collectively, these results indicate that severe AKI results in sustained activation of EGFR, which is required for reparative response of renal tubular cells initially, but eventually leads to fibrogenesis.

An intriguing finding about the gene encoding methyl-CpG binding protein 2 (MeCP2) is that the loss-of-function mutations cause Rett syndrome and duplication (gain-of-function) of MECP2 leads to another neurological disorder termed MECP2 duplication syndrome. To ensure proper neurodevelopment, a precise regulation of MeCP2 expression is critical, and any gain or loss of MeCP2 over a narrow threshold level may lead to postnatal neurological impairment. To evaluate MeCP2 dosage effects, we generated Mecp2(WT_EGFP) transgenic (TG) mouse in which MeCP2 (endogenous plus TG) is mildly overexpressed (approximately 1.5×). The TG MeCP2(WT_EGFP) fusion protein is functionally active, as cross breeding of these mice with Mecp2 knockout mice led to alleviation of major phenotypes in the null mutant mice, including premature lethality. To characterize the Mecp2(WT_EGFP) mouse model, we performed an extensive battery of behavioral tests, which revealed that these mice manifest increased aggressiveness and higher pentylenetetrazole (PTZ)-induced seizure propensity. Evaluation of neuronal parameters revealed a reduction in the number of tertiary branching sites and increased spine density in Mecp2(WT_EGFP) transgenic (TG) neurons. Treatment of TG neurons with epileptogenic compound-PTZ led to a marked increase in amplitude and frequency of calcium spikes. Based on our ex vivo and in vivo data, we conclude that epileptic seizures are manifested as the first symptom when MeCP2 is mildly overexpressed in mice.

Subconjunctival fibrosis at the surgical site determines the outcome of glaucoma surgery. Myofibroblast transformation has a significant role in fibrosis, and vascular endothelial growth factor (VEGF) is reported to trigger myofibroblast transformation by inducing transforming growth factor (TGF)-β1. In the present study, we used IHC, Western blot analysis, enzyme-linked immunosorbent assay, and electron microscopy to determine the contribution of VEGF to myofibroblast transformation in subconjunctival fibrosis after glaucoma surgery. A rabbit trabeculectomy model was generated, and VEGF stimulation or VEGF inhibition was performed during surgery. VEGF stimulation induced TGF-β1 expression in a dose-dependent manner. Down-regulation of epithelial markers (E-cadherin and β-catenin) and up-regulation of mesenchymal marker (α-smooth muscle actin) were observed in the subconjunctival layers after trabeculectomy with VEGF stimulation. Up-regulations of Smad and Snail, which play a central role in myofibroblast transformation, were observed in the conjunctival and subconjunctival layers at the site of trabeculectomy. Electron microscopy revealed changes of the conjunctival epithelial cells, especially the presence of myofilaments and increased rough endoplasmic reticulum in the cytoplasm. Myofibroblast transformation was activated by VEGF stimulation and decreased by VEGF inhibition. These findings suggest that VEGF potentially affected the TGF-β1/Smad/Snail pathway, thereby triggering myofibroblast transformation. Therapeutic approaches modulating VEGF may control myofibroblast transformation and reduce subconjunctival fibrosis after glaucoma surgery.

Syncytiotrophoblast is the multinucleated epithelium of the placenta. Although many nuclei are dispersed within the syncytioplasm, others are aggregated into specializations, referred to as true and false syncytial knots and syncytial sprouts. Nuclei within true knots display highly condensed chromatin and are thought to be aged and effete. True knots increase in frequency with gestational age. Excessive formation (Tenney-Parker change) is associated with placental pathology, and a knotting index is used to assess severity. However, this index is potentially confounded by the creation of artifactual appearances (false knots) through tangential sectioning. In addition, knots must be distinguished from syncytial sprouts, which are markers of trophoblast proliferation. Here, we distinguish among sprouts, true knots, and false knots using serial sections and perform IHC for proliferating cell nuclear antigen, upstream binding factor, RNA polymerase II, and 8-oxo-deoxyguanosine as markers of recent incorporation, transcriptional activity, and oxidative damage. Villous explants were exposed to hydrogen peroxide to test the relationship between transcriptional activity and oxidative damage. Sprouts and false knots were found to contain recently incorporated and transcriptionally active nuclei. By contrast, most nuclei within true knots are negative for transcriptional markers but positive for 8-oxo-deoxyguanosine. In vitro, we observed a negative correlation between transcriptional activity and oxidative damage. These findings demonstrate that true knots contain effete damaged nuclei and provide IHC markers for their identification.

Colorectal cancer is the second leading cause of death from cancer. Osteopontin (OPN) is a component of tumor extracellular matrix identified as a key marker of cancer progression. The estrogen-related receptor α (ERRα) has been implicated in endocrine-related cancer development and progression, possibly through modulation of cellular energy metabolism. Previous reports that ERRα regulates OPN expression in bone prompted us to investigate whether ERRα controls OPN expression in human colorectal cancer. Using a tissue microarray containing 83 tumor-normal tissue pairs of colorectal cancer samples, we found that tumor epithelial cells displayed higher staining for ERRα than normal mucosa, in correlation with elevated OPN expression. In addition, knocking down endogenous ERRα led to reduced OPN expression in HT29 colon cancer cells. Promoter analysis, inhibition of ERRα activity, and expression and mutation of potential ERRα response elements in the proximal promoter of human OPN showed that ERRα and its obligate co-activator, peroxisome proliferator-activated receptor γ co-activator-1 α, positively control human OPN promoter activity. Furthermore, chromatin immunoprecipitation experiments confirmed in vivo occupancy of the OPN promoter by ERRα in HT29 cells, suggesting that OPN is a direct target of ERRα in colorectal cancer. These findings suggest an additional mechanism by which ERRα participates in the development and progression of colorectal cancer, further supporting the relevance of targeting ERRα with antagonists as anticancer agents.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a neurodegenerative disorder caused by mutations in the tau gene. Many mutations identified in FTDP-17 have been shown to affect tau exon 10 splicing in vitro, which presumably causes pathologic imbalances in exon 10(-) [3-repeat (3R)] and exon 10(+) [4-repeat (4R)] tau expression and leads to intracellular inclusions of hyperphosphorylated tau in patient brains. However, no reports have investigated this theory using model mice with a tau intronic mutation. Herein, we generated new transgenic mice harboring the tau intron 10 + 16C→T mutation. We prepared a transgene construct containing intronic sequences required for exon 10 splicing in the longest tau isoform cDNA. Although mice bearing the construct without the intronic mutation showed normal developmental changes of the tau isoform from 3R tau to equal amounts of 3R and 4R tau, mice with the mutation showed much higher levels of 4R tau at the adult stage. 4R tau was selectively recovered in insoluble brain fractions in their old age. Furthermore, these mice displayed abnormal tau phosphorylation, synapse loss and dysfunction, memory impairment, glial activation, pretangle formation, and neuronal loss in an age-dependent manner. These findings provide the first evidence in a mouse model that a tau intronic mutation-induced imbalance of 3R and 4R tau could be a cause of tauopathy.

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355I U/L compared to 93 ± 65 IU/L in nonfibrotic), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

Patients undergoing glucocorticoid therapy for a variety of disorders, including autoimmune diseases and hematological malignancies, are at risk of developing osteonecrosis. Despite extensive research in both patients and animal models, the underlying pathogenesis remains unclear. Proposed inciting mechanisms include intravascular thrombotic occlusion, marrow fat hypertrophy, osteocyte and/or endothelial cell apoptosis, hypercoagulability, and vasoconstriction of specific arteries and arterioles supplying bone. Our laboratory has developed a model of steroid-induced osteonecrosis in Balb/cJ mice which reflects clinically relevant exposures to glucocorticoids in which treated mice develop osteonecrosis of the distal femoral epiphysis when administered 4 to 8 mg/L dexamethasone in drinking water for 6 weeks. We identified lesions in arterioles supplying this area, with the mildest occurring in knees without any evidence of osteonecrosis. However, arteriopathy was more common among mice that did versus did not develop osteonecrosis (P < 0.0001); in mice with osteonecrosis, the associated vessels showed transmural necrosis and thickening of the vessel wall progressing to the point of luminal obstruction. In the most severe cases of osteonecrosis, end-stage lesions consisted of fully occluded vessels with marrow and bone necrosis involving the entire epiphysis. We propose that a primary arteriopathy is the initiating event in the genesis of steroid-induced osteonecrosis and provides a basis for future investigation of this disease process.