Cyst-Like Osteolytic Formations in Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Augmented Sheep Spinal Fusion

Pan, H.C.,Lee, S.,Ting, K.,Shen, J.,Wang, C.,Nguyen, A.,Berthiaume, E.A.,Zara, J.N.,Turner, A.S.,Seim, H.B.,Kwak, J.H.,Zhang, X.,Soo, C. American Association of Pathologists and Bacteriol 2017 The American journal of pathology Vol.187 No.7

<P>Multiple case reports using recombinant human bone morphogenetic protein-2 (rhBMP-2) have reported complications. However, the local adverse effects of rhBMP-2 application are not well documented. In this report we show that, in addition to promoting Lumbar spinal fusion through potent osteogenic effects, rhBMP-2 augmentation promotes local cyst-like osteolytic formations in sheep trabecular bones that have undergone anterior lumbar interbody fusion. Three months after operation, conventional computed tomography showed that the trabecular bones of the rhBMP-2 application groups could fuse, whereas no fusion was observed in the control group. Micro computed tomography analysis revealed that the core implant area's bone volume fraction and bone mineral density increased proportionately with rhBMP-2 dose. Multiple cyst-Like bone voids were observed in peri-implant areas when using rhBMP2 applications, and these sites showed significant bone mineral density decreases in relation to the unaffected regions. Biomechanically, these areas decreased in strength by 32% in comparison with noncystic areas. Histologically, rhBMP-2 affected void sites had an increased amount of fatty marrow, thinner trabecular bones, and significantly more adiponectin- and cathepsin K-positive cells. Despite promoting successful fusion, rhBMP-2 use in clinical applications may result in local adverse structural alterations and compromised biomechanical changes to the bone.</P>

Down-Regulation of Dual-Specificity Phosphatase 5 in Gastric Cancer by Promoter CpG Island Hypermethylation and Its Potential Role in Carcinogenesis

Shin, S.H.,Park, S.Y.,Kang, G.H. American Association of Pathologists and Bacteriol 2013 The American journal of pathology Vol.182 No.4

Dual-specificity phosphatase 5 (DUSP5), which regulates the duration and magnitude of ERK½ phosphoactivation within the mitogen-activated protein kinase (MAPK) cascade, has recently been proposed to be a tumor suppressor. However, the epigenetic regulation of DUSP5 and its critical roles in gastric cancer (GC) remain unknown. We compared differential RNA expression profiles of GC cell lines with or without treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine. DUSP5 expression was dramatically decreased by DNA methylation. Hypermethylation of the DUSP5 promoter was detected in GC tissue samples, but not in normal healthy gastric mucosa samples. Restoring DUSP5 expression in DUSP5-silenced GC cell lines decreased their growth and colony-forming ability by causing arrest in the transition from G1 to S phase in the cell cycle as a result of dephosphorylation of ERK½ in the nucleus. Moreover, in a set of surgically resected GC cases (n = 179), GCs with DUSP5 promoter region hypermethylation (30.2%) exhibited significantly shortened survival, compared with GCs without DUSP5 methylation (P = 0.009). These results suggest that silencing of DUSP5 by promoter hypermethylation causes increased maintenance of phosphorylated ERK½, driving cell proliferation and contributing to gastric carcinogenesis. Furthermore, DUSP5 methylation may serve as a prognostic marker for GC, but this requires validation in a larger set of GC samples.

Cartilage Oligometric Matrix Protein-Angiopoietin-1 Promotes Revascularization Through Increased Survivin Expression in Dermal Endothelial Cells of Skin Grafts in Mice

Byun, S.J.,Choi, K.S.,Park, S.H.,Cho, N.W.,Hyun Yoo, C.,Yun, K.J.,Koh, Y.J.,Koh, G.Y.,So, B.J.,Yoon, K.H. American Association of Pathologists and Bacteriol 2007 The American journal of pathology Vol.171 No.5

The present study examined the effects of cartilage oligometric matrix protein angiopoietin-1 (COMP-Ang1) on the revascularization of mice skin grafts. Full-thickness skin grafts were autotransferred into BALB/c mice. The donor grafts were soaked in COMP-Ang1 protein (50 μg/ml, n = 10) or in bovine serum albumin (BSA) (50 μg/ml, n = 10) dissolved in 1 ml of sterile, phosphate-buffered saline for 5 minutes before transfer. Revascularization of the grafts was monitored using an intravital microscope on postoperative days 3, 4, and 5. Morphological and immunohistochemical analyses were performed to evaluate platelet-endothelial cell adhesion molecule-1 and survivin expression and apoptotic signal in the transplanted grafts. Grafts soaked in COMP-Ang1 (COMP-Ang1 group) showed significantly increased revascularization compared with grafts soaked in BSA (BSA group) on intravital microscopy and platelet-endothelial cell adhesion molecule-1 staining. The COMP-Ang1 group showed a significant increase of survivin expression in the endothelial cells and a reduction of apoptotic signal in comparison to the BSA group. Therefore, we believe that COMP-Ang1 provides the therapeutic benefit of enhancing the survival of vascular endothelial cells during transplantation of skin graft.

Elevated Levels of Cholesterol-Rich Lipid Rafts in Cancer Cells Are Correlated with Apoptosis Sensitivity Induced by Cholesterol-Depleting Agents

Li, Y.C.,Park, M.J.,Ye, S.K.,Kim, C.W.,Kim, Y.N. American Association of Pathologists and Bacteriol 2006 The American journal of pathology Vol.168 No.4

Lipid rafts/caveolae are membrane platforms for signaling molecules that regulate various cellular functions, including cell survival. To better understand the role of rafts in tumor progression and therapeutics, we investigated the effect of raft disruption on cell viability and compared raft levels in human cancer cell lines versus their normal counterparts. Here, we report that cholesterol depletion using methyl-β cyclodextrin caused anoikis-like apoptosis, which in A431 cells involved decreased raft levels, Bcl-xL down-regulation, caspase-3 activation, and Akt inactivation regardless of epidermal growth factor receptor activation. Cholesterol repletion replenished rafts on the cell surface and restored Akt activation and cell viability. Moreover, the breast cancer and the prostate cancer cell lines contained more lipid rafts and were more sensitive to cholesterol depletion-induced cell death than their normal counterparts. These results indicate that cancer cells contain increased levels of rafts and suggest a potential use of raft-modulating agents as anti-cancer drugs.

Heme Oxygenase-1 Expression in Murine Dendritic Cell Subpopulations

Park, D.J.,Agarwal, A.,George, J.F. American Association of Pathologists and Bacteriol 2010 The American journal of pathology Vol.176 No.6

Heme oxygenase-1 (HO-1) is a microsomal enzyme with antioxidant, antiapoptotic, and immunoregulatory functions. We studied the expression of HO-1 by bone marrow-derived dendritic cells (BMDCs) and splenic DC subpopulations under quiescent conditions or following lipopolysaccharide (LPS) stimulation. The kinetics of HO-1 expression by BMDCs depended on the conditions under which they were propagated. Expression of HO-1 in mouse BMDCs in 100 U/ml GM-CSF peaked at 16 hours after LPS treatment and maintained expression for at least 48 hours. But cultures in 800 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) showed peak expression by 16 hours that disappeared by 48 hours after LPS stimulation, similar to BMDCs cultured in both 100 U/ml GM-CSF and IL-4 (10 ng/ml). By flow cytometry, a large proportion of CD8<SUP>+</SUP> splenic DCs strongly expressed HO-1, and this population significantly increased following LPS administration in vivo. In HO-1<SUP>-/-</SUP> mice, the proportion of splenic CD8<SUP>+</SUP> DCs was significantly decreased in comparison with HO-1<SUP>+/+</SUP> mice. In addition, a unique subpopulation of MHC II<SUP>-</SUP>CD11b<SUP>+</SUP>CD11c<SUP>+</SUP> cells was prominent in HO-1<SUP>-/-</SUP> spleens. Injection of GFP-labeled HO-1<SUP>+/+</SUP> splenic DC precursors into HO-1<SUP>+/+</SUP> mice resulted in the generation of GFP<SUP>+</SUP>CD8<SUP>+</SUP> DCs in the spleen after 5 days, but GFP<SUP>+</SUP> CD8<SUP>+</SUP> DCs failed to appear in HO-1<SUP>-/-</SUP> spleens. Conversely, GFP<SUP>+</SUP>HO-1<SUP>-/-</SUP> splenic cells also generated GFP<SUP>+</SUP>CD8<SUP>+</SUP> DCs in HO-1<SUP>+/+</SUP> mice. These results show that HO-1 is involved in splenic DC differentiation, and/or the homing of CD8<SUP>+</SUP> splenic DC precursors appears to be dependent on HO-1 expression by the host.

CD137 Is Required for M Cell Functional Maturation but Not Lineage Commitment

Hsieh, E.H.,Fernandez, X.,Wang, J.,Hamer, M.,Calvillo, S.,Croft, M.,Kwon, B.S.,Lo, D.D. American Association of Pathologists and Bacteriol 2010 The American journal of pathology Vol.177 No.2

Mucosal immune surveillance depends on M cells that reside in the epithelium overlying Peyer's patch and nasopharyngeal associated lymphoid tissue to transport particles to underlying lymphocytes. M cell development is associated with B lymphocytes in a basolateral pocket, but the interactions between these cells are poorly understood. In a cell culture model of M cell differentiation, we found lymphotoxin/tumor necrosis factor α induction of CD137 (TNFRSF9) protein on intestinal epithelial cell lines, raising the possibility that CD137 on M cells in vivo might interact with CD137L expressed by B cells. Accordingly, while CD137-deficient mice produced UEA-1+ M cell progenitors in nasopharyngeal associated lymphoid tissue and Peyer's patch epithelium, they showed an abnormal morphology, including the absence of basolateral B cell pockets. More important, CD137-deficient nasopharyngeal associated lymphoid tissue M cells were defective in microparticle transcytosis. Bone marrow irradiation chimeras confirmed that while induction of UEA-1+ putative M cell precursors was not CD137-dependent, full M cell transcytosis function required expression of CD137 by radioresistant stromal cells as well as by bone marrow-derived cells. These results are consistent with a two-step model of M cell differentiation, with initial CD137-independent commitment to the M cell lineage followed by a CD137-CD137L interaction of M cells with CD137-activated B lymphocytes or dendritic cells for functional maturation.

Decreased Catalase Expression and Increased Susceptibility to Oxidative Stress in Primary Cultured Corneal Fibroblasts from Patients with Granular Corneal Dystrophy Type II

Choi, S.i.,Kim, T.i.,Kim, K.S.,Kim, B.Y.,Ahn, S.y.,Cho, H.j.,Lee, H.K.,Cho, H.S.,Kim, E.K. American Association of Pathologists and Bacteriol 2009 The American journal of pathology Vol.175 No.1

Granular corneal dystrophy type II (GCD II) is an autosomal dominant disorder characterized by age-dependent progressive accumulation of transforming growth factor-β-induced protein (TGFBIp) deposits in the corneal stroma. Several studies have suggested that corneal fibroblasts may decline with age in response to oxidative stress. To investigate whether oxidative stress is involved in the pathogenesis of GCD II, we assayed antioxidant enzymes, oxidative damage, and susceptibility to reactive oxygen species-induced cell death in primary cultured corneal fibroblasts (PCFs) from GCD II patients and healthy subjects. We found elevated protein levels of Mn-superoxide dismutase, Cu/Zn-superoxide dismutase, glutathione peroxidase, and glutathione reductase, as well as increased CAT mRNA and decreased catalase protein in GCD II PCFs. Furthermore, catalase is down-regulated in normal PCFs transfected with transforming growth factor-β-induced gene-h3. We also observed an increase in not only intracellular reactive oxygen species and H<SUB>2</SUB>O<SUB>2</SUB> levels, but also malondialdehyde, 4-hydroxynonenal, and protein carbonyls levels in GCD II PCFs. Greater immunoreactivity for malondialdehyde was observed in the corneal tissue of GCD II patients. In addition, we observed a decrease in Bcl-2 and Bcl-xL levels and an increase in Bax and Bok levels in GCD II PCFs. Finally, GCD II PCFs are more susceptible to H<SUB>2</SUB>O<SUB>2</SUB>-induced cell death. Together, these results suggest that oxidative damage induced by decreased catalase is involved in GCD II pathogenesis, and antioxidant agents represent a possible treatment strategy.

Role of CD11b⁺ Macrophages in Intraperitoneal Lipopolysaccharide-Induced Aberrant Lymphangiogenesis and Lymphatic Function in the Diaphragm

Kim, K.E.,Koh, Y.J.,Jeon, B.H.,Jang, C.,Han, J.,Kataru, R.P.,Schwendener, R.A.,Kim, J.M.,Koh, G.Y. American Association of Pathologists and Bacteriol 2009 The American journal of pathology Vol.175 No.4

Lymphatic vessels in the diaphragm are essential for draining peritoneal fluid, but little is known about their pathological changes during inflammation. Here we characterized diaphragmatic lymphatic vessels in a peritonitis model generated by daily i.p. administration of lipopolysaccharide (LPS) in mice. Intraperitoneal LPS increased lymphatic density, branching, sprouts, connections, and network formation in the diaphragm in time- and dose-dependent manners. These changes were reversible on discontinuation of LPS administration. The LPS-induced lymphatic density and remodeling occur mainly through proliferation of lymphatic endothelial cells. CD11b<SUP>+</SUP> macrophages were massively accumulated and closely associated with the lymphatic vessels changed by i.p. LPS. Both RT-PCR assays and experiments with vascular endothelial growth factor-C/D blockade and macrophage-depletion indicated that the CD11b<SUP>+</SUP> macrophage-derived lymphangiogenic factors vascular endothelial growth factor-C/D could be major mediators of LPS-induced lymphangiogenesis and lymphatic remodeling through paracrine activity. Functional assays with India ink and fluorescein isothiocyanate-microspheres indicated that impaired peritoneal fluid drainage in diaphragm of LPS-induced peritonitis mice was due to inflammatory fibrosis and massive attachment of CD11b<SUP>+</SUP> macrophages on the peritoneal side of the diaphragmatic lymphatic vessels. These findings reveal that CD11b<SUP>+</SUP> macrophages play an important role in i.p. LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm.

Role of Human Aquaporin 5 In Colorectal Carcinogenesis

Kang, S.K.,Chae, Y.K.,Woo, J.,Kim, M.S.,Park, J.C.,Lee, J.,Soria, J.C.,Jang, S.J.,Sidransky, D.,Moon, C. American Association of Pathologists and Bacteriol 2008 The American journal of pathology Vol.173 No.2

While overexpression of several aquaporins (AQPs) has been reported in different types of human cancer, the role of AQPs in carcinogenesis has not been clearly defined. Here, by immunochemistry, we have found expression of AQP5 protein in 62.8% (59/94) of resected colon cancer tissue samples as well as association of AQP5 with liver metastasis. We then demonstrated that overexpression of human AQP5 (hAQP5) induces cell proliferation in colon cancer cells. Overexpression of wild-type hAQP5 increased proliferation and phosphorylation of extracellular signal-regulated kinase-½ in HCT116 colon cancer cells whereas these phenomena in hAQP5 mutants (N185D and S156A) were diminished, indicating that both membrane association and serine/threonine phosphorylation of AQP5 are required for proper function. Interestingly, overexpression of AQP1 and AQP3 showed no differences in extracellular signal-regulated kinase-½ phosphorylation, suggesting that AQP5, unlike AQP1, may be involved in signal transduction. Moreover, hAQP5-overexpressing cells showed an increase in retinoblastoma protein phosphorylation through the formation of a nuclear complex with cyclin D1 and CDK4. Small interfering RNA analysis confirmed that hAQP5 activates the Ras signaling pathway. These data not only describe the induction of hAQP5 expression during colorectal carcinogenesis but also provide a molecular mechanism for colon cancer development through the interaction of hAQP5 with the Ras/extracellular signal-regulated kinase/retinoblastoma protein signaling pathway, identifying hAQP5 as a novel therapeutic target.

Notch1 Signaling in FIZZ1 Induction of Myofibroblast Differentiation

Liu, T.,Hu, B.,Choi, Y.Y.,Chung, M.,Ullenbruch, M.,Yu, H.,Lowe, J.B.,Phan, S.H. American Association of Pathologists and Bacteriol 2009 The American journal of pathology Vol.174 No.5

Notch1 is an evolutionarily conserved receptor that regulates cell fate, including such events as differentiation, proliferation, and apoptosis. Myofibroblast differentiation is a key feature of lung fibrosis. Found in inflammatory zone 1 (FIZZ1) has direct fibrogenic properties because of its ability to induce myofibroblast differentiation. However, the downstream signaling pathway that mediates FIZZ1 induction of myofibroblast differentiation remains unknown. The objective of this study was to investigate the involvement of Notch signaling in FIZZ1 induction of lung myofibroblast differentiation and thus explore the potential role of Notch1 in pulmonary fibrosis. The results showed that FIZZ1 increased the expression levels of activated intracellular domain of Notch1 (NIC), its ligand Jagged1, and its target gene Hes1, which were associated with elevated α-smooth muscle actin expression levels. Fibroblast α-smooth muscle actin expression is induced by the overexpression of NIC but is suppressed by the inhibition of NIC. Moreover, lung fibroblasts that were isolated from mice lacking the GDP-4-keto-6-deoxymannose3,5-epimerase-4-reductase enzyme (FX knockout) exhibited significantly reduced responsiveness to FIZZ1, which was reversed by fucose supplementation. In the absence of exogenous fucose, these FX-deficient cells exhibited defective fucosylation, which is required for Notch signaling. These knockout mice also showed impaired lung fibrosis. These findings suggest that Notch1 signaling in response to FIZZ1 may play a significant role in myofibroblast differentiation during lung fibrosis.