Yong Sun Maeng

Soluble PTK7 inhibits tube formation, migration, and invasion of endothelial cells and angiogenesis

Human PTK7 is a defective receptor protein tyrosine kinase and its expression is upregulated in various cancers including colorectal carcinomas. To determine whether PTK7 functions in angiogenesis, we have expressed and purified the extracellular domain of PTK7 (soluble PTK7; sPTK7) as a decoy receptor to counteract the effects of endogenous PTK7. Capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs) was accompanied by modulation in the PTK7 mRNA level. Neutralization of endogenous PTK7 with sPTK7 inhibited vascular endothelial growth factor (VEGF)-induced tube formation, migration, and invasion of HUVECs in a dose-dependent manner. sPTK7 reduced VEGF-induced phosphorylation of focal adhesion kinase (FAK) and paxillin, relocalization of paxillin to focal adhesions, and formation of stress fibers. Moreover, sPTK7 inhibited VEGF-induced angiogenesis in vivo. Knockdown of PTK7 using siRNA also inhibited VEGF-induced tube formation, supporting that sPTK7 specifically blocks function of the endogenous PTK7. These results demonstrate that PTK7 plays an important role not only in tube formation, migration, and invasion of endothelial cells but also in angiogenesis.

Lysosomal Trafficking of TGFBIp via Caveolae-Mediated Endocytosis

Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin αVβ3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.

Pathogenesis and treatments of TGFBI corneal dystrophies

Transforming growth factor beta-induced (TGFBI) corneal dystrophies are a group of inherited progressive corneal diseases. Accumulation of transforming growth factor beta-induced protein (TGFBIp) is involved in the pathogenesis of TGFBI corneal dystrophies; however, the exact molecular mechanisms are not fully elucidated. In this review article, we summarize the current knowledge of TGFBI corneal dystrophies including clinical manifestations, epidemiology, most common and recently reported associated mutations for each disease, and treatment modalities. We review our current understanding of the molecular mechanisms of granular corneal dystrophy type 2 (GCD2) and studies of other TGFBI corneal dystrophies. In GCD2 corneal fibroblasts, alterations of morphological characteristics of corneal fibroblasts, increased susceptibility to intracellular oxidative stress, dysfunctional and fragmented mitochondria, defective autophagy, and alterations of cell cycle were observed. Other studies of mutated TGFBIp show changes in conformational structure, stability and proteolytic properties in lattice and granular corneal dystrophies. Future research should be directed toward elucidation of the biochemical mechanism of deposit formation, the relationship between the mutated TGFBIp and the other materials in the extracellular matrix, and the development of gene therapy and pharmaceutical agents.

Heterochromatin Protein 1 Alpha (HP1α: CBX5) is a Key Regulator in Differentiation of Endothelial Progenitor Cells to Endothelial Cells

As the ability to control the differentiation of endothelial stem/progenitor cells (EPCs) into vascular endothelial cell lineages could be useful for promoting neovascularization, it is important to obtain a deeper understanding of the epigenetic mechanisms that regulate EPC differentiation and neovascularization. Heterochromatin protein 1α (HP1α) is known to be involved in the epigenetic regulation of gene silencing. However, recent reports demonstrate that HP1α can also activate gene expression during cell differentiation. In this study, microarray analysis revealed that HP1α expression was induced during EPC differentiation and is associated with the expression of outgrowing endothelial cell (OEC)‐specific protein markers. To explore the role of HP1α in the differentiation of EPCs to OECs, its expression was knocked‐down or over‐expressed in differentiating EPCs. Overexpression of HP1α promoted the differentiation and angiogenic activity of EPCs in vitro and in vivo, whereas knockdown of HP1α led to a defect in OEC migration, tube formation, and angiogenic sprouting activity. Gene expression profiling showed increased expression of angiogenic genes, including NOTCH1, cadherin‐5, and angiopoietin‐like‐2, and decreased expression of progenitor cell marker genes, including CD133, CXCR4, and C‐KIT, in HP1α‐overexpressing EPCs. Also, increased HP1α at an early stage of EPC differentiation may regulate angiogenic gene transcription by interacting with chromatin that modifies epigenetic factors such as the methyl‐CpG binding domain, Polycomb group ring finger 2, and DNA methyltransferases. Our findings demonstrate, for the first time, that HP1α plays an important role in the differentiation and angiogenic function of EPCs by regulating endothelial gene expression. Stem Cells 2015;33:1512–1522

Differential expression of nestin in normal and pre-eclamptic human placentas

Background. Nestin is a type VI intermediate filament protein originally described in neural stem cells. Recent reports have documented nestin expression in endothelium of newly formed blood vessels and suggested its role as a marker of capacity for neovascularization and angiogenesis in endothelial cells. The aim of this study was to investigate the differential expression of nestin in normal and pre‐eclamptic human placentas. Methods. Placental tissues from 12 women with severe pre‐eclampsia and 15 gestational age‐matched normotensive women were collected at the time of their cesarean section. Western blot analysis for each placental tissue was performed for nestin quantification. Immunohistochemical staining was employed to localize nestin‐positive cells and to investigate differential staining intensity in each placental cell. Results. Nestin expression was detected in all of the normal and pre‐eclamptic placental tissues by Western blotting. Compared with the normal placentas, tissues from severe pre‐eclamptic placentas showed higher expression of nestin (p<0.001). Nestin immunoreactivity was localized only to endothelial cells of chorionic villi. However, mesenchymal connective tissue cells, cytotrophoblasts, syncytiotrophoblasts, and decidual cells did not reveal any specific signal for nestin. Conclusions. We suggest that the capacity for neovascularization and angiogenesis in endothelial cell is increased in pre‐eclamptic placenta compared to that from normal pregnancy. Such changes may be a compensatory mechanism for the reduced maternofetal exchanges and long‐lasting fetal hypoxia in pre‐eclamptic pregnancy. Furthermore, these changes in endothelial cells of chorionic villi in pre‐eclamptic pregnancy may give an explanation for fetal response to pre‐eclamptic conditions.

TGF-β regulates TGFBIp expression in corneal fibroblasts via miR-21, miR-181a, and Smad signaling

Transforming growth factor-β (TGF-β)-induced gene (TGFBI) protein (TGFBIp) is associated with granular corneal dystrophy type 2 (GCD2). TGFBIp levels can affect GCD2 phenotypes, but the underlying molecular mechanisms have not been fully elucidated. We investigated the involvement of microRNA (miRNA) and TGF-β in the regulation of TGFBIp expression in corneal fibroblasts. Ectopic expression of miR-9, miR-21, and miR-181a significantly decreased TGFBIp levels. Conversely, expression of miR-21 and miR-181a was induced by TGF-β1. Expression of miR-21 was 10-fold higher than that of miR-9 and miR-181a in corneal fibroblasts. Additionally, TGF-β1 expression was significantly higher than that of TGF-β2 and TGF-β3 in corneal fibroblasts, whereas expression of all three TGF-β forms was not significantly different between wild-type (WT) and GCD2 homozygotes (HO) corneal fibroblasts. Taken together, these data indicate that TGFBIp expression is positively regulated by TGF-β, whereas TGF-β-induced miR-21 and miR-181a negatively regulate TGFBIp expression. In conclusion, TGFBIp levels in corneal fibroblasts are controlled via the coordinated activity of miR-21 and miR-181a and by Smad signaling. Pharmacologic modulation of these miRNAs and TGF-β signaling could have therapeutic potential for TGFBI-associated corneal dystrophy, including GCD2.

Disrupted cell cycle arrest and reduced proliferation in corneal fibroblasts from GCD2 patients: A potential role for altered autophagy flux

This study investigates the role of impaired proliferation, altered cell cycle arrest, and defective autophagy flux of corneal fibroblasts in granular corneal dystrophy type 2 (GCD2) pathogenesis. The proliferation rates of homozygous (HO) GCD2 corneal fibroblasts at 72 h, 96 h, and 120 h were significantly lower (1.102 ± 0.027, 1.397 ± 0.039, and 1.527 ± 0.056, respectively) than those observed for the wild-type (WT) controls (1.441±0.029, 1.758 ± 0.043, and 2.003 ± 0.046, respectively). Flow cytometry indicated a decreased G1 cell cycle progression and the accumulation of cells in the S and G2/M phases in GCD2 cells. These accumulations were associated with decreased levels of Cyclin A1, B1, and E1, and increased expression of p16 and p27. p21 and p53 expression was also significantly lower in GCD2 cells compared to the WT. Interestingly, treatment with the autophagy flux inhibitor, bafilomycin A1, resulted in similarly decreased Cyclin A1, B1, D1, and p53 expression in WT fibroblasts. Furthermore, similar findings, including a decrease in Cyclin A1, B1, and D1 and an increase in p16 and p27 expression were observed in autophagy-related 7 (Atg7; known to be essential for autophagy) gene knockout cells. These data provide new insight concerning the role of autophagy in cell cycle arrest and cellular proliferation, uncovering a number of novel therapeutic possibilities for GCD2 treatment.

Senescence of fetal endothelial progenitor cell in pregnancy with idiopathic fetal growth restriction

Objective: The aim of our study was to investigate the change of count and the status of cellular senescence in fetal endothelial progenitor cells (EPCs) obtained from the umbilical cord blood of women with fetal growth restriction (FGR). Methods: Fetal EPCs were obtained from thirty five normal and thirty pregnant women with FGR. Each EPC was characterized and counted. EPC differentiation time and outgrowth endothelial cell (OEC) colony formation assay, senescence-associated β-galactosidase (SA-β-gal) activity assay, and telomerase activity assay were performed. Results: Fetal EPC counts were significantly decreased in the FGR group compared with normal controls. In the FGR group, the EPC differentiation time was prolonged, OEC colonies were much less formed, the staining intensity of SA-β-gal was relatively increased and the telomerase activity of EPCs was significantly decreased, compared with normal pregnancy (p < 0.001 for all). Conclusions: The fetal EPCs in FGR pregnancies were decreased, functionally impaired and senescently altered.

Role of TGFBIp in wound healing and mucin expression in corneal epithelial cells

Purpose Transforming growth factor-β-induced protein (TGFBIp) is highly expressed in the cornea, and mutant TGFBIp induces corneal diseases. However, the function of TGFBIp in cornea epithelium is not fully investigated. Here, we tested the importance of TGFBIp in regulation of gene expression and corneal epithelial cell (CEC) activity. Materials and Methods The effect of TGFBIp on CEC activity was analyzed by cell migration, adhesion, proliferation and wound healing assay. Analysis of gene expression was examined by western blot and quantitative reverse transcription PCR. Results The results demonstrated that TGFBIp increased adhesion, migration, proliferation, and wound healing of CECs. Analysis of gene expression presented that TGFBIp-stimulated CECs exhibited increased expression of mucin family genes, such as MUC1, -4, -5AC, and -16. Furthermore, TGFBIp treatment increased the expression of MUC1, -4, -5AC, -7, and -16 in conjunctival epithelial cells. TGFBIp also increased the activity of intracellular signaling molecules ERK and AKT in CECs. Using pharmacologic inhibitors of ERK and AKT, we showed that the expression of mucin genes by TGFBIp is mediated by the activation of ERK and AKT signaling. Conclusion Our findings demonstrate that the locally generated TGFBIp in the cornea may contribute to wound healing of CECs by enhancing the migration, adhesion, and proliferation of CECs. In addition, our results suggest that TGFBIp has a protective effect on ocular surfaces by inducing the expression of mucin genes in corneal and conjunctival epithelial cells. These data suggest that TGFBIp is a useful therapeutic target for patients with corneal wounds.

Melatonin reduces endoplasmic reticulum stress and corneal dystrophy-associated TGFBIp through activation of endoplasmic reticulum-associated protein degradation

Endoplasmic reticulum (ER) stress is emerging as a factor for the pathogenesis of granular corneal dystrophy type 2 (GCD2). This study was designed to investigate the molecular mechanisms underlying the protective effects of melatonin on ER stress in GCD2. Our results showed that GCD2 corneal fibroblasts were more susceptible to ER stress‐induced death than were wild‐type cells. Melatonin significantly inhibited GCD2 corneal cell death, caspase‐3 activation, and poly (ADP‐ribose) polymerase 1 cleavage caused by the ER stress inducer, tunicamycin. Under ER stress, melatonin significantly suppressed the induction of immunoglobulin heavy‐chain‐binding protein (BiP) and activation of inositol‐requiring enzyme 1α (IRE1α), and their downstream target, alternative splicing of X‐box binding protein 1(XBP1). Notably, the reduction in BiP and IRE1α by melatonin was suppressed by the ubiquitin‐proteasome inhibitor, MG132, but not by the autophagy inhibitor, bafilomycin A1, indicating involvement of the ER‐associated protein degradation (ERAD) system. Melatonin treatment reduced the levels of transforming growth factor‐β‐induced protein (TGFBIp) significantly, and this reduction was suppressed by MG132. We also found reduced mRNA expression of the ERAD system components HRD1 and SEL1L, and a reduced level of SEL1L protein in GCD2 cells. Interestingly, melatonin treatments enhanced SEL1L levels and suppressed the inhibition of SEL1L N‐glycosylation caused by tunicamycin. In conclusion, this study provides new insights into the mechanisms by which melatonin confers its protective actions during ER stress. The results also indicate that melatonin might have potential as a therapeutic agent for ER stress‐related diseases including GCD2.