Jae-Chang Jung

Gold nanoparticles functionalized by gadolinium-DTPA conjugate of cysteine as a multimodal bioimaging agent.

The synthesis and characterization of gold nanoparticles coated with Gd-chelate (Au@GdL), where L is a conjugate of DTPA and cysteine, is described. These particles are obtained by the replacement of citrate from the gold nanoparticle surfaces with gadolinium chelate (GdL). The average size of Au@GdL is 14 nm with a loading of GdL reaching up to 2.9x10(3) per particles, and they demonstrate very high R1 relaxivity (approximately 10(5) mM(-1) s(-1)) as well as X-ray attenuation. The R1 relaxivity per [Gd] is 17.9 mM(-1) s(-1). The present system also exhibits macrophage-specific property, as demonstrated by histological and TEM images as well as CT and MR, rendering itself as a new class of T1 multimodal CT/MR contrast agent.

Gd‐DOTA Conjugate of RGD as a Potential Tumor‐Targeting MRI Contrast Agent

Targeted delivery of contrast agents (CAs) with specific tumor recognition sites and simultaneous monitoring of the growth and metastasis of tumors in the body is an important goal in diagnostic molecular imaging. RGD (Arg-Gly-Asp) peptide is well known to have a relatively high and specific affinity for anb3-integrin, which is over-expressed in nascent endothelial cells during angiogenesis (formation of new blood vessels) in various tumor types and not in inactive endothelial cells. The expression of an endothelial anb3-integrin has been shown to correlate with tumor grade and thus plays a significant role in diagnosis of tumors. Some progress in tumor-targeted imaging by positron emission tomography (PET) or near-infrared fluorescence (NIRF) has recently been made with the aid of RGD complexes labeled with radioactive isotopes or fluorescent tags. Despite the usefulness of PET and NIRF, their applications are rather limited because of inherent problems such as light scattering, the invasive nature of data collection, photo-bleaching, and poor resolution. Molecular magnetic resonance imaging (MRI), however, can not only overcome these restrictions, but, with the assistance of a CA that catalytically shortens the relaxation time of the protons of nearby water molecules, can also provide excellent anatomy images. Thermodynamically stable, water-soluble, and highly paramagnetic Gd complexes, each bearing a multidentate ligand and at least one coordinated water molecule, have demonstrated high relaxivity and have therefore served as versatile MRI CAs. Among the early MRI CAs approved for clinical use are Dotarem and Omniscan . These Gd complexes incorporate the macrocycle 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (DOTA) as ligand and exhibit high thermodynamic stability. The fact that DOTA can employ at least one carboxylate to form a conjugate with a peptide such as RGD provides additional advantages for the preparation of targetspecific MRI CAs. In this regard, GdACHTUNGTRENNUNG(DOTA) conjugated with RGD should be an attractive candidate as a paramagnetic MRI CA for tumor-targeting. We now therefore wish to introduce the Gd ACHTUNGTRENNUNG(DOTA) conjugate of RGD, designed to monitor the activation of anb3-integrin in tumor tissue. The synthesis initially involved conjugation of DOTA and the cyclic pentapeptide c ACHTUNGTRENNUNG(RGDYK) as described by others. The DOTA-RGD conjugate thus prepared was purified and isolated by preparative HPLC. The Gd-DOTA-RGD complex was prepared by treatment of the DOTA-RGD with GdCl3·6 H2O in water. The final product was isolated as a white solid after purification by preparative HPLC. MALDI-TOF-MS shows a peak corresponding to [M+H H2O] (m/z 1161.50; calculated MW for C43H67GdN13O16 = 1178.39). The proton relaxivities—R1 and R2—of Gd-DOTA-RGD are 7.4 0.20 mm 1 s 1 and 4.0 0.24 mm 1 s , respectively at 298 K and 64 MHz. Gd-DOTA-RGD exhibits higher longitudinal relaxivity than small-molecule MRI CAs (for the data see the Supporting Information), which may be explained in terms of slower molecular tumbling (tg) as a result of the increase in molecular weight achieved through conjugation with RGD. In [a] J.-A. Park, J.-J. Lee, Prof. Y. Chang Department of Medical and Biological Engineering Kyungpook National University Daegu 702-701 (Korea) [b] Prof. J.-C. Jung Department of Biology, Kyungpook National University Daegu 702-701 (Korea) [c] Dr. D.-Y. Yu Korea Research Institute of Bioscience and Biotechnology Daejeon 305-806 (Korea) [d] Prof. C. Oh Department of Dermatology, College of Medicine, Korea University Seoul 152-703 (Korea) [e] Prof. C. Oh, S. Ha Research Institute for Skin Image College of Medicine, Korea University Seoul 152-703 (Korea) [f] Prof. T.-J. Kim Department of Applied Chemistry, Kyungpook National University Daegu 702-701 (Korea) Fax: (+ 82) 53-950-6594 E-mail : tjkim@knu.ac.kr [g] Prof. Y. Chang Department of Diagnostic Radiology and Molecular Medicine Kyungpook National University Daegu 702-701 (Korea) Fax: (+ 82) 53-422-2677 E-mail : ychang@knu.ac.kr Supporting information for this article is available on the WWW under http ://www.chembiochem.org or from the author.

Gold nanoparticles coated with gadolinium-DTPA-bisamide conjugate of penicillamine (Au@GdL) as a T1-weighted blood pool contrast agent

The work is directed toward the synthesis and characterization of gold nanoparticles coated by Gd-chelate (Au@GdL), where L is a conjugate of diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) and penicillamine. Au@GdL is obtained by the replacement of citrate from Au@citrate with gadolinium chelate (GdL). The average size of Au@GdL is 12 nm with the loading of GdL reaching up to 3.0 × 103 per particle, and they exhibit very high r1 relaxivity (∼107 mM−1 s−1) as well as X-ray attenuation. The r1 relaxivity per [Gd] is 20.1 mM−1 s−1. The small size of the present system also exhibits a USPIO-like behavior, such as contrast enhancement in the liver, the blood pool and the lymph node, as demonstrated by histological and TEM images, as well as CT and MR, rendering itself as a new class of T1 multimodal CT/MR contrast agent with potential application to the blood pool and the lymph node.

Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF‐β isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF‐β1, ‐2, and ‐3 were weakly detected in Bowmans layer (BL). In healing corneas, TGF‐β1 was primarily deposited in the fibrin clot and the unwounded BL. TGF‐β2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF‐β3 was mainly detected in the unwound region of basal epithelial cells. α‐Smooth muscle actin (α‐SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, α‐SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF‐β2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemets membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF‐β2 were released into the posterior region of healing stroma on day 14. High levels of α‐SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF‐β2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF‐β2‐mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo. J. Cell. Biochem. 108: 476–488, 2009.

Roles of MMP/TIMP in regulating matrix swelling and cell migration during chick corneal development†

Tissue remodeling is central to embryonic development. Here, we used immunohistochemistry, Western blotting, and RT‐PCR analysis to investigate the roles of matrix metalloproteinases (MMPs) and the related “a disintegrin and metalloproteinase“ (ADAM) family proteinases in chick corneal development. While MMP‐13 was expressed in developing chick corneas from embryonic day (ED) 5 to ED 10, its inhibitor, tissue inhibitors of metalloproteinase‐1 (TIMP‐1), was expressed from ED 18 to 2 days post‐hatching (P2). Early MMP‐13 activity may be associated with degradation of type IX collagen from the primary stroma, which loosens the collagen fibrils and facilitates neural crest (NC) cell migration. The membrane‐bound and secreted forms of ADAM10 were both detected throughout corneal development, and active ADAM10 formed a cleavage complex with CD44v6, a CD44 splice variant that is a major cell surface adhesion molecule for hyaluronic acid (HA) and has been implicated in cell migration. Both CD44v6 and its ectodomain cleavage products were detected from ED 5 to ED 14, and a broad‐spectrum MMP inhibitor blocked ectodomain cleavage in cultured stromal cells. These findings suggest that ADAM10 mediates CD44v6 cleavage in the developing cornea, facilitating NC cell‐derived mesenchymal cell migration. Finally, we identified high levels of active membrane‐type 3‐MMP (MT3‐MMP) in developing corneas at ED 7, ED 14, and ED 18. MT3‐MMP takes part in MMP‐2 activation and possibly also CD44v6 shedding, suggesting that this pathway may be involved in cell migration. These findings collectively show for the first time that multiple MMPs, ADAMs, and TIMPs appear to functionally interact during corneal development. J. Cell. Biochem. 101:1222–1237, 2007.

Gadolinium complex of DO3A-benzothiazole aniline (BTA) conjugate as a theranostic agent.

A gadolinium complex of 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DO3A) and benzothiazole-aniline (BTA) of the type [Gd(DO3A-BTA)(H2O)] has been prepared for use as a single molecule theranostic agent. The kinetic inertness and r1 relaxivity (= 3.84 mM(-1) s(-1)) of the complex compare well with those of structurally analogous Gd-DOTA. The same complex is not only tumor-specific but also intracellular, enhancing MR images of cytosols and nuclei of tumor cells such as MCF-7, MDA-MB-231, and SK-HEP-1. Both DO3A-BTA and Gd(DO3A-BTA) reveal antiproliferative activities as demonstrated by GI50 and TGI values obtainable from the cell counting kit-8 (CCK-8) assays performed on these cell lines. Ex vivo and in vivo monitoring of tumor sizes provide parallel and supportive observations for such antiproliferative activities.

ADAM10 mediates N-cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina.

Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro‐form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10‐specific siRNA, the level of mature ADAM10 decreased. Phase‐contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X‐treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X‐treated cultures. N‐cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N‐cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N‐cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation. J. Cell. Biochem. 114: 942–954, 2013.

Differential expression of transforming growth factor-β in the interstitial tissue of testis during aging

Transforming growth factor‐βs (TGF‐βs) have significant effects on testis development. The pattern of TGF‐β expression in aging testis has not been established to date. We examined age‐related changes in the expression of TGF‐β and its receptors in the testis using Western blot analysis. TGF‐β1 expression increased continuously in aging rat testis, whereas no age‐associated changes were observed for TGF‐β3. Strong expression of TGF‐β2, as well as type I and II receptors was observed in 12‐month‐old testis, but following this time, expression decreased dramatically. Interestingly, TGF‐β2 and ‐β3 displayed strong and similar expression patterns in liver, regardless of age, suggesting that the down‐regulation of TGF‐β2 is testis‐specific. We observed significant induction of p53 and p21WAF1 in 18‐month‐old testis that appeared to correspond with aging. Moreover, caloric restriction (CR) prevented age‐related decrease in TGF‐β2 expression. Using immunohistochemistry, we showed that all TGF‐β1, ‐β2, and ‐β3 proteins are expressed primarily in interstitial cells, which are located in the space between adjoining seminiferous tubules. Our data collectively indicate that aging in the testis is regulated by differential expression of TGF‐β proteins, and decreased levels of TGF‐β2 contribute to the aging process.

In Vivo Effects of Preservative-free and Preserved Prostaglandin Analogs: Mouse Ocular Surface Study

Purpose Chronic use of topical hypotensive agents induces several side effects caused by preservatives. The purpose of this study was to evaluate the effects of prostaglandin analogs with varying concentrations of benzalkonium chloride (BAC), preservative-free (PF), and alternative preservatives on mouse corneal tissue. Methods Thirty-five, 8- to 10-week-old female C57BL/6 mice (five mice for each group) were used for this study. To the control group, we applied normal saline, and to each drug-treated group we applied 0.02% BAC, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), travoprost 0.004% (with 0.001% polyquad) or tafluprost 0.0015% with/without 0.001% BAC, once a day (9 p.m.) for 4 weeks. Corneal fluorescein staining was evaluated in all groups. After harvest, the corneal tissues were embedded in paraffin and then Hematoxylin-Eosin stain was performed for histopathological examination. Immunofluorescence staining was done against TNF-α, IL-6, HLA DR, pJNK, and pAkt. Results In corneal fluorescein staining, severe punctate epithelial keratitis was seen in the groups of 0.02% BAC, 0.02% BAC containing bimatoprost 0.01% and latanoprost 0.005%. The surface desquamation, irregular surface, loss of cell borders, anisocytosis and stromal shrinkage were observed in the groups of BAC-containing eye drops. Moreover, the groups treated with BAC-containing eye drops have high inflammatory markers, significantly decreased cell viability-related signal, pAkt, and higher apoptosis-inducing signal, pJNK, than the control group. On the other hand, travoprost 0.004% and PF tafluprost 0.0015% have less cellular morphologic changes, lower inflammation, and higher cellular viability than BAC-containing formulations. Conclusions Corneal damage, increased inflammation and apoptosis and low cell viability were observed in BAC-containing groups. PF or alternatively preserved glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC.

Constitutive collagenase-1 synthesis through MAPK pathways is mediated, in part, by endogenous IL-1α during fibrotic repair in corneal stroma

Collagenase‐1 is a protease expressed by active fibroblasts that is involved in remodeling of the extracellular matrix (ECM). In this study, we characterize the intracellular signaling mechanism of collagenase‐1 production by IL‐1α in subcultured normal fibroblasts (NF) from uninjured normal corneas, compared to that in repair wound fibroblasts (WF). In NF, collagenase‐1 was induced specifically after the exogenous addition of IL‐1α via activation of ERK and p38MAPK. Collagenase‐1 expression was strongly suppressed upon treatment with either a MEK or p38MAPK inhibitor. In contrast, repair WF constitutively synthesized both IL‐1α and collagenase‐1. Combined treatment with both mitogen‐activated protein kinase (MAPK) inhibitors dramatically reduced collagenase‐1 synthesis, while individual MEK1 or p38 inhibitors weakly modulated the collagenase‐1 level. The results indicate that both pathways are crucial in the regulation of collagenase‐1 synthesis. Furthermore, an IL‐1α receptor antagonist (IL‐1ra) could not abolish constitutive collagenase‐1 synthesis, even at high doses, suggesting that other cytokines/factors are additionally involved in this process. We propose that induction of collagenase‐1 by IL‐1α in both WF and NF depends on a unique combination of cell type‐specific signaling pathways. J. Cell. Biochem. 102: 453–462, 2007.