Publications by authors named "Min Hyung Kang"

32 Publications

Instant, multiscale dry transfer printing by atomic diffusion control at heterogeneous interfaces.

Sci Adv 2021 Jul 9;7(28). Epub 2021 Jul 9.

Department of Robotics Engineering, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, South Korea.

Transfer printing is a technique that integrates heterogeneous materials by readily retrieving functional elements from a grown substrate and subsequently printing them onto a specific target site. These strategies are broadly exploited to construct heterogeneously integrated electronic devices. A typical wet transfer printing method exhibits limitations related to unwanted displacement and shape distortion of the device due to uncontrollable fluid movement and slow chemical diffusion. In this study, a dry transfer printing technique that allows reliable and instant release of devices by exploiting the thermal expansion mismatch between adjacent materials is demonstrated, and computational studies are conducted to investigate the fundamental mechanisms of the dry transfer printing process. Extensive exemplary demonstrations of multiscale, sequential wet-dry, circuit-level, and biological topography-based transfer printing demonstrate the potential of this technique for many other emerging applications in modern electronics that have not been achieved through conventional wet transfer printing over the past few decades.
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http://dx.doi.org/10.1126/sciadv.abh0040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8270493PMC
July 2021

Outdoor-Useable, Wireless/Battery-Free Patch-Type Tissue Oximeter with Radiative Cooling.

Adv Sci (Weinh) 2021 05 9;8(10):2004885. Epub 2021 Mar 9.

School of Electrical Engineering and Computer Science (EECS) Gwangju Institute of Science and Technology (GIST) 123, Cheomdangwagi-ro, Bukgu Gwangju 61005 Republic of Korea.

For wearable electronics/optoelectronics, thermal management should be provided for accurate signal acquisition as well as thermal comfort. However, outdoor solar energy gain has restricted the efficiency of some wearable devices like oximeters. Herein, wireless/battery-free and thermally regulated patch-type tissue oximeter (PTO) with radiative cooling structures are presented, which can measure tissue oxygenation under sunlight in reliable manner and will benefit athlete training. To maximize the radiative cooling performance, a nano/microvoids polymer (NMVP) is introduced by combining two perforated polymers to both reduce sunlight absorption and maximize thermal radiation. The optimized NMVP exhibits sub-ambient cooling of 6 °C in daytime under various conditions such as scattered/overcast clouds, high humidity, and clear weather. The NMVP-integrated PTO enables maintaining temperature within ≈1 °C on the skin under sunlight relative to indoor measurement, whereas the normally used, black encapsulated PTO shows over 40 °C owing to solar absorption. The heated PTO exhibits an inaccurate tissue oxygen saturation (StO) value of ≈67% compared with StO in a normal state (i.e., ≈80%). However, the thermally protected PTO presents reliable StO of ≈80%. This successful demonstration provides a feasible strategy of thermal management in wearable devices for outdoor applications.
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http://dx.doi.org/10.1002/advs.202004885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132059PMC
May 2021

NFC-Based Wearable Optoelectronics Working with Smartphone Application for Untact Healthcare.

Sensors (Basel) 2021 Jan 28;21(3). Epub 2021 Jan 28.

School of Electrical Engineering and Computer Science, Gwangju Institute of Science and Technology, 123 Cheomdangwagi-ro, Buk-gu, Gwangju 61005, Korea.

With growing interest in healthcare, wearable healthcare devices have been developed and researched. In particular, near-field communication (NFC) based wearable devices have been actively studied for device miniaturization. Herein, this article proposes a low-cost and convenient healthcare system, which can monitor heart rate and temperature using a wireless/battery-free sensor and the customized smartphone application. The authors designed and fabricated a customized healthcare device based on the NFC system, and developed a smartphone application for real-time data acquisition and processing. In order to achieve compact size without performance degradation, a dual-layered layout is applied to the device. The authors demonstrate that the device can operate as attached on various body sites such as wrist, fingertip, temple, and neck due to outstanding flexibility of device and adhesive strength between the device and the skin. In addition, the data processing flow and processing result are presented for offering heart rate and skin temperature. Therefore, this work provides an affordable and practical pathway for the popularization of wireless wearable healthcare system. Moreover, the proposed platform can easily delivery the measured health information to experts for contactless/personal health consultation.
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http://dx.doi.org/10.3390/s21030878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865650PMC
January 2021

Secreted protein acidic and rich in cysteine (SPARC) knockout mice have greater outflow facility.

PLoS One 2020 4;15(11):e0241294. Epub 2020 Nov 4.

Department of Ophthalmology & Visual Sciences, University Hospitals Eye Institute, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America.

Purpose: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates intraocular pressure (IOP) by altering extracellular matrix (ECM) homeostasis within the trabecular meshwork (TM). We hypothesized that the lower IOP previously observed in SPARC -/- mice is due to a greater outflow facility.

Methods: Mouse outflow facility (Clive) was determined by multiple flow rate infusion, and episcleral venous pressure (Pe) was estimated by manometry. The animals were then euthanized, eliminating aqueous formation rate (Fin) and Pe. The C value was determined again (Cdead) while Fin was reduced to zero. Additional mice were euthanized for immunohistochemistry to analyze ECM components of the TM.

Results: The Clive and Cdead of SPARC -/- mice were 0.014 ± 0.002 μL/min/mmHg and 0.015 ± 0.002 μL/min/mmHg, respectively (p = 0.376, N/S). Compared to the Clive = 0.010 ± 0.002 μL/min/mmHg and Cdead = 0.011 ± 0.002 μL/min/mmHg in the WT mice (p = 0.548, N/S), the Clive and Cdead values for the SPARC -/- mice were higher. Pe values were estimated to be 8.0 ± 0.2 mmHg and 8.3 ± 0.7 mmHg in SPARC -/- and WT mice, respectively (p = 0.304, N/S). Uveoscleral outflow (Fu) was 0.019 ± 0.007 μL/min and 0.022 ± 0.006 μL/min for SPARC -/- and WT mice, respectively (p = 0.561, N/S). Fin was 0.114 ± 0.002 μL/min and 0.120 ± 0.016 μL/min for SPARC -/- and WT mice (p = 0.591, N/S). Immunohistochemistry demonstrated decreases of collagen types IV and VI, fibronectin, laminin, PAI-1, and tenascin-C within the TM of SPARC -/- mice (p < 0.05).

Conclusions: The lower IOP of SPARC -/- mice is due to greater aqueous humor outflow facility through the conventional pathway. Corresponding changes in several matricellular proteins and ECM structural components were noted in the TM of SPARC -/- mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241294PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641442PMC
December 2020

Formulation and in vitro/in vivo evaluation of chitosan-based film forming gel containing ketoprofen.

Drug Deliv 2017 Nov;24(1):1056-1066

a Department of Pharmacy , Chungbuk National University , Cheongju , Republic of Korea.

The film forming gel, adhered to skin surfaces upon application and formed a film, has an advantage onto skin to provide protection and continuous drug release to the application site. This study aimed to prepare a chitosan-based film forming gel containing ketoprofen (CbFG) and to evaluate the CbFG and film from CbFG (CbFG-film). CbFG were prepared with chitosan, lactic acid and various skin permeation enhancers. The physicochemical characteristics were evaluated by texture analysis, viscometry, SEM, DSC, XRD and FT-IR. To identify the mechanism of skin permeation, in vitro skin permeation study was conducted with a Franz diffusion cell and excised SD-rat and hairless mouse dorsal skin. In vivo efficacy assessment in mono-iodoacetate (MIA)-induced rheumatoid arthritis animal model was also conducted. CbFG was successfully prepared and, after applying CbFG to the excised rat dorsal skin, the CbFG-film was also formed well. The physicochemical characteristics of CbFG and CbFG-film could be explained by the grafting of oleic acid onto chitosan in the absence of catalysts. In addition, CbFG containing oleic acid had a higher skin permeation rate in comparison with any other candidate enhancers. The in vivo efficacy study also confirmed significant anti-inflammatory and analgesic effects. Consequently, we report the successful preparation of chitosan-based film forming gel containing ketoprofen with excellent mechanical properties, skin permeation and anti-inflammatory and analgesic effects.
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http://dx.doi.org/10.1080/10717544.2017.1346001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8241006PMC
November 2017

Docetaxel-loaded RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes: Drug release, cytotoxicity, and antitumor efficacy.

Int J Pharm 2017 May 21;523(1):229-237. Epub 2017 Mar 21.

College of Pharmacy, Chung-Ang University, 221 Heuksuk-dong, Dongjak-gu, Seoul 156-756, South Korea. Electronic address:

We previously synthesized the RIPL peptide (IPLVVPLRRRRRRRRC) to facilitate selective delivery into hepsin-expressing cancer cells and showed that RIPL peptide-conjugated liposomes (RIPL-L) enhanced the intracellular delivery of fluorescent probes in vitro. In this study, docetaxel-loaded RIPL-L (DTX-RIPL-L) were prepared and evaluated for in vitro drug release, cytotoxicity, and in vivo antitumor efficacy. DTX was successfully encapsulated by pre-loading, with an average encapsulation efficiency and drug loading capacity of 32.4% and 21.39±2.05 (μg/mg), respectively. A DTX release study using dialysis showed a biphasic release pattern, i.e., rapid release for 6h, followed by sustained release up to 72h. The first-order equation provided the best fit for drug release (r=0.9349). In vitro cytotoxicity was dose-dependent, resulting in IC values of 36.10 (SK-OV-3) and 48.62ng/mL (MCF-7) for hepsin-positive, and 61.12 (DU145) and 53.04ng/mL (PC-3) for hepsin-negative cell lines. Live/dead cell imaging was carried out to visualize the proportion of viable and nonviable SK-OV-3 cells. Compared to DTX solution, DTX-RIPL-L significantly inhibited tumor growth and prolonged survival time in BALB/c nude mice with SK-OV-3 cell tumors. We suggest that DTX-RIPL-L is a good candidate for efficient drug targeting to hepsin-expressing cancer cells.
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http://dx.doi.org/10.1016/j.ijpharm.2017.03.045DOI Listing
May 2017

Surface-Modification of RIPL Peptide-Conjugated Liposomes to Achieve Steric Stabilization and pH Sensitivity.

J Nanosci Nanotechnol 2017 Feb;17(2):1008-17

We have previously demonstrated that RIPL peptide-conjugated liposomes (RIPL-L) exhibited high hepsin (HPN) selectivity and enhanced intracellular drug delivery. In this study, surface modification of RIPL-L was performed to reduce plasma protein adsorption and off-target effects. For steric stabilization, distearoyl phosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)2000 was used (5% molar ratio to total lipid) to prepare PEG-RIPL-L. Further, pH-sensitive oligopeptides [(HD)4 or (HE)4] were coupled to shield the RIPL polyarginine moiety, yielding (HD)4/PEG-RIPL-L and (HE)4/PEG-RIPL-L. All liposomal vesicles had a narrow and homogenous size distribution of approximately 140–150 nm, with zeta potentials varying from −15 to 36 mV. Increased plasma stability was observed upon quantifying the protein adsorbed onto liposomes by using a micro bicinchoninic acid assay. The (HD)4- and (HE)4-coupling capacity of PEG-RIPL-L was investigated by measuring the amount of oligopeptide involved in transient ionic complexation (TIC-oligopep) and zeta potential changes. As the molar ratio of (HD)4 and (HE)4 increased, TIC-oligopep increased and zeta potential decreased. (HE)4/PEG-RIPL-L were pH-sensitive, producing 1.6-fold greater cellular uptake of FITC-dextran by LNCaP cells at pH 6.8 than at pH 7.4. This result suggested that (HE)4/PEG-RIPL-L might provide a sterically stabilized, pH-sensitive drug carrier for HPN-specific cancer targeting.
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http://dx.doi.org/10.1166/jnn.2017.12670DOI Listing
February 2017

Development of ciclopirox nail lacquer with enhanced permeation and retention.

Arch Pharm Res 2016 Jul 15;39(7):953-9. Epub 2016 Jun 15.

College of Pharmacy, Yeungnam University, 214-1 Dae-dong, Gyeongsan, 712-749, South Korea.

Onychomycosis is a prevailing disease caused by fungal infection of nails that mostly affects athletes and the elderly. Ciclopirox is approved by the US Food and Drug Administration for the topical treatment of onychomycosis. However, the desired penetration of ciclopirox into the nail bed has not been achieved via topical application for efficient treatment. Therefore, the main aim of this study was to enhance ciclopirox permeation and retention in nail by the development of a new nail lacquer formulation. We screened the effects of different solvents, alkalizing agents, and permeation enhancers on the permeation of bovine hooves by ciclopirox and its retention in human nail clippings. The results suggest that isopropyl alcohol, potassium hydroxide, and urea as the solvent, alkalizing agent, and permeation enhancer, respectively, improved the permeation of the ciclopirox nail lacquer formulation the most with high flux rates. Comparison of the final formulation and marketed product revealed enhanced retention of ciclopirox from our developed formulation in human nail clippings. Therefore, our newly developed nail lacquer may be a potentially effective formulation for the treatment of onychomycosis in humans.
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http://dx.doi.org/10.1007/s12272-016-0774-0DOI Listing
July 2016

Design of Multifunctional Liposomal Nanocarriers for Folate Receptor-Specific Intracellular Drug Delivery.

Mol Pharm 2015 Dec 6;12(12):4200-13. Epub 2015 Nov 6.

College of Pharmacy, Chung-Ang University , 221 Heuksuk-dong, Dongjak-gu, Seoul 156-756, Korea.

As a novel carrier for folate receptor (FR)-targeted intracellular delivery, we designed two types of targetable liposomal systems using Pep-1 peptide (Pep1) and folic acid as a cell-penetrating peptide (CPP) and target molecule, respectively. Folate-linked Pep1 (Fol-Pep1) was synthesized by solid phase peptide synthesis (SPPS) and verified using (1)H NMR and far-ultraviolet (UV) circular dichroism (CD). The chimeric ligand (Fol-Pep1)-modified liposome (cF-P-L) was prepared by coupling Fol-Pep1 to maleimide-derivatized liposomes at various ratios. The dual ligand (folate and Pep1)-modified liposome (dF/P-L) was prepared by separately attaching both ligands to the liposomal surface via a short (PEG2000) or long (PEG3400) linker. The physical and conformational characteristics including vesicle size, zeta potential, and the number of conjugated ligands were determined. Intracellular uptake specificities of various fluorescent probe-containing cF-P-L and dF/P-L systems were assessed using FR-positive HeLa and FR-negative HaCaT cells. Cellular uptake behavior was visualized by confocal laser scanning microscopy (CLSM). Internalization was time-dependent. Fol-Pep1 and Pep-1 cytotoxicities were negligible up to 25 μM in FR-positive and FR-negative cells. Empty cF-P-L and dF/P-L were nontoxic at the concentration used. The optimized dF3/P2(450/90) system carrying 450 PEG3400-linked folate and 90 PEG2000-linked Pep1 molecules could be a good candidate for FR-specific intracellular drug delivery.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00399DOI Listing
December 2015

Development and optimization of a self-microemulsifying drug delivery system for atorvastatin calcium by using D-optimal mixture design.

Int J Nanomedicine 2015 5;10:3865-77. Epub 2015 Jun 5.

College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.

In this study, we developed and optimized a self-microemulsifying drug delivery system (SMEDDS) formulation for improving the dissolution and oral absorption of atorvastatin calcium (ATV), a poorly water-soluble drug. Solubility and emulsification tests were performed to select a suitable combination of oil, surfactant, and cosurfactant. A D-optimal mixture design was used to optimize the concentration of components used in the SMEDDS formulation for achieving excellent physicochemical characteristics, such as small droplet size and high dissolution. The optimized ATV-loaded SMEDDS formulation containing 7.16% Capmul MCM (oil), 48.25% Tween 20 (surfactant), and 44.59% Tetraglycol (cosurfactant) significantly enhanced the dissolution rate of ATV in different types of medium, including simulated intestinal fluid, simulated gastric fluid, and distilled water, compared with ATV suspension. Good agreement was observed between predicted and experimental values for mean droplet size and percentage of the drug released in 15 minutes. Further, pharmacokinetic studies in rats showed that the optimized SMEDDS formulation considerably enhanced the oral absorption of ATV, with 3.4-fold and 4.3-fold increases in the area under the concentration-time curve and time taken to reach peak plasma concentration, respectively, when compared with the ATV suspension. Thus, we successfully developed an optimized ATV-loaded SMEDDS formulation by using the D-optimal mixture design, that could potentially be used for improving the oral absorption of poorly water-soluble drugs.
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http://dx.doi.org/10.2147/IJN.S83520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462857PMC
August 2016

Canonical wnt signaling regulates extracellular matrix expression in the trabecular meshwork.

Invest Ophthalmol Vis Sci 2014 Oct 28;55(11):7433-40. Epub 2014 Oct 28.

Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.

Purpose: Canonical Wnt signaling has emerged as a critical regulator of aqueous outflow facility and intraocular pressure (IOP). In this study, we examine the role of canonical Wnt signaling on extracellular matrix (ECM) expression in the trabecular meshwork (TM) and explore the molecular mechanisms involved.

Methods: β-catenin localization in human TM tissue was examined using immunofluorescent staining. Primary human TM cells were incubated with lithium chloride (LiCl) and the effect on active β-catenin expression was assessed by immunoblot. Adenovirus expressing a dominant-negative TCF4 mutant that lacks a β-catenin binding domain was used. Changes in the levels of the microRNA-29 (miR-29) family and ECM proteins were determined by real-time quantitative PCR and immunoblot analysis, respectively.

Results: β-catenin was expressed throughout the TM, with localization primarily to the plasma membrane. Incubation of TM cells with lithium chloride increased the expression of active β-catenin. Lithium chloride treatment upregulated miR-29b expression, and suppressed the levels of various ECM proteins under both basal and TGF-β2 stimulatory conditions. Infection of TM cells with a dominant-negative TCF4 mutant induced ECM levels without a significant change in the expression of the miR-29 family.

Conclusions: Collectively, our data identify the canonical Wnt signaling pathway as an important modulator of ECM expression in the TM and provide a mechanistic framework for its regulation of outflow facility and IOP.
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http://dx.doi.org/10.1167/iovs.13-12652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238318PMC
October 2014

Aqueous outflow: segmental and distal flow.

J Cataract Refract Surg 2014 Aug;40(8):1263-72

From Harvard-MIT Division of Health Sciences and Technology (Swaminathan), Harvard Medical School, Boston, Massachusetts, and the Department of Ophthalmology & Visual Sciences (Oh, Kang, Rhee), Case Western Reserve University, Cleveland, Ohio, USA. Electronic address:

Unlabelled: The elevated intraocular pressure (IOP) of primary open-angle glaucoma is caused by impaired outflow of aqueous humor through the trabecular meshwork. Within the juxtacanalicular region, alterations of both extracellular matrix homeostasis and the cellular tone of trabecular meshwork endothelial and the inner wall of Schlemm canal cells affect outflow. Newer pharmacologic agents that target trabecular meshwork and Schlemm canal cell cytoskeleton lower IOP. Aqueous drainage occurs nonhomogenously with greater flow going through certain portions of the TM and less going through other portions-a concept known as segmental flow, which is theoretically the result of outflow being dependent on the presence of discrete pores within Schlemm canal. The limited long-term success of trabecular meshwork bypass surgeries implicates the potential impact of resistance in Schlemm canal itself and collector channels. Additionally, others have observed that outflow occurs preferentially near collector channels. These distal structures may be more important to aqueous outflow than previously believed.

Financial Disclosure: Dr. Rhee is a consultant to Aerie Pharmaceuticals, Alcon Laboratories, Inc., Allegan, Inc., Aquesys, Inc., Glaukos Corp., Ivantis, Inc., Johnson & Johnson, Merck Sharp & Dohme Corp. and Santen, Inc., and has received research funding from Alcon Laboratories, Inc., Merck Sharp & Dohme Corp., and Ivantis, Inc. No other author has a financial or proprietary interest in any material or method mentioned.
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http://dx.doi.org/10.1016/j.jcrs.2014.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151118PMC
August 2014

TGF-β2-mediated ocular hypertension is attenuated in SPARC-null mice.

Invest Ophthalmol Vis Sci 2014 Jun 6;55(7):4084-97. Epub 2014 Jun 6.

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.

Purpose: Transforming growth factor-β2 (TGF-β2) has been implicated in the pathogenesis of primary open-angle glaucoma through extracellular matrix (ECM) alteration among various mechanisms. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates ECM within the trabecular meshwork (TM), and is highly upregulated by TGF-β2. We hypothesized that, in vivo, SPARC is a critical regulatory node in TGF-β2-mediated ocular hypertension.

Methods: Empty (Ad.empty) or TGF-β2-containing adenovirus (Ad.TGF-β2) was injected intravitreally into C57BL6-SV129 WT and SPARC-null mice. An initial study was performed to identify a stable period for IOP measurement under isoflurane. The IOP was measured before injection and every other day for two weeks using rebound tonometry. Additional mice were euthanized at peak IOP for immunohistochemistry.

Results: The IOP was stable under isoflurane during minutes 5 to 8. The IOP was significantly elevated in Ad.TGF-β2-injected (n = 8) versus Ad.empty-injected WT (n = 8) mice and contralateral uninjected eyes during days 4 to 11 (P < 0.03). The IOPs were not significantly elevated in Ad.TGF-β2-injected versus Ad.empty-injected SPARC-null mice. However, on day 8, the IOP of Ad.TGF-β2-injected SPARC-null eyes was elevated compared to that of contralateral uninjected eyes (P = 0.0385). Immunohistochemistry demonstrated that TGF-β2 stimulated increases in collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC in WT mice, but only PAI-1 and CTGF in SPARC-null mice (P < 0.05).

Conclusions: SPARC is essential to the regulation of TGF-β2-mediated ocular hypertension. Deletion of SPARC significantly attenuates the effects of TGF-β2 by restricting collagen IV and fibronectin expression. These data provide further evidence that SPARC may have an important role in IOP regulation and possibly glaucoma pathogenesis.
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http://dx.doi.org/10.1167/iovs.13-12463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4078948PMC
June 2014

AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork.

Invest Ophthalmol Vis Sci 2014 Apr 8;55(5):3127-39. Epub 2014 Apr 8.

Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.

Purpose: In this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK) affects extracellular matrix (ECM) and cellular tone in the trabecular meshwork (TM), and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice.

Methods: Human TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR), under basal or TGF-β2 stimulatory conditions. Conditioned media (CM) was probed for secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1 (TSP-1), and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα) and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT), and aqueous clearance were measured in AMPKα2-null and wild-type (WT) mice.

Results: Both AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-β2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188). Transforming growth factor-β2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology.

Conclusions: Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.
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http://dx.doi.org/10.1167/iovs.13-12755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4023495PMC
April 2014

RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delivery to hepsin-expressing cancer cells.

Eur J Pharm Biopharm 2014 Aug 2;87(3):489-99. Epub 2014 Apr 2.

College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea. Electronic address:

Background: To facilitate selective drug delivery to hepsin (Hpn)-expressing cancer cells, the RIPL peptide (IPLVVPLRRRRRRRRC; 16mer; 2.1 kDa) was synthesized as a novel cell penetrating/homing peptide (CPHP) and conjugated to a liposomal carrier.

Methods: RIPL peptide-conjugated liposomes (RIPL-Lipo) were prepared by conjugating RIPL peptides to maleimide-derivatized liposomal vesicles via the thiol-maleimide reaction. Vesicle size and zeta potential were examined using a Zetasizer. Intracellular uptake specificity of the RIPL peptide, or RIPL-Lipo, was assessed by measuring mean fluorescence intensity (MFI) after treatment with a fluorescent marker in various cell lines: SK-OV-3, MCF-7, and LNCaP for Hpn(+); DU145, PC3, and HaCaT for Hpn(-). FITC-dextran was used as a model compound. Selective translocational behavior of RIPL-Lipo to LNCaP cells was visualized by fluorescence microscopy and confocal laser scanning microscopy. Cytotoxicities of the RIPL peptide and RIPL-Lipo were evaluated by WST-1 assay.

Results: RIPL peptides exhibited significant Hpn-selectivity. RIPL-Lipo systems were of positively charged nanodispersion (165 nm in average; 6-24 mV depending on RIPL conjugation ratio). RIPL-Lipo with the conjugation of 2300 peptide molecules revealed the greatest MFI in all cell lines tested. Cellular uptake of RIPL-Lipo increased by 20- to 70-fold in Hpn(+) cells, and 5- to 7-fold in Hpn(-) cells, compared to the uptake of FITC-dextran. Cytosolic internalization of RIPL-Lipo was time-dependent: bound instantly; internalized within 30 min; distributed throughout the cytoplasm after 1 h. Cytotoxicities of RIPL peptide (up to 50 μM) and RIPL-Lipo (up to 10%) were minor (cell viability >90%) in LNCaP and HaCaT cells.

Conclusion: By employing a novel CPHP, the RIPL-Lipo system was successfully developed for Hpn-specific drug delivery.
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http://dx.doi.org/10.1016/j.ejpb.2014.03.016DOI Listing
August 2014

The effects of tenascin C knockdown on trabecular meshwork outflow resistance.

Invest Ophthalmol Vis Sci 2013 Aug 19;54(8):5613-23. Epub 2013 Aug 19.

Casey Eye Institute, Oregon Health & Science University, Portland, Oregon 97239, USA.

Purpose: Tenascin C (TNC) is a matricellular glycoprotein whose expression in adult tissue is indicative of tissue remodeling. The purpose of the current study was to determine the localization of TNC in trabecular meshwork (TM) tissue and to analyze the effects of TNC on intraocular pressure (IOP).

Methods: Human TM frontal sections were immunostained with anti-TNC and imaged by confocal microscopy. TNC mRNA and protein levels were quantitated in anterior segments perfused at physiological and elevated pressure. Short, hairpin RNA (shRNA) silencing lentivirus targeting full-length TNC (shTNC) was applied to anterior segment perfusion organ cultures. The IOPs and central corneal thickness (CCT) of wild-type, TNC(-/-), and tenascin X (TNX(-/-)) knockout mice were measured.

Results: TNC was distributed in the juxtacanalicular (JCT) region of adult human TM, predominantly in the basement membrane underlying the inner wall of Schlemm's canal. Application of shTNC lentivirus to human and porcine anterior segments in perfusion culture did not significantly affect outflow rate. Although TNC was upregulated in response to pressure, there was no difference in outflow rate when shTNC-silenced anterior segments were subjected to elevated pressure. Furthermore, IOPs and CCTs were not significantly different between TNC(-/-) or TNX(-/-) and wild-type mice.

Conclusions: TNC does not appear to contribute directly to outflow resistance. However, TNC immunolocalization in the JCT of adult human eyes suggests that certain areas of the TM are being continuously remodeled with or without an IOP increase.
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http://dx.doi.org/10.1167/iovs.13-11620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3747717PMC
August 2013

Central corneal thickness does not correlate with TonoLab-measured IOP in several mouse strains with single transgenic mutations of matricellular proteins.

Exp Eye Res 2013 Oct 24;115:106-12. Epub 2013 Jun 24.

Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA.

Accurate and reliable measurement of intraocular pressure (IOP) is crucial in the study of glaucoma using the mouse model. The purpose of this study was to determine the relationship between TonoLab-measured IOP and central corneal thickness (CCT) in mouse strains with single gene mutations of matricellular proteins. Wild-type (WT) and transgenic mouse strains with single gene mutations (KO) of thrombospondin-1 (TSP-1), thrombospondin-2 (TSP-2), osteopontin (OPN), hevin, and secreted protein acidic rich in cysteine (SPARC) were imaged at six weeks using optical coherence tomography (Stratus, Zeiss) to determine CCT. IOP was measured between 11am and 3pm using TonoLab, one week later. For all measurements, mice were anesthetized using intraperitoneal injection ketamine:xylazine. CCT and IOP were measured in 583 mice (TSP-1 n = 71 and 41, TSP-2 n = 60 and 32, OPN n = 81 and 50, hevin n = 59 and 76, SPARC n = 54 and 59, WT and KO, respectively). Mean CCT was 5-6% lower in three KO strains-TSP-1, OPN, and SPARC-compared to their corresponding WT (p = 1.55 × 10(-7), 1.63 × 10(-11), and 1.91 × 10(-7), respectively). The mean IOP was 8.3%, 6.6%, and 15.1% lower in three KO strains-TSP-1, TSP-2, and SPARC-compared to corresponding WT (p = 2.11 × 10(-5), 2.93 × 10(-3), and 3.76 × 10(-9), respectively. Linear regression of IOP versus CCT yielded no statistically significant within-strain correlations for TSP-1 (p = 0.12 and 0.073), TSP-2 (p = 0.473 and 0.92), OPN (p = 0.212 and 0.916), Hevin (p = 0.746 and 0.257), and SPARC (p = 0.080 and 0.056), reported as p-values considering a null hypothesis of zero slope (WT and KO, respectively). Neither C57-derived strains (TSP-1 and OPN) nor 129-derived strains (TSP-2, hevin, SPARC) demonstrated a correlation between mean IOP and mean CCT across different strains (p = 0.75 and p = 0.53, respectively). Taken together, these results indicate that CCT is not required to interpret TonoLab IOP readings in the mice when CCT varies 10% about the mean. This does not exclude the possibility of an IOP-CCT correlation for CCT values outside this range or for inter-strain comparisons where the mean CCT differs more than 10%.
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http://dx.doi.org/10.1016/j.exer.2013.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795815PMC
October 2013

Overexpression of SPARC in human trabecular meshwork increases intraocular pressure and alters extracellular matrix.

Invest Ophthalmol Vis Sci 2013 May 7;54(5):3309-19. Epub 2013 May 7.

Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, MA, USA.

Purpose: Intraocular pressure (IOP) regulation is largely unknown. SPARC-null mice demonstrate a lower IOP resulting from increased outflow. SPARC is a matricellular protein often associated with fibrosis. We hypothesized that SPARC overexpression would alter IOP by affecting extracellular matrix (ECM) synthesis and/or turnover in the trabecular meshwork (TM).

Methods: An adenoviral vector containing human SPARC was used to increase SPARC expression in human TM endothelial cells and perfused human anterior segments using multiplicities of infection (MOIs) 25 or 50. Total RNA from TM was used for quantitative PCR, while protein from cell lysates and conditioned media were used for immunoblot analyses and zymography. After completion of perfusion, the anterior segments were fixed, sectioned, and examined by light and confocal microscopy.

Results: SPARC overexpression increased the IOP of perfused human anterior segments. Fibronectin and collagens IV and I protein levels were elevated in both TM cell cultures and within the juxtacanalicular (JCT) region of perfused anterior segments. Collagen VI and laminin protein levels were increased in TM cell cultures but not in perfused anterior segments. The protein levels of pro-MMP-9 decreased while the kinetic inhibitors of metalloproteinases, TIMP-1 and PAI-1 protein levels, increased at MOI 25. At MOI 50, the protein levels of pro-MMP-1, -3, and -9 also decreased while PAI-1 and TIMP-1 and -3 increased. Only MMP-9 activity was decreased on zymography. mRNA levels of the collagens, fibronectin, and laminin were not affected by SPARC overexpression.

Conclusions: SPARC overexpression increases IOP in perfused cadaveric human anterior segments resulting from a qualitative change the JCT ECM. Selective decrease of MMP-9 activity is likely part of the mechanism. SPARC is a regulatory node for IOP.
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http://dx.doi.org/10.1167/iovs.12-11362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648228PMC
May 2013

Regulation of SPARC by transforming growth factor β2 in human trabecular meshwork.

Invest Ophthalmol Vis Sci 2013 Apr 5;54(4):2523-32. Epub 2013 Apr 5.

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114, USA.

Purpose: An increased aqueous level of TGF-β2 has been found in many primary open-angle glaucoma patients. Secreted Protein, Acidic, and Rich in Cysteine (SPARC)-null mice have a lower intraocular pressure. The mechanistic relationship between SPARC and TGF-β2 in trabecular meshwork (TM) is unknown. We hypothesized that TGF-β2 upregulates SPARC expression in TM.

Methods: Cultured TM cells were incubated with selective inhibitors for p38 MAP kinase (p38), Smad3, p42, JNK, RhoA, PI3K, or TGF-β2 receptor for 2 hours, and then TGF-β2 was added for 24 hours in serum-free media. Quantitative polymerase chain reaction (qPCR) and immunoblot analysis were performed. Immunofluorescent microscopy was used to determine nuclear translocation of signaling proteins. Ad5.hSPARC and Lentiviral shRNA for p38 and Smad3 were constructed, and infected human TM cells.

Results: SPARC was upregulated by TGF-β2 in the human TM cells (3.8 ± 1.7-fold, n = 6, P = 0.01 for protein and 7.1 ± 3.7-fold, n = 6, P = 0.01 for mRNA), while upregulation of SPARC had no effect on TGF-β2. TGF-β2-induced SPARC expression was suppressed by inhibitors against p38 (-40.3 ± 20.9%, n = 10, P = 0.0001), Smad3 (-56.2 ± 18.9%, n = 10, P = 0.0001), JNK (-49.1 ± 24.6%, n = 10, P = 0.0001), and TGF-β2 receptor (-83.6 ± 14.4%, n = 6, P = 0.003). Phosphorylation and translocation of Smad3, p38, and MAPKAPK2 were detected at 30 minutes and 1 hour, respectively, following TGF-β2 treatment. Phosphorylation of JNK and c-jun was detected before TGF-β2 treatment. SPARC was suppressed 31 ± 13% (n = 5, P < 0.0001) by shRNA-p38 and 41 ± 3% (n = 5, P < 0.0001) by shRNA-Smad3.

Conclusions: TGF-β2 upregulates SPARC expression in human TM through Smad-dependent (Smad2/3) or -independent (p38) signaling pathways. SPARC may be a downstream regulatory node of TGF-β2-mediated IOP elevation.
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http://dx.doi.org/10.1167/iovs.12-11474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626313PMC
April 2013

Secreted protein acidic and rich in cysteine (SPARC)-null mice exhibit more uniform outflow.

Invest Ophthalmol Vis Sci 2013 Mar 21;54(3):2035-47. Epub 2013 Mar 21.

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

Purpose: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein known to regulate extracellular matrix (ECM) in many tissues and is highly expressed in trabecular meshwork (TM). SPARC-null mice have a 15% to 20% decrease in intraocular pressure (IOP) compared to wild-type (WT) mice. We hypothesized that mouse aqueous outflow is segmental, and that transgenic deletion of SPARC causes a more uniform pattern that correlates with IOP and TM morphology.

Methods: Eyes of C57BL6-SV129 WT and SPARC-null mice were injected with fluorescent microbeads, which were also passively exposed to freshly enucleated eyes. Confocal and electron microscopy were performed. Percentage effective filtration length (PEFL) was calculated as PEFL = FL/TL × 100%, where TL = total length and FL = filtration length. IOP was measured by rebound tonometry.

Results: Passive microbead affinity for WT and SPARC-null ECM did not differ. Segmental flow was observed in the mouse eye. SPARC-null mice had a 23% decrease in IOP. PEFL increased in SPARC-null (70.61 ± 11.36%) versus WT mice (54.68 ± 9.95%, P < 0.005; n = 11 pairs), and PEFL and IOP were negatively correlated (R(2) = 0.72, n = 10 pairs). Morphologically, TM of high-tracer regions had increased separation between beams compared to low-tracer regions. Collagen fibril diameter decreased in SPARC-null (28.272 nm) versus WT tissue (34.961 nm, P < 0.0005; n = 3 pairs).

Conclusions: Aqueous outflow in mice is segmental. SPARC-null mice demonstrated a more uniform outflow pattern and decreased collagen fibril diameter. Areas of high flow had less compact juxtacanalicular connective tissue ECM, and IOP was inversely correlated with PEFL. Our data show a correlation between morphology, aqueous outflow, and IOP, indicating a modulatory role of SPARC in IOP regulation.
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http://dx.doi.org/10.1167/iovs.12-10950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3621502PMC
March 2013

Interleukin-10 promoter gene polymorphisms and susceptibility to asthma: a meta-analysis.

PLoS One 2013 15;8(1):e53758. Epub 2013 Jan 15.

Korea University College of Medicine, Seoul, Korea.

Objective: The aim of this study was to explore whether the interleukin (IL)-10 polymorphisms and their haplotypes contribute to asthma susceptibility.

Methods: MEDLINE, EMBASE and the COCHRANE library databases were utilized to identify available articles. A meta-analysis was conducted on IL-10 -1082 G/A, -819 C/T, -592 C/A polymorphisms, and their haplotypes and asthma.

Results: Eleven studies involving 2,215 asthma patients and 2,170 controls were considered in the meta-analysis. The meta-analysis revealed no association between asthma and the IL-10 -1082 G allele [Odds ratio (OR) = 0.87, 95% Confidence interval (CI) = 0.68-1.12, p = 0.28]. However, meta-analysis of the five studies in Hardy-Weinburg equilibrium produced the relationship between the IL-10 -1082 G allele and asthma (OR = 0.71, 95% CI = 0.60-0.83, p<0.0001). Stratification by ethnicity indicated an association between the IL-10 -1082 G allele and asthma in East Asians (OR = 0.74, 95% CI = 0.57-0.96, p = 0.02), but not in West Asians. Furthermore, stratification by age indicated an association between the IL-10 -1082 G allele and asthma in adults and mixed groups (OR = 0.77, 95% CI = 0.62-0.96, p = 0.02; OR = 0.67, 95% CI = 0.49-0.92, p = 0.01). No association was found between asthma and IL-10 -819 C/T and IL-10 -592 C/A polymorphisms and their haplotypes.

Conclusion: The IL-10 -1082 G/A polymorphism confers susceptibility to asthma in East Asians and in adults. However, the IL-10 -819 C/T, -592 C/A polymorphisms and their haplotypes are not associated with asthma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546046PMC
June 2013

Thrombospondin-1 (TSP1)-null and TSP2-null mice exhibit lower intraocular pressures.

Invest Ophthalmol Vis Sci 2012 Sep 28;53(10):6708-17. Epub 2012 Sep 28.

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA 02114, USA.

Purpose: Thrombospondin-1 (TSP1) and TSP2 are matricellular proteins that have been shown to regulate cytoskeleton, cell adhesion, and extracellular matrix remodeling. Both TSP1 and TSP2 are found in the trabecular meshwork (TM). In cadaver eyes with primary open-angle glaucoma (POAG), TSP1 is increased in one third of patients. We hypothesized that TSP1 and TSP2 participate in the regulation of intraocular pressure (IOP). Methods. IOPs of TSP1-null, TSP2-null mice, and their corresponding wild-type (WT) mice were measured using a commercial rebound tonometer. Fluorophotometric measurements assessed aqueous turnover. Central corneal thickness (CCT) was measured by optical coherence tomography. Iridocorneal angles were examined using light microscopy (LM), immunofluorescence (IF), and transmission electron microscopy (TEM).

Results: Average IOPs of TSP1-null and TSP2-null mice were 10% and 7% less than that of the corresponding WT mice, respectively. CCTs were 6.5% less in TSP1-null mice (P < 0.05) and 1.1% less in TSP2-null mice (P > 0.05). Fluorophotometric measurements suggest that aqueous turnover rates in TSP1-null and TSP2-null mice are greater than those of WT mice. LM of the TSP1-null and TSP2-null iridocorneal angles reveals morphology, which is indistinguishable from that of their corresponding WTs. IF revealed possible concurrent underexpression of TSP2 in TSP1-null mice and of TSP1 in TSP2-null mice. TEM revealed larger collagen fibril diameters in TSP1-null and TSP2-null mice compared with WTs.

Conclusions: TSP1-null and TSP2-null mice have lower IOPs than their WT counterparts. The rate of aqueous turnover suggests that the mechanism is enhanced outflow facility. An alteration in the extracellular matrix may contribute to this finding.
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http://dx.doi.org/10.1167/iovs.11-9013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462480PMC
September 2012

TLR3 signaling is either protective or pathogenic for the development of Theiler's virus-induced demyelinating disease depending on the time of viral infection.

J Neuroinflammation 2011 Dec 21;8:178. Epub 2011 Dec 21.

Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611, USA.

Background: We have previously shown that toll-like receptor 3 (TLR3)-mediated signaling plays an important role in the induction of innate cytokine responses to Theiler's murine encephalomyelitis virus (TMEV) infection. In addition, cytokine levels produced after TMEV infection are significantly higher in the glial cells of susceptible SJL mice compared to those of resistant C57BL/6 mice. However, it is not known whether TLR3-mediated signaling plays a protective or pathogenic role in the development of demyelinating disease.

Methods: SJL/J and B6;129S-Tlr3tm1Flv/J (TLR3KO-B6) mice, and TLR3KO-SJL mice that TLR3KO-B6 mice were backcrossed to SJL/J mice for 6 generations were infected with Theiler's murine encephalomyelitis virus (2 × 105 PFU) with or without treatment with 50 μg of poly IC. Cytokine production and immune responses in the CNS and periphery of infected mice were analyzed.

Results: We investigated the role of TLR3-mediated signaling in the protection and pathogenesis of TMEV-induced demyelinating disease. TLR3KO-B6 mice did not develop demyelinating disease although they displayed elevated viral loads in the CNS. However, TLR3KO-SJL mice displayed increased viral loads and cellular infiltration in the CNS, accompanied by exacerbated development of demyelinating disease, compared to the normal littermate mice. Late, but not early, anti-viral CD4+ and CD8+ T cell responses in the CNS were compromised in TLR3KO-SJL mice. However, activation of TLR3 with poly IC prior to viral infection also exacerbated disease development, whereas such activation after viral infection restrained disease development. Activation of TLR3 signaling prior to viral infection hindered the induction of protective IFN-γ-producing CD4+ and CD8+ T cell populations. In contrast, activation of these signals after viral infection improved the induction of IFN-γ-producing CD4+ and CD8+ T cells. In addition, poly IC-pretreated mice displayed elevated PDL-1 and regulatory FoxP3+ CD4+ T cells in the CNS, while poly IC-post-treated mice expressed reduced levels of PDL-1 and FoxP3+ CD4+ T cells.

Conclusions: These results suggest that TLR3-mediated signaling during viral infection protects against demyelinating disease by reducing the viral load and modulating immune responses. In contrast, premature activation of TLR3 signal transduction prior to viral infection leads to pathogenesis via over-activation of the pathogenic immune response.
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http://dx.doi.org/10.1186/1742-2094-8-178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293102PMC
December 2011

Coordinated regulation of extracellular matrix synthesis by the microRNA-29 family in the trabecular meshwork.

Invest Ophthalmol Vis Sci 2011 May 1;52(6):3391-7. Epub 2011 May 1.

Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114, USA.

Purpose: The microRNA-29 (miR-29) family has emerged, in various tissues, as a key modulator of extracellular matrix (ECM) homeostasis. In this study, the authors investigate the role of the miR-29 family in the regulation of ECM synthesis in the trabecular meshwork (TM) under basal and TGF-β2 stimulatory conditions.

Methods: Human TM cells were incubated with 2.5 ng/mL activated, recombinant human TGF-β2 for 24, 48, and 72 hours. A specific pharmacologic inhibitor was used to block SMAD3 function in the context of TGF-β2 stimulation. Changes in the expression of the miR-29 family were assessed by real-time PCR. The effect of miR-29 molecules and inhibitors on ECM levels was determined by immunoblot analysis.

Results: All three members of the miR-29 family were expressed in cultured TM cells. Although the incubation of TM cells with TGF-β2 induced miR-29a and suppressed miR-29b levels, no significant effect was observed on miR-29c expression. Additional studies revealed that SMAD3 modulates miR-29b expression under basal and TGF-β2 conditions. Subsequent gain- and loss-of-function experiments demonstrated that the miR-29 family functions as a critical suppressor of various ECM proteins under basal and TGF-β2 stimulatory conditions.

Conclusions: The findings derived from this study identify the miR-29 family as a critical regulator of ECM expression in the TM and suggest that its modulation by TGF-β2 may be important in controlling ECM synthesis. Together, these data provide further insight into the complex regulatory mechanisms mediating TGF-β2 signaling and ECM production in the TM.
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http://dx.doi.org/10.1167/iovs.10-6165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109034PMC
May 2011

Effect of hevin deletion in mice and characterization in trabecular meshwork.

Invest Ophthalmol Vis Sci 2011 Apr 6;52(5):2187-93. Epub 2011 Apr 6.

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA 02114, USA.

Purpose: Hevin is a matricellular protein and the result of a gene duplication of SPARC. SPARC-null mice have lower intraocular pressure (IOP). The function of hevin in trabecular meshwork (TM) is unknown. The authors hypothesized that hevin is expressed in TM and has a functional consequence on IOP.

Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting were performed to identify transcription and protein expression in TM and cultured TM cells. Toluidine blue stain was performed to compare anterior segments in wild-type (WT) and hevin-null mice. Confocal microscopy localized the structural distribution of hevin in human TM and hevin/SPARC in mouse anterior segments. IOP was measured in WT (C57BL6 × 129SvJ) and hevin-null mice using both rebound tonometry and cannulation tonometry. Central corneal thickness (CCT) was measured by ocular coherence tomography. Cultured TM cells were treated with TGF-β2 because TGF-β2 is associated with primary open-angle glaucoma.

Results: Hevin mRNA and protein were expressed in TM tissues but not in cultured TM cells. No structural differences were observed in anterior segments of WT and hevin-null mice. IOP between hevin-null (n = 46) and WT (n = 44) mice was equivalent (15.3 ± 1.92 mm Hg and 15.9 ± 2.01 mm Hg, respectively; P = 0.15). CCT was similar between hevin-null and WT mice (107.95 ± 5.06 μm and 106.76 ± 3.46 μm, respectively; P = 0.11). TGF-β2 did not induce hevin, whereas SPARC expression was induced in a dose-dependent manner in human TM cell cultures.

Conclusions: Hevin does not appear to be critical to regulating IOP. Hevin is expressed in TM but, in contrast to SPARC, does not appear to be regulated by TGF-β2.
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http://dx.doi.org/10.1167/iovs.10-5428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080182PMC
April 2011

Pep-1 Peptide-modified liposomal carriers for intracellular delivery of gold nanoparticles.

Chem Pharm Bull (Tokyo) 2011 ;59(1):109-12

College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.

A Pep-1 peptide-modified liposomal (Pep1-Lipo) carrier system was investigated to increase the intracellular delivery of gold nanoparticles (Au NPs). Au NPs with a mean diameter of 13 nm were successfully encapsulated into the inner aqueous compartment of the novel carrier using an ethanol injection technique, reserving the distinctive optical characteristics of the surface plasmon resonance peak around 530 nm. The Au NP-loaded liposomal carrier was physically characterized as 150-170 nm in size and 45 mV in zeta potential. Dark field microscopic observation demonstrated that in vitro cellular association and/or translocation of the nanoprobes into the cells was increased by Pep1-Lipo carriers compared to bare Au NPs. In conclusion, this novel liposomal formulation is a promising platform for the intracellular delivery of metallic nanoprobes including Au NPs.
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http://dx.doi.org/10.1248/cpb.59.109DOI Listing
April 2011

SPARC-null mice exhibit lower intraocular pressures.

Invest Ophthalmol Vis Sci 2009 Aug 24;50(8):3771-7. Epub 2009 Jan 24.

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

Purpose: SPARC is a matricellular protein that is highly expressed in remodeling tissues, including the trabecular meshwork and ciliary body. The hypothesis for the study was that SPARC contributes to the regulation of intraocular pressure (IOP). The IOPs of SPARC-null mice, their corresponding wild-type (WT), and heterozygous animals were compared.

Methods: Diurnal and nocturnal IOPs of C57Bl/6x129SvJ WT, SPARC-null, and heterozygous mice were measured. Fluorophotometric measurements were made to assess aqueous turnover. Central corneal thickness (CCT) was measured using histology, ultrasound biomicroscopy, and optical coherence tomography. Iridocorneal angles were examined using light microscopy (LM).

Results: During the day, the mean IOP of SPARC-null mice (n = 142, 16.9 +/- 2.4 mm Hg) was lower than that of both WT mice (n = 104, 19.9 +/- 2.9 mm Hg; P < 10(-12)), and heterozygotes (n = 38, 19.3 +/- 2.5 mm Hg; P < 10(-4)). At night, SPARC-null mice also exhibited a blunted increase in IOP in comparison to WT and heterozygous mice. CCTs were not significantly different between WT and SPARC-null mice. Heterozygous mice tended to have thicker corneas (3.4%). Fluorophotometric measurements suggest that aqueous turnover rates in SPARC-null mice are equal to if not greater than rates in WT mice. LM of the SPARC-null iridocorneal angle revealed morphology that is indistinguishable from WT.

Conclusions: SPARC-null mice have lower IOPs than do their WT counterparts with equal CCTs. The rate of aqueous turnover suggests that the mechanism is enhanced outflow resistance.
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http://dx.doi.org/10.1167/iovs.08-2489DOI Listing
August 2009

Matricellular proteins in the trabecular meshwork.

Exp Eye Res 2009 Apr 11;88(4):694-703. Epub 2008 Dec 11.

Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Boston, MA 02114, USA.

The trabecular meshwork is one of the primary tissues of interest in the normal regulation and dysregulation of intraocular pressure (IOP) that is a causative risk factor for primary open-angle glaucoma. Matricellular proteins generally function to allow cells to modulate their attachments with and alter the characteristics of their surrounding extracellular matrix (ECM). In non-ocular tissues, matricellular proteins generally increase fibrosis. Since ECM turnover is very important to the outflow facility, matricellular proteins may have a significant role in the regulation of IOP. The formalized study of matricellular proteins in trabecular meshwork is in its infancy. SPARC, thrombospondins-1 and -2, and tenascins-C and -X, and osteopontin have been localized to varying areas within the trabecular meshwork. Preliminary evidence indicates that SPARC and thrombospondin-1 play a role in the regulation of IOP and possibly the pathophysiology of glaucoma. These data show promise that matricellular proteins are involved in IOP dysregulation and are potential therapeutic targets. Further study is needed to clarify these roles.
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http://dx.doi.org/10.1016/j.exer.2008.11.032DOI Listing
April 2009

Replication of Theiler's virus requires NF-kappa B-activation: higher viral replication and spreading in astrocytes from susceptible mice.

Glia 2008 Jul;56(9):942-53

Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611, USA.

To investigate viral replication and cell-cell spreading in astrocytes, recombinant Theiler's murine encephalomyelitis virus (TMEV) expressing green fluorescent protein (GFP) during the replication was generated. GFP and TMEV proteins were processed correctly in infected cells and production of viral proteins could be tracked by fluorescent microscopy. Viral replication of both wild-type TMEV and GFP-TMEV was dependent on the activation of NF-kappaB and partially MAP kinase, based on chemical inhibition studies. Viral replication was significantly reduced in primary astrocytes from NF-kappaB1 (p105)-deficient mice compared with that from wild-type control mice, whereas cytokine production was enhanced. These results suggest an association of canonical NF-kappaB subunits in viral replication, but not cytokine production. Viral replication was also suppressed in both IKKalpha and IKKbeta-deficient mouse embryonic fibroblasts (MEFs), compared with that in wild-type MEF. However, the inhibition was significantly greater in IKKbeta-deficient MEF, suggesting that IKKbeta plays a stronger role in supporting viral replication. Interestingly, viral replication and spreading in primary astrocytes from susceptible SJL/J mice were several-fold higher than those in astrocytes from resistant C57BL/6 mice, suggesting that higher viral replication levels in astrocytes may also contribute to the viral persistence in the central nervous system (CNS) of susceptible SJL/J mice. A relatively higher level of activated NF-kappaB was found in the nuclei of virus-infected SJL astrocytes compared with C57BL/6 astrocytes suggest that the NF-kappaB activation level affects on viral replication.
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http://dx.doi.org/10.1002/glia.20668DOI Listing
July 2008

Induction of chemokine and cytokine genes in astrocytes following infection with Theiler's murine encephalomyelitis virus is mediated by the Toll-like receptor 3.

Glia 2006 Jun;53(8):858-67

Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois, USA.

Theiler's murine encephalomyelitis virus (TMEV) infection in the central nervous system (CNS) induces a demyelinating disease similar to human multiple sclerosis. TMEV infection results in activation of various chemokine and cytokine genes that are important in the initiation of an inflammatory response. We have previously shown that the production of these chemokines and cytokines in astrocytes is induced via the NF-kappaB pathway following TMEV and Coxsackie virus infection. In this study, we investigated whether the NF-kappaB-dependent inflammatory responses after TMEV infection is triggered through TLR3 and/or TLR7. The activation of NF-kappaB or IRF/ISRE, as well as the production of both MCP-1/CCL2 and IL-8/CXCL8, was observed in only TLR3-transfected HEK 293 cells, but not in TLR7-tranfected cells. The potential involvement of TLR3 in mouse embryonic fibroblasts and primary astrocytes was further investigated following transfection with wildtype or dominant negative form of TLRs and MyD88, as well as astrocytes from TLR3- and MyD88-deficient mice. Similarly, the activation of transcription factors and chemokine genes is induced in these mouse cells through primarily TLR3 signaling pathway, but not TLR7 or other MyD88-mediated pathways following TMEV infection. However, the TLR3-mediated cellular activation does not appear to affect the level of viral replication in astrocytes. These results strongly suggest that TLR3-signaling by TMEV alone is sufficient to induce the initial inflammatory cytokine responses that could be very important for the outcome of virus-induced encephalitis and/or demyelinating diseases, such as multiple sclerosis.
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http://dx.doi.org/10.1002/glia.20346DOI Listing
June 2006
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