In vivo human T cell engineering with enveloped delivery vehicles.

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR-Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody-antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

Effect of SPARC Suppression in Mice, Perfused Human Anterior Segments, and Trabecular Meshwork Cells.

Purpose: Secreted protein, acidic and rich in cysteine (SPARC) elevates intraocular pressure (IOP), increases certain structural extracellular matrix (ECM) proteins in the juxtacanalicular trabecular meshwork (JCT), and decreases matrix metalloproteinase (MMP) protein levels in trabecular meshwork (TM) endothelial cells. We investigated SPARC as a potential target for lowering IOP. We hypothesized that suppressing SPARC will decrease IOP, decrease structural JCT ECM proteins, and alter the levels of MMPs and/or their inhibitors. Methods: A lentivirus containing short hairpin RNA of human SPARC suppressed SPARC in mouse eyes and perfused cadaveric human anterior segments with subsequent IOP measurements. Immunohistochemistry determined structural correlates. Human TM cell cultures were treated with SPARC suppressing lentivirus. Quantitative reverse transcriptase polymerase chain reaction (PCR), immunoblotting, and zymography determined total RNA, relative protein levels, and MMP enzymatic activity, respectively. Results: Suppressing SPARC decreased IOP in mouse eyes and perfused human anterior segments by approximately 20%. Histologically, this correlated to a decrease in collagen I, IV, and VI in both the mouse TM and human JCT regions; in the mouse, fibronectin was also decreased but not in the human. In TM cells, collagen I and IV, fibronectin, MMP-2, and tissue inhibitor of MMP-1 were decreased. Messenger RNA of the aforementioned genes was not changed. Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting. MMP-1 activity was reduced in vitro by zymography. Conclusions: Suppressing SPARC decreased IOP in mice and perfused cadaveric human anterior segments corresponding to qualitative structural changes in the JCT ECM, which do not appear to be the result of transcription regulation.

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

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.

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

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 (r2=0.9349). In vitro cytotoxicity was dose-dependent, resulting in IC50 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.

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

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 140­150 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.

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

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.

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

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: ß-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 ß-catenin expression was assessed by immunoblot. Adenovirus expressing a dominant-negative TCF4 mutant that lacks a ß-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: ß-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 ß-catenin. Lithium chloride treatment upregulated miR-29b expression, and suppressed the levels of various ECM proteins under both basal and TGF-ß2 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.

Aqueous outflow: segmental and distal flow.

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.

TGF-ß2-mediated ocular hypertension is attenuated in SPARC-null mice.

PURPOSE: Transforming growth factor-ß2 (TGF-ß2) 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-ß2. We hypothesized that, in vivo, SPARC is a critical regulatory node in TGF-ß2-mediated ocular hypertension. METHODS: Empty (Ad.empty) or TGF-ß2-containing adenovirus (Ad.TGF-ß2) 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-ß2-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-ß2-injected versus Ad.empty-injected SPARC-null mice. However, on day 8, the IOP of Ad.TGF-ß2-injected SPARC-null eyes was elevated compared to that of contralateral uninjected eyes (P = 0.0385). Immunohistochemistry demonstrated that TGF-ß2 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-ß2-mediated ocular hypertension. Deletion of SPARC significantly attenuates the effects of TGF-ß2 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.

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

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.

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

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-ß-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR), under basal or TGF-ß2 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-ß2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188). Transforming growth factor-ß2 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.

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

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.

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

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%.

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

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.

Regulation of SPARC by transforming growth factor ß2 in human trabecular meshwork.

PURPOSE: An increased aqueous level of TGF-ß2 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-ß2 in trabecular meshwork (TM) is unknown. We hypothesized that TGF-ß2 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-ß2 receptor for 2 hours, and then TGF-ß2 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-ß2 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-ß2. TGF-ß2-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-ß2 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-ß2 treatment. Phosphorylation of JNK and c-jun was detected before TGF-ß2 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-ß2 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-ß2-mediated IOP elevation.

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

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.

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

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-ß2 because TGF-ß2 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-ß2 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-ß2.

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

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.

SPARC-null mice exhibit lower intraocular pressures.

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.