Loss of Caveolin-1 Impairs Light Flicker-Induced Neurovascular Coupling at the Optic Nerve Head.

Glaucoma is a neurodegenerative disease, which results in characteristic visual field defects. Intraocular pressure (IOP) remains the main risk factor for this leading cause of blindness. Recent studies suggest that disturbances in neurovascular coupling (NVC) may be associated with glaucoma. The resultant imbalance between vascular perfusion and neuronal stimulation in the eye may precede retinal ganglion cell (RGC) loss and increase the susceptibility of the eye to raised IOP and glaucomatous degeneration. Caveolin-1 (Cav-1) is an integral scaffolding membrane protein found abundantly in retinal glial and vascular tissues, with possible involvement in regulating the neurovascular coupling response. Mutations in Cav-1 have been identified as a major genetic risk factor for glaucoma. Therefore, we aim to evaluate the effects of Cav-1 depletion on neurovascular coupling, retinal vessel characteristics, RGC density and the positive scotopic threshold response (pSTR) in Cav-1 knockout (KO) versus wild type C57/Bl6 mice (WT). Following light flicker stimulation of the retina, Cav-1 KO mice showed a smaller increase in perfusion at the optic nerve head and peripapillary arteries, suggesting defective neurovascular coupling. Evaluation of the superficial capillary plexus in Cav-1 KO mice also revealed significant differences in vascular morphology with higher vessel density, junction density and decreased average vessel length. Cav-1 KO mice exhibited higher IOP and lower pSTR amplitude. However, there was no significant difference in RGC density between Cav-1 KO and wild type mice. These findings highlight the role of Cav-1 in regulating neurovascular coupling and IOP and suggest that the loss of Cav-1 may predispose to vascular dysfunction and decreased RGC signaling in the absence of structural loss. Current treatment for glaucoma relies heavily on IOP-lowering drugs, however, there is an immense potential for new therapeutic strategies that increase Cav-1 expression or augment its downstream signaling in order to avert vascular dysfunction and glaucomatous change.

Positive-charge tuned gelatin hydrogel-siSPARC injectable for siRNA anti-scarring therapy in post glaucoma filtration surgery.

Small interfering RNA (siRNA) therapy is a promising epigenetic silencing strategy. However, its widespread adoption has been severely impeded by its ineffective delivery into the cellular environment. Here, a biocompatible injectable gelatin-based hydrogel with positive-charge tuned surface charge is presented as an effective platform for siRNA protection and delivery. We demonstrate a two-step synthesis of a gelatin-tyramine (Gtn-Tyr) hydrogel with simultaneous charge tunability and crosslinking ability. We discuss how different physiochemical properties of the hydrogel interact with siSPARC (siRNA for secreted protein, acidic and rich in cysteine), and study the positive-charge tuned gelatin hydrogel as an effective delivery platform for siSPARC in anti-fibrotic treatment. Through in vitro studies using mouse tenon fibroblasts, the positive-charge tuned Gtn-Tyr hydrogel shows sustained siSPARC cellular internalization and effective SPARC silencing with excellent biocompatibility. Similarly, the same hydrogel platform delivering siSPARC in an in vivo assessment employing a rabbit model shows an effective reduction in subconjunctival scarring in post glaucoma filtration surgery, and is non-cytotoxic compared to a commonly used anti-scarring agent, mitomycin-C. Overall, the current siRNA delivery strategy involving the positive-charge tuned gelatin hydrogel shows effective delivery of gene silencing siSPARC for anti-fibrotic treatment. The current charge tunable hydrogel delivery system is simple to fabricate and highly scalable. We believe this delivery platform has strong translational potential for effective siRNA delivery and epigenetic silencing therapy.

Hydroxyapatite Particles Induced Modulation of Collagen Expression and Secretion in Primary Human Dermal Fibroblasts.

BACKGROUND: Hydroxyapatite (HA) [Ca5(PO4)3(OH)] is a naturally occurring calcium phosphate which makes up 60-70% of the dry weight of human bones. Nano-scale HA particles are increasingly being used as carriers for controlled and targeted delivery of bioactive agents like drugs, proteins, and nucleic acids due to their high porosity, negative charge, and biodegradability. PURPOSE: Although much effort has been devoted to understanding the delivery kinetics and effects of the payloads in such carriers, a thorough understanding of the influence of the carriers themselves is lacking. METHODS: HA particles (300 µg/mL) were administered to primary human dermal fibroblasts (HDFs). The uptake and intracellular localization of the particles were determined by flow cytometry, confocal imaging, and transmission electron microscopy (TEM). Immunological assays and PCR were performed to determine the levels of pro-inflammatory cytokines and collagens in cell lysates and media supernatant. RESULTS: The current study explores the effects of poly-dispersed HA particles on primary HDFs as a model system. The majority of the particles were determined to range between 150 and 200 nm in diameter. Upon exposure to HA suspensions, primary HDFs internalized the particles by endocytosis within 6 hours of exposure, showing maximum uptake at 72 hours following which the particles were exocytosed by 168 hours. This correlated to reduced secretion of various pro-inflammatory and pro-collagenic cytokines. Biochemical analysis further revealed a reduction in Type I collagen expression and secretion. CONCLUSION: HA particles have an immune-modulatory effect on dermal fibroblasts and reduce collagen production, which may impact the integrity of the extracellular matrix (ECM). This study demonstrates the need to consider the secondary effects of particulate carriers like HA, beyond basic cytotoxicity, in the specific tissue environment where the intended function is to be realized.

A Minimally Invasive Experimental Model of Acute Ocular Hypertension with Acute Angle Closure Characteristics.

Purpose: To describe a minimally invasive experimental model of acute ocular hypertension (OHT) with characteristics of acute angle closure (AAC). Methods: Adult C57/Bl6 mice (n = 31) were subjected to OHT in one eye using a modified circumlimbal suture technique that elevated intraocular pressure (IOP) for 30 minutes. Contralateral un-operated eyes served as controls. IOP, anterior segment optical coherence tomography, and fundus fluorescein angiography (FFA) were performed. The positive scotopic threshold response (pSTR) and a-wave and b-wave amplitudes were also evaluated. Retinal tissues were immunostained for the retinal ganglion cell (RGC) marker RBPMS and the glial marker GFAP. Results: OHT eyes developed shallower anterior chambers and dilated pupils. FFA showed focal leakage in 32.2% of OHT eyes, but in none of the control eyes. pSTR was significantly reduced at week 1 in OHT eyes compared to control eyes (57.3 ± 7.2 µV vs. 106.9 ± 24.8 µV; P < 0.05), but a- and b-waves were unaffected. GFAP was upregulated in OHT eyes but not in control eyes or eyes that had been sutured without OHT. RGC density was reduced in OHT eyes after 4 weeks (3857 ± 143.8) vs. control eyes (4469 ± 176.0) (P < 0.05). Conclusions: Our minimally invasive model resulted in acute OHT with characteristics of AAC in the absence of non-OHT-related neuroinflammatory changes arising from ocular injury alone. Translational Relevance: This model provides a valuable approach to studying specific characteristics of a severe blinding disease in an experimental setting. Focal areas of ischemia were demonstrated, consistent with clinical studies of acute angle closure patients elsewhere, which may indicate the need for further research into how this could affect visual outcome in these patients.

Design and in vitro release study of siRNA loaded Layer by Layer nanoparticles with sustained gene silencing effect.

OBJECTIVES: Clinical translation of siRNA therapeutics has been severely limited due to the lack of stable and sustained siRNA delivery systems. Furthermore, when nanocarrier systems with siRNA are administered systemically to treat diseases, insufficient doses reach the target tissue. Here we report the successful development of a new nanocarrier system for the management of fibrosis. METHODS: The new carrier has a hydroxyapatite core, with alternating layers of siRNA and a cationic peptide. The siRNA used here targets secreted protein acidic and rich in cysteine (SPARC), a key matricellular protein involved in the regulation of collagen fibrillogenesis and assembly. We have also used FRET studies to elucidate the fate of the particles inside cells, including the mechanistic details of layer-by-layer detachment. RESULTS: In vitro studies using murine conjunctiva fibroblasts show sustained release over 2 weeks, and that such released siRNA sustained SPARC knockdown without affecting cell growth, and maintained siRNA presence in the cells for at least two weeks with a single-dose treatment. Release studies of siRNA from particles in vitro gave insight on how the particles delivered prolonged gene-silencing effects. CONCLUSION: A single treatment of the layer-by-layer nanoparticle designed can achieve sustained gene silencing over 2 weeks. Localized delivery of stabilized siRNA with sustained-release capabilities opens the door for many other applications of siRNA-based gene regulation.

A simple and inexpensive physical lysis method for DNA and RNA extraction from freshwater microalgae.

In this work, a simple and inexpensive physical lysis method using a cordless drill fitted with a plastic pellet pestle and 150 mg of sterile sea sand was established for the extraction of DNA from six strains of freshwater microalgae. This lysis method was also tested for RNA extraction from two microalgal strains. Lysis duration between 15 and 120 s using the cetyltrimethyl ammonium bromide (CTAB) buffer significantly increased the yield of DNA from four microalgalstrains (Monoraphidium griffithii NS16, Scenedesmus sp. NS6, Scenedesmus sp. DPBC1 and Acutodesmus sp. DPBB10) compared to control. It was also found that grinding was not required to obtain DNA from two strains of microalgae (Choricystis sp. NPA14 and Chlamydomonas sp. BM3). The average DNA yield obtained using this lysis method was between 62.5 and 78.9 ng/mg for M. griffithii NS16, 42.2-247.0 ng/mg for Scenedesmus sp. NS6, 70.2-110.9 ng/mg for Scenedesmus sp. DPBC1 and 142.8-164.8 ng/mg for Acutodesmus sp. DPBB10. DNA obtained using this method was sufficiently pure for PCR amplification. Extraction of total RNA from M. griffithii NS16 and Mychonastes sp. NPD7 using this lysis method yielded high-quality RNA suitable for RT-PCR. This lysis method is simple, cheap and would enable rapid nucleic acid extraction from freshwater microalgae without requiring costly materials and equipment such as liquid nitrogen or beadbeaters, and would facilitate molecular studies on microalgae in general.

Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition.

BACKGROUND: To develop targeted antifibrotic therapy for glaucoma filtration surgery; this study determines the effectiveness of small interfering RNA (siRNA) to reduce in vivo secreted protein acidic and rich in cysteine (SPARC) expression using the mouse model of conjunctival scarring. METHODS: Experimental surgery was performed as described for the mouse model of conjunctival scarring. Scrambled (siScram) or Sparc (siSparc) siRNAs, loaded on layer-by-layer (LbL) nanoparticles, were injected into the conjunctiva immediately after surgery. Expression of Sparc, Col1a1, Fn1 and Mmp14 was measured by real-time PCR and immunoblotting on days 7 and 14 postsurgery. Live imaging of the operated eyes was performed using slit lamp, anterior segment-optical coherence tomography and confocal microscopy. Tissue pathology was evaluated by histochemical and immunofluorescent analyses of operated conjunctival cryosections. Tissue apoptosis was quantitated by annexin V assay. RESULTS : siSparc, delivered via expanded LbL nanoparticles, significantly inhibited Sparc transcription in both day 7 (2.04-fold) and day 14 (1.39-fold) treated tissues. Sparc suppression on day 7 was associated with a significant reduction of Col1a1 (2.52-fold), Fn1 (2.89-fold) and Mmp14 (2.23-fold) mRNAs. At the protein level, both SPARC and collagen 1A1 (COL1A1) were significantly reduced at both time points with siSparc treatment. Nanoparticles were visualised within cell-like structures by confocal microscopy, while overt tissue response or apoptosis was not observed. CONCLUSIONS : SPARC targeted therapy effectively reduced both SPARC and collagen production in the operated mouse conjunctiva. This proof-of-concept study suggests that targeted treatment of fibrosis in glaucoma surgery is safe and feasible, with the potential to extend to a range of potential genes associated with fibrosis.

Inhibition of Monocyte Chemoattractant Protein 1 Prevents Conjunctival Fibrosis in an Experimental Model of Glaucoma Filtration Surgery.

Purpose: To evaluate the effect of treatment with monocyte chemoattractant protein-1 receptor inhibitor (MCP-Ri) to maintain bleb survival and prevent fibrosis in an experimental model of glaucoma filtration surgery (GFS). Methods: GFS was performed on one eye of C57/Bl6 mice (n = 36) that was treated with MCP-Ri, mitomycin-C (MMC), or vehicle at the time of surgery. Real-time polymerase chain reaction was used to evaluate conjunctival expression of monocyte chemoattractant protein-1 (MCP-1), TGFB1, TGFB2, collagen 1a1 (Col1a1), sparc (Sparc), and fibronectin at 2 and 7 days following surgery. Anterior segment slit-lamp examination, optical coherence tomography, and confocal microscopy were performed in vivo at day 14. Eyes were processed for immunohistochemical staining of F4/80, a monocyte-macrophage marker, at day 2. In vitro experiments were also performed to compare the effect of MMC, MCP-Ri, and vehicle on the viability of mouse Tenon's fibroblasts. Results: Treatment with MCP-Ri results in a greater reduction in the percentage of F4/80-positive cells in conjunctival blebs and lesser MCP-1 gene expression following experimental GFS than MMC or control. Both MMC and MCP-Ri reduced Col1a1 and Sparc expression, but not fibronectin. TGFB1 decreased with MCP-Ri but not MMC; MMC but not MCP-Ri reduced TGFB2. MMC and MCP-Ri treatment resulted in the preservation of bleb height at day 14, as compared to control. MCP-Ri was less toxic to mouse Tenon's fibroblasts in comparison with MMC. Conclusions: Targeting MCP-1 results in prolonged bleb survival following experimental GFS with less cellular toxicity as compared to MMC. MCP inhibition could provide a safer alternative to conventional antifibrotic adjunctive treatments in GFS.

Altered Anterior Segment Biometric Parameters in Mice Deficient in SPARC.

Purpose: Secreted protein acidic and rich in cysteine (SPARC) and Hevin are structurally related matricellular proteins involved in extracellular matrix assembly. In this study, we compared the anterior chamber biometric parameters and iris collagen properties in SPARC-, Hevin- and SPARC-/Hevin-null with wild-type (WT) mice. Methods: The right eyes of 53 WT, 35 SPARC-, 56 Hevin-, and 63 SPARC-/Hevin-null mice were imaged using the RTVue-100 Fourier-domain optical coherence tomography system. The parameters measured were anterior chamber depth (ACD), trabecular-iris space area (TISA), angle opening distance (AOD), and pupil diameter. Biometric data were analyzed using analysis of covariance and adjusted for age, sex, and pupil diameter. Expression of Col1a1, Col8a1, and Col8a2 transcripts in the irises was measured by quantitative polymerase chain reaction. Collagen fibril thickness was evaluated by transmission electron microscopy. Results: Mice that were SPARC- and SPARC-/Hevin-null had 1.28- and 1.25-fold deeper ACD, 1.45- and 1.53-fold larger TISA, as well as 1.42- and 1.51-fold wider AOD than WT, respectively. These measurements were not significantly different between SPARC- and SPARC-/Hevin-null mice. The SPARC-null iris expressed lower Col1a1, but higher Col8a1 and Col8a2 transcripts compared with WT. Collagen fibrils in the SPARC- and SPARC-/Hevin-null irises were 1.5- and 1.7-fold thinner than WT, respectively. The Hevin-null iris did not differ from WT in these collagen properties. Conclusions: SPARC-null mice have deeper anterior chamber as well as wider drainage angles compared with WT. Therefore, SPARC plays a key role in influencing the spatial organization of the anterior segment, potentially via modulation of collagen properties, while Hevin is not likely to be involved.