Subretinal AAV2.COMP-Ang1 suppresses choroidal neovascularization and vascular endothelial growth factor in a murine model of age-related macular degeneration.

To assess whether Tie2-mediated vascular stabilization ameliorates neovascular age-related macular degeneration (AMD), we investigated the impact of adeno-associated virus-mediated gene therapy with cartilage oligomeric matrix protein angiopoietin-1 (AAV2.COMP-Ang1) on choroidal neovascularization (CNV), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF) in a mouse model of the disease. We treated mice with subretinal injections of AAV2.COMP-Ang1 or control (AAV2.AcGFP, AAV2.LacZ, and phosphate-buffered saline). Subretinal AAV2 localization and plasmid protein expression was verified in the retinal pigment epithelium (RPE)/choroid of mice treated with all AAV2 constructs. Laser-assisted simulation of neovascular AMD was performed and followed by quantification of HIF, VEGF, and CNV in each experimental group. We found that AAV2.COMP-Ang1 was associated with a significant reduction in VEGF levels (29-33%, p < 0.01) and CNV volume (60-70%, p < 0.01), without a concomitant decrease in HIF1-α, compared to all controls. We concluded that a) AAV2 is a viable vector for delivering COMP-Ang1 to subretinal tissues, b) subretinal COMP-Ang1 holds promise as a prospective treatment for neovascular AMD, and c) although VEGF suppression in the RPE/choroid may be one mechanism by which AAV2.COMP-Ang1 reduces CNV, this therapeutic effect may be hypoxia-independent. Taken together, these findings suggest that AAV2.COMP-Ang1 has potential to serve as an alternative or complementary option to anti-VEGF agents for the long-term amelioration of neovascular AMD.

Acridine orange leukocyte fluorography in mice.

Simultaneous non-invasive visualization of blood vessels and nerves in patients can be obtained in the eye. The retinal vasculature is a target of many retinopathies. Inflammation, readily manifest by leukocyte adhesion to the endothelial lining, is a key pathophysiological mechanism of many retinopathies, making it a valuable and ubiquitous target for disease research. Leukocyte fluorography has been extensively used in the past twenty years; however, fluorescent markers, visualization techniques, and recording methods have differed between studies. The lack of detailed protocol papers regarding leukocyte fluorography, coupled with lack of uniformity between studies, has led to a paucity of standards for leukocyte transit (velocity, adherence, extravasation) in the retina. Here, we give a detailed description of a convenient method using acridine orange (AO) and a commercially available scanning laser ophthalmoscope (SLO, HRA-OCT Spectralis) to view leukocyte behavior in the mouse retina. Normal mice are compared to mice with acute and chronic inflammation. This method can be readily adopted in many research labs.

Optimum on-time and off-time combinations for micropulse phacoemulsification in venturi vacuum mode.

PURPOSE: To measure the time to fragment removal and number of chatter events using various combinations of micropulse on times and off times (measured in milliseconds) of longitudinal ultrasound (US) using a venturi-based phacoemulsification system. SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, USA. DESIGN: Experimental study. METHODS: Pig lenses were hardened with formalin and cut into 2.0 mm cubes. The time to fragment removal (efficiency) and frequency of fragments bouncing off the tip (chatter) were measured with the venturi-based system. Micropulse longitudinal US was tested. Parameters were combinations of 5, 6, and 7 milliseconds on, with 5, 6, and 7 milliseconds off. Twenty runs each of 9 combinations were completed. RESULTS: There was a statistically significant difference between on/off duty cycle combinations. The 6 on/7 off group had higher efficiency than the 5 on/6 off and 7 on/7 off groups. Six on/5 off was more efficient than 5 on/6 off. When data were pooled and on times alone were used, 6 milliseconds on time was more efficient than 5 or 7 milliseconds. No efficiency differences in off times were found. No significant chatter differences were observed. CONCLUSIONS: Using micropulse longitudinal US in venturi vacuum mode, 6 milliseconds on was the most efficient on time. Five, 6, and 7 milliseconds off times had similar efficiency. These data suggest that the most efficient setting with lowest US energy use is 6 milliseconds on and 7 milliseconds off.

Comparison of venturi and peristaltic vacuum in phacoemulsification.

PURPOSE: To evaluate the efficiency of peristaltic-based and venturi-based vacuums. SETTING: John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Porcine lenses were hardened with formalin and cut into 2.0 mm cubes. Time to fragment removal (efficiency) and fragment bounces off the tip (chatter) were measured using a Signature machine with the ability to switch between peristaltic-based and venturi-based vacuum. Micropulse longitudinal and transversal ultrasound motions were tested. RESULTS: Venturi-based vacuum had increased efficiency and decreased chatter compared with peristaltic-based vacuum at lower vacuum levels. CONCLUSION: Use of a venturi-based vacuum, when available, may result in reduced clearance time of lens material and mitigate chatter even under noisy conditions. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Effect of increased vacuum and aspiration rates on phacoemulsification efficiency.

PURPOSE: To evaluate the effect of vacuum and aspiration rates on phacoemulsification efficiency. SETTING: John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Formalin-soaked porcine lenses were divided into 2.0 mm cubes, and 0.9 mm 30-degree beveled 20-degree bent tips were used with micropulse ultrasound (US) (6 milliseconds on and 6 milliseconds off) and a peristaltic flow system. Vacuum levels were tested at 200, 300, 400, and 500 mm Hg, and aspiration rates were tested at 20, 35, and 50 mL/min. Efficiency (time to lens removal) and chatter (number of lens fragment repulsions from the tip) were determined. RESULTS: Increasing vacuum increased efficiency only when going from 200 mm Hg to higher vacuum levels. Increasing aspiration increased efficiency at all points measured (25 mL/min versus 35 mL/min, P < .0001; 35 mL/min versus 50 mL/min, P = .012; 25 mL/min versus 50 mL/min, P < .0001). Chatter was highest at 200 mm Hg and decreased when vacuum was increased from 200 mm Hg to 300 mm Hg and up. Chatter decreased with increasing flow. CONCLUSIONS: Vacuum improved efficiency only up to 300 mm Hg and was more dependent on increasing flow. Similarly, chatter correlated with 200 mm Hg vacuum only and was more correlated with flow. Limitations of this study include use of only 1 US power modulation and hard nuclear material. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Intravitreal AAV2.COMP-Ang1 Prevents Neurovascular Degeneration in a Murine Model of Diabetic Retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.

Ceramide mediates vascular dysfunction in diet-induced obesity by PP2A-mediated dephosphorylation of the eNOS-Akt complex.

Vascular dysfunction that accompanies obesity and insulin resistance may be mediated by lipid metabolites. We sought to determine if vascular ceramide leads to arterial dysfunction and to elucidate the underlying mechanisms. Pharmacological inhibition of de novo ceramide synthesis, using the Ser palmitoyl transferase inhibitor myriocin, and heterozygous deletion of dihydroceramide desaturase prevented vascular dysfunction and hypertension in mice after high-fat feeding. These findings were recapitulated in isolated arteries in vitro, confirming that ceramide impairs endothelium-dependent vasorelaxation in a tissue-autonomous manner. Studies in endothelial cells reveal that de novo ceramide biosynthesis induced protein phosphatase 2A (PP2A) association directly with the endothelial nitric oxide synthase (eNOS)/Akt/Hsp90 complex that was concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and by dephosphorylating eNOS. Ceramide decreased the association between PP2A and the predominantly cytosolic inhibitor 2 of PP2A. We conclude that ceramide mediates obesity-related vascular dysfunction by a mechanism that involves PP2A-mediated disruption of the eNOS/Akt/Hsp90 signaling complex. These results provide important insight into a pathway that represents a novel target for reversing obesity-related vascular dysfunction.