Judd Cahoon

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.

Morpholino-mediated increase in soluble Flt-1 expression results in decreased ocular and tumor neovascularization

Background Angiogenesis is a key process in several ocular disorders and cancers. Soluble Flt-1 is an alternatively spliced form of the Flt-1 gene that retains the ligand-binding domain, but lacks the membrane-spanning and intracellular kinase domains of the full-length membrane bound Flt-1 (mbFlt-1) protein. Thus, sFlt-1 is an endogenous inhibitor of VEGF-A mediated angiogenesis. Synthetic mopholino oligomers directed against splice site targets can modulate splice variant expression. We hypothesize that morpholino-induced upregulation of sFlt-1 will suppress angiogenesis in clinically relevant models of macular degeneration and breast cancer. Methods and Findings In vivo morpholino constructs were designed to target murine exon/intron 13 junction of the Flt-1 transcript denoted VEGFR1_MOe13; standard nonspecific morpholino was used as control. After nucleofection of endothelial and breast adenocarcinoma cell lines, total RNA was extracted and real-time RT-PCR performed for sFlt-1 and mbFlt-1. Intravitreal injections of VEGFR1_MOe13 or control were done in a model of laser-induced choroidal neovascularization and intratumoral injections were performed in MBA-MD-231 xenografts in nude mice. VEGFR1_MOe13 elevated sFlt-1 mRNA expression and suppressed mbFlt-1 mRNA expression in vitro in multiple cellular backgrounds (p<0.001). VEGFR1_MOe13 also elevated sFlt/mbFlt-1 ratio in vivo after laser choroidal injury 5.5 fold (p<0.001) and suppressed laser-induced CNV by 50% (p = 0.0179). This latter effect was reversed by RNAi of sFlt-1, confirming specificity of morpholino activity through up-regulation of sFlt-1. In the xenograft model, VEGFR1_MOe13 regressed tumor volume by 88.9%, increased sFlt-1 mRNA expression, and reduced vascular density by 50% relative to control morpholino treatment (p<0.05). Conclusions Morpholino oligomers targeting the VEGFR1 mRNA exon/intron 13 junction promote production of soluble FLT-1 over membrane bound FLT-1, resulting in suppression of lesional volume in laser induced CNV and breast adenocarcinoma. Thus, morpholino manipulation of alternative splicing offers translational potential for therapy of angiogenic disorders.

Dual suppression of hemangiogenesis and lymphangiogenesis by splice-shifting morpholinos targeting vascular endothelial growth factor receptor 2 (KDR)

The KDR gene, which participates in angiogenesis and lymphangiogenesis, produces two functionally distinct protein products, membrane‐bound KDR (mbKDR) and its isoform, soluble KDR (sKDR). Since sKDR does not have a tyrosine kinase domain and does not dimerize, it is principally an antagonist of lymphangiogenesis by sequestering VEGF‐C. Alternative polyadenylation of exon 30 or intron 13 leads to the production of mbKDR or sKDR, respectively, yet the regulatory mechanisms are unknown. Here we show that an antisense morpholino oligomer directed against the exon 13‐intron 13 junction increases sKDR (suppressing lymphangiogenesis) and decreases mbKDR (inhibiting hemangiogenesis). The latent polyadenylation site in intron 13 of KDR is activated by blocking the upstream 5′ splicing site with an antisense morpholino oligomer. Intravitreal morpholino injection suppressed laser choroidal neovascularization while increasing sKDR. In the mouse cornea, subconjunctival injection of the morpholino‐inhibited corneal angiogenesis and lymphangiogenesis, and suppressed graft rejection after transplantation. Thus, this morpholino can be used for concurrent suppression of hemangiogenesis and lymphangiogenesis. This study offers new insight into the mechanisms and potential therapeutic modulation of alternative polyadenylation.—Uehara, H., Cho, YK., Simonis, J., Cahoon, J., Archer, B., Luo, L., Das, S. K., Singh, N., Ambati, J., Ambati, B. K. Dual suppression of hemangiogenesis and lymphangiogenesis by splice‐shifting morpholinos targeting vascular endothelial growth factor receptor 2 (KDR). FASEB J. 27, 76–85 (2013). www.fasebj.org

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.

Impact of micropulsed ultrasound power settings on the efficiency and chatter associated with lens-fragment removal

Purpose To determine the optimum power settings in micropulsed ultrasound (US). Setting John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, USA. Design Experimental study. Methods Pig lenses hardened to be comparable to dense human cataracts were cut into 2.0 mm cubes and removed using micropulsed longitudinal US with previously optimized settings (6 milliseconds on and 6 milliseconds off and using a 0.9 mm 30‐degree beveled bent phaco tip). The aspiration was set at 40 mL/min and the vacuum level at 550 mm Hg. Twenty lens cubes were tested with the power set from 10% to 100% in increments of 10%. Primary outcome measures were efficiency time (time to lens removal) and chatter (number of times the lens fragment visibly bounced off the tip). Results Efficiency time decreased with increasing power. There was a correlation between power and efficiency time (R2 = 0.41, P = .046), which was more substantial between 30% and 100% power (R2 = 0.71, P = .004). The mean number of chatter events did not differ significantly between power settings (R2 = 0.012, P = .1195). Conclusions There was a 5‐fold increase in efficiency between 10% power and 20% power, which likely indicates that there is a minimum power threshold for efficient breakup of the lens. Between 20% and 100% power, there was a linear, strong, and statistically significant improvement in efficiency in these lens fragments. In addition, with micropulsed US there was little chatter or microchatter throughout the power range. 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.

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.

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.

Bent versus straight tips in micropulsed longitudinal phacoemulsification

OBJECTIVE The aim of this study was to evaluate bent and straight phacoemulsification tips to determine which tip is more efficient in removal of lens fragments, using micropulsed longitudinal ultrasound in phacoemulsification. DESIGN In vitro laboratory study. METHODS The John A. Moran Eye Center Laboratories, University of Utah, Salt Lake City, Utah, was the study setting. Pig lenses hardened in a manner comparable with dense human cataracts were cut into 2-mm cubes and removed with micropulsed longitudinal ultrasound using settings previously shown to be optimally efficient (6 milliseconds on and 6 milliseconds off for a bent tip). To verify this time as most efficient for a straight tip, we also tested times of 5, 6, and 7 milliseconds time on and off. The tips were either straight or with a 20-degree bend. Twenty cubes were used for each comparative run. RESULTS For the straight tip, 6 milliseconds on (1.56 ± 0.815 seconds) was significantly more efficient than 7 milliseconds on (2.45 ± 1.56 seconds, p = 0.001) and not significantly more efficient than 5 milliseconds on (1.69 ± 0.86 seconds, p = 0.43). Five milliseconds off time (1.45 ± 0.76s) was more efficient than 6 milliseconds (2.06 ± 1.37 seconds, p = 0.004) and 7 milliseconds off (2.18 ± 1.24s, p = 0.001). The straight tip was more efficient than the bent tip (1.38 ± 0.83 versus 2.93 ± 2.14 seconds, p = 0.006). CONCLUSIONS Results are contrary to accepted common belief. Micropulsed longitudinal phacoemulsification is more efficient with a straight rather than a bent tip.

The impact of tip bevel angulation on phacoemulsification efficiency and chatter

Primary objective: To evaluate the effect of tip bevel angulation on phacoemulsification efficiency and chatter. Research design: In vitro laboratory study. Methods and procedures: Formalin-soaked porcine lenses were divided into 2 mm cubes. 0.9 mm straight 0, 15, 30, 45 beveled degree tips were used with micropulse ultrasound (6 ms on and 6 ms off ). Power was set at 100%, vacuum levels were set at 500 mmHg; and aspiration rates were set at 50 mL/min. Efficiency (time to lens removal) and chatter (number of lens fragment repulsions from the tip) were determined. Main outcomes and results: Changing the bevel angulation on a straight 0.9 mm phacoemulsification tip had no significant effect on efficiency. A 45 degree bevel was the most efficient tip overall. Chatter was seen to be significantly higher with a 15 degree tip (ANOVA, P=.0046). Conclusions: Tip bevel angulation has little effect on phacoemulsification efficiency and chatter, especially when optimized parameters are used. Limitations of this study include use of only one ultrasound power modulation and hard nuclear material.