Dallas S. Shi

Interleukin receptor activates a MYD88–ARNO–ARF6 cascade to disrupt vascular stability

The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1β (IL-1β), which in turn activate a well-described, myeloid-differentiation factor 88 (MYD88)-mediated, nuclear factor-κB (NF-κB)-dependent transcriptional pathway that results in inflammatory-cell activation and recruitment. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response. Paradoxically, the cytokines vital to a successful immune defence also have disruptive effects on endothelial cell–cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture. The mechanism of this barrier dissolution and its relationship to the canonical NF-κB pathway remain poorly defined. Here we show that the direct, immediate and disruptive effects of IL-1β on endothelial stability in a human in vitro cell model are NF-κB independent and are instead the result of signalling through the small GTPase ADP-ribosylation factor 6 (ARF6) and its activator ARF nucleotide binding site opener (ARNO; also known as CYTH2). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88–ARNO–ARF6 as a proximal IL-1β signalling pathway distinct from that mediated by NF-κB. Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation.

Proteasome function is required for platelet production

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1(fl/fl) Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1(fl/fl) Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma

Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as β-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and β-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.

Lack of CCM1 induces hypersprouting and impairs response to flow

Cerebral cavernous malformation (CCM) is a disease of vascular malformations known to be caused by mutations in one of three genes: CCM1, CCM2 or CCM3. Despite several studies, the mechanism of CCM lesion onset remains unclear. Using a Ccm1 knockout mouse model, we studied the morphogenesis of early lesion formation in the retina in order to provide insight into potential mechanisms. We demonstrate that lesions develop in a stereotypic location and pattern, preceded by endothelial hypersprouting as confirmed in a zebrafish model of disease. The vascular defects seen with loss of Ccm1 suggest a defect in endothelial flow response. Taken together, these results suggest new mechanisms of early CCM disease pathogenesis and provide a framework for further study.

Torsional power study using CENTURION phacoemulsification technology.

To evaluate the effect of varying levels of power on phacoemulsification efficiency using the CENTURION Vision System.

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.

Comparative analysis of the safety and efficacy of intracameral cefuroxime, moxifloxacin and vancomycin at the end of cataract surgery: a meta-analysis

Background Current practice methods are unclear as to the most safe and effective prophylactic pharmacotherapy and method of delivery to reduce postoperative endophthalmitis occurrence. Methods A systematic review and meta-analysis using Meta-analysis of Observational Studies in Epidemiology guidelines was performed to compare the efficacy of intracameral cefuroxime, moxifloxacin and vancomycin in preventing postphacoemulsification cataract surgery endophthalmitis. A safety analysis of intracameral antibiotics was concurrently performed. Data sources BIOSIS Previews, CINAHL, ClinicalTrials.gov, Cochrane Library, Dissertations & Theses, EMBASE, PubMed, ScienceDirect and Scopus were searched from inception to January 2017. Data were pooled using a random effects model. All articles were individually reviewed and data were extracted by two independent reviewers. Funnel plot, risk of bias and quality of evidence analyses were performed. Results Seventeen studies with over 900 000 eyes were included, which favoured the use of intracameral antibiotics at the end of cataract surgery (OR 0.20; 95% CI 0.13 to 0.32; P<0.00001). The average weighted postoperative endophthalmitis incidence rates with intracameral cefuroxime, moxifloxacin and vancomycin were 0.0332%, 0.0153% and 0.0106%, respectively. Secondary analyses showed no difference in efficacy between intracameral plus topical antibiotics versus intracameral alone (P>0.3). Most studies had low to moderate risk of bias. The safety analysis showed minimal toxicity for moxifloxacin. Dosing errors led to the majority of toxicities with cefuroxime. Although rare, vancomycin was associated with toxic retinal events. Conclusion Intracameral cefuroxime and moxifloxacin reduced endophthalmitis rates compared with controls with minimal or no toxicity events at standard doses. Additionally, intracameral antibiotics alone may be as effective as intracameral plus topical antibiotics.

Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs — ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.

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.