Pharmacologic Manipulation of Conventional Outflow Facility in Ex Vivo Mouse Eyes

PURPOSE. Mouse models are useful for glaucoma research, but it is unclear whether intraocular pressure (IOP) regulation in mice operates through mechanisms similar to those in humans. Our goal was to determine whether pharmacologic compounds that affect conventional outflow facility in human eyes exert similar effects in C57BL/6 mice. METHODS. A computerized perfusion system was used to measure conventional outflow facility in enucleated mouse eyes ex vivo. Paired eyes were perfused sequentially, either immediately after enucleation or after 3 hours storage at 4°C. Three groups of experiments examined sphingosine 1-phosphate (S1P), S1P with antagonists to S1P(1) and S1P(2) receptors, and the prostanoid EP(4) receptor agonist 3,7-dithia PGE(1). We also examined whether a 24-hour postmortem delay affected the response to 3,7-dithia prostaglandin E(1) (PGE(1)). RESULTS. S1P decreased facility by 39%, and was blocked almost completely by an S1P(2), but not S1P(1), receptor antagonist. The S1P(2) receptor antagonist alone increased facility nearly 2-fold. 3,7-dithia PGE(1) increased facility by 106% within 3 hours postmortem. By 24 hours postmortem, the facility increase caused by 3,7-dithia PGE(1) was reduced 3-fold, yet remained statistically detectable. CONCLUSIONS. C57BL/6 mice showed opposing effects of S1P(2) and EP(4) receptor activation on conventional outflow facility, as observed in human eyes. Pharmacologic effects on facility were detectable up to 24 hours postmortem in enucleated mouse eyes. Mice are suitable models to examine the pharmacology of S1P and EP(4) receptor stimulation on IOP regulation as occurs within the conventional outflow pathway of human eyes, and are promising for studying other aspects of aqueous outflow dynamics

The effects of monocytes on tumor cell extravasation in a 3D vascularized microfluidic model

Metastasis is the leading cause of cancer-related deaths. Recent developments in cancer immunotherapy have shown exciting therapeutic promise for metastatic patients. While most therapies target T cells, other immune cells, such as monocytes, hold great promise for therapeutic intervention. In our study, we provide primary evidence of direct engagement between human monocytes and tumor cells in a 3D vascularized microfluidic model. We first characterize the novel application of our model to investigate and visualize at high resolution the evolution of monocytes as they migrate from the intravascular to the extravascular micro-environment. We also demonstrate their differentiation into macrophages in our all-human model. Our model replicates physiological differences between different monocyte subsets. In particular, we report that inflammatory, but not patrolling, monocytes rely on actomyosin based motility. Finally, we exploit this platform to study the effect of monocytes, at different stages of their life cycle, on cancer cell extravasation. Our data demonstrates that monocytes can directly reduce cancer cell extravasation in a non-contact dependent manner. In contrast, we see little effect of monocytes on cancer cell extravasation once monocytes transmigrate through the vasculature and are macrophage-like. Taken together, our study brings novel insight into the role of monocytes in cancer cell extravasation, which is an important step in the metastatic cascade. These findings establish our microfluidic platform as a powerful tool to investigate the characteristics and function of monocytes and monocyte-derived macrophages in normal and diseased states. We propose that monocyte-cancer cell interactions could be targeted to potentiate the anti-metastatic effect we observe in vitro, possibly expanding the milieu of immunotherapies available to tame metastasis

Mouse models of intra-ocular pressure, with applications to glaucoma

Glaucoma is the second most common cause of blindness worldwide and is often associated with an increased intraocular pressure (IOP). IOP is determined by the dynamics of aqueous humour, the liquid filling the anterior segment of the eye. In primary-open angle glaucoma, the elevated IOP is caused by a decreased outflow facility of aqueous humour through the conventional pathway (decreased conventional facility, C). Existing glaucoma therapies aim to lower IOP, but remain inefficient because they fail to target C. As a result, there is growing interest in using the mouse to unravel the mechanisms controlling C. The mouse is a particularly powerful model because it can be routinely manipulated genetically, thereby giving insight into molecules and genes involved in determining C. However, it is still not clear whether mice are suitable surrogates for studying human C. To fill this gap, we aim to demonstrate that the mouse is a suitable model for human IOP regulation, and to then use this model to investigate key processes in IOP regulation. To achieve this aim we used an existing perfusion system to measure C in enucleated mouse eyes. First, we improved the perfusion system by including hydration and temperature control to better mimic in vivo perfusions. Secondly, selected receptor-mediated drugs were found to have similar effects on C in mice as they did in past human studies. Finally, we show, amongst other studies, anti-metabolic agents decreased C, suggesting aqueous humour outflow is metabolic dependent. We conclude that the mouse is a valid model for studying human conventional facility, yielding novel insight into the mechanisms controlling conventional facility. Importantly, this will help the design of novel efficient anti-glaucoma treatments. Notably, this project will have brought fundamental insight into mouse eye perfusions, thereby consolidating the technique for future studies.Open Acces

Aqueous humor outflow requires active cellular metabolism in mice.

Purpose: Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice. Methods: We measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2. Results: Reducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P<0.001), but reducing temperature did not affect ATP. Conclusions: Inhibiting cellular metabolism decreases outflow facility within minutes. This implies that outflow is not entirely passive, but depends partly on energy-dependent cellular processes, at least in mice. This study also suggests that there is a yet unidentified mechanism, which is strongly temperature-dependent but metabolism-independent, that is necessary for nearly half of normal outflow function in mice

eNOS, a Pressure-Dependent Regulator of Intraocular Pressure

PURPOSE. Pathology in the primary drainage pathway for aqueous humor in the eye is responsible for ocular hypertension, the only treatable risk factor in patients with glaucoma. Unfortunately, the mechanisms that regulate pressure-dependent drainage of aqueous humor and thus intraocular pressure (IOP) are unknown. To better understand one possible underlying molecular factor that regulates IOP, nitric oxide (NO), pressure-dependent drainage in transgenic mice overexpressing endothelial NO synthase (eNOS) was studied. METHODS. IOP was measured by rebound tonometry in mice, and pressure versus flow data were measured by ex vivo perfusion at multiple pressures between 8 and 45 mm Hg, using mock AH Ϯ100 M L-NAME. A subset of eyes was examined histologically using standard techniques or was assayed for fusion protein expression by Western blot analysis. RESULTS. IOP was lower (9.6 Ϯ 2.7 vs. 11.4 Ϯ 2.5 mm Hg; mean Ϯ SD; P ϭ 0.04) and pressure-dependent drainage was higher (0.0154 Ϯ 0.006 vs. 0.0066 Ϯ 0.0009 L/min/mm Hg; P ϭ 0.002) in the transgenic mice than in the wild-type animals; however, pressure-independent drainage was unaffected. The NOS inhibitor L-NAME normalized pressure-dependent drainage in transgenic animals. For IOP Ͼ35 mm Hg, the slope of the pressure-flow curve in wild-type mice increased to match that seen in transgenic mice. Shear stress in the pressure-dependent pathway at elevated pressures was calculated to be in a range known to affect eNOS expression and activity in vascular endothelia. CONCLUSIONS. Endothelial NOS overexpression lowers IOP by increasing pressure-dependent drainage in the mouse eye. Data are consistent with NO&apos;s having a mechanoregulatory role in aqueous humor dynamics, with eNOS induction at elevated IOPs leading to increased pressure-dependent outflow. (Invest Ophthalmol Vis Sci. 2011;52:9438-9444

Macrophage-Secreted TNFα and TGFβ1 Influence Migration Speed and Persistence of Cancer Cells in 3D Tissue Culture via Independent Pathways

The ability of a cancer cell to migrate through the dense extracellular matrix within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment, but the basis for their effects is not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB-dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFβ1 dual blockade as an antimetastatic strategy in solid tumors.National Institutes of Health (U.S.) (Grant U01 CA202177-01

eNOS, a Pressure-Dependent Regulator of Intraocular Pressure

A comparison of the ocular pressure–flow relationship in wild-type and transgenic mice overexpressing eNOS revealed that nitric oxide participates in a pressure-dependent feedback loop for the drainage of aqueous humor from the mouse eye

Pharmacologic Manipulation of Conventional Outflow Facility in Ex Vivo Mouse Eyes

PURPOSE. Mouse models are useful for glaucoma research, but it is unclear whether intraocular pressure (IOP) regulation in mice operates through mechanisms similar to those in humans. Our goal was to determine whether pharmacologic compounds that affect conventional outflow facility in human eyes exert similar effects in C57BL/6 mice. METHODS. A computerized perfusion system was used to measure conventional outflow facility in enucleated mouse eyes ex vivo. Paired eyes were perfused sequentially, either immediately after enucleation or after 3 hours storage at 48C. Three groups of experiments examined sphingosine 1-phosphate (S1P), S1P with antagonists to S1P 1 and S1P 2 receptors, and the prostanoid EP 4 receptor agonist 3,7-dithia PGE 1 . We also examined whether a 24-hour postmortem delay affected the response to 3,7-dithia prostaglandin E 1 (PGE 1 ). RESULTS. S1P decreased facility by 39%, and was blocked almost completely by an S1P 2 , but not S1P 1 , receptor antagonist. The S1P 2 receptor antagonist alone increased facility nearly 2-fold. 3,7-dithia PGE 1 increased facility by 106% within 3 hours postmortem. By 24 hours postmortem, the facility increase caused by 3,7-dithia PGE 1 was reduced 3-fold, yet remained statistically detectable. CONCLUSIONS. C57BL/6 mice showed opposing effects of S1P 2 and EP 4 receptor activation on conventional outflow facility, as observed in human eyes. Pharmacologic effects on facility were detectable up to 24 hours postmortem in enucleated mouse eyes. Mice are suitable models to examine the pharmacology of S1P and EP 4 receptor stimulation on IOP regulation as occurs within the conventional outflow pathway of human eyes, and are promising for studying other aspects of aqueous outflow dynamics. (Invest Ophthalmol Vis Sci. 2012;53:5838-5845