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Cell migration is important in many physiological and pathological processes. Mechanisms of two-dimensional cell migration have been investigated most commonly by evaluating rates of cell migration into linearly scratched zones on the surfaces of culture plates. Here, we present a detailed description of a simple adaptation for the well-known and popular wound closure assay, using a circular wound instead of a straight line. This method demonstrates improved precision, reproducibility, and sampling objectivity for measurements of wound sizes as compared with classic scratch assays, enabling more accurate calculations of migration rate. The added benefits of the method are simplicity and low cost as compared with commercially available assays for generating circular wounds.

The risk for developing primary open-angle glaucoma (POAG) correlates with the magnitude of ocular hypertension (OHT) and the concentration of transforming growth factor-β2 (TGFβ2) in the aqueous humor. Effective treatment of POAG requires a detailed understanding of the interaction between pressure sensing mechanisms in the trabecular meshwork (TM) and biochemical risk factors. Here, we employed molecular, optical, electrophysiological, and tonometric strategies to establish the role of TGFβ2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels and intraocular pressure (IOP) regulation in a mouse model of TGFβ2-induced OHT. TGFβ2 upregulated expression of Trpv4 and Piezo1 transcripts and time-dependently augmented functional TRPV4 activation. TRPV4 agonists induced contractility of TM-seeded hydrogels, whereas pharmacological inhibition suppressed TGFβ2-induced hypercontractility and abrogated OHT in eyes overexpressing TGFβ2. Trpv4 -deficient mice resisted TGFβ2-driven increases in IOP, but nocturnal OHT was not additive to TGFβ-evoked OHT. Our study establishes the fundamental role of TGFβ as a modulator of mechanosensing in nonexcitable cells, identifies the TRPV4 channel as the final common mechanism for TM contractility and circadian and pathological OHT, and offers insights for future treatments that can lower IOP in the sizeable cohort of hypertensive glaucoma patients that resist current treatments.

Age and elevated intraocular pressure (IOP) are the two primary risk factors for glaucoma, an optic neuropathy that is the leading cause of irreversible blindness. In most people, IOP is tightly regulated over a lifetime by the conventional outflow tissues. However, the mechanistic contributions of age to conventional outflow dysregulation, elevated IOP and glaucoma are unknown. To address this gap in knowledge, we studied how age affects the morphology, biomechanical properties and function of conventional outflow tissues in C57BL/6 mice, which have an outflow system similar to humans. As reported in humans, we observed that IOP in mice was maintained within a tight range over their lifespan. Remarkably, despite a constellation of age‐related changes to the conventional outflow tissues that would be expected to hinder aqueous drainage and impair homeostatic function (decreased cellularity, increased pigment accumulation, increased cellular senescence and increased stiffness), outflow facility, a measure of conventional outflow tissue fluid conductivity, was stable with age. We conclude that the murine conventional outflow system has significant functional reserve in healthy eyes. However, these age‐related changes, when combined with other underlying factors, such as genetic susceptibility, are expected to increase risk for ocular hypertension and glaucoma. Age and elevated intraocular pressure (IOP) are the two primary risk factors for the second leading cause of blindness in the world, glaucoma. Despite a number of age‐related changes to the IOP‐regulating tissues of the eye that should impair homeostatic function (decreased cellularity, increased pigment accumulation, increased cellular senescence and increased stiffness), we observed that IOP was stable with age. Thus, healthy eyes appear to have significant functional reserve for IOP regulation, but when combined with other underlying factors, such as genetic susceptibility, are predicted to increase risk for elevated IOP and glaucoma.

Polymorphisms in the CAV1/2 gene loci impart increased risk for primary open-angle glaucoma (POAG). CAV1 encodes caveolin-1 (Cav1), which is required for biosynthesis of plasma membrane invaginations called caveolae. Cav1 knockout mice exhibit elevated intraocular pressure (IOP) and decreased outflow facility, but the mechanistic role of Cav1 in IOP homeostasis is unknown. We hypothesized that caveolae sequester/inhibit RhoA, to regulate trabecular meshwork (TM) mechanosensing and contractile tone. Using phosphorylated myosin light chain (pMLC) as a surrogate indicator for Rho/ROCK activity and contractile tone, we found that pMLC was elevated in Cav1-deficient TM cells compared to control (131 ± 10%, n = 10, p = 0.016). Elevation of pMLC levels following Cav1 knockdown occurred in cells on a soft surface (137 ± 7%, n = 24, p < 0.0001), but not on a hard surface (122 ± 17%, n = 12, p = 0.22). In Cav1-deficient TM cells where pMLC was elevated, Rho activity was also increased (123 ± 7%, n = 6, p = 0.017), suggesting activation of the Rho/ROCK pathway. Cyclic stretch reduced pMLC/MLC levels in TM cells (69 ± 7% n = 9, p = 0.002) and in Cav1-deficient TM cells, although not significantly (77 ± 11% n = 10, p = 0.059). Treatment with the Cav1 scaffolding domain mimetic, cavtratin (1 μM) caused a reduction in pMLC (70 ± 5% n = 7, p = 0.001), as did treatment with the scaffolding domain mutant cavnoxin (1 μM) (82 ± 7% n = 7, p = 0.04). Data suggest that caveolae differentially regulate RhoA signaling, and that caveolae participate in TM mechanotransduction. Cav1 regulation of these key TM functions provide evidence for underlying mechanisms linking polymorphisms in the Cav1/2 gene loci with increased POAG risk.

Aquaporin-1 (AQP1) has been proposed as a dual water and cation channel that when upregulated in cancers enhances cell migration rates; however, the mechanism remains unknown. Previous work identified AqB011 as an inhibitor of the gated human AQP1 cation conductance, and bacopaside II as a blocker of AQP1 water pores. In two colorectal adenocarcinoma cell lines, high levels of AQP1 transcript were confirmed in HT29, and low levels in SW480 cells, by quantitative PCR (polymerase chain reaction). Comparable differences in membrane AQP1 protein levels were demonstrated by immunofluorescence imaging. Migration rates were quantified using circular wound closure assays and live-cell tracking. AqB011 and bacopaside II, applied in combination, produced greater inhibitory effects on cell migration than did either agent alone. The high efficacy of AqB011 alone and in combination with bacopaside II in slowing HT29 cell motility correlated with abundant membrane localization of AQP1 protein. In SW480, neither agent alone was effective in blocking cell motility; however, combined application did cause inhibition of motility, consistent with low levels of membrane AQP1 expression. Bacopaside alone or combined with AqB011 also significantly impaired lamellipodial formation in both cell lines. Knockdown of AQP1 with siRNA (confirmed by quantitative PCR) reduced the effectiveness of the combined inhibitors, confirming AQP1 as a target of action. Invasiveness measured using transwell filters layered with extracellular matrix in both cell lines was inhibited by AqB011, with a greater potency in HT29 than SW480. A side effect of bacopaside II at high doses was a potentiation of invasiveness, that was reversed by AqB011. Results here are the first to demonstrate that combined block of the AQP1 ion channel and water pores is more potent in impairing motility across diverse classes of colon cancer cells than single agents alone.

The risk for developing primary open-angle glaucoma (POAG) correlates with the magnitude of ocular hypertension (OHT) and the concentration of transforming growth factor-β2 (TGFβ2) in the aqueous humor. Effective treatment of POAG requires a detailed understanding of the interaction between pressure sensing mechanisms in the trabecular meshwork (TM) and biochemical risk factors. Here, we employed molecular, optical, electrophysiological, and tonometric strategies to establish the role of TGFβ2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels and intraocular pressure (IOP) regulation in a mouse model of TGFβ2-induced OHT. TGFβ2 upregulated expression of Trpv4 and Piezo1 transcripts and time-dependently augmented functional TRPV4 activation. TRPV4 agonists induced contractility of TM-seeded hydrogels, whereas pharmacological inhibition suppressed TGFβ2-induced hypercontractility and abrogated OHT in eyes overexpressing TGFβ2. Trpv4 -deficient mice resisted TGFβ2-driven increases in IOP, but nocturnal OHT was not additive to TGFβ-evoked OHT. Our study establishes the fundamental role of TGFβ as a modulator of mechanosensing in nonexcitable cells, identifies the TRPV4 channel as the final common mechanism for TM contractility and circadian and pathological OHT, and offers insights for future treatments that can lower IOP in the sizeable cohort of hypertensive glaucoma patients that resist current treatments.

The risk for developing primary open-angle glaucoma (POAG) correlates with the magnitude of ocular hypertension (OHT) and the concentration of transforming growth factor-β2 (TGFβ2) in the aqueous humor. Effective treatment of POAG requires detailed understanding of interaction between pressure sensing mechanisms in the trabecular meshwork (TM) and biochemical risk factors. Here, we employed molecular, optical, electrophysiological and tonometric strategies to establish the role of TGFβ2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels, and intraocular pressure (IOP) regulation in a mouse model of TGFβ2 -induced OHT. TGFβ2 upregulated expression of and transcripts and time-dependently augmented functional TRPV4 activation. TRPV4 agonists induced contractility of TM-seeded hydrogels whereas pharmacological inhibition suppressed TGFβ2-induced hypercontractility and abrogated OHT in eyes overexpressing TGFβ2. -deficient mice resisted TGFβ2-driven increases in IOP, but nocturnal OHT was not additive to TGFβ-evoked OHT. Our study establishes the fundamental role of TGFβ as a modulator of mechanosensing in nonexcitable cells, identifies the TRPV4 channel as the final common mechanism for TM contractility and circadian and pathological OHT, and offers insights for future treatments that can lower IOP in the sizeable cohort of hypertensive glaucoma patients that resist current treatments.

Pathologic alterations in the biomechanical properties of the Schlemm's canal (SC) inner wall endothelium and its immediate vicinity are strongly associated with ocular hypertension in glaucoma due to decreased outflow facility. Specifically, the underlying trabecular meshwork is substantially stiffer in glaucomatous eyes compared to that from normal eyes. This raises the possibility of a critical involvement of mechanotransduction processes in driving SC cell dysfunction. Yes-associated protein (YAP) has emerged as a key contributor to glaucoma pathogenesis. However, the molecular underpinnings of SC cell YAP mechanosignaling in response to glaucomatous extracellular matrix (ECM) stiffening are not well understood. Using a novel biopolymer hydrogel that facilitates dynamic and reversible stiffness tuning, we investigated how ECM stiffening modulates YAP activity in primary human SC cells, and whether disruption of YAP mechanosignaling attenuates SC cell pathobiology and increases outflow facility. We demonstrated that ECM stiffening drives pathologic YAP activation and cytoskeletal reorganization in SC cells, which was fully reversible by matrix softening in a distinct time-dependent manner. Furthermore, we showed that pharmacologic or genetic disruption of YAP mechanosignaling abrogates stiffness-induced SC cell dysfunction involving altered cytoskeletal and ECM remodeling. Lastly, we found that perfusion of the clinically-used, small molecule YAP inhibitor verteporfin (without light activation) increases outflow facility in normal mouse eyes. Collectively, our data provide new evidence for a pathologic role of aberrant YAP mechanosignaling in SC cell dysfunction and suggest that YAP inhibition has therapeutic value for treating ocular hypertension in glaucoma.

Intraocular pressure is tightly regulated by the conventional outflow tissues, preventing ocular hypertension that leads to neurodegeneration of the optic nerve, or glaucoma. Although macrophages reside throughout the conventional outflow tract, their role in regulating intraocular pressure remains unknown. Using macrophage lineage tracing approaches, we uncovered a dual macrophage ontogeny with distinct spatial organizations across the mouse lifespan. Long-lived, resident tissue macrophages concentrated in the trabecular meshwork and Schlemm's canal, whereas short-lived monocyte-derived macrophages, instead, were abundant around distal vessels. Specific depletion of resident tissue macrophages triggered elevated intraocular pressure and outflow resistance, linked to aberrant extracellular matrix turnover in the resistance-generating tissues of the trabecular meshwork. This dysregulated physiology and tissue remodeling were not observed when we depleted monocyte-derived macrophages. Results show ontogeny and tissue-specific macrophage function within the outflow tract, uncovering the integral homeostatic role of resident tissue macrophages in resistance-generating tissues whose dysfunction is responsible for glaucoma.

Age and elevated intraocular pressure (IOP) are the two primary risk factors for glaucoma, an optic neuropathy that is the leading cause of irreversible blindness. In most people, IOP is tightly regulated over a lifetime by the conventional outflow tissues. However, the mechanistic contributions of age to conventional outflow dysregulation, elevated IOP and glaucoma are unknown. To address this gap in knowledge, we studied how age affects the morphology, biomechanical properties and function of conventional outflow tissues in C57BL/6 mice, which have an outflow system similar to humans. As reported in humans, we observed that IOP in mice was maintained within a tight range over their lifespan. Remarkably, despite a constellation of age-related changes to the conventional outflow tissues that would be expected to hinder aqueous drainage and impair homeostatic function (decreased cellularity, increased pigment accumulation, increased cellular senescence and increased stiffness), outflow facility, a measure of conventional outflow tissue fluid conductivity, was stable with age. We conclude that the murine conventional outflow system has significant functional reserve in healthy eyes. However, these age-related changes, when combined with other underlying factors, such as genetic susceptibility, are expected to increase risk for ocular hypertension and glaucoma.Competing Interest StatementThe authors have declared no competing interest.Footnotes* We have added new results, reformatted figures and made additional edits to text.