Hippo signaling in the ovary and polycystic ovarian syndrome.

PURPOSE: To provide a commentary on our understanding of the role that the Hippo signaling pathway may play in patients with polycystic ovarian syndrome (PCOS) and how this understanding may impact the diagnosis of PCOS. METHODS: We assessed publications discussing the role of the Hippo signaling pathway in the ovary. In particular, we discuss how Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, has been used in treatment of patients with primary ovarian insufficiency. Furthermore, we discuss our own data on variations in Hippo signaling pathway gene expression in cumulus cells isolated from women undergoing IVF with a previous diagnosis of PCOS. RESULTS AND CONCLUSIONS: Aberrant Hippo signaling in PCOS patients is likely a contributing mechanism to the multifactorial etiology of the disease. Given the challenge of discerning the underlying etiology of oligo-ovulation in some patients, especially those with normal body mass indices, and the need for customized stimulation protocols for PCOS patients who have an increased risk of over-response and higher percentage of immature oocyte yield, it is important to identify these patients prior to treatment. Hippo gene expression fingerprints could potentially be used to more accurately define patients with PCOS. Additionally, targeting this pathway with pharmacologic agents could lead to non-surgical therapeutic options for PCOS.

ROCK2 primes the endothelium for vascular hyperpermeability responses by raising baseline junctional tension.

Rho kinase mediates the effects of inflammatory permeability factors by increasing actomyosin-generated traction forces on endothelial adherens junctions, resulting in disassembly of intercellular junctions and increased vascular leakage. In vitro, this is accompanied by the Rho kinase-driven formation of prominent radial F-actin fibers, but the in vivo relevance of those F-actin fibers has been debated, suggesting other Rho kinase-mediated events to occur in vascular leak. Here, we delineated the contributions of the highly homologous isoforms of Rho kinase (ROCK1 and ROCK2) to vascular hyperpermeability responses. We show that ROCK2, rather than ROCK1 is the critical Rho kinase for regulation of thrombin receptor-mediated vascular permeability. Novel traction force mapping in endothelial monolayers, however, shows that ROCK2 is not required for the thrombin-induced force enhancements. Rather, ROCK2 is pivotal to baseline junctional tension as a novel mechanism by which Rho kinase primes the endothelium for hyperpermeability responses, independent from subsequent ROCK1-mediated contractile stress-fiber formation during the late phase of the permeability response.

ROCK-isoform-specific polarization of macrophages associated with age-related macular degeneration.

Age is a major risk factor in age-related macular degeneration (AMD), but the underlying cause is unknown. We find increased Rho-associated kinase (ROCK) signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2-like macrophages accumulated in AMD, but not in normal eyes, suggesting that these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.

Selective ROCK2 Inhibition In Focal Cerebral Ischemia.

OBJECTIVE: Rho-associated kinase (ROCK) is a key regulator of numerous processes in multiple cell types relevant in stroke pathophysiology. ROCK inhibitors have improved outcome in experimental models of acute ischemic or hemorrhagic stroke. However, the relevant ROCK isoform (ROCK1 or ROCK2) in acute stroke is not known. METHODS: We characterized the pharmacodynamic and pharmacokinetic profile, and tested the efficacy and safety of a novel selective ROCK2 inhibitor KD025 (formerly SLx-2119) in focal cerebral ischemia models in mice. RESULTS: KD025 dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 hours from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke. INTERPRETATION: Altogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation.

Targeting the leukocyte activation cascade: getting to the site of inflammation using microfabricated assays.

This paper describes the use of microfabricated devices to study the leukocyte activation cascade (LAC). The devices consist of microchannels fabricated in polydimethylsiloxane using soft lithography. Microfluidics, used to generate physiologically relevant levels of shear flow, was achieved by the simple attachment of a syringe pump. Microchannel surfaces were modified by self-assembled monolayer (SAM) chemistries. The devices were adapted to standard 96-well tissue culture format with microchannels that could accommodate either a monolayer of endothelial cells or a SAM with immobilized chemokines. Chemotaxis was performed using linear gradients of chemokine set in a 3D matrix. Using this approach, we demonstrated robust chemotaxis of primary human leukocytes (PHLs) in response to a gradient of the chemokine CCL2. Rolling and adhesion assays performed under shear flow demonstrated that leukocyte recruitment to the substrate was highly sensitive to both biological and physical forces. CCL2 and CXCL12 treatment of PHLs dose dependently increased activation and adhesion. These actions could be inhibited by the use of peptide or small molecule antagonists. These devices provide a robust platform to perform LAC assays under in vivo-like conditions.

A small-molecule inhibitor of enterocytic microsomal triglyceride transfer protein, SLx-4090: biochemical, pharmacodynamic, pharmacokinetic, and safety profile.

First-generation microsomal triglyceride transfer protein (MTP) inhibitors were designed to inhibit hepatic MTP and provide a novel treatment of dyslipidemia. Effective at lowering low-density lipoprotein-cholesterol (LDL-C), these inhibitors also elevate liver enzymes and induce hepatic steatosis in animals and humans. MTP is highly expressed in the enterocytes, lining the lumen of the jejunum, and is critical in the production of chylomicrons assembled from lipid/cholesterol and their transfer into systemic circulation. 6-(4'-Trifluoromethyl-6-methoxy-biphenyl-2-ylcarboxamido)-1,2,3,4-tetrahydroisoquinoline-2-carboxylic acid phenyl ester (SLx-4090) (IC(50) value ∼8 nM) was designed to inhibit only MTP localized to enterocytes. In Caco-2 cells SLx-4090 inhibited apolipoprotein B (IC(50) value ∼9.6 nM) but not apolipoprotein A1 secretion. Administered orally to rats SLx-4090 reduced postprandial lipids by >50% with an ED(50) value ∼7 mg/kg. SLx-4090 was not detected in the systemic or portal vein serum of the animals (lower limit of quantitation ∼5 ng/ml) after single or multiple oral doses in fasted rodents. When coadministered with tyloxapol, SLx-4090 did not inhibit the secretion of hepatic triglycerides (TG), consistent with the absence of systemic exposure. Chronic treatment with SLx-4090 in mice maintained on a high-fat diet decreased LDL-C and TG and resulted in weight loss without the elevation of liver enzymes or an increase in hepatic fat. The compound did not result in toxicity when administered to rats for 90 days at a dose of 1000 mg/kg per day. These data support the concept that the inhibition of enterocytic MTP could serve as a useful strategy in the treatment of metabolic disorders.

Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin.

Inhibitors of Rho kinase (ROCK) are a relatively new class of drugs with potential benefits in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK inhibitors modulate many cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds that inhibit both isoforms of ROCK, ROCK1 and ROCK2. This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2 inhibitor SLx-2119 in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 h, after which total RNA was isolated and genome-wide gene-expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor, a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and connective tissue growth factor mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced connective tissue growth factor mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have antifibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments.