Endothelin A receptor drives invadopodia function and cell motility through the ß-arrestin/PDZ-RhoGEF pathway in ovarian carcinoma.

The endothelin-1 (ET-1)/ET A receptor (ETAR) signalling pathway is a well-established driver of epithelial ovarian cancer (EOC) progression. One key process promoted by ET-1 is tumor cell invasion, which requires the scaffolding functions of ß-arrestin-1 (ß-arr1) downstream of the receptor; however, the potential role of ET-1 in inducing invadopodia, which are crucial for cellular invasion and tumor metastasis, is completely unknown. We describe here that ET-1/ETAR, through ß-arr1, activates RhoA and RhoC GTPase and downstream ROCK (Rho-associated coiled coil-forming kinase) kinase activity, promoting actin-based dynamic remodelling and enhanced cell invasion. This is accomplished by the direct interaction of ß-arr1 with PDZ-RhoGEF (postsynaptic density protein 95/disc-large/zonula occludens-RhoGEF). Interestingly, ETAR-mediated invasive properties are related to the regulation of invadopodia, as evaluated by colocalization of actin with cortactin, as well as with TKS5 and MT1-MMP (membrane type 1-matrix metalloproteinase) with areas of matrix degradation, and activation of cofilin pathway, which is crucial for regulating invadopodia activity. Depletion of PDZ-RhoGEF, or ß-arr1, or RhoC, as well as the treatment with the dual ET-1 receptor antagonist macitentan, significantly impairs invadopodia function, MMP activity and invasion, demonstrating that ß-arr1/PDZ-RhoGEF interaction mediates ETAR-driven ROCK-LIMK-cofilin pathway through the control of RhoC activity. In vivo, macitentan is able to inhibit metastatic dissemination and cofilin phosphorylation. Collectively, our data unveil a noncanonical activation of the RhoC/ROCK pathway through the ß-arr1/PDZ-RhoGEF complex as a regulator of ETAR-induced motility and metastasis, establishing ET-1 axis as a novel regulator of invadopodia protrusions through the RhoC/ROCK/LIMK/cofilin pathway during the initial steps of EOC invasion.

Attachment and proliferation of bovine aortic endothelial cells onto additive modified poly(ether urethane ureas).

To better understand endothelial cell interactions with poly(ether urethane urea) (PEUU) materials, and to assess bovine aortic endothelial cell attachment, films were incubated for 24 h with BAEC in media containing 5% fetal bovine serum. Other films were allowed to incubate for 4 more days in media containing 5% fetal bovine serum without cells to assess BAEC proliferation. The assay was performed on PEUU films modified with acrylate and methacrylate polymer and copolymer additives that spanned a wide range on the hydrophobicity/hydrophilicity scale. Tissue culture polystyrene (TCPS) was used as a control. The assay showed that PEUU films loaded with Methacrol 2138F [copoly(diisopropylaminoethyl methacrylate [DI-PAM]/decyl methacrylate [DM]) (3/1)] or with its hydrophilic component, DIPAM, in homopolymer form (i.e., h-DIPAM), significantly enhanced BAEC attachment (approximately 80% of TCPS values) and proliferation (approximately 80%) when compared to unloaded PEUU films (attachment 73%; proliferation, 47%) or to PEUU films loaded with the more hydrophobic acrylate or methacrylate polymer additives (attachment, 32-69%; proliferation, 18-57%). The assay also showed that PEUU films coated with homopoly(diisopropylaminoethyl acrylate) (h-DIPAA) significantly enhanced BAEC attachment and proliferation when compared to PEUU films coated with h-decyl acrylate (h-DA); films coated with the copolymer of these two acrylates (i.e., co-[DIPAA/DA] [3/1]) showed intermediate behavior. To explain the enhancement of BAEC interaction with films loaded with Methacrol 2138F or h-DIPAM, when compared to unmodified PEUU films or to PEUU films loaded with more hydrophobic acrylate and methacrylate polymer additives, it was assumed that the additives near the surface region of the solvent swollen PEUU matrix may have migrated to, or near to, the PEUU-air interface during film formation, creating an additive enriched PEUU surface region. It is suggested that, once at this surface region, dynamic reorientation in response to an aqueous medium ensured the additives were able significantly to influence protein adsorption, and concomitant endothelial cell behavior, but only if they interacted with aqueous media more favorably than the PEUU. The ability of Methacrol and h-DIPAM additives to enhance endothelial cell behavior is argued to be the result of increased hydrophilicity. This is the result of exposed, hydrogen-bonding DIPAM moieties and increased surface flexibility, which is itself due to the hydration of unhindered Methacrol chains, which may create an additive enriched PEUU-water interfacial zone.

Protein adsorption and endothelial cell attachment and proliferation on PAPI-based additive modified poly(ether urethane ureas).

To better understand vascular interactions with poly(ether urethane urea) (PEUU) materials, protein adsorption, and endothelial cell attachment and proliferation assays were performed on a base PEUU formulation, on PEUU formulations loaded with hydrophobic and amphiphilic poly(methylene-[polyphenyl isocyanate]) (PAPI) based additives, and on PEUU formulations in which some of the polymer chains had been endcapped with either diisopropylaminoethyl (DIPAA) or decyl (DA) moieties. Protein adsorption experiments with PAPI-based additives showed that additive loaded PEUU formulations adsorbed significantly lower amounts of the studied proteins than did the unloaded PEUU. Protein adsorption to the DA and DIPAA endcapped PEUU films was found not to vary consistently from that of the unloaded PEUU film. Endothelial cell attachment and proliferation experiments with PAPI-DA and polyethylene glycol-PAPI-DA (PEG-PAPI-DA) loaded PEUU films showed that many of the films exhibited attachment and proliferation that was significantly enhanced compared to PEUU A' and that approached or equaled that of the tissue culture polystyrene control. Experiments with PAPI-DIPAA and PEG-PAPI-DIPAA loaded PEUU films exhibited attachment and proliferation data that was often below 10% of the tissue culture polystyrene control values. Experiments with the DA and DIPAA endcapped PEUU films showed endothelial cell attachment and proliferation that was statistically indistinguishable from the PEUU A' values. Contact angle analysis was carried out on the endcapped PEUU films, on the PAPI-based additive loaded PEUU films, and on PEUU A' using the sessile drop method. The advancing and receding contact angle behavior of the PAPI-based additive loaded PEUU films deviated markedly from the behavior of PEUU A', suggesting that the additives were present at the PEUU-water interface. The contact angle behavior of the endcapped PEUUs was similar to that of PEUU A', suggesting that the DA and DIPAA endcap moieties did not exist at the hydrated PEUU surface in appreciable quantities. To explain the differences in protein adsorption and endothelial cell behavior on the air side of additive loaded PEUUs when compared to the base PEUU, it was assumed that the additives near this region of the solvent swollen PEUU matrix may have migrated to, at, or near the PEUU-air interface during film formation, creating an additive enriched PEUU surface region.(ABSTRACT TRUNCATED AT 400 WORDS)