cGMP-dependent protein kinase contributes to hydrogen sulfide-stimulated vasorelaxation.

A growing body of evidence suggests that hydrogen sulfide (H₂S) is a signaling molecule in mammalian cells. In the cardiovascular system, H₂S enhances vasodilation and angiogenesis. H₂S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K(ATP)); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H₂S-induced vasorelaxation. The effect of H₂S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H₂S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H₂S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H₂S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H₂S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H₂S production) were reduced in vessels of PKG-I knockout mice (PKG-I⁻/⁻). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I⁻/⁻, suggesting that there is a cross-talk between K(ATP) and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation

Mitochondrial permeabilization engages NF-kappa B-dependent anti-tumour activity under caspase deficiency

Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the downregulation of inhibitor of apoptosis proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies

Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure.

Heart failure (HF) is a shared manifestation of several cardiovascular pathologies, including hypertension and myocardial infarction, and a limited repertoire of treatment modalities entails that the associated morbidity and mortality remain high. Impaired nitric oxide (NO)/guanylyl cyclase (GC)/cyclic guanosine-3',5'-monophosphate (cGMP) signaling, underpinned, in part, by up-regulation of cyclic nucleotide-hydrolyzing phosphodiesterase (PDE) isozymes, contributes to the pathogenesis of HF, and interventions targeted to enhancing cGMP have proven effective in preclinical models and patients. Numerous PDE isozymes coordinate the regulation of cardiac cGMP in the context of HF; PDE2 expression and activity are up-regulated in experimental and human HF, but a well-defined role for this isoform in pathogenesis has yet to be established, certainly in terms of cGMP signaling. Herein, using a selective pharmacological inhibitor of PDE2, BAY 60-7550, and transgenic mice lacking either NO-sensitive GC-1α (GC-1α-/-) or natriuretic peptide-responsive GC-A (GC-A-/-), we demonstrate that the blockade of PDE2 promotes cGMP signaling to offset the pathogenesis of experimental HF (induced by pressure overload or sympathetic hyperactivation), reversing the development of left ventricular hypertrophy, compromised contractility, and cardiac fibrosis. Moreover, we show that this beneficial pharmacodynamic profile is maintained in GC-A-/- mice but is absent in animals null for GC-1α or treated with a NO synthase inhibitor, revealing that PDE2 inhibition preferentially enhances NO/GC/cGMP signaling in the setting of HF to exert wide-ranging protection to preserve cardiac structure and function. These data substantiate the targeting of PDE2 in HF as a tangible approach to maximize myocardial cGMP signaling and enhancing therapy.British Heart Foundation Grant PG/10/077/28554

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness‐induced CCN1 activates β‐catenin nuclear translocation and signaling and that this contributes to upregulate N‐cadherin levels on the surface of the endothelium, in vitro. This facilitates N‐cadherin‐dependent cancer cell–endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness‐induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis

Recombinant Human VEGF165b Inhibits Experimental Choroidal Neovascularization

The alternative splice form of VEGF, VEGF-A165b, inhibits choroidal neovascularization at very low doses in mice, indicating that it may be an effective therapy for age-related macular degeneration, comparable with or better than existing anti-VEGF therapy

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity.

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion