Fluoxetine exerts anti-inflammatory effects on human epidermal keratinocytes and suppresses their endothelin release.

Fluoxetine is a safe antidepressant with remarkable anti-inflammatory actions; therefore, we aimed to investigate its effects on immortalized (HaCaT) as well as primary human epidermal keratinocytes in a polyinosinic-polycytidylic acid (p(I:C))-induced inflammatory model. We found that a non-cytotoxic concentration (MTT-assay, CyQUANT-assay) of fluoxetine significantly suppressed p(I:C)-induced expression and release of several pro-inflammatory cytokines (Q-PCR, cytokine array, ELISA), and it decreased the release of the itch mediator endothelins (ELISA). These effects were not mediated by the inhibition of the NF-κB or p38 MAPK pathways (western blot), or by the suppression of the p(I:C)-induced elevation of mitochondrial ROS production (MitoSOX Red labeling). Instead, unbiased activity profiling revealed that they were most likely mediated via the inhibition of the phosphoinositide 3-kinase (PI3K) pathway. Importantly, the PI3K-inhibitor GDC0941 fully mimicked the effects of fluoxetine (Q-PCR, ELISA). Although fluoxetine was able to occupy the binding site of GDC0941 (in silico molecular docking), and exerted direct inhibitory effect on PI3K (cell-free PI3K activity assay), it exhibited much lower potency and efficacy as compared to GDC0941. Finally, RNA-Seq analysis revealed that fluoxetine deeply influenced the transcriptional alterations induced by p(I:C)-treatment, and exerted an overall anti-inflammatory activity. Collectively, our findings demonstrate that fluoxetine exerts potent anti-inflammatory effects, and suppresses the release of the endogenous itch mediator endothelins in human keratinocytes, most likely via interfering with the PI3K pathway. Thus, clinical studies are encouraged to explore whether the currently reported beneficial effects translate in vivo following its topical administration in inflammatory and pruritic dermatoses.

Long-term efficacy and safety of 1% glycopyrronium bromide cream in patients with severe primary axillary hyperhidrosis: Results from a Phase 3b trial.

BACKGROUND: Primary axillary hyperhidrosis (PAHH) strongly affects the patient's quality of life. To date, topical treatment options are limited. One percent glycopyrronium bromide (GPB) showed promising efficacy and safety in a pivotal 4-week Phase 3a study. OBJECTIVES: To assess efficacy and safety of topical 1% GPB cream in patients with severe PAHH in a long-term study of 72 weeks versus baseline. METHODS: This was a long-term, open-label, Phase 3b trial for 72 weeks including 518 patients with severe PAHH. Patients were treated with 1% GPB cream once daily for 4 weeks, followed by a flexible dosing scheme (min. twice per week, max. once daily). Primary endpoint was the absolute change in sweat production from baseline to week 12. Further study endpoints included assessment of the severity of PAHH and the impact on quality of life. RESULTS: Total median sweat production decreased by 119.30 mg (-65.6%, both median) until week 12. Absolute change in sweat production from baseline to week 12 in logarithmic values was statistically significant (p < 0.0001). Patients' quality of life was improved at all study time points compared to baseline, as assessed by Hyperhidrosis Quality of Life Index and Dermatology Life Quality Index (p < 0.0001). Treatment was safe and locally well-tolerated with only few mild to moderate adverse drug reactions (ADRs). Dry mouth and application site erythema were the most common reported ADRs. CONCLUSIONS: Treatment with 1% GPB cream over 72 weeks significantly reduces sweat production and improves quality of life in patients with severe PAHH. One percent GPB cream is well-tolerated and provides an effective treatment option for long-term use in patients with severe PAHH.

Current and emerging treatments targeting the neuroendocrine system for disorders of the skin and its appendages.

The skin as a neuroendocrine organ and the role of neuroendocrine signalling in the development of disorders affecting the skin and its appendages has received increasing attention in the last years. Different neuroendocrine systems have been described in the barrier organ skin, including the thyroid system, the hypothalamic-pituitary-adrenal axis, the opioid, the endocannabinoid, the cholinergic, the secosteroidogenic and the serotonergic systems. All of these systems have been implicated in the development of skin diseases, which often have an inflammatory origin. These discoveries have led to an increase in the development of new drugs targeting components of neuroendocrine signalling pathways. Additionally, attempts have been made to repurpose already approved drugs targeting neuroendocrine signalling pathways in other organs for the treatment of skin diseases. Recently published results from preclinical and clinical studies look promising and may offer improved therapies to patients suffering from skin diseases in the near future. In this review, from a pharmaceutical point of view, we focus on recent progress in synthetic drug development of compounds targeting neuroendocrine signalling in the skin and its appendages to treat skin diseases such as atopic dermatitis, psoriasis, acne, alopecia areata and hyperhidrosis.

Small molecule drugs for the treatment of pruritus in patients with atopic dermatitis.

Chronic pruritus is a cardinal symptom of the inflammatory skin disease atopic dermatitis (AD). Pathogenic mechanisms in the periphery, spinal cord and the brain have been implicated in AD-related pruritus. Therefore, both systemic and topical administration of drugs could potentially provide relief. Despite efforts to elucidate the mechanisms behind AD-related pruritus and the relative contribution of peripheral nervous system and central nervous system (CNS), specific and successful treatment options have not yet been developed. Several small molecule drugs are currently being investigated to treat AD and AD-related pruritus. These small molecule drugs can be applied systemically but also topically, as they are able to penetrate into the skin due to their small size. Small molecule drugs specifically targeting peripheral itch transmission, e.g. peripherally selective κ-opioid receptors agonists and neurokinin 1 receptors antagonists, have so far been unable to improve AD-related pruritus when applied systemically, possibly because of the lack of CNS activity. Current evidence from clinical and preclinical trials with centrally acting or peripherally selective oral κ-opioid receptors agonists implies that CNS activity is required for an antipruritic effect. CNS activity is, however, directly associated with CNS-mediated side-effects. On the other hand, topical application of small molecules with anti-inflammatory activity such as Janus kinase inhibitors and phosphodiesterase 4 inhibitors, and also of κ-opioid receptor agonists, has shown promising results regarding their ability to reduce AD-related pruritus. In conclusion, topical application of anti-inflammatory compounds appears to be a highly promising strategy for the treatment of AD-related pruritus.

Influence of Buffers of Different pH and Composition on the Murine Skin Barrier, Epidermal Proliferation, Differentiation, and Inflammation.

The pH of the skin is tightly regulated by endogenous buffering systems. We examined the influence of buffers of different pH and composition on skin barrier repair, pH, inflammation, and epidermal thickness/proliferation/differentiation. After tape-stripping in hairless mice buffers with pH 4-7 were applied in patch test chambers. After removal of the chambers, skin pH and transepidermal water loss (TEWL) were monitored for 24 h, and biopsies were taken for histology/immunohistology. Hairless mice showed a basal skin pH of about 5.8. Following barrier disruption and application of water, the pH increased by 0.6 units; increase in pH was reduced by the pH 4 glycolate buffer, unchanged by pH 4 citrate and pH 5.5 buffers, and even increased by the pH 7 buffer. pH 5.5, pH 4 citrate, and pH 4 glycolate buffers led to a slight, while the pH 7 buffer led to a significant increase in TEWL after barrier disruption compared to water. The pH 7 buffers led to a significant increase in epidermal thickness/proliferation/differentiation and inflammation after barrier disruption, whereas buffers with pH 4 and 5.5 caused a slight increase. In conclusion, only the pH 4 glycolate buffer significantly reduced the skin barrier disruption-related increase in skin pH. This was accompanied by only slight increase in epidermal thickness and inflammation compared to water. Application of the pH 7 buffer led to a significant increase in the skin pH, TEWL, epidermal thickness, and inflammation. The results are important for the formulation of topical products for effective acidification in pathological skin conditions.

Modulators of the endocannabinoid system influence skin barrier repair, epidermal proliferation, differentiation and inflammation in a mouse model.

Endocannabinoids (ECs) are important regulators of cell signalling. Cannabinoid receptors are involved in keratinocyte proliferation/differentiation. Elevation of the endogenous cannabinoid tone leads to strong anti-inflammatory effects. Here, we explored the influence of endocannabinoid system (ECS) modulators on skin permeability barrier repair, epidermal proliferation, differentiation and inflammation in hairless mice. We used WOBE440, a selective fatty acid amide hydrolase (FAAH) inhibitor, WOL067-531, an inhibitor of endocannabinoid reuptake with no relevant FAAH activity, which both signal via cannabinoid receptor-1 and cannabinoid receptor-2 (CB-1R and CB-2R) and compared them to WOBE15 which signals via CB-2R. Barrier disruption and skin irritation were induced by tape stripping or by sodium dodecyl sulphate (SDS) patch testing. Immediately after barrier disruption, 30 µL of 0.5% WOBE440, WOL067-531 and WOBE15 solutions or the vehicle was applied topically. Barrier repair was monitored by transepidermal water loss at 1.5, 3, 5 and 7 hours. We found that barrier repair was significantly delayed by WOL067-531. A tendency for a delay was noticed for WOBE440, whereas for WOBE15, no effect was observed. Immunohistology showed that the tape-stripping-induced increase in epidermal proliferation and filaggrin expression was significantly reduced by topical applications of WOL067-531 and WOBE440, but not by WOBE15. Also, the SDS-induced inflammation, as determined by the number of inflammatory cells, was reduced by WOL067-531 and WOBE440. In summary, we showed that WOL067-531 exhibits a significant effect on skin barrier repair, epidermal proliferation/differentiation and inflammation.

Skin acidification with a water-in-oil emulsion (pH 4) restores disrupted epidermal barrier and improves structure of lipid lamellae in the elderly.

The pH of the skin surface increases with age and thus reduces epidermal barrier function. Aged skin needs appropriate skin care to counterbalance age-related pH increase and improve barrier function. This confirmatory randomized study investigated the efficacy of water-in-oil (w/o) emulsions with either pH 4 or pH 5.8 in 20 elderly subjects after 4 weeks of treatment. After the treatment, the skin was challenged with a sodium dodecyl sulphate (SDS) solution in order to analyze barrier protection properties of both formulations. The pH 4 w/o emulsion resulted in a significantly lower skin pH compared with the pH 5.8 w/o emulsion and an improved skin hydration after 4-week treatment. Further, the pH 4 emulsion led to more pronounced improvements in length of intercellular lipid lamellae, lamellar organization as well as lipid levels than the pH 5.8 emulsion. Following SDS-induced barrier damage to the skin, the pH of all test areas increased, but the area treated with the pH 4 emulsion showed the lowest increase compared with baseline. In addition, even after the SDS challenge the skin area treated with the pH 4 emulsion still maintained a significantly increased length of intercellular lipid lamellae compared with the beginning of the study. This study provides evidence that topical application of a w/o emulsion with pH 4 reacidifies the skin in elderly and has beneficial effects on skin moisturization, regeneration of lipid lamellae and lipid content. Application of a pH 4 emulsion can improve the epidermal barrier as well as the stratum corneum organization in aged skin.

Pharmacology, toxicology and clinical safety of glycopyrrolate.

The clinical use of the anticholinergic glycopyrrolate dates back to the early 1960s when it was first approved in the U.S. Since then, oral and inhalation formulations have been developed as therapeutic agents inhibiting the muscarinic acetylcholine receptor in various indications including chronic obstructive pulmonary disease (COPD), excessive salivation, and peptic ulcers. More recently, topical formulations of glycopyrrolate (GPB, also known as glycopyrronium bromide) have gained interest as a treatment option for excessive sweating (hyperhidrosis). The U.S. Food and Drug Administration (FDA) approved the first topical glycopyrronium product for the treatment of hyperhidrosis in 2018. Glycopyrrolate, as a quaternary amine, shows minimal penetration of the blood brain barrier which limits CNS side effects. In addition, lack of phototoxicity, genotoxicity and carcinogenicity makes it suitable for chronic indications. The information on the nonclinical and clinical safety profile of glycopyrronium supporting various therapeutically approved uses has been obtained from published literature, our own data as well as summary documents issued by regulatory bodies. Collectively, these data support the conclusion that the benefits of glycopyrronium generally outweigh the risks in chronic use indications that require muscarinic receptor antagonism to provide therapeutic effects.

Efficacy of hydrophilic or lipophilic emulsions containing Echinacea purpurea extract in treatment of different types of pruritus.

BACKGROUND: Pruritus reduces quality of life and may occur at different sites of the body. To alleviate pruritus, lipid replenishing and rehydration of the skin is often unsatisfactory. Thus, products with additional antipruritic effects are needed. OBJECTIVES: Antipruritic effects and cosmetic properties of two different emulsions, water-in-oil (w/o) or oil-in-water (o/w), and a shampoo containing a lipophilic Echinacea purpurea root extract (Ec.-extract) were assessed in adults suffering from pruritus. METHODS: Adults (n = 55) with pruritus of the body applied a w/o emulsion for 2 weeks. In a separate study, adults (n = 33) with a pruritic scalp applied an o/w-emulsion for 4 weeks. In a third study, shampoo (n = 34) was applied for 4 weeks. Objective (erythema, dryness, and papules) and subjective (intensity, duration, and burden of pruritus) parameters were assessed. RESULTS: Treatment with the w/o emulsion significantly reduced erythema and dryness (P < 0.0001) as well as pruritus (in 93% of participants) on the body. Treatment with the o/w-emulsion on the scalp significantly (P < 0.0001) reduced objective (erythema in 61% and dryness in 85% of participants) and subjective (85% of participants had reduced pruritus) parameters. Similar results in reduction of dryness (76% of participants) and pruritus (70 % of participants) were seen after 4 weeks of shampoo use. CONCLUSION: Independent from the type of emulsion (w/o or o/w), cosmetic products containing a proprietary Ec.-extract significantly reduced objective and subjective parameters in adults suffering from acute or chronic pruritus exhibiting excellent tolerability.

Divergent effects of a designer natriuretic peptide CD-NP in the regulation of adipose tissue and metabolism.

OBJECTIVE: Obesity is defined as an abnormal increase in white adipose tissue (WAT) and is a major risk factor for type 2 diabetes and cardiovascular disease. Brown adipose tissue (BAT) dissipates energy and correlates with leanness. Natriuretic peptides have been shown to be beneficial for brown adipocyte differentiation and browning of WAT. METHODS: Here, we investigated the effects of an optimized designer natriuretic peptide (CD-NP) on murine adipose tissues in vitro and in vivo. RESULTS: In murine brown and white adipocytes, CD-NP activated cGMP production, promoted adipogenesis, and increased thermogenic markers. Consequently, mice treated for 10 days with CD-NP exhibited increased "browning" of WAT. To study CD-NP effects on diet-induced obesity (DIO), we delivered CD-NP for 12 weeks. Although CD-NP reduced inflammation in WAT, CD-NP treated DIO mice exhibited a significant increase in body mass, worsened glucose tolerance, and hepatic steatosis. Long-term CD-NP treatment resulted in an increased expression of the NP scavenging receptor (NPR-C) and decreased lipolytic activity. CONCLUSIONS: NP effects differed depending on the duration of treatment raising questions about the rational of natriuretic peptide treatment in obese patients.

The Gq signalling pathway inhibits brown and beige adipose tissue.

Brown adipose tissue (BAT) dissipates nutritional energy as heat via the uncoupling protein-1 (UCP1) and BAT activity correlates with leanness in human adults. Here we profile G protein-coupled receptors (GPCRs) in brown adipocytes to identify druggable regulators of BAT. Twenty-one per cent of the GPCRs link to the Gq family, and inhibition of Gq signalling enhances differentiation of human and murine brown adipocytes. In contrast, activation of Gq signalling abrogates brown adipogenesis. We further identify the endothelin/Ednra pathway as an autocrine activator of Gq signalling in brown adipocytes. Expression of a constitutively active Gq protein in mice reduces UCP1 expression in BAT, whole-body energy expenditure and the number of brown-like/beige cells in white adipose tissue (WAT). Furthermore, expression of Gq in human WAT inversely correlates with UCP1 expression. Thus, our data indicate that Gq signalling regulates brown/beige adipocytes and inhibition of Gq signalling may be a novel therapeutic approach to combat obesity.

A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function.

OBJECTIVE: Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor ß (TGFß) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. METHODS: Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. RESULTS: Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPARγ and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. CONCLUSIONS: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.

Impaired border zone formation and adverse remodeling after reperfused myocardial infarction in cannabinoid CB2 receptor deficient mice.

AIMS: Reperfusion ofmyocardial infarction is associated with inflammatory reaction and subsequentmyocardial remodeling with a rapid scar formation in mice. The cannabinoid receptor CB2 has been associated with cardioprotection and regulation ofmacrophage function.Weinvestigated its role in remodeling of reperfused infarction. MAIN METHODS: One hour LAD-occlusion was followed by reperfusion over 6 h and 1, 3 and 7 days in wild-type C57/BL6J (WT) and CB2 receptor-deficient (Cnr2−/−)mice (n=8/group). Hearts were processed for functional, morphological and mRNA/protein analysis, and tissue concentration of endocannabinoidswas determined using liquid chromatography-multiple reaction monitoring. KEY FINDINGS: In contrast to a rapid formation of granulation tissue and a compacted non-transmural scar inWT mice after 7 days of reperfusion, Cnr2−/− mice showed a non-compacted transmural scar. Millar® left ventricular catheter measurements revealed a significantly worse function in Cnr2−/− mice.We found no compensatory elevation of endocannabinoid concentration in Cnr2−/− hearts. Macrophage infiltration was significantly stronger in Cnr2−/− hearts and affected also the remote septum, when compared to WT hearts.We found a cytokine-driven inflammatory response in Cnr2−/− hearts with no significant induction of chemokines. Immunohistochemistry for thrombospondin-1 revealed a dysfunctional infarction border zone formation in Cnr2−/− hearts. Cnr2−/−hearts showed no significant induction of tenascin C, collagen-Iα or lysil oxidase, thereby indicating adversemyocardial remodeling. SIGNIFICANCE: Endocannabinoids act via CB2 receptor in the modulation of inflammatory response and myocardial remodeling after infarction. CB2 receptor plays an important role in the formation of infarction border zone, collagen deposition and organization of stable scar during remodeling.

Combined inhibition of PI3Kß and PI3Kγ reduces fat mass by enhancing α-MSH-dependent sympathetic drive.

Obesity is defined as an abnormal increase in white adipose tissue and has become a major medical burden worldwide. Signals from the brain control not only appetite but also energy expenditure, both of which contribute to body weight. We showed that genetic or pharmacological inhibition of two phosphatidylinositol 3-kinases (PI3Kß and PI3Kγ) in mice reduced fat mass by promoting increased energy expenditure. This effect was accompanied by stimulation of lipolysis and the acquisition of the energy-burning characteristics of brown adipocytes by white adipocytes, a process referred to as "browning." The browning of the white adipocytes involved increased norepinephrine release from the sympathetic nervous system. We found that PI3Kß and PI3Kγ together promoted a negative feedback loop downstream of the melanocortin 4 receptor in the central nervous system, which controls appetite and energy expenditure in the periphery. Analysis of mice with drug-induced sympathetic denervation suggested that these kinases controlled the sympathetic drive in the brain. Administration of inhibitors of both PI3Kß and PI3Kγ to mice by intracerebroventricular delivery induced a 10% reduction in fat mass as quickly as 10 days. These results suggest that combined inhibition of PI3Kß and PI3Kγ might represent a promising treatment for obesity.

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors.

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of ß-adrenergic receptors. Because BAT therapies based on cold exposure or ß-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Early and transient sodium-hydrogen exchanger isoform 1 inhibition attenuates subsequent cardiac hypertrophy and heart failure following coronary artery ligation.

Na(+)/H(+) exchanger 1 (NHE-1) inhibition attenuates the hypertrophic response and heart failure in various experimental models. As the hypertrophic program is rapidly initiated following insult, we investigated whether early and transient administration of a NHE-1 inhibitor will exert salutary effects on cardiomyocyte hypertrophy or heart failure using both in vitro and in vivo approaches. Neonatal cardiomyocytes were treated with the novel, potent, and highly specific NHE-1 inhibitor BIX (N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine; 100 nM) for 1 hour in the presence of 10 µM phenylephrine, after which the cells were maintained for a further 23 hours in the absence of NHE-1 inhibition. One-hour treatment with the NHE-1 inhibitor prevented phenylephrine-induced hypertrophy, which was associated with prevention of activation of calcineurin, a key component of the hypertrophic process. Experiments were then performed in rats subjected to coronary artery ligation, in which the NHE-1 inhibitor was administered immediately after infarction for a 1-week period followed by a further 5 weeks of sustained coronary artery occlusion in the absence of drug treatment. This approach significantly attenuated left ventricular hypertrophy and improved both left ventricular systolic and diastolic dysfunction, which was also associated with inhibition of calcineurin activation. Our findings indicate that early and transient administration of an NHE-1 inhibitor bestows subsequent inhibition of cardiomyocyte hypertrophy in culture as well as cardiac hypertrophy and heart failure in vivo, suggesting a critical early NHE-1-dependent initiation of the hypertrophic program. The study also suggests a preconditioning-like phenomenon in preventing hypertrophy and heart failure by early and transient NHE-1 inhibition.

Regulation of metabolism by cGMP.

The second messenger cyclic guanosine monophosphate (cGMP) mediates the physiological effects of nitric oxide and natriuretic peptides in a broad spectrum of tissues and cells. So far, the major focus of research on cGMP lay on the cardiovascular system. Recent evidence suggests that cGMP also plays a major role in the regulation of cellular and whole-body metabolism. Here, we focus on the role of cGMP in adipose tissue. In addition, other organs important for the regulation of metabolism and their regulation by cGMP are discussed. Targeting the cGMP signaling pathway could be an exciting approach for the regulation of energy expenditure and the treatment of obesity.

A molecular mechanism for therapeutic effects of cGMP-elevating agents in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive, usually fatal disease with abnormal vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients are hyperproliferative and apoptosis-resistant and demonstrate decreased signaling in response to bone morphogenetic proteins (BMPs). Cyclic GMP-elevating agents are beneficial in PAH, but their mechanism(s) of action are incompletely understood. Here we show that BMP signaling via Smad1/5/8 requires cGMP-dependent protein kinase isotype I (PKGI) to maintain PASMCs in a differentiated, low proliferative state. BMP cooperation with cGMP/PKGI was crucial for transcription of contractile genes and suppression of pro-proliferative and anti-apoptotic genes. Lungs from mice with low or absent PKGI (Prkg1(+/-) and Prkg1(-/-) mice) exhibited impaired BMP signaling, decreased contractile gene expression, and abnormal vascular remodeling. Conversely, cGMP stimulation of PKGI restored defective BMP signaling in rats with hypoxia-induced PAH, consistent with cGMP-elevating agents reversing vascular remodeling in this PAH model. Our results provide a mechanism for the therapeutic effects of cGMP-elevating agents in PAH and suggest that combining them with BMP mimetics may provide a novel, disease-modifying approach to PAH therapy.

Increased cGMP promotes healthy expansion and browning of white adipose tissue.

With more than half a billion individuals affected worldwide, obesity has reached pandemic proportions. Development of "brown-like" or "brite" adipocytes within white adipose tissue (WAT) has potential antiobesity and insulin-sensitizing effects. We investigated the role of cyclic GMP (cGMP) signaling, focusing on cGMP-dependent protein kinase I (PKGI) in WAT. PKGI is expressed in murine WAT, primary adipocytes, and 3T3-L1. Treatment of adipocytes with cGMP resulted in increased adipogenesis, with a 54% increase in expression of peroxisome proliferator-activated receptor-γ. Lentiviral overexpression of PKGI further increased adipogenesis, whereas loss of PKGI significantly reduced adipogenic differentiation. In addition to adipogenic effects, PKGI had an antihypertrophic and anti-inflammatory effect via RhoA phosphorylation and reduction of proinflammatory adipokine expression. Moreover, PKGI induced a 4.3-fold increase in abundance of UCP-1 and the development of a brown-like thermogenic program in primary adipocytes. Notably, treatment of C57BL/6 mice with phosphodiesterase inhibitor sildenafil (12 mg/kg/d) for 7 d caused 4.6-fold increase in uncoupling protein-1 expression and promoted establishment of a brown fat cell-like phenotype ("browning") of WAT in vivo. Taken together, PKGI is a key regulator of cell size, adipokine secretion and browning of white fat depots and thus could be a valuable target in developing novel treatments for obesity.

Role of NF-κB and p38 MAPK activation in mediating angiotensin II and endothelin-1-induced stimulation in leptin production and cardiomyocyte hypertrophy.

We recently identified leptin as a downstream factor mediating the hypertrophic effects of both angiotensin II and endothelin-1 in cardiomyocytes, an effect dependent on increased leptin biosynthesis, however, the mechanism for such increased leptin production is not known. This study was designed to elucidate the mechanisms underlying angiotensin II- and endothelin-1-stimulated synthesis in cultured ventricular myocytes. The hypertrophic effects of both angiotensin II (100 nM) and endothelin-1 (10 nM) were associated with increased leptin secretion and gene expression by 40 and 50 %, and 86 and 68 %, respectively. These effects were associated with significantly increased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation by 34 and 52 %, as well as enhanced translocation of NF-κB into nuclei and also the NF-κB-DNA binding activity by 35 and 31 % induced by angiotensin II and endothelin-1, respectively. On their own, 24 h treatment with either angiotensin II or endothelin-1 increased cell surface area by 30 and 40 %, protein synthesis by 30 % and the α-skeletal actin gene by 53 and 68 %, respectively, indicating a robust hypertrophic effect whereas this was completely prevented by NF-κB inhibition. In addition, NF-κB inhibition significantly attenuated angiotensin II and endothelin-1-induced p38 MAPK activation whereas inhibition of p38 MAPK blocked both angiotensin II- and endothelin-1-induced increases in leptin secretion. The ability of both angiotensin II- and endothelin-1 to increase leptin production in cardiomyocytes and the resultant hypertrophic response are mediated by NF-κB and dependent on p38 MAPK activation.