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Growing evidence suggests that neuroinflammation is not just a consequence of neurodegeneration in pathologies such as Alzheimer's disease, Parkinson's disease, Huntington's disease or Amyotrophic lateral sclerosis, but it is rather a determinant factor, which plays a pivotal role in the onset and progression of these disorders. Neuroinflammation can affect cells and processes in the central nervous system (CNS) as well as immune cells, and might precede protein aggregation, which is a hallmark of the neurodegenerative process. Standard treatment methods are far from being able to counteract inflammation and delay neurodegeneration. Remarkably, phosphodiesterase 5 inhibitors (PDE5is), which represent potent vasoactive drugs used as a first-line treatment for erectile dysfunction (ED), display important anti-inflammatory effects through cyclic guanosine monophosphate (cGMP) level stabilization. Since PDE5 hydrolyzes cGMP, several studies positioned PDE5 as a therapeutic target, and more specifically, PDE5is as potential alternative strategies for the treatment of a variety of neurological disorders. Indeed, PDE5is can limit neuroinflammation and enhance synaptic plasticity, with beneficial effects on cognitive function and memory. The aim of this review is to provide an overview of some of the main processes underlying neuroinflammation and neurodegeneration which may be potential targets for PDE5is, focusing on sildenafil, the most extensively studied. Current strategies using PDEis for the treatment of neurodegenerative diseases will be summarized.

The phosphodiesterases type 5 (PDE5) are catalytic enzymes converting the second messenger cyclic guanosine monophosphate (cGMP) to 5' GMP. While intracellular cGMP reduction is associated with several detrimental effects, cGMP stabilization associates with numerous benefits. The PDE5 specific inhibitors, PDE5i, found their explosive fortune as first-line treatment for erectile dysfunction (ED), due to their powerful vasoactive properties. The favorable effect for ED emerged as side-effect when PDE5i were originally proposed for coronary artery disease (CAD). From that point on, the use of PDE5i captured the attention of researchers, clinicians, and companies. Indeed, PDE5-induced intracellular cGMP stabilization offers a range of therapeutic opportunities associated not only with vasoactive effects, but also with immune regulatory/anti-inflammatory actions. Chronic inflammation is acknowledged as the common link underlying most non-communicable diseases, including metabolic and cardiac diseases, autoimmune and neurodegenerative disorders, cancer. In this scenario, the clinical exploitation of PDE5i is undeniably beyond ED, representing a potential therapeutic tool in several human diseases. This review aims to overview the biological actions exerted by PDE5i, focusing on their ability as modulators of inflammation-related human diseases, with particular attention to inflammatory-related disorders, like cardiac diseases and cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Paronetto and Crescioli.)

Aging can be seen as a physiological progression of biomolecular damage and the accumulation of defective cellular components, which trigger and amplify the process, toward whole-body function weakening. Senescence initiates at the cellular level and consists in an inability to maintain homeostasis, characterized by the overexpression/aberrant expression of inflammatory/immune/stress responses. Aging is associated with significant modifications in immune system cells, toward a decline in immunosurveillance, which, in turn, leads to chronic elevation of inflammation/oxidative stress, increasing the risk of (co)morbidities. Albeit aging is a natural and unavoidable process, it can be regulated by some factors, like lifestyle and diet. Nutrition, indeed, tackles the mechanisms underlying molecular/cellular aging. Many micronutrients, i.e., vitamins and elements, can impact cell function. This review focuses on the role exerted by vitamin D in geroprotection, based on its ability to shape cellular/intracellular processes and drive the immune response toward immune protection against infections and age-related diseases. To this aim, the main biomolecular paths underlying immunosenescence and inflammaging are identified as biotargets of vitamin D. Topics such as heart and skeletal muscle cell function/dysfunction, depending on vitamin D status, are addressed, with comments on hypovitaminosis D correction by food and supplementation. Albeit research has progressed, still limitations exist in translating knowledge into clinical practice, making it necessary to focus attention on the role of vitamin D in aging, especially considering the growing number of older individuals.

Vitamin D exerts important extra-skeletal effects, exhibiting an exquisite immune regulatory ability, affecting both innate and adaptive immune responses through the modulation of immunocyte function and signaling. Remarkably, the immune function of working skeletal muscle, which is fully recognized to behave as a secretory organ with immune capacity, is under the tight control of vitamin D as well. Vitamin D status, meaning hormone sufficiency or insufficiency, can push toward strengthening/stabilization or decline of immune surveillance, with important consequences for health. This aspect is particularly relevant when considering the athletic population: while exercising is, nowadays, the recommended approach to maintain health and counteract inflammatory processes, "too much" exercise, often experienced by athletes, can increase inflammation, decrease immune surveillance, and expose them to a higher risk of diseases. When overexercise intersects with hypovitaminosis D, the overall effects on the immune system might converge into immune depression and higher vulnerability to diseases. This paper aims to provide an overview of how vitamin D shapes human immune responses, acting on the immune system and skeletal muscle cells; some aspects of exercise-related immune modifications are addressed, focusing on athletes. The crossroad where vitamin D and exercise meet can profile whole-body immune response and health., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Crescioli.)

The prostacyclin analogue iloprost is used to treat vascular alterations and digital ulcers, the early derangements manifesting in systemic sclerosis (SSc), an autoimmune disease leading to skin and organ fibrosis. Bioindicator(s) of SSc onset and progress are still lacking and the therapeutic approach remains a challenge. The T helper 1 (Th1) chemokine interferon (IFN)γ-induced protein 10 (IP-10/CXCL10) associates with disease progression and worse prognosis. Endothelial cells and fibroblasts, under Th1-dominance, release CXCL10, further enhancing SSc's detrimental status. We analyzed the effect of iloprost on CXCL10 in endothelial cells, dermal fibroblasts, and in the serum of SSc patients. Human endothelial cells and dermal fibroblasts activated with IFNγ/Tumor Necrosis Factor (TNF)α, with/without iloprost, were investigated for CXCL10 secretion/expression and for intracellular signaling cascade underlying chemokine release (Signal Transducer and Activator of Transcription 1, STAT1; Nuclear Factor kappa-light-chain-enhancer of activated B cells, NF-kB; c-Jun NH 2 -terminal kinase, JNK: Phosphatidyl-Inositol 3-kinase (PI3K)/protein kinase B, AKT; Extracellular signal-Regulated Kinase 1/2, ERK1/2). CXCL10 was quantified in sera from 25 patients taking iloprost, satisfying the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) 2013 classification criteria for SSc, and in sera from 20 SSc sex/age-matched subjects without therapy, previously collected. In human endothelial cells and fibroblasts, iloprost targeted CXCL10, almost preventing IFNγ/TNFα-dependent cascade activation in endothelial cells. In SSc subjects taking iloprost, serum CXCL10 was lower. These in vitro and in vivo data suggest a potential role of iloprost to limit CXCL10 at local vascular/dermal and systemic levels in SSc and warrant further translational research aimed to ameliorate SSc understanding/management.

The antidiabetic sodium-glucose cotransporter type 2 inhibitor (SGLT2i) empagliflozin efficiently reduces heart failure (HF) hospitalization and cardiovascular death in type 2 diabetes (T2D). Empagliflozin-cardioprotection likely includes anti-inflammatory effects, regardless glucose lowering, but the underlying mechanisms remain unclear. Inflammation is a primary event in diabetic cardiomyopathy (DCM) and HF development. The interferon (IFN)γ-induced 10-kDa protein (IP-10/CXCL10), a T helper 1 (Th1)-type chemokine, promotes cardiac inflammation, fibrosis, and diseases, including DCM, ideally representing a therapeutic target. This preliminary study aims to explore whether empagliflozin directly affects Th1-challenged human cardiomyocytes, in terms of CXCL10 targeting. To this purpose, empagliflozin dose-response curves were performed in cultured human cardiomyocytes maintained within a Th1-dominant inflammatory microenvironment (IFNγ/TNFα), and CXCL10 release with the intracellular IFNγ-dependent signaling pathway (Stat-1) was investigated. To verify possible drug-cell-target specificity, the same assays were run in human skeletal muscle cells. Empagliflozin dose dependently inhibited CXCL10 secretion (IC50 = 76,14 × 10-9 M) in association with Stat-1 pathway impairment only in Th1-induced human cardiomyocytes, suggesting drug-selective cell-type-targeting. As CXCL10 plays multifaceted functions in cardiac remodeling toward HF and currently there is no effective method to prevent it, these preliminary data might be hypothesis generating to open new scenarios in the translational approach to SGLT2i-dependent cardioprotection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Giannattasio, Citarella, Trocchianesi, Filardi, Morano, Lenzi, Ferretti and Crescioli.)

The number of aged individuals is increasing worldwide, rendering essential the comprehension of pathophysiological mechanisms of age-related alterations, which could facilitate the development of interventions contributing to "successful aging" and improving quality of life. Cardiovascular diseases (CVD) include pathologies affecting the heart or blood vessels, such as hypertension, peripheral artery disease and coronary heart disease. Indeed, age-associated modifications in body composition, hormonal, nutritional and metabolic factors, as well as a decline in physical activity are all involved in the increased risk of developing atherogenic alterations that raise the risk of CVD development. Several factors have been reported to play a role in the alterations observed in muscle and endothelial cells and that lead to increased CVD, such as genetic pattern, smoking and unhealthy lifestyle. Moreover, a difference in the risk of these diseases in women and men has been reported. Interestingly, in the past decades attention has been focused on a potential role of several pollutants that disrupt human health by interfering with hormonal pathways, and more specifically in non-communicable diseases such as obesity, diabetes and CVD. This review will focus on the potential alteration induced by Endocrine Disruptors (Eds) in the attempt to characterize a potential role in the cellular and molecular mechanisms involved in the atheromatous degeneration process and CVD progression.

Women experience a dramatical raise in cardiovascular events after menopause. The decline in estrogens is pointed to as the major responsible trigger for the increased risk of cardiovascular disease (CVD). Indeed, the menopausal transition associates with heart macro-remodeling, which results from a fine-tuned cell micro-remodeling. The remodeling of cardiomyocytes is a biomolecular response to several physiologic and pathologic stimuli, allowing healthy adaptation in normal conditions or maladaptation in an unfavorable environment, ending in organ architecture disarray. Estrogens largely impinge on cardiomyocyte remodeling, but they cannot fully explain the sex-dimorphism of CVD risk. Albeit cell remodeling and adaptation are under multifactorial regulation, vitamin D emerges to exert significant protective effects, controlling some intracellular paths, often shared with estrogen signaling. In post-menopause, the unfavorable association of hypoestrogenism-D hypovitaminosis may converge towards maladaptive remodeling and contribute to increased CVD risk. The aim of this review is to overview the role of estrogens and vitamin D in female cardiac health, speculating on their potential synergistic effect in cardiomyocyte remodeling, an issue that is not yet fully explored. Further learning the crosstalk between these two steroids in the biomolecular orchestration of cardiac cell fate during adaptation may help the translational approach to future cardioprotective strategies for women health.

Skeletal muscle damage is a common clinical manifestation of systemic sclerosis (SSc). C-X-C chemokine ligand 10 (CXCL10) is involved in myopathy and cardiomyopathy development and is associated with a more severe SSc prognosis. Interestingly, the phosphodiesterase type 5 inhibitor (PDE5i) sildenafil reduces CXCL10 sera levels of patients with diabetic cardiomyopathy and in cardiomyocytes. Here, we analyzed the levels of CXCL10 in the sera of 116 SSc vs. 35 healthy subjects and explored differences in 17 SSc patients on stable treatment with sildenafil. CXCL10 sera levels were three-fold higher in SSc vs. healthy controls, independent of subset and antibody positivity. Sildenafil treatment was associated with lower CXCL10 sera levels. Serum CXCL10 strongly correlated with the clinical severity of muscle involvement and with creatine kinase (CK) serum concentration, suggesting a potential involvement in muscle damage in SSc. In vitro , sildenafil dose-dependently reduced CXCL10 release by activated myocytes and impaired cytokine-induced Signal transducer and activator of transcription 1 (STAT1), Nuclear factor-κB (NFκB) and c-Jun N-terminal kinase (JNK) phosphorylation. This was also seen in cardiomyocytes. Sildenafil-induced CXCL10 inhibition at the systemic and human muscle cell level supports the hypothesis that PDE5i could be a potential therapeutic therapy to prevent and treat muscle damage in SSc.

Skeletal muscle cells, albeit classified as vitamin D receptor (VDR)-poor cells, are finely controlled by vitamin D through genomic and non-genomic mechanisms. Skeletal muscle constantly undergoes cell remodeling, a complex system under multilevel regulation, mainly orchestrated by the satellite niche in response to a variety of stimuli. Cell remodeling is not limited to satisfy reparative and hypertrophic needs, but, through myocyte transcriptome/proteome renewal, it warrants the adaptations necessary to maintain tissue integrity. While vitamin D insufficiency promotes cell maladaptation, restoring vitamin D levels can correct/enhance the myogenic program. Hence, vitamin D fortified foods or supplementation potentially represents the desired approach to limit or avoid muscle wasting and ameliorate health. Nevertheless, consensus on protocols for vitamin D measurement and supplementation is still lacking, due to the high variability of lab tests and of the levels required in different contexts (i.e., age, sex, heath status, lifestyle). This review aims to describe how vitamin D can orchestrate skeletal muscle cell remodeling and myogenic programming, after reviewing the main processes and cell populations involved in this important process, whose correct progress highly impacts on human health. Topics on vitamin D optimal levels, supplementation and blood determination, which are still under debate, will be addressed.

Systemic sclerosis (SSc) is a multi-system connective tissue disease characterized by the increased deposition of extracellular matrix proteins such as collagen and fibronectin. Although the pathogenesis is not completely understood, a number of studies suggest that free radicals could be the major contributors to the disease. Indeed, different studies demonstrated how oxidative stress could contribute to the fibrotic process activation at the level of the skin and visceral organs. Emerging evidences highlight the beneficial effects of sildenafil, a phosphodiesterase type 5 inhibitor (PDE5i), which protects different cell lines from the cell damage induced by reactive oxygen species (ROS). These data make sildenafil a good candidate for therapeutic treatment aimed to protect biological macromolecules against oxidative damage, thus preserving cell viability. The purpose of this study was to evaluate the sensitivity of SSc dermal fibroblasts to an oxidative insult and the ability for sildenafil to prevent/reduce the DNA damage due to ROS action. Additionally, we evaluated the capacity for sildenafil to influence redox homeostasis and cytotoxicity, as well as cell proliferation and cell cycle progression. We demonstrated that SSc fibroblasts have an increased sensitivity to a pro-oxidant environment in comparison to healthy controls. The sildenafil treatment reduced ROS-induced DNA damage, counteracted the negative effects of ROS on cell viability and proliferation, and promoted the activity of specific enzymes involved in redox homeostasis maintenance. To our knowledge, in this report, we demonstrate, for the first time, that sildenafil administration prevents ROS-induced instability in human dermal fibroblasts isolated by SSc patients. These results expand the use of PDE5i as therapeutic agents in SSc by indicating a protective role in tissue damage induced by oxidative insult.

Human perinatal stem cells (SCs) can be isolated from fetal annexes without ethical or safety limitations. They are generally considered multipotent; nevertheless, their biological characteristics are still not fully understood. The aim of this study was to investigate the pluripotency potential of human perinatal SCs as compared to human induced pluripotent stem cells (hiPSCs). Despite the low expression of the pluripotent factors NANOG, OCT4, SOX2, and C-KIT in perinatal SC, we observed minor differences in the promoters DNA-methylation profile of these genes with respect to hiPSCs; we also demonstrated that in perinatal SCs miR-145-5p had an inverse trend in comparison to these stemness markers, suggesting that NANOG, OCT4, and SOX2 were regulated at the post-transcriptional level. The reduced expression of stemness markers was also associated with shorter telomere lengths and shift of the oxidative metabolism between hiPSCs and fetal annex-derived cells. Our findings indicate the differentiation ability of perinatal SCs might not be restricted to the mesenchymal lineage due to an epigenetic barrier, but other regulatory mechanisms such as telomere shortening or metabolic changes might impair their differentiation potential and challenge their clinical application.

In the elderly, whole-body health largely relies on healthy skeletal muscle, which controls body stability, locomotion, and metabolic homeostasis. Age-related skeletal muscle structural/functional deterioration is associated with a higher risk of severe comorbid conditions and poorer outcomes, demanding major socioeconomic costs. Thus, the need for efficient so-called geroprotective strategies to improve resilience and ensure a good quality of life in older subjects is urgent. Skeletal muscle senescence and metabolic dysregulation share common cellular/intracellular mechanisms, potentially representing targets for intervention to preserve muscle integrity. Many factors converge in aging, and multifaceted approaches have been proposed as interventions, although they have often been inconclusive. Physical exercise can counteract aging and metabolic deficits, not only in maintaining tissue mass, but also by preserving tissue secretory function. Indeed, skeletal muscle is currently considered a proper secretory organ controlling distant organ functions through immunoactive regulatory small peptides called myokines. This review provides a current perspective on the main biomolecular mechanisms underlying age-dependent and metabolic deterioration of skeletal muscle, herein discussed as a secretory organ, the functional integrity of which largely depends on exercise and myokine release. In particular, muscle-derived interleukin (IL)-6 is discussed as a nutrient-level biosensor. Overall, exercise and vitamin D are addressed as optimal geroprotective strategies in view of their multi-target effects.

Gestational diabetes mellitus (GDM) is defined as "glucose intolerance that is first diagnosed during pregnancy". Mothers with GDM and their infants may experience both short and long term complications. Dietary intervention is the first therapeutic strategy. If good glycaemic control is not achieved, insulin therapy is recommended. There is no consensus on which nutritional approach should be used in GDM. In the last few years, there has been growing evidence of the benefits of a low glycaemic index (LGI) diet on diabetes and cardiovascular disease. The effect of a LGI diet on GDM incidence has been investigated as well. Several studies observed a lower incidence of GDM in LGI diet arms, without adverse maternal and fetal outcomes. The main positive effect of the LGI diet was the reduction of 2-h post-prandial glucose (PPG). Several studies have also evaluated the effect of the LGI diet in GDM treatment. Overall, the LGI diet might have beneficial effects on certain outcomes, such as 2-h PPG, fasting plasma glucose and lipid profile in patients with GDM. Indeed, most studies observed a significant reduction in insulin requirement. Overall, according to current evidence, the LGI nutritional approach is safe and it might therefore be considered in clinical care for GDM.

The term diabetic cardiomyopathy (DCM) labels an abnormal cardiac structure and performance due to intrinsic heart muscle malfunction, independently of other vascular co-morbidity. DCM, accounting for 50%-80% of deaths in diabetic patients, represents a worldwide problem for human health and related economics. Optimal glycemic control is not sufficient to prevent DCM, which derives from heart remodeling and geometrical changes, with both consequences of critical events initially occurring at the cardiomyocyte level. Cardiac cells, under hyperglycemia, very early undergo metabolic abnormalities and contribute to T helper (Th)-driven inflammatory perturbation, behaving as immunoactive units capable of releasing critical biomediators, such as cytokines and chemokines. This paper aims to focus onto the role of cardiomyocytes, no longer considered as "passive" targets but as "active" units participating in the inflammatory dialogue between local and systemic counterparts underlying DCM development and maintenance. Some of the main biomolecular/metabolic/inflammatory processes triggered within cardiac cells by high glucose are overviewed; particular attention is addressed to early inflammatory cytokines and chemokines, representing potential therapeutic targets for a prompt early intervention when no signs or symptoms of DCM are manifesting yet. DCM clinical management still represents a challenge and further translational investigations, including studies at female/male cell level, are warranted.

Management of patients presenting to the Emergency Department with chest pain is continuously evolving. In the setting of acute coronary syndrome, the availability of high-sensitivity cardiac troponin assays (hs-cTn) has allowed for the development of algorithms aimed at rapidly assessing the risk of an ongoing myocardial infarction. However, concerns were raised about the massive application of such a simplified approach to heterogeneous real-world populations. As a result, there is a potential risk of underdiagnosis in several clusters of patients, including women, for whom a lower threshold for hs-cTn was suggested to be more appropriate. Implementation in clinical practice of sex-tailored cut-off values for hs-cTn represents a hot topic due to the need to reduce inequality and improve diagnostic performance in females. The aim of this review is to summarize current evidence on sex-specific cut-off values of hs-cTn and their application and usefulness in clinical practice. We also offer an extensive overview of thresholds reported in literature and of the mechanisms underlying such differences among sexes, suggesting possible explanations about debated issues., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2019 Giulio Francesco Romiti et al.)

Phosphodiesterase type 5 inhibitors (PDE5i) (e.g., sildenafil, tadalafil, vardenafil, and avanafil) are drugs commonly used to treat erectile dysfunction, pulmonary arterial hypertension, and benign prostatic hyperplasia. PDE5i are not prohibited by the World Anti-Doping Agency (WADA) but are alleged to be frequently misused by healthy athletes to improve sporting performance. In vitro and in vivo studies have reported various effects of PDE5i on cardiovascular, muscular, metabolic, and neuroendocrine systems and the potential, therefore, to enhance performance of healthy athletes during training and competition. This suggests well-controlled research studies to examine the ergogenic effects of PDE5i on performance during activities that simulate real sporting situations are warranted to determine if PDE5i should be included on the prohibited WADA list. In the meantime, there is concern that some otherwise healthy athletes will continue to misuse PDE5i to gain an unfair competitive advantage over their competitors.

The primary aim in the treatment of autoimmune inflammatory myopathies (IMs) is to recover muscle function. The presence of immune/inflammatory cell infiltrates within muscle tissues represents the common feature of different IM subtypes, albeit a correlation between muscular damage extent and inflammation degree is often lacking. Treatments for IMs are based on life-long immunosuppressive therapy, with the well known adverse effects; recovery is incomplete for many patients. More effective therapies, with reduced side-effects, are highly desirable. Vitamin D receptor (VDR) agonists emerge to retain pleiotropic anti-inflammatory properties, since they regulate innate and adaptive immunity by switching the immune response from proinflammatory T helper 1 (Th1) type to tolerogenic T helper 2 (Th2) type dominance. In skeletal muscle cells less hypercalcemic VDR ligands target powerful mediators of inflammation, such as TNF α and TNF α driven paths, without affecting immune or muscle cells viability, retaining the potentiality to counteract Th1 driven overreactivity established by the self-enhancing inflammatory loop between immune and skeletal muscle cells. This review summarizes those features of VDR agonists as candidates in future treatment of IM.

Objective: This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1β and IL-10, vs. healthy subjects., Methods: Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC50 determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis., Results: BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls., Conclusions: Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.

Understanding the many biological extraskeletal actions of vitamin D has increased in the past decades. Indeed, vitamin D and analogue molecules, besides the classical actions on bone metabolism, exert several beneficial effects on metabolic homeostasis, heart-cardiovascular, brain, and muscle physiological functions, throughout the interaction with the specific vitamin D receptor (VDR). In particular, VDR agonists powerfully control innate and adaptive immune system with favorable effects on human health. VDR ligands act as immunomodulators that are potent enough to retain anti-inflammatory effects, even though the mechanism underlying those effects is not yet fully elucidated. VDR agonists exert a significant suppression of inflammatory processes switching the immune response from T helper 1 (Th1) to T helper 2 (Th2) dominance and counteracting the self-enhancing inflammatory loop between immune and resident cells, especially by cytokine release impairment. Those molecules are able, indeed, to reduce the release of the interferon (IFN)γ-induced 10 kDa protein IP-10/CXCL10, a powerful chemokine driving Th1-mediated inflammation. Based on their features, VDR ligands show the potentiality to be included in immunosuppressive regimens, aimed to control auto- and alloimmune Th1-driven overreactivity, occurring, for example, in autoimmune disease or graft rejection.

Phosphodiesterase type 5 inhibitors may influence human physiology, health, and performance by also modulating endocrine pathways. We evaluated the effects of a 2-day tadalafil administration on adenohypophyseal and adrenal hormone adaptation to exercise in humans. Fourteen healthy males were included in a double-blind crossover trial. Each volunteer randomly received two tablets of placebo or tadalafil (20 mg/day with a 36-h interval) before a maximal exercise was performed. After a 2-wk washout, the volunteers were crossed over. Blood samples were collected at -30 and -15 min and immediately before exercise, immediately after, and during recovery (+15, +30, +60, and +90 min) for adrenocorticotropin (ACTH), β-endorphin, growth hormone (GH), prolactin, cortisol (C), corticosterone, dehydroepiandrosterone-sulfate (DHEAS), and cortisol binding globulin (CBG) assays. C-to-CBG (free cortisol index, FCI) and DHEAS-to-C ratios were calculated. Exercise intensity, perceived exertion rate, O₂ consumption, and CO₂ and blood lactate concentration were evaluated. ACTH, GH, C, corticosterone, and CBG absolute concentrations and/or areas under the curve (AUC) increased after exercise after both placebo and tadalafil. Exercise increased DHEAS only after placebo. Compared with placebo, tadalafil administration reduced the ACTH, C, corticosterone, and FCI responses to exercise and was associated with higher β-endorphin AUC and DHEAS-to-C ratio during recovery, without influencing cardiorespiratory and performance parameters. Tadalafil reduced the activation of the hypothalamus-pituitary-adrenal axis during exercise by probably influencing the brain's nitric oxide- and cGMP-mediated pathways. Further studies are necessary to confirm our results and to identify the involved mechanisms, possible health risks, and potential clinical uses.

During kidney allograft rejection, CXC chemokine ligand 10 (CXCL10)-CXC chemokine receptor 3 (CXCR3) trafficking between peripheral blood and tissues initiates alloresponse and perpetuates a self-inflammatory loop; thus, CXCL10-CXCR3 axis could represent a pharmacologic target. In this perspective, immunosuppressors targeting graft-resident cells, beside immune cells, could be very advantageous. Vitamin D receptor (VDR) agonists exhibit considerable immunomodulatory properties. This study aimed to investigate whether elocalcitol and BXL-01-0029 could decrease the expression of CXCL10 in activated renal tubular cells in vitro and thus be useful in kidney allograft rejection treatment. Experiments were performed in human tubular renal cells stimulated with interferon-gamma + tumor necrosis factor-alpha with and without VDR agonists, tacrolimus, sirolimus, hydrocortisone, methylprednisolone, cyclosporin A and mycophenolate mofetil. CXCL10 protein secretion and gene expression were measured by ELISA and by quantitative PCR. Specific inhibitors were used to investigate intracellular pathways involved in tubular cells activation. For IC(50) determination and comparison, dose-response curves with VDR agonists, tacrolimus and mycophenolic acid were performed. Elocalcitol and BXL-01-0029 inhibited CXCL10 secretion by renal cells, without affecting cell viability, while almost all the immunosuppressors were found to be ineffective, except for tacrolimus and mycophenolate mofetil. BXL-01-0029 was the most potent drug and, notably, it was found to be capable of allowing reduction in tacrolimus-inhibitory doses. Our data suggest that BXL-01-0029 could potentially be a dose-reducing agent for conventional immunosuppressors in kidney rejection management.

It has been hypothesized that interferon (IFN) alpha and beta cause autoimmune thyroid dysfunctions by changing the Th1/Th2 balance, but the mechanisms involved are not yet known. The aims of this study were: (a) to test the effect of IFNalpha, IFNbeta and IFNgamma on the secretion of the Th1 chemokines CXCL9 and CXCL10, in "primary cultures of human thyroid follicular cells" (TFC); (b) to assess the effect of PPARgamma activation on CXCL9 and CXCL10 secretion. In TFC, CXCL9 and CXCL10 were undetectable in the supernatant. IFNgamma, IFNalpha and IFNbeta, dose dependently induced CXCL9 and CXCL10 release. TNFalpha alone had no effect. The combination of each of the IFNs with TNFalpha had a significant synergistic effect on CXCL9 and CXCL10 secretion. Treatment of TFC with rosiglitazone dose dependently inhibited the IFNs-stimulated CXCL9 and CXCL10 release. Compared with IFNalpha and IFNbeta, IFNgamma was the most potent stimulus of CXCL9 and CXCL10 secretion. In conclusion, IFNalpha, IFNbeta, IFNgamma and TNFalpha (synergistically with IFNs) dose-dependently induce the release of CXCL9 and CXCL10 by TFC, suggesting that this process may be related, at least in part, to the appearance of thyroid dysfunction during IFNs therapy. Furthermore, PPARgamma activation partially inhibits this process., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Epididymis is a sex steroid (androgen + estrogen)-sensitive duct provided with spontaneous motility, allowing sperm transport. We previously reported that the oxytocin (OT) receptor (OTR) mediates an estrogen-dependent increase in epididymal contractility. Because endothelin (ET)-1 also regulates epididymal motility, we tested its sex steroid dependence in a rabbit model. We demonstrated that estrogens up-regulate responsiveness to ET-1, which is reduced by blocking aromatase activity (letrozole, 2.5 mg/kg) or by triptorelin (2.9 mg/kg)-induced hypogonadism, whereas it is fully restored by estradiol valerate (3.3 mg/kg weekly) but not by testosterone enanthate (30 mg/kg weekly). However, changing sex steroid milieu did not affect either ET-1, its receptor gene, or protein expression. Two structurally distinct OTR-antagonists [(d(CH2)5(1), Tyr(Me)(2), Orn(8))-OT and atosiban] almost completely abolished ET-1 contractility, without competing for [125I]ET-1 binding, suggesting that OT/OTR partially mediates ET-1 action. Immunohistochemical studies in human and rabbit epididymis demonstrated that both OT and its synthesis-associated protein, neurophysin I, are expressed in the epithelial cells facing the muscular layer, suggesting local OT production. Quantitative RT-PCR demonstrated a high abundance of OT transcripts in human epididymis. OT transcript was also originally detected and partially sequenced in rabbit epididymis. To verify whether ET-1 regulates OT release, we used rabbit epididymal epithelial cell cultures. These cells expressed a high density of [125I]ET-1 binding sites and responded to ET-1 with a dose-dependent OT release. Hence, we propose that an ET-1-induced OT/OTR system activation underlies the estrogen-dependent hyperresponsiveness to ET-1. These local sources might promote the spontaneous motility necessary for sperm transport.

Chemokines are a large family of cytokines involved in the pathogenesis of inflammatory and autoimmune diseases. Among CXC chemokines, CXC chemokine ligand 10 (CXCL10) has been identified to play an important role in several endocrinological autoimmune diseases, such as Hashimoto's thyroiditis, Graves' disease, and type 1 diabetes mellitus. Although the mechanisms leading to glandular autoimmune process may be at least in part shared by different endocrine organs, the role of CXCL10 in autoimmune adrenal insufficiency is unknown. The aim of this study was to evaluate the role of CXCL10 in Addison's disease (AD). Serum CXCL10 levels were assayed in 64 patients with clinically evident autoimmune AD, 20 patients with autoimmune subclinical AD, nine patients with nonautoimmune AD, and 48 healthy volunteers. Clinically evident and subclinical AD, but not nonautoimmune AD patients, showed a significant increase in serum CXCL10 levels compared with healthy subjects: 119.9 pg/ml (range, 39.8-427.6) and 124.0 pg/ml (range, 37.0-384.7) vs. 75.6 pg/ml (range, 22.4-164.0; P < 0.001 for both groups). Comparable serum CXCL10 levels were found between patients with an isolated form of AD and patients with other autoimmune conditions associated with AD, suggesting a specific influence of the adrenal autoimmune process in determining elevated CXCL10 concentrations in such patients. No relationship was found between serum CXCL10 levels and anti-21-hydroxylase or adrenal cortex autoantibody titers or between CXCL10 levels and duration of disease. The role of CXCL10 in the adrenal gland was also evaluated in vitro in human zona fasciculata cells (hZFC). CXCL10, although not basally detected in cultured hZFC, was strongly induced by interferon-gamma and synergistically increased by TNF-alpha addition. Hydrocortisone or ACTH alone had no effect on CXCL10 secretion in hZFC, but they both significantly inhibited cytokine-induced CXCL10 secretion. Taken together, these data suggest a potential role of hZFC, through the production of CXCL10, in regulating the recruitment of specific subsets of activated lymphocytes in autoimmune AD.

Although abnormalities of the male external genitalia (MEG) are a relatively common problem, little is known concerning the molecular mechanisms that finely regulate penile development. We report here the expression of the oxytocin receptor (OTR) gene by real-time RT-PCR in human fetal tissues (11th-12th week of gestation), including the MEG. The developing penis expressed a very high level of OTR mRNA, only a half log(10) unit lower than fetal central nervous system, used as a positive control. The OTR protein is also highly expressed (western, immunohistochemistry and binding studies) and immunolocalized both in the mesenchymal body and in the surrounding blood capillaries, which will later constitute penile trabeculae and sinusoids. Binding studies using [125I]oxytocin antagonist ([125I]OTA) in cultured human fetal penile smooth muscle cells (hfPSMC) revealed the presence of specific OTR with a high capacity and affinity for oxytocin (OT) and for OTA. Increasing concentrations of OT dose-dependently induced intracellular Ca2+ mobilization. Furthermore, OTR mediated an increase in the proliferation and the migration of hfPSMC. In conclusion, we demonstrate that in the developing human MEG, OTR is highly expressed and might be involved in coordinating timely and appropriate proliferation and migration of the penile cells. Thus, OTR might represent an additional target for investigating human fetal MEG organogenesis.

Human prostate is now considered a target for vitamin D receptor (VDR) ligands, such as BXL-628. Because BXL-628 inhibited prostate growth without interfering with androgen signaling, it represents a new option for benign prostate hyperplasia (BPH) therapy. However, BPH symptoms are related not only to prostate size, but also to compensatory bladder hypertrophy and eventual overactivity. We now report that human bladder expresses VDR (determined by real-time PCR immunohistochemistry and Western blot) and responds to VDR agonists, such as the natural ligand, calcitriol, and its synthetic and less hypercalcemic derivative, BXL-628. Experiments were conducted with stromal cells derived from human bladder neck obtained at surgery from BPH patients. BXL-628 counteracted keratinocyte growth factor (KGF) and androgen-induced cell proliferation and stimulated apoptosis with a parallel reduced expression of the survival oncoprotein Bcl-2. Prolonged serum starvation time-dependently pushed bladder stromal cells to express activated myofibroblast markers, such as desmin and smoothelin, without changing other contractile-related proteins and intermediate filaments, such as vimentin. Chronic exposure to BXL-628 prevented starvation-induced cell phenotype modification. Because hypertrophy and starvation-induced bladder remodeling are supposed to underlie bladder overactivity, it is possible that BXL-628 might be helpful in reducing not only cumbersome symptoms related to prostate overgrowth, but also those related to bladder irritation.

Selective Alzheimer's disease indicator-1 (seladin-1) is a novel gene with antiapoptotic activity that is down-regulated in vulnerable brain regions in Alzheimer's disease. This gene encodes 3-beta-hydroxysterol Delta-24-reductase (DHCR24), which converts desmosterol into cholesterol. In the adrenal cortex, increased expression of seladin-1/DHCR24, which appears to be modulated by ACTH, has been recently reported in cortisol-secreting adenomas, compared with the adjacent atrophic tissue. In our study, we measured the expression level of seladin-1/DHCR24 in cortisol- (n = 18) and aldosterone-secreting (n = 16) adrenocortical adenomas, in carcinomas (n = 17), and in normal adrenal glands (n = 8) by quantitative real-time RT-PCR. The amount of seladin-1/DHCR24 mRNA was significantly reduced in carcinomas (total RNA, 2.5 +/- 0.8 pg/ micro g) compared with the other groups (P < 0.01). Western blot analysis confirmed the mRNA results. Similarly, in adrenal malignancies, significantly reduced levels of expression of the ACTH receptor gene were found. In the adrenal cancer cell line H295R and in primary cultures from adrenocortical cells, ACTH (1 nM) and forskolin (10 micro M) effectively increased seladin-1/DHCR24 expression, confirming that seladin-1/DHCR24 is modulated by the ACTH/cAMP-driven pathway. In summary, this is the first demonstration that seladin-1/DHCR24 expression is reduced in adrenal cancer, suggesting that it might be viewed as a new potential marker of adrenal malignancies.

Olfactory neurons and gonadotropin-releasing hormone (GnRH) neurons share a common origin during organogenesis. Kallmann's syndrome, clinically characterized by anosmia and hypogonadotropic hypogonadism, is due to an abnormality in the migration of olfactory and GnRH neurons. We recently characterized the human FNC-B4 cell line, which retains properties present in vivo in both olfactory and GnRH neurons. In this study, we found that FNC-B4 neurons expressed GnRH receptor and responded to GnRH with time- and dose-dependent increases in GnRH gene expression and protein release (up to 5-fold). In addition, GnRH and its analogs stimulated cAMP production and calcium mobilization, although at different biological thresholds (nanomolar for cAMP and micromolar concentrations for calcium). We also observed that GnRH triggered axon growth, actin cytoskeleton remodeling, and a dose-dependent increase in migration (up to 3-4-fold), whereas it down-regulated nestin expression. All these effects were blocked by a specific GnRH receptor antagonist, cetrorelix. We suggest that GnRH, secreted by olfactory neuroblasts, acts in an autocrine pattern to promote differentiation and migration of those cells that diverge from the olfactory sensory lineage and are committed to becoming GnRH neurons.

Cortisol secretion in adrenal Cushing's syndrome can be regulated by aberrant hormone receptors, such as gastric inhibitory polypeptide, V1 vasopressin, catecholamines, LH/human chorionic gonadotropin, and serotonin receptors. We report the case of a patient with Cushing's syndrome due to bilateral adrenal macronodular hyperplasia. Extensive in vivo testing for the presence of aberrant receptors revealed a 5-fold increase of plasma cortisol after the administration of cisapride, an agonist of the serotonin 4 (5-HT(4)) receptor. Primary cell cultures were established from adrenocortical specimens obtained at surgery, and in vitro studies also showed that cisapride determined an increase [133.7 +/- 5.5% (mean +/- SE) of baseline, considered 100%) of cortisol secretion from cultured cells. The presence of 5-HT(4) receptor transcript, and in particular of isoforms c, g, and n, was confirmed by RT-PCR, and the determination of the mRNA levels by real-time RT-PCR revealed a higher expression than in normal adrenal glands. To our knowledge, this is one of the first reports of Cushing's syndrome in which cortisol secretion is regulated mainly by the 5-HT(4) receptor, among known aberrant receptors. In addition, it is noteworthy that hypocortisolism ensued after the removal of the most enlarged adrenal gland, but the in vivo response to cisapride persisted.

The molecular mechanisms leading to adrenocortical tumorigenesis have been only partially elucidated so far. Because the pituitary hormone ACTH, via activation of the cAMP pathway, regulates both cell proliferation/differentiation and steroid synthesis in the adrenal cortex, in this study we focused on the cAMP-dependent transcription factors cAMP responsive element modulator (CREM) and cAMP responsive element binding protein (CREB). We studied CREM and CREB expression by RT-PCR in human normal adrenal cortex (n = 3), adrenocortical adenomas (n = 8), and carcinomas (n = 8). We found transcripts corresponding to the isoforms alpha, beta, gamma, and tau2 of the CREM gene in all of the normal adrenal tissues, in the adenomas, and in seven of eight carcinomas. On the other hand, mRNA for the inducible cAMP early repressor isoforms, which derive from an internal promoter of CREM gene, was detected in the normal adrenal and in seven of eight adenomas, but in only three of eight carcinomas. Similarly, CREB transcripts were readily detectable in all normal adrenals and adenomas, whereas they were not found in four of eight adrenal carcinomas. To further characterize the carcinomas, telomerase activity and the expression of the ACTH receptor gene were determined. Telomerase activity in the carcinomas resulted in levels significantly higher than in the adenomas, whereas the levels of ACTH receptor mRNA were lower in the carcinomas. No correlation was found in the carcinomas between the levels of the ACTH receptor transcript and the loss of expression of CREB/inducible cAMP early repressor, suggesting that this alteration is not secondary to an upstream disregulation at the receptor level. In conclusion, our results suggest that an alteration in cAMP signaling may be associated with malignancies of the adrenal cortex.

In a previous report, we demonstrated that in FNC-B4 cells, derived and characterized from a human fetal olfactory epithelium, both sex steroids and odorants regulate GnRH secretion. We now report the presence and biological activity of endothelin (ET)-1 in this GnRH-secreting neuronal cell. By in situ hybridization and immunohistochemistry, we found gene and protein expression of ET-1 and its converting enzyme ECE-1 in both fetal olfactory mucosa and FNC-B4 cells. The presence of authentic ET-1 in the conditioned media of FNC-B4 cells was further supported by combined RIAs and high-performance liquid chromatography studies. Experiments with radiolabeled ET-1 and ET-3 strongly indicated the presence of two classes of binding sites, corresponding to the ETA (16,500 sites/cell) and the ETB receptors (8,700 sites/cell). Functional studies, using selective analogs, indicated that these two classes of receptors subserve distinct functions in human GnRH-secreting cells. The ETA receptor subtype mediated an increase in intracellular calcium and GnRH secretion. Conversely, stimulation of the ETB subtype induced DNA synthesis and mitogen-activated protein kinase p44ERK1 expression. This is the first demonstration, in a human in vitro model, of a neuroendocrine role for ET-1 as regulator of GnRH-secreting neuron activity.