Self-Assessed Benefits of a Prostate Health Formulation on Nocturia in Healthy Males With Mild Lower Urinary Tract Symptoms: An Open Label Study.

BACKGROUND: Nocturia is the most bothersome lower urinary tract symptoms (LUTS) and can significantly reduce men's quality of life. It is often poorly managed with conventional treatments. OBJECTIVE: The purpose of this study was to evaluate the self-assessed benefits of a prostate health dietary combination formulation on mild LUTS, especially nocturia in healthy males. METHODS: In an open label clinical study, thirty healthy male subjects with mild LUTS took one daily capsule of the product for 60 days. The primary outcome was self-assessed severity of LUTS using the International Prostate Symptoms Score (IPSS) questionnaire at Day 1 (baseline), Day 30 and Day 60. Safety and compliance were also evaluated. RESULTS: At Day 60, IPSS significantly decreased from baseline by 16.3% (3.6 ± 2.1 vs. 4.3 ± 1.5, p < 0.05). Although the reduction in IPSS did not reach statistical significance at Day 30, it was mostly driven by a 30.7% decrease (p < 0.05) in the nocturia sub-score compared with baseline. While 37% of subjects reported at baseline waking up 2‒3 times/night to void, none did so after taking the study product for 60 days. Compliance was very high throughout the study. No adverse events related to the study product were reported. CONCLUSIONS: The study product might be a safe alternative for individuals willing to explore a non-conventional approach to manage their nocturia. A larger randomized placebo-controlled clinical trial is warranted to confirm these results. Clinical trial registry: Clinical Trials.gov. Registration number (September 1st, 2016): NCT02886832.

The systemic activin response to pancreatic cancer: implications for effective cancer cachexia therapy.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a particularly lethal malignancy partly due to frequent, severe cachexia. Serum activin correlates with cachexia and mortality, while exogenous activin causes cachexia in mice. METHODS: Isoform-specific activin expression and activities were queried in human and murine tumours and PDAC models. Activin inhibition was by administration of soluble activin type IIB receptor (ACVR2B/Fc) and by use of skeletal muscle specific dominant negative ACVR2B expressing transgenic mice. Feed-forward activin expression and muscle wasting activity were tested in vivo and in vitro on myotubes. RESULTS: Murine PDAC tumour-derived cell lines expressed activin-ßA but not activin-ßB. Cachexia severity increased with activin expression. Orthotopic PDAC tumours expressed activins, induced activin expression by distant organs, and produced elevated serum activins. Soluble factors from PDAC elicited activin because conditioned medium from PDAC cells induced activin expression, activation of p38 MAP kinase, and atrophy of myotubes. The activin trap ACVR2B/Fc reduced tumour growth, prevented weight loss and muscle wasting, and prolonged survival in mice with orthotopic tumours made from activin-low cell lines. ACVR2B/Fc also reduced cachexia in mice with activin-high tumours. Activin inhibition did not affect activin expression in organs. Hypermuscular mice expressing dominant negative ACVR2B in muscle were protected for weight loss but not mortality when implanted with orthotopic tumours. Human tumours displayed staining for activin, and expression of the gene encoding activin-ßA (INHBA) correlated with mortality in patients with PDAC, while INHBB and other related factors did not. CONCLUSIONS: Pancreatic adenocarcinoma tumours are a source of activin and elicit a systemic activin response in hosts. Human tumours express activins and related factors, while mortality correlates with tumour activin A expression. PDAC tumours also choreograph a systemic activin response that induces organ-specific and gene-specific expression of activin isoforms and muscle wasting. Systemic blockade of activin signalling could preserve muscle and prolong survival, while skeletal muscle-specific activin blockade was only protective for weight loss. Our findings suggest the potential and need for gene-specific and organ-specific interventions. Finally, development of more effective cancer cachexia therapy might require identifying agents that effectively and/or selectively inhibit autocrine vs. paracrine activin signalling.

Avoiding holiday seasonal weight gain with nutrient-supported intermittent energy restriction: a pilot study.

This pilot randomised controlled study evaluated the effects of a nutrient-supported intermittent energy restriction nutrition programme to prevent weight gain in healthy overweight adults during the 6-week winter holiday period between Thanksgiving and New Year. For 52 d, twenty-two overweight adults (mean age 41·0 years, BMI 27·3 kg/m2) were assigned to either the nutrition programme (n 10; two fasting days of 730 kcal/d (3050 kJ/d) of balanced shake and dietary supplements to support weight management efforts, followed by 5 d of habitual diet) or a control group (n 12; habitual diet). A significant weight loss from baseline (pre-holiday 10 d before Thanksgiving) to day 52 (post-holiday 3 January) was observed in the nutrition programme (75·0 (sd 9·8) v. 76·3 (sd 9·8) kg; P < 0·05). Body weight did not significantly change in the control group and there was no between-group difference. Increases from baseline in fasting insulin (42·9 %; P = 0·0256), updated homoeostasis model assessment (HOMA2) (43 %; P = 0·025), LDL-cholesterol (8·4 %; P = 0·0426) and total cholesterol (7·1 %; P = 0·0154) levels were also reported in the control group. In the nutrition programme group, baseline HDL-cholesterol and TAG levels measured after two fasting days increased (13 %; P = 0·0245) and decreased (22·8 %; P = 0·0416), respectively. There was no significant change in HOMA2. Between-group differences in changes in insulin levels (P = 0·0227), total cholesterol:HDL-cholesterol ratio (P = 0·0419) and HOMA2 (P = 0·0210) were significant. Overall compliance rate was 98 % and no severe adverse events were reported. These preliminary findings suggest that this intermittent energy restriction intervention might support weight management efforts and help promote metabolic health during the winter holiday season.

GDF11 induces kidney fibrosis, renal cell epithelial-to-mesenchymal transition, and kidney dysfunction and failure.

BACKGROUND: GDF11 modulates embryonic patterning and kidney organogenesis. Herein, we sought to define GDF11 function in the adult kidney and in renal diseases. METHODS: In vitro renal cell lines, genetic, and murine in vivo renal injury models were examined. RESULTS: Among tissues tested, Gdf11 was highest in normal adult mouse kidney. Expression was increased acutely after 5/6 nephrectomy, ischemia-reperfusion injury, kanamycin toxicity, or unilateral ureteric obstruction. Systemic, high-dose GDF11 administration in adult mice led to renal failure, with accompanying kidney atrophy, interstitial fibrosis, epithelial-to-mesenchymal transition of renal tubular cells, and eventually death. These effects were associated with phosphorylation of SMAD2 and could be blocked by follistatin. In contrast, Gdf11 heterozygous mice showed reduced renal Gdf11 expression, renal fibrosis, and expression of fibrosis-associated genes both at baseline and after unilateral ureteric obstruction compared with wild-type littermates. The kidney-specific consequences of GDF11 dose modulation are direct effects on kidney cells. GDF11 induced proliferation and activation of NRK49f renal fibroblasts and also promoted epithelial-to-mesenchymal transition of IMCD-3 tubular epithelial cells in a SMAD3-dependent manner. CONCLUSION: Taken together, these data suggest that GDF11 and its downstream signals are critical in vivo mediators of renal injury. These effects are through direct actions of GDF11 on renal tubular cells and fibroblasts. Thus, regulation of GDF11 presents a therapeutic target for diseases involving renal fibrosis and impaired tubular function.

Effect of vital dyes on retinal pigmented epithelial cell viability and apoptosis: implications for chromovitrectomy.

PURPOSE: To investigate the in vitro effect of vital dyes on toxicity and apoptosis in a human retinal pigment epithelial cell line. METHODS: ARPE-19 cells were exposed to brilliant blue (BBG), Evans Blue (EB), bromophenol blue (BroB), indocyanine green (ICG), infracyanine green (IfCG), light green (LG), fast green (FG), indigo carmine (IC) and Congo red (CR). Balanced salt solution was used as the control. Five different concentrations and 2 exposure times were tested. Cell viability was determined by the MTS (1-solution methyl thiazolyl tetrazolium) assay and apoptosis by Bax expression on Western blot. RESULTS: All dyes significantly reduced cell viability after 3 min of exposure at all concentrations (p < 0.01), except for BBG that was safe at concentrations up to 0.25 mg/ml and CR up to 0.05 mg/ml, while LG was safe at all concentrations. Toxicity was higher after 30 min of exposure. Expression of Bax was upregulated after all dye exposures, except BBG; ICG had the highest Bax expression (p < 0.01). CONCLUSIONS: Overall the safest dye was BBG followed by LG, IfCG, FG, CR, IC, BroB, EB and ICG. ICG was toxic at all concentrations and exposure times tested. Moreover, BBG was the only dye that did not induce apoptosis in ARPE-19 cells.

Retinal pigmented epithelial cells cytotoxicity and apoptosis through activation of the mitochondrial intrinsic pathway: role of indocyanine green, brilliant blue and implications for chromovitrectomy.

PURPOSE: To investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line. METHODS: ARPE-19 cells were exposed to brilliant blue (BriB), methyl blue (MetB), acid violet (AcV) and indocyanine green (ICG). Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL) and two exposure times (3 and 30 min) were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process. RESULTS: ICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01). BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein. CONCLUSIONS: The safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process.

Angiotensin II-induced MMP-2 activity and MMP-14 and basigin protein expression are mediated via the angiotensin II receptor type 1-mitogen-activated protein kinase 1 pathway in retinal pigment epithelium: implications for age-related macular degeneration.

Accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE) has been observed in eyes with age-related macular degeneration (AMD). RPE-derived matrix metalloproteinase (MMP)-2, MMP-14, and basigin (BSG) are major enzymes involved in the maintenance of ECM turnover. Hypertension (HTN) is a systemic risk factor for AMD. It has previously been reported that angiotensin II (Ang II), one of the most important hormones associated with HTN, increases MMP-2 activity and its key regulator, MMP-14, in RPE, inducing breakdown of the RPE basement membrane, which may lead to progression of sub-RPE deposits. Ang II exerts most of its actions by activating the mitogen-activated protein kinase (MAPK) signaling pathway. Herein is explored the MAPK signaling pathway as a potential key intracellular modulator of Ang II-induced increase in MMP-2 activity and MMP-14 and BSG protein expression. It was observed that Ang II stimulates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in RPE cells and ERK/p38 and Jun N-terminal kinase (JNK) in mice. These effects were mediated by Ang II type 1 receptors. Blockade of ERK or p38 MAPK abrogated the increase in MMP-2 activity and MMP-14 and BSG proteins in ARPE-19 cells. A better understanding of the molecular events by which Ang II induces ECM dysregulation is of critical importance to further define its contribution to the progression of sub-RPE deposits in AMD patients with HTN.

Cigarette smoke-related hydroquinone dysregulates MCP-1, VEGF and PEDF expression in retinal pigment epithelium in vitro and in vivo.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice. PRINCIPAL FINDINGS: MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo. CONCLUSION: We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD.

Nicotine increases the VEGF/PEDF ratio in retinal pigment epithelium: a possible mechanism for CNV in passive smokers with AMD.

PURPOSE: Cigarette smoking is the strongest environmental risk factor for wet age-related macular degeneration (AMD). Inappropriate expression of proangiogenic vascular endothelial growth factor (VEGF) and antiangiogenic pigment epithelium derived factor (PEDF) may cause choroidal neovascularization (CNV), a key event in wet AMD, resulting in vision loss. Nicotine (NT), a potent angiogenic agent abundant in second-hand smoke, may play a major role in the pathogenesis of wet AMD. The purpose of this study was to evaluate the expression of nicotinic acetylcholine receptors (nAchR) in retinal pigment epithelium (RPE) and determine the effects of NT on RPE-derived VEGF and PEDF expression in the context of passive smoking. METHODS: Human RPE cells were treated with NT (10(-8) M), with or without the nAchR-nonspecific antagonist hexamethonium (HXM) (10(-5) M) for 72 hours. RPE sheets were microdissected from rats exposed to NT in drinking water (100 µg/mL), with or without HXM (40 mg/kg/d, intraperitoneally), for 72 hours. Cell death was determined by cell count and proliferation by Western blot for proliferating cell nuclear antigen (PCNA). nAchR expression was examined by real-time PCR and Western blot. ERK activation was evaluated by Western blot analysis. VEGF and PEDF expression was assessed by ELISA, Western blot, and real-time PCR. RESULTS: Cultured RPE cells constitutively expressed the nAchR α3, α10, and ß1 subunits, with ß1 being the most prevalent. The nAchR α4, α5, α7, and ß2 subunits were detected in RPE sheets from rats, among which α4 is the predominant subtype. NT, which did not result in either cell death or proliferation, induced ß1 nAchR, upregulated VEGF, and downregulated PEDF expression through nAChR in ARPE-19 cells. Transcriptional activation of the nAchR α4 subunit and nAChR-mediated upregulation of VEGF and PEDF were observed in RPE from rats exposed to NT. CONCLUSIONS: NT increased the VEGF-to-PEDF ratio in the RPE through nAchR in vitro and in vivo. This alteration in the ratio may play a key role in the progression to wet AMD in passive smokers.

Cigarette smoke-related hydroquinone induces filamentous actin reorganization and heat shock protein 27 phosphorylation through p38 and extracellular signal-regulated kinase 1/2 in retinal pigment epithelium: implications for age-related macular degeneration.

Retinal pigment epithelium (RPE)-derived membranous debris named blebs, may accumulate and contribute to sub-RPE deposit formation, which is the earliest sign of age-related macular degeneration (AMD). Oxidative injury to the RPE might play a significant role in AMD. However, the underlying mechanisms are unknown. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, foodstuff, and atmospheric pollutants, induces actin rearrangement and membrane blebbing in RPE cells as well as sub-RPE deposits in mice. Here, we show for the first time that phosphorylated Heat shock protein 27 (Hsp27), a key regulator of actin filaments dynamics, is up-regulated in RPE from patients with AMD. Also, HQ-induced nonlethal oxidative injury led to Hsp27mRNA up-regulation, dimer formation, and Hsp27 phosphorylation in ARPE-19 cells. Furthermore, we found that a cross talk between p38 and extracellular signal-regulated kinase (ERK) mediates HQ-induced Hsp27 phosphorylation and actin aggregate formation, revealing ERK as a novel upstream mediator of Hsp27 phosphorylation. Finally, we demonstrated that Hsp25, p38, and ERK phosphorylation are increased in aging C57BL/6 mice chronically exposed to HQ, whereas Hsp25 expression is decreased. Our data suggest that phosphorylated Hsp27 might be a key mediator in AMD and HQ-induced oxidative injury to the RPE, which may provide helpful insights into the early cellular events associated with actin reorganization and bleb formation involved in sub-RPE deposits formation relevant to the pathogenesis of AMD.

Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia.

Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.

Protein-rich diet attenuates cyclosporin A-induced renal tubular damage in rats.

OBJECTIVE: The objective of the present study was to look at the effect of a protein-rich diet on cyclosporine A (CsA)-induced acute nephrotoxicity in rodents using markers of tubular damage. DESIGN: Female Sprague-Dawley rats were conditioned to either a standard or a casein-rich diet for 2 weeks. Then, they were given CsA intraperitoneally (25 mg/kg/24 h or an equivalent volume of vehicle (Cremophor EL; Sigma Chemical Co, St. Louis, MO) for 7 days at 7 AM. RESULTS: During CsA treatment, bodyweight, caloric consumption, water intake, and urine output were not significantly different in animals fed with the standard Rat Chow and those on the high-protein feeding. On days 1 and 7, the 24-hour urine excretion of N-acetyl-beta-d-glucosaminidase (NAG) and beta-galactosidase (beta-GAL) were significantly (P < .001) lower in CsA-treated rats on the high-protein diet than in those on the standard Rat Chow. After 7 days of treatment with CsA, no significant difference in the renal function level was found between rats fed with the standard or the casein-rich diet. The post-necrotic cellular regeneration in renal cortex was significantly lower (p<0.001) in CsA-treated rats on the high-protein than on the standard diet. In CsA-treated rats on the standard diet, immunogold labeling showed a massive and specific concentration of the drug into lysosomes of proximal tubular cells. Contrastingly, no gold particle was found over the lysosomes of animals given the rich-protein feeding. CONCLUSION: In our current experimental conditions, a protective effect of high-casein diet against CsA-induced proximal tubular damage was observed in Sprague-Dawley rats.