VEGFA165 can rescue excess steroid secretion, inflammatory markers, and follicle arrest in the ovarian cortex of High A4 cows†.

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and that vascular endothelial growth factor A would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later-stage follicles compared to controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, anti-Mullerian hormone, and A4 secretion compared to controls. In trial 2, we tested if vascular endothelial growth factor A isoforms could rescue the High A4 phenotype. High A4 (n = 5) and control (n = 5) ovarian cortex was cultured with (1) PBS, (2) VEGFA165 (50 ng/mL), (3) VEGFA165B (50 ng/mL), or (4) VEGFA165 + VEGFA165B (50 ng/mL each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids and had greater indicators of inflammation compared to controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. The VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFß secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and may promote folliculogenesis.

Dimerization of Dengue Virus E Subunits Impacts Antibody Function and Domain Focus.

Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers.IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.

Phoenixin participated in regulation of food intake and growth in spotted scat, Scatophagus argus.

Phoenixin (Pnx) is an endogenous peptide known to be involved in reproduction and food intake in rats, with two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleost. Here, pnx was cloned and was detected in all tissues of both male and female in spotted scat (Scatophagus argus), including growth axis, hypothalamus, pituitary, and liver. Real-time PCR analysis showed that pnx in the hypothalamus increased significantly after 2 d and 7 d fasting, while reduced significantly after re-feeding (P < 0.05). When pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of ghrhr (growth hormone-releasing hormone receptor) and gh (growth hormone) in the pituitary, and ghr1 (growth hormone receptor 1) in the liver increased significantly, except ghr2 (growth hormone receptor 2) incubated with 10 nM and 100 nM Pnx-20 and ghr1 incubated with 10 nM Pnx-20. Similarly, the expression of ghrhr and gh in the pituitary, as well as ghr1 and ghr2 in the liver, increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight). These results indicate that Pnx is likely to be involved in the regulation of food intake, and also regulates the growth of S. argus by increasing ghrhr and gh expression in the pituitary, ghr1 and ghr2 in the liver, and ghr1 directly in the liver.

Immune protection efficacy of FAdV-4 surface proteins fiber-1, fiber-2, hexon and penton base.

The spread of hydropericardium syndrome has recently become serious in China since 2015. There is, therefore, an urgent need for new, safe and effective vaccines that prevent the disease. Here, the immune protection induced by Escherichia coli-expressed capsid proteins of fowl adenovirus serotype 4, including fiber-1, fiber-2, penton base and hexon (loop-1 region) were compared in chickens at different inoculation amounts. According to challenge mortalities and tissue gross/micro lesion results, fiber-2 induced the best protection, followed by fiber-1 and hexon. Fiber-1 and fiber-2 provided complete protection against 105.5 TCID50 viral load challenge with 100 or 50µg doses per chicken, respectively. Penton could induce effective protection only at the high dosage of 200µg per chicken. The immunoprotective characteristics of these FAdV-4 capsid proteins may prove useful for developing subunit vaccines to control hydropericardium syndrome.

Current Insights Into Inositol Isoforms, Mediterranean and Ketogenic Diets for Polycystic Ovary Syndrome: From Bench to Bedside.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex syndrome characterized by reproductive and metabolic implications. Lifestyle changes, such as diet and exercise, are considered first-line treatment for women affected by PCOS. Pharmacologic treatments target the hormonal and metabolic dysregulations associated to the disease such as insulin resistance, anovulation, hirsutism and menstrual irregularities. OBJECTIVE: To focus on the role of inositol isoforms, as well as Mediterranean and ketogenic diets, as possible therapeutic strategies in PCOS women. METHOD: Narrative overview, synthesizing the findings of literature retrieved from searches of computerized databases. RESULTS: Accumulating evidence suggests that two inositol isoforms, myo- and D-chiro-, may play a pivotal role in re-addressing both hormonal and metabolic parameters toward homeostasis, counteracting the symptoms and signs typical of this syndrome. In addition, studies focused on Mediterranean and ketogenic diet provided positive results in patients affected by obesity and type 2 diabetes, so these dietetic regimens could represent a fascinating dietetic treatment for the management of PCOS. CONCLUSION: Both the isoforms of inositol are effective in improving ovarian function and metabolism in patients with PCOS. In spite of accumulating evidence, it is currently not possible to draw firm conclusion(s) about the efficacy of these interventions considering the severe bias due to different samples size, dose, and duration of intervention among the published studies on this topic. Furthermore, future longitudinal cohort studies along with prospective interventional trials may contribute to better clarify the role of Mediterranean and ketogenic diets in the treatment of PCOS.

Low-Dose IL-17 Therapy Prevents and Reverses Diabetic Nephropathy, Metabolic Syndrome, and Associated Organ Fibrosis.

Diabetes is the leading cause of kidney failure, accounting for >45% of new cases of dialysis. Diabetic nephropathy is characterized by inflammation, fibrosis, and oxidant stress, pathologic features that are shared by many other chronic inflammatory diseases. The cytokine IL-17A was initially implicated as a mediator of chronic inflammatory diseases, but recent studies dispute these findings and suggest that IL-17A can favorably modulate inflammation. Here, we examined the role of IL-17A in diabetic nephropathy. We observed that IL-17A levels in plasma and urine were reduced in patients with advanced diabetic nephropathy. Type 1 diabetic mice that are genetically deficient in IL-17A developed more severe nephropathy, whereas administration of low-dose IL-17A prevented diabetic nephropathy in models of type 1 and type 2 diabetes. Moreover, IL-17A administration effectively treated, prevented, and reversed established nephropathy in genetic models of diabetes. Protective effects were also observed after administration of IL-17F but not IL-17C or IL-17E. Notably, tubular epithelial cell-specific overexpression of IL-17A was sufficient to suppress diabetic nephropathy. Mechanistically, IL-17A administration suppressed phosphorylation of signal transducer and activator of transcription 3, a central mediator of fibrosis, upregulated anti-inflammatory microglia/macrophage WAP domain protein in an AMP-activated protein kinase-dependent manner and favorably modulated renal oxidative stress and AMP-activated protein kinase activation. Administration of recombinant microglia/macrophage WAP domain protein suppressed diabetes-induced albuminuria and enhanced M2 marker expression. These observations suggest that the beneficial effects of IL-17 are isoform-specific and identify low-dose IL-17A administration as a promising therapeutic approach in diabetic kidney disease.

Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice.

OBJECTIVE: Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. METHODS: Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. RESULTS: Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 µg/mg vs. 19.3 ± 5.6 µg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR). CONCLUSIONS: A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.

IL-33 isoforms: their future as vaccine adjuvants?

The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide additional diversity to their complex biological effects that participate in control and protection against different foreign pathogens. Recently, IL-33 has been identified as a proinflammatory cytokine having several different biologically active isoform products. Originally associated with Th2 immunity, new evidence now supports the role of two IL-33 isoforms to facilitate the generation of protective Th1 and CD8 T cell immunity against specific pathogens. Therefore, a better understanding of the IL-33 isoforms will inform us on how to utilize them to facilitate their development as tools as vaccine adjuvants for immune therapy.

Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating bax expression.

Delta-lactoferrin (∆Lf) is a transcription factor belonging to the lactoferrin family, the expression of which inhibits cell proliferation and leads to Skp1 and DcpS gene transactivation. In this study, we showed that ∆Lf expression also induces cell death via apoptosis in HEK 293 and MCF7 cells using a cell viability assay and DNA fragmentation. Western blot analyses showed that apoptosis was caspase-9, 7 and 8 dependent. Proteolytic cleavage of the endonuclease PARP was significantly increased. The levels of expression of Bcl family members were detected by immunochemistry and showed that the Bcl-xl/Bax and Bcl-2/Bax protein ratios were decreased. We determined that the pro-apoptotic effects of ∆Lf are mainly mediated by the activation of the mitochondria-dependent death-signaling pathway. Apoptosis induction by ∆Lf is concomitant with increased cellular levels of Bax protein. Analysis of the Bax promoter region detected a ∆Lf response element located at -155 bp from the transcription start site. Both luciferase reporter gene and chromatin immunoprecipitation assays confirmed that ∆Lf interacts in vitro and in vivo specifically with this sequence. Its deletion, realized using directed mutagenesis, totally abolished ∆Lf transcriptional activity, identifying it as a ∆Lf-responsive element. These results indicate that the Bax gene is a novel ∆Lf target. Moreover we also showed that the O-GlcNAc/P interplay, which controls ∆Lf transcriptional activity, modulates Bax transactivation.

Functional genomics lead to new therapies in follicular lymphoma.

Recent technological advances allow analysis of genomic changes in cancer in unprecedented detail. The next challenge is to prioritize the multitude of genetic aberrations found and identify therapeutic opportunities. We recently completed a study that illustrates the use of unbiased genetic screens and murine cancer models to find therapeutic targets among complex genomic data. We genetically dissected the common deletion of chromosome 6q and identified the ephrin receptor A7 (EPHA7) as a tumor suppressor in lymphoma. Notably, EPHA7 encodes a soluble splice variant that acts as an extrinsic tumor suppressor. Accordingly, we developed an antibody-based strategy to specifically deliver EPHA7 back to tumors that have lost this gene. Recent sequencing studies have implicated EPHA7 in lung cancer and other tumors, suggesting a broader therapeutic potential for antibody-mediated delivery of this tumor suppressor for cancer therapy. Together, our comprehensive approach provides new insights into cancer biology and may directly lead to the development of new cancer therapies.

Role of CD44 in tumour progression and strategies for targeting.

CD44 or hyaluronan receptor is a transmembrane receptor associated with aggressive tumour growth, proliferation, and metastasis. In normal physiology, this receptor has a crucial role in cell adhesion, inflammation, and repair processes. However, many tumour cells over-express this receptor and abuse it to become progressive and perpetual units. The article comments from common functioning of the CD44 receptor, to its diabolic multi-dimensional effects in promotion of malignant cells. It also illuminates the relations of CD44 endorsed processes with other biomolecular events in cancer progression. In an end, the review focuses comprehensively at ongoing researches to exploit the CD44 over-expression as a probable target in treatment, management, and diagnosis of malignancy.

Cellular signaling and biological functions of R-spondins.

R-spondins (RSPOs) are a family of cysteine-rich secreted proteins containing a single thrombospondin type I repeat (TSR) domain. A vast amount of information regarding cellular signaling and biological functions of RSPOs has emerged over the last several years, especially with respect to their roles in the activation of the WNT signaling pathway. The identification of several classes of RSPO receptors may indicate that this family of proteins can affect several signaling cascades. Herein, we summarize the current understanding of RSPO signaling and its biological functions, and discuss its potential therapeutic implications to human diseases.

A phase I clinical study of naked DNA expressing two isoforms of hepatocyte growth factor to treat patients with critical limb ischemia.

BACKGROUND: The purpose of the present phase I clinical trial was to evaluate the safety, tolerability, and preliminary efficacy of naked DNA therapy expressing two isoforms of hepatocyte growth factor (pCK-HGF-X7) in critical limb ischemia (CLI) patients. MATERIALS AND METHODS: Twenty-one patients with CLI were consecutively assigned to receive increasing doses (cohort I: 4 mg; cohort II: 8 mg; cohort III: 12 mg; and cohort IV: 16 mg) of pCK-HGF-X7, which was administered into the ischemic calf and/or thigh muscle at days 1 and 15. A safety and tolerability evaluation and measurement of pain severity score using a visual analog scale (VAS), ulcer status, transcutaneous oxygen (TcPO(2) ) and ankle-brachial index (ABI) were performed throughout a 3-month follow-up period. RESULTS: No serious adverse events were observed in any of the 21 patients for the 3-month follow-up period. A significant reduction in pain was observed in the treated patients, with the mean VAS decreasing from 5.95-1.64 (p < 0.001). The mean ABI value increased from 0.49-0.63 (p = 0.026) at 3-month follow-up. The mean TcPO(2) value on the dorsum of the foot, the anterior calf and posterior calf significantly increased from 28.25-39.28 mmHg (p = 0.012), from 22.00-30.63 mmHg (p = 0.046) and 32.05-47.19 mmHg (p = 0.001) at 3-month follow-up, respectively. Wound healing improvement was observed in the six of nine patients that had an ulcer at baseline. CONCLUSIONS: These results support the performance of a phase II randomized controlled trial with pCK-HGF-X7.

Leptin exacerbates sepsis-mediated morbidity and mortality.

The adipose-derived hormone leptin is well known for its contribution to energy metabolism and satiety signaling in the hypothalamus. Previous studies suggested that obesity is an independent risk factor for sepsis morbidity and mortality, and it is associated with elevated baseline levels of circulating leptin in normal, nonseptic patients. In mouse endotoxemia and cecal ligation puncture models of sepsis, we observed elevated levels of leptin and soluble leptin receptor (sLR). Exogenously administered leptin increased mortality in endotoxemia and cecal ligation puncture models and was associated with increased expression of adhesion and coagulation molecules, macrophage infiltration into the liver and kidney, and endothelial barrier dysfunction. Conversely, longform leptin receptor-deficient mice were protected from sepsis morbidity and mortality and had less endothelial dysfunction. Furthermore, an in vitro study revealed that leptin-induced endothelial dysfunction is likely mediated, at least in part, by monocytes. Moreover, administration of an sLR conferred a survival benefit. Human septic patients have increased circulating sLR concentrations, which were correlated with disease severity indices. Together, these data support a pathogenic role for leptin signaling during sepsis.

Exogenous soluble VEGF receptor-1 (sFlt-1) regulates Th1/Th2 cytokine production from normal placental explants via intracellular calcium.

OBJECTIVE: The increase of soluble VEGF-Receptor 1 (sFlt-1) is thought to contribute to the pathogenesis of preeclampsia. Soluble VEGF-Receptor 1 binds to circulating free VEGF and PLGF and this cascade is associated with endothelial dysfunction, a prominent feature of preeclampsia. Preeclampsia is also associated with excessive maternal response to pro-inflammatory stimuli manifesting as an imbalance of Th1/Th2 cytokine production at the maternal-fetal interface. Whether increased sFlt-1 expression has any effect on placental production of Th1/Th2 cytokines IL-10 and TNF-alpha is yet to be investigated. The aim of this study is to examine if exogenous sFlt-1 can regulate Th1/Th2 cytokines IL-10 and TNF-alpha production from normal placental explants via intracellular calcium release. METHODS: Placental explants were taken from the decidual surface of normal non-laboured term placentas (n = 11).Villous explants were cultured with increasing concentrations of sFlt-1. The dose effect of sFlt-1 on placental Th1 and Th2 cytokine production (TNF-alpha and IL-10) were examined. Free PLGF, VEGF and sFlt-1 concentrations in the conditioned medium were also measured. Intracellular calcium blocker, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA/AM) was applied to investigate whether the changes in cytokine concentration were mediated by intracellular free calcium. RESULTS: Placental IL-10 and TNF-alpha production were significantly increased after sFlt-1 incubation. The increase in IL-10 can be inhibited by BAPTA/AM. Soluble Flt-1 and free PLGF concentration in the conditioned medium was not changed. Free VEGF concentration in the conditioned medium was not detectable. CONCLUSION: Exogenous sFlt-1 can increase TNF-alpha and IL-10 production from normal placental explants. The change in Th1/Th2 cytokine level may be mediated by intracellular free calcium.

Neuregulin 1 is a pronociceptive cytokine that is regulated by progesterone in the spinal cord: implications for sex specific pain modulation.

Sex differences in the magnitude of response to thermal and tactile stimuli have been demonstrated in both clinical and animal studies. Females typically display lower threshold responses to painful stimuli as compared to males. We have previously observed sexually dimorphic expression of the growth factor, neuregulin 1 (NRG1) following L5 nerve root ligation (LR) in male and female rats. In the present study, we sought to determine which gonadal hormones were involved in regulating NRG1 expression following L5 nerve root ligation. We observed that expression of NRG1 mRNA and the neuregulin receptors, ErbB2 and ErbB4 in the lumbar spinal cord was facilitated by the presence of progesterone in female rats following L5 nerve root ligation. An increase in NRG1 protein and NRG1 immunoreactivity was also observed in the ipsilateral spinal cord of progesterone treated female rats as compared to ovariectomized female rats and male rats at day 14 following LR. NRG1 immunoreactivity was equally colocalized with either the astrocytic marker, GFAP, and with NeuN labeled neurons 14days following L5 nerve root ligation. Intrathecal administration of recombinant NRG1-beta1 protein significantly decreased the hindpaw tactile withdrawal threshold in male rats, ovariectomized female rats, and progesterone treated female rats. These results demonstrate a role for progesterone-dependent regulation of glial and/or neuronal neuregulin 1 in female rats in mediating sex differences in nociception. Furthermore, our results suggest that NRG1 may be involved in central sensitization during the maintenance phase, but not in the initiation of persistent pain in female rats.

HGF-MSP chimera protects kidneys from ischemia-reperfusion injury.

Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation and is related to long-term graft function. MF-1, a bifunctional hepatocyte growth factor (HGF)-macrophage-stimulating protein (MSP) (HGF-MSP) chimera was recently reported to prevent apoptosis. We therefore hypothesized that treatment with MF-1 would protect kidneys from I/R injury by inhibiting tubular epithelial apoptosis. MF-1 directly guarded cultured proximal tubular epithelial cells from hypoxia-induced necrosis and apoptosis in vitro. In addition, the therapeutic effects of MF-1 were evaluated using a rat I/R injury model in vivo. Saline-treated kidneys had increased creatinine and BUN, and exhibited tubular epithelial apoptosis with activated caspase 3 expression. In contrast, MF-1 treatment up-regulated Akt phosphorylation, and inhibited caspase 3 activation and tubular apoptosis, thereby ameliorating renal dysfunction. Of particular interest is that macrophage infiltration was suppressed in the MF-1-treated kidney. In conclusion, we identified a novel therapeutic approach using MF-1 to protect kidneys from I/R injury.

Supplemental and dietary vitamin E intakes and risk of prostate cancer in a large prospective study.

Supplemental vitamin E (alpha-tocopherol) has been linked to lower prostate cancer incidence in one randomized trial and several, although not all, observational studies. The evidence regarding dietary intake of individual vitamin E isoforms and prostate cancer is limited and inconclusive, however. We prospectively examined the relations of supplemental vitamin E and dietary intakes of alpha-, beta-, gamma-, and delta- tocopherols to prostate cancer risk among 295,344 men, ages 50 to 71 years and cancer-free at enrollment in 1995 to 1996, in the NIH-AARP Diet and Health Study. At baseline, participants completed a questionnaire that captured information on diet, supplement use, and other factors. Proportional hazards models were used to estimate relative risks (RR) and 95% confidence intervals (95% CI) of prostate cancer. During 5 years of follow-up, 10,241 incident prostate cancers were identified. Supplemental vitamin E intake was not related to prostate cancer risk (for >0-99, 100-199, 200-399, 400-799, and > or = 800 IU/d versus never use: RR, 0.97, 0.89, 1.03, 0.99, and 0.97 (95% CI, 0.87-1.07) respectively; Ptrend = 0.90). However, dietary gamma-tocopherol, the most commonly consumed form of vitamin E in the United States, was significantly inversely related to the risk of advanced prostate cancer (for highest versus lowest quintile: RR, 0.68; 95% CI, 0.56-0.84; Ptrend = 0.001). These results suggest that supplemental vitamin E does not protect against prostate cancer, but that increased consumption of gamma-tocopherol from foods is associated with a reduced risk of clinically relevant disease. The potential benefit of gamma-tocopherol for prostate cancer prevention deserves further attention.

The anti-angiogenic VEGF isoform VEGF165b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo.

Vascular endothelial growth factor (VEGF) is the principal agent that increases microvascular permeability during physiological and pathological angiogenesis. VEGF is differentially spliced to form two families of isoforms: VEGF(xxx), and VEGF(xxx)b. Whereas VEGF(165) stimulates angiogenesis, VEGF(165)b is anti-angiogenic. To determine the effect of VEGF(165)b on permeability, hydraulic conductivity (L(p)) was measured in individually perfused microvessels in the mesentery of frogs and rats. As with VEGF(165), VEGF(165)b increased L(p) in amphibian (2.4 +/- 0.3-fold) and mammalian (1.9 +/- 0.2-fold) mesenteric microvessels. A dose-response relationship showed that VEGF(165)b (EC(50), 0.65 pm) was approximately 25 times more potent than VEGF(165) (EC(50), 16 pm) in amphibian microvessels. VEGF(165) has been shown to increase permeability through VEGF receptor 2 (VEGF-R2) signalling. However, VEGF(165)b increased L(p) of frog vessels to the same extent in the presence of the VEGF-R2 inhibitor ZM323881, indicating that it does not increase permeability via VEGF-R2 signalling, and was inhibited by the VEGF receptor inhibitor SU5416 at doses that are specific for VEGF receptor 1 (VEGF-R1). VEGF(165)b, in contrast to VEGF(165), did not result in a sustained chronic increase in L(p). These results show that although VEGF(165)b is anti-angiogenic in the mesentery, it does signal in endothelial cells in vivo resulting in a transient, but not sustained, increase in microvascular L(p), probably through VEGF-R1.

Analysis of effects of connexin-mimetic peptides in rat mesenteric small arteries.

Synthetic peptides homologous to the extracellular loops of the major vascular connexins represent a novel class of gap junction blockers that have been used to assess the role of direct cellular communication in arteries and veins. However, the specificity of action of such peptides on the coupling between smooth muscle cells (SMCs) has not yet been fully characterized. Isolated third-order rat mesenteric arteries were therefore studied with respect to isometric tension (myography), intracellular Ca2+ concentration ([Ca2+]i) (Ca2+ -sensitive dyes), membrane potential, and input resistance (sharp intracellular glass electrodes). Confocal imaging was used for visualization of [Ca2+]i events in individual SMCs in the arterial wall and membrane currents (patch clamp) measured in individual SMCs isolated from the same arteries. A triple peptide combination (37,43Gap 27 + 40Gap 27 + 43Gap 26) increased intercellular resistance (measured as input resistance) in intact arterial segments without affecting the membrane conductance of individual cells and also interrupted electrical coupling between pairs of rat aortic A7r5 myocytes. In intact arterial segments, the peptides desynchronized [Ca2+]i transients in individual SMCs and abolished vasomotion without suppressing Ca2+ transients in individual cells. They also depolarized SMCs, increased [Ca2+]i, and attenuated acetylcholine-induced, endothelium-dependent smooth muscle hyperpolarization. Experiments with endothelium-denuded arteries suggested that the depolarization produced by the peptides under basal conditions was in part secondary to electrical uncoupling of the endothelium from SMCs with loss of a tonic hyperpolarizing effect of the endothelium. Taken together, the results indicate that connexin-mimetic peptides block electrical signaling in rat mesenteric small arteries without exerting major nonjunctional effects.