The impact of cardiomotor rehabilitation on endothelial function in elderly patients with chronic heart failure.

BACKGROUND: To explore the effects of cardiac exercise rehabilitation on peripheral blood endothelial progenitor cells (EPC) in elderly patients with chronic heart failure. METHODS: 80 elderly patients with chronic heart failure were selected from March 2017 to March 2019 and randomly divided into two groups (N = 40). The control group was treated routinely and walked freely for 30-60 min every day. The patients in the exercise rehabilitation group developed a cardiac exercise rehabilitation plan. Then, cardiac function and peripheral blood B-natriuretic peptide (BNP) levels in the two groups were compared. The cell viability, proliferation, apoptosis, and invasion ability of EPCs were detected. The levels of the PI3K/AKT pathway and eNOS and VEGF were compared. RESULTS: There were no significant differences in all indexes between the two groups before treatment (P > 0.05), and both improved significantly after treatment (P < 0.05). After treatment, LVEF and LVFS in the exercise rehabilitation group were significantly higher than those in the control group (P < 0.05), and LVEDD and LVESD were significantly lower than those in the control group (P < 0.05). The BNP level in the exercise rehabilitation group was significantly lower than that in the control group (P < 0.05). The cell viability, proliferation, invasion ability of EPC, and the levels of PI3K, AKT, eNOS, and VEGF mRNA and protein in the exercise rehabilitation group were significantly higher than those in the control group. Apoptosis rate was significantly lower than those in the control group (P < 0.05). CONCLUSIONS: Visceral exercise rehabilitation can improve cardiac ejection and myocardial function in elderly patients with chronic heart failure, and can promote the vitality, proliferation, and invasion of peripheral blood EPC, and promote the expression of eNOS and VEGF by upregulating the PI3K/AKT pathway to promote angiogenesis and endothelial function.

An advanced method for the immunohistochemical detection of nitric oxide synthase (NOS) in the female genital tract.

The expression of nitric oxide synthase (NOS) in male and female urogenital tissues has been investigated by using conventional light microscopical immunoperoxidase staining. We present an improved immunohistochemical method for the specific and simultaneous detection of endothelial and neuronal NOS (eNOS/nNOS) in vaginal tissue. Specific antibodies have been used in combination with the tyramide signal amplification method. We found a subepithelial meshwork of varicose nerve fibers. A subpopulation of fibers presented immunoreactivity specific for nNOS. Epithelial cells also showed cytoplasmatic labeling for nNOS. Arteries presenting signals for eNOS in their endothelial layer were found in close proximity to nNOS-positive nerve fibers.

Testosterone positively regulates functional responses and nitric oxide expression in the isolated human corpus cavernosum.

BACKGROUND: Testosterone (T) deficiency is associated with erectile dysfunction (ED). The relaxant response of T on the corporal smooth muscle through a non-genomic pathway has been reported; however, the in vitro modulating effects of T on human corpus cavernosum (HCC) have not been studied. OBJECTIVES: To compare the effects of various concentrations of T on nitric oxide (NO)-dependent and nitric oxide-independent relaxation in organ bath studies and elucidate its mode of action, specifically targeting the cavernous NO/cyclic guanosine monophosphate (cGMP) pathway. MATERIALS AND METHODS: Human corpus cavernosum (HCC) samples were obtained from men undergoing penile prosthesis implantation (n = 9). After phenylephrine (Phe) precontraction, the effects of various relaxant drugs of HCC strips were performed using organ bath at low (150 ng/dL), eugonadal (400 ng/dL), and hypergonadal (600 ng/dL) T concentrations. The penile tissue measurements of endothelial nitric oxide synthase (eNOS), neuronal (n)NOS, and phosphodiesterase type 5 (PDE5) were evaluated via immunostaining, Western blot, cGMP and nitrite/nitrate (NOx) assays. RESULTS: Relaxation responses to ACh and EFS in isolated HCC strips were significantly increased at all T levels compared with untreated tissues. The sildenafil-induced relaxant response was significantly increased at both eugonadal and hypergonadal T levels. Normal and high levels of T are accompanied by increased eNOS, nNOS, cGMP, and NOx levels, along with reduced PDE5 protein expression. CONCLUSION: This study reveals an important role of short-term and modulatory effects of different concentrations of T in HCC. T positively regulates functional activities, inhibition of PDE5 expression, and formation of cGMP and NOx in HCC. These results demonstrate that T indirectly contributes to HCC relaxation via downstream effects on nNOS, eNOS, and cGMP and by inhibiting PDE5. This action provides a rationale for normalizing T levels in hypogonadal men with ED, especially when PDE5 inhibitors are ineffective. T replacement therapy may improve erectile function by modulating endothelial function in hypogonadal men.

Aspartic acid supplementation ameliorates symptoms of diabetic kidney disease in mice.

Diabetic kidney disease (DKD) is among the most common and serious complications of both type 1 and type 2 diabetes. In this study, we used KK/Ta-Ins2Akita (KK-Akita) mice as a model of DKD and KK/Ta (KK) mice as controls to identify novel factors related to the development/progression of DKD. Capillary electrophoresis coupled with mass spectrometry analysis revealed that circulating Asp (l-aspartic acid) levels in diabetic KK-Akita mice tend to be lower than those in control KK mice. Therefore, we evaluated the effect of Asp supplementation to prevent the progression of DKD in KK-Akita mice. Mice were divided into three groups: (a) untreated KK mice (Control group), (b) untreated KK-Akita mice (DKD group), and (c) treated (double-volume Asp diet) KK-Akita mice (Tx group). Kidney sections were stained with fluorescein isothiocyanate-labeled lectins, wheat germ agglutinin (WGA), and anti-endothelial nitric oxide synthase (eNOS) antibody for evaluation of endothelial surface layer (ESL) and NO synthesis. The mesangial area and glomerular size in the DKD group were significantly larger than those in the Control group; however, there was no significant difference in those between the DKD and Tx groups. Albuminuria, the ratio of foot process effacement, and thickness of glomerular basement membrane in the Tx group were significantly lower than those in the DKD group. Furthermore, the expression levels of glomerular WGA and microvascular eNOS in the Tx group improved significantly and approached the level in the Control group. In conclusion, the improvement of albuminuria in the Tx group may be caused by the reduction of oxidative stress in the kidneys, which may lead to the subsequent improvement of glomerular ESL.

Regional Distribution of Nitric Oxide Synthase in Human Anorectal Tissue: A Pilot Study on the Potential Role for Nitric Oxide in Haemorrhoids.

OBJECTIVE: To study the distribution of nitric oxide synthase (NOS) isoforms and protein levels in human haemorrhoids and rectal tissue. METHODS: Protein expression of NOS1, NOS2 and NOS3 was compared between haemorrhoids (n=14) and normal rectal submucosa (n=6) using Western blot analysis. The localisation of all NOS isoforms to specific structures was determined by immunohistochemistry. RESULTS: Western blot analysis showed median (interquartile range) protein levels of all NOS isoforms were 1.5-2.4 times higher in haemorrhoids than rectal tissue; 121.4 (55.2-165.5) vs 50.0 (25.5-73.7) for NOS1 (p=0.020), 32.2 (23.8-140.6) vs 14.8 (9.6-34.0) for NOS2 (p=0.109), and 80.1 (62.0-139.5) vs 54.3 (48.7 -61.7) for NOS3 (p=0.015). Immunohistochemistry revealed a different distribution and location of all NOS isoforms in vascular and non-vascular structure of haemorrhoids and rectal tissues. The number of haemorrhoid specimens showing positive immunoreactivity of NOS in the vascular endothelium was significantly higher than that in rectal tissue for NOS1 (11/14 (79%) vs 1/6 (17%); p=0.018) and NOS3 (8/14 (57%) vs 0/6 (0%); p=0.042), but not for NOS2 (6/14 (43%) vs 4/6 (67%); p=0.63). CONCLUSION: Haemorrhoids have significantly higher protein levels of NOS1 and NOS3 than rectal tissue. The vascular endothelium of haemorrhoids also has significantly higher positive immunoreactivity of NOS1 and NOS3 than rectal tissue suggesting that blood vessels in haemorrhoids are exposed to higher NO concentrations than those of rectal tissue. Since haemorrhoids exhibit marked vascular dilatation and present with bleeding or swelling, a reduction in NOS - by applying NOS inhibitors - may potentially improve the symptoms of haemorrhoids.

Early Alterations of Endothelial Nitric Oxide Synthase Expression Patterns in the Guinea Pig Cochlea After Noise Exposure.

Constitutively expressed endothelial nitric oxide synthase (eNOS) is supposed to play a role in noise-induced nitric oxide (NO)-production. It is commonly known that intense noise exposure results in inducible NOS (iNOS) expression and increased NO-production, but knowledge about a contribution of the eNOS isoform is still lacking. Effects of noise exposure on eNOS immunolabeling were determined in male guinea pigs (n=24). For light microscopic analysis, 11 animals were exposed to 90 dB for 1 hr and 6 animals were used as controls. After exposure, eNOS immunostaining was performed on paraffin sections, and the staining intensities were quantified for 4 cochlear regions. For electron microscopic analysis, 2 animals were exposed for 2 hr to 90 dB and 5 animals were used as controls. The intensity of eNOS immunolabeling was found to be already comprehensively increased 1 hr after noise exposure to 90 dB. At the ultrastructural level, a clear increase in eNOS immunolabeling was found in microtubules-rich areas of cochlear cuticular structures. Hence, our findings indicate that the reticular lamina forming the endolymph-perilymph barrier at the apical side of the organ of Corti is involved in a fast intrinsic otoprotective mechanism of the cochlea.

eNose analysis of volatile chemicals from dogs naturally infected with Leishmania infantum in Brazil.

BACKGROUND: Visceral leishmaniasis (VL) in Brazil is a neglected, vector-borne, tropical parasitic disease that is responsible for several thousand human deaths every year. The transmission route involves sand flies becoming infected after feeding on infected reservoir host, mainly dogs, and then transmitting the Leishmania infantum parasites while feeding on humans. A major component of the VL control effort is the identification and euthanasia of infected dogs to remove them as a source of infection. A rapid, non-invasive, point-of-care device able to differentiate between the odours of infected and uninfected dogs may contribute towards the accurate diagnosis of canine VL. METHODOLOGY/PRINCIPAL FINDINGS: We analysed the headspace volatile chemicals from the hair of two groups of dogs collected in 2017 and 2018 using a bench-top eNose volatile organic chemical analyser. The dogs were categorised as infected or uninfected by PCR analysis of blood samples taken by venepuncture and the number of parasites per ml of blood was calculated for each dog by qPCR analysis. We demonstrated using a robust clustering analysis that the eNose data could be discriminated into infected and uninfected categories with specificity >94% and sensitivity >97%. The eNose device and data analysis were sufficiently sensitive to be able to identify infected dogs even when the Leishmania population in the circulating blood was very low. CONCLUSIONS/SIGNIFICANCE: The study illustrates the potential of the eNose to rapidly and accurately identify dogs infected with Le. infantum. Future improvements to eNose analyser sensor sensitivity, sampling methodology and portability suggest that this approach could significantly improve the diagnosis of VL infected dogs in Brazil with additional potential for effective diagnosis of VL in humans as well as for the diagnosis of other parasitic diseases.

Age-Related Impairment of Structure and Function of Iliac Artery Endothelium in Rats Is Improved by Elevated Fluid Shear Stress.

BACKGROUND Aging plays an important role in endothelial dysfunction. Fluid shear stress (FSS) can activate endothelial cells (ECs). Herein, we tested the hypothesis that this endothelial impairment could be improved by elevated FSS (EFSS) in aged rats. MATERIAL AND METHODS EFSS was created through ligation of the unilateral common iliac artery in 20-month-old rats, evaluated by measuring blood flow velocity with Doppler spectrum. The effect of FSS on aged ECs was examined by senescence-associated ß-galactosidase (SA-ß-Gal) staining, ultrastructural observation, and immunostaining and qPCR analysis of eNOS and SIRT1 expression on both the mRNA and protein levels. RESULTS (1) FSS was significantly increased in the right common iliac artery (RCIA) in rats with the ligation of the left common iliac artery (LCIA). (2) SA-ß-Gal staining was significantly attenuated by EFSS in the RCIA of aged rats. (3) Ultrastructural observation showed that ECs in the RCIA of normal aged rats became irregular and enlarged, with increasingly polypoid nuclei and fewer mitochondria, whereas ECs in the RCIA of aged rats with LCIA ligation became more prominent and contained more mitochondria. (4) eNOS and SIRT1 expression in the RCIA of aged rats with LCIA ligation was significantly upregulated compared with that in control group rats. CONCLUSIONS The present study for the first time shows that EFSS has the ability to improve age-related impairment of endothelial structure and functions.

Long-Term Aspirin Administration Has No Effect on Erectile Function: Evidence from Adult Rats and Ageing Rat Model.

As the broad spectrum pharmacological action, aspirin has been one of the most widely used medicines since its initial synthesis; however, the association between aspirin and erectile function is still controversial. We aim to explore whether long-term aspirin administration deteriorates or preserves erectile function from adult rats and ageing rat model. Twenty adult rats (10 weeks of age) and twenty ageing rats (80 weeks of age) were randomly divided into four groups as follows: Adult-Control (normal saline [NS]), Adult-Aspirin (aspirin, 10 mg/kg/d), Ageing-Control (NS), and Ageing-Aspirin (aspirin, 10 mg/kg/d) groups (n = 10 per group). For all rats, erectile function was assessed by maximum intracavernous pressure (ICP), total area under ICP curve (AUC), ICP/mean arterial pressure (MAP) ratio, and MAP. The total treatment duration was one month. Protein expression levels of cyclooxygenase-1 (COX-1), COX-2, endothelial nitric oxide synthase (eNOS), and nNOS of the corpus cavernosum were detected by Western blot. ELISA kits were used to determine 6-keto PGF1a, PGE2, TXB2, cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP) levels. Total nitric oxide (NO) concentration was measured using a fluorometric assay kit. As a result, Ageing-Control rats revealed significantly decreased ICP, AUC, and ICP/MAP ratios compared to Adult-Control rats, and these effects were accompanied by reduced eNOS protein expression and lower total NO and cGMP levels; however, no difference was found in nNOS protein expression. For adult rat groups, aspirin significantly inhibited the production of 6-keto PGF1a, PGE2, and TXB2; however, it neither changed the ICP, AUC, or ICP/ MAP ratios nor altered the protein expression of eNOS, nNOS, COX-1, and COX-2. Meanwhile, aspirin did not influence the concentrations of total NO, cAMP, or cGMP. The same tendency was also found in the ageing rat model, which confirmed that aspirin did not alter erectile function. Our data suggested that long-term aspirin administration did not strengthen or weaken erectile function in adult rats or ageing rat model. Thus, it had no impact on erectile function.

Long-term endothelial dysfunction in irradiated vessels: an immunohistochemical analysis.

BACKGROUND: Microvascular free flap reconstruction has become a standard technique in head and neck reconstructive surgery. Pre-operative radiotherapy is associated with a higher incidence of free flap malperfusion and the need for operative revision. Irradiated vessels present characteristic histomorphological and structural changes. Alterations in endothelial cells of irradiated arteries remain incompletely investigated especially with regard to long-term changes in endothelial dysfunction supporting an intraluminal pro-thrombotic and pro-inflammatory milieu. METHODS: Endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E­ and P­selectin, endothelial NO-synthase (eNOS), thrombomodulin and plasminogen activator inhibitor-1 (PAI-1) in irradiated and non-irradiated arteries was analysed using immunohistochemistry and Remmele scale grading. The average radiation dose was 58.7 ± 7.0 Gy; the time interval between end of radiation and tissue sampling was 106.0 ± 86.8 months. RESULTS: Endothelial expression of ICAM-1, VCAM-1, E­ and P­selectin as well as PAI-1 was significantly increased in previously irradiated arteries compared with non-irradiated controls, whereas thrombomodulin and eNOS expression did not show any differences. However, when comparing non-irradiated free flap arteries with irradiated arteries from the head and neck area in respective individuals, eNOS expression was significantly lower in irradiated vessels whereas ICAM-1, VCAM-1, E­/p-Selectin and PAI-1 showed significantly higher expression levels. CONCLUSION: There is ongoing endothelial dysfunction in terms of increased expression of pro-thrombotic and pro-inflammatory markers in irradiated arteries even years after radiotherapy. Treating this endothelial dysfunction might reduce the complication rates associated with microvascular free flap reconstructions in irradiated patients.

Effects of nitric oxide on steroidogenesis and apoptosis in goat luteinized granulosa cells.

The aim of this study was to investigate effects of nitric oxide (NO) on steroidogenesis and apoptosis in goat luteinized granulosa cells (LGCs). We cultured goat LGCs from healthy follicles in culture medium supplemented with the NO donor sodium nitroprusside (SNP) or the NO synthase inhibitor Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), then examined steroid synthesis, oxidative stress and apoptosis in vitro. The results showed that SNP treatment significantly increased the cGMP concentration in the LGCs (P < 0.05), whereas the l-NAME treatment significantly decreased cGMP concentration (P < 0.05). Then Inhibition of NO production significantly inhibited the expression of CYP19A1, a key gene that is involved in sex steroid hormones synthesis and is responsible for the decrease of E2. Inhibition of NO production resulted in an increased percentage of apoptosis, which was accompanied by upregulating expression levels of apoptosis-related markers BAX, CASP3 and CASP9. These data indicate that NO is required for goat LGCs steroidogenesis and cell survival. Furthermore, Inhibition of NO production decreased the expression of mitochondrial biogenesis related genes and proteins (PPARGC1A, NRF-1 and TFAM) and the mtDNA copy number. Simultaneously, inhibition of NO production suppressed the transcription and translation of SOD, GPX1, and CAT, and decreased the glutathione level and increased the 8-OHdG level. However, SNP treatment increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the anti-oxidant stress system and steroid synthesis. Together, our results indicate that NO may up-regulate the expression of PPARGC1A and its downstream factors through the cGMP pathway, thereby decreasing granulosa cell apoptosis, and may participate in the regulation of granulocyte steroid production through the mitochondrial-dependent pathway.

Ventilation-induced changes correlate to pulmonary vascular response and VEGF, VEGFR-1/2, and eNOS expression in the rat model of postnatal hypoxia.

Neonatal asphyxia occurs due to reduction in oxygen supply to vital organs in the newborn. Rapid restoration of oxygen to the lungs after a long period of asphyxia can cause lung injury and decline of respiratory function, which result from the activity of molecules that induce vascular changes in the lung such as nitric oxide (NO) and vascular endothelial growth factors (VEGF). In this study, we evaluated the pulmonary and vascular morphometry of rats submitted to the model of neonatal asphyxia and mechanical ventilation, their expression of pulmonary VEGF, VEGF receptors (VEGFR-1/VEGFR-2), and endothelial NO synthase (eNOS). Neonate Sprague-Dawley rats (CEUA #043/2011) were divided into four groups (n=8 each): control (C), control submitted to ventilation (CV), hypoxia (H), and hypoxia submitted to ventilation (HV). The fetuses were harvested at 21.5 days of gestation. The morphometric variables measured were body weight (BW), total lung weight (TLW), left lung weight (LLW), and TLW/BW ratio. Pulmonary vascular measurements, VEGFR-1, VEGFR-2, VEGF, and eNOS immunohistochemistry were performed. The morphometric analysis showed decreased TLW and TLW/BW ratio in HV compared to C and H (P<0.005). Immunohistochemistry showed increased VEGFR-2/VEGF and decreased VEGFR-1 expression in H (P<0.05) and lower eNOS expression in H and HV. Median wall thickness was increased in H, and the expression of VEGFR-1, VEGFR-2, VEGF, and eNOS was altered, especially in neonates undergoing H and HV. These data suggested the occurrence of arteriolar wall changes mediated by NO and VEGF signaling in neonatal hypoxia.

[Protective effect of urine-derived stem cells on erectile dysfunction in rats with cavernous nerve injury].

OBJECTIVE: To investigate the protective effect of human urine-derived stem cells (USCs) on erectile function and cavernous structure in rats with cavernous nerve injury (CNI). METHODS: Sixty adult male SD rats with normal sexual function were randomly divided into four groups of equal number: sham operation, bilateral CNI (BCNI) model control, phosphate buffered saline (PBS), and USC. The BCNI model was established in the latter three groups of rats by clamping the bilateral cavernous nerves. After modeling, the rats in the PBS and USC groups were treated by intracavernous injection of PBS at 200 µl and USCs at 1×106/200 µl PBS respectively for 28 days. Then, the maximum intracavernous pressure (mICP) and the ratio of mICP to mean arterial pressure (mICP/MAP) of the rats were calculated by electrical stimulation of the major pelvic ganglions, the proportion of nNOS- or NF200-positive nerve fibers in the total area of penile dorsal nerves determined by immunohistochemical staining, the levels of endothelial cell marker eNOS, smooth muscle marker α-SMA and collagen I detected by Western blot, and the smooth muscle to collagen ratio and the cell apoptosis rate in the corpus cavernosum measured by Masson staining and TUNEL, respectively. RESULTS: After 28 days of treatment, the rats in the USC group, as compared with those in the PBS and BCNI model control groups, showed significant increases in the mICP (ï¼»81 ± 9.9ï¼½ vs ï¼»31 ± 8.3ï¼½ and ï¼»33 ± 4.2ï¼½ mmHg, P <0.05), mICP/MAP ratio (0.72 ± 0.05 vs 0.36 ± 0.03 and 0.35 ± 0.04, P <0.05), the proportions of nNOS-positive nerve fibers (ï¼»11.31 ± 4.22ï¼½% vs ï¼»6.86 ± 3.08ï¼½% and ï¼»7.29 ± 4.84ï¼½% , P <0.05) and NF200-positive nerve fibers in the total area of penile dorsal nerves (ï¼»27.31 ± 3.12ï¼½% vs ï¼»17.38 ± 2.87ï¼½% and ï¼»19.49 ± 4.92ï¼½%, P <0.05), the eNOS/GAPDH ratio (0.52 ± 0.08 vs 0.31 ± 0.06 and 0.33 ± 0.07, P <0.05), and the α-SMA/GAPDH ratio (1.01 ± 0.09 vs 0.36 ± 0.05 and 0.38 ± 0.04, P <0.05), but a remarkable decrease in the collagen I/GAPDH ratio (0.28 ± 0.06 vs 0.68 ± 0.04 and 0.70 ± 0.10, P <0.05). The ratio of smooth muscle to collagen in the corpus cavernosum was significantly higher in the USC than in the PBS and BCNI model control groups (17.91 ± 2.86 vs 7.70 ± 3.12 and 8.21 ± 3.83, P <0.05) while the rate of cell apoptosis markedly lower in the former than in the latter two (3.31 ± 0.83 vs 9.82 ± 0.76, P <0.01; 3.31 ± 0.83 vs 9.75 ± 0.91, P <0.05). CONCLUSIONS: Intracavernous injection of USCs can protect the erectile function of the rat with cavernous nerve injury by protecting the nerves, improving the endothelial function, alleviating fibrosis and inhibiting cell apoptosis in the cavernous tissue.

Ginger (zingiber officinale) might improve female fertility: A rat model.

BACKGROUND: Ginger (Zingiber officinale) is a well known and extensively used antioxidant in traditional remedies. In this study, we aimed to investigate the effects of ginger powder on ovarian folliculogenesis and implantation in rats. METHODS: There were two study groups. In the 5-day treatment group (one estrous cycle), 100 mg ginger powder, 200 mg ginger powder or distilled water was given for 5 days to the three subgroups each containing seven rats. In the 10-day treatment group, same doses were given for 10 days (two estrous cycle) to the three subgroups each containing seven rats. At the end of the 5th and 10th days, ovarian volumes, ovarian weights, primordial follicles, antral follicles, atretic follicles, and corpus luteum counts were assessed. To evaluate the angiogenic effects of ginger, vascular endothelial growth factor (VEGF) and for the antioxidant effects of ginger endothelial nitric oxide synthase (eNOS) were examined in the ovaries and in the endometrium immunohistochemically. RESULTS: In the 5-day treatment group, antral follicle count and ovarian stromal VEGF were significantly high in the 100 mg ginger subgroup in comparison to the control group (p < 0.05). In the 10-day treatment group, endometrial VEGF and ovarian stromal eNOS were significantly high in the 100 mg ginger subgroup in comparison to the control group (p < 0.05). There was no statistically significant difference at 200 mg ginger dose both in 5-day and 10-day treatment groups. CONCLUSION: The increases in the antral follicle count and ovarian stromal VEGF in the 100 mg/5-day treatment subgroup indicate that ginger have positive effects on folliculogenesis in short term with low dose. Additionally, ginger may enhance implantation in rats in long term with low dose.

Antithrombotic components of Malus halliana Koehne flowers.

Flowers of Malus halliana (M. halliana) Koehne have been used as a Chinese traditional medicine to treat metrorrhagia and in our study, its chemical composition and anticoagulant effect were investigated. Five compounds were isolated and identified from M. halliana flowers, including limocitrin-3-O-glucoside (1), baohuoside Ⅱ (2), kaempferol-3-O-α-L-furan arabinoside (3), phloretin-4'-O-glycosidase (4) and afzeloside (5). Compound 1-3 were isolated for the first time from this genus. The anticoagulant effect of the compounds and extracts of M. halliana flowers were evaluated by APTT, PT, TT and FIB on plasma of rabbit in vitro. The results indicated that several fractions of M. halliana flowers and compounds 2-5 exhibited anticoagulant activity in vitro. Subsequently, afzeloside (5), the abundant component in M. halliana flowers, was investigated further for its antithrombotic effect in vivo and its antithrombotic mechanisms were evaluated on rats acute blood-stasis model. The antithrombotic effect was evaluated by WBV, PV, HCT, ESR, APTT, PT, TT, FIB, 6-keto-PGF1α, TXB2, ET-1 and eNOS in vivo. Afzeloside demonstrated inhibitory effect of thrombus formation, and its underlying antithrombotic mechanism was found to be related to the regulation of vascular endothelium active substance, activating blood flow and anticoagulant effect. Hence, we postulate that flavonoids may be the active ingredients of the plant.

Pulsatile stretch as a novel modulator of amyloid precursor protein processing and associated inflammatory markers in human cerebral endothelial cells.

Amyloid ß (Aß) deposition is a hallmark of Alzheimer's disease (AD). Vascular modifications, including altered brain endothelial cell function and structural viability of the blood-brain barrier due to vascular pulsatility, are implicated in AD pathology. Pulsatility of phenomena in the cerebral vasculature are often not considered in in vitro models of the blood-brain barrier. We demonstrate, for the first time, that pulsatile stretch of brain vascular endothelial cells modulates amyloid precursor protein (APP) expression and the APP processing enzyme, ß-secretase 1, eventuating increased-Aß generation and secretion. Concurrent modulation of intercellular adhesion molecule 1 and endothelial nitric oxide synthase (eNOS) signaling (expression and phosphorylation of eNOS) in response to pulsatile stretch indicates parallel activation of endothelial inflammatory pathways. These findings mechanistically support vascular pulsatility contributing towards cerebral Aß levels.

Ventilation-induced changes correlate to pulmonary vascular response and VEGF, VEGFR-1/2, and eNOS expression in the rat model of postnatal hypoxia

Neonatal asphyxia occurs due to reduction in oxygen supply to vital organs in the newborn. Rapid restoration of oxygen to the lungs after a long period of asphyxia can cause lung injury and decline of respiratory function, which result from the activity of molecules that induce vascular changes in the lung such as nitric oxide (NO) and vascular endothelial growth factors (VEGF). In this study, we evaluated the pulmonary and vascular morphometry of rats submitted to the model of neonatal asphyxia and mechanical ventilation, their expression of pulmonary VEGF, VEGF receptors (VEGFR-1/VEGFR-2), and endothelial NO synthase (eNOS). Neonate Sprague-Dawley rats (CEUA #043/2011) were divided into four groups (n=8 each): control (C), control submitted to ventilation (CV), hypoxia (H), and hypoxia submitted to ventilation (HV). The fetuses were harvested at 21.5 days of gestation. The morphometric variables measured were body weight (BW), total lung weight (TLW), left lung weight (LLW), and TLW/BW ratio. Pulmonary vascular measurements, VEGFR-1, VEGFR-2, VEGF, and eNOS immunohistochemistry were performed. The morphometric analysis showed decreased TLW and TLW/BW ratio in HV compared to C and H (P<0.005). Immunohistochemistry showed increased VEGFR-2/VEGF and decreased VEGFR-1 expression in H (P<0.05) and lower eNOS expression in H and HV. Median wall thickness was increased in H, and the expression of VEGFR-1, VEGFR-2, VEGF, and eNOS was altered, especially in neonates undergoing H and HV. These data suggested the occurrence of arteriolar wall changes mediated by NO and VEGF signaling in neonatal hypoxia.

Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells.

The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca2+ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca2+ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca2+ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats.

OBJECTIVE:: We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats. METHODS:: Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each) and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively). The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal's body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M) with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M) in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system. RESULTS:: Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%). Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group. CONCLUSIONS:: In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving endothelial function.