Unripe Carica papaya Fresh Fruit Extract Protects against Methylglyoxal-Mediated Aging in Human Dermal Skin Fibroblasts.

The glycolytic metabolite methylglyoxal (MGO) initiates the formation of advanced glycation end products and oxidative stress, leading to cellular senescence and skin aging. This study focuses on the anti-aging properties of unripe Carica papaya L. (UCP) fresh fruit extract on MGO-induced human dermal fibroblast senescence. We pretreated human foreskin fibroblasts with UCP before incubating them with MGO (400 µM) for 72 h. We used the glycation inhibitor aminoguanidine hydrochloride (AG) as the positive control. Senescent fibroblasts were detected using senescence-associated beta-galactosidase activity and collagen type I expression (COL1A1). We investigated the changes in the Akt, JNK/p38 mitogen-activated protein kinase (MAPK), c-Jun, and nuclear factor kappa B (NF-κB) signaling pathways using Western blotting. UCP significantly suppressed MGO-induced senescent fibroblasts (from 20.90±2.00% to 11.78±2.04%) when compared with the baseline level at 7.10±0.90% (P<0.05). While COL1A1 was diminished by 43.35±1.56% (P<0.001) in the MGO-treated fibroblasts, UCP and AG could recover COL1A1 to 63.22±4.78% and 64.39±3.34%, respectively. MGO triggered overactivation of Akt, JNK/p38 MAPK, c-Jun, and NF-κB by 2.10±0.09, 8.10±0.37, 6.60±0.29, 2.18±0.23, and 3.74±0.37 folds, respectively. UCP and AG significantly abolished these changes. Consistently, MGO increased matrix metalloproteinase-1 (MMP-1) levels by 2.58±0.04 folds, which was significantly suppressed by UCP and AG pretreatment to 1.87±0.11 and 1.69±0.07 folds, respectively. In summary, UCP controlled MGO-induced fibroblast senescence by suppressing the JNK/c-Jun/MMP and p38/NF-κB/COL1A1 pathways, similar to the action of the glycation inhibitor AG. Therefore, UCP can be considered a functional fruit for preventing and delaying skin aging.

Raphanus sativus L. var. caudatus Extract Alleviates Impairment of Lipid and Glucose Homeostasis in Liver of High-Fat Diet-Induced Obesity and Insulin Resistance in Mice.

The present study investigated the activities of Raphanus sativus L. var. caudatus extract (RS) on abnormal lipid and glucose homeostasis in a high-fat diet (HFD)-induced obesity and insulin resistance in a mouse model. Institute of Cancer Research mice were rendered obese by 16-week HFD feeding. Obese mice were administered with 100 or 200 mg/kg/d RS orally during the last 8 weeks of diet feeding. Then, the biochemical parameters were determined. The gene and protein expressions regulating lipid and glucose homeostasis in the liver were measured. This study revealed that the state of hyperglycemia, hyperleptinemia, hyperinsulinemia, and hyperlipidemia was reduced after 8 weeks of RS treatment (100 or 200 mg/kg). Administration of RS also improved insulin sensitivity and increased serum adiponectin. The liver total cholesterol and triglyceride concentrations were decreased by both doses of RS. Notably, a decrease in the expression of liver-specific genes, including sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-CoA carboxylase, was found in the RS-treated groups. Moreover, administration of RS showed a significant increase in the expression of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) proteins. These findings indicated that RS improved abnormal lipid and glucose homeostasis in the liver of obesity-associated insulin resistance mouse model, possibly through the stimulation of the AMPK/Sirt1 pathway.

Screening of potential tropical fruits in protecting endothelial dysfunction in vitro.

BACKGROUND: High consumption of antioxidant-rich fruits and vegetables reduces the endothelial damage involved in cardiovascular disease pathogenesis. OBJECTIVE: To evaluate the phytochemical content, antioxidant and scavenging activities (FRAP, ORAC, OH•, HOCl, H2O2, and O2 -), endothelial H2O2-cytoprotective effect, nitric oxide (NO) release activation potential, and endothelial wound healing properties of 10 tropical fruits, comprising pineapple, sugar apple, papaya fruit, longan, mangosteen, lychee, langsat, mango, rambutan, and guava. DESIGN: Experimental study. The experiments were conducted in vitro using endothelial cell line EA.hy926. RESULTS: The high performance liquid chromatography (HPLC) phytochemical analysis indicated the presence of gallic acid and quercetin in all fruits, along with the overall absence of ellagic acid. Chlorogenic acid was only detected in three fruits, that is, pineapple, ripe papaya, and guava. The antioxidant and scavenging activities of all fruits were concentration-dependent. Only the H2O2 scavenging activity exhibited broad positive associations with other ROS-scavenging activities. Sugar apple and unripe papaya induced a significant reduction in H2O2-induced cell death in endothelial cells while pineapple, sugar apple, longan, and langsat activated NO release. DISCUSSION: All the studied tropical fruits contained bioactive phytoantioxidants with wide ranges of antioxidant capacity and scavenging activities. The endothelial functional tests were relevant to the screening for fruits that may benefit cardiovascular health. Among the four fruits that promoted endothelial wound closure, only sugar apple and unripe papaya induced cell migration and vascular capillary-like tube formation. CONCLUSION: Sugar apple and unripe papaya are potential functional fruits that can protect against oxidative cell death and enhance endothelial wound healing.

Unripe Carica papaya Protects Methylglyoxal-Invoked Endothelial Cell Inflammation and Apoptosis via the Suppression of Oxidative Stress and Akt/MAPK/NF-κB Signals.

Methylglyoxal (MGO), a highly reactive dicarbonyl compound, causes endothelial oxidative stress and vascular complications in diabetes. Excessive MGO-induced ROS production triggers eNOS uncoupling, inflammatory responses, and cell death signaling cascades. Our previous study reported that unripe Carica papaya (UCP) had antioxidant activities that prevented H2O2-induced endothelial cell death. Therefore, this study investigated the preventive effect of UCP on MGO-induced endothelial cell damage, inflammation, and apoptosis. The human endothelial cell line (EA.hy926) was pretreated with UCP for 24 h, followed by MGO-induced dicarbonyl stress. Treated cells were evaluated for intracellular ROS/O2•- formation, cell viability, apoptosis, NO releases, and cell signaling through eNOS, iNOS, COX-2, NF-κB, Akt, MAPK (JNK and p38), and AMPK/SIRT1 autophagy pathways. UCP reduced oxidative stress and diminished phosphorylation of Akt, stress-activated MAPK, leading to the decreases in NF-kB-activated iNOS and COX-2 expression. However, UCP had no impact on the autophagy pathway (AMPK and SIRT1). Although UCP pretreatment decreased eNOS phosphorylation, the amount of NO production was not altered. The signaling of eNOS and NO production were decreased after MGO incubation, but these effects were unaffected by UCP pretreatment. In summary, UCP protected endothelial cells against carbonyl stress by the mechanisms related to ROS/O2•- scavenging activities, suppression of inflammatory signaling, and inhibition of JNK/p38/apoptosis pathway. Thus, UCP shows considerable promise for developing novel functional food and nutraceutical products to reduce risks of endothelial inflammation and vascular complications in diabetes.

Mentha cordifolia Leaf Extract Improves Hepatic Glucose and Lipid Metabolism in Obese Mice Fed with High-Fat Diet.

Mentha cordifolia (MC) is a popular herb used to flavor food in Thailand that exhibits several biological effects. The present study aimed to determine the role of MC in regulating glucose and lipid metabolism in mice fed a high-fat diet (HFD). ICR obese mice were fed an HFD (45 kcal% lard fat) for 12 weeks, with MC (100 and 200 mg/kg/d) treatment from Week 7. After treatment with MC for 6 weeks, mice showed significantly lower rates of hyperglycemia, hyperinsulinemia, hyperleptinemia, and hyperlipidemia, and increased amounts of serum adiponectin. Furthermore, in mice treated with MC, serum interleukin-6 and tumor necrosis factor alpha were significantly inhibited and liver histology results showed decreased lipid accumulation and liver triglyceride content vs. untreated mice. In addition, MC treatment was associated with smaller fat cells and lower gene expression of liver sterol regulatory element binding protein 1c, acetyl-CoA carboxylase, and fatty acid synthase. However, MC treatment was associated with higher carnitine palmitoyltransferase 1a gene expression and significantly higher rates of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in liver, but lower levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. These results indicate MC regulates glucose and lipid metabolism in a HFD-induced obese mouse model, possibly via activation of AMPK signaling pathway.

Potential Use of Amla (Phyllanthus emblica L.) Fruit Extract to Protect Skin Keratinocytes from Inflammation and Apoptosis after UVB Irradiation.

Ultraviolet B (UVB) exposure is the primary risk factor for the deadliest type of skin cancer-melanoma. Incorporating natural antioxidants in skin protection products is currently a favored research theme. For this study, we selected Phyllanthus emblica L. fruit extract (PE) to assess its potential use in dermal protection against UVB-induced keratinocyte inflammation and apoptosis. High-performance liquid chromatography (HPLC) was used to investigate PE's phytochemical constituents (ascorbic acid, ellagic acid, gallic acid, chlorogenic acid, and quercetin), while ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), total ROS, OH•, O2•-, and H2O2-scavenging activities were used to determine the antioxidant properties. PE significantly increased the cell viability (MTT assay) and reduced apoptosis (Hoechst staining) in HaCaT cells exposed to UVB (40 mJ/cm2). PE abolished oxidative stress by reducing the production of intracellular ROS, O2•- and H2O2 production. Catalase activity (but not superoxide dismutase or glutathione peroxidase activity) was enhanced in keratinocytes incubated with PE prior to UVB exposure. Western blot analysis suggested that PE inhibited cytochrome c release and inhibited the dysregulation of PI3K/Akt without any impact on p38 activation. PE attenuated the inflammatory response to UVB irradiation by inhibiting AP-1, NF-κB, and the mediator PGE2. Thus, PE is a candidate with great potential for use as an active ingredient in skin care products.

Ultraviolet B irradiation-induced keratinocyte senescence and impaired development of 3D epidermal reconstruct.

Ultraviolet B (UVB) induces morphological and functional changes of the skin. This study investigated the effect of UVB on keratinocyte senescence and the development of reconstructed human epidermis (RHE). Primary normal human keratinocytes (NHK) from juvenile foreskin were irradiated with UVB (30 mJ cm-2) and these effects were compared to NHK that underwent senescence in the late passage. UVB enhanced the accumulation of reactive oxygen species (ROS) and halted cell replication as detected by BrdU cell proliferation assay. The senescence phenotype was evaluated by beta-galactosidase (ß-gal) staining and qPCR of genes related to senescent regulation, i.e. p16INK4a, cyclin D2, and IFI27. Senescence induced by high dose UVB resulted in morphological changes, enhanced ß-gal activity, elevated cellular ROS levels and reduced DNA synthesis. qPCR revealed differential expression of the genes regulated senescence. p16INK4a expression was significantly increased in NHK exposed to UVB whereas enhanced IFI27 expression was observed only in cultural senescence. The levels of cyclin D2 expression were not significantly altered either by UVB or long culturing conditions. UVB significantly induced the aging phenotype in keratinocytes and impaired epidermal development. RHE generated from UVB-irradiated keratinocytes showed a thinner cross-sectional structure and the majority of keratinocytes in the lower epidermis were degenerated. The 3D epidermis model is useful in studying the skin aging process.

Antioxidant Properties of Unripe Carica papaya Fruit Extract and Its Protective Effects against Endothelial Oxidative Stress.

It has been proven that high consumption of fruit and vegetable lowers the risks of cardiovascular and other oxidative stress-related diseases. Here we evaluated the effects of a tropical fruit, unripe Carica papaya (UCP), on endothelial protection against oxidative damage induced by H2O2. The antioxidant properties of UCP were investigated using the assays of FRAP and ORAC and specific ROS scavenging activities (H2O2, O2 •-, OH•, HOCl). Cytoprotective property was tested in human endothelial cell line EA.hy926 with respect to cell survival, intracellular ROS levels, antioxidant enzyme activities (CAT, SOD, GPX), survival/stress signaling (AKT, JNK, p38), and nuclear signaling (Nrf2, NF-kB). UCP processed high antioxidant activity and scavenging activity against H2O2> OH•> O2 •-> HOCl, respectively. UCP improved cell survival in the milieu of ROS reduction. While SOD was increased by UCP, CAT activity was enhanced when cells were challenged with H2O2. UCP had no impact on H2O2-activated AKT, JNK, and p38 signaling but significantly decreased nuclear NF-κB levels. The overactivation of Nrf2 in response to oxidative stress was constrained by UCP. In conclusion, UCP protected endothelial cells against oxidative damage through intracellular ROS reduction, enhanced CAT activity, suppression of NF-kB, and prohibition of Nrf2 dysregulation. Thus, UCP might be a candidate for development of nutraceuticals against CVD and oxidative-related diseases and conditions.

Effects of Brassica oleracea extract on impaired glucose and lipid homeostasis in high-fat diet-induced obese mice

Objective: To examine the effect of Brassica oleracea extract (BO) on impaired glucose and lipid homeostasis in high-fat diet (HFD)-induced obese mice. Methods: Obesity of ICR mice was induced by feeding a HFD (45 kcal％ lard fat) for 16 weeks. During the last 8 weeks of study period, obese mice were additionally administered with BO (100 and 200 mg/kg/day). The metabolic parameters were determined. The gene expressions of hepatic lipogenesis were also studied. Results: After 8 weeks of treatment, BO (100 and 200 mg/kg) significantly reduced hyperglycemia and improved insulin sensitivity (P < 0.05). The serum lipid (total cholesterol, triglyceride, and non-esterified fatty acid) and hepatic triglyceride and non-esterified fatty acid were decreased (P < 0.05). The levels of insulin and leptin in serum were also decreased (P < 0.05). Moreover, the expressions of hepatic lipogenic genes including sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-CoA carboxylase were decreased by BO treatment (P < 0.05). Conclusions: These results suggest that BO is a new therapeutic agent for improving the homeostasis of glucose and lipid in HFD-induced obese mice probably by suppression of lipogenic genes in liver tissue.

Increased permeability of reconstructed human epidermis from UVB-irradiated keratinocytes.

Extrinsic (photo) aging accelerates chronologically aging in the skin due to cumulative UV irradiation. Despite recent insights into the molecular mechanisms of fibroblast aging, age-related changes of the skin barrier function have been understudied. In contrast, the constantly increasing subpopulation of aged patients causes a clinical need for effective and safe (dermatological) treatment. Herein, we reconstructed human epidermis from UVB-irradiated keratinocytes (UVB-RHE). UVB-irradiated keratinocytes show higher activity of senescence associated ß-galactosidase, less cell proliferation, and reduced viability. Higher amounts of ß-galactosidase are also detectable in UVB-RHE. Moreover, UVB-RHE release more interleukin-1α and -8 into the culture medium and present altered differentiation with a thinner stratum corneum compared to normal RHE. For the first time, the permeation of testosterone and caffeine through UVB-irradiated RHE indicate a clear influence of the UVB stress on the skin barrier function. Impaired barrier function was confirmed by the increased permeation of testosterone and caffeine as well as by the increased penetration of dendritic core-multishell nanocarriers into the constructs. Taken together, UVB-RHE emulate hallmarks of skin aging and might contribute to an improved non-clinical development of medicinal or cosmetic products.

Antiangiogenic Effects of VH02, a Novel Urea Derivative: In Vitro and in Vivo Studies.

Vascular endothelial growth factor receptor 2 (VEGFR2) is a vital target for therapeutic intervention in cancer. We have recently described a computer-based drug design for a small molecule VEGFR2 inhibitor named VH02 (1-((1-(1H-indazol-6-yl)-1H-1,2,3-triazol-4-yl)methyl)-3-(3-chloromethylphenyl)urea). This study aimed to further explore the anti-angiogenic activity of VH02 both in vitro and in vivo. The in vitro assays include cell viability, capillary-like tube formation, MMP activity, and western blot analyses of signaling through VEGFR2 while the in vivo anti-angiogenic response were performed to evaluate the effect on vascularization in Matrigel plug applied in C57BL/6L mice. VH02 reduced angiogenesis behavior of EA.hy926 including cell viability, migration, adhesion, capillary-like tube formation, and MMP-2 activity induced by VEGF. Furthermore, VH02 regulated angiogenesis by directly inhibiting VEGFR2 on Tyr1175 signaling pathway leading to the inhibition of Akt-mediated cell survival and migration. Disruption of phosphorylation at VEGFR2-Tyr1175 by VH02 abolished FAK-Tyr397 signaling but not phosphorylation of p38 MAPK. This suggests that blockade of FAK by VH02 apparently associated with reduction of endothelial cell motility. Actin cytoskeleton rearrangement was diminished by VH02 in human endothelial cells. The anti-angiogenic effect of VH02 was confirmed in the in vivo model, revealing the reduction of vascular density in Matrigel plug after VH02 treatment. Additionally, the pericyte-like cells surrounding blood vessels in the plugs were significantly reduced as well as vascular density and p-Akt intensity. Our findings indicate that VH02 successfully inhibits VEGF-induced angiogenesis both in vitro and in vivo models. The compound could be further developed as an antiangiogenesis agent for cancer therapy.

Selected activities of Citrus maxima Merr. fruits on human endothelial cells: enhancing cell migration and delaying cellular aging.

Endothelial injury and damage as well as accumulated reactive oxygen species (ROS) in aging play a significant role in the development of cardiovascular disease (CVD). Recent studies show an association of high citrus fruit intake with a lower risk of CVD and stroke but the mechanisms involved are not fully understood. This study investigated the effects of pummelo (Citrus maxima Merr. var. Tubtim Siam, CM) fruit extract on human umbilical vein endothelial cell (HUVECs) migration and aging. The freeze-dried powder of fruit extract was characterized for antioxidant capacity (FRAP assay) and certain natural antioxidants, including ascorbic acid, gallic acid, hesperidin, and naringin (HPLC). Short-term (48 h) co-cultivation of HUVECs with CM enhanced cell migration as evaluated by a scratch wound assay and Boyden chamber assay. A long-term treatment with CM for 35 days significantly increased HUVEC proliferation capability as indicated by population doubling level (PDL). CM also delayed the onset of aging phenotype shown by senescence-associated ß-galactosidase (SA-ß-gal) staining. Furthermore, CM was able to attenuate increased ROS levels in aged cells when determined by 2',7'-dichlorodihydrofluorescein diacetate (DCDHF) while eNOS mRNA expression was increased but the eNOS protein level was not changed. Thus, further in vivo and clinical studies are warranted to support the use of pummelo as a functional fruit for endothelial health and CVD risk reduction.

Protective effect of Phyllanthus emblica fruit extract against hydrogen peroxide-induced endothelial cell death.

Numerous antioxidants from natural products have been shown to lower ROS levels and enhance vascular endothelial function. The fruits of Phyllanthus emblica are well-known in possessing antioxidative properties but its role and mechanisms in the protection of vascular endothelial cells from ROS damage have not yet been established. The present study was aimed to determine the possible protective effect of P. emblica fruit extract (PE) on human EA.hy926 endothelial cell death induced by hydrogen peroxide (H2O2) and PE protective mechanisms. Following incubation of endothelial cells with 300 microM H2O2 for 2 h, cell viability was decreased to 50.65 +/- 0.94% and intracellular ROS levels was increased to 159.01% +/- 6.27% as measured by MTT assay and DCF fluorescent intensity, respectively. Cytotoxic effect of PE was not observed in the range of 0.1 to 100 microM Pretreatment with PE (20 to 100 microg/mL) for 48 h significantly ameliorated the cytotoxic effect of H2O2 and attenuated the excessive intracellular ROS formation in endothelial cells. In addition, western blot analysis revealed that PE pretreatment (40 microg/L) induced Akt phosphorylation but did not activate NF-kappaB pathway. These findings suggest that PE could effectively protect human endothelial cell death induced by H2O2 via modification of ROS-related mechanism along with activation of PI3K/Akt pathway. However the value of this plant in vivo needs further investigations in supporting them to be developed as nutraceuticals for cardiovascular disease prevention.

Phyllanthus emblica L. Enhances Human Umbilical Vein Endothelial Wound Healing and Sprouting.

Endothelial dysfunction is the hallmark of impaired wound healing and increased risk of cardiovascular disease. Antioxidants from natural sources decrease oxidative stress and protect against cellular damage caused by reactive oxygen species (ROS). In this study, we examined the antioxidant constituents and capacity of Phyllanthus emblica L. (PE) fruit in freeze-dried power form. The pharmacological properties of PE were investigated using human umbilical vein endothelial cells (HUVECs) in the aspects of endothelial cell proliferation, nitric oxide (NO) production, wound healing, cell migration, in vitro angiogenesis, and VEGF gene expression. The ASC content of PE was 1.574% + 0.046% (w/w) as determined by HPLC and the total phenolic content was 36.1% ± 0.7% gallic acid equivalent when measured by Folin-Ciocalteu assay. The FRAP assay revealed a relatively high antioxidant capacity at 3,643 + 192.5 µmole/mg. PE at 0.1 to 10 µg/mL did not significantly influence endothelial cell proliferation, but at higher concentrations PE decreased cell survival to 62%. PE significantly promoted NO production, endothelial wound closure, endothelial sprouting, and VEGF mRNA expression. Therefore, PE is a candidate for antioxidant supplement that promotes endothelial function and restores wound healing competency.

Novel VEGFR-2 kinase inhibitors identified by the back-to-front approach.

We report a novel VEGFR-2 inhibitor, developed by the back-to-front approach. Docking experiments indicated that the 3-chloromethylphenylurea motif of the lead compound occupied the back pocket of VEGFR-2 kinase. An attempt was made to enhance the binding affinity of 1 by expanding the structure to access the front pocket using a triazole linker. A library of 1,4-(disubstituted)-1H-1,2,3-triazoles were screened in silico, and one compound (VH02) was identified with an IC50 against VEGFR-2 of 0.56µM. VH02 showed antiangiogenic effects, inhibiting tube formation in HUVEC cells (EA.hy926) at 0.3µM, 13 times lower than its cytotoxic dose. These enzymatic and cellular activities suggest that VH02 has potential as a lead for further optimization.

Synthesis and evaluation of 1-(substituted)-3-prop-2-ynylureas as antiangiogenic agents.

Novel urea derivatives of alkynes have been designed, synthesized, and evaluated as potential cancer therapeutics leads. The most active 1-((3-chloromethyl)phenyl)-3-prop-2-ynylurea (1) exhibited cytotoxic effect against HELA and MCF-7 cell lines with IC(50) values of 1.55 µM and 1.48 µM, respectively. Further investigation on tube formation assay in human vein umbilical cells (HUVEC) demonstrated that 1 and methyl 4-(3-(3-ethynylureido)benzyloxy) benzoate (6) possess antiangiogenic activity, with minimum effective dose of 25 nM (for 1) and 6.25 µM (for 6). The ED(50) of 1 and 6 were found to be 0.26 µM and 17.52 µM, respectively. The results from in vitro tyrosine kinase assay indicated the EGFR inhibition of 1 over other kinases (VEGFR2, FGFR1 and PDGFRß). The cytotoxicity of 1 against EGFR overexpressing cell line A431 (IC(50) 36 nM) was comparable to that of erlotinib. The binding mode of 1 from docking simulation in the EGFR active site revealed that the urea motif formed hydrogen bonding with Lys745, Thr854 and Asp855 in hydrophobic pocket of EGFR. Compound 1 is considered as a potential lead for further optimization.

Effects of human endothelial gene polymorphisms on cellular responses to hyperglycaemia: role of NOS3 (Glu298Asp) and ACE (I/D) polymorphisms.

The functional relevance of NOS3 and ACE genetic variations to endothelial cell function is largely unstudied. Here we tested the functional relevance of the NOS3 (Glu298Asp) polymorphism and ACE (I/D) polymorphism in endothelial cells in vitro. Our hypothesis was that these genetic polymorphisms alter endothelial cell sensitivity to glucose and 3-nitrotyrosine (3NT). Genotyped HUVECs were incubated with glucose, free 3NT or a combination of these two toxicants. Significant differences in glucose-induced cell death and free 3NT-induced cell death were observed among the NOS3 genotypes. Combined glucose/3NT caused increased toxicity among the NOS3 genotypes. No differences were observed among the ACE genotypes in their responses to glucose/3NT. These data demonstrate that the NOS3 genotype may be an important predictor of, or be mechanistically involved in, endothelial vulnerability, whereas the ACE I/D genotype is apparently less important. Thus this NOS3 genetic variation may play a role in vulnerability to endothelium-dependent diabetic vascular complications.

Influence of capsicum extract and capsaicin on endothelial health.

OBJECTIVE: To examine the effect of Capsicum spp extract (CEX) and capsaicin (CAP) on endothelial nitric oxide release and protection against lipopolysaccharide (LPS)-induced cellular apoptosis. MATERIAL AND METHOD: Human umbilical vein endothelial cells (HUVEC) were isolated from newborn cords. Evaluation of cytotoxicity was performed by MTT assay. Endothelial nitric oxide (NO) production was evaluated by Griess reaction. Alteration in eNOS expression was detected by westernblot analysis. To induce oxidative stress and apoptosis, lipopolysaccharide (LPS) was coincubated with HUVEC in the presence or absence of CEX or CAP, and the vanilloid receptor blocker capsazepine (CZP). Hoechst nuclear staining was used to determine percent apoptotic nuclei. RESULTS: The highest concentrations of CEX (1000 microg/mL) and CAP (25 microM) used in the study did not induce cytotoxicity in HUVEC. Significant increase in NO release was observed when cells were incubated with CEX (100 microg/mL) and CAP (25 microM) and this effect was inhibited by CZP only in CAP treatment group. Despite enhanced NO generation was observed, western blot analysis indicated no change in eNOS expression. Interestingly, endothelial cells incubated with L-arginine (L-ARG, 1000 microg/mL) alone significantly showed increased NO production while L-ARG co-incubation abrogated CEX or CAP effects on endothelialNO generation. CEX (10 microg/mL) and CAP (1 microM) decreased apoptotic nuclei in HUVEC treated with LPS. CONCLUSION: CEX and CAP improved endothelial function and protected against LPS-induced apoptosis. Regular consumption of Capsicum spp. may promote endothelial health and reduce cardiovascular disease risk.

Cytoprotective role of Phyllanthus urinaria L. and glutathione-S transferase Pi in doxorubicin-induced toxicity in H9c2 cells.

OBJECTIVE: To examine cytoprotective effect of Phyllanthus urinaria (PU) ethanolic extract in doxorubicin (DOX)-induced toxicity. The research focus was on the mechanism of action in association with the expression and localization of glutathione-S transferase (GST) in cardiac H9c2 cells. MATERIAL AND METHOD: The presence of GST isoforms was evaluated in H9c2 cells using western blot analysis and confocal immunofluorescence visualization. Cells were then treated with DOX in the presence and absence of PU and several cytoprotective indices were evaluated, including the expression of the rate-limiting enzyme for glutathione synthesis, gamma-glutamylcysteine synthetase (gamma-GCS), manganese superoxide dismutase (MnSOD), copper-zinc SOD (CuZnSOD), and GST activity from cell lysate. The investigations for GST-mediated cytoprotection from DOX-induced oxidative damage were further carried out by SiRNA transfection and apoptosis detection using TUNEL assay. RESULTS: GST Pi (GSTP) was predominantly expressed in H9c2 cells compared with GST Alpha and GST Mu. Treatment with PU protected against the cardiotoxicity of DOX by influencing the nuclear localization of GSTP without significantly affecting the enzymatic activity. Suppression of GSTP expression by RNA interference potentiated the accumulation of DOX in the nucleus and enhanced apoptosis as evaluated by TUNEL assay. Treatment with PU had a cytoprotective effect by reducing cellular levels of DOX with enhanced nuclear localization of GSTP in myocardiac cells. CONCLUSION: The cytoprotective mechanism of PU against DOX cardiotoxicity partially involved the presence of GSTP. Thus, PU extracts may be used as an alternative source of antioxidants with distinctive mechanisms of action that may be suitable for specific types of oxidative insults.

Isolation and characterization of umbilical cord blood hematopoietic stem cells.

Umbilical cord blood (UCB) has recently represented another rich source for hematopoietic stem cells (HSCs). Recent clinical studies have shown that UCB stem cells can potentially be used in place of HSCs from bone marrow as well as in basic research in regenerative medicine. This article will describe the methods for isolation and characterization of HSCs from UCB. UCB were obtained from umbilical vessels at the time of delivery. The HSCs were isolated from UCB using a density-gradient centrifugation method, CD34-immunomagnetic separation, and finally fluorescent-activated cell sorting (FACS). Functional assays were evaluated for the ability of multipotent progenitors to differentiate to lineage-specific committed cells and heterogeneous hierarchy of pluripotent cells. Approximately 1% of CD34+ cells were isolated and sorted from mononucleated cells. Functional assays revealed that the CD34+ subpopulation gave rise to several hematopoietic cell lineages including CFU-E, BFU-E, CFU-G, CFU-M, CFU-GM, and CFU-GEMM. These cells also maintained their stemness as evaluated by primitive long-term culture initiating cells (LTC-IC). The basic methods in HSC isolation and characterization employing gradient isolation, CD34-immunomagnetic separation, FACS, and functional assays are important in the area of stem cell investigation and applications.