Angelica keiskei (Ashitaba) powder and its functional compound xanthoangelol prevent heat stress-induced impairment in sperm density and quality in mouse testes.

Recently, gradual decline in human sperm production has become a serious worldwide concern because it leads to increased rates of infertility. Endocrine disrupters, lifestyle changes, and varicocele, all of which elevate testicular temperature, are thought to be the main causes of this decline. The present study aimed to determine whether the dietary phytochemicals Angelica keiskei (Ashitaba) powder (57.5 mg/kg) and its functional component, xanthoangelol (3 mg/kg), can prevent heat stress-induced impairment in sperm density and quality in mice. Sperm parameters were analyzed 28 days after mice exposure to heat. Supplementation with Ashitaba powder completely prevented heat-induced impairment in sperm parameters, including densities of motile sperms and progressive sperms (> 25 µm/sec), and amplitude of lateral head displacement. Xanthoangelol did not exert a complete protective effect; nevertheless, it significantly prevented heat stress-induced reduction in most parameters. Both Ashitaba powder and xanthoangelol elevated the expression of the widely expressed heat shock proteins (HSPs) Hspa1a and Hsp40 and the antioxidant enzyme glutathione synthase in non-stressed testes. Ashitaba powder significantly prevented heat stress-induced reduction in the expression of Hspa1l and Hspa2, which are highly expressed in the testes and critical for fertility. Our results showed that Ashitaba powder and xanthoangelol protected testicular cells from heat stress, probably by elevating the levels of antioxidant enzymes and HSPs. Supplementation with dietary functional phytochemicals may help prevent heat stress-induced male infertility.

Role of Hydroxytyrosol-dependent Regulation of HO-1 Expression in Promoting Wound Healing of Vascular Endothelial Cells via Nrf2 De Novo Synthesis and Stabilization.

Hydroxytyrosol (HT), an olive plant (Olea europaea L.) polyphenol, has proven atheroprotective effects. We previously demonstrated that heme oxygenase-1 (HO-1) is involved in the HT dependent prevention of dysfunction induced by oxidative stress in vascular endothelial cells (VECs). Here, we further investigated the signaling pathway of HT-dependent HO-1 expression in VECs. HT dose- and time-dependently increased HO-1 mRNA and protein levels through the PI3K/Akt and ERK1/2 pathways. Cycloheximide and actinomycin D inhibited both increases, suggesting that HT-triggered HO-1 induction is transcriptionally regulated and that de novo protein synthesis is necessary for this HT effect. HT stimulated nuclear accumulation of nuclear factor E2-related factor 2 (Nrf2). This Nrf2 accumulation was blocked by actinomycin D and cycloheximide whereas HT in combination with the 26S proteasome inhibitor MG132 enhanced the accumulation. HT also extended the half-life of Nrf2 proteins by decelerating its turnover. Moreover, HO-1 inhibitor, ZnppIX and CO scavenger, hemoglobin impaired HT-dependent wound healing while CORM-2, a CO generator, accelerated wound closure. Together, these data demonstrate that HT upregulates HO-1 expression by stimulating the nuclear accumulation and stabilization of Nrf2, leading to the wound repair of VECs crucial in the prevention of atherosclerosis.

Combined treatment of hydroxytyrosol with carbon monoxide-releasing molecule-2 prevents TNF α-induced vascular endothelial cell dysfunction through NO production with subsequent NFκB inactivation.

This study investigated the atheroprotective properties of olive oil polyphenol, hydroxytyrosol (HT), in combination with carbon monoxide-releasing molecule-2 (CORM-2) that acts as a carbon monoxide donor using vascular endothelial cells (VECs). Our results showed that CORM-2 could strengthen the cytoprotective and anti-apoptotic effects of HT against TNFα-induced cellular damage by enhancing cell survival and the suppression of caspase-3 activation. While HT alone attenuated NFκBp65 phosphorylation and IκBα degradation triggered by TNFα in a dose-dependent manner, combined treatment of HT with CORM-2 but not iCORM-2 nearly completely blocked these TNFα effects. Furthermore, combined action of both compounds results in the inhibition of NFκB nuclear translocation. Results also indicate that both compounds time-dependently increased eNOS phosphorylation levels and the combination of HT with CORM-2 was more effective in enhancing eNOS activation and NO production in VECs. The NOS inhibitor, L-NMMA, significantly suppressed the combined effects of HT and CORM-2 on TNFα-triggered NFκBp65 and IκBα phosphorylation as well as decreased cell viability. Together, these data suggest that carbon monoxide-dependent regulation of NO production by the combination of HT with CORM-2 may provide a therapeutic benefit in the treatment of endothelial dysfunction and atherosclerosis.

Hydroxytyrosol induces vascular smooth muscle cells apoptosis through NO production and PP2A activation with subsequent inactivation of Akt.

Olive oil has been shown to exhibit beneficial effects in the prevention of cardiovascular diseases although its molecular mechanism still remains unclear. In the present study, we investigated the effect of hydroxytyrosol (HT), a major phenolic component in olive oil and leaves from OLEA EUROPAEA L. (Oleaceae family), on vascular smooth muscle cells (VSMCs) survival, migration, and apoptosis. HT treatment resulted in a dose-dependent decrease of cell survival and migration in the presence or absence of platelet-derived growth factor (PDGF) by inducing apoptosis of VSMCs. HT enhanced nitric oxide (NO) production in a dose-dependent manner, and the NO synthase inhibitor L-NMMA blocked HT-mediated effects on VSMCs survival. HT as well as the NO donor SNAP reduced the phosphorylation levels of Akt, suggesting that HT inactivates Akt via NO production with subsequent apoptosis of VSMCs. Moreover, HT-dependent apoptosis and reduction in the phosphorylation level of Akt were suppressed by okadaic acid, an inhibitor of protein phosphatase 2A (PP2A) that dephosphorylates Akt. In contrast, the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), an upstream activator of Akt, was not affected by HT. Together, these findings indicate that HT could induce VSMCs apoptosis through NO production and PP2A activation followed by inactivation of Akt signaling pathway.

Protein tyrosine phosphatase LMW-PTP exhibits distinct roles between vascular endothelial and smooth muscle cells.

The present study examined the cellular functions of low-molecular-weight protein tyrosine phosphatase (LMW-PTP), which consists of two active isoforms IF-1 and IF-2, in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), focusing on cell growth and migration. We transduced recombinant IF-1 and IF-2, and ribozyme targeting both isoforms using an adenovirus vector in these cells. We detected the expression of IF-1 and IF-2 in both types of cells. IF-1 as well as IF-2 inhibited PDGF-induced DNA synthesis and migration in VSMCs. In contrast, both isoforms enhanced lysophosphatidic acid-stimulated cell migration without change in DNA synthesis in ECs. Whereas there is a report indicating that reactive oxygen species-dependent inactivation of LMW-PTP regulates actin cytoskeleton reorganization during cell spreading and migration, the isoforms conversely suppressed the PDGF-induced H2O2 generation with subsequent decrease in the p38 activity in VSMCs. Catalytically inactive LMW-PTP exerted the opposite and similar effects to the wild type in ECs and in VSMCs, respectively, suggesting that substrates for the phosphatase differ between these cells. Moreover, high concentrations of glucose suppressed the expression of LMW-PTP in both cells. These data suggest that LMW-PTP negatively regulates the pathogenesis of atherosclerosis and that glucose-dependent suppression of LMW-PTP expression may promote the development of atherosclerosis in diabetics.

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation.

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.