A2AR-mediated lymphangiogenesis via VEGFR2 signaling prevents salt-sensitive hypertension.

AIMS: Excess dietary sodium intake and retention lead to hypertension. Impaired dermal lymphangiogenesis and lymphatic dysfunction-mediated sodium and fluid imbalance are pathological mechanisms. The adenosine A2A receptor (A2AR) is expressed in lymphatic endothelial cells (LECs), while the roles and mechanisms of LEC-A2AR in skin lymphangiogenesis during salt-induced hypertension are not clear. METHODS AND RESULTS: The expression of LEC-A2AR correlated with lymphatic vessel density in both high-salt diet (HSD)-induced hypertensive mice and hypertensive patients. Lymphatic endothelial cell-specific A2AR knockout mice fed HSD exhibited 17 ± 2% increase in blood pressure and 17 ± 3% increase in Na+ content associated with decreased lymphatic density (-19 ± 2%) compared with HSD-WT mice. A2AR activation by agonist CGS21680 increased lymphatic capillary density and decreased blood pressure in HSD-WT mice. Furthermore, this A2AR agonist activated MSK1 directly to promote VEGFR2 activation and endocytosis independently of VEGF as assessed by phosphoprotein profiling and immunoprecipitation assays in LECs. VEGFR2 kinase activity inhibitor fruquintinib or VEGFR2 knockout in LECs but not VEGF-neutralizing antibody bevacizumab suppressed A2AR activation-mediated decrease in blood pressure. Immunostaining revealed phosphorylated VEGFR2 and MSK1 expression in the LECs were positively correlated with skin lymphatic vessel density and A2AR level in hypertensive patients. CONCLUSION: The study highlights a novel A2AR-mediated VEGF-independent activation of VEGFR2 signaling in dermal lymphangiogenesis and sodium balance, which might be a potential therapeutic target in salt-sensitive hypertension.

[Application of polarization fluorescence to the study on the effects of oxidative stress on wheat chloroplast].

In the present paper wheat flag leaves were collected during the tasseling period, and then 1 mmol x L(-1) hydrogen peroxide was added to induce oxidative stress on leaves. In comparison, the detached leaves were also kept under drought or darkness condition for 24 h for the same purpose. Following the preparation of chloroplasts, polarization fluorescence spectroscopic method was utilized to measure fluorescence emission spectra and fluorescence excitation spectra of chloroplasts in the case of VV, VH, HV and HH, where V and H is representative of vertical polarization and horizontal polarization, respectively. Gaussian deconvolution was done on emission spectra, and the fitting data revealed that no matter whether Chla or Chlb molecules were excited upon excitation at 436 nm or 475 nm, the ratio of fluorescence peak area at 684 nm and 720 nm, i. e. A684/ A720, tends to increase slightly after oxidative stress. In addition, some useful information was available from polarization excitation spectra, where it was observed that the treatment of oxidative stress gave rise to higher ratio of excitation peak intensity between 436 nm and 475 nm (E436/E475), indicating that Chla made more contribution to PSII fluorescence emission than Chlb did. Simultaneously, the ratio of 475 nm and 600 nm (E475/E600), representing the energy transfer efficiency from Car to Chlb, was also found to be higher after the detached leaves were treated. In addition, both fluorescence polarization and viscosity were calculated in this paper, and the data showed that oxidative stress should be responsible for higher fluorescence polarization at 680 nm and higher viscosity in microenviroment. The above-mentioned phenomenon is consistent with the lipid peroxidation of unsaturated fatty acids. It also provides a simple and feasible method to study oxidative stress.

Role of the CPC sequence in the antioxidant activity of GcGAST protein in E.coli.

Gibberellic acid stimulated transcriptional protein from Gymnadenia conopsea (GcGAST) is a novel member of GA-induced cysteine-rich protein family, which shared 12 highly conserved cysteine residues with other members in C-terminal domain. In the present paper, the recombinant plasmid, as well as two mutants Serine-Proline-Cysteine (SPC) and Cysteine-Proline-Serine (CPS), were constructed to investigate for the first time the effects of the cysteines in Cysteine-Proline-Cysteine (CPC) sequence on the antioxidant activity of GcGAST protein. It was found that E.coli expressing wt GcGAST exhibited significant resistance against exogenous H(2)O(2). Similar phenomenon was observed for E.coli harboring SPC mutant. In contrast, the host cell overexpressing CPS mutant became more sensitive to H(2)O(2). Some studies on the level of inclusion body revealed that wt GcGAST and SPC mutant embedded in Inclusion bodies (IB) could effectively eliminate H(2)O(2), whereas the mutagenesis to Ser of the second Cys residue in CPC sequence gave rise to the compete loss of H(2)O(2)-eliminating ability. Fourier transform Infrared spectroscopy analysis indicated that the IB of CPS mutant contained more ß-sheet secondary structure than wt and SPC mutant. Non-reducing SDS-PAGE combined western-blotting analysis revealed that the disulfide bonds were important for the formation of IBs of wt GcGAST and SPC mutant, whereas non-reducing SDS-PAGE of resolubilized IBs showed that hydrophobic interaction favored the aggregation of IBs in CPS mutant. Taken together, these results suggested that GcGAST possessed antioxidant activity in the level of IB, which made some contribution to cellular resistance to H(2)O(2). More importantly, the second cysteine residue in CPC sequence was more essential for its antioxidant biological function.