A pollen coat-inducible autoinhibited Ca2+-ATPase expressed in stigmatic papilla cells is required for compatible pollination in the Brassicaceae.

In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca(2+) export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca(2+)-ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca(2+) transport systems suggested that ACA13 indeed functions as an autoinhibited Ca(2+) transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca(2+) export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca(2+) to the compatible pollen tube, which promotes successful fertilization.

Autologous angiogenic therapy with cultured mesenchymal stromal cells in platelet-rich plasma for critical limb ischemia.

Introduction: The prevalence of diabetes mellitus is increasing globally, including in Japan. Patients with diabetes often experience microangiopathy and macroangiopathy, which lead to difficult-to-treat foot ulcers and diabetic gangrene. Conventional cellular therapies have limited safety and are invasive. In this study, we investigated the use of cultured autologous mesenchymal stromal cells derived from the bone marrow and grown in platelet-rich plasma as a potential treatment for diabetic complications. Methods: A prospective clinical trial was conducted to assess safety as the primary endpoint and efficacy as the secondary endpoint of the aforementioned therapy in five patients with critical limb ischemia, with or without hemodialysis. Results: Five patients with critical limb ischemia were enrolled between 2016 and 2019, three of whom underwent hemodialysis. Platelet-rich plasma was obtained from 288 ± 39.6 mL of blood/patient, yielding 31.6 ± 1.67 mL of platelet-rich plasma. Bone marrow aspiration yielded 18.4 ± 4.77 mL/patient, and 4.64 ± 1.51 × 107 cells were incubated for 16 ± 2.8 days to obtain 3.26 ± 0.33 × 107 mesenchymal stromal cells. Although several adverse events were observed, none were directly attributed to cell therapy. Clinical severity, as assessed by both the Fontaine stage and Rutherford category, improved significantly following therapy. This improvement was accompanied by enhancements in the 6-min walking distance, dorsal skin perfusion pressure, ankle transcutaneous partial oxygen pressure, and ankle brachial pressure index. Conclusion: Autologous angiogenic therapy with cultured mesenchymal stromal cells derived from the bone marrow and grown in platelet-rich plasma is a safe and feasible, and was expected as a potential treatment for critical limb ischemia.

Glucuronides of phytoestrogen flavonoid enhance macrophage function via conversion to aglycones by ß-glucuronidase in macrophages.

INTRODUCTION: Flavonoids are converted to inactive metabolites like glucuronides in the gut, and circulate mainly as glucuronides in blood stream, resulting in low concentrations of active aglycones in plasma. It is therefore unclear how oral flavonoids exert their effects in tissues. We recently reported the plasma pharmacokinetics of some flavonoids and suggested the possibility that the absorbed flavonoids modified macrophage functions leading to enhance bacterial clearance. We aimed to confirm their pharmacological profiles focusing on tissue macrophages. METHODS: Pseudoinfection was induced by intradermal injection of FITC-conjugated and killed Staphylococcus aureus into the ears of mice treated with or without genistein 7-O-glucuronide (GEN7G, 1 mg/kg, i.v.). FACS analysis was performed on single cell suspensions dispersed enzymatically from the skin lesions at 6 h post pseudoinfection to evaluate phagocytic activities of monocytes/macrophages (CD11b+ Ly6G- ) and neutrophils (CD11b+ Ly6G+ ). Phagocytosis of the FITC-conjugated bacteria by four glucuronides including GEN7G was evaluated in cultures of mouse macrophages. RESULTS: After GEN7G injection, genistein was identified in the inflamed ears as well as GEN7G, and the phagocytic activity of CD11b+ Ly6G- cells was increased. GEN7G was converted to genistein by incubation with macrophage-related ß-glucuronidase. Macrophage culture assays revealed that GEN7G increased phagocytosis, and the action was dampened by a ß-glucuronidase inhibitor. Binding of aglycones to estrogen receptors (ERs), putative receptors of flavonoid aglycones, correlated to biological activities, and glucuronidation reduced the binding to ERs. An ER antagonist suppressed the increase of macrophage function by GEN7G, whereas estradiol enhanced phagocytosis as well. CONCLUSIONS: This study suggests a molecular mechanism by which oral flavonoids are carried as glucuronides and activated to aglycones by ß-glucuronidase in tissue macrophages, and contributes to the pharmacological study of glucuronides.

Safety assessment of bone marrow derived MSC grown in platelet-rich plasma.

The injection of endothelial progenitor cells and mononuclear cells derived from bone marrow at the ischemic region of peripheral artery disease patients is reported to be effective for therapeutic angiogenesis; however, these cell therapies require large amounts of bone marrow to obtain sufficient numbers of cells. To solve this problem, we attempted to culture bone-marrow-derived mesenchymal stem cells (BM-MSC), which are supposed to secrete several cytokines that promote angiogenesis. We also focused on using platelet-rich plasma (PRP) as a supplement for cell culture instead of fetal bovine serum. Human BM-MSC obtained from healthy volunteers expanded rapidly when cultured with 10% PRP prepared from their own blood. FACS analysis revealed that these cultured human MSC were homogeneous populations, and chromosomal analysis showed a normal karyotype. Moreover, the angiogenetic effect was apparent two weeks after human BM-MSC were injected into the ischemic muscle in SCID mice. Tumor formation was not detected three months after injection into SCID mice either subcutaneously or intramuscularly. To simulate clinical settings, canine BM-MSC were grown with canine PRP and injected into their ischemic muscles. We confirmed that donor cells existed in situ two and six weeks after operation without any side effects. These results suggest that cultured human BM-MSC can be a promising cell source for therapeutic angiogenesis.