Functional analysis of Sonic Hedgehog variants associated with holoprosencephaly in humans using a CRISPR/Cas9 zebrafish model.

Genetic variation in the highly conserved Sonic Hedgehog (SHH) gene is one of the most common genetic causes for the malformations of the brain and face in humans described as the holoprosencephaly clinical spectrum. However, only a minor fraction of known SHH variants have been experimentally proven to lead to abnormal function. Employing a phenotypic rescue assay with synthetic human messenger RNA variant constructs in shha-/- knockout zebrafish, we evaluated 104 clinically reported in-frame and missense SHH variants. Our data helped us to classify them into loss of function variants (31), hypomorphic variants (33), and nonpathogenic variants (40). We discuss the strengths and weaknesses of currently accepted predictors of variant deleteriousness and the American College of Medical Genetics and Genomics guidelines for variant interpretation in the context of this functional model; furthermore, we demonstrate the robustness of model systems such as zebrafish as a rapid method to resolve variants of uncertain significance.

MicroRNA-22 negatively regulates LPS-induced inflammatory responses by targeting HDAC6 in macrophages.

Dysregulation of histone deacetylase 6 (HDAC6) can lead to the pathologic states and result in the development of various diseases including cancers and inflammatory diseases. The objective of this study was to elucidate the regulatory role of microRNA-22 (miR-22) in HDAC6-mediated expression of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated macrophages. LPS stimulation induced HDAC6 expression, but suppressed miR-22 expression in macrophages, suggesting possible correlation between HDAC6 and miR-22. Luciferase reporter assays revealed that 3'UTR of HDAC6 was a bona fide target site of miR-22. Transfection of miR-22 mimic significantly inhibited LPS-induced HDAC6 expression, while miR-22 inhibitor further increased LPS-induced HDAC6 expression. LPS-induced activation of NF-κB and AP-1 was inhibited by miR-22 mimic, but further increased by miR-22 inhibitor. LPS-induced expression of pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6 was inhibited by miR-22 mimic, but further increased by miR-22 inhibitor. Taken together, these data provide evidence that miR-22 can downregulate LPS-induced expression of proinflammatory cytokines via suppression of NF-κB and AP-1 axis by targeting HDAC6 in macrophages. [BMB Reports 2020; 53(4): 223-228].

Hindsiipropane B alleviates HIV-1 Tat-induced inflammatory responses by suppressing HDAC6-NADPH oxidase-ROS axis in astrocytes.

Human immunodeficiency virus-1 (HIV-1) transactivator of transcription (Tat) is an important viral factor in neuroinflammation. Hindsiipropane B, present in Celastrus hindsii, possesses various biological mechanisms including antiinflammatory activity. In this report, we explored the regulatory activity of hindsiipropane B on HIV-1 Tat-mediated chemokine production and its mode of action in astrocytes. Hindsiipropane B significantly alleviated HIV-1 Tat-mediated production of inflammatory chemokines, CCL2, CXCL8, and CXCL10. Hindsiipropane B inhibited expression of HDAC6, which is important regulator in HIV-1 Tat-mediated chemokine production. Hindsiipropane B diminished HIV-1 Tat-mediated reactive oxygen species (ROS) generation and NADPH oxidase activation/expression. Furthermore, hindsiipropane B inhibited HIV-1 Tat-mediated signaling cascades including MAPK, NF-κB, and AP-1. These data suggest that hindsiipropane B exerts its inhibitory effects on HIV-1 Tat-mediated chemokine production via down-regulating the HDAC6-NADPH oxidase-MAPK-NF-κB/AP-1 signaling axis, and could serve as a therapeutic lead compound against HIV-1 Tat-associated neuroinflammation. [BMB Reports 2018; 51(8): 394-399].

Human podoplanin-positive monocytes and platelets enhance lymphangiogenesis through the activation of the podoplanin/CLEC-2 axis.

Emerging studies suggested that murine podoplanin-positive monocytes (PPMs) are involved in lymphangiogenesis. The goal of this study was to demonstrate the therapeutic lymphangiogenesis of human PPMs by the interaction with platelets. Aggregation culture of human peripheral blood mononuclear cells (PBMCs) resulted in cellular aggregates termed hematospheres. During 5-day culture, PPMs expanded exponentially and expressed several lymphatic endothelial cell-specific markers including vascular endothelial growth factor receptor (VEGFR)-3 and well-established lymphangiogenic transcription factors. Next, we investigated the potential interaction of PPMs with platelets that had C-type lectin-like receptor-2 (CLEC-2), a receptor of podoplanin. In vitro coculture of PPMs and platelets stimulated PPMs to strongly express lymphatic endothelial markers and upregulate lymphangiogenic cytokines. Recombinant human CLEC-2 also stimulated PPMs through Akt and Erk signaling. Likewise, platelets in coculture with PPMs augmented secretion of a lymphangiogenic cytokine, interleukin (IL)-1-ß, via podoplanin/CLEC-2 axis. The supernatant obtained from coculture was able to enhance the migration, viability, and proliferation of lymphatic endothelial cell. Local injection of hematospheres with platelets significantly increased lymphatic neovascularization and facilitated wound healing in the full-thickness skin wounds of nude mice. Cotreatment with PPMs and platelets augments lymphangiogenesis through podoplanin/CLEC-2 axis, which thus would be a promising novel strategy of cell therapy to treat human lymphatic vessel disease.

Transient blood brain barrier disruption induced by oleic acid is mediated by nitric oxide.

The blood brain barrier (BBB) maintains cerebral microenvironmental homeostasis. Transient disruption of the BBB after brain fat embolism in clinical cases and animal models has been reported but the precise mechanism underlying this occurrence is unclear. In the present study, we investigated BBB alterations in rats treated oleic acid (OA) delivered intra-arterially. Following OA treatment, transient brain edema, extravasation of Evans blue and Fluorescein isothiocyanate (FITC)-labeled dextran, and loss of laminin in the affected brain area were observed. Activation of matrix metalloproteinase (MMP)-2, -3, and -13 was found in the cerebral vessels 2 h after OA administration. Expression of intercellular adhesion molecule (ICAM)-1 in the vessels and neutrophil infiltration into the brain tissue was also observed. Inducible nitric oxide synthase (iNOS) was expressed in the neutrophils and nitrotyrosine was produced mainly in the vessels. Inhibitor of iNOS activity suppressed the loss of laminin, leakage of FITC-labeled dextran and Evans blue, and activation of MMP-2 and -13. Protein level of aquaporin (AQ)-4 was increased after OA administration but was not affected by treatment with iNOS inhibitor. In conclusion, we suggest that nitric oxide (NO) contributes to OA-induced MMP activation, BBB disruption and the development of transient brain edema.

Highly angiogenic CXCR4(+)CD31(+) monocyte subset derived from 3D culture of human peripheral blood.

Ex vivo expansion of human circulating angiogenic cells is a major challenge in autologous cell therapy for ischemic diseases. Here, we demonstrate that hematosphere-derived CXCR4(+)CD31(+) myeloid cells using peripheral blood possess robust proangiogenic capacity such as formation of vessel-like structures and tip cell-like morphology in Matrigel. We also found that CD31 positive myeloid cells are principal cellular component of hematospheres by magnetic cell sorting. Flow cytometry analysis showed that fresh peripheral blood contained 40.3 ± 15.2% of CXCR4(+)CD31(+) myeloid cells, but at day 5 of hematosphere culture, most of myeloid cells were CXCR4(+)CD31(+) by 86.9 ± 5.4%. Hematosphere culture significantly increased the production of angiogenic niche-supporting cytokines. Moreover, CD31-homophilic interaction and VEGF-VEGF receptor loop signaling were essential for sphere formation and acquisition of angiogenic capacity in hematospheres. Matrigel plug and ischemic hindlimb model provide in vivo evidence that hematosphere-derived myeloid cells have highly vasculogenic capacities, participate in new and mature vessel formation, and exert therapeutic effects on ischemic hindlimb. In conclusion, our strategy for ex vivo expansion of human CXCR4(+)CD31(+) angiogenic cells using hematospheres provides an autologous therapeutic cell source for ischemic diseases and a new model for investigating the microenvironment of angiogenesis.

New method to differentiate human peripheral blood monocytes into insulin producing cells: Human hematosphere culture.

Strategy to differentiate stem cells into insulin producing cells (IPCs) in vitro has been a promising one to get cell source of ß-cell replacement therapy for diabetes. It has been suggested that islets and neurons share features and nestin-positive cells could differentiate into IPCs. We have recently developed a three-dimensional culture system using human peripheral blood cells named as blood-born hematosphere (BBHS). Here we showed that most of BBHS were composed of nestin-positive cells. Under the four-stage differentiation protocol for IPCs, we plated nestin-positive BBHS onto fibronectin-coated dish. These cells form islet-like clusters and most of them expressed insulin. Pancreatic specific genes were turned on, such as transcription factors (Pdx-1, Ngn3 and Nkx6.1), genes related to endocrine function (Glut-2 and PC2) or ß cell function (Kir6.2, SUR1). Furthermore islet differentiation was confirmed by dithizone (DTZ) staining to detect zinc ion which binds insulin protein within the cells. Finally, IPCs derived from BBHS showed capability to secrete insulin in response to glucose stimulation. Taken together, our novel protocol successfully induced islet-like human insulin producing cells out of BBHS. This strategy of ex vivo expansion of IPCs using BBHS provides an autologous therapeutic cell source for the treatment of diabetes.

Water extract of Triticum aestivum L. and its components demonstrate protective effect in a model of vascular dementia.

Although vascular dementia is the second leading cause of dementia and often underdiagnosed, there are no drugs yet approved for the treatment of vascular dementia. In this study, it is demonstrated that water extract of Triticum aestivum L. (TALE) and some of its components have protective effects against vascular dementia-induced damage by preserving the myelin sheath and inhibiting astrocytic activation. The memory test used a vascular dementia model utilizing bilateral ligation of the carotid arteries of rats. TALE, some of its components, such as starch, total dietary fiber (TDF), arabinoxylan, beta-glucan, and degraded products of arabinoxylan, such as arabinose and xylose, were administered to the animals from day 8 to day 14, following the surgery. Twenty-one days after the surgery, the water maze test was performed for 5 days, and the time taken to find the platform during training trials (mean escape latency) was measured. The mean escape latency was decreased consistently in the TALE-, starch-, TDF-, arabinoxylan-, and arabinose-treated groups, compared with that in the vascular dementia group. To measure brain damage, Luxol fast blue staining and immunohistochemistry of myelin basic protein (MBP) were performed to observe myelin sheath in the white matter, and immunohistochemistry of glial fibrillary acidic protein (GFAP) was performed to observe the astrocytic reaction. Vascular dementia reduced the MBP level and increased the GFAP level. Arabinose effectively inhibited the MBP and GFAP change, whereas arabinoxylan inhibited the GFAP change only. These results suggest that TALE and some of its components can be used as a medicinal material for the development of neuroprotective agents against vascular dementia.

A theoretical model to predict the low-frequency sound absorption of a helmholtz resonator array.

A theoretical method based on mutual radiation impedance is proposed to compute the sound absorption performance of a Helmholtz resonator array in the low-frequency range. Any configuration of resonator arrangement can be allowed in the method, while all the resonators may or may not be identical. Comparisons of the theoretical predictions with those done by the past studies or experiments show that the present method can accurately predict the absorption performance in more general cases.