Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

[A Case of Anti-Resorptive Agents-Related Osteonecrosis of the Jaw and Extensive Loss of Maxillary Bone in a Patient of Breast Cancer].

A 74-year-old woman had a mastectomy for right breast cancer in 201X. Eight years later, the patient developed multiple bone metastases and was treated with denosumab by her previous doctor. A year after, she was diagnosed with anti-resorptive agents-related osteonecrosis of the jaw, and preservation treatment was performed. In 201X+11, the patient had difficulty walking and was admitted to our palliative care ward. A month later, her maxillary bone detached extensively and spontaneously. By applying infection preventive measures and in cooperation with the former doctor, dentist, and oral surgeon, as well as visiting the dental department in our hospital, the patient managed to continue eating what she liked. Jaw infection did not occur. The patient died of liver dysfunction due to liver metastasis 5 months following the extensive loss of the maxillary bone.

[Breast Decompression Using Automatic Pressure Reduction Control Function: Relationship between Breast Thickness, Density, and Compression Pressure].

PURPOSE: This retrospective study was conducted to evaluate whether the breast density and initial compression pressure affected the compressed breast thickness (CBT) and compression pressure after decompression using compression control function (CCF) during mammography. METHODS: Consecutive 779 mammograms obtained from 392 patients between February and October 2019 were included. The initial compression was randomly performed at 110 N-140 N. CCF was set to stop decompression when the pressure decreased to 80 N or restoration of CBT reached 3 mm. If the CCF stopped due to 3 mm or more restoration of CBT, it was defined as goal unachieved. Mammograms were classified into non-dense and dense groups. CBT, ∆P (actual compression pressure after decompression-80 N), and the ratio of goal unachieved were compared between breast density subgroups and among initial compression pressure. RESULTS: CBT was significantly different between non-dense and dense groups both at initial compression (42.3±12.1 mm vs. 27.6±9.7 mm, p<0.001) and after decompression (44.6±12.3 mm vs. 29.7±9.9 mm, p<0.001), but not different based on initial compression pressure. The higher the initial compression pressure, the higher the ∆P. When the initial compression pressure was 130 N and 140 N, ∆P was significantly higher in the non-dense group, -0.1±3.7 N vs. -1.6±2.7 N (p=0.0018) and 2.9±5.8 N vs. 0.4±3.3 N (p<0.001), respectively. Goal unachieved was significantly frequent in the non-dense group (19.6% vs. 13.1%, p=0.016). CONCLUSION: Breast density and initial compression pressure affected the decompression using CCF. Especially with lower initial compression pressure to the dense breast, decompression using CCF was successfully performed.

Gross Cystic Disease Fluid Protein-15 (GCDFP-15) Expression Characterizes Breast Mucinous Carcinomas in Older Women.

The predominant histological subtype of breast mucinous carcinoma in older women is type B (hypercellular type), and, in younger women, it is type A (hypocellular type). The characteristics of mucinous carcinomas of the same histological subtype may differ between older and younger women. This study aims to systematically clarify the pathological/immunohistochemical features of mucinous carcinomas. A total of 21 surgical cases of mucinous carcinoma (type A/B: 9/12 cases) in the older group (≥65 years) and 16 cases (type A/B: 14/2 cases) in the younger group (≤55 years) (n = 37) were included. Gross cystic disease fluid protein-15 (GCDFP-15) and eight other markers were used for immunostaining. The GCDFP-15-positive rate in the older group was high regardless of the histological subtype (type A, 77.8%; type B, 91.7%). The GCDFP-15 positivity in the older group was significantly higher than that in the younger group (p < 0.001 for Allred score). Among type A, GCDFP-15 positivity was significantly higher in the older group than in the younger group (p = 0.042 for the Allred score and p = 0.007 for the positivity rate). The present results suggest that GCDFP-15 expression characterizes mucinous carcinomas in older women.

HDAC inhibitor, MS-275, increases vascular permeability by suppressing Robo4 expression in endothelial cells.

Roundabout guidance receptor 4 (Robo4) is an endothelial-specific membrane protein that suppresses pathological angiogenesis and vascular hyperpermeability by stabilizing endothelial cells. Robo4 suppresses severe systemic inflammation induced by pathogens and endotoxins and inhibits tumor growth and metastasis, therefore serving as a potential therapeutic target. Although the regulation of Robo4 expression through transcription factors and epigenetic mechanisms has been studied, the role of histone deacetylases (HDACs) has not been explored. In the present study, we investigated the involvement of HDACs in the regulation of Robo4 expression. An HDAC inhibitor, MS-275, which inhibits HDAC1, HDAC2, and HDAC3, was found to suppress Robo4 expression in endothelial cells. Small interfering RNA (siRNA)-mediated knockdown of HDAC3, but not of HDAC1 and 2, also decreased its expression level. MS-275 downregulated the expression of the transcription factor complex GABP, in addition to suppressing Robo4 promoter activity. GABP expression was also downregulated by the siRNA against HDAC3. MS-275 decreased the transendothelial electrical resistance of a monolayer of mouse endothelial cells and increased the rate of leakage of Evans blue dye in the mouse lungs. In addition, MS-275 accelerated cell migration through the endothelial cell monolayer and augmented cell extravasation in the mouse lungs. Taken together, we demonstrated that MS-275 suppresses Robo4 expression by inhibiting HDAC3 in endothelial cells and enhances endothelial and vascular permeability. Thus, we demonstrated a novel mechanism regulating Robo4 expression and vascular permeability, which is anticipated to contribute to future therapies for infectious and inflammatory diseases.

Vascular endothelial growth factor expression in rat skin incision wound.

Vascular endothelial growth factor (VEGF) is a glycoprotein that enhances vascular permeability, induces chemotaxis and activation of monocytes/macrophages, and promotes growth of vascular endothelial cells. We report that infiltrating polymorphonuclear leukocytes in an incision wound in rat skin express VEGF from 1 day after the injury, as shown by immunohistochemistry. VEGF is also present in macrophages, fibroblast-like cells, and endothelial cells 3 and 7 days after the injury. mRNA for VEGF is statistically significantly increased in the wound area in the tissue 1 day after the skin incision compared with 3 and 7 days after the incision. The VEGF protein content in the wound tissue is statistically significantly higher in the wound than in control skin at 1 and 3 days after skin incision. Our results indicate that VEGF is produced by inflammatory cells to induce vascularization in the early stage of the wound healing process.