A microphysiological system-based potency bioassay for the functional quality assessment of mesenchymal stromal cells targeting vasculogenesis.

Mesenchymal stromal cells (MSCs) continue to be proposed for use in clinical trials to treat various diseases due to their therapeutic potential to pleiotropically influence endogenous regenerative processes, such as vasculogenesis. However, the functional heterogeneity of MSCs has hampered their clinical success and poses a significant manufacturing challenge with respect to MSC quality control. Here, we evaluated and qualified a quantitative bioassay based on an enhanced-throughput, microphysiological system to measure the specific paracrine bioactivity of MSCs to stimulate vasculogenesis as a measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages were co-cultured with human umbilical vein endothelial cells (HUVECs) and exhibited significant heterogeneity in vasculogenic potency between donors and cell passage. Using our microphysiological system (MPS)-based platform, we demonstrated that variations in MSC vasculogenic bioactivity were maintained when assayed across laboratories and operators. The differences in MSC vasculogenic bioactivity were also correlated with the baseline expression of several genes involved in vasculogenesis (hepatocyte growth factor (HGF), angiopoietin-1 (ANGPT)) or the production of matricellular proteins (fibronectin (FN), insulin-like growth factor-binding protein 7 (IGFBP7)). These findings emphasize the significant functional heterogeneity of MSCs in vasculogenic bioactivity and suggest that changes in baseline gene expression of vasculogenic or matricellular protein genes during manufacturing may affect this bioactivity. The development of a reliable and functionally relevant potency assay for measuring the specific vasculogenic bioactivity of manufactured MSCs will help to reliably assure their quality when used in appropriate clinical trials.

Determination of Blood NOTCH3 Extracellular Domain and Jagged-1 Levels in Healthy Subjects.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common genetic disorder among those responsible for hereditary strokes, and it is caused by a mutation in the NOTCH3 gene on chromosome 19. Blood biomarkers related to the Notch signaling pathway have not been investigated extensively in CADASIL. In this study, we measured the serum and plasma levels of NOTCH3 extracellular domain (N3ECD) and its ligand, Jagged-1, in 279 healthy subjects. The levels of N3ECD and Jagged-1 showed significant correlations in both serum (p < 0.0001, r = 0.2681) and plasma (p < 0.0001, r = 0.4065). The N3ECD levels were significantly higher in the serum than in plasma and tend to increase with age. In contrast, there was no significant difference between the serum and plasma levels of Jagged-1 levels. To summarize, we were able to measure N3ECD and Jagged-1 protein levels in healthy human serum and plasma. Taken together, our findings provide the basis for further studies investigating the clinical use of blood N3ECD and Jagged-1 levels for CADASIL and other Notch signaling-related diseases.

Pathogenic NOTCH3 Variants Are Frequent Among the Korean General Population.

OBJECTIVE: This study aimed to determine the frequency of pathogenic NOTCH3 variants among Koreans. METHODS: In this cross-sectional study, we queried for pathogenic NOTCH3 variants in 2 Korean public genome databases: the Korean Reference Genome Database (KRGDB) and the Korean Genome Project (Korea1K). In addition, we screened the 3 most common pathogenic NOTCH3 variants (p.Arg75Pro, p.Arg544Cys, and p.Arg578Cys) for 1,000 individuals on Jeju Island, where the largest number of patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) have been reported in Korea. RESULTS: The pathogenic NOTCH3 variant (p.Arg544Cys) was found in 0.12% of sequences in the KRGDB, and 3 pathogenic variants (p.Arg75Pro, p.Arg182Cys, and p.Arg544Cys) were present in 0.44% of the Korea1K database. Of the 1,000 individuals on Jeju Island, we found 2 cysteine-altering NOTCH3 variants (p.Arg544Cys variant in 9 and p.Arg578Cys in 1 individual) in 1.00% of the participants (95% confidence interval: 0.48%-1.83%). The presence of cysteine-altering NOTCH3 variants was significantly associated with a history of stroke (p < 0.001). DISCUSSION: Pathogenic NOTCH3 variants are frequently found in the general Korean population. Such a high prevalence of pathogenic variants could threaten the brain health of tens of thousands to hundreds of thousands of older adults in Korea.

Hypoglycemic effect of standardized Chrysanthemum zawadskii ethanol extract in high-fat diet/streptozotocin-induced diabetic mice and rats.

Chrysanthemum zawadskii (CZ) is a perennial herb belonging to the Asteraceae family. CZ is used medicinally to treat inflammatory and uterine diseases in Asia. CZ was extracted with 50% ethanol and CZ extract (CZE; at 125, 250, and 500 mg/kg body weight) was administered orally every day for 5 or 6 weeks to investigate the anti-diabetic effects in streptozotocin (STZ)-induced rats and STZ + high-fat diet (HFD)-fed mice. CZE significantly decreased fasting blood glucose levels in STZ- and STZ + HFD-induced diabetic models. In addition, glucose tolerance and insulin tolerance were improved in the STZ + HFD + CZE group by increasing insulin levels and decreasing hemoglobin A1c (HbA1c) levels in serum. Furthermore, CZE supplements decreased components of the serum lipid profile such as triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels. These results suggest that CZE may be a potential candidate for controlling hyperglycemia.

Effects of yuja peel extract and its flavanones on osteopenia in ovariectomized rats and osteoblast differentiation.

SCOPE: Yuja (Citrus junos Tanaka) possesses various health benefits, but its effects on bone health are unknown. In this study, the preventative effects of yuja peel ethanol extract (YPEE) on osteopenia were determined in ovariectomized (OVX) rats, and the mechanisms by which YPEE and its flavanones regulate osteoblastogenesis were examined in vitro. METHODS AND RESULTS: The effects of YPEE on osteoblastogenesis were investigated in MC3T3-E1 cells. YPEE promoted alkaline phosphatase (ALP) activity, mineralization, and the expression of osteoblast differentiation marker genes, such as ALP, runt-related transcription factor 2 (Runx2), and osteocalcin. YPEE and its flavanones promoted osteoblast differentiation via BMP-2-mediated p38 and the Smad1/5/8 signaling pathway. YPEE supplementation significantly decreased body weight and increased uterine weight and bone mineral density in OVX rats. Based on a micro-CT analysis of femurs, YPEE significantly attenuated osteopenia and increased trabecular volume fraction, trabecular separation, and trabecular number (p < 0.05). CONCLUSION: Dietary YPEE has a protective effect on OVX-induced osteopenia. YPEE and its flavanones promote osteoblastogenesis via the activation of the BMP/p38/Smad/Runx2 pathways. These results extend our knowledge of the beneficial effects of YPEE and provide a basis for the development of novel therapies for osteoporosis.

Pharmacokinetics of Tyrosol Metabolites in Rats.

Tyrosol is considered a potential antioxidant; however, little is known regarding the pharmacokinetics of its metabolites. To study the pharmacokinetics of tyrosol-derived metabolites after oral administration of a single dose of tyrosol, we attempted to identify tyrosol metabolites in rat plasma by using ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Two tyrosol metabolites (M1 and M2) were detected in the plasma. M1 was identified as tyrosol-4-sulfate (T4S) with an [M - H](-) ion at m/z 217. While M2 showed an [M - H](-) ion at m/z 151.0, its metabolite was not identified. Pharmacokinetic analysis of T4S and M2 showed rapid uptake after oral administration of tyrosol within 1 h. The metabolites were rapidly distributed in most organs and tissues and eliminated within 4 h. The greatest T4S deposition by tissue weight was observed in the liver, followed by the kidney and spleen, while M2 was most concentrated in the kidney followed by the liver and spleen. These findings indicate that T4S and M2 were distributed mainly in tissues with an abundant blood supply and were rapidly excreted in urine.

Pharmacokinetics, Tissue Distribution, and Anti-Lipogenic/Adipogenic Effects of Allyl-Isothiocyanate Metabolites.

Allyl-isothiocyanate (AITC) is an organosulfur phytochemical found in abundance in common cruciferous vegetables such as mustard, wasabi, and cabbage. Although AITC is metabolized primarily through the mercapturic acid pathway, its exact pharmacokinetics remains undefined and the biological function of AITC metabolites is still largely unknown. In this study, we evaluated the inhibitory effects of AITC metabolites on lipid accumulation in vitro and elucidated the pharmacokinetics and tissue distribution of AITC metabolites in rats. We found that AITC metabolites generally conjugate with glutathione (GSH) or N-acetylcysteine (NAC) and are distributed in most organs and tissues. Pharmacokinetic analysis showed a rapid uptake and complete metabolism of AITC following oral administration to rats. Although AITC has been reported to exhibit anti-tumor activity in bladder cancer, the potential bioactivity of its metabolites has not been explored. We found that GSH-AITC and NAC-AITC effectively inhibit adipogenic differentiation of 3T3-L1 preadipocytes and suppress expression of PPAR-γ, C/EBPα, and FAS, which are up-regulated during adipogenesis. GSH-AITC and NAC-AITC also suppressed oleic acid-induced lipid accumulation and lipogenesis in hepatocytes. Our findings suggest that AITC is almost completely metabolized in the liver and rapidly excreted in urine through the mercapturic acid pathway following administration in rats. AITC metabolites may exert anti-obesity effects through suppression of adipogenesis or lipogenesis.