Exploring 2-methyl-substituted vitamin K3 derivatives with potent inhibitory activity against the 3CL protease of SARS-CoV-2.

Since the outbreak of the pandemic, various anti-SARS-CoV-2 drugs have been developed. In particular, 3CL protease (3C-like protease, 3CLpro) is an attractive drug target because it is an essential enzyme for viral multiplication and is present only in viruses, not in humans. To date, 3CLpro inhibitors against SARS-CoV-2 such as nirmatrelvir and ensitrelvir have been launched as oral drugs in Japan, but there is still no potent drug against SARS-CoV-2, due to issues of in vivo absorption and stability. Recently, vitamin K3 was reported to show inhibitory activity against 3CLpro of SARS-CoV-2, and the mechanism of action was predicted to be the formation of a covalent bond between the thiol group of cysteine 145, the active center of 3CLpro, and the C-3 position of vitamin K3. Therefore, we synthesized derivatives in which the 2-methyl group of the vitamin K3 was systematically converted to other substituents and examined their inhibitory activity against 3CLpro of SARS-CoV-2. The results showed that the compounds with the sulfide structure showed an approximately 4-fold increase in activity over vitamin K3. These results indicated the possibility of creating new inhibitors based on vitamin K3 and its derivatives.

Exploring Novel Vitamin K Derivatives with Anti-SARS-CoV-2 Activity.

From our compound library of vitamin K derivatives, we found that some compounds exhibited anti-SARS-CoV-2 activity in VeroE6/TMPRSS2 cells. The common structure of these compounds was menaquinone-2 (MK-2) with either the m-methylphenyl or the 1-naphthyl group introduced at the end of the side chain. Therefore, new vitamin K derivatives having more potent anti-SARS-CoV-2 activity were explored by introducing various functional groups at the ω-position of the side chain. MK-2 derivatives with a purine moiety showed the most potent antiviral activity among the derivatives. We also found that their mechanism of action was the inhibition of RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. The chemical structures of our compounds were completely different from those of nucleic acid derivatives such as remdesivir and molnupiravir, clinically approved RdRp inhibitors for COVID-19 treatment, suggesting that our compounds may be effective against viruses resistant to these nucleic acid derivatives.

Activation of transient receptor potential channels is involved in reactive oxygen species (ROS)-dependent regulation of blood flow by (-)-epicatechin tetramer cinnamtannin A2.

Intervention trials confirmed that blood flow-mediated dilatation increases significantly after intake of astringent (-)-epicatechin (EC) oligomers (procyanidins)-rich foods, but the mechanism remains unclear. We have previously found that procyanidins can activate the sympathetic nervous and subsequently increase blood flow. Here, we examined whether procyanidin-derived reactive oxygen species (ROS) activate transient receptor potential (TRP) channels in gastrointestinal sensory nerves and consequently induce sympathoexcitation. We evaluated the redox properties of EC and its tetramer cinntamtannin A2 (A2) at pH 5 or 7, mimicking plant vacuole or oral cavity/small intestine using a luminescent probe. At pH 5, A2 or EC showed O2·- scavenging ability, but they promoted O2·- generation at pH 7. We observed blood flow in rat cremaster arterioles using laser Doppler, a single oral dose of 10 µg/kg A2 markedly increased blood flow, while EC showed little activity. This change with A2 was significantly dampened by co-administration of adrenaline blocker, ROS scavenger N-acetyl-L-cysteine (NAC), TRP vanilloid 1, or ankyrin 1 antagonist. We also performed a docking simulation of EC or A2 with the binding site of a typical ligand for each TRP channel and calculated the respective binding affinities. The binding energies were notably higher for A2 than typical ligands, suggesting that A2 is less likely to bind to these sites. ROS produced at neutral pH following the orally administered A2 to the gastrointestinal tract could activate TRP channels, triggering sympathetic hyperactivation and causing hemodynamic changes.

Effect of lonidamine derivatives on the inhibition of transformed cell area expansion.

Expansion of transformed cell area is regulated by the surrounding nontransformed cells. Lonidamine (LND) was recently found to regulate transformed cell area expansion through suppressing the cell motility of nontransformed cells; however, the structure-activity relationship between LND and this inhibitory activity has yet to be elucidated. We synthesized several LND derivatives and evaluated their inhibitory activity against the expansion of transformed cell area and found that the halogenation pattern on the benzene ring moiety, the carboxylic acid moiety, and the overall hydrophobicity of the molecule were correlated with inhibition activity. We also found that the localization of the tight junction protein, zonula occludens-1 (ZO-1), in nontransformed cells was significantly altered after treatment with the LND derivatives that displayed inhibitory activity. Further studies with LND derivatives and monitoring the localization of ZO-1 may help to develop more active compounds for suppressing transformed cell area expansion and lead to new anticancer treatments.

Naturally occurring UBIAD1 mutations differentially affect menaquinone biosynthesis and vitamin K-dependent carboxylation.

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is responsible for the biosynthesis of menaquinone-4 (MK-4), a cofactor for extrahepatic carboxylation of vitamin K-dependent (VKD) proteins. Genetic variations of UBIAD1 are mainly associated with Schnyder corneal dystrophy (SCD), a disease characterized by abnormal accumulation of cholesterol in the cornea. Results from in vitro studies demonstrate that SCD-associated UBIAD1 mutations are defective in MK-4 biosynthesis. However, SCD patients do not exhibit typical phenotypes associated with defects of MK-4 or VKD carboxylation. Here, we coupled UBIAD1's biosynthetic activity of MK-4 with VKD carboxylation in HEK293 cells that stably express a chimeric VKD reporter protein. The endogenous Ubiad1 gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. The effect of UBIAD1 mutations on MK-4 biosynthesis and VKD carboxylation was evaluated in Ubiad1-deficient reporter cells by determining the production of MK-4 or by measuring the efficiency of reporter-protein carboxylation. Our results show that the hot-spot mutation N102S has a moderate impact on MK-4 biosynthesis (retained ˜ 82% activity) but does not affect VKD carboxylation. However, the G186R mutation significantly affected both MK-4 biosynthesis and VKD carboxylation. Other mutations exhibit varying degrees of effects on MK-4 biosynthesis and VKD carboxylation. These results are consistent with in vivo results obtained from gene knock-in mice and SCD patients. Our findings suggest that UBIAD1's MK-4 biosynthetic activity does not directly correlate with the phenotypes of SCD patients. The established cell-based assays in this study provide a powerful tool for the functional studies of UBIAD1 in a cellular milieu.

A novel vitamin K derived anticoagulant tolerant to genetic variations of vitamin K epoxide reductase.

BACKGROUND: Vitamin K antagonists (VKAs), such as warfarin, have remained the cornerstone of oral anticoagulation therapy in the prevention and treatment of thromboembolism for more than half a century. They function by impairing the biosynthesis of vitamin K-dependent (VKD) clotting factors through the inhibition of vitamin K epoxide reductase (VKOR). The challenge of VKAs therapy is their narrow therapeutic index and highly variable dosing requirements, which are partially the result of genetic variations of VKOR. OBJECTIVES: The goal of this study was to search for an improved VKA that is tolerant to the genetic variations of its target enzyme. METHODS: A series of vitamin K derivatives with benzyl and related side-chain substitutions at the 3-position of 1,4-naphthoquinone were synthesized. The role of these compounds in VKD carboxylation was evaluated by mammalian cell-based assays and conventional in vitro activity assays. RESULTS: Our results showed that replacing the phytyl side-chain with a methylene cyclooctatetraene (COT) moiety at the 3-position of vitamin K1 converted it from a substrate to an inhibitor for VKD carboxylation. Strikingly, this COT-vitamin K derivative displayed a similar inhibition potency in warfarin-resistant VKOR mutations whose warfarin resistance varied more than 400-fold. Further characterization of COT-vitamin K for the inhibition of VKD carboxylation suggested that this compound targets multiple enzymes in the vitamin K redox cycle. Importantly, the anticoagulation effect of COT-vitamin K can be rescued with high doses of vitamin K1 . CONCLUSION: We discovered a vitamin K analogue that functions as a VKA and is tolerant to genetic variations in the target enzyme.

Development of Selective TGR5 Ligands Based on the 5,6,7,8-Tetrahydro-5,5,8,8-tetramethylnaphthalene Skeleton.

TGR5, a G-protein-coupled receptor (GPCR), plays an important role in several physiological functions. TGR5 activation through bile acids induces an increase in energy expenditure. Therefore, synthetic TGR5 ligands could be useful for the treatment of obesity or dyslipidemia. In this study, we designed and synthesized a set of TGR5 ligands with a 5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalene (TMN) skeleton, and evaluated their TGR5 agonistic activity. We also investigated the selectivity of the synthesized compounds for TGR5 relative to the farnesoid X receptor (FXR) and retinoic acid receptor (RAR). Our results show that compound 4 b [N-(2-chlorophenyl)-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenecarboxamide] exhibited potent TGR5 agonist activity with an IC50 value of 8.4 nM without significant cytotoxicity. In addition, compound 4 b showed only slight agonistic activity toward FXR and RAR at 1 µM treatment. These data indicate that compound 4 b is a selective TGR5 agonist, and could be a promising therapeutic agent for dyslipidemia.

Study on structure-activity relationship of vitamin K derivatives: Conversion of the naphthoquinone part into another aromatic ring and evaluation of their neuronal differentiation-inducing activity.

We synthesized novel vitamin K derivatives by converting the naphthoquinone group to benzene derivatives and benzoquinone. We evaluated their neuronal differentiation activities to investigate the effect of the quinone moiety on this process. We observed that the 1,4-quinone as well as the side chain part play important roles in neuronal differentiation. We also performed QSAR analysis to predict the compounds which would have higher differentiation activity.

Synthesis and In Vitro Evaluation of Novel Liver X Receptor Agonists Based on Naphthoquinone Derivatives.

We aimed to synthesize novel liver X receptor (LXR) agonists with potent agonist activity and subtype selectivity. Our synthetic scheme started with naphthoquinone derivatives, such as menadione and 2,3-dichloro-1,4-naphthoquinone. We introduced different substituents into the naphthoquinone structures, including aniline, piperidine, pyrrolidine, and morpholine, in one or two steps, and thus, we produced 14 target compounds. All 14 synthetic ligands were tested to determine whether they mediated LXR-mediated transcriptional activity. We investigated the transcriptional activity of each compound with two types of receptors, LXRα and LXRß. Among all 14 compounds, two showed weak LXRß-agonist activity, and two others exhibited potent LXRα-agonist activity. We also performed docking studies to obtain a better understanding of the modes of compound binding to LXR at the atomic level. In conclusion, we successfully synthesized naphthoquinone derivatives that act as LXRα/ß agonists and selective LXRα agonists.

Elucidation of the Interaction between Flavan-3-ols and Bovine Serum Albumin and Its Effect on Their In-Vitro Cytotoxicity.

Flavan-3-ols (FLs), specifically catechin and its oligomer B-type procyanidins, are suggested to potently bind to bovine serum albumin (BSA). We examined the interaction between BSA and FLs by fluorescence quenching and found the following order of binding activities to BSA: cinnamtannin A2 (A2; tetramer) > procyanidin C1 (C1; trimer) ≈ procyanidin B2 (B2, dimer) > (-)epicatechin (EC, monomer). Docking simulations between BSA and each compound at the binding site showed that the calculated binding energies were consistent with the results of our experimental assay. FLs exerted cytotoxicity at 1000 µg/mL in F11 cell culture with fetal bovine serum containing BSA. In culture containing serum-free medium, FLs exhibited significant cell proliferation at 10-4 µg/mL and cytotoxicity was observed at concentrations greater than 10 µg/mL. Results of this study suggest that interactions between polyphenols and BSA should be taken into account when evaluating procyanidin in an in vitro cell culture system.

New Aspects of Vitamin K Research with Synthetic Ligands: Transcriptional Activity via SXR and Neural Differentiation Activity.

Vitamin K is classified into three homologs depending on the side-chain structure, with 2-methyl-1,4-naphthoqumone as the basic skeleton. These homologs are vitamin K1 (phylloquinone: PK), derived from plants with a phythyl side chain; vitamin K2 (menaquinone-n: MK-n), derived from intestinal bacteria with an isoprene side chain; and vitamin K3 (menadione: MD), a synthetic product without a side chain. Vitamin K homologs have physiological effects, including in blood coagulation and in osteogenic activity via γ-glutamyl carboxylase and are used clinically. Recent studies have revealed that vitamin K homologs are converted to MK-4 by the UbiA prenyltransferase domain-containing protein 1 (UBIAD1) in vivo and accumulate in all tissues. Although vitamin K is considered to have important physiological effects, its precise activities and mechanisms largely remain unclear. Recent research on vitamin K has suggested various new roles, such as transcriptional activity as an agonist of steroid and xenobiotic nuclear receptor and differentiation-inducing activity in neural stem cells. In this review, we describe synthetic ligands based on vitamin K and exhibit that the strength of biological activity can be controlled by modification of the side chain part.

UBIAD1 Plays an Essential Role in the Survival of Pancreatic Acinar Cells.

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) is a vitamin K2 biosynthetic enzyme. We previously showed the lethality of this enzyme in UBIAD1 knockout mice during the embryonic stage. However, the biological effects of UBIAD1 deficiency after birth remain unclear. In the present study, we used a tamoxifen-inducible systemic UBIAD1 knockout mouse model to determine the role of UBIAD1 in adult mice. UBIAD1 knockout resulted in the death of the mice within about 60 days of administration of tamoxifen. The pancreas presented with the most prominent abnormality in the tamoxifen-induced UBIAD1 knockout mice. The pancreas was reduced remarkably in size; furthermore, the pancreatic acinar cells disappeared and were replaced by vacuoles. Further analysis revealed that the vacuoles were adipocytes. UBIAD1 deficiency in the pancreatic acinar cells caused an increase in oxidative stress and autophagy, leading to apoptotic cell death in the tamoxifen-induced UBIAD 1 knockout mice. These results indicate that UBIAD1 is essential for maintaining the survival of pancreatic acinar cells in the pancreas.

Eldecalcitol is more effective in promoting osteogenesis than alfacalcidol in Cyp27b1-knockout mice.

Calcium (Ca) absorption from the intestinal tract is promoted by active vitamin D (1α,25D3). Vitamin D not only promotes Ca homeostasis, but it also inhibits bone resorption and promotes osteogenesis, thus playing a role in the maintenance of normal bone metabolism. Because 1α,25D3 plays an important role in osteogenesis, vitamin D formulations, such as alfacalcidol (ALF) and eldecalcitol (ELD), are used for treating osteoporosis. While it is known that, in contrast to ALF, ELD is an active ligand that directly acts on bone, the reason for its superior osteogenesis effects is unknown. Cyp27b1-knockout mice (Cyp27b1-/-mice) are congenitally deficient in 1α,25D3 and exhibit marked hypocalcemia and high parathyroid hormone levels, resulting in osteodystrophy involving bone hypocalcification and growth plate cartilage hypertrophy. However, because the vitamin D receptor is expressed normally in Cyp27b1-/-mice, they respond normally to 1α,25D3. Accordingly, in Cyp27b1-/-mice, the pharmacological effects of exogenously administered active vitamin D derivatives can be analyzed without being affected by 1α,25D3. We used Cyp27b1-/-mice to characterize and clarify the superior osteogenic effects of ELD on the bone in comparison with ALF. The results indicated that compared to ALF, ELD strongly induces ECaC2, calbindin-D9k, and CYP24A1 in the duodenum, promoting Ca absorption and decreasing the plasma concentration of 1α,25D3, resulting in improved osteogenesis. Because bone morphological measurements demonstrated that ELD has stronger effects on bone calcification, trabecular formation, and cancellous bone density than ALF, ELD appears to be a more effective therapeutic agent for treating postmenopausal osteoporosis, in which cancellous bone density decreases markedly. By using Cyp27b1-/-mice, this study was the first to succeed in clarifying the osteogenic effect of ELD without any influence of endogenous 1α,25D3. Furthermore, ELD more strongly enhanced bone mineralization, trabecular proliferation, and cancellous bone density than did ALF. Thus, ELD is expected to show an effect on postmenopausal osteoporosis, in which cancellous bone mineral density decreases markedly. In the future, this study may enable the development of next-generation active vitamin D derivatives with higher affinity for bone than ELD.

Comparison of the sympathetic stimulatory abilities of B-type procyanidins based on induction of uncoupling protein-1 in brown adipose tissue (BAT) and increased plasma catecholamine (CA) in mice.

OBJECTIVES: We previously found that elevated energy expenditure following a single oral dose of flavan 3-ols (FL), a mixture of catechins and B type procyanidins, is caused by sympathetic nerve activation. In the present study, we compared the activity of the FL components (-)-epicatechin (EC; monomer), procyanidin B2 (B2; dimer), procyanidin C1 (C1; trimer), cinnamtannin A2 (A2; tetramer), and more than pentamer fraction (P5). METHODS: Male ICR mice were treated with a single oral dose of FL, EC, B2, C1, A2, or P5. The animals were sacrificed and blood and brown adipose tissue (BAT) sampled. The plasma catecholamine (CA) levels and BAT uncoupling protein (UCP)-1 mRNA expression were determined. RESULTS: A single dose of 10 mg/kg FL significantly increased plasma CA and UCP-1 mRNA levels. B2, C1, and A2, but not EC and P5 (all at 1 mg/kg), significantly increased plasma adrenaline levels. Plasma noradrenaline was significantly elevated by B2 and A2, but not by EC, C1, or P5. UCP-1 mRNA levels were significantly increased by C1 and P5. In the dose response study of A2, 10-3 mg/kg A2 increased UCP-1 mRNA levels significantly, but not 10-2 and 10-1 mg/kg A2. In addition, combination treatment with 10-1 mg/kg A2 and yohimbine, an α2 adrenalin blocker, remarkably increased UCP-1 mRNA levels. CONCLUSION: These results suggest that FL and its components, except EC, increase UCP-1 mRNA and plasma CA with varying efficacy.

Synthesis of novel vitamin K derivatives with alkylated phenyl groups introduced at the ω-terminal side chain and evaluation of their neural differentiation activities.

Vitamin K is an essential cofactor of γ-glutamylcarboxylase as related to blood coagulation and bone formation. Menaquinone-4, one of the vitamin K homologues, is biosynthesized in the body and has various biological activities such as being a ligand for steroid and xenobiotic receptors, protection of neuronal cells from oxidative stress, and so on. From this background, we focused on the role of menaquinone in the differentiation activity of progenitor cells into neuronal cells and we synthesized novel vitamin K derivatives with modification of the ω-terminal side chain. We report here new vitamin K analogues, which introduced an alkylated phenyl group at the ω-terminal side chain. These compounds exhibited potent differentiation activity as compared to control.

Determination of Menadione by Liquid Chromatography-Tandem Mass Spectrometry Using Pseudo Multiple Reaction Monitoring.

This study aimed to develop a menadione (MD) determination method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a pseudo multiple reaction monitoring (MRM) technique, wherein two quadrupoles are used to monitor the same ion. Detection limits of 40 and 2 pg were obtained for MD and its deuterium-labeled form, respectively, whereas MD intra- and inter-assay coefficient of variation values were determined as 5.4 - 8.2%, with the corresponding recoveries equaling 90.5 - 109.6%. The developed method enables determination of MD in urine, plasma, cell extract, and culture media, demonstrating that pseudo multiple reaction monitoring can achieve quantification of compounds forming no suitable product ions, such as MD.

Synthesis of Novel Synthetic Vitamin K Analogues Prepared by Introduction of a Heteroatom and a Phenyl Group That Induce Highly Selective Neuronal Differentiation of Neuronal Progenitor Cells.

We synthesized novel vitamin K2 analogues that incorporated a heteroatom and an aromatic ring in the side chain and evaluated their effect on the selective differentiation of neuronal progenitor cells into neurons in vitro. The results showed that a menaquinone-2 analogue bearing a p-fluoroaniline had the most potent activity, which was more than twice as great as the control. In addition, the neuronal selectivity was more than 3 times greater than the control.

Substitution at the C-3 Position of Catechins Has an Influence on the Binding Affinities against Serum Albumin.

It is known that catechins interact with the tryptophan (Trp) residue at the drug-binding site of serum albumin. In this study, we used catechin derivatives to investigate which position of the catechin structure strongly influences the binding affinity against bovine serum albumin (BSA) and human serum albumin (HSA). A docking simulation showed that (-)-epigallocatechin gallate (EGCg) interacted with both Trp residues of BSA (one at drug-binding site I and the other on the molecular surface), mainly by π-π stacking. Fluorescence analysis showed that EGCg and substituted EGCg caused a red shift of the peak wavelength of Trp similarly to warfarin (a drug-binding site I-specific compound), while 3-O-acyl-catechins caused a blue shift. To evaluate the binding affinities, the quenching constants were determined by the Stern-Volmer equation. A gallate ester at the C-3 position increased the quenching constants of the catechins. Against BSA, acyl substitution increased the quenching constant proportionally to the carbon chain lengths of the acyl group, whereas methyl substitution decreased the quenching constant. Against HSA, neither acyl nor methyl substitution affected the quenching constant. In conclusion, substitution at the C-3 position of catechins has an important influence on the binding affinity against serum albumin.

Nongenomic effects of 1α,25-dihydroxyvitamin D3 on cartilage formation deduced from comparisons between Cyp27b1 and Vdr knockout mice.

The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25D3), plays an important role in the maintenance of calcium (Ca) homeostasis, bone formation, and cell proliferation and differentiation via nuclear vitamin D receptor (VDR). It is formed by the hydroxylation of vitamin D at the 1α position by 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1) in the kidney. However, Cyp27b1-/- mice, deficient in CYP27B1, and VDR-deficient mice (Vdr-/-) have not been extensively examined, particularly in a comparative framework. To clarify the physiological significance of 1α,25D3 and VDR, we produced Cyp27b1-/- mice and compared their phenotypes with those of Vdr-/- mice. Cyp27b1-/- mice exhibited hypocalcemia, growth defects, and skeletogenesis dysfunction, similar to Vdr-/- mice. However, unlike Cyp27b1-/- mice, Vdr-/- mice developed alopecia. Cyp27b1-/- mice exhibited cartilage mass formation and had difficulty walking on hindlimbs. Furthermore, a phenotypic analysis was performed on Cyp27b1-/- mice provided a high Ca diet to correct for the Ca metabolic abnormality. In addition, the effects of 1α,25D3 that are not mediated by Ca metabolic regulatory activity were investigated. Even when the blood Ca concentration was corrected, abnormalities in growth and cartilage tissue formation did not improve in Cyp27b1-/- mice. These results suggested that 1α,25D3 directly controls chondrocyte proliferation and differentiation. Using Cyp27b1-/- mice produced in this study, we can analyze the physiological effects of novel vitamin D derivatives in the absence of endogenous 1α,25D3. Accordingly, this study provides a useful animal model for the development of novel vitamin D formulations that are effective for the treatment and prevention of osteoporosis.

The impact of theaflavins on systemic-and microcirculation alterations: The murine and randomized feasibility trials.

Theaflavins are polyphenols found in black tea; their physiological activities were not well investigated. The present study in rats evaluated the influence of theaflavins on circulation. In addition, an intervention pilot study examined the influence of a theaflavin drink on postprandial hemodynamic change. In an animal study, a single oral dose of theaflavin rich fraction (TF, 10mg/kg) caused transient increase in mean blood pressure (MBP) and heart rate (HR). TF also elevated cremastric blood flow significantly, and the magnitude of this effect was in this order: theaflavin 3'-O-gallate (TF2B) >>theaflavin-3-O-gallate (TF2A) >>theaflavin (TF1)=theaflavin-3, 3'-di-O-gallate (TF3). In addition, these hemodynamic alterations in mammals totally disappeared when pretreated with carvedilol as an adrenaline blocker. We also treated 10-mg/kg/day TF to the rats for 2 weeks. At the end of the ingestion period, MBP was reduced significantly, and aortic eNOS level was elevated by the repeated ingestion of TF compared with distilled water. In the intervention trial, blood pressure of the volunteers was increased significantly 2 and 4h after ingestion of the TF drink (45mg/drink) compared with before treatment. A significant difference was observed in FMD between the placebo and theaflavin groups 4h after ingestion. These results suggested that theaflavin has potent activity to alter hemodynamics in both murine and healthy subjects. Further studies is needed to elucidate the details; however, the results of animal study suggested that the possible involvement of sympathetic nervous system in the hemodynamic changes caused by TF.