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Abstract Here we report the effects of low-intensity pulsed ultrasound (LIPUS) on symptoms in peripheral arterial disease patients with Buerger disease. A double-blinded and randomized study with active and inactive LIPUS was conducted. We assessed symptoms in leg circulation during a 24-week period of LIPUS irradiation in 12 patients with Buerger disease. Twelve patients without LIPUS irradiation served as controls. The pain intensity on visual analog score was significantly decreased after 24-week LIPUS treatment. Skin perfusion pressure was significantly increased in patients who received LIPUS treatment. There was no significant difference in symptoms and perfusion parameters in the control group. No severe adverse effects were observed in any of the patients who underwent LIPUS treatment. LIPUS is noninvasive, safe and effective option for improving symptoms in patients with Buerger disease.

Abstract Mesenchymal stem cells (MSCs) exert their anti-inflammatory and anti-fibrotic effects by secreting various humoral factors. Interferon-gamma (IFN-γ) can enhance these effects of MSCs, and enhancement of regulatory T (Treg) cell induction is thought to be an underlying mechanism. However, the extent to which Treg cell induction by MSCs pretreated with IFN-γ (IFN-γ MSCs) ameliorates renal fibrosis remains unknown. In this study, we investigated the effects of Treg cell induction by IFN-γ MSCs on renal inflammation and fibrosis using an siRNA knockdown system. Administration of IFN-γ MSCs induced Treg cells and inhibited infiltration of inflammatory cells in ischemia reperfusion injury (IRI) rats more drastically than control MSCs without IFN-γ pretreatment. In addition, administration of IFN-γ MSCs more significantly attenuated renal fibrosis compared with control MSCs. Indoleamine 2,3-dioxygenase (IDO) expression levels in conditioned medium from MSCs were enhanced by IFN-γ pretreatment. Moreover, IDO1 knockdown in IFN-γ MSCs reduced their anti-inflammatory and anti-fibrotic effects in IRI rats by reducing Treg cell induction. Our findings suggest that the increase of Treg cells induced by enhanced secretion of IDO by IFN-γ MSCs played a pivotal role in their anti-fibrotic effects. Administration of IFN-γ MSCs may potentially be a useful therapy to prevent renal fibrosis progression.

Abstract The purpose of this study was to evaluate the effects of administration of overnight 1 mg dexamethasone on vascular function in patients with nonfunctioning adrenal adenomas (NFA). Flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID) were measured to assess vascular function in 22 patients with NFA who had hypertension and/or diabetes mellitus (DM) and 272 patients without adrenal incidentalomas who had hypertension and/or DM (control patients with hypertension and/or DM). FMD and NID were measured in the morning before and after administration of 1 mg of dexamethasone at 2300 h in 18 patients with NFA. There were no significant differences in FMD and NID between control patients with hypertension and/or DM and patients with NFA who had hypertension and/or DM (3.4 ± 2.8% vs. 2.9 ± 1.9% and 11.5 ± 5.7% vs. 11.4 ± 4.3%, P = 0.46, and P = 0.99, respectively). There were no significant differences in vascular function between control patients with hypertension and/or DM and patients with NFA who had hypertension and/or DM even after adjustment for cardiovascular risk factors. Overnight 1 mg dexamethasone increased FMD from 2.4 ± 1.9% to 5.3 ± 3.2% (P

Abstract Background Contrast-induced nephropathy (CIN) is a major clinical problem associated with acute kidney injury during hospitalization. However, effective treatments for CIN are currently lacking. Mesenchymal stem cells (MSCs) have protective effects against kidney injury by suppressing inflammation and fibrosis. We previously showed that MSCs cultured in serum-free medium (SF-MSCs) enhance their anti-inflammatory and anti-fibrotic effects. However, whether SF-MSCs potentiate their anti-apoptotic effects is unknown. Here, we investigated the effects of SF-MSCs on a CIN mouse model. Methods To create CIN model mice, we removed right kidney at first. One week later, the left renal artery was clamped for 30 min to cause ischemia–reperfusion injury, and mice were injected with iohexol. Then the kidney received 10 Gy of irradiation, and MSCs or SF-MSCs were injected immediately. At 24 h post-injection, mice were sacrificed, and their blood and kidneys were collected to evaluate renal function, DNA damage, and apoptosis. In addition, apoptosis was induced in HEK-293 cells by irradiation and cells were treated with conditioned medium from MSCs or SF-MSCs. Results Treatment of CIN model mice with SF-MSCs markedly improved renal function compared with MSCs treatment. Cleaved caspase-3 levels and TUNEL-positive cell numbers were strongly suppressed in CIN model mice treated with SF-MSCs compared with the findings in those treated with MSCs. γH2AX levels, a chromosome damage marker, were reduced by MSCs and further reduced by SF-MSCs. In addition, cleaved caspase-3 in irradiated HEK-293 cells was more strongly suppressed by conditioned medium from SF-MSCs than by that from MSCs. Secretion of epidermal growth factor (EGF) was enhanced by culturing MSCs in serum-free medium. Knockdown of EGF by siRNA attenuated the inhibitory effects of SF-MSCs on CIN-induced renal dysfunction and tubular apoptosis. Conclusions These findings strongly suggest that SF-MSCs improve CIN in model mice by exerting anti-apoptotic effects in a paracrine manner. Thus, SF-MSCs represent a potential novel therapy for CIN. Graphical Abstract

Abstract Background Fibrosis is a common histological feature in the process from chronic organ injury to organ failure. Chronic tissue injury causes inflammatory cell infiltration into the injured tissue. The persistence of this inflammatory cell infiltration leads to fibrosis and organ failure. Adipose-derived mesenchymal stem cells (ASCs) have received much attention as a regenerative therapeutic tool to prevent progression from organ injury to failure. Subcutaneous abdominal adipose tissue is divided into superficial and deep layers by a superficial fascia. Adipose tissue easily collected by liposuction is usually obtained from a deep layer, so ASCs derived from a deep layer are generally used for regenerative medicine. However, no research has been conducted to investigate differences in the therapeutic effects of ASCs from the superficial and deep layers (Sup-ASCs and Deep-ASCs, respectively). Therefore, we compared the therapeutic potencies of Sup-ASCs and Deep-ASCs. Methods ASCs were isolated from superficial and deep subcutaneous abdominal adipose tissues collected from patients who underwent breast reconstruction. We first compared cell characteristics, such as morphology, cell proliferation, cell surface markers, adipogenic and osteogenic differentiation, cell senescence markers, and expression of coagulation and anticoagulant factors between Sup-ASCs and Deep-ASCs. Furthermore, we compared their ability to promote polarization of M2 macrophages and to inhibit transforming growth factor (TGF)-β/Smad signaling using THP-1 cells and TGF-β1 stimulated HK-2 cells incubated with conditioned media from Sup-ASCs or Deep-ASCs. In in vivo experiments, after renal ischemia–reperfusion injury (IRI) procedure, Sup-ASCs or Deep-ASCs were injected through the abdominal aorta. At 21 days post-injection, the rats were sacrificed and their left kidneys were collected to evaluate fibrosis. Finally, we performed RNA-sequencing analysis of Sup-ASCs and Deep-ASCs. Results Sup-ASCs had greater proliferation and adipogenic differentiation compared with Deep-ASCs, whereas both ASC types had similar morphology, cell surface markers, senescence markers, and expression of coagulation and anticoagulant factors. Conditioned media from Sup-ASCs and Deep-ASCs equally promoted polarization of M2 macrophages and suppressed TGF-β/Smad signaling. Moreover, administration of Sup-ASCs and Deep-ASCs equally ameliorated renal fibrosis induced by IRI in rats. RNA-sequencing analysis revealed no significant difference in the expression of genes involved in anti-inflammatory and anti-fibrotic effects between Sup-ASCs and Deep-ASCs. Conclusions These results indicate that both Sup-ASCs and Deep-ASCs can be used effectively and safely as an intravascular ASC therapy for organ injury.

The effects of Covid-19 vaccines on vascular function are still controversial. We evaluated the effects of BNT162b2 vaccine (BioNTech and Pfizer) on endothelial function assessed by flow-mediated vasodilation (FMD) and vascular smooth muscle function assessed by nitroglycerine-induced vasodilation (NID). This study was a prospective observational study. A total of 23 medical staff at Hiroshima University Hospital were enrolled in this study. FMD and NID were measured before vaccination and two weeks and six months after the 2nd dose of vaccination. FMD was significantly smaller two weeks after the 2nd dose of vaccination than before vaccination (6.5±2.4% and 8.2±2.6%, p = 0.03). FMD was significantly larger at six months than at two weeks after the 2nd dose of vaccination (8.2±3.0% and 6.5±2.4%, p = 0.03). There was no significant difference between FMD before vaccination and that at six months after the 2nd dose of vaccination (8.2±2.6% to 8.2±3.0%, p = 0.96). NID values were similar before vaccination and at two weeks, and six months after vaccination (p = 0.89). The BNT162b2 Covid-19 vaccine temporally impaired endothelial function but not vascular smooth muscle function, and the impaired endothelial function returned to the baseline level within six months after vaccination.

Mesenchymal stem cells (MSCs) have attracted a great deal of interest as a therapeutic tool for renal fibrosis. Although both adipose-derived and bone marrow-derived MSCs (ADSCs and BMSCs, respectively) suppress renal fibrosis, which of these two has a stronger therapeutic effect remains unclear. This study aimed to compare the antifibrotic effects of ADSCs and BMSCs extracted from adipose tissue and bone marrow derived from the same rats. When cultured in serum-containing medium, ADSCs had a more potent inhibitory effect than BMSCs on renal fibrosis induced by ischemia-reperfusion injury in rats. ADSCs and BMSCs cultured in serum-free medium were equally effective in suppressing renal fibrosis. Mice infused with ADSCs (serum-containing or serum-free cultivation) had a higher death rate from pulmonary embolism than those infused with BMSCs. In vitro, mRNA levels of tissue factor, tumor necrosis factor-α-induced protein 6 and prostaglandin E synthase were higher in ADSCs than in BMSCs, while that of vascular endothelial growth factor was higher in BMSCs than in ADSCs. Although ADSCs had a stronger antifibrotic effect, these findings support the consideration of thromboembolism risk in clinical applications. Our results emphasize the importance of deciding between ADSCs and BMSCs based upon the target disease and culture method.

Abstract It is well known that white blood cell (WBC) count is an independent predictor of cardiovascular events. However, associations of WBC count and WBC subtypes with endothelial function assessed by flow-mediated vasodilation (FMD) and vascular smooth muscle function assessed by nitroglycerine-induced vasodilation (NID) are unclear. The aim of this study was to determine the relationships of WBC count and WBC subtypes with vascular function assessed by FMD and NID. A total of 1351 subjects in whom FMD and NID had been measured were recruited from Hiroshima University Vascular Registry. Mean values were 3.7 ± 2.8% for FMD and 11.8 ± 5.9% for NID. WBC was not correlated with FMD or NID. NID was significantly correlated with lymphocytes in univariate analysis but not with other hematologic parameters. In multiple linear regression analyses, NID was not correlated with lymphocytes. In all subgroups including subgroups of age, gender, body mass index, hypertension, dyslipidemia, diabetes mellitus, smoking and tertile of WBC count, WBC count was not correlated with FMD or NID. WBC count and WBC subtypes were not associated with endothelial function assessed by FMD or vascular smooth muscle function assessed by NID. WBC count and vascular function assessed by FMD and NID may reflect different aspects of atherosclerosis. Clinical Trial Registration Information: URL for Clinical Trial: http://www.umin.ac.jp Registration Number for Clinical Trial: UMIN000039512.

BackgroundThe purpose of this study was to evaluate the effects of exposure to radiation caused by an atomic bomb in atomic bomb survivors on vascular function and vascular structure and to evaluate the relationships of radiation dose from the atomic bomb with vascular function and vascular structure in atomic bomb survivors.MethodsFlow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID) as indices of vascular function, brachial-ankle pulse wave velocity (baPWV) as an index of vascular function and vascular structure, and brachial artery intima-media thickness (IMT) as an index of vascular structure were measured in 131 atomic bomb survivors and 1,153 control subjects who were not exposed to the atomic bomb. Ten of the 131 atomic bomb survivors with estimated radiation dose in a cohort study of Atomic Bomb Survivors in Hiroshima were enrolled in the study to evaluate the relationships of radiation dose from the atomic bomb with vascular function and vascular structure.ResultsThere was no significant difference in FMD, NID, baPWV, or brachial artery IMT between control subjects and atomic bomb survivors. After adjustment of confounding factors, there was still no significant difference in FMD, NID, baPWV, or brachial artery IMT between control subjects and atomic bomb survivors. Radiation dose from the atomic bomb was negatively correlated with FMD (ρ = −0.73, P = 0.02), whereas radiation dose was not correlated with NID, baPWV or brachial artery IMT.ConclusionThere were no significant differences in vascular function and vascular structure between control subjects and atomic bomb survivors. Radiation dose from the atomic bomb might be negatively correlated with endothelial function.

Abstract We divided the 466 subjects into two groups based on information on sitting time on a non-working day and evaluated flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID). FMD was smaller in subjects with sitting time on a non-working day of ≥6 h/day than in subjects with sitting time on a non-working day of

ABSTRACT Aims/Introduction Diabetes mellitus is associated with endothelial dysfunction. However, it is still controversial as to whether antidiabetic drug treatment affects endothelial function. The purpose of this study was to evaluate the relationships of the hemoglobin A1c (HbA1c) level with flow‐mediated vasodilation (FMD) and nitroglycerine‐induced vasodilation (NID) in patients with type 2 diabetes mellitus who are receiving antidiabetic drugs. Materials and Methods The FMD was measured in 866 patients with type 2 diabetes mellitus who were receiving antidiabetic drugs (625 men and 241 women; mean age: 62 ± 10 years). The patients were divided into four groups according to HbA1c levels:

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.

Abstract Background Mesenchymal stromal cells (MSCs) are a potential therapeutic tool for pulmonary fibrosis. However, ex vivo MSC expansion using serum poses risks of harmful immune responses or unknown pathogen infections in the recipients. Therefore, MSCs cultured in serum-free media (SF-MSCs) are ideal for clinical settings; however, their efficacy in pulmonary fibrosis is unknown. Here, we investigated the effects of SF-MSCs on bleomycin-induced pulmonary inflammation and fibrosis compared to those of MSCs cultured in serum-containing media (S-MSCs). Methods SF-MSCs and S-MSCs were characterized in vitro using RNA sequence analysis. The in vivo kinetics and efficacy of SF-MSC therapy were investigated using a murine model of bleomycin-induced pulmonary fibrosis. For normally distributed data, Student’s t test and one-way repeated measures analysis of variance followed by post hoc Tukey’s test were used for comparison between two groups and multiple groups, respectively. For non-normally distributed data, Kruskal–Wallis and Mann–Whitney U tests were used for comparison between groups, using e Bonferroni’s correction for multiple comparisons. All tests were two-sided, and P

Abstract A body shape index (ABSI) was proposed for estimation of abdominal adiposity. ABSI has been reported to have associations with cardiovascular risk factors and cardiovascular events. However, there is no information on the association between ABSI and endothelial function. We examined cross-sectional associations of ABSI with endothelial function in 8823 subjects (6773 men and 2050 women). Subjects with a lower quartile of flow-mediated vasodilation (FMD) were defined as subjects having endothelial dysfunction. Pearson’s correlation coefficient analysis revealed that ABSI was negatively correlated with FMD (men, r = − 0.23, P = 0.003; women, r = − 0.32, P

Abstract Background Mesenchymal stem cells (MSCs) repair injured tissue in a paracrine manner. To enhance their therapeutic properties, preconditioning with various factors has been researched. We have previously showed that MSCs cultured in serum-free medium (SF-MSCs) promote their immunosuppressive ability, thereby enhancing their anti-fibrotic effect. Here, we examined whether serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of MSCs on renal fibrosis in rats with ischemia–reperfusion injury (IRI). Methods SF-MSCs were incubated under 1% O2 conditions (hypo-SF-MSCs) or 21% O2 conditions (normo-SF-MSCs) for 24 h before collection. After IRI procedure, hypo-SF-MSCs or normo-SF-MSCs were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were killed and their kidneys were collected to evaluate inflammation and fibrosis. In in vitro experiments, we investigated whether hypo-SF-MSCs enhanced secretion of anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from hypo-SF-MSCs or normo-SF-MSCs. Results Normo-SF-MSCs showed attenuation of senescence, which increased their proliferative capacity. Although no significant difference in cellular senescence was found between normo-SF-MSCs and hypo-SF-MSCs, hypo-SF-MSCs further increased their proliferative capacity compared with normo-SF-MSCs. Additionally, administration of hypo-SF-MSCs more strongly ameliorated renal fibrosis than that of normo-SF-MSCs. Moreover, although hypo-SF-MSCs strongly attenuated infiltration of inflammatory cells compared with the control rats, which were treated with PBS, this attenuation was almost equal between normo-SF-MSCs and hypo-SF-MSCs. In vitro experiments revealed that hypo-SF-MSCs more significantly inhibited transforming growth factor (TGF)-β/Smad signaling compared with normo-SF-MSCs. Moreover, hypoxic preconditioning increased hepatocyte growth factor (HGF) secretion even under serum-free conditions, whereas knockdown of HGF in hypo-SF-MSCs attenuated inhibition of TGF-β/Smad signaling. Conclusions These results indicate that administration of ex vivo-expanded, hypoxia-preconditioned SF-MSCs may be a useful cell therapy to prevent renal fibrosis.

Abstract Background Mesenchymal stem cells (MSCs) provide potential treatments for peritoneal fibrosis. However, MSCs cultured in media containing serum bring risks of infection and other problems. In this study, we compared the effect of human MSCs in serum-free medium (SF-MSCs) on peritoneal fibrosis with that of MSCs cultured in medium containing 10% fetal bovine serum (10%MSCs). Methods Peritoneal fibrosis was induced by intraperitoneally injecting 0.1% chlorhexidine gluconate (CG). SF-MSCs or 10%MSCs were intraperitoneally administered 30 min after the CG injection. Ten days after the CG and MSC injections, we performed histological analyses and peritoneal equilibrium testing. In the in vitro experiments, we used transforming growth factor (TGF)-β1-stimulated human peritoneal mesothelial cells incubated in conditioned medium from MSCs to examine whether the SF-MSCs showed enhanced ability to produce antifibrotic humoral factors. Results Histological staining showed that the SF-MSCs significantly suppressed CG-induced cell accumulation and thickening compared with that of the 10%MSCs. Additionally, the SF-MSCs significantly inhibited mesenchymal cell expression, extracellular matrix protein deposition and inflammatory cell infiltration. Peritoneal equilibration testing showed that compared with administering 10%MSCs, administering SF-MSCs significantly reduced the functional impairments of the peritoneal membrane. The in vitro experiments showed that although the conditioned medium from MSCs suppressed TGF-β1 signaling, the suppression did not significantly differ between the SF-MSCs and 10%MSCs. Conclusions Serum-free culture conditions can enhance the antifibrotic abilities of MSCs by suppressing inflammation. Administering ex vivo expanded SF-MSCs may be a potential therapy for preventing peritoneal fibrotic progression.

Heart failure (HF) is associated with endothelial dysfunction. Vascular function per se plays an important role in cardiac function, whether it is a cause or consequence. However, there is no information on vascular function in patients with wild-type transthyretin amyloid cardiomyopathy (ATTRwt-CM). The purpose of this study was to evaluate vascular function in patients with ATTRwt-CM. We measured flow-mediated vasodilation (FMD) as an index of endothelial function and nitroglycerine-induced vasodilation (NID) as an index of vascular smooth muscle function and brachial artery intima-media thickness (bIMT) and brachial-ankle pulse wave velocity (baPWV) as indices of arterial stiffness in 22 patients with ATTRwt-CM and in 22 one-by-one matched control patients using vascular function confounding factors. FMD was significantly greater in patients with ATTRwt-CM than in the controls (5.4 ± 3.4% versus 3.5 ± 2.4%, p = 0.038) and the N-terminal pro-brain natriuretic peptide (NT-proBNP) level was significantly greater in patients with ATTRwt-CM than in the controls (2202 ± 1478 versus 470 ± 677 pg/mL, p < 0.001). There were no significant differences in NID, bIMT or baPWV between the two groups. There was a significant relationship between NT-proBNP and FMD in patients with ATTRwt-CM (r = 0.485, p = 0.022). NT-proBNP showed no significant relationships with NID, bIMT or baPWV. Conclusions: Endothelial function was preserved in patients with ATTRwt-CM. Patients with ATTRwt-CM may have compensatory effects with respect to endothelial function through elevation of BNP.

Abstract Mesenchymal stem cells (MSCs) administered for therapeutic purposes can be activated by interferon-γ (IFN-γ) secreted from natural killer cells in injured tissues and exert anti-inflammatory effects. These processes require a substantial period of time, leading to a delayed onset of MSCs’ therapeutic effects. In this study, we investigated whether pretreatment with IFN-γ could potentiate the anti-fibrotic ability of MSCs in rats with ischemia–reperfusion injury (IRI) and unilateral ureter obstruction. Administration of MSCs treated with IFN-γ strongly reduced infiltration of inflammatory cells and ameliorated interstitial fibrosis compared with control MSCs without IFN-γ treatment. In addition, conditioned medium obtained from IFN-γ-treated MSCs decreased fibrotic changes in cultured cells induced by transforming growth factor-β1 more efficiently than that from control MSCs. Most notably, secretion of prostaglandin E2 from MSCs was significantly increased by treatment with IFN-γ. Increased prostaglandin E2 in conditioned medium obtained from IFN-γ-treated MSCs induced polarization of immunosuppressive CD163 and CD206-positive macrophages. In addition, knockdown of prostaglandin E synthase weakened the anti-fibrotic effects of MSCs treated with IFN-γ in IRI rats, suggesting the involvement of prostaglandin E2 in the beneficial effects of IFN-γ. Administration of MSCs treated with IFN-γ might represent a promising therapy to prevent the progression of renal fibrosis.

Abstract It is established that smoking is a major risk factor of atherosclerosis. Endothelial dysfunction occurs in the initial step in the pathogenesis of atherosclerosis and plays a critical role in the development of atherosclerosis. The purpose of this study was to evaluate the association between smoking status and endothelial function in detail in men. We measured flow-mediated vasodilation (FMD) in 2209 Japanese men including 1181 men who had never smoked and 1028 current smokers. All of the participants were divided into five groups by smoking pack-years: never smoker group (= 0), light smoker group (> 0 to 10), moderate smoker group (> 10 to 20), heavy smoker group (> 20 to 30) and excessive smoker group (> 30). FMD significantly decreased in relation to pack-years (6.6 ± 3.4% in the never smoker group, 6.8 ± 3.0% in the light smoker group, 6.5 ± 2.9% in the moderate smoker group, 5.9 ± 2.9% in the heavy smoker group, and 4.9 ± 2.7% in the excessive smoker group; P

Abstract Background Mesenchymal stem cells (MSCs) have been reported to promote the regeneration of injured tissue via their paracrine abilities, which are enhanced by hypoxic preconditioning. In this study, we examined the therapeutic efficacy of hypoxia-preconditioned MSCs on renal fibrosis and inflammation in rats with ischemia-reperfusion injury (IRI). Methods MSCs derived from rats and humans were incubated in 1% O2 conditions (1%O2 MSCs) for 24 h. After IRI, 1%O2 MSCs or MSCs cultured under normoxic conditions (21%O2 MSCs) were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were sacrificed and their kidneys were analyzed. In in vitro experiments, we examined whether 1%O2 MSCs enhanced the ability to produce anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from MSCs. Results Administration of rat 1%O2 MSCs (1%O2 rMSCs) attenuated renal fibrosis and inflammation more significantly than rat 21%O2 MSCs. Notably, human 1%O2 MSCs (1%O2 hMSCs) also attenuated renal fibrosis to the same extent as 1%O2 rMSCs. Flow cytometry showed that 1%O2 hMSCs did not change human leukocyte antigen expression. Further in vitro experiments revealed that conditioned medium from 1%O2 MSCs further suppressed TGF-β1-induced fibrotic changes in HK-2 cells compared with 21%O2 MSCs. Hypoxic preconditioning enhanced vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) secretion. Interestingly, VEGF knockdown in 1%O2 MSCs attenuated HGF secretion and the inhibition of TGF-β1-induced fibrotic changes in HK-2 cells. In addition, VEGF knockdown in 1%O2 hMSCs reduced the anti-fibrotic effect in IRI rats. Conclusions Our results indicate that hypoxia-preconditioned MSCs are useful as an allogeneic transplantation cell therapy to prevent renal fibrosis and inflammation.

Hypoxia preconditioning enhances the paracrine abilities of mesenchymal stem cells (MSCs) for vascular regeneration and tissue healing. Implantation of hypoxia-induced mesenchymal stem cells (hi-MSCs) may further improve limb perfusion in a murine model of hindlimb ischemia. This study is aimed at determining whether implantation of hi-MSCs is an effective modality for improving outcomes of treatment of ischemic artery diseases. We evaluated the effects of human bone marrow-derived MSC implantation on limb blood flow in an ischemic hindlimb model. hi-MSCs were prepared by cell culture under 1% oxygen for 24 hours prior to implantation. A total of 1×105 MSCs and hi-MSCs and phosphate-buffered saline (PBS) were intramuscularly implanted into ischemic muscles at 36 hours after surgery. Restoration of blood flow and muscle perfusion was evaluated by laser Doppler perfusion imaging. Blood perfusion recovery, enhanced vessel densities, and improvement of function of the ischemia limb were significantly greater in the hi-MSC group than in the MSC or PBS group. Immunochemistry revealed that hi-MSCs had higher expression levels of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor A than those in MSCs. In addition, an endothelial cell-inducing medium showed high expression levels of vascular endothelial growth factor, platelet endothelial cell adhesion molecule-1, and von Willebrand factor in hi-MSCs compared to those in MSCs. These findings suggest that pretreatment of MSCs with a hypoxia condition and implantation of hi-MSCs advances neovascularization capability with enhanced therapeutic angiogenic effects in a murine hindlimb ischemia model.

Objective The aim of this study was to determine the relationship of haemoglobin A1c (HbA1c) level with flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID) in patients with type 2 diabetes.Design Cross-sectional study.Setting 22 university hospitals and affiliated clinics in Japan.Participants 1215 patients with type 2 diabetes including 349 patients not taking antidiabetic drugs.Measures We evaluated FMD and HbA1c level. All patients were divided into four groups based on HbA1c level:

In medical and occupational settings, ionizing irradiation of the circulatory system occurs at various dose rates. We previously found sparing and enhancing dose protraction effects for aortic changes in wild-type mice at 6 months after starting irradiation with 5 Gy of photons. Here, we further analyzed changes at 12 months after stating irradiation. Irrespective of irradiation regimens, irradiation little affected left ventricular function, heart weight, and kidney weight. Irradiation caused structural disorganizations and intima-media thickening in the aorta, along with concurrent elevations of markers for proinflammation, macrophage, profibrosis, and fibrosis, and reductions in markers for vascular functionality and cell adhesion in the aortic endothelium. These changes were qualitatively similar but quantitatively less at 12 months than at 6 months. The magnitude of such changes at 12 months was not smaller in 25 fractions (Frs) but was smaller in 100 Frs and chronic exposure than acute exposure. The magnitude at 6 and 12 months was greater in 25 Frs, smaller in 100 Frs, and much smaller in chronic exposure than acute exposure. These findings suggest that dose protraction changes aortic damage, in a fashion that depends on post-irradiation time and is not a simple function of dose rate.

The ischemia–reperfusion injury (IRI) of rat kidneys is used as a model of acute kidney injury. Salt-sensitive hypertension occurs in rats after IRI, and the distal nephrons play important roles in the development of this condition. We investigated the role of the mineralocorticoid receptor (MR) in the progression of IRI-induced salt-sensitive hypertension in rats. Fourteen days after right-side nephrectomy, IRI was induced by clamping the left renal artery, with sham surgery performed as a control. IRI rats were provided with normal water or water with 1.0% NaCl (IRI/NaCl), or they were implanted with an osmotic mini-pump to infuse vehicle or aldosterone (IRI/Aldo). Esaxerenone, a non-steroidal MR blocker (MRB), was administered to IRI/NaCl and IRI/Aldo rats for 6 weeks. MR expression increased by day 7 post-IRI. Blood pressure and urinary protein excretion increased in IRI/NaCl and IRI/Aldo rats over the 6-week period, but these effects were negated by MRB administration. The MRB attenuated the expression of the gamma-epithelial sodium channel (ENaC) and renal damage. The ENaC inhibitor, amiloride, ameliorated hypertension and renal damage in IRI/NaCl and IRI/Aldo rats. Our findings thus showed that MR upregulation may play a pivotal role in ENaC-mediated sodium uptake in rats after IRI, resulting in the development of salt-sensitive hypertension in response to salt overload or the activation of the renin–angiotensin–aldosterone system.

We evaluated the relationship of daily coffee intake with endothelial function assessed by flow-mediated vasodilation and vascular smooth muscle function assessed by nitroglycerine-induced vasodilation in patients with hypertension. A total of 462 patients with hypertension were enrolled in this cross-sectional study. First, we divided the subjects into two groups based on information on daily coffee intake: no coffee group and coffee group. The median coffee intake was two cups per day in the coffee group. There were significant differences in both flow-mediated vasodilation (2.6 ± 2.8% in the no coffee group vs. 3.3 ± 2.9% in the coffee group, p = 0.04) and nitroglycerine-induced vasodilation (9.6 ± 5.5% in the no coffee group vs. 11.3 ± 5.4% in the coffee group, p = 0.02) between the two groups. After adjustment for confounding factors, the odds ratio for endothelial dysfunction (OR: 0.55, 95% CI: 0.32–0.95) and the odds ratio for vascular smooth muscle dysfunction (OR: 0.50, 95% CI: 0.28–0.89) were significantly lower in the coffee group than in the no coffee group. Next, we assessed the relationship of the amount of daily coffee intake with vascular function. Cubic spline curves revealed that patients with hypertension who drank half a cup to 2.5 cups of coffee per day had lower odds ratios for endothelial dysfunction assessed by flow-mediated vasodilation and vascular smooth muscle dysfunction assessed by nitroglycerine-induced vasodilation. Appropriate daily coffee intake might have beneficial effects on endothelial function and vascular smooth muscle function in patients with hypertension.

Hypoxia is a common pathway to the progression of end-stage kidney disease. Retinoic acid-inducible gene I (RIG-I) encodes an RNA helicase that recognizes viruses including SARS-CoV2, which is responsible for the production of interferon (IFN)-α/β to prevent the spread of viral infection. Recently, RIG-I activation was found under hypoxic conditions, and klotho deficiency was shown to intensify the activation of RIG-I in mouse brains. However, the roles of these functions in renal inflammation remain elusive. Here, for in vitro study, the expression of RIG-I and IFN-α/β was examined in normal rat kidney (NRK)-52E cells incubated under hypoxic conditions (1% O2). Next, siRNA targeting RIG-I or scramble siRNA was transfected into NRK52E cells to examine the expression of RIG-I and IFN-α/β under hypoxic conditions. We also investigated the expression levels of RIG-I and IFN-α/β in 33 human kidney biopsy samples diagnosed with IgA nephropathy. For in vivo study, we induced renal hypoxia by clamping the renal artery for 10 min in wild-type mice (WT mice) and Klotho-knockout mice (Kl−/− mice). Incubation under hypoxic conditions increased the expression of RIG-I and IFN-α/β in NRK52E cells. Their upregulation was inhibited in NRK52E cells transfected with siRNA targeting RIG-I. In patients with IgA nephropathy, immunohistochemical staining of renal biopsy samples revealed that the expression of RIG-I was correlated with that of IFN-α/β (r = 0.57, P

Various therapeutic strategies for angiogenesis are performed to improve symptoms in patients with critical limb ischemia (CLI). Pre-clinical studies have shown that low-intensity pulsed ultrasound (LIPUS) exposure induces angiogenesis. LIPUS may be a new stratergy for treatment of CLI. The purpose of this pilot trial was to evaluate outcomes in patients with CLI who were treated with LIPUS. Fourteen patients with CLI, who were not candidates for angioplasty or surgical revascularization, were enrolled in this study. Historical control data were obtained from the Hiroshima University PAD database. The primary endpoints were major amputation and death. The outcomes were compared in 16 lower limbs of the 14 patients with CLI who were treated with LIPUS and in 14 lower limbs of 14 patients with CLI as historical controls. All patients were followed for after 5 years after treatment with LIPUS. The mean duration of LIPUS exposure in the LIPUS group was 381± 283 days. During the 5-year follow-up periods, there were 3 major amputations and 7 deaths in the LIPUS group and there were 14 major amputations and 7 deaths in the historical control group. The overall amputation-free survival rate was significantly higher in patients who were treated with LIPUS than in historical controls. There was no significant difference between overall mortality-free survival rates in the LIPUS group and historical control group. LIPUS is a noninvasive option for therapeutic angiogenesis with the potential to reduce the incidence of major amputations in patients with CLI.

Background Upstroke time is the transit time from the nadir to peak of the waveform of pulse volume recording. The purpose of this study was to determine whether upstroke time at the ankle is a useful vascular marker for detecting patients with advanced atherosclerosis in combination with ankle‐brachial index (ABI). Methods and Results We measured upstroke time and ABI in 2313 subjects (mean age, 61.2±15.3 years). The prevalence of coronary artery disease (CAD) was significantly higher in patients with prolonged upstroke time (≥180 ms) than in subjects with normal upstroke time (

Objectives The purpose of this study was to evaluate the relationship between high-density lipoprotein cholesterol (HDL-C) levels and endothelial function in women.Design Cross-sectional study.Setting 22 university hospitals and affiliated clinics in Japan.Participants 1719 Japanese women aged 17–90 years who were not receiving lipid-lowering therapy.Measures We evaluated flow-mediated vasodilation (FMD) and serum levels of HDL-C. All participants were divided into four groups by HDL-C level: low HDL-C (

Light to moderate alcohol consumption has protective effects on all-cause death and coronary artery disease in women. It is thought that light to moderate alcohol consumption has a beneficial effect on vascular function in women. We measured flow-mediated vasodilation (FMD) in 702 women aged 17-86 years who provided information on alcohol consumption. We divided the subjects into four groups: non-drinkers (0 g/week), light drinkers (>0 to 140 g/week), moderate drinkers (>140 to 280 g/week) and heavy drinkers (>280 g/week). There was no significant difference in FMD among the four groups. Multivariate regression analysis revealed that alcohol consumption in non-drinkers and light drinkers was not an independent predictor of FMD (β = -0.001, P = 0.98). We compared 50 moderate drinkers and 50 non-drinkers matched for age and medical histories and 22 heavy drinkers and 22 non-drinkers in matched pair analysis. There was no significant difference in FMD between moderate drinkers and non-drinkers (8.2±4.3% vs. 8.1±3.5, P = 0.91), while FMD in heavy drinkers was significantly lower than that in non-drinkers (5.9±2.5% vs. 8.9±3.5%, P = 0.002). These findings suggest that heavy alcohol consumption is associated with endothelial dysfunction but that light to moderate alcohol consumption is not associated with endothelial dysfunction in women. Clinical trial registration information This study was approved by principal authorities and ethical issues in Japan (University Hospital Medical Information Network UMIN000012952, 01/12/2009). www.umin.ac.jp/.

Background Diagnostic criteria of flow‐mediated vasodilation (FMD), an index of endothelial function, and nitroglycerin‐induced vasodilation (NID), an index of vascular smooth muscle function, of the brachial artery have not been established. The purpose of this study was to propose diagnostic criteria of FMD and NID for normal endothelial function and normal vascular smooth muscle function. Methods and Results We investigated the cutoff values of FMD and NID in subjects with (risk group) and those without cardiovascular risk factors or cardiovascular diseases (no‐risk group) in 7277 Japanese subjects (mean age 51.4±10.8 years) from the Flow‐Mediated Dilation Japan study and the Flow‐Mediated Dilatation Japan Registry study for analysis of the cutoff value of FMD and in 1764 Japanese subjects (62.2±16.1 years) from the registry of Hiroshima University Hospital for analysis of the cutoff value of NID. Receiver‐operator characteristic curve analysis of FMD to discriminate subjects in the no‐risk group from patients in the risk group showed that the optimal cutoff value of FMD to diagnose subjects in the no‐risk group was 7.1%. Receiver‐operator characteristic curve analysis of NID to discriminate subjects in the no‐risk group from patients in the risk group showed that the optimal cutoff value of NID to diagnose subjects in the no‐risk group was 15.6%. Conclusions We propose that the cutoff value for normal endothelial function assessed by FMD of the brachial artery is 7.1% and that the cutoff value for normal vascular smooth muscle function assessed by NID of the brachial artery is 15.6% in Japanese subjects. Clinical Trial Registration www.umin.ac.jp Unique identifiers: UMIN000012950, UMIN000012951, UMIN000012952, and UMIN000003409

The purpose of this study was to evaluate whether heart failure with mildly reduced ejection fraction (HFmrEF) is associated with vascular dysfunction and whether vascular function predicts future deterioration of LVEF in patients with HFmrEF. We evaluated endothelial function assessed by flow-mediated vasodilation (FMD) and vascular smooth muscle function assessed by nitroglycerine-induced vasodilation (NID) in 69 patients with HFmrEF and 426 patients without HF and evaluated the future deterioration of LVEF, defined as a decrease in LVEF to p < 0.01). Multivariate Cox proportional hazard analysis revealed that NID of

During medical (therapeutic or diagnostic) procedures or in other settings, the circulatory system receives ionizing radiation at various dose rates. Here, we analyzed prelesional changes in the circulatory system of wild-type mice at six months after starting acute, intermittent, or continuous irradiation with 5 Gy of photons. Independent of irradiation regimens, irradiation had little impact on left ventricular function, heart weight, and kidney weight. In the aorta, a single acute exposure delivered in 10 minutes led to structural disorganizations and detachment of the aortic endothelium, and intima-media thickening. These morphological changes were accompanied by increases in markers for profibrosis (TGF-β1), fibrosis (collagen fibers), proinflammation (TNF-α), and macrophages (F4/80 and CD68), with concurrent decreases in markers for cell adhesion (CD31 and VE-cadherin) and vascular functionality (eNOS) in the aortic endothelium. Compared with acute exposure, the magnitude of such aortic changes was overall greater when the same dose was delivered in 25 fractions spread over 6 weeks, smaller in 100 fractions over 5 months, and much smaller in chronic exposure over 5 months. These findings suggest that dose protraction alters vascular damage in the aorta, but in a way that is not a simple function of dose rate.

Background: Although malnutrition and bone fracture are both major complications in patients undergoing hemodialysis, their association has not been clarified. The aim of our study was to clarify the association between the geriatric nutritional risk index (GNRI), an indicator of nutritional status, and the incidence of bone fractures in patients undergoing hemodialysis. Methods: We included 1342 registered patients undergoing hemodialysis and performed a post hoc analysis. We divided patients into the high GNRI group (≥92), considered to have a low risk of malnutrition, and the low GNRI group (GNRI groups was evaluated by the Kaplan–Meier method. Cox proportional hazards models were used to identify the risk factors for fractures requiring hospitalization. All results were stratified by sex. Results: New bone fractures developed in 108 (8.0%) patients in 5 years of follow-up. Bone fractures occurred more frequently in the low GNRI group compared with the high GNRI group (HR: 3.51, 95% CI: 1.91–6.42, p < 0.01 in males; HR: 2.47, 95% CI: 1.52–4.03, p < 0.01 in females). A low GNRI was significantly associated with an increased incidence of bone fractures, even after adjustment for covariates. However, the serum levels of calcium, phosphate, parathyroid hormone, and alkaline phosphatase were not associated with the incidence of bone fractures. Conclusions: A low GNRI is an independent risk factor for bone fractures in patients undergoing hemodialysis. Early intervention for the low GNRI group may be important in preventing the occurrence of fractures.

Abstract We investigated (1) the relationship between low-density lipoprotein cholesterol (LDL-C) and vascular function in patients receiving and those not receiving statin therapy and (2) optimal level of LDL-C for maintenance of vascular function. Flow-mediated vasodilation (FMD) and nitroglycerine-induced vasodilation (NID) were inversely correlated with LDL-C in the 957 statin naïve subjects but not in the 392 subjects receiving statin therapy. In statin naïve subjects, non-high LDL-C (≤100 mg/dL) was independently associated with a decrease in adjusted odds ratio of the low tertile of FMD (OR: 0.62, 95% CI: 0.45–0.85; P = 0.003) and NID (OR: 0.69, 95% CI: 0.50–0.96; P = 0.03). Adjusted odds ratio of the low tertile of FMD was significantly lower in the low LDL-C group (≤70 mg/dL) (OR: 0.47, 95% CI, 0.27–0.81; P = 0.006) and in the moderate LDL-C group (70.1–100 mg/dL) (OR: 0.66, 95% CI, 0.48–0.94; P = 0.02) than in the high LDL-C group (>100 mg/dL). There was no significant difference in FMD between the low LDL-C group and moderate LDL-C group. There were significant relationships of FMD and NID with LDL-C levels in statin naïve subjects. In a general population, LDL-C of ≤100 mg/dL may be the optimal target level for maintenance of endothelial function.

BackgroundLow-grade albuminuria has been considered a predictor of cardiovascular mortality. We investigated the relationship between high-normal albuminuria and subclinical atherosclerosis in non-diabetic men with estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2.MethodsIn this cross-sectional study, 1,756 men with eGFR ≥60 mL/min/1.73 m2 and urine albumin-to-creatinine ratio (UACR) ResultsMedian UACR was 4.8 mg/g (interquartile range, 3.6-6.9 mg/g). Compared with subjects with low-normal UACR (ConclusionsOur results indicate that high-normal albuminuria is associated with both carotid IMT and plaque formation in the non-diabetic male population with eGFR ≥60 mL/min/1.73 m2.

KLOTHO deficiency is associated with the progression of kidney dysfunction, whereas its overexpression exerts renoprotective effects. Oxidative stress suppresses KLOTHO expression in renal epithelial cells but upregulates microRNA-200c (miR-200c) in human umbilical vein endothelial cells. In this study, we investigated whether oxidative stress-induced miR-200c is implicated in KLOTHO downregulation in human renal tubular epithelium (HK-2) cells. HK-2 cells were stimulated with hydrogen peroxide (H2O2) to examine the effect of oxidative stress. A luciferase reporter containing the KLOTHO 3'-UTR was used to investigate the effect of miR-200c on KLOTHO mRNA metabolism. The expressions of KLOTHO, oxidative stress markers, and miR-200c were determined in human kidney biopsy specimens. H2O2 suppressed KLOTHO expression without a reduction in KLOTHO mRNA levels but upregulated miR-200c expression. Similarly, transfection of a miR-200c mimic reduced KLOTHO levels and luciferase activity without a reduction in KLOTHO mRNA levels. In contrast, transfection of a miR-200c inhibitor maintained KLOTHO expression. Immunofluorescent assay revealed KLOTHO was present in the cytosol and nuclei of HK-2 cells. In human kidney biopsies, KLOTHO expression was inversely correlated with levels of oxidative stress markers (8-hydroxy-2'-deoxyguanosine: ρ = -0.38, P = 0.026; 4-hydroxy-2-hexenal: ρ = -0.35, P = 0.038) and miR-200c (ρ = -0.34, P = 0.043). Oxidative stress-induced miR-200c binds to the KLOTHO mRNA 3'-UTR, resulting in reduced KLOTHO expression.

Mesenchymal stem cells (MSCs) are a potential therapeutic tool for preventing the progression of acute kidney injury (AKI) to chronic kidney disease (CKD). Herein, we investigated the localization and maintenance of engrafted human bone marrow-derived MSCs in rats subjected to a renal ischemia-reperfusion injury (IRI) and compared the effectiveness of two intravascular injection routes via the renal artery or inferior vena cava. Renal artery injection of MSCs was more effective than intravenous injection at reducing IRI-induced renal fibrosis. Additionally, MSCs injected through the renal artery persisted in injured kidneys for over 21 days, whereas MSCs injected through the inferior vena cava survived for less than 7 days. This difference may be attributed to the antifibrotic effects of MSCs. Interestingly, MSCs injected through the renal artery were localized primarily in glomeruli until day 3 post-IRI, and they decreased in number thereafter. In contrast, the number of MSCs localized in tubular walls, and the interstitium increased gradually until day 21 post-IRI. This localization change may be related to areas of damage caused by IRI because ischemia-induced AKI leads to tubular cell damage. Taken together, these findings suggest renal artery injection of MSCs may be useful for preventing the progression of AKI to CKD.

An elevation of serum low-density lipoprotein cholesterol (LDL-C) levels has been associated with endothelial dysfunction in statin naïve subjects. However, there is no information on endothelial function in subjects with extremely low levels of LDL-C. The purpose of the present study was to determine the relationship of LDL-C levels, especially low levels of LDL-C, with endothelial function. Endothelial function assessed by flow-mediated vasodilation (FMD) measurement and LDL-C levels were evaluated in 7120 subjects without lipid-lowering therapy. We divided the subjects into five groups by LDL-C levels: p < 0.001 and p = 0.004, respectively). The FMD values in the LDL-C of p = 0.570). A significant benefit was not found in subjects with low LDL-C levels from the aspect of endothelial function.

There has been a recent upsurge of interest in the effects of ionizing radiation exposure on the circulatory system, because a mounting body of epidemiological evidence suggests that irradiation induces cardio- and cerebrovascular disease at a much lower dose and lower dose rate than previously considered. The goal of our project is to determine whether dose protraction alters radiation effects on the circulatory system in a mouse model. To this end, the use of wild-type mice is pivotal albeit without manifestation of vascular diseases, because disease models (e.g., apolipoprotein E-deficient mice) are prone to hormetic responses following protracted exposures. As such, here, we first set out to analyze prelesional changes in the descending thoracic aorta of wild-type mice up to six months after a single acute exposure to 0 or 5 Gy of 137Cs γ-rays. Scanning electron microscopy demonstrated that irradiation facilitated structural disorganizations and detachment of the aortic endothelium. The Miles assay with an albumin-binding dye Evans Blue revealed that irradiation enhanced vascular permeability. Immunofluorescence staining showed that irradiation led to partial loss of the aortic endothelium (evidenced by a lack of adhesion molecule CD31 and 4′,6-diamidino-2-phenylindole (DAPI) signals), a decrease in endothelial nitric oxide synthase and adherens junction protein (vascular endothelial (VE)-cadherin) in the aortic endothelium, along with an increase in inflammation (tumor necrosis factor (TNF)-α) and macrophage (F4/80) markers in the aorta. These findings suggest that irradiation produces vascular damage manifested as endothelial cell loss and increased vascular permeability, and that the decreased adherens junction and the increased inflammation lead to macrophage recruitment implicated in the early stage of atherosclerosis.

Background The usefulness of vascular function tests for management of patients with a history of coronary artery disease is not fully known. Methods and Results We measured flow‐mediated vasodilation (FMD) and brachial–ankle pulse wave velocity (baPWV) in 462 patients with coronary artery disease for assessment of the predictive value of FMD and baPWV for future cardiovascular events in a prospective multicenter observational study. The first primary outcome was coronary events, and the second primary outcome was a composite of coronary events, stroke, heart failure, and sudden death. During a median follow‐up period of 49.2 months, the first primary outcome occurred in 56 patients and the second primary outcome occurred in 66 patients. FMD above the cutoff value of 7.1%, derived from receiver‐operator curve analyses for the first and second primary outcomes, was significantly associated with lower risk of the first (hazard ratio, 0.27; 95% confidence interval, 0.06–0.74; P=0.008) and second (hazard ratio, 0.32; 95% confidence interval, 0.09–0.79; P=0.01) primary outcomes. baPWV above the cutoff value of 1731 cm/s was significantly associated with higher risk of the first (hazard ratio, 1.86; 95% confidence interval, 1.01–3.44; P=0.04) and second (hazard ratio, 2.19; 95% confidence interval, 1.23–3.90; P=0.008) primary outcomes. Among 4 groups stratified according to the combination of cutoff values of FMD and baPWV, stepwise increases in the calculated risk ratio for the first and second primary outcomes were observed. Conclusions In patients with coronary artery disease, both FMD and baPWV were significant predictors of cardiovascular events. The combination of FMD and baPWV provided further cardiovascular risk stratification. Clinical Trial Registration URL: www.umin.ac.jp. Unique identifier: UMIN000012950.

Deubiquitinating enzymes (DUBs) remove ubiquitin from their substrates and, together with ubiquitin ligases, play an important role in the regulation of protein expression. Although transforming growth factor (TGF)-β1-Smad signaling is a central pathway of renal fibrosis, the role of DUBs in the expression of TGF-β receptors and Smads during the development of renal fibrosis remains unknown. In this study, we investigated whether PR-619, a pan-DUB inhibitor, suppresses fibrosis in mice with unilateral ureteral obstruction (UUO) and TGF-β1-stimulated normal rat kidney (NRK)-49F cells, a rat renal fibroblast cell line. Either the vehicle (dimethyl sulfoxide) or PR-619 (100 μg) was intraperitoneally administered to mice after UUO induction once a day for 7 days. Administration of PR-619 attenuated renal fibrosis with downregulation of mesenchymal markers, extracellular matrix proteins, matrix metalloproteinases, apoptosis, macrophage infiltration, and the TGF-β1 mRNA level in UUO mice. Although type I TGF-β receptor (TGF-βRI), Smad2, Smad3, and Smad4 protein expression levels were markedly increased in mice with UUO, administration of PR-619 suppressed only Smad4 expression but not TGF-βRI, Smad2, or Smad3 expression. PR-619 also had an inhibitory effect on TGF-β1-induced α-smooth muscle actin expression and reduced Smad4 levels in NRK-49F cells. Our results indicate that PR-619 ameliorates renal fibrosis, which is accompanied by the reduction of Smad4 expression.

Transforming growth factor-β1 (TGF-β1) is a major mediator of peritoneal fibrosis and reportedly affects expression of the H3K4 methyltransferase, SET7/9. SET7/9-induced H3K4 mono-methylation (H3K4me1) critically activates transcription of fibrosis-related genes. In this study, we examined the effect of SET7/9 inhibition on peritoneal fibrosis in mice and in human peritoneal mesothelial cells (HPMCs). We also examined SET7/9 expression in nonadherent cells isolated from the effluent of peritoneal dialysis (PD) patients. Murine peritoneal fibrosis was induced by intraperitoneal injection of methylglyoxal (MGO) into male C57/BL6 mice over 21 days. Sinefungin, a SET7/9 inhibitor, was administered subcutaneously just before MGO injection (10 mg/kg). SET7/9 expression was elevated in both MGO-injected mice and nonadherent cells isolated from the effluent of PD patients. SET7/9 expression was positively correlated with dialysate/plasma ratio of creatinine in PD patients. Sinefungin was shown immunohistochemically to suppress expression of mesenchymal cells and collagen deposition, accompanied by decreased H3K4me1 levels. Peritoneal equilibration tests showed that sinefungin attenuated the urea nitrogen transport rate from plasma and the glucose absorption rate from the dialysate. In vitro, sinefungin suppressed TGF-β1-induced expression of fibrotic markers and inhibited H3K4me1. These findings suggest that inhibiting the H3K4 methyltransferase SET7/9 ameliorates peritoneal fibrosis.

BackgroundFlow‐mediated vasodilation (FMD) of the brachial artery has been used for the assessment of endothelial function. Considering the mechanism underlying the vasodilatory response of the brachial artery to reactive hyperemia, hyperemic shear stress (HSS), a stimulus for FMD; nitroglycerine‐induced vasodilation (NID), an index of endothelium‐independent vasodilation; and baseline brachial artery diameter (BAD) are also involved in vasodilatory response. The purpose of this study was to investigate the interrelationships among FMD, HSS, NID, baseline BAD, and cardiovascular risk factors. Methods and ResultsWe measured FMD, HSS, NID, and baseline BAD simultaneously in 1033 participants (633 men and 400 women; mean age: 58.6±17.0 years). Framingham risk score was negatively correlated with FMD, HSS, and NID and was positively correlated with baseline BAD. HSS and NID were positively correlated with FMD, and baseline BAD was negatively correlated with FMD. In participants with normal NID, FMD was correlated with HSS, NID, and baseline BAD, all of which were independent variables of FMD in multivariate analysis. In participants with impaired NID, FMD was correlated with NID and baseline BAD, both of which were independent variables of FMD in multivariate analysis, but there was no association between FMD and HSS. ConclusionsNID and baseline BAD were independent variables of FMD regardless of the status of endothelium‐independent vasodilation, whereas there was a significant association between FMD and HSS in participants with normal NID but not in those with impaired NID. The influence of HSS on FMD seems to be dependent on the status of endothelium‐independent vasodilation.

Activity of H3K9 histone methyltransferase G9a is reportedly induced by transforming growth factor-β1 (TGF-β1) and plays an important role in the progression of cancer and fibrosis. In this study, we investigated whether inhibition of G9a-mediated H3K9 methylation attenuates peritoneal fibrosis in mice and human peritoneal mesothelial cells (HPMCs). Nonadherent cells of peritoneal dialysis (PD) patients were isolated from PD effluent to examine expression of G9a. Peritoneal fibrosis was induced by peritoneal injection of methylglyoxal (MGO) in male C57/B6 mice for 3 weeks. BIX01294, a G9a inhibitor, was administered by subcutaneous injection. Effects of BIX01294 on MGO-induced pathological and functional changes in mice were evaluated by immunohistochemistry and a peritoneal equilibration test. HPMCs were isolated from human omentum, and the inhibitory effect of BIX01294 on TGF-β1-induced fibrotic changes was investigated in the HPMCs by western blotting. G9a was upregulated in nonadherent cells of human PD effluent, the peritoneum of MGO-injected mice, and TGF-β1-stimulated HPMCs. BIX01294 significantly reduced the submesothelial zone thickness and cell density in MGO-injected mice. Immunohistochemical staining revealed that BIX01294 treatment decreased not only mono-methylation of H3K9 (H3K9me1), but also the number of mesenchymal cells, accumulation of collagen, and infiltration of monocytes. In addition to the pathological changes, BIX01294 reduced the level of TGF-β1 in peritoneal fluid and improved peritoneal functions. Furthermore, BIX01294 inhibited TGF-β1-induced fibrotic changes along with suppression of H3K9me1 in HPMCs. Therefore, inhibition of H3K9 methyltransferase G9a suppresses peritoneal fibrosis through a reduction of H3K9me1.

BackgroundA new device for automatic measurement of flow‐mediated vasodilation (FMD) using an oscillometric method has been developed to solve technical problems of conventional FMD measurement. This device measures enclosed zone FMD (ezFMD). The purpose of this study was to evaluate the prognostic value of endothelial function assessed by ezFMD for future cardiovascular events. Methods and ResultsWe measured ezFMD in 272 participants who underwent health‐screening examinations. First, we investigated cross‐sectional associations between ezFMD and cardiovascular risk factors, and then we assessed the associations between ezFMD and first major cardiovascular events (death from cardiovascular causes, stroke, and coronary revascularization). Univariate regression analysis revealed that ezFMD was significantly correlated with age, triglycerides, glucose, smoking pack‐years, estimated glomerular filtration rate, high‐sensitivity C‐reactive protein, and Framingham risk score. During a median follow‐up period of 36.1 months (interquartile range 18.8–40.1 months), 12 participants died (6 from cardiovascular causes), 3 had stroke, 8 had coronary revascularization, and 10 were hospitalized for heart failure. There was no episode of acute coronary syndrome during the study period. Participants were divided into tertiles (low, intermediate, and high) based on ezFMD. Kaplan–Meier curves for first major cardiovascular events among the 3 groups were significantly different (P=0.004). After adjustment for cardiovascular risk factors, the low group was significantly associated with an increased risk of first major cardiovascular events compared with the high group (hazard ratio 6.47; 95% CI 1.09–125.55; P=0.038). ConclusionsThese findings suggest that endothelial function assessed by ezFMD may be useful as a surrogate marker of future cardiovascular events. Clinical Trial RegistrationURL: https://upload.umin.ac.jp. Unique identifier: UMIN000004902.

Recent studies have reported increases of methylglyoxal (MGO) in peritoneal dialysis patients, and that MGO-mediated inflammation plays an important role in the development of peritoneal fibrosis through production of transforming growth factor-β1 (TGF-β1). Linagliptin, a dipeptidyl peptidase-4 inhibitor, exerts anti-inflammatory effects independent of blood glucose levels. In this study, we examined whether linagliptin suppresses MGO-induced peritoneal fibrosis in mice. Male C57/BL6 mice were divided into three groups: control, MGO injection plus saline, and MGO injection plus linagliptin (n = 6 per group). Peritoneal fibrosis was induced by daily intraperitoneal injection of saline containing 40 mmol/L MGO for 21 days. Saline was administered intraperitoneally to the control group. Linagliptin (10 mg/kg) or saline were administrated by once-daily oral gavage from 3 weeks before starting MGO injections. Immunohistochemical staining revealed that linagliptin suppressed expression of α-smooth muscle actin and fibroblast-specific protein-1, deposition of type I and III collagen, and macrophage (F4/80) infiltration. Peritoneal equilibration testing showed improved peritoneal functions in mice treated with linagliptin. Peritoneal injection of MGO increased plasma levels of glucagon-like peptide-1 (GLP-1) in mice, and a further increase was observed in linagliptin-treated mice. Although MGO increased plasma glucose levels, linagliptin did not decrease plasma glucose levels. Moreover, linagliptin reduced the TGF-β1 concentration in the peritoneal fluid of MGO-treated mice. GLP-1 receptor (GLP-1R) was expressed in monocytes/macrophages and linagliptin suppressed GLP-1R expression in MGO-injected mice. These results suggest that oral administration of linagliptin ameliorates MGO-induced peritoneal fibrosis.

OBJECTIVE:Rho-associated kinase (ROCK) signaling pathway has been shown to mediate various cellular functions including cell proliferation, migration, adhesion, apoptosis, and contraction, all of which may be involved in pathogenesis of atherosclerosis. Endogenous nitric oxide (NO) is well known to have an anti-atherosclerotic effect, whereas the exogenous NO-mediated cardiovascular effect still remains controversial. The purpose of this study was to evaluate the effect of exogenous NO on ROCK activity in vascular smooth muscle cells (VSMCs) in vitro and in vivo. METHODS:VSMCs migration was evaluated using a modified Boyden chamber assay. ROCK activities were measured by Western blot analysis in murine and human VSMCs and aorta of mice treated with or without angiotensin II (Ang II) and/or sodium nitroprusside (SNP), an NO donor. RESULTS:Co-treatment with SNP inhibited the Ang II-induced cell migration and increases in ROCK activity in murine and human VSMCs. Similarly, the increased ROCK activity 2 weeks after Ang II infusion in the mouse aorta was substantially inhibited by subcutaneous injection of SNP. CONCLUSIONS:These findings suggest that administration of exogenous NO can inhibit ROCK activity in VSMCs in vitro and in vivo.

Aldosterone-salt treatment induces not only hypertension but also extensive inflammation that contributes to fibrosis in the rat kidney. However, the mechanism underlying aldosterone-salt-induced renal inflammation remains unclear. Pyroptosis has recently been identified as a new type of cell death that is accompanied by the activation of inflammatory cytokines. We hypothesized that aldosterone-salt treatment could induce inflammation through pyroptosis and that mizoribine, an effective immunosuppressant, would ameliorate the renal inflammation that would otherwise cause renal fibrosis. Ten days after recovery from left uninephrectomy, rats were given drinking water with 1% sodium chloride. The animals were divided into three groups (n = 7 per group): (1) vehicle infusion group, (2) aldosterone infusion group, or (3) aldosterone infusion plus oral mizoribine group. Aldosterone-salt treatment increased the expression of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 and caspase-1, and also increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. However, the oral administration of mizoribine attenuated these alterations. Furthermore, mizoribine inhibited hypertension and renal fibrosis, and also attenuated the aldosterone-induced expression of serum/glucocorticoid-regulated kinase and α epithelial sodium channel. These results suggest that caspase-1 activation plays an important role in the development of inflammation induced by aldosterone-salt treatment and that it functions as an anti-inflammatory strategy that protects against renal injury and hypertension.

BACKGROUND: The purpose of this study was to determine whether autologous mesenchymal stem cells (MSCs) implantation improves endothelial dysfunction in a rabbit ischemic limb model. METHODS: We evaluated the effect of MSC implantation on limb blood flow (LBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and sodium nitroprusside (SNP), an endothelium-independent vasodilator, in rabbits with limb ischemia in which cultured MSCs were implanted (n = 20) or saline was injected as a control group (n = 20). LBF was measured using an electromagnetic flowmeter. A total of 10(6) MSCs were implanted into each ischemic limb. RESULTS: Histological sections of ischemic muscle showed that capillary index (capillary/muscle fiber) was greater in the MSC implantation group than in the control group. Laser Doppler blood perfusion index was significantly increased in the MSC implantation group compared with that in the control group. LBF response to ACh was greater in the MSC group than in the control group. After administration of N(G)-nitro-L-arginine, a nitric oxide synthase inhibitor, LBF response to ACh was similar in the MSC implantation group and control group. Vasodilatory effects of SNP in the two groups were similar. CONCLUSIONS: These findings suggest that MSC implantation induces angiogenesis and augments endothelium-dependent vasodilation in a rabbit ischemic model through an increase in nitric oxide production.