Alteration of Piezo1 signaling in type 2 diabetic mice: focus on endothelium and BKCa channel.

Piezo1 mechanosensitive ion channel plays a important role in vascular physiology and disease. This study aimed to elucidate the altered signaling elicited by Piezo1 activation in the arteries of type 2 diabetes. Ten- to 12-week-old male C57BL/6 (control) and type 2 diabetic mice (db-/db-) were used. The second-order mesenteric arteries (~ 150 µm) were used for isometric tension experiments. Western blot analysis and immunofluorescence staining were performed to observe protein expression. Piezo1 was significantly decreased in mesenteric arteries of type 2 diabetic mice compared to control mice, as analyzed by western blot and immunofluorescence staining. Piezo1 agonist, Yoda1, concentration-dependently induced relaxation of mesenteric arteries in both groups. Interestingly, the relaxation response was significantly greater in control mice than in db-/db- mice. The removal of endothelium reduced relaxation responses induced by Yoda1, which was greater in control mice than db-/db- mice. Furthermore, the relaxation response was reduced by pre-treatment with various types of K+ channel blockers in endothelium-intact arteries in control mice. In endothelium-denuded arteries, pre-incubation with charybdotoxin, an Ca2+-activated K+ channel (BKCa channel) blocker, significantly attenuated Yoda1-induced relaxation in db-/db- mice, while there was no effect in control mice. Co-immunofluorescence staining showed co-localization of Piezo1 and BKCa channel was more pronounced in db-/db- mice than in control mice. These results indicate that the vascular responses induced by Piezo1 activation are different in the mesenteric resistance arteries in type 2 diabetic mice.

Investigating the Cardiovascular Benefits of Dapagliflozin: Vasodilatory Effect on Isolated Rat Coronary Arteries.

Dapagliflozin, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, is an antidiabetic medication that reduces blood glucose. Although it is well known that dapagliflozin has additional benefits beyond glycemic control, such as reducing blood pressure and lowering the risk of cardiovascular events, no sufficient research data are available on the direct effect of dapagliflozin on cardiovascular function. Thus, in this study, we investigated the direct vascular effect of dapagliflozin on isolated rat coronary arteries. The left descending coronary arteries of 13-week-old male Sprague Dawley rats were cut into segments 2-3 mm long and mounted in a multi-wire myography system to measure isometric tension. Dapagliflozin effectively reduced blood vessel constriction induced by U-46619 (500 nM) in coronary arteries regardless of the endothelium. Treatment with an eNOS inhibitor (L-NNA, 100 µM), sGC inhibitor (ODQ, 5 µM), or COX inhibitor (indomethacin, 3 µM) did not affect the vasodilation induced by dapagliflozin. The application of a Ca2+-activated K+ channel (KCa) blocker (TEA, 2 mM), voltage-dependent K+ channel (KV) blocker (4-AP, 2 mM), ATP-sensitive K+ channel blocker (KATP) glibenclamide (3 µM), and inward-rectifier K+ channel (KIR) blocker (BaCl2, 30 µM) did not affect the dapagliflozin-induced vasodilation either. The treatment with dapagliflozin decreased contractile responses induced by the addition of Ca2+, which suggested that the extracellular Ca2+ influx was inhibited by dapagliflozin. Treatment with dapagliflozin decreased the phosphorylation level of the 20 kDa myosin light chain (MLC20) in vascular smooth muscle cells. In the present study, we found that dapagliflozin has a significant vasodilatory effect on rat coronary arteries. Our findings suggest a novel pharmacologic approach for the treatment of cardiovascular diseases in diabetic patients through the modulation of Ca2+ homeostasis via dapagliflozin administration.

Ezetimibe Induces Vasodilation in Rat Mesenteric Resistance Arteries through Inhibition of Extracellular Ca2+ Influx.

Ezetimibe is a lipid-lowering agent that selectively inhibits cholesterol absorption by binding to the Niemann-Pick C1-like 1 (NPC1L1) protein. Although it is well known that administration of ezetimibe in hypercholesterolemia patients reduces the risk of cardiovascular events through attenuation of atherosclerosis, studies on the direct effect of ezetimibe on vascular function are not sufficient. The aim of the present study was to investigate the vascular effects of ezetimibe in rat mesenteric arteries. In the present study, 12-week-old male Sprague Dawley rats were used. After the rats were sacrificed, the second branches of the mesenteric arteries were isolated and cut into 2-3 mm segments and mounted in a multi-wire myography system to measure isometric tension. Ezetimibe reduced vasoconstriction induced by U46619 (500 nM) in endothelium-intact and endothelium-denuded arteries. Ezetimibe-induced vasodilation was not affected by the endothelial nitric oxide synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 300 µM) or the non-selective potassium channel blocker, tetraethylammonium (TEA, 10 mM). Moreover, ezetimibe also completely blocked the contraction induced by an increase in external calcium concentration. Ezetimibe significantly reduced vascular contraction induced by L-type Ca2+ channel activator (Bay K 8644, 30 nM). Treatment with ezetimibe decreased the phosphorylation level of 20 kDa myosin light chain (MLC20) in vascular smooth muscle cells. In the present study, we found that ezetimibe has a significant vasodilatory effect in rat mesenteric resistance arteries. These results suggest that ezetimibe may have beneficial cardiovascular effects beyond its cholesterol-lowering properties.

Vasorelaxant Effect of Trachelospermi caulis Extract on Rat Mesenteric Resistance Arteries.

BACKGROUND: Trachelospermi caulis (T. caulis) has been used as a traditional herbal medicine in Asian countries. Although it is well known that T. caulis has beneficial effects, no sufficient research data are available on the cardiovascular effect of T. caulis. We investigated whether T. caulis extract has vascular effects in rat resistance arteries in this study. METHODS: To examine whether T. caulis extract affects vascular reactivity, we measured isometric tension of rat mesenteric resistance arteries using a multi-wire myograph system. T. caulis extract was administered after arteries were pre-contracted with high K+ (70 mM) or phenylephrine (5 µM). Vanillin, a single active component of T. caulis, was used to treat mesenteric arteries. RESULTS: T. caulis extract caused vascular relaxation in a concentration-dependent manner, which was endothelium-independent. To further identify the mechanism, we incubated the arteries in Ca2+-free solution containing high K+, followed by a cumulative administration of CaCl2 (0.01-2.0 mM) with or without T. caulis extract (250 µg/mL). The treatment of T. caulis extract decreased contractile responses induced by the addition of Ca2+, which suggested that the extracellular Ca2+ influx was inhibited by the T. caulis extract. Moreover, an active compound of T. caulis extract, vanillin, also induced vasodilation in mesenteric resistance arteries. CONCLUSION: T. caulis extract and its active compound, vanillin, concentration-dependently induced vascular relaxation in mesenteric resistance arteries. These results suggest that the administration of T. caulis extract could help decrease blood pressure.

Vanillin Induces Relaxation in Rat Mesenteric Resistance Arteries by Inhibiting Extracellular Ca2+ Influx.

Vanillin is a phenolic aldehyde, which is found in plant species of the Vanilla genus. Although recent studies have suggested that vanillin has various beneficial properties, the effect of vanillin on blood vessels has not been studied well. In the present study, we investigated whether vanillin has vascular effects in rat mesenteric resistance arteries. To examine the vascular effect of vanillin, we measured the isometric tension of arteries using a multi-wire myograph system. After the arteries were pre-contracted with high K+ (70 mM) or phenylephrine (5 µM), vanillin was administered. Vanillin induced concentration-dependent vasodilation. Endothelial denudation or treatment of eNOS inhibitor (L-NNA, 300 µM) did not affect the vasodilation induced by vanillin. Treatment of K+ channel inhibitor (TEA, 10 mM) or sGC inhibitor (ODQ, 10 µM) or COX-2 inhibitor (indomethacin, 10 µM) did not affect the vanillin-induced vasodilation either. The treatment of vanillin decreased the contractile responses induced by Ca2+ addition. Furthermore, vanillin significantly reduced vascular contraction induced by BAY K 8644 (30 nM). Vanillin induced concentration-dependent vascular relaxation in rat mesenteric resistance arteries, which was endothelium-independent. Inhibition of extracellular Ca2+ influx was involved in vanillin-induced vasodilation. Treatment of vanillin reduced phopsho-MLC20 in vascular smooth muscle cells. These results suggest the possibility of vanillin as a potent vasodilatory molecule.

Obesity-induced Vitamin D Deficiency Contributes to Lung Fibrosis and Airway Hyperresponsiveness.

Vitamin D (VitD) has pleiotropic effects. VitD deficiency is closely involved with obesity and may contribute to the development of lung fibrosis and aggravation of airway hyperresponsiveness (AHR). We evaluated the causal relationship between VitD deficiency and the lung pathologies associated with obesity. In vivo effects of VitD supplementation were analyzed using high-fat diet (HFD)-induced obese mice and TGF-ß1 (transforming growth factor-ß1) triple transgenic mice. Effects of VitD supplementation were also evaluated in both BEAS-2B and primary lung cells from the transgenic mice. Obese mice had decreased 25-OH VitD and VitD receptor expressions with increases of insulin resistance, renin and angiotensin-2 system (RAS) activity, and leptin. In addition, lung pathologies such as a modest increase in macrophages, enhanced TGF-ß1, IL-1ß, and IL-6 expression, lung fibrosis, and AHR were found. VitD supplementation to HFD-induced obese mice recovered these findings. TGF-ß1-overexpressing transgenic mice enhanced macrophages in BAL fluid, lung expression of RAS, epithelial-mesenchymal transition markers, AHR, and lung fibrosis. VitD supplementation also attenuated these findings in addition to the attenuation of the expressions of TGF-ß1, and phosphorylated Smad-2/3 in lung. Supplementing in vitro-stimulated BEAS-2B and primary lung cells with VitD inhibited TGF-ß1 expression, supporting the suppressive effect of VitD for TGF-ß1 expression. These results suggest that obesity leads to VitD deficiency and worsens insulin resistance while enhancing the expression of leptin, RAS, TGF-ß1, and proinflammatory cytokines. These changes may contribute to the development of lung fibrosis and AHR. VitD supplementation rescues these changes and may have therapeutic potential for asthma with obesity.

Efficacy of transdermal immunotherapy with biodegradable microneedle patches in a murine asthma model.

BACKGROUND: House dust mite (HDM) is a well-known cause of asthma. Allergen-specific immunotherapy (AIT) can only modify the natural course of the disease. Conventional routes of HDM AIT are subcutaneous or sublingual. Subcutaneous immunotherapy (SCIT) has a disadvantage of systemic hypersensitive reaction, and the sublingual immunotherapy has a disadvantage of local allergic reaction and low drug adherence. OBJECTIVE: To overcome the weak points of conventional AIT, we developed a HDM loaded biodegradable microneedle patch (MNP) for transdermal immunotherapy (TDIT). We aim to demonstrate the efficacy of TDIT in murine asthma model triggered by HDM compared with conventional SCIT. METHODS: To make HDM asthma mouse model, 5-week-old BALB/c female mice were sensitized and challenged by intranasal administration of HDM. The mice were divided into 5 groups: sham, asthma, low (10 µg) and high dose (100 µg) SCIT, and TDIT (10 µg). To make HDM loaded MNP, droplet-born air blowing method was used. Airway hyperresponsiveness and allergic inflammation markers were analysed by bronchoalveolar lavage fluid, immunohistochemistry, serum immunoglobulin (Ig) analysis, and lung cytokine assays. RESULTS: Airway hyperresponsiveness was ameliorated by TDIT. Eosinophilic inflammation in bronchoalveolar lavage was improved without adverse reactions. Reduction of Th2 (IL-4, IL-5, and IL-13) cytokines, and HDM-specific IgE, induction of Treg (IL-10, TGF-ß), Th1 (IFN-γ) cytokines were observed. Eosinophilic infiltration, goblet cell hyperplasia, and subepithelial fibrosis were also alleviated by TDIT. These changes were more significant in the TDIT group than in subcutaneous AIT group. CONCLUSION: In conclusion, HDM loaded biodegradable TDIT is a novel treatment option to treat asthma which showed more effectiveness and may have better safety profiles than conventional SCIT.

Potential of serum soluble CD93 as a biomarker for asthma in an ovalbumin-induced asthma murine model.

BACKGROUND: CD93 is a membrane-associated glycoprotein, which can be released in a soluble form (sCD93) into the serum. CD93 has received renewed attention as a candidate biomarker of inflammation in various inflammatory and immune-mediated diseases, including asthma. OBJECTIVE: We aimed to evaluate the effects of airway inflammation on CD93 levels in murine models. METHODS: We established an ovalbumin (OVA)-induced acute asthma murine model (OVA model) and a lipopolysaccharide (LPS)-induced airway inflammation murine model (LPS model). Dexamethasone was administered by gavage to attenuate the airway inflammation. RESULTS: The OVA model demonstrated typical allergic asthma features with increased airway hyper-responsiveness, inflammatory cell infiltration, increased Th2 cytokine levels, compared to the control group. CD93 levels were decreased in lung homogenates and, respiratory epithelial cells, whereas serum sCD93 levels were increased in the OVA model, as compared to the control group. Dexamethasone reversed these effects of OVA. In contrast, in the LPS model, CD93 levels were not affected in neither respiratory epithelial cells nor serum. CONCLUSIONS: Our findings demonstrate the potential of using sCD93 as a biomarker for allergic asthma.

Fine particulate matter aggravates smoking induced lung injury via NLRP3/caspase-1 pathway in COPD.

BACKGROUND: Exposure to noxious particles, including cigarette smoke and fine particulate matter (PM2.5), is a risk factor for chronic obstructive pulmonary disease (COPD) and promotes inflammation and cell death in the lungs. We investigated the combined effects of cigarette smoking and PM2.5 exposure in patients with COPD, mice, and human bronchial epithelial cells. METHODS: The relationship between PM2.5 exposure and clinical parameters was investigated in patients with COPD based on smoking status. Alveolar destruction, inflammatory cell infiltration, and pro-inflammatory cytokines were monitored in the smoking-exposed emphysema mouse model. To investigate the mechanisms, cell viability and death and pyroptosis-related changes in BEAS-2B cells were assessed following the exposure to cigarette smoke extract (CSE) and PM2.5. RESULTS: High levels of ambient PM2.5 were more strongly associated with high Saint George's respiratory questionnaire specific for COPD (SGRQ-C) scores in currently smoking patients with COPD. Combined exposure to cigarette smoke and PM2.5 increased mean linear intercept and TUNEL-positive cells in lung tissue, which was associated with increased inflammatory cell infiltration and inflammatory cytokine release in mice. Exposure to a combination of CSE and PM2.5 reduced cell viability and upregulated NLRP3, caspase-1, IL-1ß, and IL-18 transcription in BEAS-2B cells. NLRP3 silencing with siRNA reduced pyroptosis and restored cell viability. CONCLUSIONS: PM2.5 aggravates smoking-induced airway inflammation and cell death via pyroptosis. Clinically, PM2.5 deteriorates quality of life and may worsen prognosis in currently smoking patients with COPD.

Effects of Particulate Matter 10 Inhalation on Lung Tissue RNA expression in a Murine Model.

BACKGROUND: Particulate matter 10 (PM10; airborne particles <10 µm) inhalation has been demonstrated to induce airway and lung diseases. In this study, we investigate the effects of PM10 inhalation on RNA expression in lung tissues using a murine model. METHODS: Female BALB/c mice were affected with PM10, ovalbumin (OVA), or both OVA and PM10. PM10 was administered intranasally while OVA was both intraperitoneally injected and intranasally administered. Treatments occurred 4 times over a 2-week period. Two days after the final challenges, mice were sacrificed. Full RNA sequencing using lung homogenates was conducted. RESULTS: While PM10 did not induce cell proliferation in bronchoalveolar fluid or lead to airway hyper-responsiveness, it did cause airway inflammation and lung fibrosis. Levels of interleukin 1ß, tumor necrosis factor-α, and transforming growth factor-ß in lung homogenates were significantly elevated in the PM10-treated group, compared to the control group. The PM10 group also showed increased RNA expression of Rn45a, Snord22, Atp6v0c-ps2, Snora28, Snord15b, Snora70, and Mmp12. Generally, genes associated with RNA splicing, DNA repair, the inflammatory response, the immune response, cell death, and apoptotic processes were highly expressed in the PM10-treated group. The OVA/PM10 treatment did not produce greater effects than OVA alone. However, the OVA/PM10-treated group did show increased RNA expression of Clca1, Snord22, Retnla, Prg2, Tff2, Atp6v0c-ps2, and Fcgbp when compared to the control groups. These genes are associated with RNA splicing, DNA repair, the inflammatory response, and the immune response. CONCLUSION: Inhalation of PM10 extensively altered RNA expression while also inducing cellular inflammation, fibrosis, and increased inflammatory cytokines in this murine mouse model.

Soluble CD93 in allergic asthma.

CD93 has been shown critical roles in inflammatory and immune diseases. However, in allergic asthma, the potential roles of soluble CD93 (sCD93) have not been well studied. We conducted house dust mite (HDM) stimulation with Der p 1 in BEAS-2B and U937 cells, followed by treatment with dexamethasone or small interfering RNA against CD93. A HDM-induced murine allergic asthma model was also established. We estimated the power of sCD93 to predict allergic asthma in a retrospective post-hoc analysis containing 96 human samples. HDM-stimulated BEAS-2B cells showed increased mRNA expression levels of IL-6, IL-8, IL-33, TSLP, and CD93. The CD93 level in culture supernatants steadily increased for 24 h after allergen stimulation, which was significantly suppressed by both dexamethasone and CD93 silencing. CD93 silencing increased IL-6 and TSLP, but not IL-33 levels in culture supernatants. HDM-induced asthma mice showed significant airway hyperresponsiveness and inflammation with Th2 cytokine activation, along with decreased CD93 expression in bronchial epithelial cells and lung homogenates but increased serum CD93 levels. The sCD93 level in asthma patients was significantly higher than that in healthy controls and could predict asthma diagnosis with moderate sensitivity (71.4%) and specificity (82.4%) (AUC = 0.787, P < 0.001). The level of sCD93 which has potential role to predict asthma significantly increased after HDM stimulation via IL-6 and TSLP in vitro and in vivo.

Empagliflozin and Dulaglutide are Effective against Obesity-induced Airway Hyperresponsiveness and Fibrosis in A Murine Model.

Patients with asthma with obesity experience severe symptoms, are unresponsive to conventional asthma treatment, and lack proper pharmacotherapy. Empagliflozin and dulaglutide, developed for diabetes, reduce weight, decrease insulin resistance, and exert additive effects. We evaluated the efficacy of empagliflozin, dulaglutide, and their combination on obesity-induced airway hyperresponsiveness (AHR) and lung fibrosis using a murine model. We assigned C57BL/6J mice to five groups: control, high-fat diet (HFD), and HFD with empagliflozin, dulaglutide, or both. Mice received a 12-week HFD, empagliflozin (5 days/week, oral gavage), and dulaglutide (once weekly, intraperitoneally). Both drugs significantly attenuated HFD-induced weight increase, abnormal glucose metabolism, and abnormal serum levels of leptin and insulin, and co-treatment was more effective. Both drugs significantly alleviated HFD-induced AHR, increased macrophages in bronchoalveolar lavage fluid (BALF), and co-treatment was more effective on AHR. HFD-induced lung fibrosis was decreased by both drugs alone and combined. HFD induced interleukin (IL)-17, transforming growth factor (TGF)-ß1, and IL-1ß mRNA and protein expression, which was significantly reduced by empagliflozin, dulaglutide, and their combination. Tumour necrosis factor (TNF)-α and IL-6 showed similar patterns without significant differences. HFD-enhanced T helper (Th) 1 and Th17 cell differentiation was improved by both drugs. Empagliflozin and dulaglutide could be a promising therapy for obesity-induced asthma and showed additive effects in combination.

Insulin resistance mediates high-fat diet-induced pulmonary fibrosis and airway hyperresponsiveness through the TGF-ß1 pathway.

Prior studies have reported the presence of lung fibrosis and enhanced airway hyperresponsiveness (AHR) in mice with high-fat-diet (HFD)-induced obesity. This study evaluated the role of TGF-ß1 in HFD-induced AHR and lung fibrosis in a murine model. We generated HFD-induced obesity mice and performed glucose and insulin tolerance tests. HFD mice with or without ovalbumin sensitization and challenge were also treated with an anti-TGF-ß1 neutralizing antibody. AHR to methacholine, inflammatory cells in the bronchoalveolar lavage fluid (BALF), and histological features were evaluated. Insulin was intranasally administered to normal diet (ND) mice, and in vitro insulin stimulation of BEAS-2b cells was performed. HFD-induced obesity mice had increased insulin resistance, enhanced AHR, peribronchial and perivascular fibrosis, and increased numbers of macrophages in the BALF. However, they did not have meaningful eosinophilic or neutrophilic inflammation in the lungs compared with ND mice. The HFD enhanced TGF-ß1 expression in the bronchial epithelium, but we found no differences in the expression of interleukin (IL)-4 or IL-5 in lung homogenates. Administration of the anti-TGF-ß1 antibody attenuated HFD-induced AHR and lung fibrosis. It also attenuated goblet cell hyperplasia, but did not affect the AHR and inflammatory cell infiltration induced by OVA challenge. The intranasal administration of insulin enhanced TGF-ß1 expression in the bronchial epithelium and lung fibrosis. Stimulating BEAS-2b cells with insulin also increased TGF-ß1 production by 24 h. We concluded that HFD-induced obesity-associated insulin resistance enhances TGF-ß1 expression in the bronchial epithelium, which may play an important role in the development of lung fibrosis and AHR in obesity.

Biological validation of plant-derived anti-human colorectal cancer monoclonal antibody CO17-1A.

We validated expression and biological activities of plant-derived monoclonal antibody (MAb(P)) CO17-1A for its efficacy in cancer immunotherapy. PCR and immunoblot analyses demonstrated insertion and expression of heavy and light chains of MAb CO17-1A in transgenic plants, respectively. Confocal analysis revealed that MAb(P) CO17-1A was accumulated throughout the cytoplasm near the outer membrane, suggesting its secretion to the outer membrane via a default pathway. Cell ELISA analysis confirmed that the MAb(P) CO17-1A heavy and light chains in crude plant leaf samples assembled to specifically bind SW948 human colorectal carcinoma cells. Flow cytometry analysis showed that the Fc domains of both the purified MAb(P) and the mammalian-derived MAb (MAb(M)) evidenced similar binding activity to the FcgammaRI receptor (CD64). The biological activities of both MAbs were similar, although the glycosylation pattern of MAb(P) CO17-1A is distinct from that of MAb(M). These results point to the potential use of MAb(P) CO17-1A for colorectal cancer immunotherapy.