Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Detrusor Hyperactivity with Impaired Contractility via Transient Potential Vanilloid Channels: A Rat Model for Ovarian Hormone Deficiency.

This study explores low-intensity extracorporeal shock wave therapy (LiESWT)'s efficacy in alleviating detrusor hyperactivity with impaired contractility (DHIC) induced by ovarian hormone deficiency (OHD) in ovariectomized rats. The rats were categorized into the following four groups: sham group; OVX group, subjected to bilateral ovariectomy (OVX) for 12 months to induce OHD; OVX + SW4 group, underwent OHD for 12 months followed by 4 weeks of weekly LiESWT; and OVX + SW8 group, underwent OHD for 12 months followed by 8 weeks of weekly LiESWT. Cystometrogram studies and voiding behavior tracing were used to identify the symptoms of DHIC. Muscle strip contractility was evaluated through electrical-field, carbachol, ATP, and KCl stimulations. Western blot and immunofluorescence analyses were performed to assess the expressions of various markers related to bladder dysfunction. The OVX rats exhibited significant bladder deterioration and overactivity, alleviated by LiESWT. LiESWT modified transient receptor potential vanilloid (TRPV) channel expression, regulating calcium concentration and enhancing bladder capacity. It also elevated endoplasmic reticulum (ER) stress proteins, influencing ER-related Ca2+ channels and receptors to modulate detrusor muscle contractility. OHD after 12 months led to neuronal degeneration and reduced TRPV1 and TRPV4 channel activation. LiESWT demonstrated potential in enhancing angiogenic remodeling, neurogenesis, and receptor response, ameliorating DHIC via TRPV channels and cellular signaling in the OHD-induced DHIC rat model.

Up-regulated small-conductance calcium-activated potassium currents contribute to atrial arrhythmogenesis in high-fat feeding mice.

AIMS: Metabolic syndrome (MetS) is associated with arrhythmias and cardiovascular mortality. Arrhythmogenesis in MetS results from atrial structural and electrical remodelling. The small-conductance Ca2+-activated K+ (SK) currents modulate atrial repolarization and may influence atrial arrhythmogenicity. This study investigated the regulation of SK current perturbed by a high-fat diet (HFD) to mimic MetS. METHODS AND RESULTS: Thirty mice were divided into two groups that were fed with normal chow (CTL) and HFD for 4 months. Electrocardiography and echocardiography were used to detect cardiac electrical and structure remodelling. Atrial action potential duration (APD) and calcium transient duration (CaTD) were measured by optical mapping of Langendorff-perfused mice hearts. Atrial fibrillation (AF) inducibility and duration were assessed by burst pacing. Whole-cell patch clamp was performed in primarily isolated atrial myocytes for SK current density. The SK current density is higher in atrial myocytes from HFD than in CTL mice (P ≤ 0.037). The RNA and protein expression of SK channels are increased in HFD mice (P ≤ 0.041 and P ≤ 0.011, respectively). Action potential duration is shortened in HFD compared with CTL (P ≤ 0.015). The shortening of the atrial APD in HFD is reversed by the application of 100 nM apamin (P ≤ 0.043). Compared with CTL, CaTD is greater in HFD atria (P ≤ 0.029). Calcium transient decay (Tau) is significantly higher in HFD than in CTL (P = 0.001). Both APD and CaTD alternans thresholds were higher in HFD (P ≤ 0.043), along with higher inducibility and longer duration of AF in HFD (P ≤ 0.023). CONCLUSION: Up-regulation of apamin-sensitive SK currents plays a partial role in the atrial arrhythmogenicity of HFD mice.

Efficiency comparison of an isoeugenol-derivated compound, eugenosedin-A, with glibenclamide and pioglitazone in protecting cardiovascular dysfunction of diabetic SHR.

This study was to investigate three agents possible protective effect against DM-induced cardiovascular dysfunction in spontaneously hypertensive rats (SHR). Control group was fed normal diet, DM group was injected with STZ/NA and fed high fat diet (HFD), and treatment groups were given STZ/NA, fed HFD, and then oral gavaged with eugenosedin-A (Eu-A), glibenclamide (Gli), or pioglitazone (Pio) 5 mg/kg/per day for 4-week, respectively. Eu-A, Gli, and Pio clearly ameliorated the changes of body weight, cardiac weight, and the biochemical parameters, cardiovascular disorders and inflammation. Like Gli and Pio, Eu-A may be effectively to control DM and the cardiovascular dysfunction.

Sodium Glucose Cotransporter 2 (SGLT2) Inhibitor Ameliorate Metabolic Disorder and Obesity Induced Cardiomyocyte Injury and Mitochondrial Remodeling.

Sodium-glucose transporter 2 inhibitors (SGLT2is) exert significant cardiovascular and heart failure benefits in type 2 diabetes mellitus (DM) patients and can help reduce cardiac arrhythmia incidence in clinical practice. However, its effect on regulating cardiomyocyte mitochondria remain unclear. To evaluate its effect on myocardial mitochondria, C57BL/6J mice were divided into four groups, including: (1) control, (2) high fat diet (HFD)-induced metabolic disorder and obesity (MDO), (3) MDO with empagliflozin (EMPA) treatment, and (4) MDO with glibenclamide (GLI) treatment. All mice were sacrificed after 16 weeks of feeding and the epicardial fat secretome was collected. H9c2 cells were treated with the different secretomes for 18 h. ROS production, Ca2+ distribution, and associated proteins expression in mitochondria were investigated to reveal the underlying mechanisms of SGLT2is on cardiomyocytes. We found that lipotoxicity, mitochondrial ROS production, mitochondrial Ca2+ overload, and the levels of the associated protein, SOD1, were significantly lower in the EMPA group than in the MDO group, accompanied with increased ATP production in the EMPA-treated group. The expression of mfn2, SIRT1, and SERCA were also found to be lower after EMPA-secretome treatment. EMPA-induced epicardial fat secretome in mice preserved a better cardiomyocyte mitochondrial biogenesis function than the MDO group. In addition to reducing ROS production in mitochondria, it also ameliorated mitochondrial Ca2+ overload caused by MDO-secretome. These findings provide evidence and potential mechanisms for the benefit of SGLT2i in heart failure and arrhythmias.

The Iridoid Glycoside Loganin Modulates Autophagic Flux Following Chronic Constriction Injury-Induced Neuropathic Pain.

Autophagy facilitates the degradation of organelles and cytoplasmic proteins in a lysosome-dependent manner. It also plays a crucial role in cell damage. Whether loganin affects autophagy in chronic constriction injury (CCI)-induced neuropathic pain remains unclear. We investigated the neuroprotective effect of loganin on the autophagic-lysosomal pathway in the rat CCI model. Sprague-Dawley rats were divided into sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was intraperitoneally injected once daily, and rats were sacrificed on day 7 after CCI. This study focused on the mechanism by which loganin modulates autophagic flux after CCI. CCI enhanced the autophagic marker LC3B-II in the ipsilateral spinal cord. The ubiquitin-binding protein p62 binds to LC3B-II and integrates into autophagosomes, which are degraded by autophagy. CCI caused the accumulation of p62, indicating the interruption of autophagosome turnover. Loganin significantly attenuated the expression of Beclin-1, LC3B-II, and p62. Double immunofluorescence staining was used to confirm that LC3B-II and p62 were reduced by loganin in the spinal microglia and astrocytes. Loganin also lessened the CCI-increased colocalization of both proteins. Enhanced lysosome-associated membrane protein 2 (LAMP2) and pro-cathepsin D (pro-CTSD) in CCI rats were also attenuated by loganin, suggesting that loganin improves impaired lysosomal function and autophagic flux. Loganin also attenuated the CCI-increased apoptosis protein Bax and cleaved caspase-3. Loganin prevents CCI-induced neuropathic pain, which could be attributed to the regulation of neuroinflammation, neuronal autophagy, and associated cell death. These data suggest autophagy could be a potential target for preventing neuropathic pain.

Potential Actions of Baicalein for Preventing Vascular Calcification of Smooth Muscle Cells In Vitro and In Vivo.

Vascular calcification (VC) is associated with cardiovascular disease. Baicalein, a natural flavonoid extract of Scutellaria baicalensis rhizome has several biological properties which may inhibit VC. We investigated whether baicalein suppresses Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP-2) and upregulates smooth muscle 22-alpha (SM22-α) and alpha-smooth muscle actin (α-SMA). In an in vitro experiment, primary rat aortic vascular smooth muscle cells (VSMCs) were pretreated with 0.1, 1, and 5 µM baicalein, followed by ß-glycerophosphate (ß-GP) to induce calcification. In an in vivo experiment, VC was generated by vitamin D3 plus nicotine (VDN) administration to male Sprague Dawley (SD) rats randomly assigned into a control group, a VC group, a VC group pretreated with baicalein, and a baicalein alone group. Each group comprised 10 rats. Left ventricular (LV) morphology, function and performance were assessed by echocardiography. Calcium content was measured by Alizarin red S staining and alkaline phosphatase (ALP) activity assays. Apoptotic VSMCs were detected by flow cytometry. Protein levels and superoxide changes were evaluated using Western blotting and immunofluorescence assays respectively. Plasma malondialdehyde (MDA) was assayed. Baicalein pretreatment significantly reduced calcium content in calcified VSMCs (p < 0.001) as well as in VC rat aortic smooth muscle (p < 0.001). Additionally, ALP activity was decreased in calcified VSMCs and VC rat aortic smooth muscle (p < 0.001). Apoptosis was significantly attenuated by 1 µM baicalein pretreatment in calcified VSMCs. Runx2 and BMP-2 expressions were downregulated by the baicalein in calcified VSMCs. Baicalein pretreatment increased typical VSMCs markers SM22-α and α-SMA in calcified VSMCs. Baicalein pretreatment was associated with adverse changes in LV morphometry. Markers of oxidative stress declined, and endogenous antioxidants increased in VC rats pretreated with baicalein. Baicalein mitigates VC through the inhibition of Runx2/BMP-2 signaling pathways, enhancement of vascular contractile phenotype and oxidative stress reduction. However, our study is of basic experimental design; more advanced investigations to identify other molecular regulators of VC and their mechanisms of action is required.

Characteristics of Ventricular Electrophysiological Substrates in Metabolic Mice Treated with Empagliflozin.

Empagliflozin (EMPA) is a sodium-glucose transporter 2 (SGLT2) inhibitor that functions as a new-generation glucose-lowering agent and has been proven to be beneficial for patients with cardiovascular diseases. However, the possible benefits and mechanisms of its antiarrhythmic effects in cardiac tissue have not yet been reported. In this study, we elucidated the possible antiarrhythmic effects and mechanisms of EMPA treatment in cardiac tissues of metabolic syndrome (MS) mice. A total of 20 C57BL/6J mice (age: 8 weeks) were divided into four groups: (1) control group, mice fed a standard chow for 16 weeks; (2) MS group, mice fed a high-fat diet for 16 weeks; (3) EMPA group, mice fed a high-fat diet for 12 weeks and administered EMPA at 10 mg/kg daily for the following 4 weeks; and (4) glibenclamide (GLI) group, mice fed a high-fat diet for 12 weeks and administered GLI at 0.6 mg/kg daily for the following 4 weeks. All mice were sacrificed after 16 weeks of feeding. The parameters of electrocardiography (ECG), echocardiography, and the effective refractory period (ERP) of the left ventricle were recorded. The histological characteristics of cardiac tissue, including connexin (Cx) expression and fibrotic areas, were also evaluated. Compared with the MS group, the ECG QT interval in the EMPA group was significantly shorter (57.06 ± 3.43 ms vs. 50.00 ± 2.62 ms, p = 0.011). The ERP of the left ventricle was also significantly shorter in the EMPA group than that in the GLI group (20.00 ± 10.00 ms vs. 60.00 ± 10.00 ms, p = 0.001). The expression of Cx40 and Cx43 in ventricular tissue was significantly lower in the MS group than in the control group. However, the downregulation of Cx40 and Cx43 was significantly attenuated in the EMPA group compared with the MS and GLI groups. The fibrotic areas of ventricular tissue were also fewer in the EMPA group than that in the MS group. In this study, the ECG QT interval in the EMPA group was shorter than that in the MS group. Compared with the MS group, the EMPA group exhibited significant attenuation of downregulated connexin expression and significantly fewer fibrotic areas in ventricles. These results may provide evidence of possible antiarrhythmic effects of EMPA.

Low-Intensity Extracorporeal Shock Wave Therapy Promotes Bladder Regeneration and Improves Overactive Bladder Induced by Ovarian Hormone Deficiency from Rat Animal Model to Human Clinical Trial.

Postmenopausal women with ovary hormone deficiency (OHD) are subject to overactive bladder (OAB) symptoms. The present study attempted to elucidate whether low-intensity extracorporeal shock wave therapy (LiESWT) alters bladder angiogenesis, decreases inflammatory response, and ameliorates bladder hyperactivity to influence bladder function in OHD-induced OAB in human clinical trial and rat model. The ovariectomized (OVX) for 12 months Sprague-Dawley rat model mimicking the physiological condition of menopause was utilized to induce OAB and assess the potential therapeutic mechanism of LiESWT (0.12 mJ/mm2, 300 pulses, and 3 pulses/second). The randomized, single-blinded clinical trial was enrolled 58 participants to investigate the therapeutic efficacy of LiESWT (0.25 mJ/mm2, 3000 pulses, 3 pulses/second) on postmenopausal women with OAB. The results revealed that 8 weeks' LiESWT inhibited interstitial fibrosis, promoted cell proliferation, enhanced angiogenesis protein expression, and elevated the protein phosphorylation of ErK1/2, P38, and Akt, leading to decreased urinary frequency, nocturia, urgency, urgency incontinence, and post-voided residual urine volume, but increased voided urine volume and the maximal flow rate of postmenopausal participants. In conclusion, LiESWT attenuated inflammatory responses, increased angiogenesis, and promoted proliferation and differentiation, thereby improved OAB symptoms, thereafter promoting social activity and the quality of life of postmenopausal participants.

Low Intensity Extracorporeal Shock Wave Therapy as a Novel Treatment for Stress Urinary Incontinence: A Randomized-Controlled Clinical Study.

Background and Objectives: To evaluate the effects of low intensity extracorporeal shock wave therapy (LiESWT) on stress urinary incontinence (SUI). Materials and Methods: This investigation was a multicenter, single-blind, randomized-controlled trial study. Sixty female SUI patients were randomly assigned to receive LiESWT with 0.25 mJ/mm2 intensity, 3000 pulses, and 3 pulses/s, once weekly for a 4-week (W4) and 8-week (W8) period, or an identical sham LiESWT treatment without energy transmission. The primary endpoint was the changes in urine leakage as measured by a pad test and validated standardized questionnaires, while the secondary endpoint was the changes in a 3-day urinary diary among the baseline (W0), the W4 and W8 of LiESWT, and 1-month (F1), 3-month (F3), and 6-month (F6) follow-up after LiESWT. Results: The results showed that 4 weeks of LiESWT could significantly decrease urine leakage based on the pad test and validated standardized questionnaire scores, as compared to the sham group. Moreover, 8 weeks of LiESWT could significantly reduce urine leakage but increase urine volume and attenuate urgency symptoms, which showed meaningful and persistent improvement at W8, F1, F3, and F6. Furthermore, validated standardized questionnaire scores were significantly improved at W8, F1, F3, and F6 as compared to the baseline (W0). Conclusions: Eight weeks of LiESWT attenuated SUI symptoms upon physical activity, reduced urine leakage, and ameliorated overactive bladder symptoms, which implied that LiESWT significantly improved the quality of life. Our findings suggested that LiESWT could serve as a potentially novel and non-invasive treatment for SUI.

The beneficial effects of angiotensin-converting enzyme II (ACE2) activator in pulmonary hypertension secondary to left ventricular dysfunction.

Pulmonary hypertension (PH) is a lethal and rapidly progressing disorder if left untreated, but there is still no definitive therapy. An imbalance between vasoconstriction and vasodilation has been proposed as the mechanism underlying PH. Among the vasomediators of the pulmonary circulation is the renin-angiotensin system (RAS), the involvement of which in the development of PH has been proposed. Within the RAS, angiotensin-converting enzyme 2 (ACE2), which converts angiotensin (Ang) II into Ang-(1-7), is an important regulator of blood pressure, and has been implicated in cardiovascular disease and PH. In this study, we investigated the effects of the ACE2 activator diminazene aceturate (DIZE) on the development of PH secondary to left ventricular dysfunction. A model of PH secondary to left ventricular dysfunction was established in 6-week-old Wistar rats by ascending aortic banding for 42 days. The hemodynamics and pulmonary expression of ACE, Ang II, ACE2, Ang-(1-7), and the Ang-(1-7) MAS receptor were investigated in the early treatment group, which was administered DIZE (15 mg/kg/day) from days 1 to 42, and in the late treatment group, administered DIZE (15 mg/kg/day) from days 29 to 42. Sham-operated rats served as controls. DIZE ameliorated mean pulmonary artery pressure, pulmonary arteriolar remodeling, and plasma brain natriuretic peptide levels, in addition to reversing the overexpression of ACE and up-regulation of both Ang-(1-7) and MAS, in the early and late treatment groups. DIZE has therapeutic potential for preventing the development of PH secondary to left ventricular dysfunction through ACEII activation and the positive feedback of ANG-(1-7) on the MAS receptor. A translational study in humans is needed to substantiate these findings.

Effects of Secretome from Fat Tissues on Ion Currents of Cardiomyocyte Modulated by Sodium-Glucose Transporter 2 Inhibitor.

Sodium-glucose transporter 2 (SGLT2) inhibitors were shown to decrease mortality from cardiovascular diseases in the EMPA-REG trial. However, the effects of empagliflozin (EMPA) for cardiac arrhythmia are not yet clarified. A total of 20 C57BL/6J mice were divided into four groups: (1) The control group were fed standard chow, (2) the metabolic syndrome (MS) group were fed a high-fat diet, (3) the empagliflozin (EMPA) group were fed a high-fat diet and empagliflozin 10 mg/kg daily, and (4) the glibenclamide (GLI) group were fed a high-fat diet and glibenclamide 0.6 mg/kg daily. All mice were sacrificed after 16 weeks of feeding. H9c2 cells were treated with adipocytokines from the pericardial and peripheral fat from the study groups. The delayed-rectifier potassium current (IK) and L-type calcium channel current (ICa,L) were measured by the whole-cell patch clamp techniques. Adipocytokines from the peripheral and pericardial fat tissues of mice with MS could decrease the IK and increase the ICa,L of cardiomyocytes. After treating adipocytokines from pericardial fat, the IK in the EMPA and GLI groups were significantly higher than that in the MS group. The IK of the EMPA group was also significantly higher than the GLI group. The ICa,L of the EMPA and GLI groups were significantly decreased overload compared with that of the MS group. However, there was no significant difference of IK and ICa,L among study groups after treating adipocytokines from peripheral fat. Adipocytokines from pericardial fat but not peripheral fat tissues after EMPA therapy attenuated the effects of IK decreasing and ICa,L increasing in the MS cardiomyocytes, which may contribute to anti-arrhythmic mechanisms of sodium-glucose transporter 2 (SGLT2) inhibitors.

Decreased Ambient Oxygen Tension Alters the Expression of Endothelin-1, iNOS and cGMP in Rat Alveolar Macrophages.

Background: Hypoxia plays an important role in the vascular tone of pulmonary circulation via the vasculature and parenchymal tissue. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, plays a role in inflammation in mononuclear cells. Nitric oxide synthase (NOS), which generates nitric oxide (NO)/cyclic 3', 5'-monophosphate (cGMP), is coexpressed with ET-1 in many cell types. The aim of this study was to assess whether hypoxia induces the production of ET-1 and associated expression of NOS, NO/cGMP and chemokines in rat alveolar macrophages (AMs). Methods: NR8383 cells were cultured under hypoxic (1% oxygen) conditions for 0, 2, 4, 8 and 12 hours. Levels of ET-1, inducible NOS (iNOS), phosphorylated iNOS (p-iNOS), nitrite/nitrate (NOx), cGMP and monocyte chemoattractant protein-1 (MCP-1) were measured. Results: ET-1, p-iNOS, NOx, and cGMP increased significantly in AMs after 4 hours of hypoxia (p < 0.05). ET-1 and MCP-1 mRNA increased after 8 hours (p < 0.05). The protein expression of ET-1, MCP-1, and p-iNOS increased in a time-dependent manner, while iNOS expression decreased with time. Conclusions: The changes in ET-1, p-iNOS, and the NO/cGMP pathway in AMs may help elucidate the mechanisms in the hypoxic lung. Understanding changes in the endothelin axis in hypoxic AMs is a crucial first step to unravel its role in pulmonary circulation.

KMUP-1 Ameliorates Ischemia-Induced Cardiomyocyte Apoptosis through the NO⁻cGMP⁻MAPK Signaling Pathways.

To test whether KMUP-1 (7-[2-[4-(2-chlorophenyl) piperazinyl]ethyl]-1,3-dimethylxanthine) prevents myocardial ischemia-induced apoptosis, we examined KMUP-1-treated H9c2 cells culture. Recent attention has focused on the activation of nitric oxide (NO)-guanosine 3', 5'cyclic monophosphate (cGMP)-protein kinase G (PKG) signaling pathway triggered by mitogen-activated protein kinase (MAPK) family, including extracellular-signal regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 in the mechanism of cardiac protection during ischemia-induced cell-death. We propose that KMUP-1 inhibits ischemia-induced apoptosis in H9c2 cells culture through these pathways. Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and apoptotic evaluation was conducted using DNA ladder assay and Hoechst 33342 staining. The level of intracellular calcium was detected using - Fura2-acetoxymethyl (Fura2-AM) staining, and mitochondrial calcium with Rhod 2-acetoxymethyl (Rhod 2-AM) staining under fluorescence microscopic observation. The expression of endothelium NO synthase (eNOS), inducible NO synthase (iNOS), soluble guanylate cyclase α1 (sGCα1), PKG, Bcl-2/Bax ratio, ERK1/2, p38, and JNK proteins were measured by Western blotting assay. KMUP-1 pretreatment improved cell viability and inhibited ischemia-induced apoptosis of H9c2 cells. Calcium overload both in the intracellular and mitochondrial sites was attenuated by KMUP-1 pretreatment. Moreover, KMUP-1 reduced intracellular reactive oxygen species (ROS), increased plasma NOx (nitrite and nitrate) level, and the expression of eNOS. Otherwise, the iNOS expression was downregulated. KMUP-1 pretreatment upregulated the expression of sGCα1 and PKG protein. The ratio of Bcl-2/Bax expression was increased by the elevated level of Bcl2 and decreased level of Bax. In comparison with the ischemia group, KMUP-1 pretreatment groups reduced the expression of phosphorylated extracellular signal-regulated kinases ERK1/2, p-p38, and p-JNK as well. Therefore, KMUP-1 inhibits myocardial ischemia-induced apoptosis by restoration of cellular calcium influx through the mechanism of NO-cGMP-MAPK pathways.

Elucidating Mechanisms of Bladder Repair after Hyaluronan Instillation in Ketamine-Induced Ulcerative Cystitis in Animal Model.

Ketamine-induced ulcerative cystitis (KIC) initially damaged the bladder mucosa and induced contracted bladder thereafter. Hyaluronan (hyaluronic acid; HA) instillation to the bladder has been used to treat KIC. The present study investigated bladder injury by urothelial defect and HA degeneration and bladder repair by urothelium proliferation and differentiation. This work was based on the hypothesis that HA treatment altered the bladder urothelial layer and the expression of hyaluronan-metabolizing enzymes and/or HA receptors in KIC. Cystometrogram study and tracing analysis of voiding behavior revealed that the ketamine-treated rats exhibited significant bladder hyperactivity with an increase in micturition frequency and a decrease in bladder capacity. The expression of inflammatory and fibrosis markers was also increased in the ketamine-treated group. Moreover, ketamine administration decreased the expression of urothelial barrier-associated protein, altered HA production, and induced abnormal urothelial differentiation, which might attribute to urothelial lining defects. However, HA instillation ameliorated bladder hyperactivity, lessened bladder mucosa damage, and decreased interstitial fibrosis. HA instillation also improved the level of HA receptors (CD44, Toll-like receptor-4, and receptor for HA-mediated motility) and HA synthases 1 to 3 and decreased the expression of hyaluronidases in the urothelial layer of bladder, resulting in enhanced mucosal regeneration. These findings suggested that HA could modulate inflammatory responses, enhance mucosal regeneration, and improve urothelial lining defects in KIC.

B-type natriuretic peptide prevents postnatal closure of ductus arteriosus by both vasodilation and anti-remodeling in neonatal rats.

The physiologic process of postnatal ductus arteriosus (DA) closure consists of vasoconstriction followed by vascular remodeling. We have recently reported that B-type natriuretic peptide (BNP), a potent vasodilator, also has anti-remodeling effects in pulmonary vasculature. However, its effects on DA have not been elucidated. We investigated whether BNP can prevent DA closure, and if so, the underlying mechanisms. Using in vivo studies, we examined effects of BNP (10 mg/kg, ip at birth) on DA closure in neonatal rats within 4 h after birth. We found that in control rats, the DA spontaneously closed at 4 h with a decreased DA diameter, enhanced intimal thickening, and luminal occlusion. BNP prevented DA closure at 4 h with a preserved DA diameter, attenuated intimal thickening, and preserved luminal patency. Ex vivo, BNP attenuated oxygen-induced vasoconstriction of isolated DA rings of newborn rats. These vasodilating effects were blunted by Rp-8-Br-PET-cGMPS, a cGMP inhibitor. In vitro, BNP inhibited angiotensin II (Ang II)-induced proliferation and migration of DA smooth muscle cells (DASMCs). BNP inhibited Ang II-induced mitochondrial reactive oxygen species (ROS) production and calcium overload in DASMCs. Finally, BNP inhibited Ang II-induced ERK1/2 activation. These in vitro effects were antagonized by Rp-8-Br-PET-cGMPS. In conclusion, BNP prevents postnatal DA closure by both vasodilation and anti-remodeling through the cGMP pathway. The mechanisms underlying anti-remodeling effects include anti-poliferation and anti-migration, with attenuation of mitochondrial ROS production and intracellular calcium and ERK1/2 signaling. Therefore, the BNP/cGMP pathway can be a promising therapeutic target for clinical management of DA patency.

KMUP-1, a GPCR Modulator, Attenuates Triglyceride Accumulation Involved MAPKs/Akt/PPARγ and PKA/PKG/HSL Signaling in 3T3-L1 Preadipocytes.

Xanthine-based KMUP-1 was shown to inhibit phosphodiesterases (PDEs) and modulate G-protein coupled receptors (GPCRs) to lower hyperlipidemia and body weight. This study further investigated whether KMUP-1 affects adipogenesis and lipolysis in 3T3-L1 preadipocytes. KMUP-1 (1⁻40 µM) concentration-dependently attenuated Oil Red O (ORO) staining and decreased triglyceride (TG) accumulation, indicating adipogenesis inhibition in 3T3-L1 cells. In contrast, the ß-agonist ractopamine increased ORO staining and TG accumulation and adipogenesis. KMUP-1 (1⁻40 µM) also reduced MAPKs/Akt/PPARγ expression, PPARγ1/PPARγ2 mRNA, and p-ERK immunoreactivity at the adipogenesis stage, but enhanced hormone sensitive lipase (HSL) immunoreactivity at the lipolysis stage. Addition of protein kinase A (PKA) or protein kinase G (PKG) antagonist (KT5720 or KT5728) to adipocytes did not affect HSL immunoreactivity. However, KMUP-1 did increase HSL immunoreactivity and the effect was reduced by PKA or PKG antagonist. Simvastatin, theophylline, caffeine, and sildenafil, like KMUP-1, also enhanced HSL immunoreactivity. Phosphorylated HSL (p-HSL) was enhanced by KMUP-1, indicating increased lipolysis in mature 3T3-L1 adipocytes. Decreases of MAPKs/Akt/PPARγ during adipogenesis contributed to inhibition of adipocyte differentiation, and increases of PKA/PKG at lipolysis contributed to HSL activation and TG hydrolysis. Taken together, the data suggest that KMUP-1 can inhibit hyperadiposity in 3T3-L1 adipocytes.

Exogenous Heat Shock Cognate Protein 70 Suppresses LPS-Induced Inflammation by Down-Regulating NF-κB through MAPK and MMP-2/-9 Pathways in Macrophages.

Heat shock cognate protein 70 (HSC70), a molecular chaperone, is constitutively expressed by mammalian cells to regulate various cellular functions. It is associated with many diseases and is a potential therapeutic target. Although HSC70 also possesses an anti-inflammatory action, the mechanism of this action remains unclear. This current study aimed to assess the anti-inflammatory effects of HSC70 in murine macrophages RAW 264.7 exposed to lipopolysaccharides (LPS) and to explain its pathways. Mouse macrophages (RAW 264.7) in 0.1 µg/mL LPS incubation were pretreated with recombinant HSC70 (rHSC70) and different assays (Griess assay, enzyme-linked immune assay/ELISA, electrophoretic mobility shift assay/EMSA, gelatin zymography, and Western blotting) were performed to determine whether rHSC70 blocks pro-inflammatory mediators. The findings showed that rHSC70 attenuated the nitric oxide (NO) generation, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) expressions in LPS-stimulated RAW264.7 cells. In addition, rHSC70 preconditioning suppressed the activities and expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Finally, rHSC70 diminished the nuclear translocation of nuclear factor-κB (NF-κB) and reduced the phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and phosphatidylinositol-3-kinase (PI3K/Akt). We demonstrate that rHSC70 preconditioning exerts its anti-inflammatory effects through NO production constriction; TNF-α, and IL-6 suppression following down-regulation of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and MMP-2/MMP-9. Accordingly, it ameliorated the signal transduction of MAPKs, Akt/IκBα, and NF-κB pathways. Therefore, extracellular HSC70 plays a critical role in the innate immunity modulation and mechanisms of endogenous protective stimulation.

Theophylline-Based KMUP-1 Improves Steatohepatitis via MMP-9/IL-10 and Lipolysis via HSL/p-HSL in Obese Mice.

KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) has been reported to cause hepatic fat loss. However, the action mechanisms of KMUP-1 in obesity-induced steatohepatitis remains unclear. This study elucidated the steatohepatitis via matrix metallopeptidase 9 (MMP-9) and tumor necrosis factor α (TNFα), and related lipolysis via hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) by KMUP-1. KMUP-1 on steatohepatitis-associated HSL/p-HSL/ATGL/MMP-9/TNFα/interleukin-10 (IL-10) and infiltration of M1/M2 macrophages in obese mice were examined. KMUP-1 was administered by oral gavage from weeks 1-14 in high-fat diet (HFD)-supplemented C57BL/6J male mice (protection group) and from weeks 8-14, for 6 weeks, in HFD-induced obese mice (treatment group). Immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining of tissues, oil globules number and size, infiltration and switching of M1/M2 macrophages were measured to determine the effects on livers. IL-10 and MMP-9 proteins were explored to determine the effects of KMUP-1 on M1/M2 macrophage polarization in HFD-induced steatohepatitis. Long-term administration of KMUP-1 reversed HFD-fed mice increased in body weight, sGOT/sGPT, triglyceride (TG) and glucose. Additionally, KMUP-1 decreased MMP-9 and reactive oxygen species (ROS), and increased HSL/p-HSL and IL-10 in HFD mice livers. In conclusion, KMUP-1, a phosphodiesterase inhibitor (PDEI), was shown to reduce lipid accumulation in liver tissues, suggesting that it could be able to prevent or treat steatohepatitis induced by HFD.

VLDL from Metabolic Syndrome Individuals Enhanced Lipid Accumulation in Atria with Association of Susceptibility to Atrial Fibrillation.

Metabolic syndrome (MetS) represents a cluster of metabolic derangements. Dyslipidemia is an important factor in MetS and is related to atrial fibrillation (AF). We hypothesized that very low density lipoproteins (VLDL) in MetS (MetS-VLDL) may induce atrial dilatation and vulnerability to AF. VLDL was therefore separated from normal (normal-VLDL) and MetS individuals. Wild type C57BL/6 male mice were divided into control, normal-VLDL (nVLDL), and MetS-VLDL (msVLDL) groups. VLDL (15 µg/g) and equivalent volumes of saline were injected via tail vein three times a week for six consecutive weeks. Cardiac chamber size and function were measured by echocardiography. MetS-VLDL significantly caused left atrial dilation (control, n = 10, 1.64 ± 0.23 mm; nVLDL, n = 7, 1.84 ± 0.13 mm; msVLDL, n = 10, 2.18 ± 0.24 mm; p < 0.0001) at week 6, associated with decreased ejection fraction (control, n = 10, 62.5% ± 7.7%, vs. msVLDL, n = 10, 52.9% ± 9.6%; p < 0.05). Isoproterenol-challenge experiment resulted in AF in young msVLDL mice. Unprovoked AF occurred only in elderly msVLDL mice. Immunohistochemistry showed excess lipid accumulation and apoptosis in msVLDL mice atria. These findings suggest a pivotal role of VLDL in AF pathogenesis for MetS individuals.

Xanthine-based KMUP-1 improves HDL via PPARγ/SR-B1, LDL via LDLRs, and HSL via PKA/PKG for hepatic fat loss.

The phosphodiesterase inhibitor (PDEI)/eNOS enhancer KMUP-1, targeting G-protein coupled receptors (GPCRs), improves dyslipidemia. We compared its lipid-lowering effects with simvastatin and explored hormone-sensitive lipase (HSL) translocation in hepatic fat loss. KMUP-1 HCl (1, 2.5, and 5 mg/kg/day) and simvastatin (5 mg/kg/day) were administered in C57BL/6J male mice fed a high-fat diet (HFD) by gavage for 8 weeks. KMUP-1 inhibited HFD-induced plasma/liver TG, total cholesterol, and LDL; increased HDL/3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)/Rho kinase II (ROCK II)/PPARγ/ABCA1; and decreased liver and body weight. KMUP-1 HCl in drinking water (2.5 mg/200 ml tap water) for 1-14 or 8-14 weeks decreased HFD-induced liver and body weight and scavenger receptor class B type I expression and increased protein kinase A (PKA)/PKG/LDLRs/HSL expression and immunoreactivity. In HepG2 cells incubated with serum or exogenous mevalonate, KMUP-1 (10(-7)∼10(-5) M) reversed HMGR expression by feedback regulation, colocalized expression of ABCA1/apolipoprotein A-I/LXRα/PPARγ, and reduced exogenous geranylgeranyl pyrophosphate/farnesyl pyrophosphate (FPP)-induced RhoA/ROCK II expression. A guanosine 3',5'-cyclic monophosphate (cGMP) antagonist reversed KMUP-1-induced ROCK II reduction, indicating cGMP/eNOS involvement. KMUP-1 inceased PKG and LDLRs surrounded by LDL and restored oxidized LDL-induced PKA expresion. Unlike simvastatin, KMUP-1 could not inhibit (14)C mevalonate formation. KMUP-1 could, but simvastatin could not, decrease ROCK II expression by exogenous FPP/CGPP. KMUP-1 improves HDL via PPARγ/LXRα/ABCA1/Apo-I expression and increases LDLRs/PKA/PKG/HSL expression and immunoreactivity, leading to TG hydrolysis to lower hepatic fat and body weight.