Optimizing myocardial cell protection with xanthine derivative KMUP-3 potentiates autophagy through the PI3K/Akt/eNOS axis.

BACKGROUND: Autophagy can have either beneficial or detrimental effects on various heart diseases. Pharmacological interventions improve cardiac function, which is correlated with enhanced autophagy. To assess whether a xanthine derivative (KMUP-3) treatment coincides with enhanced autophagy while also providing cardio-protection, we investigated the hypothesis that KMUP-3 treatment activation of autophagy through PI3K/Akt/eNOS signalling offered cardioprotective properties. METHODS: The pro-autophagic effect of KMUP-3 was performed in a neonatal rat model targeting cardiac fibroblasts and cardiomyocytes, and by assessing the impact of KMUP-3 treatment on cardiotoxicity, we used antimycin A-induced cardiomyocytes. RESULTS: As determined by transmission electron microscopy observation, KMUP-3 enhanced autophagosome formation in cardiac fibroblasts. Furthermore, KMUP-3 significantly increased the expressions of autophagy-related proteins, LC3 and Beclin-1, both in a time- and dose-dependent manner; moreover, the pro-autophagy and nitric oxide enhancement effects of KMUP-3 were abolished by inhibitors targeting eNOS and PI3K in cardiac fibroblasts and cardiomyocytes. Notably, KMUP-3 ameliorated cytotoxic effects induced by antimycin A, demonstrating its protective autophagic response. CONCLUSION: These findings enable the core pathway of PI3K/Akt/eNOS axis in KMUP-3-enhanced autophagy activation and suggest its principal role in safeguarding against cardiotoxicity.

Proteasome inhibitor bortezomib prevents proliferation and migration of pulmonary arterial smooth muscle cells.

Pulmonary vascular remodeling is a key pathological process of pulmonary arterial hypertension (PAH), characterized by uncontrolled proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Bortezomib (BTZ) is the first Food and Drug Administration (FDA)-approved proteasome inhibitor for multiple myeloma treatment. Recently, there is emerging evidence showing its effect on reversing PAH, although its mechanisms are not well understood. In this study, anti-proliferative and anti-migratory effects of BTZ on PASMCs were first examined by different inducers such as fetal bovine serum (FBS), angiotensin II (Ang II) and platelet-derived growth factor (PDGF)-BB, while potential mechanisms including cellular reactive oxygen species (ROS) and mitochondrial ROS were then investigated; finally, signal transduction of ERK and Akt was examined. Our results showed that BTZ attenuated FBS-, Ang II- and PDGF-BB-induced proliferation and migration, with associated decreased cellular ROS production and mitochondrial ROS production. In addition, the phosphorylation of ERK and Akt induced by Ang II and PDGF-BB was also inhibited by BTZ treatment. This study indicates that BTZ can prevent proliferation and migration of PASMCs, which are possibly mediated by decreased ROS production and down-regulation of ERK and Akt. Thus, proteasome inhibition can be a novel pharmacological target in the management of PAH.

Vasculoprotective Potential of Baicalein in Angiotensin II-Infused Abdominal Aortic Aneurysms through Inhibiting Inflammation and Oxidative Stress.

Aortic wall inflammation, abnormal oxidative stress and progressive degradation of extracellular matrix proteins are the main characteristics of abdominal aortic aneurysms (AAAs). The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome dysregulation plays a crucial role in aortic damage and disease progression. The first aim of this study was to examine the effect of baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) on AAA formation in apolipoprotein E-deficient (ApoE-/-) mice. The second aim was to define whether baicalein attenuates aberrant vascular smooth muscle cell (VSMC) proliferation and inflammation in VSMC culture. For male ApoE-/- mice, a clinically relevant AAA model was randomly divided into four groups: saline infusion, baicalein intraperitoneal injection, Angiotensin II (Ang II) infusion and Ang II + baicalein. Twenty-seven days of treatment with baicalein markedly decreased Ang II-infused AAA incidence and aortic diameter, reduced collagen-fiber formation, preserved elastic structure and density and prevented smooth muscle cell contractile protein degradation. Baicalein inhibited rat VSMC proliferation and migration following the stimulation of VSMC cultures with Ang II while blocking the Ang II-inducible cell cycle progression from G0/G1 to the S phase in the synchronized cells. Cal-520 AM staining showed that baicalein decreased cellular calcium in Ang II-induced VSMCs; furthermore, a Western blot assay indicated that baicalein inhibited the expression of PCNA and significantly lowered levels of phospho-Akt and phospho-ERK, along with an increase in baicalein concentration in Ang II-induced VSMCs. Immunofluorescence staining showed that baicalein pretreatment reduced NF-κB nuclear translocation in Ang II-induced VSMCs and furthered the protein expressions of NLRP3 while ASC and caspase-1 were suppressed in a dose-dependent manner. Baicalein pretreatment upregulated Nrf2/HO-1 signaling in Ang II-induced VSMCs. Thus, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining showed that its reactive oxygen species (ROS) production decreased, along with the baicalein pretreatment. Our overall results indicate that baicalein could have therapeutic potential in preventing aneurysm development.

Soluble CD93 lectin-like domain sequesters HMGB1 to ameliorate inflammatory diseases.

Rationale: CD93, a C-type lectin-like transmembrane glycoprotein, can be shed in a soluble form (sCD93) upon inflammatory stimuli. sCD93 effectively enhances apoptotic cell clearance and has been proposed as an inflammatory disease biomarker. The function of sCD93 involved directly in inflammation remains to be determined. Herein, we attempted to examine the hypothesis that sCD93 might sequester proinflammatory high-mobility group box 1 protein (HMGB1), exerting anti-inflammatory properties. Methods: Different forms of soluble recombinant human CD93 (rCD93) were prepared by a mammalian protein expression system. rCD93-HMGB1 interaction was assessed using co-immunoprecipitation and solid-phase binding assays. Effects of soluble rCD93 were evaluated in HMGB1-induced macrophage and vascular smooth muscle cells (VSMC) activation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, CaCl2-induced and angiotensin II-infused abdominal aortic aneurysm (AAA) formation and ovariectomized-induced osteoporosis in mice. Results: Protein binding studies revealed that soluble rCD93, via the lectin-like domain (D1), can bind to HMGB1 and intercept HMGB1-receptor interaction. Soluble rCD93 containing D1 inhibited HMGB1-induced proinflammatory cytokine production and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation in macrophages and VSMCs, thereby attenuating CaCl2-induced and angiotensin II-infused AAA models. During osteoclastogenesis, RANKL stimulated HMGB1 secretion that promoted RANKL-induced osteoclastogenesis in return. Soluble rCD93 containing D1 impeded RANKL-induced osteoclastogenic marker gene expression and intracellular MAPK/NF-κB signaling, thereby mitigating ovariectomized-induced osteoporosis. Conclusion: These findings demonstrate the therapeutic potential of soluble recombinant CD93 containing D1 in inflammatory diseases. Our study highlights a novel anti-inflammatory mechanism, i.e., sequestration of HMGB1, through which sCD93 prevents HMGB1-receptor interaction on effector cells and alleviates inflammation.

Diosgenin Attenuates Myocardial Cell Apoptosis Triggered by Oxidative Stress through Estrogen Receptor to Activate the PI3K/Akt and ERK Axes.

Cardiovascular diseases in post-menopausal women are on a rise. Oxidative stress is the main contributing factor to the etiology and pathogenesis of cardiovascular diseases. Diosgenin, a member of steroidal sapogenin, is structurally similar to estrogen and has been shown to have antioxidant effects. Therefore, we aimed to investigate the effects of diosgenin in preventing oxidation-induced cardiomyocyte apoptosis and assessed its potential as a substitute substance for estrogen in post-menopausal women. Apoptotic pathways and mitochondrial membrane potential were measured in H9c2 cardiomyoblast cells and neonatal cardiomyocytes treated with diosgenin for 1[Formula: see text]h prior to hydrogen peroxide (H2O2) stimulation. H2O2-stimulated H9c2 cardiomyoblast cells displayed cytotoxicity and apoptosis via the activation of both Fas-dependent and mitochondria-dependent pathways. Additionally, it led to the instability of the mitochondrial membrane potential. However, the H2O2-induced H9c2 cell apoptosis was rescued by diosgenin through IGF1 survival pathway activation. This led to the recovery of the mitochondrial membrane potential by suppressing the Fas-dependent and mitochondria-dependent apoptosis. Diosgenin also inhibited H2O2-induced cytotoxicity and apoptosis through the estrogen receptor interaction with PI3K/Akt and extracellular regulated protein kinases 1/2 activation in myocardial cells. In this study, we confirmed that diosgenin attenuated H2O2-induced cytotoxicity and apoptosis through estrogen receptors-activated phosphorylation of PI3K/Akt and ERK signaling pathways in myocardial cells via estrogen receptor interaction. All results suggest that H2O2-induced myocardial damage is reduced by diosgenin due to its interaction with estrogen receptors to decrease the damage. Herein, we conclude that diosgenin might be a potential substitute substance for estrogen in post-menopausal women to prevent heart diseases.

Anti-Inflammatory and Anti-oxidative Effects of Puerarin in Postmenopausal Cardioprotection: Roles of Akt and Heme Oxygenase-1.

During menopause, the sharp decline in estrogen levels leads to an increased risk of cardiovascular disease in women. The inflammatory response and oxidative stress are reportedly involved in the development of cardiovascular disorders postmenopause. In this study, we evaluated the cardioprotective effects of puerarin, a phytoestrogen derived from the root of Pueraria lobate, and investigated its underlying molecular mechanisms. Puerarin alleviated cytotoxicity and the production of reactive oxygen species (ROS) in lipopolysaccharide (LPS)- and hydrogen peroxide-stimulated H9c2 cardiomyoblasts. Puerarin scavenges free radicals and reduces apoptosis, thereby suppressing NADPH oxidase-1 and Bax activation to attenuate the production of ROS and restore Bcl-2 expression. Additionally, puerarin inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, and nitric oxide production and decreased the hypertrophic phenotype under LPS stimulation. Treatment with puerarin reduced the levels of malondialdehyde and restored glutathione levels when facing oxidative stress. Mechanistically, puerarin inhibited both the LPS-induced Toll-like receptor 4/NF-[Formula: see text]B and mitogen-activated protein kinase signaling pathways. Furthermore, it reversed both the LPS-mediated downregulation of Akt activation and heme oxygenase-1 (HO-1) expression. The cardioprotective effects of puerarin were abolished by inhibitors of Akt and HO-1 and the estrogen receptor antagonist fulvestrant (ICI). This indicated that the estrogen receptor mediated by these two molecules plays important roles in conferring the anti-inflammatory and anti-oxidative functions of puerarin. These results demonstrate the therapeutic potential of puerarin for treating heart disease in postmenopausal women through Akt and HO-1 activation.

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.

Xanthine Derivative KMUP-1 Attenuates Experimental Periodontitis by Reducing Osteoclast Differentiation and Inflammation.

Periodontitis is an inflammatory disease of gum that may predispose to serious systemic complications such as diabetes and cardiovascular diseases. Activation of macrophages and osteoclasts around periodontal tissue can accelerate gum inflammation. In addition, alteration of cyclic nucleotide levels is associated with the severity of periodontitis. Our previous study has shown that KMUP-1, a xanthine derivative exhibiting phosphodiesterase inhibition and soluble guanylyl cyclase activation, can inhibit lipopolysaccharide (LPS)-induced inflammation and receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclastogenesis. This study was aimed to investigate whether KMUP-1 could attenuate periodontitis both in vitro and in vivo. In vitro, the protective effect of KMUP-1 on inflammation and osteoclastogenesis was investigated in RANKL-primed RAW264.7 cells treated by Porphyromonas gingivalis LPS (PgLPS). The results showed that KMUP-1 attenuated PgLPS-induced osteoclast differentiation as demonstrated by decreased TRAP-positive multinuclear cells and TRAP activity. This reduction of osteoclast differentiation by KMUP-1 was reversed by KT5823, a protein kinase G inhibitor. Similarly, pro-inflammatory cytokine levels induced by PgLPS were inhibited by KMUP-1 in a dose-dependent manner whereas reversed by KT5823. Mechanistically, suppression of MAPKs, PI3K/Akt, and NF-κB signaling pathways and decrease of c-Fos and NFATc1 expression in osteoclast precursors by KMUP-1 may mediate its protective effect. In vivo, two models of periodontitis in rats were induced by gingival injections of PgLPS and ligature placement around molar teeth, respectively. Our results showed that KMUP-1 inhibited alveolar bone loss in both rat models, and this effect mediated at least partly by reduced osteoclastogenesis. In conclusion, our study demonstrated the therapeutic potential of KMUP-1 on periodontitis through suppression of inflammation and osteoclast differentiation.

In Vitro Evaluation of the Anti-Inflammatory Effect of KMUP-1 and In Vivo Analysis of Its Therapeutic Potential in Osteoarthritis.

Osteoarthritis is a degenerative arthropathy that is mainly characterized by dysregulation of inflammatory responses. KMUP-1, a derived chemical synthetic of xanthine, has been shown to have anti-inflammatory and antioxidant properties. Here, we aimed to investigate the in vitro anti-inflammatory and in vivo anti-osteoarthritis effects of KMUP-1. Protein and gene expressions of inflammation markers were determined by ELISA, Western blotting and microarray, respectively. RAW264.7 mouse macrophages were cultured and pretreated with KMUP-1 (1, 5, 10 µM). The productions of TNF-α, IL-6, MMP-2 and MMP- 9 were reduced by KMUP-1 pretreatment in LPS-induced inflammation of RAW264.7 cells. The expressions of iNOS, TNF-α, COX-2, MMP-2 and MMP-9 were also inhibited by KMUP-1 pretreatment. The gene expression levels of TNF and COX families were also downregulated. In addition, KMUP-1 suppressed the activations of ERK, JNK and p38 as well as phosphorylation of IκBα/NF-κB signaling pathways. Furthermore, SIRT1 inhibitor attenuated the inhibitory effect of KMUP-1 in LPS-induced NF-κB activation. In vivo study showed that KMUP-1 reduced mechanical hyperalgesia in monoiodoacetic acid (MIA)-induced rats OA. Additionally, KMUP-1 pretreatment reduced the serum levels of TNF-α and IL-6 in MIA-injected rats. Moreover, macroscopic and histological observation showed that KMUP-1 reduced articular cartilage erosion in rats. Our results demonstrated that KMUP-1 inhibited the inflammatory responses and restored SIRT1 in vitro, alleviated joint-related pain and cartilage destruction in vivo. Taken together, KMUP-1 has the potential to improve MIA-induced articular cartilage degradation by inhibiting the levels and expression of inflammatory mediators suggesting that KMUP-1 might be a potential therapeutic agent for OA.

The Preventive Effects of Xanthohumol on Vascular Calcification Induced by Vitamin D3 Plus Nicotine.

Vascular calcification (VC) is highly prevalent in patients with atherosclerosis, chronic kidney disease, diabetes mellitus, and hypertension. In blood vessels, VC is associated with major adverse cardiovascular events. Xanthohumol (XN), a main prenylated chalcone found in hops, has antioxidant effects to inhibit VC. This study aimed to investigate whether XN attenuates VC through in vivo study. A rat VC model was established by four weeks oral administration of vitamin D3 plus nicotine in Sprague Dawley (SD) rats. In brief, 30 male SD rats were randomly divided into three groups: control, 25 mg/kg nicotine in 5 mL corn oil and 3 × 105 IU/kg vitamin D3 administration (VDN), and combination of VDN with 20 mg/L in 0.1% ethanol of XN (treatment group). Physiological variables such as body and heart weight and drinking consumption were weekly observed, and treatment with XN caused no differences among the groups. In comparison with the control group, calcium content and alkaline phosphatase (ALP) activity were increased in calcified arteries, and XN treatment reduced these levels. Dihydroethidium (DHE) and 2',7'-dichloroflurescin diacetate (DCFH-DA) staining to identify Superoxide and reactive oxygen species generation from aorta tissue showed increased production in VDN group compared with the control and treatment groups. Hematoxylin eosin (HE) and Alizarin Red S staining were determined to show medial vascular thickness and calcification of vessel wall. Administration of VDN resulted in VC, and XN treatment showed improvement in vascular structure. Moreover, overexpression of osteogenic transcription factors bone morphogenetic protein 2 (BMP-2) and runt-related transcription factor 2 (Runx2) were significantly suppressed by XN treatment in VC. Moreover, downregulation of vascular phenotypic markers alpha-smooth muscle actin (α-SMA) and smooth muscle 22 alpha (SM22α) were increased by XN treatment in VC. Furthermore, XN treatment in VC upregulated nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions. Otherwise, Kelch-like ECH-associated protein 1 (Keap1) was alleviated by XN treatment in VC. In conclusion, our findings suggested that XN enhances antioxidant capacity to improve VC by regulating the Nrf2/Keap1/HO-1 pathway. Therefore, XN may have potential effects to decrease cardiovascular risk by reducing VC.

Extracellular heat shock protein HSC70 protects against lipopolysaccharide-induced hypertrophic responses in rat cardiomyocytes.

We have recently shown that exogenous administration of extracellular heat shock protein HSC70, a previously recognized intracellular chaperone protein, can protect against LPS-induced cardiac dysfunction through anti-inflammatory actions. However, whether it can also exert anti-hypertrophic effect is unknown. The present study was aimed to investigate the efficacy of HSC70 against cardiac hypertrophy and its underlying molecular mechanisms. Cardiomyocytes were isolated from the cardiac ventricles of neonatal Wistar rats and LPS (1 µg/mL) was used to induce the hypertrophic responses. We found that HSC70 (0.1, 1 and 5 µg/mL) pretreatment attenuated LPS-induced cardiomyocyte hypertrophy dose-dependently. In addition, HSC70 mitigated LPS-induced inflammatory mediators including TNF-α, IL-6, NO, iNOS and COX-2, with down-regulated protein expression of MMP-2 and MMP-9. Moreover, HSC70 repressed LPS-induced signaling of MAPK and Akt. Finally, HSC70 inhibited NF-κB subunit p65, and the DNA binding activity of NF-κB. Taken together, these findings suggest that in vitro HSC70 can exert anti-hypertrophic effects through inhibition of pro-inflammatory mediators, which are potential mediated by the down-regulation of MAPK, Akt and NF-κB signaling pathways. In conclusion, extracellular HSC70 may be a novel pharmacologic strategy in the management of cardiac hypertrophy.