Taohong Siwu decoction enhances the chemotherapeutic efficacy of doxorubicin by promoting tumor vascular normalization.

BACKGROUND: Instead of completely suppressing blood vessels inside tumors, vascular normalization therapy is proposed to normalize and prune the abnormal vasculature in tumor microenvironment (TME) to acquire a normal and stable blood flow and perfusion. The theoretical basis for the use of "blood-activating and stasis-resolving" formulas in Traditional Chinese Medicine to treat cancer is highly consistent with the principle of vascular normalization therapy, suggesting the potential application of these traditional formulas in vascular normalization therapy. PURPOSE: To study the underlying mechanisms of a classical "blood-activating and stasis-resolving" formula, Taohong Siwu decoction (TSD), in enhancing the efficacy of chemotherapy for breast cancer treatment. STUDY DESIGN: HUVECs and transgenic zebrafish embryos were used as the major model in vitro. A 4T1 mouse breast cancer model was applied to study tumor vasculature normalization of TSD and the combination effects with DOX. RESULTS: Our data showed that TSD exhibited anti-angiogenic potential in HUVECs and transgenic zebrafish embryos. After 20 days treatment, TSD significantly normalized the tumor vasculature by remodeling vessel structure, reducing intratumoral hypoxia and vessel leakage, and promoting vessel maturation and blood perfusion in 4T1 breast tumor-bearing mice. Moreover, the anti-tumor efficacy of doxorubicin liposome in 4T1 breast tumors was significantly improved by TSD, including the suppression of tumor cell proliferation, angiogenesis, hypoxia, and the increase of cell apoptosis, which is likely through the vascular normalization induced by TSD. TSD also shifted the macrophage polarization from M2 to M1 phenotype in TME during the combination therapy, as evidenced by the reduced number of CD206+ macrophages and increased number of CD86+ macrophages. Additionally, TSD treatment protected against doxorubicin-induced cardiotoxicity in animals, as evidenced by the reduced cardiomyocytes apoptosis and improved heart function. CONCLUSION: This study demonstrated for the first time that TSD as a classical Chinese formula can enhance the drug efficacy and reduce the side effects of doxorubicin. These findings can support that TSD could be used as an adjuvant therapy in combination with conventional chemotherapy for the future breast cancer treatment.

Protective effects of Amauroderma rugosum on dextran sulfate sodium-induced ulcerative colitis through the regulation of macrophage polarization and suppression of oxidative stress.

BACKGROUND: Amauroderma rugosum (AR) is a medicinal mushroom commonly used to treat inflammation, gastric disorders, epilepsy, and cancers due to its remarkable anti-inflammatory and anti-oxidative properties. This study was designed to evaluate the pharmacological effects of AR and its underlying mechanism of action against ulcerative colitis (UC) in vitro and in vivo. METHODS: A UC mouse model was established by administration of dextran sulfate sodium (DSS). AR extract was administered intragastrically to mice for 7 days. At the end of the experiment, histopathology, macrophage phenotype, oxidative stress, and inflammatory status were examined in vivo. Furthermore, RAW 264.7, THP-1, and Caco-2 cells were used to elucidate the mechanism of action of AR in vitro. RESULTS: AR extract (0.5-2 mg/mL) significantly suppressed lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-induced M1 macrophage (pro-inflammatory) polarization in both RAW 264.7 and THP-1 cells. LPS-induced pro-inflammatory mediators (nitric oxide, TNF-α, IL-1ß, MCP-1, and IL-6) were reduced by AR extract in a concentration-dependent manner. Similarly, AR extract downregulated MAPK signaling activity in LPS-stimulated RAW 264.7 cells. AR extract elicited a concentration-dependent increase in the mRNA expression of M2 (anti-inflammatory) phenotype markers (CD206, Arg-1, Fizz-1, and Ym-1) in RAW 264.7 cells. Moreover, AR extract suppressed DSS-induced ROS generation and mitochondrial dysfunction in Caco-2 cells. The in vivo experiment revealed that AR extract (200 mg/kg) increased colon length compared to the DSS-treated group. In addition, disease activity index, spleen ratio, body weight, oxidative stress, and colonic inflammation were markedly improved by AR treatment in DSS-induced UC mice. Finally, AR suppressed M1 and promoted M2 macrophage polarization in UC mice. CONCLUSION: The AR extract protected against DSS-induced UC by regulating macrophage polarization and suppressing oxidative stress. These valuable findings suggest that adequate intake of AR can prevent and/or treat UC.

Characterization of a polysaccharide from Amauroderma rugosum and its proangiogenic activities in vitro and in vivo.

Amauroderma rugosum (AR), also known as "Blood Lingzhi" in Chinese, is a basidiomycete belonging to the Ganodermataceae family. Four polysaccharide fractions were systematically isolated and purified from AR. Subsequently, their compositions were examined and analyzed via high-performance gel permeation chromatography (HPGPC), analysis of the monosaccharide composition, Fourier-transform infrared spectroscopy (FT-IR), and 1H nuclear magnetic resonance (NMR). The zebrafish model was then used to screen for proangiogenic activities of polysaccharides by inducing vascular insufficiency with VEGF receptor tyrosine kinase inhibitor II (VRI). The third fraction of AR polysaccharides (PAR-3) demonstrated the most pronounced proangiogenic effects, effectively ameliorating VRI-induced intersegmental vessel deficiency in zebrafish. Concurrently, the mRNA expression levels of vascular endothelial growth factor (VEGF)-A and VEGF receptors were upregulated by PAR-3. Moreover, the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) were also stimulated by PAR-3, consistently demonstrating that PAR-3 possesses favorable proangiogenic properties. The activation of the Akt, ERK1/2, p38 MAPK, and FAK was most likely the underlying mechanism. In conclusion, this study establishes that PAR-3 isolated from Amauroderma rugosum exhibits potential as a bioresource for promoting angiogenesis.

Anti-metastatic effects of AGS-30 on breast cancer through the inhibition of M2-like macrophage polarization.

AGS-30, a new andrographolide derivative, showed significant anticancer and anti-angiogenic characteristics. However, its role in controlling macrophage polarization and tumor immune response is unknown. Thus, the main goals of this study are to investigate how AGS-30 regulates macrophage polarization and how it suppresses breast cancer metastasis. AGS-30 inhibited IL-4 and IL-13-induced RAW 264.7 and THP-1 macrophages into M2-like phenotype. However, AGS-30 did not affect the LPS and IFN-γ-induced polarization of M1-like macrophages. AGS-30 reduced the mRNA expressions of CD206, Arg-1, Fizz-1, Ym-1, VEGF, IL-10, MMP2, and MMP9 in M2-like macrophages in a concentration-dependent manner. In contrast, andrographolide treatment at 5 µM did not affect M1-like and M2-like macrophage polarization. The conditioned medium from M2-like macrophages increased 4T1 breast cancer cell migration and invasion, whereas AGS-30 inhibited these effects. In the 4T1 breast tumor xenograft mice, the tumor volume and weight were reduced without affecting body weight after receiving AGS-30. AGS-30 treatment also reduced lung and liver metastasis, with reduced STAT6, CD31, VEGF, and Ki67 protein expressions. Moreover, the tumors had considerably fewer M2-like macrophages and Arg-1 expression, but the proportion of M1-like macrophages and iNOS expression increased after AGS-30 treatment. Same results were found in the tail vein metastasis model. In conclusion, this study shows that AGS-30 inhibits breast cancer growth and metastasis, probably through inhibiting M2-like macrophage polarization. Our findings suggest that AGS-30 may be a potential immunotherapeutic alternative for metastatic breast cancer.

Medulla Tetrapanacis water extract alleviates inflammation and infection by regulating macrophage polarization through MAPK signaling pathway.

Medulla Tetrapanacis (MT) is a commonly used herb to promote lactation and manage mastitis in lactating mothers. However, its anti-inflammatory and anti-bacterial effects are currently unknown. We hypothesized that MT water extract possesses anti-inflammatory and anti-bacterial effects by modulating macrophage polarization to reduce the release of inflammatory mediators and phagocytosis via inactivation of MAPKs pathways. The chemical composition of the MT water extract was analyzed by UPLC-Orbitrap-mass spectrometry. The anti-inflammatory and anti-bacterial properties of the MT water extract were examined using LPS-stimulated inflammation and Staphylococcus aureus infection model in RAW 264.7 cells, respectively. The underlying mechanism of action of the MT water extract was also investigated. We identified eight compounds by UPLC-Orbitrap-mass spectrometry that are abundant within the MT water extract. MT water extract significantly suppressed LPS-induced nitric oxide, TNF-α and IL-6 secretion in RAW 264.7 cells which was accompanied by the promotion of macrophage polarization from pro-inflammatory towards anti-inflammatory phenotypes. MT water extract significantly suppressed the LPS-induced MAPK activation. Finally, MT water extract decreased the phagocytic capacity of the RAW 264.7 cells against S. aureus infection. MT water extract could suppress LPS-induced inflammation by promoting macrophages towards an anti-inflammatory phenotype. In addition, MT also inhibited the growth of S. aureus.

Glycyrrhizic Acid-Lipid Framework Nanovehicle Loading Triptolide for Combined Immunochemotherapy.

Despite the acknowledged advantages of combined immunochemotherapy for tumor treatment, the high efficiency of co-delivery of these combined agents into the targeted tumor tissue is still challenging. Herein, based on a "three-birds-with-one-stone" strategy, a facile glycyrrhizic acid (GL)-lipid hybrid nanoplatform loading triptolide (TP/GLLNP) is designed to better address the dilemma. Differing from the traditional liposomes with dual-drug co-delivery NPs, GL with a cholesterol-like structure is primarily employed to construct the lipid membrane skeleton of the GL-based lipid nanoparticle (GLLNP), and then triptolide (TP) is readily loaded in the lipid bilayer of GLLNP. The fabricated GLLNP possessed similar drug loading efficacy, particle size, and storage stability; none of the hemolysis; even higher membrane fluidity; and lower absorbed opsonin proteins compared with the conventional liposomes. Compared to TP-loaded traditional liposomes (TP/Lipo), TP/GLLNP exhibits significantly enhanced cellular uptake, cytotoxicity, and apoptosis of HepG2 cells. In addition, GLLNP could ameliorate tumor immunosuppression by promoting tumor-associated macrophage polarization from M2 to M1 phenotype. Furthermore, enhanced retention and accumulation in the tumor area of GLLNP could be found. As expected, TP/GLLNP displayed synergistic anti-hepatocellular carcinoma efficacy in vivo. In conclusion, this study provides an inspirational strategy to combine the anti-HCC benefits of GL and TP using a novel dual-drug co-delivery nanosystem.

Garcinone E suppresses breast cancer growth and metastasis by modulating tumor-associated macrophages polarization via STAT6 signaling.

Cancer metastasis remains the most common cause of death in breast cancer patients. Tumor-associated macrophages (TAMs) are a novel therapeutic target for the treatment of metastatic breast cancer. Despite the good anti-cancer activity of garcinone E (GE), there are no reports on its therapeutic effects on breast cancer metastasis. The objective of this study was to examine the anti-cancer effects of GE on metastatic breast cancer. RAW 264.7 and THP-1 cells were polarized to M2 macrophages by IL-4/IL-13 in vitro. A 4T1 mouse breast cancer model and the tail vein breast cancer metastasis model were used to explore the effect of GE on breast cancer growth and metastasis in vivo. In vitro studies showed that GE dose-dependently suppressed IL-4 + IL-13-induced expression of CD206 in both RAW 264.7 cells and differentiated THP-1 macrophages. However, GE did not affect the LPS + IFN-γ-induced polarization to the M1-like macrophages in vitro. GE inhibited the expression of the M2 macrophage specific genes in RAW 264.7 cells, and simultaneously impaired M2 macrophage-induced breast cancer cell proliferation and migration, and angiogenesis. In animal studies, GE significantly suppressed tumor growth, angiogenesis, and lung metastasis in 4T1 tumor-bearing mice, without causing toxicity. In both tumor and lung tissues, the proportion of M2-like TAMs was significantly decreased while the proportion of M1-like TAMs was markedly increased by GE treatment. Mechanistically, GE inhibited phosphorylation of STAT6 in vitro and in vivo. Our results demonstrate for the first time that GE suppresses breast cancer growth and pulmonary metastasis by modulating M2-like macrophage polarization through the STAT6 signaling pathway.

Study on the mechanism of Tong-Qiao-Huo-Xue decoction regulating apoptosis via ASK1/MKK4/JNK pathway in MCAO/R rats.

BACKGROUND: Activation of blood stasis is a crucial aspect of stroke treatment, and the Tong-Qiao-Huo-Xue-Decoction (TQHXD) formula is commonly utilized for this purpose. However, the mechanism underlying the protective effects of TQHXD against cerebral ischemia-reperfusion (I/R) injury is unclear. PURPOSE: Identification of the TQHXD components responsible for its protective effects and determination of their mode of action against cerebral I/R injury. METHODS: Gas chromatography (GC) and high-performance liquid chromatography (HPLC) were carried out to determine the active aspects of TQHXD. The active components and targets of TQHXD were looked up in the TCMSP and HERB databases; the Genecards, OMIM, TTD, and DrugBank databases were used to identify targets related to cerebral infarction; and the intersecting targets were obtained. The drug-ingredient-target-disease network and PPI network were subsequently built using Cytoscape 3.7.1 and STRING websites. Autodock VINA was used to perform molecular docking between the core target ASK1 and the active components of TQHXD detected by HPLC and GC. After successfully creating a rat model of middle cerebral artery occlusion (MCAO), the therapeutic effect of TQHXD was observed using triphenyltetrazolium and hematoxylin-eosin staining. We used Tunel-NeuN staining and transmission electron microscopy (TEM) to quantify hippocampal apoptosis. RT-qPCR and western blotting were used to detect protein and mRNA expression, respectively. RESULTS: HPLC and GC identified six active ingredients. Network pharmacology analyses were performed to test 66 intersection targets, including ASK1, MKK4, and JNK. Ferulic acid, HSYA, ligustilide, paeoniflorin, and muscone all displayed high binding affinity with ASK1 in molecular docking studies. The neuroprotective effects of TQHXD in I/R rats were demonstrated in the experimental models. In comparison with the model group, TQHXD decreased the apoptosis rate and reduced the protein levels of p-ASK1, caspase 3, p-MKK4, CytC, p-c-Jun, Bax/Bcl-2, and p-JNK, while considerably increasing the mRNA levels of Bcl-2 and decreasing those of Bax. CONCLUSION: By controlling the ASK1/MKK4/JNK pathway, TQHXD protects neurons from I/R damage and prevents apoptosis. Thus, TQHXD may be effective for the treatment of ischemic stroke. And the mechanism behind these therapeutic actions of TQHXD is supported by this research.

The role of p53 in the alternation of vascular functions.

Ageing is a risk factor for many degenerative diseases. Cardiovascular diseases (CVDs) are usually big burdens for elderly, caregivers and the health system. During the aging process, normal functions of vascular cells and tissue progressively lost and eventually develop vascular diseases. Endothelial dysfunction, reduced bioavailability of endothelium-derived nitric oxide are usual phenomena observed in patients with cardiovascular diseases. Myriad of studies have been done to investigate to delay the vascular dysfunction or improve the vascular function to prolong the aging process. Tumor suppressor gene p53, also a transcription factor, act as a gatekeeper to regulate a number of genes to maintain normal cell function including but not limited to cell proliferation, cell apoptosis. p53 also crosstalk with other key transcription factors like hypoxia-inducible factor 1 alpha that contribute to the progression of cardiovascular diseases. Therefore, in recent three decades, p53 has drawn scientists' attention on its effects in vascular function. Though the role of tumor suppressor gene p53 is still not clear in vascular function, it is found to play regulatory roles and may involve in vascular remodeling, atherosclerosis or pulmonary hypertension. p53 may have a divergent role in endothelial and vascular muscle cells in those conditions. In this review, we describe the different effects of p53 in cardiovascular physiology. Further studies on the effects of endothelial cell-specific p53 deficiency on atherosclerotic plaque formation in common animal models are required before the therapeutic potential can be realized.

Contribution of ENT4 to adenosine uptake in AC16 human cardiomyocytes under simulated ischemic conditions and its potential role in cardioprotection.

BACKGROUND: Nucleoside transporters are crucial in regulating the functions of adenosine. This study investigated the contribution of equilibrative nucleoside transporter (ENT) type 4 to adenosine transport in cardiomyocytes under simulated ischemic conditions and whether the inhibition of ENT4 could protect cardiomyocytes against ischemia-reperfusion injury. METHODS: AC16 human cardiomyocytes were used to create a model to simulate ischemia/reperfusion injury. ENT4 activity was inhibited by decynium-22 or specific siRNA against ENT4. The protein expressions of nucleoside transporters were measured by western blot analysis. The transport activity was studied by [3?H]adenosine uptake. The cell injury was studied by biochemical assays. RESULTS: The [3?H]adenosine uptake in AC16 cells was predominantly mediated by ENTs. ENT1 to ENT4 were present in AC16 cells and their protein expression levels were comparable in normal and ischemic conditions. Decynium-22 or siRNA against ENT4 did not affect the adenosine uptake in AC16 cells under normal conditions but could inhibit the adenosine uptake in AC16 cells by 28% under ischemic conditions. In addition, the cell viability and lactate dehydrogenase release of AC16 cells under ischemia conditions could be reduced by decynium-22 or siRNA against ENT4. CONCLUSION: The cell culture model has suggested that ENT4 may play a role in adenosine transport in cardiomyocytes under ischemic conditions. Inhibition or downregulation of ENT4 may be a potential approach for cardioprotection but this notion should be further validated using animal model.

Tong-Qiao-Huo-Xue decoction activates PI3K/Akt/mTOR pathway to reduce BMECs autophagy after cerebral ischemia/reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Tong-Qiao-Huo-Xue Decoction (TQHXD) is a traditional classic Chinese Medicinal Formula (CMF) used for clinical treatment of ischemic stroke. TQHXD leads to improvement in the symptoms of the acute period of cerebral infarction and recovery period after stroke. Our previous studies also showed that TQHXD produced a significant protective effect on the brain after cerebral ischemia-reperfusion (I/R) injury. It is reported that autophagy is closely related to ischemic brain injury; however, the functional contribution of TQHXD to brain microvascular endothelial cell (BMEC) autophagy and its underlying mechanism remains unclear. AIM OF THE STUDY: The purpose of this study was to investigate the effects and mechanism of TQHXD in inhibiting cerebral ischemia-induced endothelial autophagy. MATERIALS AND METHODS: The high-performance liquid chromatography (HPLC) fingerprint of the chemical constituents from TQHXD was established for the quality control, and the Longa method was used to evaluate the efficacy of TQHXD in rats with middle cerebral artery occlusion (MCAO). The expression of LC3 was determined by immunofluorescence double staining. To evaluate the protective effects of TQHXD-containing cerebrospinal fluid (CSF) on BMECs injured by oxygen-glucose deprivation and reperfusion, cell survival rate was determined using the CCK-8 assay and cell apoptosis was determined by fluorescein isothiocyanate (FITC)-Annexin V/PI. Autophagy was detected using transmission electron microscopy. RESULTS: The results showed that TQHXD-CSF significantly ameliorated oxygen-glucose deprivation/reperfusion (OGD/R)-induced injury in BMECs. Confocal microscopy and Western blot results showed that TQHXD-CSF reduced autophagy-related protein expression and autophagosome number. The results of the western blotting indicated that TQHXD-CSF caused a marked increase in the phosphorylation of protein kinase B and phosphoinsotide-3 kinase (Akt/p-Akt and PI3K/p-PI3K, respectively) and their expression levels were down-regulated after treatment with pathway inhibitor, ZSTK474. Furthermore, in a MCAO model in rats, TQHXD markedly increased p-PI3K, p-Akt and p-mTOR, whereas the autophagy related proteins decreased. CONCLUSIONS: Taken together, these findings demonstrate that TQHXD protects against ischemic insult by inhibiting autophagy through the regulation of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway and that TQHXD may have therapeutic value for protecting BMECs from cerebral ischemia.

Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters.

Equilibrative nucleoside transporters (ENTs) play a vital role in nucleotide synthesis, regulation of adenosine function and chemotherapy. Current inhibitors of ENTs are mostly ENT1-selective. Our previous study has demonstrated that 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) is a novel inhibitor of ENTs, which is more selective to ENT2 than to ENT1. The present study aimed to screen a series of FPMINT analogues and study their structure-activity relationship. Nucleoside transporter-deficient cells transfected with cloned human ENT1 and ENT2 were used as in vitro models. The results of the [3H]uridine uptake study showed that the replacement of the naphthalene moiety with the benzene moiety could abolish the inhibitory effects on ENT1 and ENT2. The addition of chloride to the meta position of this benzene moiety could restore only the inhibitory effect on ENT1 but had no effect on ENT2. However, the addition of the methyl group to the meta position or the ethyl or oxymethyl group to the para position of this benzene moiety could regain the inhibitory activity on both ENT1 and ENT2. The presence of a halogen substitute, regardless of the position, in the fluorophenyl moiety next to the piperazine ring was essential for the inhibitory effects on ENT1 and ENT2. Among the analogues tested, compound 3c was the most potent inhibitor. Compound 3c reduced V max of [3H]uridine uptake in ENT1 and ENT2 without affecting K m. The inhibitory effect of compound 3c could not be washed out. Compound 3c did not affect cell viability, protein expression and internalization of ENT1 and ENT2. Therefore, similar to FPMINT, compound 3c was an irreversible and non-competitive inhibitor. Molecular docking analysis also showed that the binding site of compound 3c in ENT1 may be different from that of other conventional inhibitors. It is expected that structural modification may further improve its potency and selectivity and lead to the development of useful pharmacological agents.

Hydroxysafflor Yellow a Promotes Angiogenesis in Rat Brain Microvascular Endothelial Cells Injured by Oxygen-glucose Deprivation/reoxygenation(OGD/R) through SIRT1-HIF-1α-VEGFA Signaling Pathway.

OBJECTIVE: Angiogenesis led by brain microvascular endothelial cells (BMECs) contributes to the remission of brain injury after brain ischemia reperfusion. In this study, we investigated the effects of hydroxysafflor yellow A(HSYA) on angiogenesis of BMECs injured by OGD/R via SIRT1-HIF-1α-VEGFA signaling pathway. METHODS: The OGD/R model of BMECs was established in vitro by OGD for 2h and reoxygenation for 24h. At first, the concentrations of vascular endothelial growth factor (VEGF), Angiopoietin (ang) and platelet-derived growth factor (PDGF) in supernatant were detected by ELISA, and the proteins expression of VEGFA, Ang-2 and PDGFB in BMECs were tested by western blot; the proliferation, adhesion, migration (scratch healing and transwell) and tube formation experiment of BMECs; the expression of CD31 and CD34 were tested by immunofluorescence staining. The levels of sirtuin1(SIRT1), hypoxia-inducible factor-1α (HIF-1α), VEGFA mRNA and protein were tested. RESULTS: HSYA up-regulated the levels of VEGF, Ang and PDGF in the supernatant of BMECs under OGD/R, and the protein expression of VEGFA, Ang-2 and PDGFB was increased; HSYA could significantly alleviate the decrease of cell proliferation, adhesion, migration and tube formation ability of BMECs during OGD/R; HSYA enhanced the fluorescence intensity of CD31 and CD34 of BMECs during OGD/R; HSYA remarkably up-regulated the expression of SIRT1, HIF-1α, VEGFA mRNA and protein after OGD/R, and these increase decreased after SIRT1 was inhibited. CONCLUSION: SIRT1-HIF-1α-VEGFA signaling pathway is involved in HSYA improves angiogenesis of BMECs injured by OGD/R.

Relaxation effect of narirutin on rat mesenteric arteries via nitric oxide release and activation of voltage-gated potassium channels.

Narirutin is one of the most common flavanones found in citrus fruits. The vascular effects of its analogues naringenin and naringin have been reported but its effects on the cardiovascular system are largely unknown. In this study, relaxation effect of narirutin and its mechanisms of action were investigated by measuring isometric tension in rat mesenteric arteries. Patch-clamping was also used to study the effect of narirutin on potassium channels in vascular smooth muscle cells. Moreover, its effects on phosphorylation of endothelial nitric oxide synthase, cAMP level and phosphodiesterase activity in rat mesenteric arteries were studied by Western blot and biochemical assays. The results showed that pre-incubation of rat mesenteric arteries with narirutin had no influence on acetylcholine-induced endothelial-dependent relaxation. However, narirutin caused a direct concentration-dependent relaxation in rat mesenteric arteries. This relaxation effect was comparable to that of narirutin's structural analogue naringenin. Narirutin-induced relaxation was reduced by the removal of endothelium, NG-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor), and 4-aminopyridine (a voltage-gated potassium channel blocker). In addition, narirutin increased the phosphorylation of endothelial nitric oxide synthase and increased the voltage-dependent potassium current in mesenteric arterial smooth muscle cells. These effects were abolished by protein kinase A inhibitor. Furthermore, narirutin could increase cAMP level and inhibit phosphodiesterase activity in rat mesenteric arteries. In conclusion, narirutin has vasorelaxing effect and the mechanism involves the inhibition of phosphodiesterase, which increases intracellular cAMP, thereby stimulating the endothelial nitric oxide synthase and activating the voltage-gated potassium channels in vascular smooth muscle cells.

Factor XII blockade inhibits aortic dilatation in angiotensin II-infused apolipoprotein E-deficient mice.

Abdominal aortic aneurysm (AAA) is an important cause of mortality in older adults. Chronic inflammation and excessive matrix remodelling are considered important in AAA pathogenesis. Kinins are bioactive peptides important in regulating inflammation. Stimulation of the kinin B2 receptor has been previously reported to promote AAA development and rupture in a mouse model. The endogenous B2 receptor agonist, bradykinin, is generated from the kallikrein-kinin system following activation of plasma kallikrein by Factor XII (FXII). In the current study whole-body FXII deletion, or neutralisation of activated FXII (FXIIa), inhibited expansion of the suprarenal aorta (SRA) of apolipoprotein E-deficient mice in response to angiotensin II (AngII) infusion. FXII deficiency or FXIIa neutralisation led to decreased aortic tumor necrosis factor-α-converting enzyme (TACE/a disintegrin and metalloproteinase-17 (aka tumor necrosis factor-α-converting enzyme) (ADAM-17)) activity, plasma kallikrein concentration, and epithelial growth factor receptor (EGFR) phosphorylation compared with controls. FXII deficiency or neutralisation also reduced Akt1 and Erk1/2 phosphorylation and decreased expression and levels of active matrix metalloproteinase (Mmp)-2 and Mmp-9. The findings suggest that FXII, kallikrein, ADAM-17, and EGFR are important molecular mediators by which AngII induces aneurysm in apolipoprotein E-deficient mice. This could be a novel pathway to target in the design of drugs to limit AAA progression.

Pro-angiogenic effects of Ilexsaponin A1 on human umbilical vein endothelial cells in vitro and zebrafish in vivo.

BACKGROUND: Ilexsaponin A1 is the major bioactive ingredient of Ilex pubescens Hook. et Arn. This plant has been conventionally used in Traditional Chinese Medicine for the treatment of cardiovascular diseases including stroke, coronary arterial disease, and peripheral vascular diseases. PURPOSE: To investigate the pro-angiogenic effect of Ilexsaponin A1 and its mechanism of action. STUDY DESIGN: Human umbilical vein endothelial cells (HUVECs) and transgenic zebrafish Tg(fli1:EGFP) were employed as an in vitro and in vivo model respectively. METHODS: Pro-angiogenic effects of Ilexsaponin A1 were examined by assessing endothelial cell proliferation, migration, invasion and tube formation. The mechanism of pro-angiogenic effects was investigated by measuring the expression level of various signalling proteins. Furthermore, vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor II (VRI)-induced vascular insufficient transgenic zebrafish model was used to confirm the results of the HUVECs results in vivo. RESULTS: Ilexsaponin A1 significantly promoted cell proliferation, migration, invasion and tube formation in HUVECs, and rescued blood vessel loss in VRI-induced vascular insufficient zebrafish. Ilexsaponin A1 upregulated p-Akt, p-mTOR, p-Src, p-FAK, p-MEK, and p-Erk1/2 in HUVECs. CONCLUSION: This study showed that Ilexsaponin A1 exhibits pro-angiogenic activity in HUVECs and VRI-induced vascular insufficient zebrafish, probably by activating Akt/mTOR, MAPK/ERK and Src- and FAK-dependent signalling pathways. The findings suggest that Ilexsaponin A1 and probably I. pubescens, a major source of Ilexsaponin A1, could be developed as a potential therapeutic agent for preventing or treating cardiovascular diseases and/or other diseases related to vascular insufficiency.

Wnt Signaling Pathway Inhibitor Sclerostin Inhibits Angiotensin II-Induced Aortic Aneurysm and Atherosclerosis.

OBJECTIVE: Sclerostin (SOST) has been identified as an important regulator of bone formation; however, it has not been previously implicated in arterial disease. The aim of this study was to assess the role of SOST in aortic aneurysm (AA) and atherosclerosis using human samples, a mouse model, and in vitro investigations. APPROACH AND RESULTS: SOST protein was downregulated in human and mouse AA samples compared with controls. Transgenic introduction of human SOST in apolipoprotein E-deficient (ApoE-/-) mice (SOSTTg .ApoE-/-) and administration of recombinant mouse Sost inhibited angiotensin II-induced AA and atherosclerosis. Serum concentrations of several proinflammatory cytokines were significantly reduced in SOSTTg .ApoE-/- mice. Compared with controls, the aortas of mice receiving recombinant mouse Sost and SOSTTg .ApoE-/- mice showed reduced matrix degradation, reduced elastin breaks, and preserved collagen. Decreased inflammatory cell infiltration and a reduction in the expression of wingless-type mouse mammary virus integration site/ß-catenin responsive genes, including matrix metalloproteinase-9, osteoprotegerin, and osteopontin, were observed in the aortas of SOSTTg .ApoE-/- mice. SOST expression was downregulated and the wingless-type mouse mammary virus integration site/ß-catenin pathway was activated in human AA samples. The cytosine-phosphate-guanine islands in the SOST gene promoter showed significantly higher methylation in human AA samples compared with controls. Incubation of vascular smooth muscle cells with the demethylating agent 5-azacytidine resulted in upregulation of SOST, suggesting that SOST is epigenetically regulated. CONCLUSIONS: This study identifies that SOST is expressed in the aorta and downregulated in human AA possibly because of epigenetic silencing. Upregulating SOST inhibits AA and atherosclerosis development, with potential important implications for treating these vascular diseases.

Mouse models of intracranial aneurysm.

Subarachnoid hemorrhage secondary to rupture of an intracranial aneurysm is a highly lethal medical condition. Current management strategies for unruptured intracranial aneurysms involve radiological surveillance and neurosurgical or endovascular interventions. There is no pharmacological treatment available to decrease the risk of aneurysm rupture and subsequent subarachnoid hemorrhage. There is growing interest in the pathogenesis of intracranial aneurysm focused on the development of drug therapies to decrease the incidence of aneurysm rupture. The study of rodent models of intracranial aneurysms has the potential to improve our understanding of intracranial aneurysm development and progression. This review summarizes current mouse models of intact and ruptured intracranial aneurysms and discusses the relevance of these models to human intracranial aneurysms. The article also reviews the importance of these models in investigating the molecular mechanisms involved in the disease. Finally, potential pharmaceutical targets for intracranial aneurysm suggested by previous studies are discussed. Examples of potential drug targets include matrix metalloproteinases, stromal cell-derived factor-1, tumor necrosis factor-α, the renin-angiotensin system and the ß-estrogen receptor. An agreed clear, precise and reproducible definition of what constitutes an aneurysm in the models would assist in their use to better understand the pathology of intracranial aneurysm and applying findings to patients.

Role of the angiotensin converting enzyme 1/angiotensin II/angiotensin receptor 1 axis in interstitial collagenase expression in human carotid atheroma.

BACKGROUND AND AIM: Angiotensin II (AII) receptor 1 (ATR1) and angiotensin converting enzyme 1 (ACE1) blockers have been shown to reduce acute cardiovascular events in patients, improve plaque stability and modify matrix metalloproteinase (MMP) expression. However, the role of the ACE1/AII/ATR1 axis in interstitial collagenase regulation has not been fully explored. In this study, we investigated the effect of ATR1 and ACE1 blockade on the expression and activity of MMP-1, -8 and -13 in human carotid atheroma. METHODS: Atheroma samples (n = 24) were obtained from patients undergoing carotid endarterectomy. The effects of ATR1 (irbesartan), ACE1 (quinapril), ACE2 (DX600) and MMP (GM6001) blockade on the expression of AII, the interstitial collagenases and soluble elastin fragments were investigated in explant culture supernatants. Paired atheroma samples were incubated with intervention or media control for 4 days. Protein levels (AII, MMP-1, -8, -13 and soluble elastin) were determined by ELISA. RESULTS: ATR1, but not ACE1, blockade significantly reduced MMP-1 and -8 concentrations in atheroma supernatants. ACE2 blockade significantly increased MMP-1 and -8 concentrations in atheroma supernatants. AII concentration in atheroma supernatants significantly increased after ATR1, ACE1 and ACE2 blockade. Release of soluble elastin fragments increased after ATR1 and ACE1 blockade, but was not changed by an MMP inhibitor. CONCLUSIONS: Our findings suggest that ATR1 blockade alters AII, MMP-1, MMP-8 expression and a marker of elastin degradation in human atheroma, but that the elastin degradation response is not MMP driven. This data contributes to the recognised ability of ATR1 blockade to modify plaque stability.