Dipeptidyl peptidase-4 inhibitor improves neovascularization by increasing circulating endothelial progenitor cells

BACKGROUND AND PURPOSE

Current methods used to treat critical limb ischaemia (CLI) are hampered by a lack of effective strategies, therefore, therapeutic vasculogenesis may open up a new field for the treatment of CLI. In this study we investigated the ability of the DPP-4 inhibitor, sitagliptin, originally used as a hypoglycaemic agent, to induce vasculogenesis in vivo.

EXPERIMENTAL APPROACH

Sitagliptin were administered daily to C57CL/B6 mice and eGFP transgenic mouse bone marrow-transplanted ICR mice that had undergone hindlimb ischaemic surgery. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neovasculogenesis and circulating levels of endothelial progenitor cells (EPCs) respectively. Cell surface markers of EPCs and endothelial NOS (eNOS) in vessels were studied.

KEY RESULTS

Sitagliptin elevated plasma glucagon-like peptide-1 (GLP-1) levels in mice subjected to ischaemia, decreased plasma dipeptidyl peptidase-4 (DPP-4) concentration, and augmented ischaemia-induced increases in stromal cell-derived factor-1 (SDF-1) in a dose-dependent manner. Blood flow in the ischaemic limb was significantly improved in mice treated with sitagliptin. Circulating levels of EPCs were also increased after sitagliptin treatment. Sitagliptin also enhanced the expression of CD 34 and eNOS in ischaemic muscle. In addition, sitagliptin promoted EPC mobilization and homing to ischaemic tissue in eGFP transgenic mouse bone marrow-transplanted ICR mice.

CONCLUSION AND IMPLICATIONS

Circulating EPC levels and neovasculogenesis were augmented by the DPP-4 inhibitor, sitagliptin and this effect was dependent on an eNOS-related pathway in a mouse model of hindlimb ischaemia. The results indicate that oral administration of sitagliptin has therapeutic potential as an inducer of vasculogenesis.     

Atorvastatin attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells via inhibiting NADPH oxidase-related oxidative stress-triggered p38MAPK signaling

Aim:

To examine the effect of atorvastatin on homocysteine (Hcy)-induced reactive oxygen species (ROS) production and apoptosis in human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were cultured with Hcy (0.1−5 mmol/L) in the presence or absence of atorvastatin (1−100 μmol//L) or various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI, 10 μmol/L), the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 μmol/L) and antioxidants N-acetyl cysteine (NAC, 1 mmol/L). Cell apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometry. ROS were detected by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Hcy-induced expression of p38MAPK protein was measured by Western blotting analysis.

Results:

Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 μmol/L. Atorvastatin (10 μmol/L) significantly suppressed Hcy (1 mmol/L for 30 min) induced ROS accumulation (3.17±0.33 vs 4.34±0.31, P<0.05). Atorvastatin (10 μmol/L) also antagonized Hcy (1 mmol/L for 30 min) induced activation of NADPH oxidase (2.57±0.49 vs 3.33±0.6, P<0.05). Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7±0.1 vs 2.22±0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580.

Conclusion:

Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function.     

Ginsenoside Rg1 promotes endothelial progenitor cell migration and proliferation

Aim:

To investigate the effect of ginsenoside Rg1 on the migration, adhesion, proliferation, and VEGF expression of endothelial progenitor cells (EPCs).

Methods:

EPCs were isolated from human peripheral blood and incubated with different concentrations of ginsenoside Rg1 (0.1, 0.5, 1.0, and 5.0 μmol/L) and vehicle controls. EPC migration was detected with a modified Boyden chamber assay. EPC adhesion was determined by counting adherent cells on fibronectin-coated culture dishes. EPC proliferation was analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In vitro vasculogenesis was assayed using an in vitro vasculogenesis detection kit. A VEGF-ELISA kit was used to measure the amount of VEGF protein in the cell culture medium.

Results:

Ginsenoside Rg1 promoted EPC adhesion, proliferation, migration and in vitro vasculogenesis in a dose- and time-dependent manner. Cell cycle analysis showed that 5.0 μmol/L of ginsenoside Rg1 significantly increased the EPC proliferative phase (S phase) and decreased the resting phase (G₀/G₁ phase). Ginsenoside Rg1 increased vascular endothelial growth factor production.

Conclusion:

The results indicate that ginsenoside Rg1 promotes proliferation, migration, adhesion and in vitro vasculogenesis.     

Salidroside exerts angiogenic and cytoprotective effects on human bone marrowderived endothelial progenitor cells via Akt/mTOR/p70S6K and MAPK signalling pathways

Background and Purpose

With the increase of age, increased susceptibility to apoptosis and senescence may contribute to proliferative and functional impairment of endothelial progenitor cells (EPCs). The aim of this study was to investigate whether salidroside (SAL) can induce angiogenic differentiation and inhibit oxidative stress-induced apoptosis in bone marrow-derived EPCs (BM-EPCs), and if so, through what mechanism.

Experimental Approach

BM-EPCs were isolated and treated with different concentrations of SAL for up to 4 days. Cell proliferation, migration and tube formation ability were detected by DNA content quantification, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively.

Key Results

Treatment with SAL promoted cellular proliferation and angiogenic differentiation of BM-EPCs, and increased VEGF and NO secretion, which in turn mediated the enhanced angiogenic differentiation of BM-EPCs. Furthermore, SAL significantly attenuated hydrogen peroxide (H₂O₂)-induced cell apoptosis, reduced the intracellular level of reactive oxygen species and restored the mitochondrial membrane potential of BM-EPCs. Moreover, SAL stimulated the phosphorylation of Akt, mammalian target of rapamycin and p70 S6 kinase, as well as ERK1/2, which is associated with cell migration and capillary tube formation. Additionally, SAL reversed the phosphorylation of JNK and p38 MAPK induced by H₂O₂ and suppressed the changes in the Bax/Bcl-xL ratio observed after stimulation with H₂O₂.

Conclusions and Implications

These findings identify novel mechanisms that regulate EPC function and suggest that SAL has therapeutic potential as a new agent to enhance vasculogenesis as well as protect against oxidative endothelial injury.     

FM19G11 reverses endothelial dysfunction in rat and human arteries through stimulation of the PI3K/Akt/eNOS pathway,independently of mTOR/HIF-1α activation

Background and Purpose

FM19G11 up-regulates mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α) and PI3K/Akt pathways, which are involved in endothelial function. We evaluated the effects of FM19G11 on defective endothelial vasodilatation in arteries from rats and humans and investigated the mechanisms involved.

Experimental Approach

Effects of chronic in vivo administration of FM19G11 on aortic endothelial vasodilatation were evaluated together with ex vivo treatment in aortic and mesenteric arteries from control and insulin-resistant rats (IRR). Its effects on vasodilator responses of penile arteries (HPRAs) and corpus cavernosum (HCC) from men with vasculogenic erectile dysfunction (ED) (model of human endothelial dysfunction) were also evaluated. Vascular expression of phosphorylated-endothelial NOS (p-eNOS), phosphorylated-Akt (p-Akt) and HIF-1α was determined by immunodetection and cGMP by elisa.

Key Results

Chronic administration of FM19G11 reversed the impaired endothelial vasodilatation in IRR. Ex vivo treatment with FM19G11 also significantly improved endothelium-dependent vasodilatation in aorta and mesenteric arteries from IRR. These effects were accompanied by the restoration of p-eNOS and cGMP levels in IRR aorta and were prevented by either NOS or PI3K inhibition. p-Akt and p-eNOS contents were increased by FM19G11 in aortic endothelium of IRR. FM19G11-induced restoration of endothelial vasodilatation was unaffected by mTOR/HIF-1α inhibitors. FM19G11 also restored endothelial vasodilatation in HPRA and HCC from ED patients.

Conclusions and Implications

Stimulation of the PI3K/Akt/eNOS pathway by FM19G11 alleviates impaired NO-mediated endothelial vasodilatation in rat and human arteries independently of mTOR/HIF-1α activation. This pharmacological strategy could be beneficial for managing pathological conditions associated with endothelial dysfunction, such as ED.     

Icaritin suppresses the proliferation of human osteosarcoma cells in vitro by increasing apoptosis and decreasing MMP expression

Aim： To explore whether icaritin, a prenylflavonoid derivative of the Chinese tonic herb Epimedium, could suppress the proliferation of human osteosarcoma cells in vitro, and to elucidate the mechanisms of the action.
Methods： Human osteosarcoma SaOS2 cell line was used in the present study. The proliferation of the cells was examined using MTT assay and immunofluorescence DAPI staining. Cell motility was studied with the scratch assay. Cell apoptosis was determined by Annexin V-FITC and PI double staining using flow cytometry. Western blotting and RT-PCR were used to measure the expression of mRNAs and proteins in the cells.

Results： Icaritin （5–15 μmol/L） suppressed the proliferation of SaOS2 cells in vitro in a dose-dependent manner. Furthermore, the cell motility was significantly decreased after exposure to icaritin. Moreover, icaritin （5 μmol/L） time-dependently induced the apoptosis of SaOS2 cells, markedly suppressed MMP-2 and MMP-9 expression, upregulated caspase-3 and caspase-9 expression, and increased the level of cleaved caspase-3 in the cells. Co-exposure to the caspase-3 inhibitor zVAD-fmk （10 μmol/L） compromised the icaritin-induced caspase-3 expression and apoptosis in SaOS2 cells.

Conclusion： Icaritin suppresses the proliferation of SaOS2 human osteosarcoma cells by increasing apoptosis and downregulating MMP expression.     

Resveratrol reduces endothelial progenitor cells senescence through augmentation of telomerase activity by Akt-dependent mechanisms

Background and purpose:

Recent studies have shown that resveratrol increased endothelial progenitor cells (EPCs) numbers and functional activity. However, the mechanisms remain to be determined. Previous studies have demonstrated that increased EPC numbers and activity were associated with the inhibition of EPC senescence, which involves activation of telomerase. Therefore, we investigated whether resveratrol inhibits the onset of EPC senescence through telomerase activation, leading to potentiation of cellular activity.

Experimental approach:

After prolonged in vitro cultivation, EPCs were incubated with or without resveratrol. The senescence of EPCs were determined by acidic β-galactosidase staining. The bromo-deoxyuridine incorporation assay or a modified Boyden chamber assay were employed to assess proliferative or migratory capacity, respectively. To further examine the underlying mechanisms of these effects, we measured telomerase activity and the phosphorylation of Akt by western blotting.

Key results:

Resveratrol dose dependently prevented the onset of EPCs senescence and increased the proliferation and migration of EPCs. The effect of resveratrol on senescence could not be abolished by eNOS inhibitor or by an oestrogenic receptor antagonist. Resveratrol significantly increased telomerase activity and Akt phosphorylation. Pre-treatment with the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, significantly attenuated resveratrol-induced telomerase activity.

Conclusions and implications:

Resveratrol delayed the onset of EPC senescence and this effect was accompanied by activation of telomerase through the PI3K-Akt signalling pathway. The inhibition of EPCs senescence by resveratrol might protect EPCs against dysfunction induced by pathological factors in vivo and improve EPC functional activities in a way that may be important for cell therapy.     

Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549

Aim:

To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.

Methods:

A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.

Results:

Treatment of A549 cells with CrTX (25–200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC₅₀=78 μg/mL). Treatment with CrTX (25 μg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 μg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G₁ arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 μg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels.

Conclusion:

CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo.     

Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogenactivated protein kinase in mouse lung

Aim:

To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling.

Methods:

Male C57BL/J6 (wild-type, WT) or eNOS knockout mice (eNOS^−/−) were exposed to mechanical ventilation (MV) with low (LV_T, 7 mL/kg) and high tidal volume (HV_T, 20 mL/kg) for 0−4 h. A subset of WT mice was administered the specific inhibitors of PI3K (100 nmol/L Wortmannin [Wort], ip) or of p38 MAPK (SB203580, 2 mg/kg, ip) 1 h before MV. Cultured type II alveolar epithelial cells C10 were exposed to 18% cyclic stretch for 2 h with or without 20 nmol/L Wort pretreatment. At the end of the experiment, the capillary leakage in vivo was assessed by extravasation of Evans blue dye (EBD), wet/dry weight ratio and lung lavage protein concentration. The lung tissue and cell lysate were also collected for protein and histological review.

Results:

MV decreased PI3K/Akt phosphorylation and eNOS expression but increased phospho-p38 MAPK expression along with a lung leakage of EBD. Inhibitions of phospho-Akt by Wort worsen the lung edema, whereas inhibition of p38 MAPK kinase restored activation of Akt together with alleviated capillary leakage. eNOS^−/− mice showed an exacerbated lung edema and injury. The stretched C10 cells demonstrated that Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38.

Conclusion:

Our results indicate that PI-3K/Akt/eNOS pathway has significant protective effects in VALI by preventing capillary leakage, and that there is a cross-talk between PI3K/Akt and p38 MAPK pathways in vascular barrier dysfunction resulting from VALI.     

Atorvastatin prevents amyloid-13 peptide oligomer- induced synaptotoxicity and memory dysfunction in rats through a p38 MAPK-dependent pathway

Aim： To investigate whether atorvastatin treatment could prevent Aβ1-42 oligomer （AβO）-induced synaptotoxicity and memory dysfunction in rats, and to elucidate the mechanisms involved in the neuroprotective actions of atorvastatin.
Methods： SD rats were injected with AβOs （5 nmol, icv）. The rats were administrated with atorvastatin （10 mg·kg^-1·d^-1, po） for 2 consecutive weeks （the first dose was given 5 d before AβOs injection）. The memory impairments were evaluated with Morris water maze task. The expression of inflammatory cytokines in the hippocampus was determined using ELISA assays. The levels of PSD-95 and p38MAPK proteins in rat hippocampus were evaluated using Western blot analysis. For in vitro experiments, cultured rat hippocampal neurons were treated with AβOs （50 nmol/L） for 48 h. The expression of MAP-2 and synaptophysin in the neurons was detected with immunofluorescence.
Results： The AβO-treated rats displayed severe memory impairments in Morris water maze tests, and markedly reduced levels of synaptic proteins synaptophysin and PSD-95, increased levels of inflammatory cytokines （IL-1β, IL-6 and TNF-α） and p38MAPK activation in the hippocampus. All these effects were prevented or substantially attenuated by atorvastatin administration. Pretreatment of cultured hippocampal neurons with atorvastatin （1 and 5 μmol/L） concentration-dependently attenuated the AβO-induced synaptotoxicity, including the loss of dendritic marker MAP-2, and synaptic proteins synaptophysin and PSD-95. Pretreatment of the cultured hippocampal neurons with the p38MAPK inhibitor SB203580 （5 μmol/L） blocked the AβO-induced loss of synaptophysin and PSD-95.
Conclusion： Atorvastatin prevents AβO-induced synaptotoxicity and memory dysfunction through a p38MAPK-dependent pathway.     

γ-Secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway

Aim： Trans-4-[4-（3-adamantan-1-yl-ureido）-cyclohexyloxy]-benzoic acid （t-AUCB） is a soluble epoxide hydrolase inhibitor that suppresses glioblastoma cell growth in vitro. The aim of this study was to examine whether the γ-secretase inhibitor N-[N-（3,5-difluorophenacetyl）-l-alanyl]-S-phenylglycine t-butyl ester （DAPT） could sensitize glioma cells to t-AUCB-induced apoptosis.
Methods： Both U251 and U87 human glioblastoma cell lines were tested. Cell growth was assessed using the cell counting kit-8. Cell apoptosis was detected with caspase-3 activity assay kits and flow cytometry. The protein levels in the p38 MAPK/MAPKAPK2/Hsp27 pathway in the cells were analyzed using Western blots.
Results： Pretreatment with DAPT （2 μmol/L） substantially potentiated the growth inhibition caused by t-AUCB （200 μmol/L） in U251 and U87 cells. Furthermore, pretreatment with DAPT markedly increased t-AUCB-induced apoptosis of U251 and U87 cells. T-AUCB alone did not significant affect caspase-3 activity in the cells, but t-AUCB plus DAPT pretreatment caused significant increase of caspase-3 activity. Furthermore, pretreatment with DAPT completely blocked t-AUCB-induced phosphorylation of p38 MAPK, MAPKAPK2 and Hsp27 in the cells.
Conclusion： The γ-secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway, suggesting that the combination of t-AUCB and DAPT may be a potentially effective strategy for the treatment of glioblastoma.     

Effects of angiotensin receptor blockers on endothelial nitric oxide release: the role of eNOS variants

AIM

Angiotensin II receptor blockers (ARBs) improve endothelial cell (EC)-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide (NO) synthase (eNOS) function. To investigate this question, we tested the effects of various ARBs on NO release in ECs from multiple donors, including those with eNOS genetic variants linked to higher cardiovascular risk.

METHODS

The effects of ARBs (losartan, olmesartan, telmisartan, valsartan), at 1 µm, on NO release were measured with nanosensors in human umbilical vein ECs obtained from 18 donors. NO release was stimulated with calcium ionophore (1 µm) and its maximal concentration was correlated with eNOS variants. The eNOS variants were determined by a single nucleotide polymorphism in the promoter region (T-786C) and in the exon 7 (G894T), linked to changes in NO metabolism.

RESULTS

All of the ARBs caused an increase in NO release as compared with untreated samples (P < 0.01, n = 4–5 in all eNOS variants). However, maximal NO production was differentially influenced by eNOS genotype. Olmesartan increased maximal NO release by 30%, which was significantly greater (P < 0.01, n = 4–5 in all eNOS variants) than increases observed with other ARBs.

CONCLUSIONS

The ARBs differentially enhanced NO release in ECs in a manner influenced by eNOS single nucleotide polymorphisms. These findings provide new insights into the effects of ARBs on EC-dependent vasodilation and eNOS function.     

KMUP-3 attenuates ventricular remodelling after myocardial infarction through eNOS enhancement and restoration of MMP-9/TIMP-1 balance

BACKGROUND AND PURPOSE

Previously, 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1, 3-dimethylxanthine (KMUP-3) has been shown to induce aortic smooth muscle relaxation through K_ATP channel opening and endothelial nitric oxide synthase (eNOS) enhancement. We further investigated whether KMUP-3 protects against myocardial remodelling after myocardial infarction (MI), and whether KMUP-3 increases the expression of eNOS in MI rats.

EXPERIMENTAL APPROACH

Wistar rats were randomly allocated into three groups: MI (n= 10), MI + KMUP-3 group (n= 10) and sham group (n= 10). MI was induced by ligation of the left anterior descending coronary artery. After recovery, the MI + KMUP-3 group received KMUP-3 (0.3 mg·kg⁻¹·day⁻¹) infusion for 4 weeks, while the MI and sham group received vehicle only. To further confirm that the effect of KMUP-3 is dependent on eNOS, KMUP-3 was applied in the culture of transforming growth factor-β-stimulated human cardiac fibroblasts.

KEY RESULTS

KMUP-3 treatment attenuated cardiac hypertrophy post-MI and improved cardiac function. The fibrotic area was reduced by KMUP-3 both in central-, peri- and non-infarction areas. KMUP-3 enhanced the expression of eNOS and tissue inhibitor of metalloproteinase-1 (TIMP-1), but reduced matrix metalloproteinase-9 (MMP-9) expression. In vitro, the activities of KMUP-3 were blocked by pretreatment with the eNOS inhibitor N^ω-nitro-L-arginine methyl ester.

CONCLUSIONS AND IMPLICATIONS

The K_ATP channel opener KMUP-3 preserved cardiac function after MI by enhancing the expression of eNOS. In addition, KMUP-3 restored the myocardial MMP-9/TIMP-1 balance and attenuated ventricular remodelling by an eNOS-dependent mechanism.     

Glucagon-like peptide-1 activates endothelial nitric oxide synthase in human umbilical vein endothelial cells

Aim:

To investigate the effects of glucagon-like peptide-1 (GLP-1) on endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVECs), and elucidate whether GLP-1 receptor (GLP-1R) and GLP-1(9–36) are involved in these effects.

Methods:

HUVECs were used. The activity of eNOS was measured with NOS assay kit. Phosphorylated and total eNOS proteins were detected using Western blot analysis. The level of eNOS mRNA was quantified with real-time RT-PCR.

Results:

Incubation of HUVECs with GLP-1 (50–5000 pmol/L) for 30 min significantly increased the activity of eNOS. Incubation of HUVECs with GLP-1 (500–5000 pmol/L) for 5 or 10 min increased eNOS phosphorylated at ser-1177. Incubation with GLP-1 (5000 pmol/L) for 48 h elevated the level of eNOS protein, did not affect the level of eNOS mRNA. GLP-1R agonists exenatide and GLP-1(9–36) at the concentration of 5000 pmol/L increased the activity, phosphorylation and protein level of eNOS. GLP-1R antagonist exendin(9–39) or DPP-4 inhibitor sitagliptin, which abolished GLP-1(9–36) formation, at the concentration of 5000 pmol/L partially blocked the effects of GLP-1 on eNOS.

Conclusion:

GLP-1 upregulated the activity and protein expression of eNOS in HUVECs through the GLP-1R-dependent and GLP-1(9–36)-related pathways. GLP-1 may prevent or delay the formation of atherosclerosis in diabetes mellitus by improving the function of eNOS.     

Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

Background and Purpose

Emerging evidence suggests a selective up-regulation of arginase I in diabetes causing coronary artery disease; however, the mechanisms behind this up-regulation are still unknown. Activated p38 MAPK has been reported to increase arginase II in various cardiovascular diseases. We therefore tested the role of p38 MAPK in the regulation of arginase I and II expression and its effect on endothelial dysfunction in diabetes mellitus.

Experimental Approach

Endothelial function was determined in septal coronary (SCA), left anterior descending coronary (LAD) and mesenteric (MA) arteries from healthy and streptozotocin-induced diabetic Wistar rats by wire myographs. Arginase activity and protein levels of arginase I, II, phospho-p38 MAPK and phospho-endothelial NOS (eNOS) (Ser¹¹⁷⁷) were determined in these arteries from diabetic and healthy rats treated with a p38 MAPK inhibitor in vivo.

Key Results

Diabetic SCA and MA displayed impaired endothelium-dependent relaxation, which was prevented by arginase and p38 MAPK inhibition while LAD relaxation was not affected. Arginase I, phospho-p38 MAPK and eNOS protein expression was increased in diabetic coronary arteries. In diabetic MA, however, increased expression of arginase II and phospho-p38 MAPK, increased arginase activity and decreased expression of eNOS were observed. All these effects were reversed by p38 MAPK inhibition.

Conclusions and Implications

Diabetes-induced activation of p38 MAPK causes endothelial dysfunction via selective up-regulation of arginase I expression in coronary arteries and arginase II expression in MA. Therefore, regional differences appear to exist in the arginase isoforms contributing to endothelial dysfunction in type 1 diabetes mellitus.     

Lactoferrin inhibits apoptosis through insulin-like growth factor I in primary rat osteoblasts

Aim： Excessive apoptosis of osteoblasts is the major cause of low bone mass, and bovine lactoferrin （bLF）, an iron-binding glycoprotein, might protect osteoblastic cells from apoptosis induced by serum withdrawal. The aim of this study was to elucidate the mechanisms underlying the anti-apoptotic action of bLF in rat osteoblasts in vitro. Methods： Primary rat osteoblasts were incubated in the presence of varying concentrations of bLF for 24 h. The expression of insulin-like growth factor I （IGF-I） and IGF-I receptor （IGF-IR） was measured uisng RT-PCR and Western blotting. Cell apoptosis was examined with flow cytometry. siRNAs targeting IGF-I was used in this study.

Results： Treatment of bLF （0.1–1000 μg/mL） dose-dependently increased the expression of IGF-I and IGF-IR in the osteoblasts. Treatment with bLF （10, 100 μg/mL） markedly inhibited the osteoblast apoptosis （with the rate of total apoptosis of 70% at 10 μg/mL）, but the high concentration of bLF （1000 μg/mL） significantly promoted the osteoblast apoptosis. Knockdown of the IGF-I gene in osteoblasts with siRNA markedly increased the osteoblast apoptosis.

Conclusion： Lactoferrin （10 and 100 μg/mL） effectively inhibits apoptosis of primary rat osteoblasts by upregulating IGF-I expression.     

OSU-CG5, a novel energy restriction mimetic agent,targets human colorectal cancer cells in vitro

Aim： Energy-restriction mimetic agents （ERMAs） are small-molecule agents that target various aspects of energy metabolism, which has emerged as a promising approach in cancer therapy. In the current study, we tested the ability of OSU-CGS, a novel ERMA, to tar- get human colorectal cancer （CRC） in vitro. Methods： Two human CRC cell lines （HCT-116 and Caco-2） were tested. Cell viability was assessed using MTT assay. Caspase-3/7 activities were measured using Caspase-GIo 3/7 assay kit. Western blot analysis was used to measure the expression of relevant pro- teins in the cells. Glucose consumption of the cells was detected using glucose uptake cell-based assay kit. Results： OSU-CG5 dose-dependently inhibited HCT-116 and Caco-2 cell proliferation with the ICso values of 3.9 and 4.6 μmol/L, respec- tively, which were 20-25-fold lower than those of resveratrol, a reference ERMA. Both OSU-CG5 （5, 10, and 20 μmol/L） and resvera- trol （50, 100, and 200 μmol/L） dose-dependently increased caspase-3/7 activity and PARP level in the cells. Furthermore, both OSU- CG5 and resveratrol induced dose-dependent energy restriction in the cells： they suppressed glucose uptake and Akt phosphoryla- tion, decreased the levels of p-mTOR and p-pTOS6K, increased the levels of ER stress response proteins GRP78 and GADD153, and increased the level of β-TrCP, which led to the downregulation of cyclin D1 and Spl. Conclusion： OSU-CG5 exhibits promising anti-cancer activity against human CRC cells in vitro, which was, at least in part, due to energy restriction and the consequent induction of ER stress and apoptosis.     

Myosin light chain kinase is responsible for high proliferative ability of breast cancer cells via antiapoptosis involving p38 pathway

Aim:

To investigate whether myosin light chain kinase (MLCK) contributed to the high proliferative ability of breast cancer cells.

Methods:

Soft agar colony formation on the MCF-7 and LM-MCF-7 cell lines was determined. The cell cycles of MCF-7 and LM-MCF-7 were detected using flow cytometry analysis. Western blot analysis was performed to detect the expression levels of p-ERK1/2, total-ERK1/2, p-p38, total p38, p-JNK, total-JNK, survivin, Bcl-2, p-MLC, caspase-9, cleaved caspase-9, and MLCK. After treatment with adriamycin (ADR), ML-7 and SB203580, apoptosis was examined using flow cytometry analysis and Annexin V-FITC fluorescence microscopy.

Results:

The breast cancer LM-MCF-7 cell line with high metastasis potential (a metastitic sub-clone of MCF-7) had higher anti-apoptosis ability relative to MCF-7 cells in response to adriamycin treatment (apoptosis rate: 6.76% vs 28.58%, P<0.05). Moreover, the expression level of MLCK was upregulated and the level of phosphorylated p38 (p-p38) was decreased in LM-MCF-7 cells. Flow cytometry analysis showed that ML-7, selective inhibitor of MLCK, could induce apoptosis of the LM-MCF-7 cells, in which the level of p-p38 was increased. Meanwhile, the expression levels of Bcl-2 and survivin were downregulated, while the caspase-9 was upregulated suggesting that the cells were undergone apoptosis. Flow cytometry analysis showed that SB203580, an inhibitor of p38, abolished ML-7-induced apoptosis, which resulted in the upregualtion of Bcl-2 and survivin, and downregulation of caspase-9, suggesting that Bcl-2, survivin and caspase-9 are downstream effectors of p38.

Conclusion:

MLCK is responsible for high proliferative ability of breast cancer cells through anti-apoptosis, in which p38 pathway was involved.     

Hydrogen sulfide inhibits homocysteine-induced endoplasmic reticulum stress and neuronal apoptosis in rat hippocampus via upregulation of the BDNF-TrkB pathway

Aim： Homocysteine （Hcy） can elicit neuronal cell death, and hyperhomocysteinemia is a strong independent risk factor for Alzheimer’s disease. The aim of this study was to examine the effects of hydrogen sulfide （H2S） on Hcy-induced endoplasmic reticulum （ER） stress and neuronal apoptosis in rat hippocampus.
Methods： Adult male SD rats were intracerebroventricularly （icv） injected with Hcy （0.6 μmol/d） for 7 d. Before Hcy injection, the rats were treated with NaHS （30 or 100 μmol·kg^-1·d^-1, ip） and/or k252a （1 μg/d, icv） for 2 d. The apoptotic neurons were detected in hippocampal coronal slices with TUNEL staining. The expression of glucose regulated protein 78 （GRP78）, C/EBP homologous protein （CHOP）, cleaved caspase-12, and BDNF in the hippocampus were examined using Western blotting assays. The generation of H2S in the hippocampus was measured with the NNDPD method.
Results： Hcy markedly inhibited the production of endogenous H2S and increased apoptotic neurons in the hippocampus. Further-more, Hcy induced ER stress responses in the hippocampus, as indicated by the upregulation of GRP78, CHOP, and cleaved caspase-12. Treatment with the H2S donor NaHS increased the endogenous H2S production and BDNF expression in a dose-dependent manner, and significantly reduced Hcy-induced neuronal apoptosis and ER stress responses in the hippocampus. Treatment with k252a, a specific inhibitor of TrkB （the receptor of BDNF）, abolished the protective effects of NaHS against Hcy-induced ER stress in the hippocampus.
Conclusion： H2S attenuates ER stress and neuronal apoptosis in the hippocampus of Hcy-treated rats via upregulating the BDNF-TrkB pathway.