The acute exposure effects of inhaled nickel nanoparticles on murine endothelial progenitor cells

Abstract

Introduction: The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures, such as increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species.

Methods: Following an acute whole body inhalation exposure to 500?µg/m³ of nickel nanoparticles for 5?h, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs) and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure.

Results and conclusions: Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.     

Circulating endothelial progenitor cells are reduced in hemodialysis patients

SUMMARY

Objective: The risk of cardiovascular disease in hemodialysis patients is far greater than in the general population. Endothelial progenitor cells (EPCs) circulating in the peripheral blood contribute to neovascularization in the ischemic tissue. EPCs are considered to be included in CD34 positive (CD34⁺) or AC133 positive (AC133⁺) mononuclear cells (MNCs). This study's aim was to determine the number and functional activity of EPCs in hemodialysis patients and age-matched control subjects.

Methods: The numbers of CD34⁺ MNCs and AC133⁺ MNCs in the peripheral blood were quantified by flow cytometry. The peripheral blood EPCs were also examined by an in vitro culture assay. The levels of serum vascular endothelial growth factor (VEGF) were measured by sandwich enzyme immunoassay.

Results: The numbers of CD34⁺ MNCs and AC133⁺ MNCs were significantly reduced by 56% and 49%, respectively, in hemodialysis patients (n?=?50) compared with control subjects (n?=?36). The number of EPCs determined by the culture assay was also significantly reduced by 41% in hemodialysis patients compared with control i subjects. Multivariate analysis revealed that none of the atherosclerotic risk factors were independent predictors of reduced CD34⁺ MNC counts. The serum VEGF levels in hemodialysis patients were not different from those in control subjects and did not correlate with CD34⁺ MNC counts.

Conclusion: Circulating EPCs are significantly reduced in hemodialysis patients, which might be related to impaired neovascularization and cardiovascular disease in these patients.     

Impact of prior chronic statin therapy and high-intensity statin therapy at discharge on circulating endothelial progenitor cell levels in patients with acute myocardial infarction: a prospective observational study

Background

Endothelial progenitor stem cells (EPCs) are mobilized to the peripheral circulation in response to myocardial ischemia, playing a crucial role in vascular repair. Statins have been shown to stimulate EPCs. However, neither the impact of previous statin therapy on EPC response of acute myocardial infarction (AMI) patients nor the effect of post-AMI high-intensity statin therapy on the evolution of circulating EPC levels has yet been addressed. Therefore, we aimed to compare circulating EPC levels between patients receiving long-term statin therapy before the AMI and statin-naive patients and to assess the impact of high-intensity statin therapy at discharge on the evolution of circulating EPCs post-AMI.

Methods

This is a prospective observational study of 100 AMI patients. Circulating EPCs (CD45dimCD34?+?KDR?+?cells) and their subpopulation coexpressing the homing marker CXCR4 were quantified by the high-performance flow cytometer FACSCanto II in whole blood, in two different moments: within the first 24 h of admission and 3 months post-AMI. Patients were followed up clinically for 2 years.

Results

Patients previously treated with statins had significantly higher levels of EPCs coexpressing CXCR4 (1.9?±?1.4 vs. 1.3?±?1.0 cells/1,000,000 events, p?=?0.031) than statin-naive patients. In addition, the subanalysis of diabetics (N?=?38) also revealed that patients previously on statins had significantly greater numbers of both CD45dimCD34?+?KDR?+?CXCR4+ cells (p?=?0.024) and CD45dimCD34?+?KDR?+?CD133+ cells (p?=?0.022) than statin-naive patients. Regarding the evolution of EPC levels after the AMI, patients not on a high-intensity statin therapy at discharge had a significant reduction of CD45dimCD34?+?KDR?+?and CD45dimCD34?+?KDR?+?CXCR4+ cells from baseline to 3 months follow-up (p?=?0.031 and p?=?0.005, respectively). However, patients discharged on a high-intensity statin therapy maintained circulating levels of all EPC populations, presenting at 3 months of follow-up significantly higher EPC levels than patients not on an intensive statin therapy. Moreover, the high-intensity statin treatment group had significantly better clinical outcomes during the 2-year follow-up period than patients not discharged on a high-intensity statin therapy.

Conclusion

Chronic statin therapy prior to an AMI strongly enhances the response of EPCs to myocardial ischemia, even in diabetic patients. Furthermore, high-intensity statin therapy after an AMI prevents the expected decrease of circulating EPC levels during follow-up. These results reinforce the importance of an early and intensive statin therapy in AMI patients.     

Effects of ACE inhibition on circulating endothelial progenitor cells, vascular damage, and oxidative stress in hypertensive patients

Purpose

The pathogenic role of angiotensin-converting enzyme (ACE) inhibition in hypertensive patients regarding endothelial progenitor-cell (EPC) function is still poorly understood. The aim of the study was to evaluate EPC number, function, and relationship to carotid intima media thickness (IMT) progression.

Methods

We studied 36 newly diagnosed mildly hypertensive patients free of cardiovascular disease and related risk factors without prior or concurrent therapy with ACE inhibitors. Patients were randomized to receive enalapril 20?mg/day (n?=?18) or zofenopril 30?mg/day (n?=?18). EPC number and migrating capacity, plasma nitrite and nitrate (NOx), and isoprostane concentrations were evaluated. Carotid IMT was determined by ultrasonography at baseline and after 1 and 5?years of follow-up.

Results

EPC number increased during the follow-up, with no statistical differences between treatment groups. There was an inverse correlation between circulating EPCs and IMT increase over time. Plasma NOx decreased during the study without evident differences between treatment groups. Isoprostanes decreased more markedly in zofenopril-treated patients. Multiple linear regression model demonstrated that carotid IMT was significantly inversely correlated with EPC but not with migratory cells after adjusting for confounders.

Conclusions

The study demonstrated that EPC levels increased during the follow-up in both groups of newly diagnosed hypertensive patients treated with ACE inhibitors. These drugs prevented progression of vascular damage, with an inverse correlation between circulating EPC levels and IMT values.     

Preparation,characterization, and pharmacokinetics study of capsaicin via hydroxypropyl-beta-cyclodextrin encapsulation

Context: Capsaicin (CAP) is an effective drug in the treatment of pain and cancer. However, its practical administration has been limited due to poor aqueous solubility and bioavailability, as well as strong gastrointestinal irritation.

Objective: The objective of this study is to improve the solubility and oral bioavailability of CAP by reducing irritation via hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion complex formulation, in vitro and in vivo analysis.

Materials and methods: The complex (CAP-HP-β-CD) was developed via the magnetic stirring method and characterized using ultraviolet (UV) absorption spectrometry, infrared radiation (IR) spectroscopy, and differential scanning calorimetry (DSC). Rats were treated with CAP (90?mg?×?kg^?1) or CAP-HP-β-CD (corresponding to 90?mg?×?kg^?1 CAP) by gavage, and all the plasma samples were analyzed with high performance liquid chromatography (HPLC).

Results: The results of UV, IR, and DSC showed that an acceptable CAP-HP-β-CD (encapsulation efficiency, 75.83%; drug loading, 7.44%) was formulated. In vitro release study of CAP-HP-β-CD revealed that the cumulative release of CAP from HP-β-CD encapsulation was obviously enhanced (above 80% increases). Similarly, the in vivo pharmacokinetics parameters also increased, C_max (from 737.94 to 1117.57?ng?×?mL^?1), AUC_0– (from 5285.9 to 7409.8?ng?×?h?×?mL^?1) or relative bioavailability (139.38%). The gastric irritation bioassay further showed that the CAP-HP-β-CD was far better than free CAP.

Discussion and conclusion: CAP exhibited significant aqueous solubility and oral bioavailability, as well as minimal irritation effect after forming inclusion complex with HP-β-CD. Therefore, these findings could provide an equally important alternative option for the clinical use of CAP.     

Decreased mobilization of endothelial progenitor cells contributes to impaired neovascularization in diabetes

• 1 Circulating bone marrow (BM)‐derived endothelial progenitor cells (EPCs) play an important role in neovascularization. In the present study, we investigated the mechanisms underlying the reduction in circulating EPCs in a mouse model of diabetes induced by streptozotocin.
• 2 Compared with non‐diabetic controls, diabetic mice had reduced circulating EPCs (0.59 ± 0.11 vs 0.94 ± 0.21%, respectively; P < 0.01) and increased plasma endothelial microparticles (18 642 ± 6809 vs 5692 ± 1862/mL, respectively; P < 0.01). In a mouse bone marrow (BM) transplantation model, increased adhesion of transplanted BM cells to aortas of diabetic mice was observed compared with control (900 ± 194 vs 431 ± 109 cells/mm², respectively; P < 0.01).
• 3 Following hindlimb ischaemia, diabetic mice exhibited suppressed EPC mobilization, a reduction in the expected increase in capillary density and suppressed restoration of transcutaneous oxygen pressure in the ischaemic tissue. Diabetic mice also showed impaired ischaemia‐induced upregulation of vascular endothelial growth factor (VEGF), hypoxia‐inducible factor (HIF)‐1α and interleukin‐1β, an exaggerated increase in matrix metalloproteinase (MMP)‐2 and ‐9 and a suppressed increase in tissue inhibitor of matrix metalloproteinase (TIMP)‐1. On multivariate analysis, VEGF expression was the only independent factor related to circulating EPC count.
• 4 In conclusion, the data indicate that the decrease in basal circulating EPCs in diabetes may be attributable, in part, to consumptive loss of EPCs due to increased endothelial damage. Impairment of ischaemia‐induced EPC mobilization in the diabetic mouse model is associated with altered HIF‐1α/VEGF and MMP/TIMP regulation and represents a novel mechanism underlying defective postischaemic neovascularization in diabetes.

     

The acute respiratory exposure by intratracheal instillation of Sprague–Dawley rats with diesel particulate matter induces retinal thickening

Context: Adverse health effects of ambient particulate matter (PM) have been demonstrated in humans, mostly in terms of respiratory and cardiovascular events. However, whether ambient particle could affect the eyes had not been fully revealed.

Objective: This study investigated the effect of acute respiratory exposure to PM on eyes.

Methods: Diesel exhaust product (DEP) of 200?mg/l was given endotracheally in Sprague–Dawley rats for 1?h (n?=?12) and compared to normal control (n?=?4). We enucleated eyes and histologically evaluated. Immunohistochemical stains for CD34 (Dako, Glostrup, Denmark, 1:50) and Ki-67 (DakoCytomation, Glostrup, Denmark, 1:150) were performed to evaluate new vessel formation and proliferation activity.

Results: After endotracheal DEP exposure, the thickness of retina was significantly increased to 258?±?96?μm in DEP group, while that of control was 113?±?9?μm (p?=?0.025). Among the retinal structure, inner plexiform, inner and outer nuclear and rod/cone cell layers were significantly thickened (p?=?0.00, 0.017, 0.004, 0.001, respectively). The outer plexiform layer of DEP group showed a tendency of thickening, but statistically insignificant. The afferent fiber and ganglion cell layer showed no thickness difference between two groups, but prominent capillaries with congestion were noted in DEP group. Neither neovascularization nor increased proliferation was demonstrated on CD34 and Ki-67 immunohistochemical staining in DEP group.

Conclusion: This study shows that the acute respiratory exposure of ambient PM increased retinal thickness, especially inner plexiform, inner and outer nuclear and rod/cone cell layers. We thought that increase of retinal thickness in DEP group resulted in hypoxia-induced edema.     

Intravitreal bevacizumab effects on VEGF levels in distant organs: an experimental study

Purpose: The aim of this study was to determine the effects of single-dose intravitreal bevacizumab on the levels of vascular endothelial growth factor (VEGF) in serum and distant organs.

Methods: Adult New Zealand albino rabbits (n?=?40) were divided into experimental and control groups. Experimental rabbits received a single 0.05?ml intravitreal injection of 1.25?mg bevacizumab (Avastin) into the right eye, and control rabbits (n?=?8) received no injection. Following injection, group 1 rabbits (n?=?8) were sacrificed on day 1, group 2 rabbits (n?=?8) on day 7, group 3 rabbits (n?=?8) on day 14, and group 4 rabbits (n?=?8) on day 28; control rabbits were sacrificed on day 28. After sacrifice, samples of brain, heart, liver, kidney and blood were collected. Levels of VEGF in serum and tissue were measured using enzyme-linked immunosorbent assay. The presence of bevacizumab was evaluated by immunofluorescence staining in tissues.

Results: Positive bevacizumab immunoreactivity was observed in brain, heart and kidney. Serum VEGF levels significantly decreased in groups 3 and 4 compared with controls (p?p?Conclusions: Intravitreal bevacizumab not only may escape from the blood-retinal barrier and enter the general circulation, but also may be disseminated to distant organs. Our study demonstrates that a single dose of intravitreally injected bevacizumab decreases VEGF levels in serum and liver.     

Effects of electroacupuncture on endothelial function and circulating endothelial progenitor cells in patients with cerebral infarction

This study evaluated the effects of electroacupuncture (EA) on endothelial function and endothelial progenitor cells (EPC) in patients with cerebral infarction. In a randomized, placebo‐controlled, crossover study, 20 patients with cerebral infarction were randomized into two treatment groups: EA or placebo. Before and after each intervention, pulse amplitude tonometry (PAT) was used to assess endothelial function and peripheral blood was analyzed for the number of EPCs. Circulating EPCs were quantified by flow cytometry as CD45^lowCD34⁺KDR2⁺ cells. Plasma vascular endothelial growth factor (VEGF) and interleukin (IL)‐10 levels were measured. Seven days later, crossover was performed on each group, with each group receiving the other treatment using the same protocol. The PAT hyperemia ratio ranged from 1.57 ± 0.41 to 2.04 ± 0.51 after EA, representing a significant improvement (P = 0.002); however, there was no improvement in the placebo group (P = 0.48). Circulating EPCs, as measured by flow cytometry, increased to 110.6 ± 74.3/100 μL in the EA group (P = 0.001) but did not change in the placebo group (45.9 ± 35.3/100 μL, P = 0.08). The increases in the number of EPCs and the PAT ratio after treatment were correlated (r = 0.78, P < 0.001). Plasma VEGF levels increased with EA compared to baseline (261.2 ± 34.0 vs 334.9 ± 80.5 pg/mL, P = 0.003). The number of circulating EPCs was positively correlated with plasma levels of VEGF (r = 0.50, P = 0.02). In conclusion, EA induced improvement of EPC levels and the PAT ratio in patients with cerebral infarction.     

24-Week study on the use of collagen hydrolysate as a dietary supplement in athletes with activity-related joint pain

ABSTRACT

Background: Collagen hydrolysate is a nutritional supplement that has been shown to exert an anabolic effect on cartilage tissue. Its administration appears beneficial in patients with osteoarthritis.

Objective: To investigate the effect of collagen hydrolysate on activity-related joint pain in athletes who are physically active and have no evidence of joint disease.

Design and setting: A prospective, randomized, placebo-controlled, double-blind study was conducted at Penn State University in University Park, Pennsylvania. Parameters including joint pain, mobility, and inflammation were evaluated with the use of a visual analogue scale during a 24-week study phase.

Study participants: Between September 2005 and June 2006, 147 subjects who competed on a varsity team or a club sport were recruited. Data from 97 of 147 subjects could be statistically evaluated.

Intervention: One hundred and forty-seven subjects (72 male, 75 female) were randomly assigned to two groups: a group (n?=?73) receiving 25?mL of a liquid formulation that contained 10?g of collagen hydrolysate (CH-Alpha)^* and a group (n?=?74) receiving a placebo, which consisted of 25?mL of liquid that contained xanthan.

Main outcome measures: The primary efficacy parameter was the change in the visual analogue scales from baseline during the study phase in relation to the parameters referring to pain, mobility, and inflammation.

Results: When data from all subjects (n?=?97) were evaluated, six parameters showed statistically significant changes with the dietary supplement collagen hydrolysate (CH) compared with placebo: joint pain at rest, assessed by the physician (CH vs. placebo (–1.37?±?1.78 vs. –0.90?±?1.74 (?p?=?0.025)) and five parameters assessed by study participants: joint pain when walking (–1.11?±?1.98 vs. –0.46?±?1.63, p?=?0.007), joint pain when standing (–0.97?±?1.92 vs. –0.43?±?1.74, p?=?0.011), joint pain at rest (–0.81?±?1.77 vs. –0.39?±?1.56, p?=?0.039), joint pain when carrying objects (–1.45?±?2.11 vs. –0.83?±?1.71, p?=?0.014) and joint pain when lifting (–1.79?±?2.11 vs. –1.26?±?2.09, p?=?0.018). When a subgroup analysis of subjects with knee arthralgia (n?=?63) was performed, the difference between the effect of collagen hydrolysate vs. placebo was more pronounced. The parameter joint pain at rest, assessed by the physician, had a statistical significance level of p?=?0.001 (–1.67?±?1.89 vs. –0.86?±?1.77), while the other five parameters based on the participants’ assessments were also statistically significant: joint pain when walking (?p?=?0.003 (– 1.38?±?2.12 vs. – 0.54?±?1.65)), joint pain when standing (?p?=?0.015 (–1.17?±?2.06 vs. –0.50?±?1.68)), joint pain at rest with (?p?=?0.021 (–1.01 ±1.92 vs. –0.47?±?1.63)), joint pain when running a straight line (?p?=?0.027 (–1.50?±?1.97 vs. –0.80?±?1.66)) and joint pain when changing direction (?p?=?0.026 (–1.87?±?2.18 vs. –1.20?±?2.10)).

Conclusion: This was the first clinical trial of 24-weeks duration to show improvement of joint pain in athletes who were treated with the dietary supplement collagen hydrolysate. The results of this study have implications for the use of collagen hydrolysate to support joint health and possibly reduce the risk of joint deterioration in a high-risk group. Despite the study's size and limitations, the results suggest that athletes consuming collagen hydrolysate can reduce parameters (such as pain) that have a negative impact on athletic performance. Future studies are needed to support these findings.     

Montelukast prevents vascular endothelial dysfunction from internal combustion exhaust inhalation during exercise

Associations between high particulate matter (PM) pollution and increased morbidity and mortality from coronary heart disease have been identified. This study assessed leukotriene (LT) participation in PM-induced vascular endothelial dysfunction. Ten healthy males exercised 4 times for 30?min in both high PM (550,286?±?42,004 particles·cm^?3) and low PM (4571?±?1922 particles·cm^?3) after ingesting placebo (PL) or 10?mg montelukast (MK; half-life 3–6?h), a leukotriene receptor antagonist. Brachial artery flow-mediated dilation (FMD) was measured pre- and 30?min, 4?h, 24?h post-exercise. No basal brachial artery vascoconstriction was evident from high PM exercise. High PM blunted FMD, whereas high PM MK, low PM PL, and low PM MK demonstrated normal FMD (p < .003). Change in FMD (pre- to post-exercise) for high PM PL was different than for high PM MK, low PM PL, and low PM MK at 30?min post-exercise (p?<?.007). At 4?h, high PM MK FMD blunting increased (p?=?.1). At 24?h, high PM FMD blunting persisted (p?<?.05); no difference was observed between high PM PL or MK treatment, but was different that low PM PL/MK treatments (p?<?.05). MK blocked high PM post-exercise FMD blunting and maintained normal response, suggesting that leukotrienes are involved in PM-initiated vascular endothelial dysfunction.     

The effects of smoking on corneal endothelial cells: a cross-sectional study on a population from Isfahan,Iran

Purpose: The aim of this study is to compare the effect of smoking in corneal endothelial cell number and morphology by specular microscopy on a non-smoker population.

Methods: Our cross-sectional study was performed on 150 participants from a non-smoker population. Non-contact specular microscopy (Tomey Corporation Inc., Nagoya, Japan) was performed in the center of the cornea of all subjects. The cell density (CD), average cell size (AVG), percent of hexagonality (HEX%) and central corneal thickness (CCT) were calculated and compared in both groups.

Results: Totally, 76 eyes of 76 smokers and 74 eyes of 74 non-smokers were enrolled in the study from 2015 to 2016. The mean age of smokers and non-smokers were 48.61?±?17.04 and 46.39?±?13.02, respectively. The mean number of pack/year among the smokers was 17.36?±?14.68. Also, the mean values of AVG and CD were significantly different for these two groups (p?=?0.011 and p?=?0.039, respectively). Other corneal endothelial variables did not show a significant difference between smokers and non-smokers (p?>?0.05). However, smokers with severe nicotine dependency had significantly greater AVG and lower CD in comparison with the non-smokers (p?=?0.004 and p?=?0.013, respectively).

Conclusion: Our study showed that smoking can cause significant changes in some of the corneal endothelial variables, but not all of them.     

Acute and chronic in vivo effects of exposure to nicotine and propylene glycol from an E-cigarette on mucociliary clearance in a murine model

Objective: To determine the effect of an acute (1 week) and chronic (3 weeks) exposure to E-cigarette (E-cig) emissions on mucociliary clearance (MCC) in murine lungs.

Methods: C57BL/6 male mice (age 10.5?±?2.4 weeks) were exposed for 20?min/day to E-cigarette aerosol generated by a Joyetech 510-T^® E-cig containing either 0% nicotine (N)/propylene glycol (PG) for 1 week (n?=?6), or 3 weeks (n?=?9), or 2.4% N/PG for one week (n?=?6), or 3 weeks (n?=?9), followed by measurement of MCC. Control mice (n?=?15) were not exposed to PG alone, or N/PG. MCC was assessed by gamma camera following aspiration of ^99mtechnetium aerosol and was expressed as the amount of radioactivity removed from both lungs over 6?hours (MCC6hrs). Venous blood was assayed for cotinine levels in control mice and in mice exposed for 3-weeks to PG alone and N/PG.

Results: MCC6hrs in control mice and in mice acutely exposed to PG alone and N/PG was similar, averaging (±1 standard deviation) 8.6?±?5.2%, 7.5?±?2.8% and 11.2?±?5.9%, respectively. In contrast, chronic exposure to PG alone stimulated MCC6hrs (17.2?±?8.0)% and this stimulation was significantly blunted following chronic exposure to N/PG (8.7?±?4.6)% (p?Conclusions: In this murine model, a chronic, daily, 20?min-exposure to N/PG, but not an acute exposure, slowed MCC, compared to exposure to PG alone and led to systemic absorption of nicotine.     

The effect and safety of intravitreal injection of ranibizumab and bevacizumab on the corneal endothelium in the treatment of diabetic macular edema

Objective: To investigate the effect and safety of intravitreal injection (IVI) of bevacizumab and ranibizumab on corneal endothelial cell count and morphology in patients with diabetic macular edema.

Materials and methods: A total of 60 eyes from 60 consecutive patients who received 0.5?mg/0.05?ml IVIs of bevacizumab (n?=?30, IVB group) or 1.25?mg/0.05?ml ranibizumab (n?=?30, IVR group) for three consecutive months were investigated prospectively. Specular microscopy was performed to evaluate endothelial cell count, the percentage of hexagonal cells (pleomorphism), and the coefficient of variation of the cell size (polymegathism); optical biometry was performed to evaluate central corneal thickness. Results before injection and 1 month after the first and third injections were compared.

Results: The groups were matched for age (p?=?0.11) and gender (p?=?0.32). There was no significant difference in endothelial cell count (IVB group, p?=?0.66; IVR group, p?=?0.74), pleomorphism (IVB group, p?=?0.44; IVR group, p?=?0.88) and polymegathism (IVB group, p?=?0.21; IVR group, p?=?0.24) before injection or 1 month after the first and third injections. There was also no difference in central corneal thickness (IVB group, p?=?0.15; IVR group, p?=?0.58) before injection or 1 month after the first and third injections.

Conclusion: Monthly 1.25?mg/0.05?ml IVIs of bevacizumab or 0.5?mg/0.05?ml of ranibizumab for three consecutive months in the treatment of diabetic macular edema does not affect corneal morphology and has no harmful effects on the endothelium.     

Identification and quantification of metabolites of arachidonic acid from cultures of endothelial cells by HPLC-MS2

Epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) are oxidative products of arachidonic acid, some of which participate in the regulation of vascular tone. Little is known about the production of EETs and HETEs in cultures of endothelial cells. This paper reports an assay for the simultaneous quantification of isomers of EETs and HETEs from endothelial cell culture supernatants by employing solid-phase extraction and liquid chromatography-mass spectrometry. The method enabled measurement of 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 8-HETE, 11-HETE, 12-HETE and 15-HETE. The metabolites were chromatographically separated by reversed-phase HPLC and identified by negative ESI tandem mass spectrometry and this method was used to investigate the metabolism of arachidonic acid with an endothelial cell line. For quantification, the sum of signal intensities of characteristic fragment ions was used. The detection limits for 5,6-EET and of other EET and HETE isomers were 2.0, 0.64 and 8?ng?ml^?1 culture medium, respectively. The precision of the method was determined with spiked culture medium (three concentrations, n?=?5) and the average RSD ranged from 6.0 to 24.2%. The dynamic range was 0.6–23.5?ng?ml^?1 culture medium for EETs and 8.0–200?ng?ml^?1 for HETEs. Arachidonic acid was mainly metabolised to HETEs with product levels ranging from 59.3 to 460?ng 10^?6 cells. The median of 8,9-EET and 14,15-EET was 14.5 and 17.7?ng 10^?6 cells, respectively, whereas 5,6-EET and 11,12-EET were below 2?ng 10^?6 cells in a 5-min incubation assay at a 30?µM arachidonic acid substrate concentration.     

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.     

Angelica sinensis polysaccharides inhibit endothelial progenitor cell senescence through the reduction of oxidative stress and activation of the Akt/hTERT pathway

Abstract

Context: Angelica sinensis (Oliv.) Diels (Apiaceae) polysaccharides (ASP) may play a key role in anti-ischemic activity. However, the anti-atherosclerotic activity and mechanism are unknown.

Objective: This study investigated the protective effects of ASP against ox-LDL-induced senescence of EPCs and explored its underlying molecular mechanisms.

Materials and methods: Mononuclear cells were isolated from bone marrow (BM) of SD rats and differentiated to EPCs. EPCs were exposed to oxidized low-density lipoprotein (ox-LDL, 10?µg/mL, 24?h) and incubated with or without high-dose (100?µg/mL, 48?h) or low-dose (20?µg/mL, 48?h) ASP. Another group of EPCs was pre-treated with Wortmannin (100?nM, 45?min), a PI3K/Akt inhibitor. EPC senescence, telomerase activity, and superoxide anion levels were assessed using SA-β-galactosidase staining, telomerase PCR-ELISA analysis, and DHE staining, respectively. The expression of related proteins, including Akt, p-Akt, hTERT, p-hTERT, and gp91phox, were detected using western blot.

Results: EPCs (47.3%) were SA-β-gal positive after treatment by ox-LDL, additionally, ox-LDL significantly increased superoxide anion levels (375% versus 100%), and inhibited telomerase activity (42% versus 100%). However, the pro-senescent effect of ox-LDL was attenuated about three-fold (16.7%), superoxide anion levels were decreased more than two-fold (148%), and telomerase activity was recovered partly (88% versus 42%) in the EPCs when treated with ASP (100?µg/mL). The immunoblotting confirmed that ASP attenuated inhibition of phosphorylation of Akt and hTERT induced by ox-LDL and down-regulated increased the expression of gp91-phox. Moreover, some effects of ASP were partially abrogated in the presence of Wortmannin.

Discussion: Ox-LDL induced senescence of EPCs via inhibition of telomerase activity, which was influenced by oxidative stress and the Akt/hTERT pathway. The inhibition of EPC senescence by ASP could be important for potential therapeutics.

Conclusion: Treatment of EPCs with ASP remarkably attenuates the harmful effects of ox-LDL via augmentation of Akt/hTERT phosphorylation and inhibition of oxidative stress.     

Pharmacokinetic interaction of entinostat and lapatinib following single and co-oral administration in rats

Abstract

1.?Entinostat, also known as SNDX-275 or MS-275, is a novel, potent, orally bioavailable, class I selective histone deacetylase inhibitor. Pre-clinical data has show that MS-275 can enhance the activity of lapatinib in HER²⁺ metastatic inflammatory and non-inflammatory breast cancer. This study examined whether oral administration of MS-275 to the rats with lapatinib led to any pharmacokinetic interactions.

2.?To evaluate pharmacokinetic interaction of MS-275 and lapatinib in rat, a sensitive and simple LC-MS method was developed to simultaneously determine MS-275 and lapatinib in rat plasma with carbamazepine as internal standard (IS). Eighteen rats were divided randomly into three groups, lapatinib group (lapatinib 15?mg/kg, n?=?8), MS-275 group (MS-275 15?mg/kg, n?=?8) and co-administration group (MS-275 15?mg/kg and lapatinib 15?mg/kg, n?=?8).

3.?There was no statistical pharmacokinetics difference for MS-275 in MS-275 group and co-administration group; the lapatinib could not influence the pharmacokinetic profile of MS-275 in rats. However, there is a statistical pharmacokinetics difference between lapatinib in the lapatinib group and co-administration group, when co-oral administration MS-275 with lapatinib, AUC increased from 2375.5 to 9900.3?ng/mL h (p?<?0.05), C_max increased from 538.0 to 2578.2?ng/mL (p?<?0.01), CL decreased from 6.2 to 1.7?L/h/kg (p?<?0.01).

4.?These data indicate MS-275 could obviously influence the pharmacokinetic profile of lapatinib in rats, which might cause drug–drug interactions in humans when using lapatinib with MS-275. Further investigations should be carried out to elucidate the synergistic mechanisms between the two drugs.     

Concentration-time extrapolation of short-term inhalation exposure levels: dimethyl sulfide,a case study using a chemical-specific toxic load exponent

Abstract

Objective: Dimethyl sulfide (DMS, CAS 75-18-3) is an industrial chemical. It is both an irritant and neurotoxicant that may be life-threatening because of accidental release. The effects of DMS on public health and associated public health response depend on the exposure concentration and duration. However, currently, public health advisory information exists for only a 1?h exposure duration, developed by the American Industrial Hygiene Association (AIHA). In the present work, the AIHA-reviewed data were computationally extrapolated to other common short-term durations.

Methods: The extrapolation was carried out using the toxic load equation, Cⁿ?×?t?=?TL, where C and t are exposure concentration and duration, TL is toxic load, and n is a chemical-specific toxic load exponent derived in the present work using probit meta-analysis. The developed threshold levels were vetted against the AIHA database of clinical and animal health effects induced by DMS.

Results: Tier-1 levels were derived based on human exposures that resulted in an easily detectable odor, because DMS is known to have a disagreeable odor that may cause nausea. Tier-2 levels were derived from the lower 95% confidence bounds on a benchmark concentration that caused 10% incidence (BMCL₁₀) of coma in rats during a 15?min inhalation exposure to DMS. Tier-3 levels were based on a BMCL₀₅ for mortality in rats.

Conclusion: Emergency responders and health assessors may consider these computationally derived threshold levels as a supplement to traditional chemical risk assessment procedures in instances where AIHA developed public health advisory levels do not exist.