Sacubitril/valsartan reversal of left ventricular remodeling is associated with improved hemodynamics in resistant hypertension.

BACKGROUND: Sacubitril/valsartan (S/V) has been shown to be an effective antihypertensive drug combination. However, its therapeutic effects on blood pressure (BP), hemodynamics, and left ventricular (LV) remodeling in resistant hypertension (RHTN) remain unclear. METHODS: Eighty-six patients completed this self-control study, during which olmesartan was administered within the first 8 weeks (phase 1), followed by S/V within the second 8 weeks (phase 2), with nifedipine and hydrochlorothiazide taken as background medications. Office BP, echocardiography, and hemodynamics assessment using impedance cardiography were performed at baseline and at the eighth and sixteenth weeks. RESULTS: The reduction in office BP was larger in phase 2 than in phase 1 (19.59/11.66 mmHg vs. 2.88/1.15 mmHg). Furthermore, the treatment in phase 2 provided greater reductions in systemic vascular resistance index (SVRI) and thoracic blood saturation ratio (TBR), with differences between the two phases of -226.59 (-1212.80 to 509.55) dyn·s/cm5/m2 and -0.02 (-0.04 to 0.02). Switching from olmesartan to S/V also significantly reduced E/E', LV mass index, LV end-diastolic volume index, and LV end-systolic volume index (all P < 0.05). Decreases in arterial stiffness, SVRI, and TBR were correlated with changes in indicators of LV remodeling (all P < 0.05). This correlation persisted even after adjusting for confounders including changes in BP. CONCLUSIONS: Switching from olmesartan to S/V effectively lowered BP and reversed ventricular remodeling in RHTN. In addition, hemodynamic improvement was also observed. Changes in hemodynamics played an important role in reversing LV remodeling of S/V, and were independent of its antihypertensive effect.

Hypertension and cystatin C account for sex differences in serum homocysteine levels in acute coronary syndrome subjects with normal serum creatinine.

BACKGROUND: Hyperhomocysteinemia is one of cardiovascular disease risk factors and fasting homocysteine levels are significantly elevated in male compared to female acute coronary syndrome (ACS) patients with normal renal function. However, it is not known the sex related determinants of plasma homocysteine levels in ACS subjects without renal dysfunction. METHODS: A total of 165 ACS participants with normal plasma creatinine who underwent coronary angiography were included in the present study. Clinical parameters, homocysteine, fasting glucose and lipid profile, hemoglobin, white blood cell, platelets, creatinine, cystatin C, blood urea nitrogen, uric acid (UA), and albumin were measured. Multivariate linear regression analyses were used to recognize the predictive factors for homocysteine. RESULTS: The levels of plasma homocysteine were significantly higher in men than in women (P < 0.0001). In males, homocysteine (log10) was positively associated with hypertension (r = 0.569, P < 0.001), creatinine (r = 0.367, P < 0.001) and cystatin C (log10) (r = 0.333, P = 0.001). In females, homocysteine (log10) was positively correlated with age (r = 0.307, P = 0.107), hypertension (r = 0.456, P < 0.001), creatinine (r = 0.341, P = 0.008), cystatin C (log10) (r = 0.429, P = 0.001) and UA (r = 0.569, P < 0.001) whereas was negatively associated with LDL-C (r = - 0.298, P = 0.021) and ApoB (r = - 0.273, P = 0.033). Parameters up to statistical significance in males or females were incorporated into the stepwise linear regression models. In men, hypertension (P < 0.001) and creatinine (P = 0.031) were independently related to homocysteine. Most of the variability of homocysteine levels in males were only determined by hypertension. In women, cystatin C (log10) (P = 0.004) and hypertension (P = 0.005) were independently related to homocysteine (log10). Plasma cystatin C had a higher explanatory value than hypertension in females. CONCLUSIONS: Hypertension and cystatin C could explain most of the sex differences in serum homocysteine levels in ACS subjects with normal serum creatinine. This finding suggested the importance of making different strategies in males and females to manage hyperhomocysteinemia effectively in ACS subjects without renal dysfunction.

Enhanced external counterpulsation ameliorates endothelial dysfunction and elevates exercise tolerance in patients with coronary artery disease.

Purpose: Enhanced external counterpulsation (EECP) is a new non-drug treatment for coronary artery disease (CAD). However, the long-term effect of EECP on endothelial dysfunction and exercise tolerance, and the relationship between the changes in the endothelial dysfunction and exercise tolerance in the patients with coronary heart disease are still unclear. Methods: A total of 240 patients with CAD were randomly divided into EECP group (n = 120) and control group (n = 120). All patients received routine treatment of CAD as the basic therapy. Patients in the EECP group received 35 1-h daily sessions of EECP during 7 consecutive weeks while the control group received the same treatment course, but the cuff inflation pressure was 0-10 mmHg. Peak systolic velocity (PSV), end diastolic velocity (EDV), resistance index (RI), and inner diameter (ID) of the right carotid artery were examined using a Color Doppler Ultrasound and used to calculate the fluid shear stress (FSS). Serum levels of human vascular endothelial cell growth factor (VEGF), vascular endothelial cell growth factor receptor 2 (VEGFR2), and human angiotensin 2 (Ang2) were determined by enzyme-linked immunosorbent assay (ELISA). Exercise load time, maximal oxygen uptake (VO2max ), metabolic equivalent (METs), anaerobic threshold (AT), peak oxygen pulse (VO2max/HR) were assessed using cardiopulmonary exercise tests. Results: After 1 year follow-up, the EDV, PSV, ID, and FSS were significantly increased in the EECP group (P < 0.05 and 0.01, respectively), whereas there were no significant changes in these parameters in the control group. The serum levels of VEGF and VEGFR2 were elevated in the EECP and control groups (all P < 0.05). However, the changes in VEGF and VEGFR2 were significantly higher in the EECP group than in the control group (P < 0.01). The serum level of Ang2 was decreased in the EECP group (P < 0.05) and no obvious changes in the control group. As for exercise tolerance of patients, there were significant increases in the exercise load time, VO2max, VO2max/HR, AT and METs in the EECP group (all P < 0.05) and VO2max and METs in the control group (all P < 0.05). Correlation analyses showed a significant and positive correlations of VEGF and VEGFR2 levels with the changes in FSS (all P < 0.001). The correlations were still remained even after adjustment for confounders (all Padjustment < 0.001). Linear regression displays the age, the medication of ACEI (angiotensin-converting enzyme inhibitors) or ARB (angiotensin receptor blockers), the diabetes and the changes in VEGF and VEGFR2 were positively and independently associated with the changes in METs after adjustment for confounders (all Padjustment < 0.05). Conclusion: The data of our study suggested that EECP is a useful therapeutic measurement for amelioration of endothelial dysfunction and long-term elevation of exercise tolerance for patients with coronary heart disease. Clinical trial registration: [http://www.chictr.org.cn/], identifier [ChiCTR1800020102].

Matrices and Affinity Ligands for Antibody Purification and Corresponding Applications in Radiotherapy.

Antibodies have become an important class of biological products in cancer treatments such as radiotherapy. The growing therapeutic applications have driven a demand for high-purity antibodies. Affinity chromatography with a high affinity and specificity has always been utilized to separate antibodies from complex mixtures. Quality chromatographic components (matrices and affinity ligands) have either been found or generated to increase the purity and yield of antibodies. More importantly, some matrices (mainly particles) and affinity ligands (including design protocols) for antibody purification can act as radiosensitizers or carriers for therapeutic radionuclides (or for radiosensitizers) either directly or indirectly to improve the therapeutic efficiency of radiotherapy. This paper provides a brief overview on the matrices and ligands used in affinity chromatography that are involved in antibody purification and emphasizes their applications in radiotherapy to enrich potential approaches for improving the efficacy of radiotherapy.

Elevated BTG2 improves the radiosensitivity of non-small cell lung cancer (NSCLC) through apoptosis.

BACKGROUND: To identify radio-responsive genes and explore the biological function of encoded proteins in non-small cell lung cancer (NSCLC). METHODS: Radio-responsive genes in irradiated H460 cells were screened from microarray data deposited in the Gene Expression Omnibus (GEO) database. A quantitative real time polymerase chain reaction assay was used to detect the expression of candidate radio-responsive genes in irradiated cells. CCK-8 assay, EDU assay, clone formation assay, immunofluorescence and flow cytometry were conducted to evaluate the biological function of B cell translocation gene 2 (BTG2) in NSCLC. RESULTS: Bioinformatic analysis using GES20549 showed that BTG2 was a radio-responsive gene in irradiated H460 cells. The mRNA expression level of BTG2 was lower in H460 cells compared with that in BEAS-2B normal lung epithelial cells. BTG2 expression was elevated upon IR exposure, in a dose-dependent but not a time-dependent manner. CCK-8 and EDU assays revealed that BTG2 overexpression inhibited the growth rate of irradiated cells. Clone formation showed that elevated BTG2 promoted DNA damage of irradiated H460 cells. The number of γ-H2AX foci induced by DNA damage was also markedly increased upon BTG2 overexpression. Flow cytometry showed that BTG2 increased IR-induced cell apoptosis. CONCLUSIONS: BTG2 may be a novel radio-responsive factor and a promising therapeutic target for radiotherapy of NSCLC.

Increased activation of cGAS-STING pathway enhances radiosensitivity of non-small cell lung cancer cells.

BACKGROUND: Radiotherapy is an effective therapeutic approach widely used clinically in non-small cell lung cancer (NSCLC), but radioresistance remains a major challenge. New and effective radiosensitizing approaches are thus urgently needed. The activation of DNA-sensing cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has become an attractive therapeutic target, but the relationship between activation of cGAS-STING pathway and radiosensitization of NSCLC cells remains unknown. METHODS: Considering low expression of cGAS-STING pathway genes in NSCLC, including STING, we used an activator (STING agonist, dimeric amidobenzimidazole [diABZI]) of cGAS-STING pathway and increased activation factor (DNA double strand breaks) of cGAS-STING pathway to respectively reinforce the activation of cGAS-STING pathway in NSCLC cells. We then investigated the effect of increased activation of cGAS-STING pathway on the proliferation of H460 and A549 cells by CCK-8 and colony formation assays, and revealed the underlying mechanism. RESULTS: We found that both diABZI and the increased DNA double strand breaks could sensitize NSCLC cells to irradiation. Mechanically, our results showed that the increased activation of cGAS-STING pathway enhanced radiosensitivity by promoting apoptosis in NSCLC cells. CONCLUSION: Taken together, we concluded that diABZI could be used as a radiosensitizer in NSCLC cells, and targeting the activation of cGAS-STING pathway has a potential to be a new approach for NSCLC radiosensitizing.

High fluid shear stress prevents atherosclerotic plaque formation by promoting endothelium denudation and synthetic phenotype of vascular smooth muscle cells.

Low blood fluid shear stress (SS) promotes vascular remodeling and atherosclerosis; however, the effects of high (H)SS on vascular remodeling and atherogenesis is not fully clarified. The major goal of this study was to investigate the role of HSS in atherosclerotic plaque formation. A perivascular SS modifier was implanted in the right carotid artery of apolipoprotein E (ApoE)­/­ mice to induce HSS, whereas the left carotid artery represented undisturbed (U)SS as a control in vivo. In vitro modeling used human umbilical vein endothelial cells and vascular smooth muscle cells exposed to HSS (2.5 Pa) using a parallel­plate flow system. The results demonstrated that there were no plaque formations or endothelial cells in the HSS regions of the carotid artery in ApoE­/­ mice. The number of umbilical vein endothelial cells was markedly decreased in a time­dependent manner in HSS. HSS significantly decreased α­smooth muscle actin and increased osteopontin protein expression levels compared with USS in vascular smooth muscle cells (P<0.05). In addition, HSS significantly increased the protein expression levels of collagen α1(XVIII) chain/endostatin and matrix metalloproteinase­8 in vascular smooth muscle cells. These data indicated that HSS may prevent atherosclerotic plaque formation through endothelium denudation and contractile­to­synthetic phenotypic conversion of smooth muscle cells.

Deletion of P110δ promotes the development of myocarditis in ApoE­deficient mice by increasing mononuclear cell peritoneal infiltration.

Phosphoinositide 3-kinase catalytic subunit δ isoform (P110δ) is mainly expressed in white blood cells. It is involved in T and B lymphocyte differentiation, maturation and the neutrophil chemotaxis process. Apolipoprotein E (ApoE) is an arginine­rich alkaline protein, which is present in plasma chylomicron, low­density lipoprotein and very low­density lipoprotein. The present study aimed to determine the effects of P110δ deletion on myocarditis in ApoE­/­ mice. A mouse model of ApoE and P110δ double deletion was initially constructed; hematoxylin and eosin (H&E) staining was performed to detect the histological alterations in the mouse myocardium. Systolic and diastolic alterations, and alterations in the left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were examined by electrocardiogram. Blood cell of ApoE and P110δ double mice was used to detect changes in white blood cells and monocytes. Western blotting was used to detect the expression levels of apoptosis­associated proteins, whereas flow cytometry was used to detect the percentage of apoptosis. Morphological alterations in myocardial cells were observed under a microscope. The results of polymerase chain reaction demonstrated that double deletion mice were successfully constructed. H&E staining revealed that cells in the ApoE­/­ mice were spindle­shaped; however, the nuclei were smaller in the double deletion mice. There was no change in cardiac contraction in normal mice; however, in double deletion mice, the systolic and diastolic contractions were markedly reduced. LVFS and LVEF were decreased compared with in the control group. Blood cell analysis indicated that the content of white blood cells and monocytes in the experimental group was significantly higher than that in the control group. Western blotting demonstrated that the expression levels of apoptotic proteins in double deletion mice were significantly higher compared with in the control group. Flow cytometry revealed that the apoptotic ratio was increased in double deletion mice compared with in the control group (42 vs. 21%). These findings suggested that deletion of P110δ may induce monocyte peritoneal infiltration and increase apoptosis, thus promoting the development of myocarditis.

[Prokaryotic expression, purification and characterization of tissue inhibitor of metalloproteinase-2].

Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-ß-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.

Acetylpuerarin protects against OGD-induced cell injury in BV2 microglia by inhibiting HMGB1 release.

High mobility group box 1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic injury, initiates inflammatory response and aggravates brain tissue damage. Acetylpuerarin (AP), an acetylated derivative of puerarin, was reported to protect against cerebrovascular ischemia-reperfusion injury in rats through anti-inflammation. In the present study, we aim to investigate whether AP inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated BV2 microglia. BV2 microglia viability after OGD with or without AP was measured by CCK-8 assay, apoptosis of BV2 microglia was determined by Hoechst 33258 staining and FITC-Annexin V/7-AAD staining. HMGB1 protein level and release was detected by western blotting and immunofluorescent FITC-staining. The results demonstrated that AP significantly rescued OGD-induced cell death and apoptosis in a dose-dependent manner. AP inhibited OGD-induced HMGB1secretion at the level of nuclear to cytoplasmic translocation, decreased cytoplasmic HMGB1 at protein level, and the effects showed dose-dependent. The findings suggest that AP can protect against OGD-induced cellular injury in BV2 microglia by inhibition of HMGB1 release.

Bile salt liposomes for enhanced lymphatic transport and oral bioavailability of paclitaxel.

Paclitaxel (PTX), a BCS class IV drug that is characterized by its poor solubility and is a substrate for P-glycoprotein, is one of the most widely used antineoplastic agents. However, oral administration of PTX for chemotherapy is highly challenging. The aim of this study was to develop bile-salt liposomes (BS-Lips) to enhance the absorption of PTX and thus improve its therapeutic outcome. The BS-Lips were prepared by the thin-film hydration method and characterized in terms of particle size and morphology. Drug release and in vitro stability in simulated gastrointestinal fluids and in media of different pH values were evaluated, as well as in vivo performance, including antitumor activity and pharmacokinetics in rats, with the plasma concentrations determined by a HPLC method. The PTX-loaded BS-Lips were successfully prepared with a diameter of approximately 150 nm and an entrapment efficiency of greater than 90 percent. Moreover, the BS-Lips were not affected by gastrointestinal enzymes or pH alternation, as evident from the unchanged particle size and the drug retained in BS-Lips after 6 h incubation. The insertion of bile salt into the lipid layer of liposomes increased the lymphatic transport of PTX by twofold. Importantly, BS-Lips increased the oral bioavailability of PTX by 2.5 and 4-fold, respectively, compared with conventional liposomes (Lips) and Taxol (free drug), thereby displaying a better inhibition of tumor growth that was similar to the group injected intravenously with Taxol. In conclusion, the BS-Lips represent promising vehicles for the oral delivery of PTX, thereby enabling an intravenous-to-oral switch for cancer chemotherapy.

Enhanced oral bioavailability of acetylpuerarin by poly(lactide-co-glycolide) nanoparticles optimized using uniform design combined with response surface methodology.

Acetylpuerarin (AP), an acetylated derivative of puerarin, shows brain-protective effects in animals. However, AP has low oral bioavailability because of its poor water solubility. The objective of this study was to design and develop poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) to enhance the oral bioavailability of AP. The NPs were prepared using a solvent diffusion method optimized via uniform design (UD) combined with response surface methodology (RSM) and characterized by their morphology, particle size, zeta (ζ)-potential, encapsulation efficiency (EE), drug loading (DL), and in vitro drug release. A pharmacokinetic study was conducted in Wistar rats administered a single oral dose of 30 mg/kg AP. The optimized NPs were spherical and uniform in shape, with an average particle size of 145.0 nm, a polydispersity index (PI) of 0.153, and a ζ-potential of -14.81 mV. The release of AP from the PLGA NPs showed an initial burst release followed by a sustained release, following Higuchi's model. The EE and DL determined in the experiments were 90.51% and 17.07%, respectively. The area under the plasma concentration-time curve (AUC0-∞) of AP-PLGA-NPs was 6,175.66±350.31 h ng/mL, which was 2.75 times greater than that obtained from an AP suspension. This study showed that PLGA NPs can significantly enhance the oral bioavailability of AP.

Polysorbate 80-coated PLGA nanoparticles improve the permeability of acetylpuerarin and enhance its brain-protective effects in rats.

OBJECTIVES: Acetylpuerarin (AP) is an acetylated derivative of puerarin (PUE). The study aimed to prepare polysorbate 80-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles to improve the permeability of AP across the blood-brain barrier (BBB) and enhance its brain-protective effects. METHODS: AP-loaded PLGA nanoparticles (AP-PLGA-NPs) were prepared using a solvent diffusion methodology. The NPs were characterized. The pharmacokinetics, tissue distributions and brain-protective effects of AP-PLGA-NPs were evaluated in animals. KEY FINDINGS: AP-PLGA-NPs were successfully prepared with a mean particle size of 145.0 nm and a zeta potential of -14.81 mV. The in-vitro release of AP from the PLGA-NPs showed a biphasic release profile. AP was metabolized into PUE in rats. The AUC0-∞ values of AP and PUE for AP-PLGA-NPs were 2.90- and 2.29-fold as great as those for AP solution, respectively. The values of the relative targeting efficiency in the brain were 2.40 and 2.58 for AP and PUE, and the ratios of peak concentration were 1.91 and 1.89 for AP and PUE, respectively. Compared with the crude drug, AP-PLGA-NPs showed better brain-protective effects in rats. CONCLUSION: Polysorbate 80-coated PLGA-NPs can improve the permeability of AP cross the BBB and enhance its brain-protective effects in rats.

Simultaneous determination of acetylpuerarin and puerarin in rat plasma by liquid chromatography-tandem mass spectrometry: Application to a pharmacokinetic study following intravenous and oral administration.

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous determination of acetylpuerarin (AP) and its major metabolite puerarin (PUE) in rat plasma using genistein as the internal standard (IS). Plasma samples were pretreated by protein precipitation with a mixture of methanol and acetonitrile. Chromatographic separation was performed on a CAPCELL PAK C18 MGШ column with a mixture of 0.1% formic acid in water and methanol (35:65, v/v) as the mobile phase. The analytes were detected using a tandem mass spectrometer in the positive ionization and multiple-reaction monitoring mode. The ion transition of m/z 669.4→627.3, 417.5→297.6 and 271.3→153.0 was utilized to quantify AP, PUE and the IS, respectively. The calibration curves showed good linearity over the plasma concentration range of 1-2000ng/mL for AP and 2.5-5000ng/mL for PUE. The intra- and inter-day precisions (RSD %) for each analyte were less than 6.91%, and the accuracies ranged from -2.17% to 2.93%. The validated LC-MS/MS method was further successfully applied to a pharmacokinetic study of AP and PUE in rats following intravenous and oral administration.

[The gene expression of Th1/Th2 cytokines and its role in hypopharyngeal carcinoma].

OBJECTIVE: To observe the mRNA expression of Th1/Th2 cytokines in hypopharyngeal neoplasms and to explore the pathogenesis of it. METHOD: The gene expression of Th1/Th2 cytokines in patients with hypopharyngeal neoplasms was detected by RT-PCR. RESULT: The cytokines expression of Th2 was significantly stronger than Th1 in tissue of hypopharyngeal carcinoma. CONCLUSION: The dominant expression of Th2 cytokines in hypopharyngeal neoplasms may inhibit the human immunity and enhance the growth and metastasis of hypopharyngeal carcinoma.