Single-cell RNA sequencing unveils unique transcriptomic signatures of endothelial cells and role of ENO1 in response to disturbed flow.

Flow patterns exert significant effects on vascular endothelial cells (ECs) to lead to the focal nature of atherosclerosis. Using a step flow chamber to investigate the effects of disturbed shear (DS) and pulsatile shear (PS) on ECs in the same flow channel, we conducted single-cell RNA sequencing analyses to explore the distinct transcriptomic profiles regulated by DS vs. PS. Integrated analysis identified eight cell clusters and demonstrated that DS induces EC transition from atheroprotective to proatherogenic phenotypes. Using an automated cell type annotation algorithm (SingleR), we showed that DS promoted endothelial-to-mesenchymal transition (EndMT) by inducing the transcriptional phenotypes for inflammation, hypoxia responses, transforming growth factor-beta (TGF-ß) signaling, glycolysis, and fatty acid synthesis. Enolase 1 (ENO1), a key gene in glycolysis, was one of the top-ranked genes in the DS-induced EndMT cluster. Pseudotime trajectory analysis revealed that the kinetic expression of ENO1 was significantly associated with EndMT and that ENO1 silencing repressed the DS- and TGF-ß-induced EC inflammation and EndMT. Consistent with these findings, ENO1 was highly expressed in ECs at the inner curvature of the mouse aortic arch (which is exposed to DS) and atherosclerotic lesions, suggesting its proatherogenic role in vivo. In summary, we present a comprehensive single-cell atlas of ECs in response to different flow patterns within the same flow channel. Among the DS-regulated genes, ENO1 plays an important role in DS-induced EC inflammation and EndMT. These results provide insights into how hemodynamic forces regulate vascular endothelium in health and disease.

Integrin-YAP/TAZ-JNK cascade mediates atheroprotective effect of unidirectional shear flow.

The Yorkie homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1), effectors of the Hippo pathway, have been identified as mediators for mechanical stimuli. However, the role of YAP/TAZ in haemodynamics-induced mechanotransduction and pathogenesis of atherosclerosis remains unclear. Here we show that endothelial YAP/TAZ activity is regulated by different patterns of blood flow, and YAP/TAZ inhibition suppresses inflammation and retards atherogenesis. Atheroprone-disturbed flow increases whereas atheroprotective unidirectional shear stress inhibits YAP/TAZ activity. Unidirectional shear stress activates integrin and promotes integrin-Gα13 interaction, leading to RhoA inhibition and YAP phosphorylation and suppression. YAP/TAZ inhibition suppresses JNK signalling and downregulates pro-inflammatory genes expression, thereby reducing monocyte attachment and infiltration. In vivo endothelial-specific YAP overexpression exacerbates, while CRISPR/Cas9-mediated Yap knockdown in endothelium retards, plaque formation in ApoE-/- mice. We also show several existing anti-atherosclerotic agents such as statins inhibit YAP/TAZ transactivation. On the other hand, simvastatin fails to suppress constitutively active YAP/TAZ-induced pro-inflammatory gene expression in endothelial cells, indicating that YAP/TAZ inhibition could contribute to the anti-inflammatory effect of simvastatin. Furthermore, activation of integrin by oral administration of MnCl2 reduces plaque formation. Taken together, our results indicate that integrin-Gα13-RhoA-YAP pathway holds promise as a novel drug target against atherosclerosis.

Recombinant Expression and Bioactivity Comparison of Four Typical Fungal Immunomodulatory Proteins from Three Main Ganoderma Species.

BACKGROUND: More than a dozen of fungal immunomodulatory proteins (FIPs) have been identified to date, most of which are from Ganoderma species. However, little is known about the similarities and differences between different Ganoderma FIPs' bioactivities. In the current study, two FIP genes termed FIP-gap1 and FIP-gap2 from G. applanatum, along with LZ-8 and FIP-gsi, another two representative Ganoderma FIP genes from G. lucidum and G. sinense were functionally expressed in Pichia. Subsequently, bioactivities of four recombinant Ganoderma FIPs were demonstrated and compared. RESULTS: All the four Ganoderma FIP genes could be effectively expressed in P. pastoris GS115 at expression levels ranging from 197.5 to 264.3 mg L- 1 and simply purified by one step chromatography using HisTrap™ FF prepack columns. Amino acid sequence analysis showed that they all possessed the FIP conserved fragments. The homologies of different Ganoderma FIPs were from 72.6 to 86.4%. In vitro haemagglutination exhibited that FIP-gap1, FIP-gsi and LZ-8 could agglutinate human, sheep and mouse red blood cells but FIP-gap2 agglutinated none. Besides, the immunomodulation activities of these Ganoderma FIPs were as: rFIP-gap2 > rFIP-gap1 > rLZ-8 and rFIP-gsi in terms of proliferation stimulation and cytokine induction on murine splenocytes. Additionally, the cytotoxic activity of different FIPs was: rFIP-gap1 > rLZ-8 > rFIP-gsi > rFIP-gap2, examined by their inhibition of three human carcinomas A549, Hela and MCF-7. CONCLUSIONS: Taken together, four typical Ganoderma FIP genes could be functionally expressed in P. pastoris, which might supply as feasible efficient resources for further study and application. Both similarities and differences were indeed observed between Ganoderma FIPs in their amino acid sequences and bioactivities. Comprehensively, rFIP-gaps from G. applanatum proved to be more effective in immunomodulation and cytotoxic assays in vitro than rLZ-8 (G. lucidum) and rFIP-gsi (G. sinense).

MicroRNA mediation of endothelial inflammatory response to smooth muscle cells and its inhibition by atheroprotective shear stress.

RATIONALE: In atherosclerotic lesions, synthetic smooth muscle cells (sSMCs) induce aberrant microRNA (miR) profiles in endothelial cells (ECs) under flow stagnation. Increase in shear stress induces favorable miR modulation to mitigate sSMC-induced inflammation. OBJECTIVE: To address the role of miRs in sSMC-induced EC inflammation and its inhibition by shear stress. METHODS AND RESULTS: Coculturing ECs with sSMCs under static condition causes initial increases of 4 anti-inflammatory miRs (146a/708/451/98) in ECs followed by decreases below basal levels at 7 days; the increases for miR-146a/708 peaked at 24 hours and those for miR-451/98 lasted for only 6 to 12 hours. Shear stress (12 dynes/cm(2)) to cocultured ECs for 24 hours augments these 4 miR expressions. In vivo, these 4 miRs are highly expressed in neointimal ECs in injured arteries under physiological levels of flow, but not expressed under flow stagnation. MiR-146a, miR-708, miR-451, and miR-98 target interleukin-1 receptor-associated kinase, inhibitor of nuclear factor-κB kinase subunit-γ, interleukin-6 receptor, and conserved helix-loop-helix ubiquitous kinase, respectively, to inhibit nuclear factor-κB signaling, which exerts negative feedback control on the biogenesis of these miRs. Nuclear factor-E2-related factor (Nrf)-2 is critical for shear-induction of miR-146a in cocultured ECs. Silencing either Nrf-2 or miR-146a led to increased neointima formation of injured rat carotid artery under physiological levels of flow. Overexpressing miR-146a inhibits neointima formation of rat or mouse carotid artery induced by injury or flow cessation. CONCLUSIONS: Nrf-2-mediated miR-146a expression is augmented by atheroprotective shear stress in ECs adjacent to sSMCs to inhibit neointima formation of injured arteries.

Different modes of endothelial-smooth muscle cell interaction elicit differential ß-catenin phosphorylations and endothelial functions.

ß-Catenin phosphorylation plays important roles in modulating its functions, but the effects of different phosphorylated forms of ß-catenin in response to heterocellular interaction are unclear. Here we investigated whether distinct modes of phosphorylation on ß-catenin could be triggered through heterocellular interactions between endothelial cells (ECs) and smooth muscle cells (SMCs), and the consequent modulation of EC functions. ECs were cocultured with SMCs to initiate direct contact and paracrine interaction. EC-SMC coculture induced EC ß-catenin phosphorylations simultaneously at tyrosine 142 (Tyr142) and serine 45/threonine 41 (Ser45/Thr41) at the cytoplasm/nuclei and the membrane, respectively. Treating ECs with SMC-conditional medium induced ß-catenin phosphorylation only at Ser45/Thr41. These findings indicate that different phosphorylation effects of EC-SMC coculture were induced through heterocellular direct contact and paracrine effects, respectively. Using specific blocking peptides, antagonists, and siRNAs, we found that the ß-catenin Tyr142-phosphorylation was mediated by connexin 43/Fer and that the ß-catenin Ser45/Thr41-phosphorylation was mediated by SMC-released bone morphogenetic proteins through VE-cadherin and bone morphogenetic protein receptor-II/Smad5. Transfecting ECs with ß-catenin-Tyr142 or -Ser45 mutants showed that these two phosphorylated forms of ß-catenin modulate differential EC function: The Tyr142-phosphorylated ß-catenin stimulates vascular cell-adhesion molecule-1 expression to increase EC-monocytic adhesion, but the Ser45/Thr41-phosphorylated ß-catenin attenuates VE-cadherin-dependent junction structures to increase EC permeability. Our findings provide new insights into the understanding of regulatory complexities of distinct modes of ß-catenin phosphorylations under EC-SMC interactions and suggest that different phosphorylated forms of ß-catenin play important roles in modulating vascular pathophysiology through different heterocellular interactions.

[Changes in Phytoplankton Community Structure in Qingcaosha Reservoir Based on Time Series Analysis].

Qingcaosha Reservoir is one among the important reservoirs and drinking water sources in Shanghai. Samples were collected from the reservoir every month from 2014 to 2021 to analyze phytoplankton community structure and water environmental factors to provide a reasonable reference for urban reservoir operation management, water resource protection, and development and utilization. The results showed that 561 species of phytoplankton were identified from eight phyla in 8a, mainly diatomata, chlorophyta, and cyanophyta, accounting for 34.94%, 34.58%, and 17.65% of the total species, respectively. A total of 26 dominant species were present in four phyla, and cyanobacteria accounted for 50%. Diatoms and green algae were the dominant species, cyanobacteria was the absolute dominant species, and other phyla accounted for a low proportion in the community structure. The Qingcaosha reservoir had the tendency of transforming into a cyanobacteria-type reservoir. The major dominant genera of chlorophyta were Scenedesmus, Ankistrodesmusc, and Chlorellaceae. The dominant genera of the phylum cyanobacteria were Merismopediaceae, Microcystaceae, Aphanocapsa, and Pseudanabaenaceae. The major dominant genera of the diatoms were Cyclotella, Melosira, and Aulacoseira. The dominant genus of xanthophyta was Tribonemataceae. Phytoplankton abundance ranged from 8.391×105 to 2.115×107 cells·L-1, with an average of 6.345×106 cells·L-1. The biomass of phytoplankton varied from 0.113 to 11.903 mg·L-1, with an average of 1.538 mg·L-1. The maximum abundance occurred in summer, and the maximum biomass occurred in spring. In spatial distribution, the maximum biomass and abundance appeared in the reservoir. Redundancy analysis （RDA） of phytoplankton community structure and water environmental factors showed that water temperature （WT）, dissolved oxygen （DO）, and nutrient salts （TN, TP） were important environmental factors affecting phytoplankton community structure, and significant changes occurred in 2014-2017 and 2018-2021. From 2018 to 2021, cyanobacteria disappeared and cyanobacteria dominated the reservoir and even changed to cyanobacteria-type reservoirs. From 2016 to 2021, half of the dominant species were cyanobacteria, and the cyanobacteria abundance accounted for the highest proportion during this period. The reasons for the extinction of xanthophyta were speculated to be the increase in phosphorus concentration and water temperature, and the reasons for the dominant position of cyanophyta, to be the rise of water level, water temperature, and alkaline water. Reservoirs use filter-feeding fish to control algal overgrowth； however, filter-feeding fish do not filter all algae and not all of their filter-feeding algae is easily digestible. In this study, it was observed that the size of digestible algae biomass in the four seasons was in the order of spring > summer > autumn > winter. RDA analysis of silver carp, bighead carp, and digestible algae showed that the biomass of digestible algae was positively correlated with that of silver carp and bighead carp in spring, autumn, and winter. These results suggest that the digestibility of algae changed the resource use efficiency of filter-feeding fish and led to changes in phytoplankton community structure. The phytoplankton community structure was directly affected by the descending effect of fish and indirectly affected by the digestibility of algae.

Mechanical regulation of epigenetics in vascular biology and pathobiology.

Vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) are constantly exposed to haemodynamic forces, including blood flow-induced fluid shear stress and cyclic stretch from blood pressure. These forces modulate vascular cell gene expression and function and, therefore, influence vascular physiology and pathophysiology in health and disease. Epigenetics, including DNA methylation, histone modification/chromatin remodelling and RNA-based machinery, refers to the study of heritable changes in gene expression that occur without changes in the DNA sequence. The role of haemodynamic force-induced epigenetic modifications in the regulation of vascular gene expression and function has recently been elucidated. This review provides an introduction to the epigenetic concepts that relate to vascular physiology and pathophysiology. Through the studies of gene expression, cell proliferation, angiogenesis, migration and pathophysiological states, we present a conceptual framework for understanding how mechanical force-induced epigenetic modifications work to control vascular gene expression and function and, hence, the development of vascular disorders. This research contributes to our knowledge of how the mechanical environment impacts the chromatin state of ECs and VSMCs and the consequent cellular behaviours.

Molecular switch for cold acclimation -- anatomy of the cold-inducible promoter in plants.

In plants, a promoter is essential to drive the transcription and expression of genes under stress conditions. The cold-regulated promoter is an important molecular switch involved in transcriptional regulation of a dynamic network of genes associated with cold acclimation processes. However, the structure and functions of the cold-regulated promoter are ambiguous. In this review, we first describe the common type and structures of the cold-regulated promoter, such as the core promoter and transcription factor binding sites, and then discuss the synergistic actions of promoter elements and cold-regulated genes. We also describe the transcriptional responses and cross-talk among cold-regulated genes in the ICE-CBF-COR cold-response pathway. Many stress-inducible genes are known to be regulated by endogenous abscisic acid (ABA), which accumulates during osmotic and cold stress. We discuss the regulation of promoters of cold-inducible genes in ABA-dependent and ABA-independent regulatory systems. We also describe the cross-talk among gene networks regulated by different cis-acting regulatory elements. Finally, we propose potential further research on, and practical applications of, the cold-regulated promoter.

Roles of microRNAs in atherosclerosis and restenosis.

Atherosclerosis is commonly appreciated to represent a chronic inflammatory response of the vascular wall, and its complications cause high mortality in patients. Angioplasty with stent replacement is commonly performed in patients with atherosclerotic disease. However, the restenosis usually has a high incidence rate in angioplasty patients. Although the pathophysiological mechanisms underlying atherosclerosis and restenosis have been well established, new signaling molecules that control the progress of these pathologies have continuously been discovered. MicroRNAs (miRs) have recently emerged as a novel class of gene regulators that work via transcriptional degradation and translational inhibition or activation. Over 30% of genes in the cell can be directly regulated by miRs. Thus, miRs are recognized as crucial regulators in normal development, physiology and pathogenesis. Alterations of miR expression profiles have been revealed in diverse vascular diseases. A variety of functions of vascular cells, such as cell differentiation, contraction, migration, proliferation and inflammation that are involved in angiogenesis, neointimal formation and lipid metabolism underlying various vascular diseases, have been found to be regulated by miRs. This review summarizes current research progress and knowledge on the roles of miRs in regulating vascular cell function in atherosclerosis and restenosis. These discoveries are expected to present opportunities for clinical diagnostic and therapeutic approaches in vascular diseases resulting from atherosclerosis and restenosis.

[Study on the toxic reaction induced by single dose of qingkailing injection].

OBJECTIVE: To study the toxic reaction induced by Qingkailing Injection. METHODS: Kunming mice were injected single dose of Qingkailing Injection via tail vein and observed for 14 days to test the toxic reaction of the drug. According to Chinese Pharmacopoeia, hemolysis tests were conducted on the injections of different lots and each ingredient of the injection. RESULTS: Toxic reaction induced by single dose of injection--with dose increasing,mice quickly showed different responses such as hypodynamia, convulsion, syncope and even death after injection. In the high dose group, blood routine detection showed that mice have lower counts of RBC, WBC and lower content of hemoglobin; The pulmonary pathological sections of dead mice showed significant hyperemia. And there were no significant difference in the contents of serum electrolyte (K+, Na+, Ca2+) between normal saline control group and Qingkailing injection group. Hemolysis test in vitro--Honeysuckle extraction (significantly) and gardenia extraction which were components of Qing-kailing injection caused hemolysis in certain dose; While gardenia, pearl shell and isatis root extraction caused RBC agglutination. With higher concentration, the Qingkailing injections of different lot caused different degree of hemolysis. There was no significant difference in the hemolysis test in vitro between the group of Balb/C mice which were sensitized by Qingkailing injection or not. CONCLUSION: In clinical practice some adverse reactions induced by Qingkailing injection occurred concomitantly with acute hemolysis within vessels, which might be caused by honeysuckle and gardenia. And the hemolysis was independent of allergy.

Mechanoresponsive Smad5 Enhances MiR-487a Processing to Promote Vascular Endothelial Proliferation in Response to Disturbed Flow.

MicroRNAs (miRs) and bone morphogenetic protein receptor-specific Smads are mechano-responsive molecules that play vital roles in modulating endothelial cell (EC) functions in response to blood flow. However, the roles of interplay between these molecules in modulating EC functions under flows remain unclear. We elucidated the regulatory roles of the interplay between miR-487a and Smad5 in EC proliferation in response to different flow patterns. Microarray and quantitative RT-PCR showed that disturbed flow with low and oscillatory shear stress (OS, 0.5 ± 4 dynes/cm2) upregulates EC miR-487a in comparison to static controls and pulsatile shear stress (12 ± 4 dynes/cm2). MiR-487a expression was higher in ECs in the inner curvature (OS region) than the outer curvature of the rat aortic arch and thoracic aorta and also elevated in diseased human coronary arteries. MiR-487a expression was promoted by nuclear phospho-Smad5, which bound to primary-miR-487a to facilitate miR-487a processing. Algorithm prediction and luciferase reporter and argonaute 2-immunoprecipitation assays demonstrated that miR-487a binds to 3'UTR of CREB binding protein (CBP) and p53. Knockdown and overexpression of miR-487a decreased and increased, respectively, phospho-Rb and cyclin A expressions through CBP and p53. A BrdU incorporation assay showed that miR-487a enhanced EC proliferation under OS in vitro and in disturbed flow regions of experimentally stenosed rat abdominal aorta in vivo. These results demonstrate that disturbed flow with OS induces EC expression of miR-487a through its enhanced processing by activated-Smad5. MiR-487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR-487 may serve as an important molecular target for intervention against disturbed flow-associated vascular disorders resulting from atherosclerosis.

Structural properties and antioxidant activities of polysaccharide from fruit bodies of Pholiota nameko.

A polysaccharide named PNP was extracted and purified from Pholiota nameko. The total sugar content of PNP was 95.29% and the molecular weight was 1.89 × 103 kDa. The structural features of PNP were investigated by the combination of chemical and instrumental analysis such as UV spectrophotometer, specific rotation determination, FT-IR, methylisation analysis and Congo red. The results showed that the optical rotation of PNP was +120° and that it had a triple-helical structure. Besides, PNP was mainly composed of glucose and mannose at the molar ratio of 4.24:1.00. The backbone of PNP was composed of (1→3)-linked-Glc and (1→3)-linked-Man whereas the branches of (1→3,6)-linked- Glc, (1→3,6)-linked-Man and T- Glc. Consistenting with the results of UV-Vis spectra, FT-IR spectroscopy and 1H NMR, indicated that PNP was a complex of polysaccharides and polyphenols. In vitro antioxidant results suggested that PNP was processed with certain scavenging capacity.

Structural characterisation and ACE-inhibitory activities of polysaccharide from Gastrodia elata Blume.

The structural properties and Angiotensin-I converting enzyme (ACE) inhibition activities of a polysaccharide (PGE) extracted from Gastrodia elata Blume were investigated. PGE was extracted using hot water and purified by Sephadex G-200 followed by ultra-filtration. The structural characterisation of PGE was analysed by FT-IR, NMR spectroscopy, specific rotation determination, periodate oxidation-smith degradation, methylation analysis, GC-MS and Congo red test. The results revealed that PGE was composed by glucose, with an average molecular weight of 1.54 × 103 kDa. The structure of PGE was 1→3 and 1→4,6-branched-glucopyranose that had a linear backbone of (1 → 4)-linked-d-glucopyranose (Glcp). ACE-inhibitory activity results showed that PGE was efficient to inhibit ACE and the IC50 value was 0.66 mg/mL.

Comparison of infection rate with tunneled vs standard external ventricular drainage: A prospective, randomized controlled trial.

OBJECTIVES: A prospective, blinded, randomized trial was performed to evaluate the incidence rates of external ventricular drainage (EVD)-related infection (ERI) after tunneled EVD (T-EVD) and standard EVD (S-EVD). PATIENTS AND METHODS: From February 2018 to February 2019, all adult patients admitted to the Union Hospital Neurosurgery Center for EVD placement were eligible for inclusion. After the application of strict exclusion criteria, all enrolled patients were randomly divided into two groups. The patients in Group A received S-EVD, and the remaining patients in Group B received T-EVD. A linear incision was made for T-EVD. The distal end of the catheter was inserted approximately 5 cm until cerebrospinal fluid was readily obtained, and then the catheter was tunneled approximately 4-5 cm from the insertion point. Finally, an external CSF drainage system was connected to the catheter. For the S-EVD patients, we secured the catheter at the original incision site after insertion, and an external CSF drainage system was also connected to the catheter. The rates of ERI were compared between the two patient groups. The odds ratios and χ² test were used to analyze the results. RESULTS: One hundred twenty patients were randomly divided into two groups and underwent EVD placement. Among them, 60 patients in Group A received S-EVD, and 60 patients in Group B received T-EVD. Finally, 51 patients in Group A and 50 patients in Group B met all of the study inclusion/exclusion criteria and were thus eligible for inclusion in the evaluation of ERI rates. All clinical features of the two groups were similar. A total of 12 patients' (11.9%) CSF cultures were positive for infection. Ten (19.6%) patients who underwent S-EVD had CSF-positive cultures, while only 2 (4.0%) patients who underwent T-EVD had CSF-positive cultures (P = 0.034). Additionally, 8 patients in Group A and 1 patient in Group B were complicated with CSF leakage (P = 0.039). CONCLUSIONS: Compared to S-EVD, T-EVD, when performed according to a previously established perioperative management protocol, resulted in lower infection and CSF leakage rates. We recommend that T-EVD should be preferentially performed when surgeons determine whether a catheter can be removed within 10 days, and the catheter used for EVD should be removed as soon as permitted by the clinical circumstances.

[Effect of Glycyrrhiza inflata and Daphne genkwa on permeabilities of rhodamine 123, a P-glycoprotein substrate across rat jejunum membranes in vitro].

OBJECTIVE: To investigate the modulation of Glycyrrhiza inflata and Daphne genkwa on the permeability characteristics of rhodamine 123 (R123), one P-glycoprotein (P-gp) substrate, across the jejunum membranes. And then approach the possible permeability mechanism of the drugs after co-administration of G. inflata and D. genkwa in gastrointestinal tract. METHOD: The permeability of R123 or fluorescein sodium (CF) via Wistar rat jejunum membranes was evaluated by in vitro diffusion chamber system after oral administration of four different decoctions and 0.9% sodium chloride (20 mL x kg(-1)) for 1 week. And the concentration of R123 or CF was determined by the fluorospectrophotometry. The apparent permeability coefficient (P(app)) was calculated by the equation P(app) = dQ/d(t) x (1/A x C0), where P(app) was expressed in cm/s, dQ/dT was the slope of the linear portion of the permeation curves, A was the diffusion area, and C0 was the initial concentration of rebamipide in the donor side, and then compare their differences were compared with control group. RESULT: After oral administration of G. inflata decoction, D. genkwa decoction and decoction of the combination of the previous decoctions, the absorptive directed transport of R123 was significantly increased (P < 0.05, compared with control group). On the other hand, D. genkwa could also decrease the permeability of secretory directed transport (P(app) = 2.98 +/- 0.59), while no action of G. inflata was found on the secretory transport of R123 ( P(app) = 5.24 +/- 3.98) across the jejunum tissues, while P(app) of control group was 4.38 +/- 1.18. Meanwhile, G. inflata had no effect on transport of CF across the jejunum tissues, though the other three groups could decrease the permeability of CF, as compared with control group. CONCLUSION: G. inflata may slightly inhibit P-glycoprotein function in the intestinal membrane, while D. genkwa may be a relatively strong inhibitor of P-gp. For another, some compositions in D. genkwa inhibit P-gp function, and some others strengthen the tight junction between cells in the intestinal membrane to decrease permeability of CF. As the inhibitory action to P-gp was enhanced by combination of G. inflata and D. genkwa, based on the results, it may be one of the mechanisms of creating toxicity once co-administration of G. inflata and D. genkwa.

MicroRNA-21 and Venous Neointimal Hyperplasia of Dialysis Vascular Access.

BACKGROUND AND OBJECTIVES: There is increasing evidence that microRNAs (miRNAs) play crucial roles in the regulation of neointima formation. However, the translational evidence of the role of miRNAs in dialysis vascular access is limited. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: miRNA expression in tissues was assessed by using venous tissues harvested from ten patients on dialysis who received revision or removal surgery, and ten patients who were predialysis and received creation surgery of arteriovenous fistulas served as controls. To extend these findings, 60 patients who received angioplasty of dialysis access were enrolled and the levels of circulating miRNAs were determined before and 2 days after angioplasty. Clinical follow-up was continued monthly for 6 months. The primary outcome of angioplasty cohort was target lesion restenosis within 6 months after angioplasty. RESULTS: In the surgery cohort, the expressions of miR-21, miR-130a, and miR-221 were upregulated in stenotic tissues, whereas those of miR-133 and miR-145 were downregulated. In situ hybridization revealed similar expression patterns of these miRNAs, localized predominantly in the neointima region. Twenty eight patients in the angioplasty cohort developed restenosis within 6 months. The levels of circulating miR-21, miR-130a, miR-221, miR-133, and miR-145 significantly increased 2 days after angioplasty. Kaplan-Meier plots showed that patients with an increase of miR-21 expression level >0.35 have a higher risk of patency loss (hazard ratio, 4.45; 95% confidence interval, 1.68 to 11.7). In a multivariable analysis, postangioplasty increase of miR-21 expression was independently associated with restenosis (hazard ratio, 1.20; 95% confidence interval, 1.07 to 1.35 per one unit increase of miR-21 expression level; P=0.001). CONCLUSIONS: Certain miRNAs are differentially expressed in the stenotic venous segments of dialysis accesses. An increase in blood miR-21 level with angioplasty is associated with a higher risk of restenosis.

Induction of microRNA-10a using retinoic acid receptor-α and retinoid x receptor-α agonists inhibits atherosclerotic lesion formation.

BACKGROUND AND AIMS: MicroRNA (miR)-10a is a shear-regulated miR with the lowest expression in vascular endothelial cells (ECs) in athero-susceptible regions with oscillatory shear stress (OS). The aim of this study is to elucidate the relationship between EC miR-10a and atherosclerosis and develop a hemodynamics-based strategy for atherosclerosis treatment. METHODS: A combination of in vitro flow system and in vivo experimental animals was used to examine the functional roles of EC miR-10a and its clinical applications in atherosclerosis. RESULTS: En face staining showed that EC miR-10a is down-regulated in the inner curvature (OS region) of aortic arch in rats. Co-administration with retinoic acid receptor-α (RARα)- and retinoid X receptor-α (RXRα)-specific agonists rescued EC miR-10a expression in this OS region. These effects of OS and RARα/RXRα-specific agonists on EC miR-10a expression were confirmed by the in vitro flow system, and were modulated by the RARα-histone deacetylases complex, with the consequent modulation in the downstream GATA6/vascular cell adhesion molecule (VCAM)-1 signaling cascade. Animal studies showed that miR-10a levels are decreased in both aortic endothelium of atherosclerotic lesions and blood plasma from apolipoprotein E-deficient (ApoE-/-) mice. In vivo induction of EC miR-10a by administration of RARα/RXRα-specific agonists protects ApoE-/- mice from atherosclerosis through inhibition of GATA6/VCAM-1 signaling and inflammatory cell infiltration. CONCLUSIONS: Our findings indicate that down-regulation of miR-10a in aortic endothelium and blood serum is associated with atherosclerosis, and miR-10a has potential to be developed as diagnostic molecule for atherosclerosis. Moreover, EC miR-10a induction by RARα/RXRα-specific agonists is a potential hemodynamics-based strategy for atherosclerosis treatment.

The inhibition of TNF-alpha-induced E-selectin expression in endothelial cells via the JNK/NF-kappaB pathways by highly N-acetylated chitooligosaccharides.

Chitooligosaccharides (COS) have been shown to regulate various cellular and biological functions. However, the effect of COS on inflammatory responses of the cells remains unclear. We investigated the regulatory effect of highly N-acetylated COS (NACOS) on tumor necrosis factor-alpha (TNF-alpha)-induced endothelial cell (EC) E-selectin expression, which is crucial for leukocyte recruitment. ECs were kept as controls or pre-treated with NACOS for different times, and then stimulated with TNF-alpha for 4h. The results show that pre-treating ECs with NACOS inhibited the TNF-alpha-induced E-selectin expression in a dose- and time-dependent manner. This NACOS-mediated inhibition in E-selectin expression was regulated at the transcriptional level, but not due to changes in mRNA stability. Stimulation of ECs with TNF-alpha-induced rapid increases in the phosphorylation of their mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinase (ERK), c-Jun-NH2-terminal kinase (JNK), and p38 MAPK]; the inhibitor for JNK (i.e., SP600125), but not those for ERK (i.e., PD98059) and p38 MAPK (i.e., SB203580), attenuated this TNF-alpha-induced E-selectin expression. Pre-treating ECs with NACOS inhibited the TNF-alpha-induced JNK activation, suggesting that JNK was involved in the inhibitory effect of NACOS on TNF-alpha-induced E-selectin expression. Pre-treating ECs with NACOS inhibited the TNF-alpha-induced p65 and p50 mRNA expressions. Gel shifting and chromatin immunoprecipitation assays showed that NACOS blocked the TNF-alpha-induced increases in the binding activity and in vivo promoter binding of nuclear factor-kappaB (NF-kappaB) in ECs. Our findings provide a molecular mechanism by which NACOS inhibit TNF-alpha-induced E-selectin expression in ECs, and a basis for using NACOS in pharmaceutical therapy against inflammation.

Structural characterization and inhibition on α-glucosidase activity of acidic polysaccharide from Annona squamosa.

The crude polysaccharide (TASP3) was extracted from the fruit pulp of Annona squamosa and then isolated and purified by the combination of grading-alcoholic precipitation and Sephadex G-200. The structure of purified polysaccharide (GASP3-3-I) was determined based on the physicochemical and instrumental analyses. The results indicated that GASP3-3-I was an acidic heteropolysaccharide and its average molecular weight was 2.28×106Da. The monosaccharide composition was analysed by GC-MS and ion chromatography, respectively. It was revealed that GASP3-3-I was consisted of rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid with a molar ratio of 5.06:45.5:5.26:0.63:6.09:31.76:0.49:5.19. Moreover, periodate oxidation reaction, Smith degrading reaction, methylation, FT-IR and NMR were used to conduct the structural characterization of GASP3-3-I. The results indicated that glycosyl residues of GASP3-3-I were mainly composed of (1→) l-arabinose, (1→6), (1→3) and (1,3→6) d-galactose, (1→) d-xylose, (3→) and (3→6) d-glucose, (1→2) l-rhamnose. The α-glucosidase inhibitory activity assay showed that GASP3-3-I had a certain inhibition on α-glucosidase activity.

Characterization and lymphocyte proliferation activity of an oligosaccharide degraded from Astragalus polysaccharide.

An Astragalus oligosaccharide (AOS) degraded from Astragalus polysaccharide (APS) and purified by membrane dialysis and silicon gel chromatography is studied in this paper. The structural features of AOS were investigated by a combination of chemical and instrumental analysis, such as monosaccharide analysis, periodate oxidation-Smith degradation, methylation analysis, electrospray ionization mass spectrometry (ESI-MS), Fourier transform infrared (FT-IR) spectrometry and nuclear magnetic resonance (NMR). The results indicated that AOS is an octasaccharide that consists of (3→)-linked-Rha, (1→3)-linked-Rha, (1→3,4)-linked-Araf, (1→3)-linked-Gal, terminal-linked-Gal and terminal-linked-Glc. The effects of AOS on cyclophosphamide-induced immunosuppression were determined by various studies, such as the proliferation of nucleated marrow, red blood cell (RBC) and white blood cell (WBC) populations, growth of the spleen and thymus, and increases in hemoglobin (HGB) concentration and granulocyte-macrophage colony stimulating factor (GM-CSF) level. The results indicated that AOS can restore cyclophosphamide-induced immunosuppression by stimulating the secretion of GM-CSF, which promoted the differentiation of progenitor cells after proliferation.