Stimulated NO Production Research Articles

Endothelial Hemoglobin Alpha Mediates Iron Regulation of Endothelial Nitric Oxide

Iron deficiency anemia (IDA) is a common comorbidity in cardiovascular disease. Previous clinical studies have found iron deficiency anemia (IDA) increases endothelial NO signaling and attributed this change to the reduction in circulating hemoglobin. However, it has been shown in ex vivo preparations that endothelial NO signaling is unaffected by the presence of erythrocytes suggesting changes in circulating hemoglobin do not alter NO signaling. Our lab has previously shown the hemoglobin-α (Hbα) is expressed in the endothelium of small arteries and scavenges NO. We hypothesized endothelial Hbα, rather than blood hemoglobin is responsible for the increased NO signaling in IDA. We first tested this hypothesis in a mouse model of IDA in which we were able to replete vascular iron stores. Male and female C57BL/6 mice were fed an iron deficient (2-6 ppm Fe) or nutrient matched control diet (48 ppm Fe) beginning at weaning. IDA mice underwent phlebotomies (10% blood volume) at 9 and 11 weeks old. To restore vascular iron stores, a subset of IDA mice received a single i.p. injection of iron dextran (FeDex; 20 mg/kg) at 12 weeks. All measurements were taken one week later. As expected, IDA mice had low blood hemoglobin, and FeDex did not rescue the anemia but did rescue vascular iron stores as measured by ferritin light chain protein. To investigate NO signaling, we used laser speckle contrast imaging to measure changes in in vivo blood flow in response to the NO synthase inhibitor L-NAME. The magnitude of the response to L-NAME is an indicator of NO signaling. IDA mice exhibited an exaggerated response to L-NAME indicating increased NO signaling. FeDex treatment restored the L-NAME response back to control levels suggesting reduced vascular iron stores, rather than the anemia itself increased NO signaling. In order to investigate the role of endothelial Hbα, we repeated these studies in endothelial specific Hbα knockout mice (Hba1fl/fl-Cdh5-CreERT2+). Lack of endothelial Hbα did not prevent the increased response to L-NAME in IDA mice but did prevent the rescue by FeDex. This suggests a model in which endothelial Hba participates in regulating NO in response to iron but not the increased NO in IDA. Because Hbα does not explain the initial increase in NO, we have begun investigating other anemia-associated NO stimulators. Erythropoetin (EPO) is known to stimulate NO production in endothelial cells and is elevated in our anemia model regardless of FeDex treatment. Future work will investigate the role of EPO in regulating NO in IDA. University of Virginia Basic and Translational Research Training Grant T32 007284 (LSD, MAL, SAL); NIH HL088554; LaunchPad; Lipedema Foundation; AHA Predoctoral Fellowship (MAL). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Distinct effects of orexin A on spontaneous and evoked synaptic currents in the rat nucleus tractus solitarius

Orexins are produced in hypothalamic areas and orexin-containing neurons are distributed in widespread areas of the central nervous system. Orexins regulate several physiological functions such as arousal, food intake and autonomic control. The presence of orexin-containing neuron terminals and orexin receptors has been confirmed in the nucleus tractus solitarius (NTS), which receives primary afferent fibers from peripheral organs including baroreceptors. However, the neuronal effects of orexin-1 receptor (OX1R) activation were not examined. Here, we aimed to determine the effects of OX1R activation on excitatory synaptic transmission. OX1R activation increased the frequency of spontaneous excitatory synaptic currents (sEPSCs). This effect was blocked by the prior application of L-NAME. In contrast, the amplitude of evoked excitatory postsynaptic currents (eEPSCs) was suppressed by OX1R activation, and this effect was prevented by a cannabinoid receptor 1 blocker, AM251, but not by the pretreatment with L-NAME. Altogether, these results suggest that OX1R activation increases sEPSCs frequency by stimulating NO production, whereas it inhibits eEPSCs by releasing endocannabinoids in the NTS. Thus, OX1R activation had distinct effects on spontaneous and evoked excitatory synaptic transmissions in the NTS.

The ABA-LANCL1/2 Hormone-Receptors System Protects H9c2 Cardiomyocytes from Hypoxia-Induced Mitochondrial Injury via an AMPK- and NO-Mediated Mechanism.

Abscisic acid (ABA) regulates plant responses to stress, partly via NO. In mammals, ABA stimulates NO production by innate immune cells and keratinocytes, glucose uptake and mitochondrial respiration by skeletal myocytes and improves blood glucose homeostasis through its receptors LANCL1 and LANCL2. We hypothesized a role for the ABA-LANCL1/2 system in cardiomyocyte protection from hypoxia via NO. The effect of ABA and of the silencing or overexpression of LANCL1 and LANCL2 were investigated in H9c2 rat cardiomyoblasts under normoxia or hypoxia/reoxygenation. In H9c2, hypoxia induced ABA release, and ABA stimulated NO production. ABA increased the survival of H9c2 to hypoxia, and L-NAME, an inhibitor of NO synthase (NOS), abrogated this effect. ABA also increased glucose uptake and NADPH levels and increased phosphorylation of Akt, AMPK and eNOS. Overexpression or silencing of LANCL1/2 significantly increased or decreased, respectively, transcription, expression and phosphorylation of AMPK, Akt and eNOS; transcription of NAMPT, Sirt1 and the arginine transporter. The mitochondrial proton gradient and cell vitality increased in LANCL1/2-overexpressing vs. -silenced cells after hypoxia/reoxygenation, and L-NAME abrogated this difference. These results implicate the ABA-LANCL1/2 hormone-receptor system in NO-mediated cardiomyocyte protection against hypoxia.

Ultrasonic-Assisted Extraction of Codonopsis pilosula Glucofructan: Optimization, Structure, and Immunoregulatory Activity.

In recent years, multiple edible polysaccharides from Codonopsis pilosula were mainly isolated with high average molecular weights and exhibited various bioactivities, but it was proven that low-molecular-weight polysaccharides could exert stronger activities due to the superior water solubility and permeability. In the present study, the water-soluble polysaccharide C. pilosula with low molecular weight was isolated under ultrasonic assistance at 30 °C, the extraction process was optimized via response surface method (RSM), and the structure and immunoregulatory activity were further investigated. The maximum yield (4.86%) for crude polysaccharides (cCPPs) was obtained under following parameters: ultrasonic power of 370 W, liquid/material ratio of 33 mL/g, ultrasonic time of 81 min. Subsequently, the cCPPs were further purified through dialysis and Sephadex G-25 column to acquire purified polysaccharide (CPPs). Structural analysis indicated that CPPs was a glucofructan (average molecular weight of 4.23 × 103 Da) with (2→1)-β-D-Fruf and (1→)-α-D-Glcp as the backbone branched by (2→6)-β-D-Fruf. Additionally, CPPs could enhance immunoregulatory function by stimulating NO production and cytokine (IL-6 and TNF-α) secretion of RAW264.7 macrophages dose-dependently, which presented no cytotoxic effects. These data suggest that CPPs have the potential to be used as a nutritional dietary compound and natural immunostimulant supplement in the food industry.

Nitric oxide synthesis stimulated by arachidonic acid accumulation via PPARα in acetylcholine-stimulated gastric mucous cells.

What is the central question of this study? Arachidonic acid (AA) stimulates NO production in antral mucous cells without any increase in [Ca2+ ]i . Given that the intracellular AA concentration is too low to measure, the relationship between AA accumulation and NO production remains uncertain. Is AA accumulation a key step for NO production? What is the main finding and its importance? We demonstrated that AA accumulation is a key step for NO production. The amount of AA released could be measured using fluorescence-HPLC. The intracellular AA concentration was maintained at <1μM. Nitric oxide is produced by AA accumulation in antral mucous cells, not as a direct effect of [Ca2+ ]i . In the present study, we demonstrate that NO production is stimulated by an accumulation of arachidonic acid (AA) mediated via peroxisome proliferation-activated receptorα (PPARα) and that the NO produced enhances Ca2+ -regulated exocytosis in ACh-stimulated antral mucous cells. The amount of AA released from the antral mucosa, measured by fluorescence high-performance liquid chromatography (F-HPLC), was increased by addition of ionomycin (10 μM) or ACh, suggesting that AA accumulation is stimulated by an increase in [Ca2+ ]i . The AA production was inhibited by an inhibitor of cytosolic phospholipaseA2 (cPLA2-inhα). GW6471 (a PPARα inhibitor) and cPLA2-inhα inhibited NO synthesis stimulated by ACh. Moreover, indomethacin, an inhibitor of cyclooxygenase, stimulated AA accumulation and NO production. However, acetylsalicylic acid did not stimulate AA production and NO synthesis. An analogue of AA (AACOCF3) alone stimulated NO synthesis, which was inhibited by GW6471. In antral mucous cells, indomethacin enhanced Ca2+ -regulated exocytosis by increasing NO via PPARα, and the enhancement was abolished by GW6471 and cPLA2-inhα. Thus, AA produced via PLA2 activation is the key step for NO synthesis in ACh-stimulated antral mucous cells and plays important roles in maintaining antral mucous secretion, especially in Ca2+ -regulated exocytosis.

In vitro Anti-colorectal Cancer Potential of the Medicinal Mushroom Ganoderma neo-japonicum Imazeki in Hyperglycemic Condition: Impact on Oxidative Stress, Cell Cycle and Apoptosis

Clinical efficacy of chemotherapy is often compromised by diabetogenic glucose on colorectal cancer (CRC). High glucose has been shown to diminish the cytotoxicity of anticancer drugs. The issue can potentially be addressed with natural products. Recently, we revealed that Ganoderma neo-japonicum exhibits inhibitory activities against human colonic carcinoma cells. In this study, the impacts of hexane fraction (Hex, sterol-enriched) and chloroform fraction (Chl, terpenoid-enriched) were further elucidated. The cellular responses, including oxidative stress, cell cycle, and apoptosis were compared between the presence of normal glucose (NG, 5.5 mM) and high glucose (HG, 25 mM). HG promoted cell viability with concomitant elevation of GSH level. Both Hex and Chl fractions stimulated NO production, in addition, induced cell cycle arrest. The apoptotic effect of Hex fraction was glucose-dependent, but Chl fraction triggered apoptosis with an equivalent extent in NG and HG conditions. Overall, the active fractions from G. neo-japonicum show therapeutic potential in managing hyperglycemia-associated CRC.

PF-429242, a Subtilisin Inhibitor, Is Effective in vitro Against Leishmania infantum.

PF-429242 is an inhibitor of subtilisin, an important protease found in Leishmania. However, studies regarding the effect of PF-429242 on Leishmania are scarce. In this work we evaluated the antileishmanial effect of PF-429242 against Leishmania infantum and the mechanism involved in the death of the parasite. PF-429242 had low toxicity against mammalian cells (peritoneal macrophages) (CC50 = 189.07 μM) and presented activity against L. infantum promastigotes (IC50 = 2.78 μM) and intracellular amastigotes (IC50 = 14.07 μM), indicating selectivity toward the parasite. Transmission electron microscopy (TEM), as well as staining of L. infantum promastigotes with MitoTracker® Red, rhodamine 123 and MitoSOX, revealed that the mitochondria was a potential target of PF-429242. In addition, PF-429242 caused an accumulation of neutral lipids in promastigotes, which was demonstrated by Nile Red staining and TEM, and induced oxidative stress (H2DCFDA staining). Furthermore the formation of autophagic vacuoles in L. infantum promastigotes was observed by MDC staining and TEM. However, the killing induced by PF-429242 in L. infantum promastigotes appeared to be unrelated to apoptosis and/or necrosis as there was no phosphatidylserine externalization, DNA fragmentation or alterations in the permeability of the parasite plasma membrane, as assessed by annexin V-FITC, TUNEL and propidium iodide staining, respectively. The morphological and ultrastructural evaluation of the promastigotes by optical microscopy, scanning electron microscopy (SEM) and TEM, revealed the presence of parasites with flagellar defects. TEM analysis of the intracellular amastigotes indicated that mitochondrial damage and autophagy could also be involved in the death of these forms after treatment with PF-429242. In addition, PF-429242 treatment stimulated NO production from infected macrophage, but only at a high concentration (100 μM), as well as an increase of TNF levels after treatment with 10 μM of PF-429242. The compound did not stimulate ROS or IL-10 production. Together, these data highlight the antileishmanial potential of PF-429242, inducing several cellular alterations in the parasite, such as mitochondrial damage, neutral lipids accumulation, oxidative stress and autophagy which culminate in the death of L. infantum, as well as modulating host cellular responses that favor the development of an immune response against the parasite.

Semi-synthetic diversification of coronarin D, a labdane diterpene, under Ugi reaction conditions

The prevalence of 5-hydroxydihydrofuran-2(3H)-one moiety in natural products is exploited for the first time using coronarin D, a labdane diterpene, to afford Ugi reaction product 1a and interrupted Ugi product 2a. The potential of the Ugi reaction was further extended to l-phenylalanine, 2-aminopyridine, and d-glucosamine, which afforded Ugi reaction products 3a–f, 4, and 5a–d, respectively. Cytotoxicity studies in RAW cells reveal that compounds 3e and 5b were non-toxic up to 50 µM, and these compounds were able to reduce the LPS stimulated NO production in RAW cells in par with the standard anti-inflammatory drug dexamethasone.

Molecular networking uncovers steroidal saponins of Paris tengchongensis

Based on a method combining the LC-MS/MS molecular networking strategy with the conventional means of phytochemical research, the chemical constituents and the availability of Paris tengchongensis, a new species found in 2017 from Yunnan Province, were investigated for the first time. The molecular networking showed that this species contained the characteristic steroidal glycosides of the genus Paris by comparison of those of Paris polyphylla var. yunnanensis. Furthermore, the detailed investigation on the 80% EtOH extract of its rhizomes resulted to the isolation of twenty steroidal glycosides including three new spirostane-type saponins, named paristengosides A-C (1–3). Their structures were confirmed by spectroscopic analyses (HRMS and NMR) and chemical methods. The new isolates were evaluated for their cytotoxicities against two human cancer cell lines (HEL and MDA-MB-231), anti-inflammatory effects on a lipopolysaccharide (LPS)-stimulated NO production model in RAW264.7 macrophages, anti-AChE, and antimicrobial activities. The results from the molecular networking and the investigation on the chemical constituents suggested that P. tengchongensis can be used as a potential resource of Rhizoma Paridis.

A Polysaccharide-Rich Fraction Derived from Oviductus ranae with Antioxidant and Immunomodulatory Potentials

Oviductus ranae, an animal-based traditional Chinese medicinal material listed in Chinese Pharmacopeia is widely consumed as tonic, approximately twenty Oviductus ranae-related functional foods have been approved on the market by National Medical Products Administration in China. Proteins are well-defined bioconstituents in Oviductus ranae, little information is availabe on its polysaccharides. In present work, a Polysaccharide-Rich fraction (POR-3) was fractionated from Oviductus ranae based on antioxidant activity-guided isolation and its structure was partially characterized as well as its immunomodulatory potential was explored. POR-3 belonged to a heteropolysaccharide-protein fraction mainly consisted of Man, GlcN, GalA, Gal and Fuc at a molar ratio of 1: 2.28: 2.40: 3.00: 2.23 with binding proteins content of 6.42±0.16%. POR-3 possessed polydisperse property with MW of 999.586 kDa and PDI of 4.09. The UV and FT-IR analysis demonstrated that structures of POR-3 correspond with the characteristics of polysaccharide-protein complex. POR-3 elicited higher scavenging capacities against hydroxyl and DPPH radicals and exerted potent in vitro immunomodulatory activity via promoting macrophages phagocytosis and via stimulating NO production as well as appeared no proliferation on macrophages. POR-3 possesses high potentials to be consumed as a medicine or functional food with antioxidant and immune modulator properties.

Identification, expression profiling and functional characterization of interleukin 11a ortholog from redlip mullet Liza haematocheila: Insight into its roles in the inflammation and apoptosis regulation

Interleukin 11 (IL-11) is a secretory cytokine with pleotropic properties, including anti-inflammatory and anti-apoptotic functions. This study aimed to functionally characterize a teleostean IL-11a ortholog from redlip mullet (LhIL-11a) through bioinformatic analysis, transcriptional expression profiling and protein function assays. The deduced LhIL-11a protein sequence is 200 amino acids long, with a predicted molecular weight of 23.168 kDa. Multiple sequence alignment indicates that LhIL-11a has a typical four-bundle architecture of α-helixes as observed in other IL-11s. The identity–similarity matrix show a higher identity between LhIL-11a and other fish IL-11a sequences. Phylogenetic analysis demonstrated that LhIL-11a falls within a clade including other fish counterparts. In the tissue distribution analysis, the highest constitutive expression of LhIL-11a mRNA was observed in the mullet gastrointestinal tract and brain tissues. Following the challenges with LPS, poly I:C and Lactococcus garvie, the transcription levels of LhIL-11a were significantly upregulated in both PBCs and liver. In the biological functional assay, recombinant LhIL-11a protein showed strong activities of suppressing pro-inflammatory cytokines and apoptotic gene expression in mullet kidney cells and reducing LPS stimulated NO production in murine macrophage cells. Overall, the findings in this study provide the experimental clues to understanding the functional roles of fish IL-11a in inflammation and apoptosis regulation during host defense against invading microbial pathogens.

A new spirocyclic cycloartane triterpenoid from Souliea vaginata

The 70% ethanol extract of the whole plant of Souliea vaginata was purified by multi-chromatographic methods including macroporous resin,silica gel,Sephadex LH-20,and C18-reversed-phase column chromatography. A new spirocyclic cycloartane triterpenoid was isolated and identified as( 16 R*,20 R*,23 S*,24 R*,25 S*)-16,23: 23,26-diepoxy-15α,24,25-trihydroxy-9,19-cycloart-3β-O-β-D-xylopyranoside( 1),and named as soulieoside S. Its planar structure and relative configuration were determined by spectroscopic techniques including 2 D NMR and HRESI-MS. As one of the main components of S. vaginata,compound 1 was evaluated for its anti-inflammatory activity by a lipopolysaccharide( LPS)-stimulated NO production model in RAW264. 7 macrophages,but it didn't show NO production inhibitory effect.

Molecular networking-based strategy for the discovery of polyacetylated 18-norspirostanol saponins from Trillium tschonoskii maxim.

Nine undescribed polyacetylated 18-norspirostanol saponins, trilliumosides A‒J, were obtained after a guidance based on a molecular networking strategy from the rhizomes of Trillium tschonoskii. Their structures were established by analysis of comprehensive spectroscopic data and chemical methods after their isolation in pure form. All isolated saponins were evaluated for their cytotoxicities against five selected human cancer cell lines (Huh7,A549,MCF-7,HepG2, and MOLT-4) and anti-inflammatory effects on a lipopolysaccharide (LPS)-stimulated NO production model in RAW264.7 macrophages. Trilliumoside D showed significant cytotoxicity against MOLT-4 cell lines with an IC50 value of 1.0 ± 0.1 μM, whereas trilliumosides H and I displayed remarkable anti-inflammatory effects on NO production with inhibitory rates of 56.3 ± 1.5 and 56.2 ± 2.2% at the concentration of 1.0 μM, respectively.

Exogenous l-ARGININE does not stimulate production OF NO or cGMP within the rat corporal smooth muscle cells in culture

Background and aimNitric oxide (NO) is the intracellular chemical responsible for initiating a penile erection. Despite conflicting clinical data, it continues to be publicized and promoted that orally administered l-arginine, the putative substrate for NO, enhances the erectile response presumably by stimulating NO production by the corporal tissues resulting in an increase in cGMP production. To shed light on this issue, an in vitro study was conducted to explore the effect of direct exogenous administration of l-arginine as well as its precursor and metabolite, l-citrulline, on the NO-cGMP pathway within the cavernosal smooth muscle (CSM) cell. Materials and methodsCSM cells obtained from 8 to 10 week old Sprague-Dawley rats were grown in Dulbecco media with 20% fetal calf serum and then incubated with or without l-arginine (L-ARG) or l-citrulline (L-CIT) in a time course and dose-response manner. Sildenafil (0.4 mM), IBMX (1 mM), l-NAME (3 μM), ODQ (5 μM) and Deta Nonoate (10 μM) were used as either inhibitors or stimulators of the NO-cGMP pathway. mRNA and protein were extracted and used for the determination of the phosphodiesterase 5 (PDE5). PDE5 activity was determined by luminometry. cGMP content was determined by ELISA. Nitrite formation, an indicator of NO production, was measured in the cell culture media by a colorimetric assay. The cationic (CAT-1) and neutral (SNAT-1) amino acid transporters for L-ARG and L-CIT, respectively, were determined by Western blot. ResultsWhen compared to untreated CSM cells, incubation with 0.25–4.0 mM of L-ARG or 0.3–4.8 mM of L-CIT anywhere between 3 and 24 h did not result in any additional nitrite or cGMP production. The addition of l-NAME, IBMX or ODQ to these L-ARG and L-CIT treated cells did not alter these results. L-CIT but not L-ARG increased PDE5 mRNA and protein content as well as the activity of the PDE5 enzyme. Both CAT-1 and SNAT-1 were expressed in the CSM cells. ConclusionsThis in vitro study demonstrates that exogenous administration of L-ARG or L-CIT failed to stimulate production of either NO or cGMP by the corporal CSM cells. A re-evaluation of the presumptive role of the exogenous administration of L-ARG in improving the synthesis of NO at least at the level of the CSM cells appears warranted.

Xylose rich heteroglycan from flaxseed gum mediates the immunostimulatory effects on macrophages via TLR2 activation

Immunostimulatory activity of the flaxseed gum neutral fraction (NFG) was investigated. NFG was characterized as a xylose rich heteroglycan through monosaccharide composition analysis, FT-IR, methylation/GC–MS, and 1D/2D-NMR. NFG stimulated NO production and phagocytic activity of macrophages. Secretion of interleukin-6, interleukin-1β, and tumor necrosis factor-α (254.7 pg/mL, 2.5 ng/mL, and 42.9 pg/mL, respectively) was significantly induced by NFG. Mitogen-activated protein kinases of JNK and P38 were activated by NFG with increased phosphorylation of JNK and P38, while NO production was reduced to 6.05 and 4.42 μM by JNK and P38 inhibitor, respectively. Nuclear factor-κB signaling pathway was also activated by NFG with the suppression of IκBα and up-regulation of phosphorylation of IκBα and nuclear factor-κB P65. Toll like receptor-2 was the molecular target of NFG and responsible for the activation of down-stream signaling pathways. Thus, NFG from flaxseed gum may potentially be used as a natural immunomodulator in functional foods.

Muscarinic M5 receptors trigger acetylcholine-induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cβ (PLCβ) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.

Characterization, sulfated modification and bioactivity of a novel polysaccharide from Millettia dielsiana

Millettia dielsiana is a traditional herb medicine to treat rheumatic arthritis and gynecological diseases. In this study, a novel polysaccharide (MDP1) with a molecular weight of 139.54 kDa was isolated from Millettia dielsiana by DEAE-52 cellulose chromatography. Its sulfated derivative (S-MDP1) was prepared by the chlorosulfonic acid-pyridine method. Structural analysis indicated that MDP1 was a pectic polysaccharide and did not exhibit a triple helical conformation. In vitro, MDP1 and S-MDP1 had protective effect on H2O2-induced PC12 cells death. Immunological assay showed that MDP1 and S-MDP1 significantly stimulated NO production and phagocytic activity in RAW264.7 cells, and promoted lymphocyte proliferation. Anticoagulant assay indicated that S-MDP1 effectively prolonged activated partial thromboplastin time (APTT) and thrombin time (TT), but not prothrombin time (PT), and MDP1 prolonged APTT and TT weakly. These results implied that MDP1 and S-MDP1 had the potential to be used as antioxidant, immunomodulatory and anticoagulant agents.

Type 2 diabetes is associated with loss of HDL endothelium protective functions.

Aims/HypothesisOne of the hallmarks of diabetes is impaired endothelial function. Previous studies showed that HDL can exert protective effects on endothelium stimulating NO production and protecting from inflammation and suggested that HDL in obese people with diabetes and dyslipidemia may have lower endothelial protective function. We aimed to investigate whether type 2 diabetes impairs HDL endothelium protective functions in people with otherwise normal lipid profile.MethodsIn a case-control study (n = 41 per group) nested in the Cooper Center Longitudinal Study we tested the ability of HDL to protect endothelium by stimulating endothelial nitric oxide synthase activity and suppressing NFκB-mediated inflammatory response in endothelial cells. In parallel we measured HDL protein composition, sphinogosine-1-phosphate and P-selectin.ResultsDespite similar levels of plasma HDL-C the HDL in individuals with type 2 diabetes lost almost 40% of its ability to stimulate eNOS activity (P<0.001) and 20% of its ability to suppress TNFα-dependent NFκB-mediated inflammatory response in endothelial cells (P<0.001) compared to non-T2D controls despite similar BMI and lipid profile (HDL-C, LDL-C, TC, TG). Significantly, the ability of HDL to stimulate eNOS activity was negatively associated with plasma levels of P-selectin, an established marker of endothelial dysfunction (r = −0.32, P<0.001). Furthermore, sphingosine-1-phosphate (S1P) levels were decreased in diabetic plasma (P = 0.017) and correlated with HDL-mediated eNOS activation.Conclusions/InterpretationsCollectively, our data suggest that HDL in individuals with type 2 diabetes loses its ability to maintain proper endothelial function independent of HDL-C, perhaps due to loss of S1P, and may contribute to development of diabetic complications.

Synthesis of novel (−)-epicatechin derivatives as potential endothelial GPER agonists: Evaluation of biological effects

To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (−)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO production, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking studies of the novel Epi derivatives on GPER conformers at 14 ns and 70 ns demostrated favorable thermodynamic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC) treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strategy to potentially isolate and identify novel Epi receptors that may account for its biological activity.

Pazopanib-Induced Hypertension in Patients With Renal Cell Carcinoma Is Associated With Low Urine Excretion of NO Metabolites.

Drugs targeting VEGF (vascular endothelial growth factor) are often associated with rapid development of hypertension by a yet not fully understood mechanism. VEGF is expressed in renal epithelial cells and stimulates NO production. In the renal medulla, inhibition of NO formation by local L-NAME or by impaired endothelin-1 leads to hypertension. The present study was designed to test the hypothesis that VEGF receptor inhibitor treatment leads to hypertension through decreased renal medullary formation of NO and endothelin-1. With a single-center prospective observational design, patients with metastatic renal cell carcinoma (n=27) treated with the receptor tyrosine kinase inhibitor pazopanib were included in the study. Home blood pressure was measured, and plasma and urine samples were collected at baseline and after 4 and 8 weeks of treatment. After 4 weeks, systolic and diastolic blood pressures increased, whereas heart rate decreased significantly; urine protein/creatinine ratio increased significantly, whereas estimated glomerular filtration rate was unchanged. Urine nitrite/nitrate (NOx) and cGMP/creatinine ratios decreased significantly, whereas urine endothelin-1/creatinine ratio and FENa+ were unchanged. In plasma, NOx, cGMP, and brain natriuretic peptide decreased significantly, whereas endothelin-1 was significantly elevated. Blood leukocyte count decreased significantly with unchanged CRP (C-reactive protein). In summary, pazopanib treatment of patients with advanced renal cell carcinoma is associated with hypertension, proteinuria, myelosuppression, and decreased urine and plasma NO metabolites. Results are compatible with a significant role of reduced renal medullary NO bioavailability in VEGF inhibitor-induced hypertension.