bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Polydeoxynucleotide Derived Drug Defibrotide Inhibits Nucleosome Generation in Whole Blood. Implications in the Management of VOD
Authors: Fareed Jawed , Hoppensteadt Debra , Walborn Amanda , Phan Trung , Spatz Stephen , Walenga Jeanine , Jeske Walter 
Published: November 2023

IntroductionDefibrotide (Defitelio, Jazz Pharmaceutical, Palo Alto, CA) is a single stranded deoxyribonucleic acid derived antithrombotic and anti‐ischemic drug of mammalian tissue origin. This agent produces its pharmacological effects through profibrinolytic, antithrombotic, hemodynamic, and anti‐inflammatory actions. In April 2016, defibrotide was approved for the treatment of hepatic veno‐occlusive disease (VOD) after hematopoietic stem cell transplantation (HSCT). VOD represents a catastrophic complication of HSCT and is often fatal due to multi‐organ failure. Defibrotide is able to resolve the VOD and is well‐tolerated. Hepatic cellular apoptosis and cell death in VOD results in the generation of nucleosomes which contribute to the propagation of this disease by enhancing thrombotic and vascular complications. It is hypothesized that defibrotide may reduce the generation of nucleosomes.Materials and MethodsDefibrotide was obtained in powdered form (Gentium, Villa Guardia, Italy) and dissolved in saline to obtain working concentrations of 10 mg/mL. Fresh citrated whole blood was collected from normal healthy donors. To study the effect of defibrotide on nucleosome generation, blood samples were supplemented with this agent at varying concentrations of 0–100 ug/mL. Saline was used as a control. Whole blood samples were incubated with E. coli and S. enterica lipopolysaccharide B at concentrations o ranging from 0 to1000 ng/mL for 15 minutes at 37 degrees Celsius to trigger the generation of nucleosomes. A commercially available ELISA PLUS, Cell Death Assay (Roche Diagnostics, Mannheim, Germany) was used to measure plasma nucleosome levels.ResultsBoth the E. coli and S. enterica lipopolysaccharides produced a concentration‐dependent increase in the generation of nucleosomes. At 1 μg/mL, the nucleosome levels were in the range of 40–70 AU/mL (47 ± 11). Defibrotide inhibited the generation of nucleosomes with an IC50 of 35 μg/mL and produced almost a complete inhibition at a concentration of 100 μg/mL. The level of nucleosome generated in different normal individuals showed wide variations, however the degree of inhibition by defibrotide was comparable in all plasmas.DiscussionThese studies suggest that beside other pharmacological effects, defibrotide may blunt the generation of nucleosomes which may contribute to its observed therapeutic effects in HSCT associated VOD. Since defibrotide is a single stranded DNA derived agent it may compete with the DNA component of nucleosome and/or interact with the histones. These studies also suggests the importance of the measurement of nucleosomes in patients with VOD for both the diagnostic and prognostic purposes.
Identification of a novel metformin‐methylglyoxal imidazolinone metabolite in T2DM patients and its potential roles in alleviating T2DM complications
Authors: Hargraves Tiffanie Leann , Kinsky Owen R. , Monks Terrence J. , Lau Serrine S. 
Published: November 2023

Reactive dicarbonyls, such as methylglyoxal (MG), are elevated in type‐2 diabetes mellitus (T2DM) patients. These endogenous electrophiles covalently modify proteins, which may contribute to diabetic complications. The T2DM first‐line therapy, metformin (MF), significantly reduces diabetic endpoints and mortality more effectively than other anti‐hyperglycemic agents. Based upon its structure, we examined whether MF directly scavenges MG as an alternative mechanism of drug efficacy, in addition to its antigluconeogenesis properties. We synthesized and characterized a MF/MG imidazolinone product (IMZ) by 13C, 1H and HMBC NMR, LC‐MS/MS and X‐ray diffraction. We measured MF and the IMZ product in human urine using a LC/MS multiple reaction monitoring (MRM) method. IMZ was detected in >90 MF‐treated T2DM patients, with increased levels of MF directly correlating with elevations in IMZ. Urinary MF was detected in the range of 0.17 μM ‐ 23.0 mM. Simultaneously the IMZ concentration was measured between 18.8 nM and 4.3 μM. Since reported plasma concentrations of MG are in the range of 40 nM to 4.5 μM, levels of IMZ production may be of therapeutic significance. Since the I1‐imidazoline receptor is a novel target for drug development for disorders associated with T2DM we examined the ability of IMZ to modulate the AMPK, AKT, and ERK1/2 signaling pathways via western blot, following treatment of PC12 cells with IMZ. Combination treatment of insulin and IMZ increased the phosphorylation of AMPK, AKT, and ERK1/2 above levels seen with insulin treatment alone. The results reveal that IMZ may enhance insulin action in the insulin‐dependent AMPK, AKT, and ERK1/2 pathways. In summary, the concordance of IMZ concentration provides evidence that metformin is scavenging MG, thereby reducing detrimental protein modifications, which may play a role in the reduction of diabetic complications. Further, IMZ has the potential to enhance insulin action, and ongoing research may indicate IMZ has potential as a therapeutic for treating insulin resistance.Support or Funding InformationR24DK0090958, T32ES007091, P30ES006694
The Fungicidal Potential of Dimethyloxaglycine (DMOG) in Infected Bone Marrow Macrophages of C57 BL6‐J mice
Authors: Lopez Abdon , Buesing William , Tweedle Jamie , Deep George 
Published: November 2023

Histoplasma capsulatum, a dimorphic fungal pathogen, can be found in all regions of the United States and is the most common pulmonary fungal infection in immune‐depressed patients. HIF‐1 is a heterodimeric transcription factor, composed of Hif‐1βand HIF‐1α, whose expression is regulated by oxygen, and regulates metabolic and immune response genes. Dimethyloxaglycine(DMOG), inhibits hydroxylases and induces HIF‐1‐dependent transcription.AIMTo investigate the fungicidal potential of DMOG in infected bone marrow macrophages of C57 BL6‐J mice and its possible mechanism dependence in HIF‐1α.MethodsBone marrow was harvested from HIF‐1α knock‐out and wild type (WT) mice, and then, cultured with GM‐CSF. Bone Marrow macrophages(BMm) were plated in 24, 48 and 96 well‐plates, with 1 × 106, 5 × 105 and 1 × 105 cells per well respectively, and treated 6hrs prior to H.capsulatum infection. BMm were treated with: Medium), Vehicle (DMSO) or 0.1% DMOG. After 24, 48 and 72hrs post infection, supernatant was collected, BMms were lysed, transferred to culture plates, and fungal burden was counted after a week of incubation at 29°C. Cytokine levels were measured by ELISA, and PCR.ResultsIn the 48 well plates, WT‐BMm treated with DMOG had a higher tendency of fungal clearance. A high expression of IL‐1β and a low expression of GM‐CSF at the protein level was observed in this group. At the transcriptional level, we saw an increase of TGF‐β.ConclusionDMOG enhances H. capsulatum clearance by macrophages, through a mechanism that may stabilize HIF‐1α and induces changes in cytokines expression.
Vascular Activation and Development of Carotid Intima‐Media Thickness in Postmenopausal Women Is Influenced by Pregnancy History
Authors: Miller Virginia M , Garovic Vesna D , Mielke Michelle M , Bailey Kent R , Lahr Brian , White Wendy M , Jayachandran Muthuvel 
Published: November 2023

ObjectivePreeclampsia (PE) is a hypertensive pregnancy disorder that represents a sex‐specific risk factor for future cardiovascular disease in affected women. This study evaluated the associations of a history of PE, conventional cardiovascular risk factors and vascular activation (defined by activation of blood elements and blood‐borne microvesicles (MV)) with carotid intima‐media thickness (CIMT) in menopausal women.MethodsCaucasian women residents of Olmsted County, MN who delivered between 1976 and 1982 were identified from review of their medical records and matched for parity and age at index birth: 40 with a history of preeclampsia (PE) and 40 with a history of normotensive pregnancy (NP). Blood clinical chemistries and 38 measures of intravascular activation including differential cell counts, platelet aggregation and secretion, cell‐cell interactions, and blood‐borne MV were measured in venous blood. CIMT was measured by B‐mode ultrasound. The study was approved by the IRB at Mayo Clinic and all women gave written informed consent.ResultsWomen averaged 60 years of age at the time of testing. CIMT was greater in the PE [0.80 mm (0.75, 0.85); median (25th, 75th percentile)] compared to the NP [0.73 mm (0.70, 0.78)] group (P=0.004). Body mass index, waist circumference, fasting insulin, high‐sensitive C‐reactive protein and present day hypertension were greater in the PE group. After adjusting for potential confounding factors, the odds of having a higher CIMT value was more than 3‐fold higher for women with a history of PE [OR 3.46 (1.38, 8.68); P=0.008]. Principal components (PC) analysis identified 7 components that accounted for 63% of the variability in measures of intravascular activation. The global test for association of intravascular activation with CIMT (P=0.009) was only significant among women with a history of NP. PC#4, which conveyed measures of whole blood platelet aggregation and platelet interactions with granulocytes and monocytes, positively associated with CIMT in the overall group after adjustment for PE status (partial ρ=0.290; P=0.009). PC#6, consisting of measures of platelet activation with platelet count and interactions between monocytes and the vascular endothelium, also showed a significant interaction with pregnancy history (P=0.014).ConclusionA history of preeclampsia accompanied by metabolic and pro‐coagulant/‐inflammatory intravascular cellular activation contributes to progression of CIMT in menopausal women.Support or Funding InformationThis work was funded by grants from the NIH AG44170, AG034676, UL1 TR000135 and the Mayo Foundation.
The effects of membrane cholesterol on the mechanics of vascular smooth muscle cell
Authors: Hong Zhongkui , Childs Josh , Sanyour Hanna 
Published: November 2023

As the major cellular component of blood vessel wall, vascular smooth muscle cell (VSMC) plays important roles at each stage of atherosclerosis by proliferation and migration toward the inner layer of blood vessel wall. It is also known that cholesterol is not only the major factor involved in fatty deposition in the atherosclerotic lesion, but also plays an important role as a regulator of cell mechanics, spreading, and migration. A further complication is the mechanical environment of the vascular wall, which undergoes progressive stiffening with aging and hypertension and thus accelerates development of atherosclerosis. Therefore, it is important to investigate the combined effect of cholesterol and mechanical environment on VSMC and determine how it alters VSMC functional characteristics such as cell stiffness and adhesion. VSMCs used in this study were enzymatically isolated from the descending aorta of male Sprague‐Dawley rats and cultured in DMEM with 10% FBS in a humidified incubator at 37°C and 5% CO2. Cell adhesion was measured for 50–70% confluent cells with an atomic force microscope (AFM) system and the fibronectin (FN) or N‐cadherin coated stylus AFM probes at 100 nm/sec retraction speed. Cell stiffness was tested with spherical AFM probe (5μm diameter) at 1 μm/sec indentation speed. Cells were treated with 5mM Methyl‐β‐cyclodextrin for 1 h for cholesterol depletion prior to the AFM experiment. Our results showed that cholesterol depletion significantly increased N‐cadherin mediated cell adhesion by ~20% and adhesion probability by ~35%, respectively. While α5β1/FN adhesion force significantly decreased upon membrane cholesterol depletion. Moreover, our results suggested that cholesterol depletion significantly reduced VSMCs stiffness by 47% compared to control cells. All together, these results reveal that membrane cholesterol is a key regulator for cellular stiffness and integrin/cadherin‐mediated cell adhesion. The knowledge that we obtained in this project may lead to a novel therapeutic strategy that could potentially control and block VSMC migration and prevent the formation of atherosclerotic plaque by regulating integrin and/or cadherin mediated cell adhesion.Support or Funding Information15SDG25420001 (AHA)
Substrate Properties Affect Thrombin Generation Kinetics
Authors: Laljee Sehar , Rubenstein David A , Yin Wei 
Published: November 2023

Platelet activation and coagulation play critical roles in hemostasis. It is well established that altered flow and shear stress can cause platelet activation. We recently reported that flow and shear stress can also affect platelet thrombin generation kinetics. In the present study, we aimed to investigate how different substrates, i.e., tissue factor, von Willebrand factor, and vascular wall endothelial cells, affect thrombin generation kinetics under shear flow. Fresh human platelets were exposed to physiological (0.1–1.2 Pa pulsatile) or pathological (0.05 – 0.3 Pa pulsatile) shear stress in the presence of prothrombin, factor Xa, and calcium in a cone and plate shearing device. The wells were precoated with tissue factor or von willebrand factor, or covered with a monolayer of confluent human coronary artery endothelial cells (untreated or activated with liposaccharide ‐ LPS). Thrombin generation was quantified using Chromozym‐TH absorbance at 405 nm. The results demonstrated that with the presence of tissue factor, shear stress magnitude hardly affected thrombin generation rate. When von Willebrand factor was available, thrombin generation rate increased by approximately 21% under normal pulsatile shear stress (compared to no shear conditions), which was further increased by low pathological shear stress to 34% (compared to no shear condition). When platelets were exposed to shear stress in the presence of healthy (untreated) coronary artery endothelial cells, thrombin generation rate decreased by about 1/3 under normal shear stress. However, when endothelial cells were pre‐activated by LPS, thrombin generation rate increased under all conditions (no shear, normal shear and low pathological shear). But pathological low shear stress did not cause significant changes in thrombin generation rate compared to normal pulsatile shear stress. These results are different from what we reported earlier, that with the presence of coagulation factors (i.e., prothrombin, factor Xa, and calcium), low pulsatile shear stress induced faster thrombin generation compared to normal shear stress. It was also observed that under normal pulsatile shear stress, thrombin generation rate almost doubled when the plate was coated with von Willebrand factor, compared to tissue factor. When endothelial cells were activated, thrombin generation rate increased to about 2.5 folds of that seen with tissue factor. Similar trend was observed under low pathological shear stress. These results suggest that substrate properties could affect thrombin generation rate greatly. Therefore, to quantify thrombin generation kinetics, not only shear stress (magnitude and exposure time) and the presence/concentration of coagulation factors, but also substrate properties need to be considered.Support or Funding InformationThis study is supported by an American Heart Association Grant‐in‐Aid Award (16GRNT30440002).
Platelet functional effects induced by fluorescent labeling via transgenic‐ and antibody‐based approaches
Authors: Da Qi , Rumbaut Rolando 
Published: November 2023

BackgroundROSA26‐Enhanced Yellow Fluorescent Protein (R26R‐EYFP) mice have been widely used for cell lineage tracing and tracking specific cells in vivo. To visualize platelets in vivo, we bred R26R‐EYFP mice with Platelet Factor 4‐Cre (PF4‐Cre) mice to generate mice that specifically expressed EYFP in platelets. To test possible effects of genetic manipulation and EYFP expression on platelet function, we compared platelet aggregation and adhesion under flow of platelets expressing EYFP from these mice (Cre+, or R26R‐EYFP/PF4‐Cre(+)) with that of littermate controls (Cre‐, or R26R‐EYFP‐Cre/(−)) and C57BL/6 mice. We also assessed platelet function changes induced by antibody labeling of platelets from C57BL/6 mice with DyLight 488 anti‐Glycoprotein Ibβ (x‐488).MethodsAnimal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). We studied platelet aggregation and adhesion under flow from transgenic mice with either fluorescent platelets (Cre+) or littermate controls with non‐fluorescent platelets (Cre−). C57BL/6 (BL/6) mice were included as an additional control. Blood was collected into either Ethylenediaminetetraacetic acid for imaging flow cytometry, Acid Citrate Dextrose Solution for platelet aggregometry, or D‐Phenylalanyl‐prolyl‐arginyl Chloromethyl Ketone for platelet adhesion under flow. Washed platelets were subjected to thrombin or collagen mediated aggregation. Platelet adhesion under flow was assayed by perfusing anticoagulated blood over a Type III collagen‐coated surface in a microfluidic parallel‐plate flow system, and platelet adhesion was quantified under bright field illumination. Statistical comparisons were performed with paired or non‐paired Student's t‐ test, as appropriate.ResultsImaging flow cytometry of blood derived from Cre+ mice confirmed that EYFP expression was specific for platelets. Platelet aggregometry demonstrated that platelets from Cre+ mice had enhanced aggregation responses to low concentrations of thrombin and collagen when compared with platelets from BL/6 mice (50±14% vs. 34±18% for 0.01U/mL thrombin, p<0.05; 42±19% vs. 10±6.7% for 0.5μg/mL collagen, p<0.01). At higher concentrations of thrombin (0.02U/mL) and collagen (1μg/mL), platelet aggregation was comparable (58±12% vs. 60±14%, and 65±8% vs. 65±9%, respectively, N.S.). Both strains of transgenic mice (Cre+ and Cre−) demonstrated enhanced platelet adhesion under flow as compared to BL/6 mice. In contrast to the genetic fluorescent protein labeling method, x‐488 antibody labeling of platelets from BL/6 mice did not induce significant differences in platelet adhesion under these conditions.ConclusionsGenetic fluorescent protein labeling of platelets with ROSA26‐EYFP induces greater alterations in platelet function ex vivo than antibody labeling with x‐488.Support or Funding InformationSupport: VA/BLR&D Merit Review Grant 1I01BX002551 & NIH/NHLBI R01 HL116524
TRPV4 channels regulate vascular integrity through stabilization of VE‐Cadherin junctions
Authors: Cappelli Holly C , Amin Vibhatsu , Sharma Priya , Kanugula Anantha K , Adapala Ravi K , Meszaros J. Gary , Paruchuri Sailaja K , Thodeti Charles K 
Published: November 2023

Abnormal angiogenesis is a hallmark of pathological conditions characterized by leaky immature vessels. Most of the studies on pathological angiogenesis including vascular normalization strategies are focused on the inhibition of pro‐angiogenic growth factor signaling, which has been met with limited success. However, there are few studies on endogenous regulators and/or mechanical signaling in the regulation of pathological angiogenesis. We have recently found mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) to be functionally low in tumor‐derived endothelial cells which exhibit aberrant mechanosensitivity and abnormal angiogenesis that may be imparted due to high basal Rho activity. Further, we demonstrated that tumors grown in TRPV4KO animals show enhanced growth as well as leaky immature vasculature. In the present study, we investigated the molecular mechanisms by which TRPV4 modulates vascular integrity. First, immunostaining revealed that normal EC exhibited distinct VE‐cadherin expression at the cell‐cell contacts in vitro which is significantly reduced in TRPV4KO EC. Next, we measured vascular growth in varying stiffness Matrigel plugs (700–900 Pa) implanted in WT and TRPV4KO mice. Interestingly, we found increased vascular growth with increasing stiffness from TRPV4KO mice. In contrast, Matrigel plugs from WT mice showed no significant vascular growth with increasing stiffness. Surprisingly, we found a significant stiffness‐dependent reduction in VE‐cadherin positive vessels in Matrigel plugs from TRPV4KO mice compared to WT mice, despite an increase in vascular growth. Further, using an in vivo tumor model with WT and TRPV4KO mice, we found a significant increase in VE‐cadherin negative vessels in TRPV4KO mice compared to their WT counterparts. Functionally, we found tumor vessels are leaky which enhanced Lewis lung carcinoma (LLC) tumor metastasis to lung in TRPV4KO mice. Taken together, our findings suggest mechanosensitive ion channel TRPV4 regulates vessel integrity by maintaining VE‐cadherin expression at cell‐cell contacts and identifies TRPV4 as a novel target for vascular normalization therapies.Support or Funding InformationThis work is supported by American Heart Association (AHA) Grant‐in‐aid (14GRNT20380935), NIH‐R15CA202847, and start‐up funds from NEOMED (CKT).
Prunella vulgaris attenuates diabetic renal injury by suppressing high glucose‐induced mesangial fibrosis and inflammation
Authors: Namgung Seung , Joo Yoon Jung , Han Han Byung Hyuk , Sik Choi Eun , Hyeon Hong Mi , Jung Lee Yun , Gill Kang Dae , Sub Lee Ho 
Published: November 2023

Prunella vulgaris, well‐known traditional medicinal plant, is used for the cure of abscess, scrofula, hypertension and urinary diseases. The present study was designed to investigate the beneficial effects of aqueous extract of Prunellae vulgaris (APV) on high glucose (HG)‐promoted human mesangial cell and STZ‐induced diabetic rats. Pretreatment of APV (1‐50 μg/ml) attenuated 25 mM HG‐induced inflammatory marker such as intracellular cell adhesion molecule‐1 (ICAM‐1) and monocyte chemoattractant protein‐1 (MCP‐1) in mesangial cells. Additionally, APV suppressed transforming growth factor α1 (TGF‐α1) and Smad‐2/4 expression, whereas increased Smad‐7 expression level. Activation of p38 has been implicated in TGF‐α1‐stimulated collagen mRNA expression, whereas ERK has been associated with fibronectin accumulation. APV not only inhibits the Smad pathway, but also elicits inhibitory effects on ERK signaling. Connective tissue growth factor (CTGF) and collagen IV, fibrosis biomarkers, were significantly decreased by APV. Moreover, APV inhibited activation and translocation of nuclear factor kappa B (NF‐κB) in HG‐stimulated mesangial cells. Sprague‐Dawley rats were divided into five groups: control, STZ, STZ were fed on APV (100 or 300 mg/kg/day) and STZ with aminoguanidine (AG) (100 mg/kg/day) for 8 weeks respectively. The renal tissue in the APV group exhibited clear signs of inflammation compared with the control group. Additionally, APV increased nephrin expression in STZ‐induced diabetic kidney. Glomerulus TGF‐α1 expression in immunohistochemical staining was significantly decreased by APV. These findings suggest that APV have protective effect against diabetic nephropathy including inflammation and fibrosis through disturbing TGF‐α/Smad signaling.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2008‐0062484).