bluuebunny/crossref_metadata_2025 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

Introduction

Defibrotide (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 Methods

Defibrotide 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.

Results

Both 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.

Discussion

These 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 Information

R24DK0090958, 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.

AIM

To investigate the fungicidal potential of DMOG in infected bone marrow macrophages of C57 BL6‐J mice and its possible mechanism dependence in HIF‐1α.

Methods

Bone 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.

Results

In 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.

Conclusion

DMOG 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

Objective

Preeclampsia (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.

Methods

Caucasian 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.

Results

Women 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).

Conclusion

A history of preeclampsia accompanied by metabolic and pro‐coagulant/‐inflammatory intravascular cellular activation contributes to progression of CIMT in menopausal women.

Support or Funding Information

This work was funded by grants from the NIH AG44170, AG034676, UL1 TR000135 and the Mayo Foundation.
Fibrinogen and its Soluble Fibrin as Mediators of Erythrocyte Aggregation
Authors: WeberFishkin Samantha , Clark Richard AF , Galanakis Dennis , Irizarry Brandon A , Young Amy L , Frame Mary (Molly)
Published: November 2023

Erythrocyte (RBC) aggregation in blood or in plasma is a reversible phenomenon with an unknown mechanism. However there is evidence that it is induced, at least in part, by fibrinogen. RBC aggregation leads to vascular occlusion or flow diversion and shunting of oxygenated blood. If fibrinogen/fibrin ultimately becomes the major occlusive moiety, as one of us has observed in peri‐burn tissue at 48h post‐burn, the occlusion is likely to be irreversible. Previous studies exploring fibrinogen induced aggregation used Millipore fibrinogen. However, based on our preliminary observation of fibrinogen‐induced aggregation, we propose that the same concentration of fibrinogen across different company brands will induce aggregation of variable extent. Millipore fibrinogen was compared with isolated soluble fibrin‐depleted fibrinogen (FgD) and with soluble fibrin‐rich fibrinogen (FgR) (human). Using a spectrophotometer (Bio‐Rad SmartSpec 3000), focused on wavelengths of 540, 560, and 580nm, corresponding to a peak in oxy, deoxy, and oxygenated hemoglobin state in the RBCs, each fibrinogen isolated was tested at varying concentrations (1, 2, and 4mg/mL) and temperatures (32, 37, and 45°C) with washed human RBC. Across all temperatures and wavelengths, FgR fibrinogen dissolved in phosphate buffered saline (PBS) had a higher percent transmission, which correlates to an increased RBC aggregation, than both Millipore fibrinogen and FgD fibrinogen. Additionally, FgR induced the most aggregation at the highest tested concentration, 4mg/mL. Using this increased aggregation at 4mg/mL of FgR, we then compared to PBS and autologous platelet poor plasma. Across all temperatures, the magnitude of RBC aggregation is higher in plasma than FgR, which induces higher aggregation than PBS alone. The results demonstrated that fibrinogen‐induced RBC aggregation varies with the levels of its soluble fibrin/fibrinogen complex. By extension, these complexes clearly enhance RBC aggregation, intimating an in vivo role particularly in pathologic conditions that increase their levels.
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 Information

15SDG25420001 (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 Information

This 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

Background

ROSA26‐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).

Methods

Animal 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.

Results

Imaging 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.

Conclusions

Genetic 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 Information

Support: VA/BLR&D Merit Review Grant 1I01BX002551 & NIH/NHLBI R01 HL116524
Endothelial Foxo1 and Foxo3a Synergistically Repress Skeletal Muscle Vascular Growth and Blood Flow Recovery in a Mouse Model of Hind Limb Ischemia.
Authors: Nwadozi Emmanuel , DeCiantis Matthew , Haas Tara
Published: November 2023

Neovascularization in peripheral artery disease (PAD) is impaired despite upregulation of factors that promote blood vessel growth which suggest a concomitant increase of anti‐angiogenic repressors. We previously showed that depletion of FoxO proteins (FoxO1/3a/4) induced angiogenesis and enhanced blood flow recovery in a mouse model of PAD. The purpose of this study is to independently assess the roles of endothelial FoxO1 and synergistic actions of FoxO 1 & 3a in a mouse model of PAD. Mice with tamoxifen‐inducible endothelial specific deletions of FoxO1 (PDGFBCreERT2+;FoxO1L/L, FoxO1Δ) and FoxO1/3a (PDGFBCreERT2+;FoxO1/3aL/L, FoxO1/3Δ), or control (PDGFBCreERT2‐, FoxO1L/L) mice, underwent unilateral ligation of the common femoral artery (at ~8–9 weeks of age) to induce a substantial (~80%) reduction of blood flow in the ligated (LIG) compared to the contralateral (CON) leg. Blood flow recovery was analyzed by laser doppler imaging at baseline, then on days 4, 7, 10 and 14 post‐ligation. Muscle histology was conducted after 14 days. FoxO1/3Δ blood flow (LIG/CON leg perfusion ratio) was recovered to basal levels (1.10 ±0.07; n=6) and significantly greater than FoxO1Δ (0.73 ±0.05; P<0.01, n=5) and FoxO1L/L (0.65 ±0.03; P<0.001, n=5) mice 14 days post‐ligation. Neither FoxO1 nor FoxO1/3 deletion altered capillary density (CD) in the control leg. In contrast, CON leg capillary‐to‐fibre ratio (C: F) in FoxO1/3Δ mice (1.02 ±0.04) was significantly lower than FoxO1L/L (1.48 ±0.08) and FoxO1Δ (1.44 ±0.09) mice (P <0.05). Ischemic muscle was characterized by marked cellular infiltration, deposition of fibrous and adipose tissue and myofibre atrophy. Capillary density was significantly increased in ischemic muscles from FoxO1Δ (CON: 746 ±40.15 vs LIG: 1218 ±187.4 capillaries/mm2; P =0.03) and FoxO1/3aΔ (CON: 743.2 ±39.35 vs LIG: 1661 ±158.5 capillaries/mm2; P =0.005) mice. C: F was increased ~25% in ischemic muscle from FoxO1/3Δ mice (1.28 ±0.05; P <0.05) but was unaltered in FoxO1L/L (1.46 ±0.19) or FoxO1Δ mice (1.55 ±0.22). Our data suggest that the combined effects of FoxO1 & 3a repress vascular growth and blood flow recovery in ischemic muscles more than the independent effects of FoxO1. Funded by CIHR.
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 Information

This 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 Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2008‐0062484).
Oncostatin M Induces Barrier Dysfunction in Human Brain Microvascular Endothelium via Snail‐Dependent Repression of TAMPs
Authors: Wertz Travis S , Hoettels Brian A , Tawara Ken , Oxford Julia T , Jorcyk Cheryl L , Beard Richard S
Published: November 2023

Homeostasis of barrier integrity in brain microvessels is maintained by dynamic regulation of endothelial cell‐cell adhesive proteins. During aberrant inflammatory responses, a host of factors act upon the endothelium to modify these proteins and diminish barrier integrity, leading to leakage of plasma components, neurotoxic substances, and potentially edema. The pleiotropic cytokine oncostatin M (OSM) is one such factor, which has been shown to be elevated in several inflammatory conditions, such as multiple sclerosis, that are associated with increased microvascular leakage. Indeed, recent evidence suggests that OSM may directly induce brain endothelial cell barrier dysfunction, but the mechanisms remain unclear. The objective of this study was to determine the effect of OSM on the barrier integrity of human brain microvascular endothelial cells (BMVECs). First, we confirmed that BMVECs were responsive to OSM by measuring activation of STAT3, which is known to be downstream of OSM receptor signaling in other cell types. Indeed, OSM triggered phosphorylation of STAT3 at Y705 in BMVECs as early as 15 minutes following stimulation. Next, we identified that treating BMVEC monolayers with OSM promoted barrier dysfunction, as demonstrated by time‐and‐dosage‐dependent decreases of transendothelial electrical resistance (TER). Concomitant with TER changes, OSM induced a significant decrease in the tight‐junction‐associated MARVEL proteins (TAMPs) occludin and tricellulin. Interestingly, OSMs effect on downregulation of endothelial cell‐cell junctional proteins appears to be exclusive to TAMPs, as there were no noticeable changes in claudin‐ 1, 3, 5, 12 or the adherens junction protein VE‐cadherin. Since it has previously been identified in epithelial cells that occludin can be negatively regulated by the transcription factor SNAI1, or Snail, we evaluated a potential role of Snail in OSM‐mediated barrier dysfunction of BMVECs. At baseline, there was minimal detection of Snail; however, OSM induced a robust upregulation of Snail protein preceding the onset of barrier dysfunction. Finally, siRNA‐mediated knockdown of SNAI1 both increased baseline levels of TAMPs and attenuated OSM‐induced TAMP downregulation and BMVEC barrier dysfunction. In conclusion, elevated OSM induces human brain endothelial barrier dysfunction in a Snail‐dependent manner, and further exploration of this signaling pathway in brain endothelium holds multiple facets of therapeutic potential.

Support or Funding Information

Supported by The Birt Family and NIH grants: P20 GM109095 and 2P20GM103408.
Ginsenoside Rb1 protects against ischemia/reperfusion‐induced myocardial injury via energy metabolism regulation mediated by RhoA signaling pathway
Authors: Han Jing‐Yan , Cui Yuan‐Chen , Pan Chun‐Shui , Yan Li , Hu Bai‐He , Chang Xin , Liu Yu‐Ying , Sun Kai , Li Quan , Fan Jing‐Yu
Published: November 2023

Cardiac ischemia and reperfusion (I/R) injury remains a challenge for clinicians. Ginsenoside Rb1 (Rb1) has been reported to have the ability to attenuate I/R injury, but its effect on energy metabolism during cardiac I/R and the underlying mechanism remain unknown. In this study, we detected the effect of Rb1 on rat myocardial blood flow, myocardial infarct size, cardiac function, velocity of venule red blood cell, myocardial structure and apoptosis, energy metabolism and change in RhoA signaling pathway during cardiac I/R injury. In addition, the binding affinity of RhoA to Rb1 was detected using surface plasmon resonance (SPR). Results showed that Rb1 treatment at 5 mg/kg/h protected all the cardiac injuries induced by I/R, including damaged myocardial structure, decrease in myocardial blood flow, impaired heart function and microcirculation, cardiomyocyte apoptosis, myocardial infarction and release of myocardial cTnI. Rb1 also inhibited the activation of RhoA signaling pathway and restored the production of ATP during cardiac I/R. Moreover, SPR assay showed that Rb1 was able to bind to RhoA in a dose‐dependent manner. These results indicate that Rb1 may prevent I/R‐induced cardiac injury by regulation of RhoA signaling pathway, and may serve as a potential regime to improve percutaneous coronary intervention outcome.

Support or Funding Information

APS
Adipose Tissue – Skeletal Muscle Crosstalk: Obese Human Subcutaneous and Visceral Adipose Tissues Both Supress Muscle Insulin Signalling in Men and Women
Authors: Sarr Ousseynou , Strohm Rachel J. , MacDonald Tara L. , Reed John K. , Foute‐Nelong Jules , Dyck David J. , Mutch David M.
Published: November 2023

Adipose tissue is an important endocrine organ that communicates with other peripheral tissues via the secretion of proteins, termed adipokines. In particular, altered secretion of specific adipokines (e.g., TNF‐α and adiponectin) has previously been shown to regulate skeletal muscle insulin sensitivity. For the current study, we established a human adipose tissue – muscle crosstalk model to investigate the impact of different adipose tissue depots on skeletal myocyte insulin signaling, and if this differs between men and women. Visceral (VAT) and subcutaneous (SAT) adipose tissue samples were collected from obese men and women during laparoscopic bariatric surgery. Adipose tissue samples were subsequently cultured for 48h and secretion media was collected. Secretion media was then transferred on to human skeletal myocytes derived from healthy, non‐diabetic male and female donors for 24h. Content of insulin signaling proteins (phosphorylated and total AKT) in mature myocytes incubated with either control M199 media or secretion media from VAT or SAT was assessed using Western Blotting in basal and insulin‐stimulated conditions (100uU/well). Adipokines in secretion media were measured with a multiplex immunoassay. In men, VAT had higher interleukin‐6 gene expression compared to SAT (p=0.004), while no differences were seen in women (p=0.271). Adiponectin and TNF‐α gene expression were not different between VAT or SAT for men or women. Secretion media collected from male SAT and VAT reduced p‐AKTThr308 activation in insulin‐stimulated myocytes compared to controls (p <0.01). Interestingly, myocytes incubated with VAT secretion media, compared to SAT secretion media, showed a significant reduction in p‐AKTSer473 in men after insulin stimulation (p<0.01). Secretion media collected from female VAT and SAT caused significant reductions in p‐AKTThr308 and p‐AKTSer473 activation following insulin stimulation, compared to controls (p<0.01). Secretion media from VAT or SAT for men or women did not affect total‐AKT levels. Independent of sex, 14 out of 18 detected adipokines (i.e., a panel consisting of cytokines, chemokines and growth factors) were more abundant in VAT secretion media compared to SAT secretion media (p<0.01). Only interleukin‐21 levels showed a sex difference, with higher levels detected in secretion media from female adipose tissue (p=0.003). In conclusion, SAT and VAT secretion media from obese men and women suppress insulin signaling in myocytes to a similar extent. These results reveal the power of this human adipose tissue – muscle model to investigate crosstalk between these two tissues.

Support or Funding Information

This work was supported by the Natural Sciences and Engineering Research Council of Canada.
Partitioning Resistance to Plasma Fluid within Endothelial Glycocalyx
Authors: Curry Fitzroy E.
Published: November 2023

We tested the hypothesis that the resistance to fluid movement within the endothelial glycocalyx is better described by models with an inner semi‐periodic structure and an outer less porous layer, likely stabilized by hyaluronic acid. Previous detailed hydrodynamic modeling has demonstrated that periodic structures close to the membrane, characteristic of membrane‐bound glycoproteins in a quasi‐periodic array extending 200–300nm from the endothelial membrane surface and having a significant resistance to water flow (Darcy coefficient (Kw) in the range 10−13 to 10−14cm2 s) are sufficient to describe both the selectivity and permeability of the glycocalyx to macromolecules and an effective lubrication layer mechanism for red cells. However, a thicker glycocalyx of similar uniform composition does not provide the same consistent description of both permeability and red cell mechanics (Annals of Biomedical Eng. 40: 828,2011). The problem is resolved if the outer layers of the glycocalyx (thickness L) have less resistance to water flows. For example, the criterion for an effective lubrication layer (L/Kw0.5 >100) is met with a outer glycocalyx thickness up to 1 micron but with a hydraulic resistance more that an order of magnitude less than the inner core. These observations are consistent with the conclusion that the fraction of the circulating plasma volume that partitions within the glycocalyx is much smaller than estimates based on the characteristics of red cell flows, total glycocalyx thickness, and glycocalyx volume (for review and clinical significance see Chapters 2 and 3 of Perioperative Fluid Management (E. Farag and A. Kurz Eds) Springer 2016)
Aortic‐Banding and Ovariectomy Independently Promote Coronary Artery Stiffness and Structural Remodeling
Authors: Ouyang An , Hiemstra Jessica , Olver T. Dylan , Edwards Jenna C. , Ivey Jan R. , Thorne Pamela K. , Emter Craig A. , Fleenor Bradley S.
Published: November 2023

Recent work by our laboratory has demonstrated coronary artery stiffening associated with structural remodeling in male aortic‐banded mini‐swine, a model of pressure‐overload heart failure exhibiting characteristics of human heart failure with preserved ejection fraction (HFpEF). The prevalence of HFpEF increases dramatically in women after menopause, and the interaction between the natural decrease in sex hormones with aging and hypertension on coronary artery stiffness is poorly understood. Thus, we hypothesized aortic banding in female mini‐swine will promote mechanical stiffness of coronary arteries, which will be worsened by the loss of female sex hormones following ovariectomy (OVX). Additionally, we hypothesized increased vascular stiffness would be associated with changes to arterial structural remodeling proteins, specifically increased collagen I deposition, decreased elastin, and increased advanced glycation end products (AGEs). Female Yucatan mini‐swine (8 months old) were divided into four groups: Intact (INT, n=7), intact‐aortic banded (AB INT, n=7), ovariectomy (OVX, n=6) and aortic banded with ovariectomy (AB OVX, n=7). After 24 weeks, arterial stiffness was assessed in the left circumflex (LCX) coronary artery by ex vivo mechanical testing using a multi‐wire Myograph system. Extracellular matrix components collagen I, elastin, and AGEs protein expressions were assessed in coronary cross‐sections by immunohistochemical staining. Mechanical testing of the LCX demonstrated an increase in the collagen elastic modulus (CEM) in AB INT and AB OVX groups compared to INT and OVX only groups (main effect of AB, P < 0.05), which was associated with greater arterial collagen expression (main effect of AB, P < 0.05). Compared with INT, the elastin elastic modulus (EEM) was reduced in AB INT, OVX and AB OVX groups (604±46 vs. 382±42, 371±43, 357 ±32 kPa, respectively, P < 0.05) that was also associated with reductions in coronary elastin expression (22±2 vs. 14±1, 14±1, 15±1 AU, respectively, P < 0.05). AGEs accumulation in the LCX was greater in AB INT, OVX and AB OVX groups compared with INT (20±2, 25±2, 21±3 vs. 13±1 AU, respectively, P < 0.05). In summary, these findings indicate aortic banding and ovariectomy promote coronary artery stiffness and structural remodeling by independent mechanisms in a translational female swine model of pressure overload HF with potential relevance to human HFpEF.
Changes in Plasma β‐Adrenergic Receptor Kinase‐1 Following Left Ventricular Assist Device Placement
Authors: Denfeld Quin , Lee Christopher S. , Woodward William R. , Habecker Beth A.
Published: November 2023

As a result of chronic sympathetic stimulation, β‐adrenergic receptor kinase‐1 (βARK1) is upregulated in human heart failure (HF). It is unknown, however, how plasma βARK1 changes following unloading of the heart through left ventricular assist device (LVAD) therapy, and moreover, the relationship between plasma βARK1 and patient‐oriented outcomes (i.e. health‐related quality of life (HRQOL)). The aim of this study was to quantify changes in plasma βARK1 expression following LVAD placement. This was a secondary analysis of plasma samples collected from a subset of patients with HF pre‐ and post‐ (one‐, three‐, and six‐months) LVAD placement. βARK1 was measured using a commercially‐available ELISA kit. In a sample of patients (n = 10) with known improvements in HRQOL over 6 months, average βARK1 was 11.8±5.5 pg/mL (picograms/milliliter, ±SEM) at baseline (pre‐LVAD). βARK1 was increased at 1 month (40.4±16.3 pg/mL) and 3 months (38.4±11.4 pg/mL) but decreased closer to baseline at 6 months (20.9±6.2 pg/mL). In a small subset of patients with known improvements in HRQOL, these results indicate βARK1 significantly increases following LVAD placement but returns close to baseline six months post‐LVAD placement.

Support or Funding Information

National Institutes of Health/National Heart, Lung, & Blood Institute 5T32HL094294 (Denfeld); National Institutes of Health/National Institute of Nursing Research R01NR013492 (Lee); National Institutes of Health/National Heart, Lung, & Blood Institute HL093056 (Habecker)
The superposition of work on a pathologic microenvironmental stiffness may regulate actin assembly via phosphorylation and sarcomeric redistribution of PKCɛ
Authors: Mkrtschjan Michael , Wondmegegn Admasu , Arena Anthony , Li Jieli , Russell Brenda
Published: November 2023

Moderate exercise benefits the heart, but chronic demand leads to failure. Thus, acute and chronic loading may drive cell contractility and cell hypertrophy differentially. We test the hypothesis that, in response to changes in demand, the actin assembly is regulated through PKCɛ signaling to alter cell hypertrophy. Cultured neonatal rat ventricular myocytes (NRVMs) were chronically stressed by increased stiffness of the microenvironment, resembling tissue fibrosis in diseased hearts. Physiologic or pathologic work load was mimicked by one‐hour or 24‐hour treatment with 2.5 μM isoproterenol, respectively. NRVMs were plated on polyacrylamide (PAA) or polydimethylsiloxane (PDMS) substrata mimicking the stiffness of normal (10 kPa) or diseased (100 kPa) hearts, in addition to plating directly on glass (>1 GPa). Cell area increased with increasing stiffness following three‐day culture but not with 24‐hour isoproterenol treatment. Contractility was assessed by beat frequency and time to peak tension by line‐scan kymographs, increasing with substrata stiffness and also with one or 24‐hour isoproterenol treatment. Actin dynamics, as assessed by fluorescence recovery after photobleaching (FRAP) using actin‐GFP, were altered by changes in work load. Both chronic stiffness and one‐hour isoproterenol treatment increased kFRAP to much higher values of ≈0.00075 s−1 on glass or on 100 kPa with isoproterenol treatment compared to untreated cells on 100 kPa, ≈0.0004 s−1 (P<0.05, n≥10 cells). To seek a mechanistic basis for regulation of actin assembly and cell hypertrophy, GFP‐PKCɛ positive NRVMs were observed using time lapse imaging over 1 hour. PKCɛ translocated to the sarcomere within minutes, was sustained for approximately 30 minutes, and declined by one hour. Immunostaining confirmed that translocated PKCɛ is localized with alpha‐actinin at the Z‐band. In order to study whether PKCɛ phosphorylation was necessary, we used a phospho‐specific PKCɛ antibody on fixed cells, which showed an increased Z‐disk localization between alpha‐actinin and phospho‐PKCɛ at 15 min as quantified by Pearson's coefficient (P<0.05), but this difference dissipated by 1 hour. Since the PKCɛ translocation was transient and actin assembly was sustained, hypertrophy may be triggered by the arrival of phospho‐PKCɛ at the Z‐band. The superposition of work on a pathologic microenvironmental stiffness may regulate actin assembly and maladaptive hypertrophy by localization of signaling molecules. This basic research may help understanding of hypertrophy found in heart failure in many cardiac disease states.

Support or Funding Information

HL 62426.
Endothelial specific epithelial sodium channel activation prompts vascular stiffness in female mice
Authors: Jia Guanghong , Aroor Annayya R , Barron Brady J , Manrique Camila , Lastra Guido , Whaley‐Connell Adam , Meininger Gerald A , Jaisser Frederic , Sowers James R
Published: November 2023

Our research suggests that enhanced endothelium mineralocorticoid receptor (ECMR) signaling induces endothelium epithelial sodium channel (EnNaC) activation to promote vascular stiffness. This occurs, in part, by reduction of endothelial nitric oxide (NO) synthase (eNOS) activity and bioavailable NO. This study demonstrated that consumption of a Western Diet (WD) containing excess fat and refined sugar for 16 weeks increases aldosterone level and MR activation resulting in impaired NO mediated vascular relaxation and aortic stiffness in females. These abnormalities were prevented by low doses of the MR antagonist, spironolactone. As ECMR KO also prevented these abnormalities we posited that ECMR/EnNaC activation was critical. Accordingly, we hypothesized that EC‐specific EnNaC activation would mediate vascular stiffness and impair flow‐mediated vasodilation through reduction of bioavailable NO. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a very low dose of amiloride (1 mg/kg/day) for 16 weeks. Female EnNaC KO (EnNaC−/−) and wild‐type littermate females were treated with aldosterone (250 μg/kg/day) via osmotic minipumps for 3 weeks. Amiloride, an antagonist for EnNaC for 16 weeks, could significantly attenuate WD‐induced increases in aortic stiffness in vivo as measured by pulse wave velocity and in vitro endothelial stiffness measured by atomic force microscopy. Meanwhile, amiloride further improved flow mediated dilation in mesenteric arteries and endothelium‐dependent relaxation with acetylcholine (10−9–10−4 mol/L) stimulation in wire myograph. Importantly, EnNaC KO mice also prevent aldosterone‐induced endothelium stiffness and impairment of endothelium‐dependent relaxation. These data suggest that the sequence of activated ECMR leading to cell specific EnNaC activation and signaling decreases bioavailable NO. This cascade plays a key role in the development of vascular stiffness and dysfunction.

Support or Funding Information

NIH (R01 HL73101‐01A and R01 HL107910‐01) and the Veterans Affairs Merit System (0018) to Dr. Sowers