bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Variation in 12‐Lox Regulates the Efficacy of ω‐3 and ω‐6 Fatty Acids to Suppress Platelet Activation
Authors: Shil Swarna , Hawley Megan , Li Cecilia , Holinstat Michael 
Published: November 2023

Individuals with type 2 diabetes mellitus (T2DM), a metabolic disorder characterized by insulin resistance and hyperglycemia, are at an increased risk for a thrombotic event, the underlying pathology of strokes or heart attacks. Concurrently, T2DM patients are resistant to antiplatelet therapy, the current standard of care to reduce the morbidity and mortality associated with cardiovascular disease (CVD). Therefore, novel approaches to inhibit platelet activation are required to treat CVD in diabetics. One such approach is through the consumption of polyunsaturated fatty acids (PUFAs) – ω‐3 fatty acids (fish oils) or the ω‐6 fatty acid dihomo‐γ‐linolenic acid (DGLA). In platelets, 12(S)‐lipoxygenase (12‐LOX) is a key enzyme primarily responsible for the oxidation of PUFAs. Recent studies have shown that 12‐LOX was responsible for the anti‐platelet effects of DGLA. Interestingly, there have been 4 single‐nucleotide polymorphisms (SNPs) in the gene encoding 12‐LOX that impact its enzymatic activity. We hypothesize that these selective 12‐LOX SNPs will influence the ability of ω‐3 and ω‐6 fatty acids to suppress platelet activation. The activation of platelets isolated from diabetic (n=44) and healthy (n=45) patients following 2 months of PUFA supplementation was measured by RAP‐1 activation, integrin activation, calcium flux, and platelet aggregation to determine if genetic variants in 12‐LOX influenced the ability of PUFAs to inhibit platelet activation in diabetics versus healthy individuals. We found that there is a correlation between 12‐LOX genotype and platelet inhibition and a significant difference in diabetic versus healthy individuals.Support or Funding InformationNIH HL114405
Novel Peptide Micellar Nanoformulation for Treatment of Thrombotic Complications of Sepsis
Authors: Bae Misuk , Rusu Luiza , Castellon Maricela , Du Xiaoping , Onyuksel Hayat , Minshall Richard D 
Published: November 2023

Sepsis is one of the leading causes of death in hospital intensive care units in the U.S. Despite supportive care, microvascular thrombosis and disseminated intravascular coagulation in septic patients leads to ~40% mortality. Since there are no anti‐thrombotic drugs for sepsis on the market, new drugs are desperately needed. Interestingly, the plasma level of von Willebrand factor (vWF), the glue to which platelets adhere to form a clot, is elevated in septic patients and high levels predict poor outcome. We recently showed Gα12 is required for αSNAP‐dependent vWF secretion from Weibel Palade bodies. We identified a 6 aa sequence in the N‐terminus of Gα12, generated a cell permeable myristoylated alpha‐SNAP Binding Domain peptide, Myr‐SBD, and showed that this blocked thrombin and shear‐stress induced vWF secretion from human endothelial cells in vitro. In addition, intravenous delivery of Myr‐SBD to mice at the time of cecal ligation and puncture (CLP) surgery reduced vWF levels, decreased microvascular thrombosis in the kidney, and increased survival. Here, we extend these initial studies using a well‐characterized sterically‐stabilized simple micelle (SSM) system to generate a nanoformulation of Myr‐SBD peptide for in vivo delivery. Pilot pharmacokinetic and efficacy parameters of Myr‐SBD SSM were investigated by fluorescence spectrometry in vitro and in vivo. Fluorescent probe‐labeled Myr‐SBD (Myr‐SBD‐PDAM), prepared by modifying the carboxylic acid group of Myr‐SBD via PDAM conjugation was confirmed by fluorescence spectrometry. Myr‐SBD‐PDAM SSM was prepared by incubating the peptide with DSPE‐PEG2000 using a film‐rehydration method. Following SSM preparation, micelle size and morphology assessed by electron microscopy (TEM) and light scattering (DLS) revealed uniform micelles of 14.1 ± 2.7 nm in diameter. Uptake and intracellular accumulation of Myr‐SBD‐PDAM SSM was confirmed by fluorescence and confocal microscopy. Intravenous delivery of Myr‐SBD SSM to mice after CLP surgery improved survival compared to the untreated CLP group. Thus, nanoformulation of Myr‐SBD peptide may enable in vivo delivery for treatment of thrombotic complications of severe inflammatory response syndrome.Support or Funding InformationThis work was supported by MPI R01 HL125356 (RDM, XD), the Michael Reese Research and Education Foundation (RDM), and AHA (LR).
Apoptosis and autophagy in human vein grafts replacing occluded coronary arteries in atherosclerosis
Authors: Bortier Hilde Elisa , Kockx Mark , De Meyer Guido 
Published: November 2023

Atherosclerosis is a chronic disease. For many decades vein grafts are used to replace occluded and suboccluded segments of coronary arteries. Restenosis is still a major problem today, as this chronic disease does not disappear when the occluded segments are replaced by autologous vein grafts. What are the cellular characteristics of this restenosis? Vein grafts obtained during surgical reinterventions were processed for light microscopy, SEM, TEM, immunohistochemistry (nuclear marker, smooth muscle cell marker, ..) and TUNEL (apoptosis).The cellular characteristics are accumulation of foam cells at the luminal side and smooth muscle cell death in the layer underneath. The remaining smooth muscle cells show nuclear fragmentation or apoptosis and remnants of cytoplasmic fragmentation or autophagy. Attempts are being made to develop drugs that might prevent the accumulation of foam cells and smooth muscle cell death in the aim to prevent the redevelopment of atherosclerotic plaques in the vein grafts.
Microvascular Content and Insulin Sensitivity within Skeletal Muscle and Adipose Tissue Differs between Male and Female Mice on a High‐fat Diet
Authors: Abdifarkoosh Ghoncheh , Rudnicki Martina , Roudier Emilie , Haas Tara L. 
Published: November 2023

Capillary networks within skeletal muscle and adipose tissue have a critical role in the delivery of energy sources and the maintenance of proper tissue function. A reduction in capillary density is correlated with impairments in whole body metabolic functions, as occurs in obesity. It is known that the susceptibility to develop obesity‐associated metabolic disturbances differs between males and females. However, studies investigating these sex‐related differences have largely overlooked the potential contribution of capillary content. The goal of this investigation is to determine whether there are sex differences in skeletal muscle and adipose tissue microvascular content and to relate these to insulin signaling in normal and obese mice. Male mice fed a high‐fat (HF) diet for 16 weeks exhibited reduced systemic insulin sensitivity compared to females who maintained a response similar to the normal‐chow (NC) group. Insulin stimulation increased levels of phosphorylated Akt (pAkt) protein equivalently in the skeletal muscle of male and female NC mice. With HF diet, males had significantly lower insulin‐stimulated pAkt levels vs. females (0.7 vs. 1.9, P<0.0001). Capillary‐to‐fiber ratio was not different in the skeletal muscle of HF male vs. female mice (1.5 vs. 1.4, n=3 ns). Ex vivo insulin stimulation of visceral adipose tissue increased pAkt in HF females but not in HF males. Microvascular content in visceral adipose was not different between NC males and females. In contrast, HF males had reduced, but HF females maintained, their adipose vascular content. This was further supported by higher mRNA levels of vascular marker CD31 (0.17 vs. 0.12, p<0.05) and pro‐angiogenic factor VEGF (0.14 vs. 0.07, p<0.05) in HF females vs. HF males. Our data suggest that capillary content and a pro‐angiogenic environment is maintained in the adipose of HF‐fed females, which in turn may be linked with maintained adipose insulin sensitivity.Support or Funding InformationCIHR
Endothelial Cell Barrier Protein Expression Differs by Organ and Sex
Authors: Kemp Scott S. , Huxley Virginia H , Schramm Christine , Sieveking Steven W 
Published: November 2023

In both humans and animals, while functional sex differences have been observed, the mechanisms responsible for these differences remains poorly understood. Hypotheses include exposure to reproductive hormones, DNA preprogramming, or a combination of the two. Our goal is to elucidate the molecular mechanisms responsible for observed sex differences in basal and activated barrier function of adult rat microvessels.To this end we focused on cells isolated from age‐matched reproductively mature male and female rats (Sprague Dawley) to test the null hypothesis that protein expression does not differ by sex. Vascular cells were isolated from aorta and skeletal muscle to facilitate answering our question and compare our findings with previously published and observed data. Six markers associated with barrier function were chosen for study, CD 31 (PECAM‐1), αvβ3 (Integrin), N‐cadherin (CDH2), NCAM1 (CD 56), VCAM‐1 (CD 106), and ICAM‐1 (CD 54). Using flow cytometry we compared the difference in expression of these markers in sex‐verified (SRY gene expression) macro‐ and microvascular cells maintained in identical conditions of culture (Passage 8).Our current data suggests sex differences in aortic EC barrier proteins: αvβ3, VCAM, NCAM and NCAD, with little to no sex difference observed in ICAM and CD 31. Sex differences have also been observed in the skeletal muscle EC barrier proteins: ICAM, VCAM, NCAM, with little to no difference observed in αvβ3, NCAD and CD 31. Organ differences have been observed between skeletal muscle and aortic EC barrier proteins in ICAM, VCAM, NCAM and NCAD, with little difference observed in αvβ3, and CD 31.Given that the proteins forming the junctional complex and regulating adhesion to the extracellular matrix differ between males and females we possess an initial understanding of one mechanism whereby barrier function may vary by sex under basal conditions. Future studies will focus on the 2nd messenger pathways linked to these proteins and expression of proteins in these cells following exposure to vasoactive components shown to alter barrier function in a sexually dimorphic manner.Support or Funding InformationNIH R01 DK095501
Capillary endothelial Gq protein‐coupled receptors and PIP2 toggle signaling between TRPV4 and Kir2 channels in the brain
Authors: Harraz Osama F , Longden Thomas A , Dabertrand Fabrice , Nelson Mark T 
Published: November 2023

The ion channel composition of capillaries is not known. Using freshly dissociated capillary endothelial cells (cECs) from mouse brain, we measured currents through strong inward rectifier K+ (Kir2.1) channels and through transient receptor potential vanilloid (TRPV4) channels. We have proposed that Kir2.1 and TRPV4 channels are involved in electrical and calcium signaling, respectively, to transmit signals from cECs to upstream arterioles. We tested the hypothesis that these two channel types are functionally linked through phosphatidylinositol 4,5‐bisphosphate (PIP2). We found that intracellular PIP2 or ATP had opposite effects on TRPV4 and Kir2.1 currents, with PIP2 or ATP suppressing TRPV4 channel activity and conversely being essential for Kir2.1 activity. The level of PIP2 is controlled by the activity of Gq protein‐coupled receptors (GqPCRs), which hydrolyze PIP2 to produce inositol 1,4,5‐triphosphate (IP3) and diacyglycerol (DAG). We therefore tested the effects of prostaglandin E2 (PGE2; an agonist of the GqPCR EP1), which has been implicated in neurovascular coupling, on TRPV4 and Kir2.1 currents in cECs, with the expectation of divergent effects on the two conductances. Indeed, when measured simultaneously in the same cECs, PGE2 increased TRPV4 currents with half‐time (t0.5) of 3.5 minutes and decreased Kir2.1 currents with a t0.5 of 5.3 minutes. In conclusion, the present study supports the concept that PIP2 tonically inhibits TRPV4 channels and activates Kir2.1 channels in the brain capillary endothelium. GqPCR activation therefore acts as a molecular switch that alters the balance between electrical (Kir2.1) and Ca2+ (TRPV4) signaling, which might change the signaling modality to upstream penetrating arterioles, and would have profound effects on the control of blood flow into the brain.Support or Funding InformationSupported by the NIH, Fondation Leducq and EC Horizon 2020 grants.
Signal Inference: A Method to Locate and Track Calcium Waves with Statistical Confidence
Authors: Billings Eric , Socha Matthew J , Domeier Timothy L , Balaban Robert S , Segal Steven S 
Published: November 2023

Optical imaging studies of cell signaling pose a recurring challenge to track signals relative to the topology of the cell structures. Image analysis platforms require the user to visually assess which signals merit further study and define them as geometric regions of interest (ROIs) ‐ throwing away information outside the ROI. Tools for segmentation, the process of identifying cellular structures in an image, let the user subjectively identify biological structures with pixel resolution. Thus, the experimentalist is challenged to objectively control for observer bias. We have developed a method to avoid manual ROI generation and use the entire data set to extract the physiology of calcium (Ca2+) signaling within and between vascular endothelial cells. We hypothesize that signal tracking and segmentation could be made rigorous and semi‐automatic by requiring signals to be continuous in time and contiguous in space. This reduces the analysis of the pixel covariance matrix to tracking contiguous 2D foci with high covariance. We test if this is sufficient to 1) identify cell structure; 2) track Ca2+ signals; and 3) provide statistical confidence metrics. The algorithm was developed using data from confocal fluorescence imaging of cytosolic Ca2+ release and reuptake in freshly isolated endothelial cell tubes following local stimulation with acetylcholine (ACh) from a micropipette (1 μm tip, 1–2s). Results were tested with biological replicates under varied technical (scan rate, gain, amplification) and physiological conditions (stimulus dosage, location) and compared to manual segmentation and tracking. We illustrate a novel method for robust signal tracking with a minimum of user settings under different dose and gain settings. The method segmented cells by identifying foci with high spatial correlation. Individual foci overlap 78.9±18.1% of the area of manually segmented cell (n=5). Foci shape was also reproducible across different conditions (high/low gain for two ACh dosages). The foci boundaries for one preparation under four acquisition conditions were within 2.2 μm along 70.8% of the boundary (5 cells). This approach provided multiple metrics for each time course including the number, size, shape, rate and behavior of detected optical signals and cell structures. Typical run time for a single dataset (256 × 1024 pixels and 1054 frames) was less than 30 minutes on a (16GB RAM) computer.Support or Funding InformationNIH F32HL107050, K01AG041208, R37HL041026
Increased propensity for cerebral microhemorrhages in a IGF‐1 deficient mouse model of accelerated vascular aging
Authors: Tarantini Stefano , Valcarcel‐Ares Marta Noa , Yabluchanskiy Andriy , Springo Zsolt , Fulop Gabor , Ashpole Nicole , Gautam Tripti , Giles Cory , Wren Jonathan D. , Sonntag William E. , Csiszar Anna , Ungvari Zoltan 
Published: November 2023

Clinical and experimental studies show that aging exacerbates hypertension‐induced cerebral microhemorrhages (CMHs), which progressively impair neuronal function. There is growing evidence that aging promotes IGF‐1 deficiency, which compromises multiple aspects of cerebromicrovascular and brain health. To determine the role of IGF‐1 deficiency in the pathogenesis of CMHs, we induced hypertension in mice with liver‐specific knockdown of IGF‐1 (Igf1f/f + TBG‐Cre‐AAV8) and control mice by angiotensin‐II plus L‐NAME treatment. In IGF‐1 deficient mice the same level of hypertension led to significantly earlier onset and increased incidence and neurological consequences of CMHs, as compared to control mice, as shown by neurological examination, gait analysis and histological assessment of CMHs in serial brain sections. Previous studies showed that in aging increased oxidative stress‐mediated MMP activation importantly contributes to the pathogenesis of CMHs. Thus, it is significant that hypertension‐induced cerebrovascular oxidative stress and MMP activation were increased in IGF‐1 deficient mice. We found that IGF‐1 deficiency, by impairing hypertension‐induced adaptive media hypertrophy and extracellular matrix remodeling, also perturbs arteriolar remodeling processes, which act to restore circumferential stress, preventing mechanical damage to the vascular wall. Collectively, IGF‐1 deficiency promotes the pathogenesis CMHs, mimicking the aging phenotype, which likely contribute to its deleterious effect on cognitive function. Therapeutic strategies that up‐regulate IGF‐1 signaling in the cerebral vessels and/or reduce microvascular oxidative stress and MMP activation may be useful for the prevention of CMHs, protecting neurocognitive function in high‐risk elderly patients.Support or Funding InformationThis work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging
Spatio‐Temporal Analysis of Intracellular and Intercellular Calcium Signaling in Microvascular Endothelial Cell Tubes
Authors: Socha Matthew J , Billings Eric , Domeier Timothy L , Balaban Robert S , Segal Steven S 
Published: November 2023

Intracellular Ca2+ concentration is an essential component of endothelial cell‐dependent regulation of vasomotor control of tissue blood flow. Coordination of vasodilation in microvascular resistance networks requires intercellular signaling in a direction opposite to blood flow to dilate upstream branches (e.g., feed arteries). We hypothesized that individual endothelial cells function both as elementary operating units of intracellular Ca2+ signaling and as elements of a syncytium in propagating intercellular Ca2+ signals. Intact endothelial cell tubes (n=3) were freshly isolated from superior epigastric arteries of male C57BL/6 mice (3–6 months), loaded with Ca2+ indicator dye (fluo‐4, 10 μM), and superfused continuously at 3 ml/min in a laminar flow stream of physiological salt solution (PSS, 25°C). The center of a tube was stimulated locally with acetylcholine (ACh) using microiontophoresis (1 μm tip, 800 nA, 2 s) to initiate Ca2+ signals that were monitored both upstream and downstream from the site of stimulation. Ca2+ responses were analyzed using a novel time series image analysis to define and track signals both within and across multiple cells. Individual cells behaved as operational units within the larger syncytium of the endothelium. Thus, individual cells exhibited intracellular Ca2+ signals that propagated longitudinally at 19.9±9.5 μm/s and laterally at 15.6±4.5 μm/s (P<0.05). Moreover, Ca2+ waves propagated from cell to cell along the intact endothelium at 23.4±0.7 μm/s in the upstream direction, opposite to the flow of PSS. In contrast, Ca2+ responses spread in the same direction as PSS flow (i.e., downstream) at > 100 μm/s, suggesting a combination of intercellular Ca2+ waves and convection of ACh. These findings resolve the ability of individual endothelial cells to generate bidirectional intracellular Ca2+ waves while engaging their neighbors in signaling as a functional syncytium along the vessel axis. The greater rate of Ca2+ wave propagation upstream along the intact endothelium vs the mean intracellular propagation rate suggests a role for feed‐forward signaling between cells that is readily distinguished from agonist convection and receptor activation occurring downstream from the site of initiating Ca2+ signaling. These data provide a reference for determining how vascular disease may affect endothelial Ca2+ signaling within and among cellular elements of the functional syncytium and thereby influence vasomotor control.Support or Funding InformationNIH F32HL107050, K01AG041208, R37HL041026
Impact of Endothelial‐Specific FoxO1 Depletion on the Development of Metabolic Dysfunction in Obesity
Authors: Rudnicki Martina , Abdifarkosh Ghoncheh , Roudier Emilie , Nwadozi Emmanuel , Haas Tara 
Published: November 2023

Background and hypothesisImpaired vascular growth and remodeling is implicated in the disruption of adipose tissue structure and function in obesity, leading to systemic metabolism dysfunction. As Forkhead Box O1 (FoxO1) transcription factor regulates endothelial cell homeostasis and restrains angiogenesis, we investigated the role of endothelial cell FoxO1 in the development of obesity using endothelium FoxO1‐deficient mice.Experimental designTo generate endothelial cell‐specific FoxO1 deficient mice (FoxO1iEC‐D), conditional FoxO1L/L floxed mice were bred with mice expressing the tamoxifen‐inducible, Pdgfb promoter‐driven creERT2 recombinase. Cre‐mediated recombination was induced with 5 consecutive intraperitoneal (i.p.) injections of 200 μL of tamoxifen (15 mg/mL) at 4–5 weeks of age. Cre−;FoxO1L/L littermates were used as control mice in this study. FoxO1 mRNA levels were analyzed in endothelial cells isolated from epidydimal white adipose tissue (eWAT) by qPCR quantification. To verify the effects of EC‐FoxO1 on diet‐induced obesity, male FoxO1iEC‐D mice and matched control mice (6–8 weeks‐old, n=10/group) were fed a normal chow (NC, 10.5% fat) or high‐fat (HF) diet (58% fat) for 16 weeks.ResultsCre‐mediated recombination resulted in 50% reduction of FoxO1 mRNA levels in endothelial cells of FoxO1iEC‐D mice. Although food intake of FoxO1iEC‐D mice consuming a HF diet did not significantly differ from controls through 16 weeks, decreased body weight gain and adiposity were observed in FoxO1iEC‐D mice at the end of the diet period. In addition, HF FoxO1iEC‐D mice had lower blood pressure and fasting blood glucose levels, and improved response to i.p. glucose tolerance test but no significant changes in i.p. insulin tolerance test. No significant changes were detected on triglycerides levels in serum, liver, muscle and heart with FoxO1 deletion. Whole‐mount staining and CD31 mRNA analysis indicated increased vascular density in the eWAT of HF FoxO1iEC‐D mice, which was not associated with significant changes in the mRNA levels of browning markers UCP‐1 and PRDM16. However, gene expression analysis of eWAT showed increased levels of PPARγ and PCG1α and reduced mRNA levels of leptin in HF FoxO1iEC‐D mice compared with HF control mice. To test if EC‐FoxO1 depletion affects adipose insulin action, ex vivo stimulation of eWAT with insulin was performed and the Akt phosphorylation status was analyzed by Western Blot. While insulin‐stimulated Akt phosphorylation was impaired by HF in control mice, no diet effect was observed in the eWAT from FoxO1iEC‐D mice, indicating that endothelial‐FoxO1 deletion contributes to maintained insulin sensitivity of adipose tissue.ConclusionOur data suggest that endothelial FoxO1 deletion improves adipose tissue structure and function and these influences may contribute to alleviating systemic metabolic dysfunction in response to high‐fat in mice.Support or Funding InformationCIHR