bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vivo imaging of infection‐induced cogulopathy in the microcirculation
Authors: Jenne Craig N , Davis Rachelle P , McDonald Braedon 
Published: November 2023

Patients with systemic infection often develop disseminated intravascular coagulation (DIC), a condition leading to clot formation throughout the vasculature. This systemic coagulation can impair blood flow within the tissue microvasculature and has been associated with organ damage and dysfunction. Infection also triggers the release of Neutrophil Extracellular Traps (NETs) within the vasculature. NETs are comprised of extracellular DNA covered in a number of nuclear and antimicrobial proteins. This immune response is designed to catch and kill pathogens, but also has the potential to bind platelets and initiate coagulation.Currently, most studies are limited to the histological analysis of tissue sections or in vitro biochemical assays, providing only snapshots of these complex and dynamic processes. As such the specific interaction between NETs, platelets and coagulation are poorly understood. Using intravital microscopy (IVM), can directly visualize NET formation and fibrin deposition in real‐time, in the blood vessels of live mice following infection. Additionally, we have developed a novel imaging protocol using an enzyme‐activatable fluorescent probe to track the time and location of thrombin activation within the vasculature of live mice. Importantly, IVM allows us to understand not only the complex interactions between the pathogen, immunity and coagulation, but to also the effect of these interactions on vascular perfusion and tissue damage. Following i.v. challenge with bacteria, we measure significant NET deposition within the liver sinusoids. These NETs in turn activate thrombin leading to fibrin deposition, vascular occlusion and tissue damage. Importantly, treatments that prevent NET formation, break‐down NETs within the vasculature, or block key NET components significantly inhibit thrombin activation, improve perfusion and attenuate tissue damage.Support or Funding InformationThis work is supported by grants from the Critical Care Strategic Clinical Network and the Natural Sciences and Engineering Research Council of Canada (NSERC)
Multicolor Two‐Photon Intravital Microscopy Reveals Monocyte Patrolling and Macrophage Migration in Atherosclerosis
Authors: McArdle Sara , Ray Nilanjan , Ley Klaus 
Published: November 2023

Atherosclerosis is an underlying cause of two of the leading causes of death in the world, heart attacks and strokes. It is a chronic inflammatory disease characterized by plaque build‐up in large and medium arteries. During disease progression, monocytes in the blood invade the arterial wall, where they can differentiate into macrophages and dendritic cells. Together, these cells phagocytose lipids, clear apoptotic cell debris, present antigen to T cells, and produce pro‐ and anti‐inflammatory cytokines. Both monocyte recruitment to the plaque and macrophage function within the plaque require cell motion. The purpose of this work is to study monocyte and macrophage movement in the context of atherosclerosis.We developed a system for imaging fluorescent leukocytes in atherosclerotic arteries in vivo. The major challenge in intravital imaging of large arteries is the motion artifacts due to the expansion of the arterial wall with the heartbeat. This system utilizes cardiac triggering and novel image post‐processing algorithms to remove these motion artifacts and allow for cell motion quantification. Apoe−/− Cx3cr1GFP/+ Cd11cYFP mice were generated to visualize labeled monocytes and macrophages. Monocytes were observed patrolling the endothelium of atherosclerotic arteries and the motion characteristics were quantified. Macrophages were seen actively probing their local environment as well as migrating through the plaque. Finally, these macrophages were extensively phenotyped by cell surface markers to link observed motion to known subsets and function.Support or Funding InformationThis work was funded by NIH R01 (115232) to K.L. and AHA (#11PRE7580009), HHMI (56005681), and NHLBI (T32HL105373‐03) to S.M.
The Effect of Splenectomy on the Recruitment of Inflammatory Cells in the Cerebral Microvasculature
Authors: Seifert Hilary , Russell Janice , Granger Daniel Neil 
Published: November 2023

Splenectomy decreases inflammatory cell recruitment to the brain following sterile cerebral inflammation that results from stroke. Similarly it is known that non‐sterile cerebral inflammation like systemic lipopolysaccharide (LPS) causes rapid recruitment of leukocytes in the cerebral venules. It is not known if the protection afforded by splenectomy is specific to stroke or if removal of the spleen can protect the brain against systemic LPS. The objective of this study was to determine if splenectomy alters the recruitment of adherent leukocytes and platelets in the cerebral microvasculature following systemic LPS administration. C57Bl/6 mice underwent either splenectomy or sham splenectomy two weeks prior to an intra‐peritoneal injection of LPS (10 ug/mouse). Four hours after LPS injection, mice were imaged with intravital video microscopy. Adherent platelets, labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and leukocytes, labeled with rhodamine G6, were quantified. Peripheral blood counts were also measured in all groups. LPS significantly increased the number of adherent leukocytes and platelets in cerebral venules. This response was not altered by splenectomy. LPS also caused a significant decrease in circulating leukocytes, which was unchanged by splenectomy. There was no affect on circulating platelets. These results demonstrate that splenectomy does not protect the brain from non‐sterile cerebral inflammation and the protective effects of splenectomy on the inflamed brain appear to be specific to stroke.Support or Funding InformationThis work was supported by a fellowship from the Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center‐Shreveport
The Effect of Media Type on Nerve Presence in Cultured Microvascular Networks
Authors: Hodges Nicholas A. , Barr Ryan W. , Murfee Walter L. 
Published: November 2023

Neurovascular patterning represents an emerging area of microvascular research. While overlapping molecular signals highlight links between angiogenesis and neurogenesis, advancing our understanding of their spatial and temporal coordination is limited by a lack of in vitro models that contain both systems. Such a potential model is the rat mesentery culture model, which our laboratory has recently introduced as an ex vivo tool to investigate cellular dynamics during angiogenesis and lympangiogenesis in an intact microvascular network scenario. The objective of this study was to evaluate the effect of media type on the maintenance of nerve presence in cultured networks. Mesenteric tissue windows were harvested from adult male Wistar rats and cultured for 3 days in the following media groups: neural basal media (NBM, n = 4 tissues), NBM + nerve growth factor (NGF, n = 7 tissues), and NBM + NGF + 20% fetal bovine serum (FBS, n = 7 tissues). Tissues were immunohistochemically labeled for PECAM (endothelial cell marker) and class III‐β tubulin (nerve marker). Control tissues (n=7) were immediately immunolabeled after harvesting with no culture. The ratio of network feeding arterioles with observable positive class III‐β tubulin nerve alignment in control tissues was significantly increased compared to each of the three cultured groups (Control = 0.83 ± 0.09; NBM = 0.04 ± 0.04; NBM + NGF = 0.33 ± 0.14; NBM + NGF + 20% FBS = 0.40 ± 0.12). These results suggest that the presence of nerves is reduced in cultured networks. The possibility for media type influences on nerve maintenance supports the potential for the future development of the rat mesentery culture model as a unique ex vivo tool to investigate angiogenesis, neurogenesis, and neurovascular patterning simultaneously.Support or Funding InformationNIH 5‐P20GM103629‐04
Caveolin‐3 plays a critical role in autophagy after ischemia reperfusion
Authors: Kassan Adam , Pham Uyen N. , Cho Eunbyul , Nguyen Quynh My , Patel Piyush P. , Roth David M. , Head Brian P. , Patel Hemal H. 
Published: November 2023

Autophagy is a dynamic recycling process responsible of the breakdown of misfolded proteins and damaged organelles, providing nutrients and energy for cellular renovation and homeostasis. Loss of autophagy is associated with cardiovascular diseases. Caveolin‐3 (Cav‐3), a muscle specific isoform, is a structural protein within caveolae and is critical to stress adaptation in the heart. However, it remains unknown whether Cav‐3 plays a role in regulating autophagy to modulate cardiac stress response. In the present study, we used HL‐1 cells, a cardiac muscle cell line, with stable Cav‐3 knockdown (Cav‐3 KO) to study the impact of Cav‐3 in regulation of autophagy in the setting of simulated ischemia‐reperfusion injury (I/R). Cav‐3 co‐immunoprecipitated with beclin‐1, showing an interaction of caveolin with autophagy related proteins. We found that Cav‐3 KO cells have a decreased expression of autophagy markers (beclin‐1, light chain (LC3)‐II) after ischemia and I/R when compared to WT cells. Moreover, Cav‐3 KO cells showed increased cell death and higher level of apoptotic proteins (cleaved caspase‐3 and cytochrome c) with suppressed mitochondrial function in response to ischemia and I/R. Taken together, these results indicate that Cav‐3 regulates autophagy to modulate stress adaptation.Support or Funding Information5 T32 MH 19934‐20
Novel Finding of Mitochondrial Biogenesis During Cardiac Surgery: Marker or Mediator of Ischemia/Reperfusion (I/R) Injury, or an Element of the Homeostatic Intracellular Repair Response (HIR2)
Authors: Andres Allen , Sengstock David , Jahania Salik , Dabir Reza , Tucker Kyle , Gottlieb Roberta , Mentzer Robert 
Published: November 2023

BACKGROUNDWe recently demonstrated that the homeostatic intracellular repair response (HIR2) is activated during cardiac surgery. Part of HIR2 involves brisk mitochondrial turnover in the heart; a process that comprises both mitophagy and mitochondrial biogenesis. We have previously demonstrated that mitophagy is an important element of ischemic preconditioning. However, it is unknown whether mitochondrial biogenesis occurs in the setting of I/R and how it might contribute to cardioprotection or injury.STUDY DESIGNAutophagy and mitochondrial turnover were assessed in 20 patients undergoing coronary artery bypass or valve surgery requiring cardiopulmonary bypass (CPB). Biopsies of right atrial appendage obtained before initiation and after weaning from CPB were processed for whole tissue lysate, mitochondria‐enriched heavy membrane, and cytosolic fractions. Samples were analyzed for autophagy by immunoblotting for LC3, Beclin‐1, ATG5‐12, and p62. Mitochondrial turnover was assessed by monitoring Tom70, Cox4, and Parkin. Cytosolic cytochrome c was examined as a marker of injury.RESULTSHeart surgery resulted in increased autophagy indicated by depletion of autophagy proteins after bypass. Parkin increased in the mitochondrial fraction and decreased in the cytosol. We observed a marked increase in mitochondrial proteins Tom70 and Cox4, both of which positively correlated with cross‐clamp and bypass time. Cytosolic cytochrome c consistently increased after CPB and correlated with increased mitochondrial mass.CONCLUSIONSThese findings provide evidence for the first time in humans that coordinated mitophagy and biogenesis occur in response to I/R. Intriguingly, increased cytosolic cytochrome c after bypass paralleled the increase in mitochondrial mass, raising the concern that mitochondrial biogenesis may contribute to I/R injury. Whether mitochondrial biogenesis is beneficial or a liability in the setting of I/R remains to be clarified. Thus, strategies designed to amplify HIR2 and/or modulate mitochondrial biogenesis during cardiac stress may represent a novel approach to myocardial protection in patients undergoing heart surgery.Support or Funding InformationNIH: P01 HL112730
Multi‐scale Modeling of Collecting Lymphatic Vessels: From Ion Channel Activity to Lymph Transport
Authors: Moshkforoush Arash , Kapela Adam , Tsoukias Nikolaos 
Published: November 2023

Exact physical, molecular and cellular mechanisms underlying the transport of the lymph within the lymphatic system is highly complicated, and for the most part is poorly understood. This proposed research outlines our recent effort in developing a multiscale computational model of collecting lymphatic vessel segments (lymphangions), in which the transport function at macroscale level can be analyzed by integrating underlying mechanisms at cellular and molecular levels. This outlined approach can provide a useful platform to study the function or dysfunction of the collecting lymphatic vessels in normal and diseased states, respectively. Detailed mathematical expressions of electrophysiological properties of major ionic channels, membrane currents and intracellular stores present in a single lymphatic smooth muscle cell (LSMC) has been derived from independent experimental studies in literature. The single LSMC model predicts the detailed dynamics of calcium and membrane potential at the whole cell level via integrating individual membrane and intracellular components. A model of the lymphangion wall is constructed via connecting LSMCs through the incorporation of homocellular gap junctions between the neighboring cells. This multicellular model has the potential to lay bare on complex intra‐ and intercellular electrical signaling and propagating ionic waves. The detailed electrophysiological model of the lymphangion wall is combined with a biomechanics and fluid dynamics model to form a mathematical model of a section of the collecting lymphatic vessel (i.e. a series of lymphangions separated by unidirectional valves). This model allows us to study the real‐time relationship between the changes in the diameter and pressure of the vessel segments and changes in the concentration of ionic species. The single LSMC model successfully predicts calcium and membrane current oscillations at physiological amplitude and frequencies without the presence of an agonist. The multicellular model suggests that the chloride current is the major contributor in synchronizing the LSMC spontaneous contractions within one lymphangion, while the presence of pressure‐sensitive channels accounts for anti‐phase contractions observed in neighboring lymphangions. This proposed model is a novel attempt that synergistically takes into account the biomechanics of the lymphatic vessel wall, fluid dynamics, and the electrophysiology of major ionic species in the cells constituting the lymphatic vessels.Support or Funding InformationSupported by National Institutes of Health grant HL121778
Mitochondria Modulates Calcium Pulsars Kinetics in Native Endothelial Cells from Mouse Mesenteric Arteries
Authors: Toussaint Fanny , Béziau Delphine , Charbel Chimène , Blanchette Alexandre , Mayer Gaétan , Ledoux Jonathan 
Published: November 2023

Endothelial control of vascular tone is modulated by local calcium (Ca2+) signaling events like Ca2+ pulsars, an IP3R‐dependant Ca2+ release restricted to myoendothelial projections (MEP). Aside from the recent report of Ca2+ pulsars being modulated by CaMKII, mechanisms regulating endothelial spontaneous Ca2+ release remain unidentified. Mitochondria are important modulators of intracellular Ca2+ dynamics in various cell types and, we hypothesized mitochondria may control endothelial Ca2+ pulsars. High‐speed confocal microscopy was used to investigate mitochondria's influence on endothelial Ca2+ pulsar in cut‐open mouse mesenteric arteries (en face configuration). Preventing mitochondrial Ca2+ uptake by uncoupling with FCCP uncovered a new Ca2+ pulsar site population that had a lower frequency (≈+30%) but larger diffusional spread (+57%) than controls. Inhibition of mitochondrial Ca2+ uptake via the uniporter using Ru360 increased the spread of Ca2+ pulsars (≈+30%), though kinetics were not significantly altered. Significantly, inhibition of mitochondrial Ca2+ export (CGP37157, CsA) increased the number of active Ca2+ pulsar sites, each with a decreased bursting frequency. In summary, these results suggest that mitochondrial uptake and release of Ca2+ acts as a critical local modulator of Ca2+ pulsar kinetics and may therefore be crucial to endothelial control of vascular tone.Support or Funding InformationFRQS, HSFC, CFI, SQHA and CIHR
Inhibition Of Inward‐Rectifier K+ Channels In Vascular Smooth Muscle Restores Myogenic Tone Development In Mouse Bladder Arterioles
Authors: Tykocki Nathan R. , Heppner Thomas J. , Nelson Mark T. 
Published: November 2023

The urinary bladder is highly vascularized, and chronic decreases in blood flow are linked to bladder dysfunction. While increases in intraluminal pressure constrict most arterioles (“myogenic tone”), mouse bladder arterioles do not exhibit a myogenic response to pressure. We examined the electrophysiological and vasoactive properties of mouse bladder feed arterioles to determine why myogenic tone is absent. In isolated pressurized bladder arterioles, smooth muscle cell membrane potential was hyperpolarized at 20 mmHg (−72.8±1.4 mV), and did not change significantly when intraluminal pressure was increased to 80 mmHg (−74.3±2.2 mV). However, increasing extracellular K+ from 5.9 mM to 60 mM caused a significant depolarization (−22.7±0.2 mV) at 80 mmHg, which was accompanied by vessel constriction. Given that inward‐rectifier K+ (KIR) channels can have a strong influence on membrane potential in smooth muscle cells, membrane potential was again measured in the presence or absence of the KIR channel blocker BaCl2 (100 μM). In the absence of BaCl2, smooth muscle cell membrane potential remained hyperpolarized at both 20 to 80 mmHg (−70.4±1.9 mV and −72.2±3.1 mV, respectively). In the presence of BaCl2, membrane potential depolarized significantly at both 20 mmHg (−56.2±3.1 mV) and 80 mmHg (−43.3±2.0 mV). To determine if KIR channel inhibition could restore myogenic tone, diameter of isolated pressurized bladder arterioles was measured at increasing pressures (10–80 mmHg) in the presence and absence of 100 μM BaCl2. BaCl2 had no effect on vessel diameter at 20 and 40 mmHg, but resulted in the development of significant myogenic tone at both 60 and 80 mmHg (25.5±9.1% and 41.5±3.9% constriction, respectively). These data support the concept that, in urinary bladder arterioles, KIR channel conductance cannot be overcome by pressure‐induced depolarizing conductances, and thus smooth muscle cell membrane potential remains hyperpolarized and the arterioles do not constrict as intraluminal pressure increases. This represents a novel adaptation to maintain blood flow into the bladder during filling.Support or Funding InformationSupported by NIH R37DK053832 and K01DK103840.
PDE6 Isoforms Are Expressed in Native Endothelium but not in Cultured Cell Lines
Authors: Blanchette Alexandre , El Jetlawi Fatma , Toussaint Fanny , Charbel Chimene , Comtois Philippe , Ledoux Jonathan 
Published: November 2023

Nitric oxide (NO) is a major component of endothelial regulation of vascular homeostasis.. Diffusion of NO to the underlying vascular smooth muscle cells (VSMCs) will trigger Cyclic GMP (cGMP) production through activation of soluble guanylate cyclase leading to myocyte relaxation. As a second messenger central to numerous signalling pathways, proper control of cGMP level is critical. Although modulation of cGMP levels by phosphodiesterases (PDEs) is well established in smooth muscle through PDE5, few studies have focused in cGMP regulation in endothelial cells. Amongst cGMP‐specific PDEs, PDE5 and PDE9 have been reported in VSMCs and ECs. PDE6, a retina‐restricted cGMP‐specific PDE, has considerable similarities with PDE5, including overlapping. The current study establishes the expression of PDE6 isoforms in endothelial cells and their impact on local cGMP levels. Interestingly, all PDE6 isoforms (α, β and γ) were found in endothelial cell from mouse mesenteric arteries. Complementary approaches provide compelling evidences of the expression of PDE6 isoforms at mRNA and protein levels in native endothelial cells. However, PDE6a and b were not detected in endothelial cell lines. Subcellular distribution of PDE6 and 5 was sought using confocal microscopy. Whilst PDE5‐ and PDE6γ‐associated immunofluorescence was homogeneously distributed, PDE6α and PDE6β appeared preferentially localized within the perinuclear region. Mathematical modeling demonstrated the influence of PDE localization on local cGMP concentrations. Indeed, subcellular endothelial cGMP level following a NO pulse is unsymmetrical, with the peri‐nuclear region showing very little increase in cGMP. These results strongly suggest that PDEs, especially PDE6α and PDE6β, might have an significant impact on cGMP‐mediated cellular pathways in native endothelium through the fine‐tuning of cGMP microdomains.Support or Funding InformationSupported by FICM
Lipid composition of the myoendothelial junction allows for compartmentalized eNOS function
Authors: Biwer Lauren , Taddeo Evan , Hoehn Kyle , Straub Adam , Isakson Brant 
Published: November 2023

Cell communication within the wall of resistance arteries is a key contributor to vascular tone. In resistance arteries that are responsible for blood pressure regulation, the endothelial cell (EC) can project through the internal elastic lamina and make contact with the neighboring smooth muscle cell (SMC), forming a myoendothelial junction (MEJ). Our lab has previously shown that endothelial nitric oxide synthase (eNOS) is present in the apical EC and the basal EC (i.e, the MEJ). To test if the apical and MEJ pools of eNOS acted in sync or separately, we stimulated an in vitro co‐culture of human SMC and EC with 50uM phenylephrine (PE). This increases MEJ eNOS phosphorylation (eNOS‐P) at S1177 but not in the rest of the EC. Conversely, 10uM bradykinin (BK) increases EC eNOS‐P but does not affect MEJ eNOS‐P. Inhibiting gap junctions abrogates the MEJ eNOS‐P seen after PE, but has no effect on BK stimulated eNOS‐P. We hypothesized that differential lipid composition between the apical EC and MEJ may account for the differential localization and function of eNOS. What we found is that the lipid composition of co‐cultured EC & SMC is unique and suggests protein kinase C (PKC) localization at the MEJ. Indeed, PKC activity is significantly higher at the MEJ than the rest of the EC. Because PKC activity also relies on ER calcium release, we used the inhibitors thapsigargin and xestospongin C which caused significant decreases in MEJ eNOS‐P after PE. Furthermore, pharmacological inhibition of PKC significantly decreases MEJ eNOS‐P. To test for a functional effect, application of tumor necrosis factor alpha significantly increases leukocyte adhesion. When BK was added, this significantly decreased the percentage of adherent calcein green loaded THP‐1 monocytes while PE had no effect. Conversely, cGMP levels increase 5 minutes after PE and this increase is abrogated with gap junction inhibition. Underscoring the role of the MEJ in this pathway, SMC grown in flasks (without the presence of EC and thus, no MEJ formation) do not increase cGMP production after PE. Thus, we hypothesize that the unique, local lipid composition of the MEJ facilitates the greater PKC activity and distinct activation and function of MEJ eNOS versus the apical eNOS.Support or Funding InformationAmerican Heart Association National Institute of Health