bluuebunny/crossref_metadata_2025 huggingface dataset:
Osteopontin Attenuates Changes in Cerebrovascular Structure‐Function Relations Induced by Subarachnoid Hemorrhage in Rat Brain
Authors: Kim Dahlim , Doan Coleen , Carreon Desirelys , Budbazar Enkhjargal , Zhang John H , Pearce William J
Published: November 2023

Subarachnoid hemorrhage (SAH) is a common injury involving a complex and incompletely understood etiology. The present study explores the hypothesis that brain injury caused by SAH involves major changes in the structure and function of small cerebral arteries, in addition to its well‐known effects on neurons, glia and capillary endothelium. To evaluate this hypothesis, we studied the effects of SAH via endovascular perforation of the bifurcation of the anterior and middle cerebral arteries (MCA) in the adult rat brain. Artery samples were harvested 24 hours post‐injury for assessment of vascular structure and function. To test if the 44 kDa extracellular matrix protein Osteopontin (OPN) could serve as a vasoprotectant, some animals were treated with OPN via intranasal administration (5 μg in 50 μl PBS) at 1 hour post‐injury. As indicated by MLCK‐MLC20 colocalization quantified via confocal microscopy, SAH caused a significant 32% increase in contractile differentiation. This effect of SAH was attenuated by 66% (11% increase) following treatment with OPN. Simultaneously, SAH decreased the magnitude of the cytosolic calcium response to potassium depolarization by 28 nM (19%), and this effect was reversed to an increase of 41nM (49%) after OPN treatment. Additionally, SAH caused a passive dilation of 13 μM compared to untreated Sham control arteries, and after OPN treatment, this dilation was reduced to only 4 μM (69% reduction). Together, these results illustrate that SAH induces vascular injury that is largely prevented by intranasal treatment with OPN. Specifically, SAH promotes passive dilation and increased contractile differentiation, but compromises regulation of cytosolic calcium; these effects are consistent with the development of cerebral vasospasm and/or loss of cerebral autoregulation, both of which are well‐known consequences of SAH. Interestingly, OPN can bind and activate multiple integrin receptors, can promote inflammation via neutrophil recruitment, and can interact with other vasotrophic growth factors such as VEGF. It is not clear which, if any or all of these factors contribute to its neuroprotective profile. The present study strongly suggests that OPN is also vasoprotective within the context of SAH, which may contribute significantly to its neuroprotective efficacy.

Support or Funding Information

These studies were supported by an NIH Program Project grant from NINDS (NS082184), and an NIH R01 grant also from NINDS (NS076945).
Decreased Glucocorticoid Exposure In Utero Alters Cerebral Artery Development And Reduces Ischemia‐Induced Vascular Injury In The Neonatal Rat Brain
Authors: Durrant Lara , Jackson Patsy Naomi , Carreon Desirelys , Doan Coleen , Pearce William
Published: November 2023

INTRODUCTION

Abundant evidence suggests an important role of glucocorticoids (GC) in cerebral injury produced by ischemia, particularly in neonates. The majority of this evidence is focused on the role of GC in neuronal, glial, and endothelial acute responses to injury. In addition to involvement in acute responses, however, GC also play a critical role in cerebrovascular growth and development in utero. Elevation of maternal GC during pregnancy can dramatically alter cerebrovascular structure‐function relations in offspring. The present study examines the hypothesis that depressed glucocorticoid exposure in utero also programs the developing cerebrovasculature, thereby altering the postnatal cerebrovascular responses to ischemia.

METHODS

The experimental design included four groups: 1) Sham Operated; 2) Ischemia; 3) Sham + Metyrapone; and 4) Ischemia + Metyrapone. Metyrapone (MET), an inhibitor of the enzyme 11‐β hydroxylase that synthesizes corticosterone, was administered in the drinking water at 0.5 mg/ml from day 11 of gestation until birth. At postnatal day 9, rat pups were anesthetized with isoflurane and underwent unilateral carotid ligation, were allowed 24 hours to recover, and then were exposed for 90 minutes to 8% hypoxia. All pups were sacrificed at postnatal days 11–12 after which middle cerebral arteries were harvested for myography and confocal analysis.

RESULTS

When compared to Sham arteries, ischemia increased passive diameters by up to 25 μm (15%, P<0.05) and MET reduced this effect to 7 μm (4%, NS). Calculations of incremental compliance at 50 mmHg in Ischemic arteries revealed a decrease in compliance by −0.04 μm/mmHg (−43%, P<0.05), and these changes were ablated after MET and averaged 0.01 μm/mmHg (12 %, NS). Ischemia increased pressure‐induced myogenic changes in Ischemic artery diameter at 40 mmHg by up to 16 μm (62 %, P<0.05); treatment with MET reduced this effect to only 2 μm (6 %, NS). Ischemia increased the diameter changes induced by potassium at 60 mm Hg by 25 μm (73%, P<0.05), and this effect at 60 mm Hg was converted by MET to a decrease of −11 μm (−23%, P<0.05). Ischemia elevated the potassium‐induced increases in cytosolic calcium at 80 mm Hg by 25% (P<0.05) and MET reduced this effect to −11% (P<0.05). Finally, ischemia increased myofilament calcium sensitivity at 20 mm Hg by 49% (P<0.05), but after MET this change averaged −5% (NS).

CONCLUSIONS

These results support the hypothesis that glucocorticoids play a critical role during cerebrovascular development. In addition, this data demonstrates that decreased glucocorticoid exposure during pregnancy may decrease the vulnerability of the fetal vasculature to postnatal ischemic injury.

Support or Funding Information

These studies were supported by an NIH Program Project grant from NINDS (NS082184) and an NIH R01 grant also from NINDS (NS076945).
Progressive Loss of Cerebrovascular Reactivity and Nitric Oxide Bioavailability with Ischemic Stroke in Obese Zucker Rats
Authors: Brooks Steven D , Branyan Kayla , DeVallance Evan , Asano Shinichi , Hu Hang , Ren Xuefang , Rottgen Trey , Frisbee Jefferson C , Chantler Paul D
Published: November 2023

Ischemic stroke occurs when occlusion of a cerebral blood vessel leads to hypoperfusion, ischemia, and infarction of neuronal tissue. In the immediate aftermath of a stroke, maintenance of cerebral blood flow to penumbra is essential to help recover damaged neurons and protect surrounding neurons from insult, and throughout the post‐stroke recovery process, adequate blood flow is necessary to promote recovery and rehabilitation. As such, pre‐existing and stroke‐induced impairments in cerebrovascular reactivity may lead to larger infarcts and worsened long term recovery. The purpose of this study was to evaluate vascular reactivity at 24 hours and 15 days post‐stroke, in the ipsilateral and contralateral hemispheres of rodents with and without comorbid metabolic syndrome. Strokes were induced for 1 hour by transient middle cerebral artery occlusion (tMCAO) in 17 week old lean and obese Zucker rats (LZR, OZR). At either 24 hours post‐occlusion, or 15 days post‐stroke, animals were sacrificed and the ipsilateral (IL) and contralateral (CL) MCAs were removed and cannulated in an ex vivo microvessel preparation. MCA reactivity was assessed in response to acetylcholine. Aortic ring segments were analyzed for nitric oxide (NO) production by DAF assay, and infarct size was measured by TTC stain. In LZR, both IL and CL MCA reactivity at 24 hr. was severely impaired compared to non‐stroke control (over 50% reduction). At 15 days, reactivity decreased another 20% in IL MCA, but remained stable in CL‐MCA. LZR NO bioavailability at 24 hr. was reduced 35% compared to non‐stroke control, which remained constant at 15 days. In OZR, the vascular impairments were more pronounced at 24 hr, and worsened significantly at 15 days. At 24 hr, IL‐MCA reactivity was reduced 80% compared to control, while CL‐MCA was reduced 60%. By 15 days, IL‐MCA reactivity was completely ablated, and CL‐MCA reactivity had decreased another 35%. NO bioavailability for OZR at 24 hr was comparable to LZR stroke, but by 15 days had fallen another 50%. Rates of stroke mortality and infarct size were higher for OZR at both time points as well. These results suggests that ischemic stroke causes severe attenuation of NO bioavailability and cerebrovascular reactivity in both the IL and CL hemispheres of OZR at 24 hrs post‐stroke, with further loss of both at 15 days. While stroke lead to decreased reactivity and NO bioavailability in LZR, degree of impairment was less severe, and remained stable over time. Therefore, the comorbid presence of metabolic syndrome in OZR may increase stroke severity and drive progressive decline of cerebrovascular reactivity following ischemic stroke.

Support or Funding Information

American Heart Association Predoctoral Fellowship,

NIH COBRE grant
Effect of focal cerebral ischemia on BKCa and TRPV4 in rat middle cerebral artery
Authors: Murphy Timothy V , Nguyen Hong L , Grayson T Hilton , Jones Nicole M , Sandow Shaun L
Published: November 2023

Large‐conductance Ca2+‐sensitive K+ channels (BKCa) are prime regulators of vascular smooth muscle cell membrane potential and contraction and may be coupled to transient receptor potential vanillioid‐4 (TRPV4) channels. This study examined the expression and function of BKCa and TRPV4 in rat middle‐cerebral arteries (MCA) in stroke. Male Sprague‐Dawley rats were subject to focal cerebral ischemic stroke by localized bolus injection of endothelin‐1 (120 pmol). Controls received saline (0.9%). Five days later, animals were euthanized and the MCAs removed. Immunofluorescence studies showed increased BKCa expression (α‐ and β1‐subunits) in the smooth muscle cells of the arteries from rats with stroke. BKCa was also expressed in the endothelium of MCA from stroke rats, in contrast with arteries from controls. TRPV4 was expressed in both endothelial and smooth muscle cells of the MCA, and expression in both cell types was increased in arteries from stroke rats. Pressurized (80 mmHg) arteries developed myogenic tone, the degree of myogenic tone was not different between arteries from control and stroke rats. The BKCa blocker iberiotoxin (0.1 μM) constricted arteries from stroke rats to greater extent than control MCA. The TRPV4 activator GSK1016790A (GSK) caused concentration‐dependent dilation of MCAs from control and stroke rats; there was no effect of stroke on responses to GSK. In both control and stroke rats, MCA dilation induced by GSK was inhibited by the TRPV4 blocker HC‐067047 (0.3 μM) but not by iberiotoxin. These studies suggest increased BKCa expression and activity contribute to modulation of MCA tone post‐stroke. TRPV4 do not induce dilation of rat MCA through activation of BKCa.

Support or Funding Information

This work was supported by the Brain Foundation.
Heterogeneous Impact of ROCK2 on Carotid and Cerebrovascular Function
Authors: De Silva T. Michael , Kinzenbaw Dale , Modrick Mary L , Reinhardt Lindsey D. , Faraci Frank
Published: November 2023

Rho kinase (ROCK) is a serine‐threonine kinase that has been implicated in numerous physiological and pathophysiological processes, including regulation of vascular function. Two isoforms of ROCK (1 and 2) have been identified, and both are expressed in vascular cells. Due to a lack of isoform selective inhibitors or relevant genetic models, the role of individual ROCK isoforms in regulating vascular function is not well understood. In the present study, we examined the importance of ROCK2 in relation to vessel function. We used several approaches and models. First, overnight incubation of carotid arteries from C57Bl6/J mice with the direct RhoA activator CN‐03 or angiotensin II (Ang II) impaired endothelium‐dependent, nitric oxide (NO)‐induced relaxation. Endothelium‐independent dilation was not impaired by these treatments. CN‐03‐ and Ang II‐induced endothelial dysfunction was prevented by Y‐27632 (an inhibitor of both ROCK isoforms) or the selective ROCK2 inhibitor SLX‐2119. In fresh untreated vessels (no incubation), SLX‐2119 had little effect on contraction of carotid arteries to U46619, phenylephrine, serotonin or vasopressin. Second, in a resistance vessel (basilar arteries), SLX‐2119 inhibited constriction to Ang II by almost 90% without significantly affecting constriction to KCl or serotonin. Third, ROCK has been identified as an important mediator of myogenic tone. In isolated pressurized brain parenchymal arterioles (baseline diameter = 17±2 μm), SLX‐2119 inhibited myogenic tone in a concentration‐dependent manner. Lastly, SLX‐2119 treatment dilated small pial arterioles in vivo, an effect that was augmented by inhibition of NO synthase. Our findings suggest that ROCK2 has major, but heterogeneous effects on vascular tone. ROCK2 plays a prominent role in regulating endothelial function and select vasoconstrictor responses in resistance vessels (myogenic tone and constriction to Ang II). In contrast, ROCK2 appears to play a much smaller role in vasoconstriction to several other receptor‐mediated agonists. Collectively, these data support the concept that aberrant ROCK2 signaling may be a key contributor to select aspects of large and small vessel disease.

Support or Funding Information

Supported by the NIH (HL‐62984, HL‐113863), the Department of Veteran's Affair's (BX001399), the Fondation Leducq, and the NHMRC (1053786).
Detection of TRPV4 channel current‐like activity in Fawn Hooded Hypertensive (FHH) rat cerebral arterial muscle
Authors: Gebremedhin Debebe , Harder David Rae
Published: November 2023

The FHH rat strain is associated impaired pressure‐dependent myogenic tone and exaggerated cerebral arterial muscle KCa channel current opening. In the present study we examined if the FHH rat cerebral arterial muscle cells exhibit TRPV4 channel current‐like activity. Western blot analysis and RT‐PCR revealed expression of protein and transcript for TRPV4 channel in cerebral arteries of FHH rat strain. Immunostaining using the TRPV4 channel antibody detected presence of TRPV4 channel protein in freshly isolated FHH rat cerebral arterial myocytes. Patch clamp recording of single‐channel current activity in FHH rat cerebral arterial myocytes identified presence of a non‐selective cation channel current with unitary conductances of ~96 pS and ~84.6 pS at depolarizing and hyperpolarizing potentials, respectively, that were sensitive to inhibition by the TRPV4 channel inhibitor RT‐1734 (5 μM) or HC 067074 (0.2 μM) and activated by the potent TRPV4 channel agonist GSK‐101690A (100 nM). These findings indicate that the FHH rat cerebral arterial myocytes appear to express TRPV4 channel current‐like activity that could have relevance for the regulation of calcium movement and calcium‐dependent mechanisms in these cells.

Support or Funding Information

This work is supported by grants from NIH (R01 NIH/NHLBI, HL033833 and HL105997)
Steady‐state Changes in Cerebral Blood Flow between a Sitting and Standing Position Across the Menstrual Cycle
Authors: Favre Michelle , Reyes Levy , Fox Apollonia , Serrador Jorge
Published: November 2023

The purpose was to determine if steady‐state changes in cerebral blood flow between a sitting and standing position are affected across the menstrual cycle in healthy women with naturally cycling hormones (NOC) compared to women on combined oral contraceptives (OC). Eleven (6 NOC and 5 OC) healthy, women (mean age 25.3 years) were tested during menstruation (M), the late follicular phase (LF), and the mid‐luteal phase (ML). All participants performed sit‐to‐stand maneuvers on each visit while measuring beat‐by‐beat blood pressure, heart rate, end‐tidal CO2, and transcranial Doppler ultrasonography of the anterior and middle cerebral arteries. In addition, five of the women (3NOC, 2 OC) underwent ultrasonography of the internal carotid artery in the sitting position to measure global cerebral blood flow. Results indicate a significant effect (p= 0.02) of oral contraceptives on mean arterial pressure(MAP) in the sitting position (NOC: 74.4 ± 4.4 vs OC: 92.4 ± 4.8 mmHg), but no effect of menstrual cycle phase. Despite significant differences in baseline MAP, there was no difference in the ability to maintain MAP at baseline levels when standing. In contrast, changes in cerebral flow velocity (CFV) in the middle cerebral artery trended towards significance (P=0.097) between the NOC and OC groups. In the NOC group, the decrease in CFV was greatest during the late follicular phase (M: −2.1 ± 3.0; LF: −6.4 ± 2.9; ML: −3.9 ± 3.5 %) while in the OC group, the decrease was least during late follicular phase (M: −6.2 ±3.3; LF: −3.7 ± 3.2; ML: −6.0 ± 3.8 %). This appeared to be due to a lack of cerebrovasodilation in the NOC group during the late follicular phase, which was also trending towards significance (P=0.082). There was also no significant effect of oral contraceptives or menstrual cycle phase on the change in heart rate between steady‐state sitting and standing. Despite the trend toward greater drops in CFV in the NOC group during late follicular phase, there was no difference in seated internal carotid blood flow between groups or across the phases of the menstrual cycle. Due to the small number of subjects currently enrolled in the study, we must be cautious in the interpretation of the data. These data suggest that in women who are not on oral contraceptives, cerebral blood flow may be slightly lower when standing during the late follicular phase, but overall baseline global brain blood flow unaffected. In addition, it appears that women on oral contraceptives had higher blood pressures. Futurework is being performed to confirm these findings in a larger group of women. This study was conducted in compliance with the Declaration of Helsinki.

Support or Funding Information

This work was supported by the War Related Illness and Injury Study Center and Deptartment of Veteran Affairs.
SHEAR STRESS INCREASES SIRTUIN 3 EXPRESSION AND PROMOTES MITOCHONDRIAL FUNCTIONS IN ENDOTHELIAL CELLS
Authors: Wang Ling Danny
Published: November 2023

Hemodynamic shear flow‐induced atheroprotective responses in endothelial cells (ECs) have been intensively studied. However, the effects of shear flow on mitochondrial biology and its function in ECs remain unclear. Sirtuin 3 (SIRT 3), a member of Sirtuins family proteins that belong to a class of NAD+‐dependent deacetylases, is predominantly located in the mitochondria. Our previous studies have shown that SIRT 3 promotes mitochondrial homeostasis in ECs. In the present study, we studied the shear effects on SIRT 3 expression and mitochondrial homeostasis in ECs. Cultured ECs were subjected to a constant shear flow with shear stress of 12 dynes/cm2 generated by a parallel‐plate flow chamber system. Shear stress increased SIRT 3 expression and NAD+/NADPH ratio. Mitochondrial biogenesis, dynamics and functions were analyzed from sheared‐ECs. Shear flow promoted mitochondrial biogenesis as shown by the elevated levels of PGC‐1α, mitochondrial transcriptional factor A (TFAM) and mitochondrial proteins. Shear‐exposed ECs showed an increment of mitochondrial fusion revealed by the expansion of mitochondrial interconnected tubular formation. This phenomenon was coupled with an increased expression of fusion proteins (MFN2, OPA1) but a decreased fission protein (FIS1). Consistently, an increase of S637 phosphorylation and a decreased S616 phosphorylation of Drp1 were observed, indicating that shear promotes mitochondrial fusion process. Shear flow increased the expression of mitochondrial antioxidant enzymes MnSOD2, TRX2, PRX3 and PRX5. As a consequence, mitochondrial functions of sheared ECs were enhanced as revealed by a decreased mitochondrial ROS levels and an increased mitochondrial membrane potential and ATP production. Our results clearly demonstrate that ECs under shear stress increase SIRT3 expression and activity that directly or indirectly promote mitochondrial homeostasis. Thus, shear flow is essential for maintaining mitochondrial homeostasis in vascular ECs.

Support or Funding Information

SUPPORTED BY MINISTER OF SCIENCE AND TECHNOLOGY, TAIWAN
Elevated endothelial microparticles induce inflammation to impair endothelial function in patients with congenital heart diseases
Authors: Ou Jingsong , Lin Ze‐Bang , Ci Hong‐Bo , Ou Zhi‐Jun , Li Yan , Liu Dong‐Hong , Wang Zhi‐Ping
Published: November 2023

Objective

We have demonstrated that endothelial microparticles (EMPs) increased in mitral valve diseases and impaired valvular endothelial cells function. Inflammation is one of the major risk factors in cardiac surgery. In this study, we investigate whether EMPs increase in congenital heart diseases and the effects of EMPs on inflammation and endothelial function.

Methods

The level of plasma EMPs in 20 patients with atrial septal defect (ASD), 23 patients with ventricular septal defect (VSD), and in 30 healthy subjects were analyzed by flow cytometry. EMPs generated from human umbilical vascular endothelial cells (HUVECs) were injected into C57BL6 mice for 6 hours, or cultured with HUVECs with/without P38 MAPK siRNAs. The expression and/or phosphorylation of endothelial nitric oxide synthase (eNOS), P38 and caveolin‐1 in mice heart and/or cultured HUVECs were determined. The nitric oxide (NO) generation in mice hearts was measured. The production of tumor necrosis factor‐a (TNF‐a) and interleukin (IL)‐6 in cultured HUVECs were detected.

Results

EMPs were significantly elevated in patients with ASD and VSD, especially in those with pulmonary hypertension. EMPs increased caveolin‐1 expression and P38 phosphorylation, decreased eNOS phosphorylation and NO production in hearts. EMPs stimulated P38 expression and TNF‐a and IL‐6 release, which could be inhibited by P38 MAPK siRNAs in cultured HUVECs.

Conclusions

Our study demonstrated that EMPs were increased in patients with congenital heart diseases. The increased of EMPs could induce inflammation to impair endothelial function. P38 MAPK pathway may play a vital role in EMPs induced inflammation and provide a therapeutic target.

Support or Funding Information

This study was supported by the National Natural Science Foundation of China (81170271, 81370370 and Distinguished Young Scholar 81325001), the Changjiang Scholars Program from Ministry of Education of China, the Guangdong Pearl River Scholars Program, 973 project (2009CB522104) and International Cooperation Project (2015DFA31070) from the Ministry of Science and Technology of China, Guangdong Natural Science Fund Committee, China (2015A030312009), Sun Yat‐Sen University Clinical Research 5010 Program; Program of National Key Clinical Specialties, the Science and Technology Research for the Returned Overseas Chinese Scholars from Ministry of Human Resources and Social Security of China (2011).
Vascular effects of alpha hemoglobin stabilizing protein deletion
Authors: Biwer Lauren , Lechauve Christophe , Billaud Marie , Weiss Michell , Isakson Brant
Published: November 2023

Hemoglobin H is a tetramer made up of 2 alpha globin subunits and 2 beta globin subunits. The alpha globin subunit requires chaperone activity by alpha hemoglobin stabilizing protein (AHSP) in order to properly associate with hemoglobin beta. In addition to erythrocytes, alpha globin is expressed in endothelial cells (EC), specifically in the small projections of the EC that make contact with the neighboring smooth muscle cells (SMC), known as myoendothelial junctions (MEJ). Immuno‐transmission electron microscopy indicates that AHSP is expressed in EC and the MEJ but not SMC of C57Bl/6 resistance arteries. There is no expression of vascular AHSP in the global AHSP knockout mice. Correlating with its role as a chaperone protein for alpha globin, 32% of AHSP is found in the EC while 60% is found in the MEJ. The percent distribution of alpha globin beads in the EC and MEJ is 35% and 60%, respectively. In AHSP −/−mice, we found that alpha globin “un‐polarizes” from the MEJ and is more equivalently spread throughout the EC and MEJ (47% and 41.5%). To determine if AHSP is regulating alpha globin distribution, we immunoprecipitated eNOS and blotted for alpha globin. In a concentration‐dependent manner, AHSP competed off eNOS for binding to alpha globin. This correlated with data from AHSP−/− mice where an increase in alpha globin‐eNOS interaction was found via PLA signal. We hypothesized that the increase in alpha globin/eNOS interaction may then alter available nitric oxide. Indeed, using radiotelemetery blood pressure measurements, we found that mean arterial pressure in AHSP−/− mice was significantly increased. These results hint that deletion of AHSP may strengthen the interaction between alpha globin and eNOS resulting in altered blood pressure stasis.

Support or Funding Information

American Heart Association

National Institute of Health
Endothelial Adherens Junctions and Endothelial Dysfunction in Aging Arteries
Authors: Chang Fumin , Flavahan Sheila , Flavahan Nicholas A
Published: November 2023

Aging causes a systemic deterioration of the structure and function of the entire arterial network resulting in organ injury, dysfunction and failure. Endothelial dysfunction, including diminished NO activity, is thought to be a key contributor to pathological vascular aging. The normal protective phenotype of arterial endothelial cells is initiated in the immediate postnatal period and appears to be dependent on increased engagement and signaling of VE‐cadherin at Adherens Junctions (Flavahan S et al, 2013, Am J Physiol Heart Circ Physiol. 305(3):H321–9). The aim of the present study was to determine whether this process might be disrupted in aging arteries and contribute to aging‐induced endothelial dysfunction. Aortas and tail arteries were isolated from young (3 to 4 months) and old (22–24 months old) F344 rats and processed for immunofluorescent, biochemical and functional analysis. Arterial function was assessed using pressure myographs at a constant transmural pressure of 60 mmHg. During constriction with phenylephrine (to 70% of baseline), acetylcholine (1 to 1000 nM) caused dilatation that was significantly reduced in old compared to young arteries. Inhibition of NO synthase with LNAME markedly inhibited responses to acetylcholine, and there was no significant difference in the magnitude of the LNAME‐resistant dilatation between young and old arteries. There was also no significant difference in dilatation to the NO donor, DEA‐NONOate (1 to 1000 nM) between young and old arteries. These results are consistent with the presence of endothelial dysfunction and reduced endothelial NO activity in aging arteries. Immunofluorescent labeling followed by LSM analysis of VE‐cadherin demonstrated the presence of intense protein staining at endothelial Adherens Junctions in young arteries. This staining was significantly reduced in old arteries, which demonstrated a significant increase in intracellular localization of the protein. Immunoblot analysis confirmed a significant reduction in cell surface expression of VE‐cadherin in old compared to young arteries. A function‐blocking antibody to VE‐cadherin, which inhibits VE‐cadherin engagement and disrupts endothelial Adherens Junctions, significantly inhibited dilatation to acetylcholine in young but not in old arteries. A control antibody had no significant effects. After inhibition of VE‐cadherin engagement with the function‐blocking antibody, there was no longer any significant different in dilatation to acetylcholine between young and old arteries. These results demonstrate that there is morphological disruption of Adherens Junctions and reduced cell surface expression of VE‐cadherin in aging endothelial cells. Furthermore, the functional role of Adherens Junctions in contributing to endothelial dilatation is selectively lost in old arteries and likely contributes to endothelial dysfunction in these blood vessels.
The Role of Epigenetic Mechanisms and Matrix Remodeling Enzymes in Oxidative Stress Induced Loss of the Microvascular Endothelial Glycocalyx
Authors: Ali Mohamed M , Mahmoud Abeer M , Phillips Shane A
Published: November 2023

The integrity of endothelial glycocalyx is crucial for normal endothelial function and endothelial response to shear stress. Endothelial glycocalyx also tethers and concentrates the extracellular superoxide dismutase (ecSOD) which protects the endothelium against the oxidative damage of reactive oxygen species (ROS). Degradation of endothelial glycocalyx results in endothelial dysfunction and vascular remodeling. The proteolytic enzymes Matrix Metalloproteinases (MMPs) are capable of disrupting endothelial cell surface proteins such as syndecans resulting in derangements of endothelial glycocalyx. We sought to 1) test the role of MMPs and oxidative stress in disrupting the endothelial glycobiology 2) to determine the role of oxidative stress in the epigenetic regulation of this response. To this end, we experimentally induced oxidative stress in human adipose microvascular endothelial cells (HAMECs) in the presence or absence of exogenous hydrogen peroxide (H2O2, 2×10−4 mol/L; 4 hours) and endogenous oxidative stress buthionine sulfoximine (BSO; 10−3 mol/L) and MMP inhibitors. We found that either H2O2 or BSO, induced a 2–3 fold increase in MMPs (MMP1, MMP2 and MMP3) and a 3–5 fold increase in ADAMs (ADAM10 and ADAM17). Consequently, there was a reduction in protein levels of syndecan‐1 and ecSOD in the total cell lysate and increases in levels of syndecan‐1 ectodomain and ecSOD in cell culture media (4 folds and 5 folds, respectively) detected by immunoprecipitation and Western blotting. The upregulation of MMPS was accompanied by a decline of the mRNA and protein levels of their inhibitors, tissue inhibitors of MMPs (TIMPs; 2–3 fold). Interestingly, we found that the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) (5×10−6 mol/L) normalized MMP expression and restored TIMP 1 and TIMP 3 after H2O2 or BSO. Pharmacological inhibition of MMPs using marimastat (50 μM) and lisinopril (10 μM) mitigated the effect of oxidative stress on the glycocalyx. Using immunofluorescent labeled wheat germ agglutinin, we found that lisinopril and marimastat effectively restored the cell surface density of heparan sulfate glycosaminoglycans (HS‐GAGs), Syndecans and ecSOD on the endothelial cell surface. Lastly, we found that lisinopril decreased HDAC mRNA expression induced by H2O2 suggesting an epigenetic mechanism of lisinopril on responses to oxidative stress. In conclusion, our findings suggest that oxidative stress induced loss of the endothelial glycocalyx involves epigenetic regulation of matrix metalloproteinases that results in the shedding of ecSOD and syndecans. These data shed light on a therapeutically targetable epigenetic regulatory mechanism by which oxidative stress may induce vascular remodeling and endothelial dysfunction.

Support or Funding Information

NIH RO1s HL095701, HL095701‐01A2S (SAP), American Heart Association Grant 15POST24480172 (AMM)
Xanthine Oxidase Inhibition Decreases Vascular Stiffness in Western Diet Fed Male and Female Mice
Authors: Aroor Annayya , Habibi Javad , Jia Guanghong , Manrique Camila , Sun Zhe , Martinez‐Lemus Luis , Meininger Gerald , Sowers James
Published: November 2023

Endothelial and arterial stiffness play a central role in the development and progression of cardiovascular disease. Obesity and diabetes are associated with development of premature arterial stiffness. We have recently reported that male and female mice fed a diet high in fat and refined carbohydrates including fructose (western diet – WD) develop premature cardiac and vascular stiffness. In this regard, excess uric acid formed from excess fructose ingestion is emerging as a risk factor for vascular stiffness and vascular injury. We hypothesized that increased tissue xanthine oxidase activity and increased hepatic production of uric acid is associated with increased vascular stiffness and vascular dysfunction in WD fed males and females. Four week‐old C57BL6/J male and female mice were fed a WD with excess fat (46%) and fructose (17.5%) for 16 weeks with or without access to allopurinol in the drinking water (125mg/L), a potent xanthine oxidase inhibitor. Plasma uric acid levels were increased in plasma from WD male and female mice and these levels were normalized by allopurinol. Endothelial stiffness as determined by atomic force microscopy, was significantly increased in WD fed mice and this was significantly decreased by allopurinol administration. Increased endothelial stiffness was associated with increased fibrosis and vascular oxidative stress as determined by 3‐nitrotyrosine staining and this was prevented by allopurinol. Allopurinol treatment significantly attenuated WD induced increases in xanthine oxidase activity in aorta. Allopurinol also improved WD induced impairment of endothelial mediated vascular relaxation. WD feeding was associated with increased macrophage inflammatory response (M1 polarization) in aorta that was ameliorated by allopurinol. Taken together, these findings support that increased production of uric acid and enhanced xanthine oxidase activity in aorta promote vascular oxidative stress, macrophage inflammatory responses and vascular fibrosis/stiffness. We posit that increased cardiovascular xanthine oxidase activity associated with consumption of a WD plays a central role in the increased oxidative stress, inflammation and fibrosis.

Support or Funding Information

NIH R01 HL107910‐01 for J.R.S and the Veterans Affairs Merit System (Grant 0018) for J.R.S
Recovery of Ischemic Infarction by Trientine‐induced Copper Redistribution in the Heart of Rhesus Monkeys
Authors: Han Pengfei , Ding Xueqin , Fu Chunyan , Zhang Xianyu , Zhang Wenjing , Xiao Ying , Wang Hongge , Sun Xiaorong , Fan Suzhen , Kang Y. James
Published: November 2023

Persistent myocardial ischemic insult leads to copper (Cu) efflux from the heart and Cu elevation in the plasma, in association with significantly decreased capillary density. Cu is essential for angiogenesis. We observed that Cu‐trientine complex increased Cu uptake by primary cultures of neonatal rat cardiomyocytes subjected to hypoxia. The present study was undertaken to test the hypothesis that Cu redistribution to the heart by trientine can improve recovery of myocardial ischemic infarction. Male Rhesus monkeys of 2–3 years old were subjected to coronary artery ligation to induce myocardial ischemia. One year after the operation, myocardial infarction and dysfunction were detected by MRI and echocardiography. Trientine at a dose of 18 mg/kg a day was administered orally for eight weeks. At the end of the dose regiment, Cu concentrations were significantly elevated in the infarct area and border zone of the ischemic hearts, along with a decrease in plasma Cu concentrations compared to the untreated group. The angiogenesis factors including vascular endothelial growth factor (VEGF), VEGF receptor‐1 (VEGFR‐1), and other relevant factors were activated by the trientine treatment. Along with these changes, myocardial infarct size was significantly decreased and the density of myocardial microvessels was significantly increased. In addition, cardiac function was significantly improved, as evidenced by increased ejection fraction (EF) value. This study thus demonstrates that trientine is capable of redistributing Cu to the heart under ischemic conditions leading to a recovery of myocardial injury and dysfunction in Rhesus monkeys.

Support or Funding Information

This study is upported by National Science Foundation of China (81230004 and 81300109).
Radiofrequency Renal Denervation Protects the Ischemic Myocardium
Authors: Gao Juan , Polhemus David J , Smart Frank , Lefer David J , Kapusta Daniel R
Published: November 2023

The sympathetic nervous system plays a critical role in the pathogenesis of acute myocardial infarction (AMI) and heart failure. This study tested whether pre‐conditioning with radiofrequency renal artery ablation (RF‐ABL) protects the heart against myocardial ischemia/reperfusion (MI/R) in the setting of hypertension. Spontaneously hypertensive rats (SHR) received either bilateral RF‐ABL (n=14) or Sham‐ABL (n=14) (Biosense Webster Stockert 70 RF generator). Four weeks following treatment, hypertensive SHR were subjected to 30 minutes of left coronary artery occlusion. After 24 hours reperfusion, the myocardial infarct size/area‐at‐risk (AAR) were determined. In addition, the left ventricle (LV) was collected for measurement of nitric oxide (NO) metabolites, nitrite (NO2−) and S‐nitrosothiol (RSNO), and oxidative stress. RF‐ABL produced a significant decrease in myocardial infarct size per AAR compared to Sham‐ABL (26.8 vs. 43.9%) and lower plasma cardiac troponin‐I (6.8 vs. 10.3 ng/ml). Cardiac NO2− levels increased from 2.6 to 3.2 nmol/mg and RSNO levels increased from 0.5 to 1.1 nmol/mg following RF‐ABL. Myocardial oxidative stress was markedly attenuated as measured by 8‐Isoprostane, carbonyl content, and MDA levels. Greater transcription (RT‐PCR) of antioxidants, SOD1 and GPX‐1 were also observed following RF‐ABL. These findings translated to a significant increase in 24‐hr survival of RF‐ABL (13 of 14) as compared to Sham‐ABL (8 of 14) SHR to MI/R. Together, these findings demonstrate that in hypertensive SHR, RF‐ABL produced a sustained increase in myocardial NO bioavailability and attenuation of oxidative stress, both of which can contribute to preconditioning the myocardium against subsequent MI/R injury. These findings provide new insights into the remote cardioprotective effects of RF‐ABL in the setting of AMI.

Support or Funding Information

Biosense Webster IIS‐175 to DRK & FS; P30 GM103514 to DRK. 1R01 HL092141, 1R01 HL093579, 1U24 HL 094373, & 1P20 HL113452 to DJL.
Hemodynamics and Premature Ventricular Beats in Conscious Rats with Acute Myocardial Infarction Treated with Pyridostigmine Bromide
Authors: Santos‐Almeida Fernanda Machado , Salgado Helio Cesar , Fazan Rubens
Published: November 2023

We examined the effect of the acetyl‐cholinesterase inhibitor pyridostigmine bromide (PYR) on hemodynamics and arrhythmias in conscious rats after acute myocardial infarction (MI). Male Wistar rats were anesthetized (ketamine/xylazine) and implanted with subcutaneous electrocardiogram (ECG) electrodes, and catheters into the left femoral artery and vein. At the following day, under inhalatory anesthesia (isoflurane), animals were subjected to coronary artery ligation to induce acute MI in the anterior wall of the left ventricle. Control rats were submitted to fictitious coronary ligation (sham surgery). 20 and 120 min after MI, with the animals already recovered from anesthesia, they were treated with PYR (0.25 mg/kg, iv), or saline. The arterial pressure (AP) and ECG were continuously recorded between the third and fourth hour after acute MI. AP as found similar among groups (92±7, 92±10 and 97±8 mmHg). Heart rate (HR) was markedly higher in non‐treated (saline) infarcted rats (442±15 bpm) as compared to control (non‐infarcted) counterparts (389±10 bpm). PYR significantly reduced HR of infarcted rats (414±9 bpm), but it remained higher than non‐infarcted control rats. In addition, infarcted rats treated with PYR showed lower number of premature ventricular beats (0 to 99 events per hour) as compared to non‐treated group (1 to 169 events per hour). In conclusion, the treatment with PYR, on the first hours after MI, showed a protective effect preserving hemodynamics (bradycardic effect without changing AP), and also reducing the incidence of ventricular arrhythmias.

Support or Funding Information

FAPESP, CNPq, FAEPA
Effect of Caffeine on Cardiac Tissue Metabolism in Rabbits
Authors: Alada Akinola Abdulrasak , Nabofa Williams Ejide
Published: November 2023

Caffeine has been reported to influence skeletal muscle tissue metabolism at rest and during exercise (Goldstein et al 2010; Graham et al 1994). This study was designed to investigate the effect of caffeine on cardiac tissue metabolism in the rabbit.

The study was carried out on adult male New Zealand rabbits (weighing 1.8–2.2Kg) divided into 3 groups (n=5). Group I rabbits were given 0.2ml/Kg of normal saline and served as control while groups II and III rabbits were administered with 2mg/Kg and 6mg/kg of caffeine respectively for 28 days. Blood samples (3.0ml/rabbit) were collected by retro orbital puncture to determine serum Myeloperoxidase (MPO) activity and nitrite concentration. The animals were sacrificed by cervical dislocation and cardiac tissue biopsies were collected on dry ice for biochemical and immunohistochemical analyses. Cardiac tissue glycogen concentration was determined with the aid of anthrone reagent. Cardiac tissue Carnitine Palmitol Transferase 1 (CPT1) activity and cAMP concentrations were determined by immunohistochemistry and colorimetry techniques respectively, with assay kits obtained from Biovision inc.

The results showed that caffeine at 2mg/kg and 6mg/kg significantly inhibited MPO activity from 0.72±0.05 to 0.164±0.045 and 0.46±0.12 U/L respectively (p<0.05). Caffeine at 2mg/kg had no significant effect on serum nitrite concentration but at 6mg/Kg, caffeine significantly increased nitrite concentration from 28.01±6.53 to 45.25±3.88μM (p<0.05). Also caffeine at 2mg/kg and 6mg/kg increased cardiac tissue glycogen concentration from 15.62±0.73 to 40.69±6.35 and 38.82±6.91mg/100g, cAMP concentrations from 5.097±0.29 to 6.840±0.26 and 6.36±0.14 pmol/well and CPT1 activity from 18.3 to 20 and 25.2% respectively (p<0.05).

In conclusion, this study demonstrates that caffeine exhibits strong effects on cardiac tissue metabolism thereby causing cardioprotection, cardiac hyperemia, glycogen sparing, increased cAMP concentration and cardiac utilization of fatty acids as source of energy.
Influence of the Cardiomyocyte Circadian Clock on Autophagy in the Heart
Authors: He Lan , Brewer Rachel , Shanmugam Gobinath , Rajasekaran Namakkal Soorappan , Darley‐Usmar Victor , Chatham John C , Zhang Jianhua , Young Martin E
Published: November 2023

Background

In addition to its roles during physiologic (e.g., fasting) and pathologic (e.g., ischemia) states, autophagy is critical for cellular ‘housekeeping’, playing an important role in the normal turnover of damaged proteins and organelles (e.g., mitochondria). Over the course of the day, cells/organs are subjected to profound fluctuations in energy supply/demand, as well as exposure to environmental stresses known to induce protein/organelle dysfunction. This is particularly true for the heart, whose workload and oxidative metabolism dramatically increase during the awake (versus sleep) period. Recent studies have shown that the cardiomyocyte circadian clock directly modulates myocardial processes in a time‐of‐day‐dependent fashion, including metabolism and contractile function. The importance of the cardiomyocyte circadian clock is highlighted by observations that genetic disruption of this mechanism in mice (i.e., cardiomyocyte‐specific BMAL1 knockout; CBK) results in age‐onset cardiomyopathy and premature death.

Hypothesis

We hypothesize that the cardiomyocyte circadian clock directly regulates autophagy in the heart (which might be important for the normal turnover of proteins/organelles).

Methods and Results

Consistent with time‐of‐day‐dependent oscillations in autophagy, the LC3II/I ratio and p62 protein levels both oscillate in the mouse heart over the course of the day; these oscillations occur in an antiphase manner (i.e., LC3II/I peaks during the light/sleep phase, whereas p62 peaks during the dark/awake phase). Somewhat surprisingly, time‐of‐day‐dependent oscillations in LC3II/I and p62 persist in CBK hearts. However, p62 protein levels are chronically elevated, whereas p62 mRNA are chronically decreased, in CBK hearts, suggesting a potential negative impact of circadian clock disruption on autophagic flux. Fasting‐induced stimulation of autophagy was therefore next investigated in control and CBK hearts. Despite persistent food withdrawal, autophagy markers exhibited time‐of‐day‐dependent oscillations in the fasted state; in the case of LC3II/I, oscillations in this marker were antiphase in hearts of fed versus fasted control mice. Importantly, fasting‐induced alterations in LC3II/I were significantly attenuated in CBK hearts. Key regulators of autophagy were subsequently investigated, revealing that phosphorylation of mTOR (marker of activation) was chronically elevated in CBK hearts, relative to littermate controls.

The cardiomyocyte circadian clock appears to be an important regulator of cardiac autophagy. We speculate that chronic impairment of autophagy in the heart following disruption of the cardiomyocyte circadian clock contributes to cardiomyopathy in CBK mice.

Support or Funding Information

UAB School of Medicine Multi‐Investigator Pilot Grant;NIH R01 HL123574;NIH R01 HL122975
1,25‐Dihydroxyvitamin D Regulates Energy Substrate Metabolism to Reduce Triacylglycerol Accumulation in 3T3‐L1 Adipocytes
Authors: Larrick Brienna M , Kee‐Hong Kim , Donkin Shawn S , Teegarden Dorothy
Published: November 2023

It is well established that vitamin D is sequestered in adipose tissue. Many studies have demonstrated vitamin D regulation of adipocyte differentiation, inflammation, and energy metabolism, highlighting the multifaceted role that vitamin D plays in adipose tissue physiology. However, whether vitamin D impacts lipid metabolism in terminally differentiated adipocytes is not yet known. To study this, 3T3‐L1 adipocytes were differentiated for 9 days, followed by stimulation with 1,25‐dihydroxyvitamin D (1,25(OH)2D, 10 nM) or vehicle for the times indicated. All data are normalized to protein content, determined using the bicinchoninic acid (BCA) assay. We previously demonstrated that 1,25(OH)2D, the bioactive vitamin D metabolite, stimulates a 21% reduction in TAG accumulation in differentiated 3T3‐L1 adipocytes after 4 days (p=0.01). This occurs despite a significant increase in fatty acid uptake, assessed using BODIPY FL C16, following 1–4 days of treatment (p<0.01), and with concomitant stimulation of PKA‐dependent lipolysis (p<0.01). In our current studies, we demonstrate that 1,25(OH)2D stimulates a 2.5‐fold increase in complete fatty acid oxidation (p<0.01), assessed by quantifying the production of 14CO2 from [1‐14C] palmitic acid. Further, 1,25(OH)2D stimulates slight inhibition of de novo fatty acid synthesis, evidenced by a 10% reduction in [13C2]acetate incorporation into palmitic, palmitoleic, stearic, and oleic acids (p=0.03), determined by liquid chromatography‐mass spectrometry (LC‐MS). Interestingly, D‐[U‐13C]glucose incorporation into these fatty acids was reduced by 30% in response to 1,25(OH)2D (p<0.01), indicating a reduced contribution of glucose as a substrate for de novo lipogenesis. Consistent with these results, the mRNA expression of pyruvate carboxylase (PC) is reduced by 40% in response to 1,25(OH)2D (p<0.01). PC catalyzes the carboxylation of pyruvate to form the tricarboxylic acid (TCA) cycle intermediate oxaloacetate, which may then be used to synthesize acetyl‐CoA and glycerol for triacylglycerol synthesis. In summary, 1,25(OH)2D stimulates fatty acid turnover in 3T3‐L1 adipocytes, characterized by stimulation of PKA‐dependent lipolysis and fatty acid uptake. Further, 1,25(OH)2D stimulates fatty acid oxidation, while reducing glucose utilization as a substrate for fatty acid synthesis, possibly through reduced flux of pyruvate into the TCA cycle. Collectively, these changes in lipid metabolism may contribute to the reduction in TAG accumulation that is stimulated by 1,25(OH)2D, and may be protective against excessive fat mass accumulation and associated metabolic disorders.

Support or Funding Information

Supported by the Purdue University Center for Cancer Research: Obesity and Cancer Discovery Group (CA023168) and a Project Development Team within the ICTSI NIH/NCRR (UL1TR001108).