bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron exposure reduces viability and plasticity of brain microvascular endothelial cells after hypoxic injury (670.2)
Authors: Flores Julio , Abdelsaid Mohammed , Ergul Adviye
Published: June 2021

Diabetes increases the risk, causes bleeding into the brain, and worsens functional outcomes after ischemic stroke. We have previously shown that diabetic Goto‐Kakizaki (GK) rats have increased yet dysfunctional angiogenesis, profuse bleeding into the brain and poor vascular repair if subjected to stroke. The effect of bleeding on endothelial cell survival and repair is unknown. Here, we test the hypothesis that iron exposure reduces brain microvascular endothelial cell (BMVEC) viability and plasticity after ischemia/reperfusion in diabetes. To test this hypothesis, BMVECs isolated from Wistar (non‐diabetic) and GK rats were treated with 0.1 mM iron (III) sulfate, incubated 6 hours in hypoxia, and reoxygenated overnight. The cells were subjected to cell proliferation, migration and tube formation assays. GK BMVECs showed increased cell proliferation, migration and tube length under normoxia. Hypoxia reduced all three measures of angiogenesis for GK BMVECs. In addition, iron treatment further reduced migration and tube length of GK BMVECs exposed to hypoxia while it did not have an effect on control cells. These results suggest that iron has a deleterious effect on survival and angiogenic properties of GK BMVECs. More work is needed to investigate the exact mechanism of iron‐induced impairments but our work identifies iron chelation as a possible novel therapy to improve recovery after ischemic stroke.
Dedicator of cytokinesis 2, a novel regulator for vascular smooth muscle cell phenotype modulation and vascular remodeling (670.3)
Authors: Guo Xia , Cui Xiao‐Bing , Wang Jia‐Ning , Shi Ning , Fukui Yoshinori , Chen Shi‐You
Published: June 2021

The objective of this study is to determine the role of dedicator of cytokinesis 2 (DOCK2) in vascular smooth muscle cell (VSMC) phenotypic modulation and vascular remodeling. Platelet‐derived growth factor‐BB (PDGF‐BB) induced DOCK2 expression while modulating VSMC phenotype as indicated by reduced VSMC marker expression. DOCK2 overexpression inhibited while DOCK2 knockout enhanced SMC marker gene expression in primary VSMC. Mechanistically, DOCK2 regulated VSMC phenotype through suppression myocardin/serum response factor (SRF)‐mediated transcription of VSMC marker genes. DOCK2 blocked myocardin, but not SRF expression and inhibited myocardin‐activated α‐SMA promoter activity. DOCK2 was also essential for PDGF‐BB‐induced VSMC proliferation and migration. DOCK2 stimulated VSMC migration via induction of focal adhesion contact and stress fiber formation. By using both rat carotid artery balloon‐injury and mouse ligation injury models, we observed that DOCK2 was highly induced in injury‐induced neointima. Knockdown of DOCK2 dramatically inhibited the neointima formation by 60%. Most importantly, knockout of DOCK2 in mice markedly blocked ligation‐induced intimal hyperplasia. These data demonstrate that DOCK2 plays an important role in VSMC phenotypic modulation and vascular lesion formation following vascular injury.

Grant Funding Source: HL093429 and HL107526 to Shi‐You Chen, Pre‐doctoral Fellowship Award 12PRE12030180 to Xia Guo
Neutrophil depletion: beneficial or detrimental to post‐ischemic hind limb recovery in Cx40‐/‐ vs. WT mice? (670.8)
Authors: Good Miranda , Barendrick Lindsay , Oulton Jeremy , Burt Janis
Published: June 2021

Connexins (Cx) regulate vascular function, remodeling, and the inflammatory response in various vascular diseases. Recovery of post‐ischemic hindlimb use and perfusion is significantly impaired in mice with global deletion of Cx40 (Cx40‐/‐), and infiltrated activated macrophages were significantly more numerous in the ischemic limbs of these mice 14 days post‐injury as compared to wild type (WT) mice. Here we begin to explore whether the inflammatory response in the Cx40‐/‐ mice is detrimental to its post‐ischemic recovery process. At both 8 hours and 24 hours post‐femoral artery ligation (FAL), Cx40‐/‐ mice had a significantly larger number of infiltrated neutrophils in the ischemic gastrocnemius muscle compared to WT mice. This increased infiltration did not, however, lead to any differences in number of infiltrated activated macrophages at 3 days post‐ischemic injury in Cx40‐/‐ and WT mice. Additionally, no differences in Cx37 or Cx40 expression were detected as a consequence of ischemia in WT and Cx40‐/‐ mice, despite genotype‐specific differences in Cx37 localization. To assess the impact of excessive neutrophil infiltration on post‐ischemic recovery of limb use and perfusion, we neutrophil depleted mice by injecting 500µg of anti‐m Ly‐6G antibody (clone: RB6‐8C5) 1 day prior to, day of, and 1 day post‐surgery. Preliminary data indicate that neutrophil depletion did not significantly improve or impair recovery of limb use in WT or Cx40‐/‐ mice; however, a trend indicative of impaired limb perfusion was observed in neutrophil depleted WT, but not Cx40‐/‐ mice. These results suggest that neutrophil infiltration may support post‐ischemic recovery in WT mice, but does not improve or diminish recovery of Cx40‐/‐ mice.

Grant Funding Source: Supported by HL058732
The potent growth promoter lysophosphatidic acid is reduced in plasma following the administration of the HDL mimetic peptide 4F (671.11)
Authors: Yadegari Brian , Hough Greg , Safar Sheila , Barseghyan Zarin
Published: June 2021

Introduction. Lysophosphatidic acid is a potent growth promoter that acts through high affinity G‐protein coupled receptors including the six receptors termed: LPA1 to LPA6. Because of its ability to stimulate cell proliferation, aberrant LPA‐signaling has been linked to cancer in numerous ways. Our group previously reported that feeding a high fat high cholesterol diet (Western diet) to ApoE deficient mice would increase the circulating levels of lysophosphatidic acid. HDL mimetic peptide 4F has been shown to have an extremely high affinity for lysophosphatidic acid and this might be used to lower the levels of this inflammatory molecule. Objective: We sought to determine if administering 4F to ApoE deficient mice on a Western diet would affect the levels of circulating lysophophosphatidic acid. Methods: Female ApoE deficient mice, 4 to 5 months of age (n=20 per group), were maintained on a Western diet for 8 weeks. One group also received 4F in drinking water at 100 ug per ml. Following 8 weeks, the mice were fasted overnight, blood was removed under anesthesia from the retro orbital sinus, and plasma was prepared and stored at ‐80°C. Lysophospholipid acid determination was done using solid phase extraction and LC‐ESI‐MS/MS, while isomers C20:4 and C18:2 were quantified. Results: Administration of 4F to the ApoE mice on a Western diet resulted in a significant reduction in the circulating levels of lysophospholipid C20:4 and C18:2 (p<0.01 for both). The level of lysophospholipid 18:0 did not show any detectable changes between the two groups. Conclusion: The data indicates that the HDL mimetic peptide 4F has the ability to reduce the Western diet induced increase in plasma levels of lysophospholipids involved in inflammatory reactions. This may be a useful consideration in helping reduce atherogenic or oncogenic conditions.
The effects of splenic denervation on atherosclerosis in ApoE‐/‐ mice (671.8)
Authors: Yang Li Jin , Ulleryd Marcus , Micallef Peter , Hua Sansan , Johansson Maria
Published: June 2021

There is a close interplay between the nervous and the immune system and the spleen is important for this interaction. We hypothesize that splenic denervation will cause the spleen to lose its immunological regulation and worsen inflammation. Our aim is to investigate the effects of splenic denervation on atherosclerosis in ApoE‐/‐ mice. The spleen of ApoE‐/‐ mice were denervated using 10% phenol. Four weeks after surgery, splenic noradrenaline content was decreased with 74% (p<0.001) in denervated compared to sham‐operated control mice. Atherosclerosis, evaluated in serial sections of the aortic root, was increased in denervated compared to controls (p<0.05). Splenic pro‐inflammatory CD11b+Ly6Chigh monocytes were increased in denervated vs control mice (p<0.05) analyzed by flow cytometry. Lack of nerve signaling to the spleen accelerates atherosclerosis in ApoE‐/‐ mice, and increase pro‐inflammatory CD11b+Ly6Chigh monocytes in the spleen. Thus, we propose that lack of nerve signaling to the spleen impairs the immunological regulation of the spleen and accelerates atherosclerosis.
Ischemic stroke‐induced increases in circulating microparticles promote leukocyte adhesion in intact microvessels (672.3)
Authors: Petrides Pete , Tan Zhenjun , Xu Sulei , Guo Ge , Huber Jason , Rosen Charles , He Pingnian
Published: June 2021

Studies in both stroke patients and experimental animals indicate an association between ischemic stroke and post stroke peripheral inflammation, but the mechanisms are poorly understood. Our ongoing study indicated that circulating microparticles (MPs), the small vesicles released from the cell membrane upon activation, were significantly elevated in both patients and animals with diabetes, and increased circulating MPs with phosphatidylserine (PS) exposure on their outer membrane played important roles in the development of diabetes‐associated microvascular complications. The objective of this study was to investigate whether local ischemic stroke produced increased circulating MPs and determine their impact on post stroke‐associated peripheral vascular inflammation. Acute cerebral ischemia was induced by occlusion of the right middle cerebral artery (MCAO) in 3‐4 month old rats using an autologous blood clot. Functional assessment, lesion volume, and hemispheric swelling were evaluated at 24h after MCAO. Plasma MPs 24h after stroke induction were quantified and characterized using flow cytometry. The number of PS exposed MPs in stroke plasma, identified by Annexin V binding, significantly increased from 5.0 x 103 (normal rats) to 30 x 103 per µl. Antibodies directly against cell specific antigens were used to differentiate the MP cell origin. Results showed that 67% of the increased stroke plasma MPs were derived from platelets. Perfusion of individually cannulated normal rat mesenteric microvessels with isolated stroke MPs for 30 min followed by 10 min of resumed blood flow induced a 4‐fold increase in leukocyte adhesion compared to baseline. These results indicate that stroke‐induced increased circulating MPs serve as mediators capable of disseminating local inflammation to remote vasculatures, and therefore, promoting post stroke systemic complications.

Grant Funding Source: Supported by HL56237, DK097391, NS061954
Vasodilation in mature arterialized collateral capillaries increases flow in ischemic arterial trees (674.10)
Authors: Gouin Kenneth , Hellstrom Sara , Cutts Josh , Cardinal Trevor
Published: June 2021

During ischemic events, natural bypass routes such as collateral arterioles can redirect blood flow to the ischemic zone. However, reperfusion of the ischemic arterial tree can also occur in animal models that lack arteriole collaterals, such as Balb/C mice. Collateral capillaries associated with the ischemic arterial tree enlarge and recruit smooth muscle cells in response to an increased pressure gradient. The arterialized collateral capillaries (ACC) must also be able to dilate and constrict in response to the changing metabolic needs of the tissue fed by them. By 21 days after the onset of ischemia, ACCs begin to dilate and constrict in response to physiological stimuli, however it has yet to be shown if the vasodilation of ACCs will increase blood flow in the ischemic arterial tree. To test whether vasodilation of the ACCs will increase blood flow in the ischemic arterial tree, we used laser speckle flowmetry at 21 days post‐ligation of the lateral cranial feed artery of the spinotrapezius muscle. We captured laser speckle images of the entire muscle to determine blood velocities at rest and after application of sodium nitroprusside (SNP). SNP increased blood velocity in the ischemic arterial tree by 60 ± 7% above res2ting velocity. Similarly, in unoperated vasculatures, blood velocity in arterial trees increased by 47 ± 5% above resting velocity. Comparable blood velocity increases between operated and unoperated vasculatures suggest that the ACCs are able to not only increase blood flow to the ischemic arterial tree, but also adequately respond to tissue metabolic demand. These results indicate that supporting the function of ACCs may be a potential therapeutic option for chronic ischemic diseases and their associated complications.
Effects of brief ischemia on oxygen consumption, tissue oxygenation, and blood flow during recovery from ischemia in rat skeletal muscle (674.13)
Authors: Dodhy Sami , Golub Aleksander , Pittman Roland
Published: June 2021

The effects of brief ischemia (5, 15, 30, and 60 s duration; Isch) on interstitial PO2 (PISFO2), arteriolar PO2 (PaO2), blood flow (Q) and O2 consumption (VO2) during the recovery period was observed in exteriorized spinotrapezius muscles of 29 male Sprague‐Dawley rats (284 ± 20 g) for intravital microscopy. Phosphorescence quenching microscopy was used to obtain PISFO2, PaO2 and VO2. Arteriolar blood flow was calculated from arteriolar diameter and centerline RBC velocity obtained from an Optical Doppler Velocimeter. Ischemia was initiated by rapidly pressurizing an airbag attached to the microscope objective for a selected ischemic duration. The average baseline value of arteriolar blood flow was 5.8±0.8 µl/min; as expected, reactive hyperemia was observed during the recovery from Isch. Flow recovery (dQ/dt) was fastest [3.88±0.64 (µl·min‐1)/s] after 60 seconds of Isch. Average baseline PISFO2 was 62±4 mmHg; PISFO2 decreased to 75%, 34%, 10% and 2% of baseline following 5, 15, 30 and 60 s of Isch, respectively. Up to 30 s of ischemia, the rate of recovery of PISFO2 steadily increased as the ischemic duration increased; however, it dropped off following 60 s of ischemia. The drop in the rate of recovery of PISFO2 despite an increase in flow recovery following 60 s of ischemia is suggestive of a period of imbalance during which the rate at which the tissue was consuming oxygen temporarily exceed the rate at which it could be supplied. The rate of recovery of PISFO2 was fastest (4.2±0.7 mmHg/s) following 30 s of Isch. Average baseline VO2 was 120±19 nl O2/cm3·s and it increased above baseline during the early part of the recovery period. The time course of recovery of PISFO2 represented a balance between O2 delivery, proportional to local arteriolar blood flow, and local VO2.

Grant Funding Source: NHLBI grant HL18292
Effects of oxygen affinity and viscosity of a hemoglobin‐based oxygen carrier (HBOC‐201) on systemic and microcirculatory variables in a top‐load rat model (674.2)
Authors: Song Bjorn , Nugent William , Moon‐Massat Paula , Pittman Roland
Published: June 2021

The effects of oxygen affinity (P50) and viscosity on arteriolar vasoactivity and tissue oxygen tension were explored by administering a polymerized bovine hemoglobin‐based oxygen carrier (HBOC) in a dose‐response fashion to normovolemic rats. The P50 and viscosity, respectively, of the parent compound (HBOC‐201) and its modifications (MP50 and LP50A) were as follows: 40 mmHg and 3.0 cP (HBOC‐20l); 18 mmHg and 4.4 cP (MP50); and 17 mmHg and 12.1 cP (LP50A). Anesthetized male Sprague‐Dawley rats (N = 32) were randomized to receive one of the HBOC solutions, and were administered with four infusions that increased in concentration for each dose (2, 22, 230 and 780 mg/kg, IV). Data were compared to rats receiving an equivalent volume for each of the four infusions (0.4, 0.4, 3.8, 13.1 ml/kg, IV) of iso‐oncotic 5.9% human serum albumin (HSA). HBOC‐201, MP50 and HSA produced significant increases in MAP after Doses 3 and 4, whereas a similar increase began at a lower dose (Dose 2) with LP50A. There were no significant changes in arteriolar diameters at any dose for any group. Interstitial PO2 remained unchanged for HBOC‐201, MP50 and HSA, but LP50A caused a significant decrease compared to baseline after Doses 3 and 4. In this healthy rat top‐load model, there was no evidence that HBOC‐201 would perform better with increased oxygen affinity (40 to 18 mmHg) or viscosity (3.0 to 4.4 cP).

Grant Funding Source: Supported by BUMED congressional funding WUN 604771N.9737.001.A0315, and in part by R01 HL18292
2,3‐Diphosphoglyceric acid increase in carotenoid‐treated hypercholesterolemic rabbits (674.4)
Authors: Beard Elizabeth , Andry Jeffrey
Published: June 2021

2,3‐Diphosphoglyceri c Acid Increase in Carotenoid Treated Atherogenic Rabbits Jeffrey Jordan Andry Diminished oxygen diffusivity in blood may be induced by hypercholesterolemia in rabbits and is thought to play a major role in the increased arterial permeability that supports atherogenesis. Hypercholesterolemic rabbits treated with the carotenoid Crocetin (CR) (extracted from saffron) for at least 4 months, has been found to increase oxygen diffusivity, lower blood cholesterol (CHO) and reduce atherogenesis. This short term study focuses on the ability of Crocetin and B Carotene (BC) treatment of hypercholesterolemic rabbits to alter their blood levels of 2,3‐Diphosphoglyceric Acid (DPG) , a component of the glycolytic sequence which causes oxygen release from hemoglobin. Nine New Zealand Strain, male rabbits (1.4 Kg av wt ) were fed an Atherogenic Diet (AD) , ad libitum, of 92% Purina Rabbit Chow, 6% Corn Oil and 2% Cholesterol, for 5 weeks. The animals were divided into 3 equal groups and injected im. on alternate days as follows: I. Control (CO) Group ‐ 0.2 ml saline, II. CR Group ‐ 2mg. CR/ 0.2ml saline and III. BC Group‐ 2mg BC/0.2ml saline. DPG assays on whole blood samples after 3 weeks of treatment averaged an increase of 8.6% DPG in blood of CR treated rabbits vs CO values. After 5 weeks, of treatment, the average DPG level was 31.9% higher in the blood samples of CR treated rabbits, and for BC samples 2.6% higher compared to the CO DPG value. Average CHO assays of blood plasma gave substantially higher values in the carotenoid treated rabbits ( 36.1% for CR and 31.5% for BC assays) compared to the CO plasma values. The increase in 2,3 DPG in the blood of CR treated rabbits vs CO assays was significant after only 5 weeks of study despite substantial hypercholesterolemia found in that group. There was slightly lowered early atherogenesis found on examination of the aortae of the CR group vs CO aortae at autopsy.
A new model for metabolically‐linked regulation of local blood flow (674.5)
Authors: Golub Aleksander , Pittman Roland
Published: June 2021

In skeletal muscle an increase in O2 consumption over a wide range evokes a proportional increase in LBF. The regulation of LBF ensures matching the delivery of materials with their rate of consumption. The mechanism by which this happens is thought to operate according to the metabolic hypothesis of LBF control, whereby an increase in tissue hypoxia (caused by an imbalance between O2 demand and supply) stimulates the production of metabolites by the hypoxic cells which further open blood vessels and lead to increased blood flow and O2 supply. The drawback of this model is the inevitable maintenance of tissue hypoxia. An alternative mechanism to explain the matching of LBF to demand in O2 and nutrients is proposed which depends on the interaction of two naturally occurring signaling radicals, nitric oxide (NO), constantly produced by endothelial cells, and superoxide (O2‐), produced primarily by the parenchymal cells. These two highly reactive molecules interact in the interstitium at a rate controlled by diffusion to determine the level of the vasodilator NO. The proposed mechanism is based on the concept that functional activity is the normal physiological state for a tissue; the corresponding normal state for its vasculature is dilation. The goal of regulation is maintaining a sufficient, but not excessive, supply of O2 and substrates to the cells. This mechanism explains why the vasodilation signal is generated by microvessels, and how the parenchymal cells block this signal when LBF provides a sufficient supply of O2 and substrates. Production of O2‐ by membrane NAD(P)H oxidase depends on the balance of glycolysis and oxidative phosphorylation via the availability of NADH and NAD(P)H in the cytosol and the interstitial PO2. Thus, the coupling of tissue metabolic activity with LBF is achieved.

Grant Funding Source: Supported by NHLBI HL18292
Impaired skeletal muscle fatigue resistance in metabolic syndrome is associated with alterations in microvascular hematocrit levels and variability (675.3)
Authors: Butcher Joshua , Stanley Shyla , Brooks Steven , Chantler Paul , Wu Fan , Frisbee Jefferson
Published: June 2021

Vascular dysfunction in metabolic syndrome is associated with poor performance of in situ skeletal muscle. Prior to impaired function in skeletal muscle itself, this reflects a failure to effectively perfuse working muscle. While altered vascular reactivity, arteriolar wall stiffness and microvessel rarefaction have been implicated, the accumulated impact on mass transport/exchange is unknown. Using tracer washout and existing models for microvascular hematocrit (HMV) and permeability‐surface area product (PS), we determined these parameters for in situ skeletal muscle of lean (LZR) and obese Zucker (OZR) rats. HMV in OZR was less than for LZR; treatment with adrenergic blockers or anti‐oxidants improved HMV slightly, but imposition of all agents restored HMV fully. PS, reduced in OZR vs. LZR, was not impacted by intervention. Using in situ cremaster muscle, HMV was determined in individual microvessels. Similar to above, HMV was reduced in OZR vs. LZR, although variability was higher; with post‐intervention changes mirroring that from washout experiments. These data suggest a low, spatially variable, HMV and global loss in surface area are contributors to poor muscle performance in OZR.

Grant Funding Source: Supported by National Institutes of Health and American Heart Association
Thrombospondin‐1 null mice exhibit enhanced microvessel endothelium dependent vasodilation, reduced adrenergic sensitivity (676.10)
Authors: Mandler W. Kyle , Nurkiewicz Timothy , Olfert I. Mark
Published: June 2021

Thrombospondin‐1 null mice exhibit enhanced microvessel endothelium dependent vasodilation, reduced adrenergic sensitivity The extracellular matrix protein thrombospondin ‐1 (TSP‐1), is an antagonist of nitric oxide‐mediated vascular relaxation by preventing activation of guanylate cyclase and other downstream targets. Our objective is to determine the effects loss of TSP‐1 loss on microvascular reactivity.

Arteriolar responses to acetylcholine, phenylephrine, and adenosine, were assessed in skeletal muscle microvasculature of global TSP‐1 null (KO) and wildtype (WT) littermate mice using intravital microscopy with the gluteus maximus.

KO animals demonstrated 10.7%, 18.6%, 16.5% greater vasodilatory response to acetylcholine concentrations of 10‐6, 10‐4, and 10‐2M (p < 0.05). WT vessels demonstrated more constriction than KO at the highest concentration of phenylephrine, 10‐3M (p < 0.05). KO vessels demonstrated greater vasodilatory capacity (p < 0.05), to 10‐2M adenosine. WT animals had a 23.8% greater (p < 0.05) vasoconstriction in response to the highest dose of phenylephrine, 10‐3M. KO animals demonstrated a 23.5% higher (p < 0.05) maximal dilatory capacity in response to 10‐2M adenosine than WT. These data provide evidence that TSP‐1 is an important physiologic regulator of skeletal muscle microvascular reactivity.
A novel ex vivo tissue culture assay for determining the effects of anti‐tumor drugs on angiogenesis (676.14)
Authors: Azimi Mohammad , Mathur Aditi , Mondal Debasis , Murfee Walter
Published: June 2021

Tumor growth is highly dependent upon angiogenesis and the interplay between the tumor stroma and the microvasculature. A challenge in evaluating anti‐tumor therapies is the inability to decouple treatment effects on cancer cells versus the microvascular network. Recently, our laboratory demonstrated that the “rat mesentery culture” model could be used for time‐lapse comparison of intact microvascular networks before and after angiogenesis induction. The objective of this study was to demonstrate that this model could be used to evaluate the anti‐angiogenic effects for a given drug treatment. Mesenteric windows were harvested from adult male Wistar rats and cultured for 3 days in serum containing medium (MEM + 10% Fetal Bovine Serum). Explants were exposed to increasing concentrations of Nelfinavir (Viracept™) (0 ‐ 4.5 μM) and Curcumin (0 ‐ 4.5 μM). Nelfinavir and Curcumin were selected for the study because Nelfinavir causes endothelial dysfunction and Curcumin, an anti‐oxidant and NF‐κB inhibitory dietary supplement, is known to have anti‐angiogenic effects. Preliminary studies also suggest that the combination of Nelfinavir/Curcumin produce synergistic anti‐cancer effects. Labeling tissues with FITC conjugated BSI‐lectin on Day 0 and Day 3 identified endothelial cells along all vessels. Results showed significant dose‐responsive effects of Nelfinavir/Curcumin combinations in suppressing both vessel segment density and the number of capillary sprouts per vascular area. Our findings establish the rat mesentery culture model as a valuable ex vivo tool for the rapid and reproducible screening of drug effects on angiogenesis in an intact network scenario.

Grant Funding Source: Supported by Tulane Center for Aging and NIH 5‐P20GM103629‐02
Zipper‐interacting protein kinase is a key regulator of vascular smooth muscle tone with implications in development of hypertension (676.18)
Authors: Turner Sara , Haystead Timothy , MacDonald Justin
Published: June 2021

Essential hypertension is linked to increased contractile tone in the resistance vasculature and affects ~25% of adults, increasing their risk for more severe cardiovascular disease. Recently, zipper‐interacting kinase (ZIPK) was linked to essential hypertension in animal models of the disease. We possess a novel inhibitor of ZIPK that lacks off‐target effects against other contractile kinases and is uniquely situated to investigate the role of ZIPK in normal and pathological vascular smooth muscle function. Hypothesis: ZIPK contributes to the Ca2+ sensitization of vascular smooth muscle, which is in turn accentuated in hypertensive disease states. We used isolated ex vivo vessels with pressure myography, along with the ZIPK inhibitor HS38, to investigate cerebral vessels isolated from human biopsies and spontaneously hypertensive rats (SHR). ZIPK was expressed in human cerebral vessels and contributes to the myogenic response to pressure. Treatment with HS38 resulted in a ~60% reduction in the magnitude of myogenic contractions over the 60‐120mmHg pressure range. Moreover, when early‐stage SHR were compared, we found the myogenic response was enhanced with an increase in the contribution of ZIPK. Based on these findings, we conclude that ZIPK is a critical factor in the development of essential hypertension, and represents a unique and viable, therapeutic target in humans.
Maximal local thermal hyperemia in able‐bodied and spinal cord injured humans (677.10)
Authors: Zhao Joan , Trbovich Michelle , Wu Yubo , McMahan C. , Kellogg Dean
Published: June 2021

Paraplegic spinal cord injury alters local thermal hyperemic responses. We hypothesized that a similar effect would be observed in tetraplegia. 6 able‐bodied (AB), 5 paraplegic (PARA), and 5 tetraplegic (TETRA) age and gender matched subjects participated. Skin blood flow (SkBF) was monitored by laser‐Doppler flowmetry (LDF) from forearm and calf sites. Local skin temperature (Tloc) was controlled with combined LDF/local heating probe holders. Tloc was measured with thermocouples placed between the probe holders and the skin. Mean arterial pressure (MAP) was recorded by Finapres. After instrumentation, Tloc was increased slowly from normothermic levels to 42°C and maintained at that temperature for at least 25 minutes to cause maximal local vasodilation. Cutaneous vascular conductance was calculated as CVC=LDF/MAP and analyzed by ANOVA. Results: At Tloc=42°C, CVC measured from forearm skin was 47±7mV in AB, 49±8mV in PARA, and 42±8mV in TETRA. These responses did not statistically differ among groups. At Tloc=42°C, CVC measured from calf skin was 26±7mV in AB, 50±8mV in PARA, and 42±8mV in TETRA. These responses differed between AB and PARA (p=0.04) and tended to differ between AB and TETRA (p=0.15). Within groups, forearm responses tended to be greater than calf responses in AB (p=0.06), but not in PARA and TETRA groups. We conclude that spinal cord injury alters maximal SkBF levels in the calf, but not the forearm. In AB persons, maximal SkBF tends to be lower in the calf than the forearm (supported by NIH Grant R03NS083961).

Grant Funding Source: Supported by NIH R03NS083961
Human CaV1.2/CaV3.x channels mediate paradoxical vasomotor responses in the human cerebral circulation (677.11)
Authors: Harraz Osama , Visser Frank , Brett Suzanne , Zechariah Anil , Watson Tim , Menon Bijoy , Starreveld Yves , Welsh Donald
Published: June 2021

This study examined which voltage‐gated Ca2+ channels are expressed in the human cerebral circulation and investigated their electrical and functional properties. Using cerebral arteries collected from patients undergoing resection surgeries, quantitative PCR and Western blot analyses revealed the expression of both L‐ and T‐type channels in the smooth muscle cell layer. Analogous to rodent, we observed both CaV1.2 (L) and CaV3.2 (T); intriguingly, CaV3.1 isoform was replaced in humans with CaV3.3 (T). Patch‐clamp electrophysiology displayed a robust whole‐cell Ba2+ current whose amplitude varied inversely with the patients’ age. This inward current was subdivided into L‐ and T‐type components using standard pharmacological/electrical approaches. The T‐type conductance was further separated into CaV3.2 and CaV3.3 components by exploiting their differential sensitivity to Ni2+. Vessel myography revealed a key role for CaV1.2 and CaV3.3 channels in mediating cerebral arterial constriction with the former and latter predominating at depolarized and hyperpolarized voltages, respectively. Like the rodent, human CaV3.2 appears to drive a negative feedback response that antagonizes myogenic tone development. In conclusion, this study is the first to document three distinct Ca2+ channel isoforms in human cerebral arteries along with their unique contribution to arterial tone development.
Characterization of vascular endothelial and smooth muscle cell pathways and their contributions to myogenic and agonist‐mediated control (677.16)
Authors: Pradhan Ranjan , Lee Pilhwa , Beard Daniel , Carlson Brian
Published: June 2021

Vascular smooth muscle cell contraction and relaxation is central to the local regulation of blood flow to tissue. At the arteriolar level, interactions between vascular endothelial cells and smooth muscel cells play critical role in determining the level of vascular tone. During mechanical and/or agonist stimulations, vascular Ca2+ dynamics are finely adjusted via a number of signaling pathways and the Ca2+ levels in the smooth muscle cells determine the level of contraction. To quantify how cellular Ca2+ dynamics are transformed into changes in vascular tone, we have constructed a cellular‐based model of isolated resistance vessel by integrating models of vessel wall mechanics, smooth muscle force generation, smooth muscle Ca2+ handling and electrophysiology with an endothelial electrophysiology and Ca2+ handling model. The dose‐response vasodilation and vasoconstriction data of different sizes of vessels in response to pharmacological vasoconstrictors and vasodilators, measured using a novel isovolumic myograph developed by Lu and Kassab are analyzed. The model simulates how changes in intraluminal pressure are used to maintain a constant vessel diameter during application of phenylephrine and acetylcholine. In order to simulate these sets of experimental data the model captures integrated vessel behavior including ionic (Ca2+, K+, Na+, etc.), IP3 and nitric oxide myoendothelial transport. Model analysis shows that α‐adrenergic pathways in smooth muscle and NO pathways in endothelium are sufficient to capture the experimentally observed phenylephrine and acetylcholine mediated responses.

Grant Funding Source: NIH U01 HL118738, NSF CBET 1133260 and NIH P50‐GM094503
Effects of exercise on vasomotor function in angiotensin II‐induced hypertension (677.2)
Authors: Pham Duc , Thalji Nassir , Hagler Michael , Zhang Bin , Fujimoto Hirokazu , LeBrasseur Nathan , Roos Carolyn , Miller Jordan
Published: June 2021

In angiotensin II‐induced hypertension, activation of NADPH oxidase is a major contributor to reactive oxygen species generation and endothelial dysfunction. As exercise has been associated with the augmentation of antioxidant enzyme expression in endothelial cells, we hypothesized that exercise would improve endothelial function in mice angiotensin II‐induced hypertension. Three‐month old received either saline or a pressor dose of angiotensin II (1000 ng/kg/min) using subcutaneous osmotic minipumps, and were housed in cages with or without a running wheel. Endothelium‐dependent relaxation in response to acetylcholine was studied in carotid arteries using isolated organ chamber baths. Gene expression was examined using qRT‐PCR. In angiotensin II‐infused sedentary mice, endothelium‐dependent relaxation was significantly impaired, and was partially improved when NADPH oxidase was inhibited by apocynin. mRNA levels of SOD1, SOD2, SOD3, and NOX2 were increased in aorta, and mRNA levels of NOX4 and NOS3 were substantially decreased. In contrast to our hypothesis, endothelium‐dependent relaxation was not improved in angiotensin II‐infused exercised mice housed with running wheels, despite marked increases in physical activity over the two week time period. We conclude that angiotensin II‐induced hypertension results in profound endothelial dysfunction that is not attenuated by voluntary wheel running exercise. Ongoing work is examining molecular mechanisms underlying these observations, as well as evaluation of the protective effects of regimented exercise of varying intensities.
Hemodynamic consequences of spatially‐dependent sympathetic regulation in skeletal muscle arteriolar trees (678.14)
Authors: Al‐Khazraji Baraa , Saleem Amani , Goldman Daniel , Jackson Dwayne
Published: June 2021

The sympathetic nervous system (SNS) elicits vasoconstriction on skeletal muscle arterioles via release of neurotransmitters norepinephrine (NE; acting on α1‐ and α2R), neuropeptide Y (NPY; acting on Y1R), and adenosine‐triphosphate (ATP; acting on P2X1R). Previous studies have shown that the level of α‐adrenoreceptor modulation is dependent on vessel branch order. However, to date, no studies have investigated the integrated roles of α1R, α2R, Y1R, and P2X1R modulation on arteriolar network hemodynamics. Thus, we evaluated the effects of phenylephrine (PE; α1R agonist), UK 14,304 (UK; α2R agonist), NPY, and ATP on arteriolar diameters in continuously branching arteriolar trees (1st order to 5th order; 1A‐5A). In male rats, concentration‐response curves (PE and ATP: 10‐9‐10‐4M; UK: 10‐9‐10‐5M; NPY: 10‐13‐10‐8M) of arteriolar diameter changes were evaluated in the gluteus maximus muscle (n蠅5 rats/group). Like others, we observed that α1R and α2R control is greatest at large (1A‐2A) arterioles, with declining sensitivity in subsequent branch orders approaching capillaries. Beyond this, we have observed that NPY and ATP elicit the greatest constrictor effects in arterioles closest to capillaries, with declining effects in descending branch orders approaching feed arterioles. Using our theoretical flow model, simulations assessed total flow and red blood cell (RBC) flow heterogeneity within the network using our experimental pharmacological data. Using our experimental inputs, we are the first to illustrate that spatial differences in ligand sensitivity enable the SNS to modify bulk tissue blood flow and RBC distribution differentially in branching networks.

Grant Funding Source: NSERC
Interleukin‐1β induces brain microvascular endothelial cell hyperpermeability through matrix metalloproteinase‐9 (678.17)
Authors: Alluri Himakarnika , Anasooya Shaji Chinchusha , Wiggins‐Dohlvik Katie , Oakley Ryan , Stagg Hayden , Davis Matthew , Tharakan Binu
Published: June 2021

Interleukin‐1β induces Brain Microvascular Endothelial Cell Hyperpermeability through Matrix metalloproteinase‐9 Himakarnika Alluri, Chinchusha Anasooya Shaji, Katie Wiggins‐Dohlvik, Ryan Oakley, Hayden W. Stagg, Matthew L. Davis, Binu Tharakan. Surgery, Texas A&M University Health Science Center & Baylor Scott and White Health, Temple, TX, USA.

Brain microvascular hyperpermeability is a major contributor of vasogenic brain edema that occurs following traumatic and ischemic injuries and involves upregulation of interleukin‐1 beta (IL‐1β). Our objective was to determine if IL‐1β mediates blood‐brain barrier (BBB) hyperpermeability via Matrix Metalloproteinases (MMPs)‐induced tight junction (TJ) disruption. Rat brain microvascular endothelial cell (RBMEC) monolayers were exposed to various concentrations of IL‐1β and the optimal concentration and time to induce permeability were determined. RBMEC monolayers were exposed to IL‐1β alone or following an MMP inhibitor (GM6001; 1hour) and MMP‐9 siRNA. MMP‐9 activity was measured following IL‐1β exposure. TJ integrity and cytoskeletal assembly were studied using zonula occludens‐1 (ZO‐1) immunofluorescence and rhodamine phalloidin staining for f‐actin respectively. IL‐1β increased monolayer permeability and MMP‐9 activity significantly (p<0.05) and induced TJ disruption and actin stress fiber formation. GM6001 and MMP‐9 siRNA attenuated IL‐1β‐induced hyperpermeability significantly (p<0.05). An MMP‐9 specific inhibitor (MMP inhibitor‐1) protected both TJ integrity and actin cytoskeletal assembly. These findings suggest that up‐regulation of IL‐1β promotes activation of MMP‐9 leading to BBB breakdown and microvascular hyperpermeability.
Aerobic exercise training enhances hydrogen peroxide mediated flow induced dilation of resistance arteries following exertion in overweight adults (678.8)
Authors: Robinson Austin , Szczurek Mary , Bian Jing , Phillips Shane
Published: June 2021

Exercise training protects against exertion‐induced microvasculature dysfunction. The purpose of this study was to determine if aerobic exercise training (AT) in overweight adults confers a protective effect. We hypothesized 1) that AT is protective and 2) H2O2 mediates this effect against microvasculature dysfunction. Eleven normotensive, overweight and obese adults (BMI 蠅25) enrolled in AT (30‐40 min. at 75% HRmax; 3 x per week) for 8 weeks, with a matched control group. Gluteal biopsies were performed at rest and following exertion at wk. 8 (6‐8 sets leg press at 10RM). Resistance arteries (RAs) were dissected from biopsies and cannulated for measures of reactivity to flow and acetylcholine induced dilation (FID; ACh) with and without the H2O2 scavenger, PEG‐CAT (500 U/ml) or the NOS inhibitor L‐NAME (10‐4 M). Nitric oxide (NO) and H2O2 production in RAs was assessed via fluorescent microscopy. FID and ACh dilations were impaired following exertion in control subjects. In contrast, FID and ACh were preserved following exertion in AT. PEG‐CAT significantly reduced FID (59 ± 8%; P< 0.01) and AChD (66 ± 6%; P< 0.01 post exertion in the AT group. Flow‐ induced NO was reduced and H2O2 production was increased in RAs of the AT subjects following exertion (H2O2:NO ↑ 2.4fold P< 0.05). We conclude that 1) AT is protective against exertion induced microvasculature dysfunction and 2) H2O2 contributes to this protective effect.

Grant Funding Source: Supported by NHLBI K23HL85614, RO1HL095701, and HL095701‐01A2S
Vitamin D attenuates proangiogenic properties of pericytes through increased production of VEGF (679.1)
Authors: Jamali Nasim , Sorenson Christine , Sheibani Nader
Published: June 2021

Retinal neovascularization is associated with pathogenesis of several major eye diseases including retinopathy of prematurity. Vitamin D compounds inhibit the growth of a variety of tumors perhaps through inhibition of angiogenesis. We showed that the active form of vitamin D, calcitriol (1, 25‐dihydroxyvitamin D3), is a potent inhibitor of retinal neovascularization during oxygen‐induced ischemic retinopathy. However, the identity of the molecular mechanisms and cellular targets remain unknown. We showed that calcitriol did not affect pro‐angiogenic properties of endothelial cells (EC). Pericytes (PC), play important roles during angiogenesis, vascular maturation and stabilization of newly forming blood vessels. Here we investigated the impact of calcitriol on retinal PC function. We show that PC express significantly higher level of vitamin D receptor compared with EC, and that calcitriol decreased proliferation and migration of PC, and caused G0/G1 arrest without affecting apoptosis. We also observed alterations in PC adhesion and expression of various matrix proteins. In addition, PC in the presence of calcitriol expressed increased level of VEGF, and antagonism of VEGF signaling by soluble VEGF‐R1 (sFlt‐1) restored proangiogenic properties of PC incubated with calcitriol. Together, our results suggest an important role for PC as target of vitamin D action and attenuation of angiogenesis.

Grant Funding Source: Supported by: RC4 EY021357, P30 EY016665, P30 CA014520, RPB, ADA 1‐10‐BS‐160, and RRF
SFLT1/PIGF angiogenic relationship in the experimental model of preeclampsia induced by sympathetic activation in the mouse (679.5)
Authors: Oros‐Pantoja Rigoberto , Román‐Aguilar Victoria , Martínez‐Carrillo Beatriz E. , Valdés‐Ramos Roxana , Rivera‐Hernández Daniel M.
Published: June 2021

It has been reported that angiogenic factors involved during pregnancy, are altered during the pathogenesis of preeclampsia. Despite some conflicting results, the majority of studies indicate an increase of soluble factor‐like tyrosine kinase 1 (sFlt‐1) with reduced placental growth factor (PIGF). However, many doubts still prevail about the angiogenic response and its relation to this disease. The purpose was to determine plasma levels of sFlt‐1 and PIGF in healthy pregnant CD1 mice (n = 7 / duplicate) and a group of pregnant mice subjected to chronic cold stress (Walking on cold platform 0°C/20 min/day) for 11 days (from day 7 to 17), a validated model for experimental preeclampsia in rats. Results: the group subjected to cold stress showed decreased levels of sFlt‐1 in respect to control group (ct 7137±487 < st 9742±846 pg/mL; p<0.001 Student t). PIGF levels increased significantly with stress (ct 3.88±1.5 < st 40.20±4.2 pg/mL; p<0.001 Student t). Besides we quantified plasma glucose levels, which increased in a non significant in the stress group (163±44 ct < st 174.6 ± 54 mg/dL). Moreover, stressed mice showed a non significant decrease in weight at 17 day of gestation (53.1 ± 4.4 ct > st 51.9±8.3 g). These data may indicate that the results are polarized to a sympathetic nervous system response. In our work, the angiogenic factors behaved contrary to several reports. This could be due to release of Neuropeptide Y by sympathetic activation. This compound has been related to cause pro‐angiogenic effects. These may indicate that the diagnostic criteria of preeclampsia, not necessarily are associated with endothelial and / or angiogenic dysfunction.
Leptin‐induced hypertension is independent of the sympathetic nervous system in obese female agouti mice (680.12)
Authors: Huby Anne‐Cecile , Belin De Chantemele Eric
Published: June 2021

The recent epidemic of obesity induced a three‐fold increase in the prevalence of cardiovascular disease (CVD) in premenopausal women. However, the mechanisms contributing to obesity‐related CVD in females of reproductive age remain completely unknown. We used obese agouti mice (Ag), lacking the anorexigenic effect of leptin, to test the hypothesis that leptin, the factor linking metabolic and CVD in men, triggers hypertension and vascular dysfunction in obese females. Conscious blood pressure (BP, telemetry) recording revealed that obesity elevated BP in both males (Lean: 99±5 vs. Ag: 125±2 mmHg) and females (Lean: 104±1 vs. Ag: 121±1 mmHg). Chronic leptin receptor inhibition reduced BP (AgMale: 117±2 and AgFemale: 112±5 mmHg) in both sexes supporting a role for leptin in the elevated BP. We assessed sympathetic tone by measuring BP response to ganglionic blockade and reported a bigger drop in BP in obese male mice only (LeanMale: ‐29±3 vs. AgMale: ‐44±4% vs. LeanFemale: ‐28±6 vs. AgFemale: ‐25±5%) supporting a higher sympathetic contribution to BP control in male mice only. In addition, male only displayed an increased vascular adrenergic tone. Females showed a reduction. Obesity induced adrenal hyperplasia, raised aldosterone levels (Lean Male: 415±73 vs. AgMale: 76±46 vs. LeanFemale: 545±71 vs. AgFemale: 1152±204) and induced an exaggerated increased in CYP11B2 expression in obese female mice only. Collectively these data suggest that obesity‐induced hypertension is leptin mediated, independent of sympatho‐activation and likely triggered by elevated aldosterone levels in obese female mice. These data highlight the need for sex‐based therapy.
Estrogen may protect female Akita mice against development of type I diabetes and parasympathetic dysfunction of the heart (680.16)
Authors: Jin Hongwei , Georgescu Serban , Naggar Jack , Welzig Michael , Aronovitz Mark , Zhang Yali , Park Ho‐Jin , Galper Jonas
Published: June 2021

Male Akita mouse harboring a mutation in the Ins‐2 gene, is an established animal model for the secondary effects of type I diabetes. Beginning around 3‐4 weeks of age male Akita mice develop marked hyperglycemia, hypoinsulinemia, polydipsia and polyuria. Female Akita mice develop a moderate hyperglycemia after sexual maturation. Ovariectomized (Ovx) female Akita animals implanted with a placebo (P) or 17β‐estradiol (E2) pellet were compared with the Sham‐operated female Akitas (Sham) or wild‐type (WT) mice. After 13 weeks, blood glucose increased from 268±24 mg/dl in Sham to 536±27 mg/dl in Ovx+P (mean±SEM, p<0.01) while decreasing significantly in Ovx+E2 to 125±5 mg/dl, compared to the WT (130±4 mg/dl). Compared with Sham, the Ovx+P group demonstrated increased polydipsia, glycosuria and proteinuria which disappeared completely in the Ovx+E2 group. Furthermore, Ovx+P diabetic mice demonstrated a significant decrease in the negative chronotropic response to carbamylcholine when compared with WT and Sham animals, as characterized by a smaller drop in the response to carbamylcholine (309±14 beats in Ovx+P vs. 406±11 beats in WT and 359±17 beats in Sham; p<0.01) and a decreased duration of bradycardia (4.9±0.5 min. in Ovx+P vs. 26.4±3.8 min. in WT and 17.4±2.5 min. in Sham; p<0.05). The pancreas of female Akita mice showed a reduced islet count and a decreased mean islet area, which could be reversed by estrogen. Thus estrogen may protect female Akita mice against development of the diabetic phenotype and the parasympathetic dysfunction of the heart. This animal model might be used to investigate gender specific effects of diabetes in the cardiovascular system.

Grant Funding Source: NIH