bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between vascular smooth muscle cell stiffness and integrin‐mediated collagen adhesion in response to specific vasoconstrictors and vasodilators
Authors: Reeves Kimberley Jayne , Hong Zhongkui , Sun Zhe , Li Zhaohui , Meininger Gerald Alan
Published: June 2021

Vascular smooth muscle cells (VSMCs) regulate vessel diameter by transferring force to the extracellular matrix and surrounding cells. A previous study demonstrated that integrin‐mediated adhesion to fibronectin (FN) is regulated in parallel with VSMC contractile activation in response to vasodilators and vasoconstrictors. Aim of this study was to determine whether collagen I (CNI) adhesion was also altered in parallel during changes in VSMC contractile activation in response to vasoconstrictors and vasodilators. VSMCs were isolated from the rat cremaster skeletal muscle feed arteriole and maintained in culture for 5–12 days without passage. Adhesion and cell stiffness were measured using atomic force microscopy (AFM). AFM probes with microsphere‐attached tips were functionalized with CNI (1mg\ml). VSMC stiffness and adhesion were assessed by bringing the AFM probe into cyclic contact with the cell membrane (0.1 Hz) before and after treatment (n=10/group) with angiotensin II (ANG; 10−6) or adenosine (ADO; 10−4). Results indicate that ANG increased both VSMC stiffness and CNI adhesion, whereas adenosine decreased VSMC stiffness and adhesion. Results support the hypothesis that integrin adhesion is dynamically up regulated in VSMC during contractile activation and down regulated during relaxation. (1P01HL095486 to G.A.M.)
Gap junction blockage in chorioallantoic membrane networks
Authors: Maibier Martin , Nitzsche Bianca , Reglin Bettina E. , Höpfner Michael , Secomb Timothy W. , Pries Axel R.
Published: June 2021

Background

Studies using theoretical models have revealed the necessity of information transfer along blood vessels, most likely mediated via gap junctions, to achieve efficient blood flow distribution in microvascular networks. Its absence might lead to malperfusion. Here we studied the effects of gap junction blockade on vessel diameter in chick chorioallantoic membrane (CAM) networks by intravital microscopy.

Methods

Blockers used were carbenoxolone (175 μM, CBX), palmitoleic acid (100 μM, PA) and GAP27 (1000 μM). Maximal vasodilation (acetylcholine 10 μM, adenosine 100 μM, papaverine 200 μM, sodium nitroprusside 10 μM) allowed examining the relative contribution of changes in vessel structure and vascular tone.

Results

After 3h, diameters of arteries and veins decreased to 0.55 and 0.54 (relative to PBS control; CBX), 0.77 and 0.81 (PA) and 0.82 and 0.76 (GAP27). For CBX, about half (59%, 46%) of the diameter reduction was caused by structural adaptation, the rest by an increase in vascular tone. After 6h, diameter reduction was unchanged (0.55 and 0.45) but mainly caused by structural adaptation (96% and 88%).

Conclusion

The present findings show the importance of gap junction based communication for developing microvascular networks. Gap junction blockade causes a substantial decrease in vessel diameter with an initial increase in vascular tone and a subsequent change of vessel structure.
Effect of mast cell activation on immune cell trafficking towards mesenteric lymphatic vessels and its aging‐associated alterations
Authors: Chatterjee Victor , Gashev Anatoliy
Published: June 2021

Aging impairs mesenteric lymph flow, which is crucial for fluid and macromolecule homeostasis, fat absorption, and immune function. Previously, we had demonstrated that mast cells line mesenteric lymphatic vessels (MLV) with a greater degree of basal activation of mast cells in aged mesentery. The number of intact mast cells available to react acutely to inflammatory stimuli was decreased with age. In this study we treated live mesenteric tissue isolated from adult and aged F‐344 rats for 2 hours with mast cell activators and performed whole mount IHC and vital dye staining to identify immune cell recruitment towards MLV after mast cell activation. Number of immune cells near MLV was counted and compared between treatments and ages. With greater density of mast cells near MLV, we demonstrated that mesenteric mast cell activation by Compound 48/80 & Substance P resulted in migration of MHC II positive cells and eosinophils towards MLV. This effect was reduced in cromolyn pre‐injected rats. The immune cell recruitment towards MLV after mast cell activation was reduced in aged tissues. We link these findings to our previous report of lesser number of intact mast cells available for initiating an acute immune response in aged mesentery. These findings serve as first step in study of the aging‐associated mechanisms that potentially link mast cells and disordered immune function in the elderly. NIH R01 AG030578, HL094269.
Relating Lymphatic Vessel Mechanical Properties Measured in Vitro to Regulation of Interstitial Fluid Volume in Vivo
Authors: Pariseau Hugo , Franks Jonathan D. , Lipinski Andrew E. , Dongaonkar Ranjeet M. , Quick Christopher M.
Published: June 2021

Although lymphatic vessels are structured similarly to veins, lymphangions, the segments of lymphatic vessels between valves, behave like ventricles. Their spontaneous contraction propels lymph from the low‐pressure interstitium to higher‐pressure veins. Lymphangion pumping in vivo affects transmural pressure and flow, and the afterload of each lymphangion forms the preload of the next. Because lymphangions are sensitive to mechanical stimuli, the basic mechanisms influencing function are studied in vitro, where inlet and outlet pressures can be rigorously controlled. However, the relevance of in vitro results for clinical research has yet to be established. We therefore developed a mathematical model to determine the impact of the interaction of a lymphangion with its environment on interstitial fluid volume. We first characterized microvascular filtration into the interstitium with the Starling‐Landis equation. Lymph flow from the interstitium into the lymphatic network leading to the lymphangion of interest was characterized by modifying the Drake‐Laine equation. The lymphangion itself can be modeled empirically from in vitro data. Lymphangion outlet pressure was assumed to equal central venous pressure. With the ability to simulate diverse edemagenic conditions, our novel approach translates basic lymphatic research into clinically relevant edema research.
Basal nitric oxide production in mouse collecting lymphatics does not enhance contractile activity
Authors: Scallan Joshua P. , Davis Michael J.
Published: June 2021

The mechanisms governing spontaneous contractions in lymphatic vessels are incompletely understood. Accruing evidence in vivo suggests that basal levels of nitric oxide (NO) enhance lymphatic contraction amplitude, despite its well‐defined actions to inhibit contractile activity at moderate doses. Testing this hypothesis, we developed a method to isolate and cannulate collecting lymphatic vessels from the popliteal fossae of wild‐type (WT, n=12) and eNOS−/− mice (n=10). Diastolic diameter, contraction amplitude, and frequency were measured as a function of known pressure (1–10 cmH2O) in the absence of flow, first without then with the NOS inhibitor, L‐NAME. Vessels exhibited robust spontaneous contractions (53% of resting diameter) modulated by pressure, similar to other mammalian species. Contrary to current thought, inhibition of eNOS with L‐NAME (100μM) significantly increased the ejection fraction of WT vessels at low filling pressures (by 28% at 1–3 cmH2O, P<0.05); likewise genetic deletion of eNOS significantly (P<0.05) increased the contraction amplitude (by 45%) and ejection fraction (by 33%). Thus, when pressure and flow are controlled, basal NO production solely depresses lymphatic contractile activity. These data highlight isolated mouse lymphatic vessels as a powerful new tool enabling genetic models to be applied to lymphatic physiology for the first time. (NIH R01HL089784)
Endothelial and Leukocyte Interactions in Oxidative Stress
Authors: Chen Juan , Kavdia Mahendra
Published: June 2021

Oxidative stress can lead to significant interactions between endothelium and leukocyte, which is implicated in the pathogenesis of many diseases. In this study, we analyzed the interactions of human umbilical vein endothelial cell (HUVEC) and human acute monocytes leukemia cell (THP‐1) line under the treatment of high glucose and hydrogen peroxide (H2O2). Cells were treated with high glucose (25 mM) and/or H2O2 (10, 50, 100 μM) for 24 hours. ROS levels and cell adhesion were measured using dihydroethidium fluorescence and calcein‐AM fluorescence via microscopy and micro‐plate reader. The ROS levels increased with the increase concentration of high glucose and H2O2 treated HUVEC. The adhesion of normal glucose treated THP‐1 cells on HUVEC increased with the increase concentration of high glucose and H2O2 treated HUVEC. High glucose treated THP‐1 cells can increase the adhesion of THP‐1 and HUVEC compared with normal glucose treated THP‐1. These results show us that both high glucose and H2O2 can increase the ROS levels and the adhesion of THP‐1 and HUVEC cells. Supported by NIH grant # R01 HL084337.
A potential role for autocrine VEGF signaling in endothelial cell function
Authors: Nwadozi Emmanuel , Uchida Cassandra , Gee Eric , Liu Sammy T.K. , Haas Tara L.
Published: June 2021

Paracrine‐derived vascular endothelial growth factor (VEGF) is established as an indispensable contributor to the angiogenic cascade. Autocrine VEGF, in contrast, is solely attributed to endothelial cell survival signaling, and its potential regulation of other endothelial cell functions has not been studied. We hypothesized that the deletion of VEGF would disrupt endothelial cell migration and shear stress adaptation. Endothelial cells were isolated from transgenic mice having VEGF exon 3 flanked by loxP sites (VEGFL/L). Cells were transduced with adeno‐Cre recombinase to induce VEGF deletion, or adeno‐β‐galactosidase as a control. When subjected to a scrape migration assay, cells with Cre induced VEGF deletion (VEGFΔ) exhibited impaired cell motility compared VEGFL/L cells (12% compared to 64% wound closure after 48 hours; p<0.05). Shear stress stimulation (15 dynes; 2hr) caused significant increases in phospho‐Akt in VEGFL/L cells, which was repressed in VEGFΔ cells (2.4 vs. 1.5 fold above static, respectively). Shear‐induced increases in phospho‐p38MAPK were unaltered in VEGFΔ cells (3.9 vs 3.3 fold above static, respectively). Shear‐induced endothelial cell alignment (15 dynes; 24hr) also was repressed in VEGFΔ cells. Taken together these results provide novel evidence that autocrine VEGF contributes to multiple aspects of endothelial cell function.
The impact of outward remodeling on vasodilation in skeletal muscle resistance arteries
Authors: Gallagher Ryan , Cardinal Trevor R
Published: June 2021

Collateral enlargement following arterial occlusion is thought to rescue downstream tissues from severe ischemic insult in patients with ischemic disease, such as peripheral arterial occlusive disease. However, in animal models, collateral‐dependent hyperemia is reduced after arterial occlusion, suggesting that vasodilation may be impaired. Impaired vasodilation would compromise the effectiveness of collaterals, as ischemia occurs during periods of increased metabolic demand. To assess the impact of collateral enlargement on vasodilation in resistance arteries, we ligated the femoral arteries of young, otherwise healthy mice to induce outward remodeling in the collateral circuit. Outward remodeling transiently impairs functional vasodilation, which is reduced at day‐7 (62 ± 3% vs. 93 ± 10%, p < 0.05), but improves by day‐28 (101 ± 4% vs. 115 ± 10%). Vascular reactivity studies indicate the impaired vasodilation is due to impaired smooth muscle‐dependent responses during outward remodeling, as both endothelial‐ and smooth muscle‐dependent vasodilation is impaired at day‐7 (100 ± 16% vs. 149 ± 15%, p < 0.05 and 103 ± 15% vs. 125 ± 15%, p < 0.05, respectively), but not at day‐28 (136 ± 24% vs. 142 ± 20% and 146 ± 18% vs. 124 ± 10%). Studies examining the cellular mechanism of the day‐7 impairment and the impact of impaired vasodilation on hemodynamics are currently underway.
Vascular Endothelial Growth Factor‐A signaling in Bone Marrow‐Derived Endothelial Progenitor Cells Exposed to Hypoxia
Authors: Hoffmann Brian Robert , Wagner Jordan R. , Prisco Anthony R. , Greene Andrew S.
Published: June 2021

Bone marrow‐derived endothelial progenitor cells (BM‐EPCs) are stimulated by vascular endothelial growth factor A (VEGF‐A) and other pro‐angiogenic factors. VEGF‐A, a diffusible mitogen derived from the vasculature, plays a critical role in angiogenesis and an understanding of its signaling is valuable for treatment of peripheral vascular disease. During physiological angiogenesis, an increase in VEGF‐A expression stimulates endothelial tube formation and increases micro‐vessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF‐A are seen in oxygen deprived epithelial and endothelial cells. This study explores the influence of hypoxia on VEGF‐A signaling in rat BM‐EPCs through a novel approach for large‐scale mapping of signaling pathways utilizing VEGF‐A coupled to magnetic epoxy resin, cross‐linking, and tandem mass spectrometry. Signaling network analysis revealed proteins related to calcium signaling, nitric oxide signaling, cell cycle regulation, and inflammatory responses. Additionally, changes in BM‐EPC angiogenic tube formation in response to VEGF‐A during hypoxia was explored in conjunction with the pathway analysis. This study verified numerous known VEGF‐A pathway components and revealed several previously unidentified mechanisms of VEGF‐A signaling in BM‐EPCs during hypoxia that may be important for vascular regeneration.
Connexin43 Modulates Endothelial Wound Healing, Cell Proliferation and Locomotion
Authors: Chen Cheng‐Hung , Mayo Jamie N. , Bearden Shawn E.
Published: June 2021

We previously reported that homocysteine (Hcy) impairs wound healing and increases connexin 43 (Cx43) levels in endothelial cells by activating metabotropic glutamate receptor 5 (mGluR5). Here, we tested the hypothesis that Cx43 regulates wound closure, cell proliferation, and locomotion in cerebral endothelium. We confirmed that Hcy increases Cx43 levels via an mGluR5‐dependent mechanism by Western blotting and specific pharmacology. We then used siRNA and DNA transfection to knockdown and overexpress Cx43 levels in cerebral microvascular endothelial cells. Cells were studied by time lapse confocal microscopy in a scrape wound model and in an open field migration/proliferation assay. Increasing expression of Cx43 significantly impaired wound closure rate (% of closure at 3 days: siRNA 100±0 vs. Control 86.7±4.2 vs. DNA 50±4.5, n=6, p<0.05 all pairwise). Knockdown of Cx43 expression increased cell proliferation (siRNA 116.6±10.3 vs. Control 33.4±1.7 & DNA 16.5±2.1 cells/mm2, n=6, p<0.05), locomotion (siRNA 10.6±0.6 vs. Control 6.6±0.4 & DNA 5.9±0.5 μm/hr, n=10, p<0.05), and net speed (siRNA 4.9±0.3 vs. Control 2.5±0.3 & DNA 1.6±0.4 μm/hr, n=10, p<0.05). Cx43 regulated the cycling and number of cellular protrusions/retractions and the efficiency of migration in a dose dependent manner. Cx43 is a key regulator of migratory dynamics and growth in cerebral endothelium. Support: HL106548; S11–28U.
The Pro‐atherosclerotic Mechanism of Lysosomal Free Cholesterol Accumulation in CD38−/− Macrophages
Authors: Zhang Fan , Li Pin‐Lan
Published: June 2021

Our previous study demonstrated that deficiency of CD38‐NAADP signaling led to macrophage lysosomal free cholesterol accumulation and atherosclerosis in CD38−/− mice. The present study was designed to explore the underlining mechanism of lysosomal free cholesterol buildup in atherogenesis. By fluorescence microscopic analysis, we found that lysosomal Ca2+ release responses were significantly attenuated and lysosomal lumen acidity markedly decreased upon the inhibition of CD38‐NAADP signaling by silencing CD38, bafilomycin A1 (Baf, 100 μM), or NED‐19 (10 μM) in primary peritoneal CD38+/+ mouse macrophages on oxLDL (10 μg/ml). Consistently, reduced lysosomal Ca2+ release responses and declined lysosomal lumen acidity were also observed in CD38−/− macrophages treated with oxLDL. The secretion of IL‐1β from CD38+/+ macrophages was significantly increased with the inhibition of CD38/NAADP. Furthermore, the elevated plasma IL‐1β was observed in atherosclerotic CD38−/− mice on the Western diet. These results for the first time indicate that lysosomal Ca2+ depletion, lumen acidity decrease, and the increased secretion of proinflammatory factors may be the atherogenic mechanism of lysosomal free cholesterol buildup in the CD38‐NAADP signaling deficient macrophages (Supported by R01HL091464, NIH R01HL115068 and R01HL057244).
Reversal of ATP‐Induced NLRP3 Inflammasome Activation and Lipids Deposition in Macrophages from Mice Lacking Apoptosis‐associated Speck‐like Protein (ASC) Gene
Authors: Li Xiang , Zhang Yang , Xia Min , Boini Krishna M. , Li Pin‐Lan
Published: June 2021

Although NLRP3 inflammasome activation in macrophages (Mφ) has been shown to be critical for the development of atherosclerosis upon atherogenic stimuli, it remains unknown whether activated NLRP3 inflammasomes by other non‐atherogenic stimuli are also involved in atherogenesis. The present study tested a hypothesis that activation of NLRP3 inflammasomes by ATP, which is generally non‐atherogenic, enhances the migration of Mφ and accelerates the foam cell formation. We first demonstrated that extracellular ATP at 2–7.5 mM markedly increased the formation and activation of NLRP3 inflammasomes in LPS‐primed bone marrow macrophages (BMMs) from wild type mice, which was confirmed by colocalization of NLRP3, ASC and caspase‐1, activation of caspase‐1, and enhanced IL‐1β production. In BMMs from ASC−/−mice, the formation and activation of NLRP3 inflammasomes in Mφ were substantially attenuated. By oil red staining, ASC+/+BMMs were found to have dramatic lipids accumulation after ATP pretreatment. Further confocal microscopic analysis of colocalization of filipin‐stained intracellular cholesterol and lysosomal marker Lamp‐1 demonstrated that large amount of cholesterol was accumulated in lysosomes of ASC+/+BMMs pretreated by ATP, which was not observed in ASC−/−BMMS. In addition, ATP‐induced NLRP3 inflammasomes activation increased migration of ASC+/+BMMS, which was markedly attenuated in ASC−/−BMMs. However, the effects of ATP on Mφ migration were not dependent on IL‐1β. These results suggest that activation of NLRP3 inflammasomes even by non‐atherogenic stimuli also remarkably increases the susceptibility of Mφ to lipid deposition and their migration ability, which may result in migration of Mφ into arterial wall, where they form foam cells and ultimately induce atherosclerosis (supported by NIH grants HL057244, HL091464 and HL075316).
Aquaporin 1 does not contribute to the progression of abdominal aortic aneurysm in mice
Authors: Stubbe Jane , Johansen Søren H. , Hansen Pernille B.L. , Bie Peter , Jensen Boye L.
Published: June 2021

Abdominal aortic aneurysm (AAA) is an asymptomatic chronic dilatation of the abdominal aorta observed predominantly in elderly males. The disease is usually first discovered at the time of rupture and often has a lethal outcome. The mechanism of aneurysm progression is not well characterized, but includes extensive vascular remodeling. Aquaporin 1 (AQP1) is necessary for endothelial‐ and vascular smooth muscle cell migration in tumors and in hypoxic conditions. Thus, we hypothesized that deletion of AQP1 attenuates the formation and progression of abdominal aortic aneurysms. 10–12 weeks old male ApoE KO and AQP1‐ApoE dKO mice were fed western diet and given angiotensin II (Ang II, 1 μg/kg/min) via subcutaneous osmotic mini pumps for 28 days in order to increase blood pressure and induce AAA formation. At 28 days mice were euthanized and aorta dissected. As expected plasma Ang II concentration did not differ between ApoE KO and AQP1‐ApoE dKO mice (403 ± 96 pg/ml vs. 370 ± 74 pg/ml, n=10–11, p=0.8). There was no difference in the incidence of AAA formation (64% vs. 61%). AAA sizes were similar in ApoE KO and AQP1‐ApoE dKO mice, as measured by outer abdominal diameter (1.45 ± 0.18 mm vs. 1.25 ± 0.18 mm, n = 10–11, p=0.4), and by wet weight of the abdominal aorta (15.4 ± 3.7 mg vs. 14.7 ± 4.3 mg, n= 10–11, p=0.9). It is concluded that in ApoE KO mice on western diet, AQP1 does not contribute significantly to incidence and progression of abdominal aorta aneurysm.

Supported by Alfred Andersen grant and Director Jacob Madsen & Olga Madsen's grant
Nutrient Overload Causes Rapid Accumulation of Myeloperoxidase in the Mesenteric Fat.
Authors: Preston Kyle , Rom Inna , Scalia Rosario
Published: June 2021

Infiltration of adipose tissue by inflammatory cells is associated with adipocyte dysfunction and insulin resistance in obesity. Recent publications support an early role for neutrophils in this process. Neutrophils are the main source of the oxidant myeloperoxidase (MPO). Accordingly, we hypothesized a role for MPO in the adipocyte dysfunction induced by nutrient overload. C57BL/6 wild‐type mice were given either a control or a high‐fat meal (HFM, 60% fat, 20% carbohydrate, 20% protein) by gavage. Mesenteric and subcutaneous fat pads were isolated from mice at 1 and 3 hours post‐feeding to measure MPO content and activity by western blot analysis and colorimetric assay, respectively. Intravital microscopy of the mesenteric fat microcirculation was also used to monitor postprandial kinetics of leukocyte‐endothelium interactions (LEI). High‐fat meals increased MPO activity only in mesenteric fat pads with peak values observed at 1 h post‐feeding (p<0.001 versus control). Moreover, MPO activity remained elevated at 3 hours post‐feeding in the stromal vascular fraction of the visceral fat (p< 0.01 versus control). In the mesenteric fat microcirculation, LEI were elevated with peak rolling and adhesion occurring at 1 hour post‐feeding. Taken together these data demonstrate that nutrient overload causes a rapid activation of neutrophil trafficking and accumulation of MPO in the visceral fat.
Role of hydrogen peroxide and epoxyeicosatrienoic acids in arachidonic acid‐induced dilation of human coronary arterioles
Authors: Zinkevich Natalya S , Gutterman David D , Zhang David X
Published: June 2021

Arachidonic acid (AA) induces potent dilation of human coronary arterioles (HCAs) through an endothelium‐dependent hyperpolarization (EDH) mechanism. However, the mediators responsible for this EDH‐related dilation in HCAs remain unknown. This study is designed to test whether AA‐induced dilation involves hyperpolarizing factors hydrogen peroxide (H2O2) and epoxyeicosatrienoic acids (EETs). Coronary arterioles (~150 μm ID), isolated from atrial tissues from patients with coronary artery disease (CAD), were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In arterioles constricted with endothelin‐1, AA elicited concentration‐dependent dilation (maximal dilation: 71±3%, n=3), and this dilation was not affected by H2O2 scavenger catalase (64±3%). The EET antagonist 14,15‐EEZE also failed to inhibit AA‐induced dilation (64±6% vs. 70±7% of control, n=5). Unexpectedly, treatment of arterioles with cell‐permeable PEG‐catalase resulted in enhanced dilation to AA (95±4% vs. 69±3% of control, n=4). This enhanced dilation was largely inhibited by 14,15‐EEZE (52±5% vs. 87±6% of control, n=5). Together, these data suggest that HCAs produce endogenous H2O2, which does not contribute to AA‐induced dilation but inhibits EET‐mediated component of AA dilation. It remains to be determined whether AA dilation in HCAs involves factors other than H2O2 and EETs.
Nitric Oxide (NO)‐mediated vasodilation is controlled by pannexin‐1‐formed channels
Authors: Puebla Mariela , Gaete Pablo , Lillo Mauricio , Figueroa Xavier
Published: June 2021

Membrane channels formed by pannexin proteins (Panxs 1–3) are expressed in the vascular wall. However, the function of these channels in endothelial cells remains to be determined. We evaluated the participation of Panx‐1 in the NO‐mediated control of vasomotor tone in isolated mesenteric resistance arteries of rat. The expression of and spatial interaction between Panx‐1, endothelial NO synthase (eNOS) and caveolin‐1 (Cav‐1) were analyzed by immunofluorescence and proximity ligation assay (Duolink® II). Panx‐1 and Cav‐1 were mainly detected in the endothelium, but also in smooth muscle cells. As expected, eNOS was observed exclusivity in endothelial cells in co‐localization with Cav‐1. In addition, Panx‐1 was spatially associated with Cav‐1 and eNOS, which suggests a functional relation between these proteins. Consistent with this notion, blockade of Panx‐1‐formed channels with probenecid enhanced the NO‐dependent vasodilation induced by acetylcholine and increased the level of the activating phosphorylation of eNOS at serine 1177.These results indicate that Panx‐1 participates in the NO‐mediated control of vasomotor tone through the interaction with eNOS in signaling microdomains known as caveolae.

Proyects: Anillo ACT‐71, Fondecyt 1100850, 1111033.
Distinct potentials in the myoendothelial junction
Authors: Brasen J. Christian , Holstein‐Rathlou Niels‐Henrik
Published: June 2021

Myoendothelial junctions (MEJ) bridge the smooth muscle cells (SMCs) and endothelial cells (ECs) in small arteries. In most vessels they project from the EC through the internal elastic lamina and into the SMC, where gap junctions connect the MEJ with the SMC. MEJs are characterised by a long thin neck and on top a “head”, which can be coupled to SMCs with gap junctions. We have used a spatiotemporal model to simulate electrical coupling between the two cell types. The model shows that the MEJ could be electrically isolated from both the EC and the SMC due to the long thin neck and that there could be levels of cations and anions in the MEJ that are different from the EC and SMC bulk levels. Local Ca2+ domains in the MEJ can activate an outward K+ current in the MEJ and we found that activation of less than 10–20 K+ channels (e.g. IK channels) is sufficient to create an electrical potential of 1–5 mV between the SMC and MEJ. This suggests that the MEJ should be considered as a unique structure with properties distinct from those of the smooth muscle cells or of the endothelial cells proper. Supported by The Danish Council for Independent Research ‐ Medical Sciences.
Increase in renal sympathetic nerve activity and hypertension in rats with chronic femoral artery occlusion
Authors: Yang Hsiao Tung , King Louise T , Terjung Ronald L , Hasser Eileen M
Published: June 2021

Patients with peripheral arterial insufficiency (PAI) show augmented sympathoexcitation (SE) (higher blood pressure (BP) and heart rate (HR)) during graded physical activity. Chronically elevated SE is detrimental in cardiovascular control and may contribute to the higher morbidity and mortality in PAI patients. In rats with femoral artery occlusion (FemOccl), a model of PAI, BP is elevated at both rest or during exercise. We hypothesized that the increased afferent activity from the hind limbs contributes to the increased BP and possibly via an altered sympathetic nerve activity (SNA) in FemOccl rats. 24 adult male SD rats received FemOccl for 3 to 5 wks. Arterial BP, heart rate and renal SNA responses to sciatic nerve afferent stimulation were monitored. Under Inactin anesthesia, baseline BP was 119±1.6 in Control (n=12) and 130±4.4 mmHg (n=11, p<0.025) in FemOccl rats. Baseline renal SNA was also about 38% higher in FemOccl rats (p<0.05). Stimulation (100 – 600 uA, 1 ms, 15 Hz) of the central end of the cut right sciatic nerve produced significantly greater increases in renal SNA in the FemOccl group compared to Control (p<0.05). Our preliminary observations imply that the central nerve system (CNS) control circuit remodels to enhance SE, and contribute to the hypertension after FemOccl. This raises the potential that an altered CNS control could contribute to a chronic SE state.
Effect of sinoaortic denervation on blood pressure and heart rate of hypertensive and control rats monitored by telemetry
Authors: Santos Fernando , Moreira Edson Dias , Mostarda Cristiano Teixeira , Barbosa Maikon , Irigoyen Maria Claudia
Published: June 2021

The aim of this study was to evaluate the effects of sinoaortic denervation (SAD) on systolic blood pressure (SBP) and heart rate (HR), and possible changes in circadian cycle. Wistar (C) and spontaneously hypertensive (H) male rats were submitted to SAD and monitored for two weeks by radio transmitter implanted directly into the abdominal aorta. The acute period (48hs) after SAD procedure was characterized by SBP increase in C (39%) and in H (17%). Surprisingly, a decrease in SBP in H (12%) below baseline levels was observed nine hours after the surgery and was maintained during the next 48 hours. In contrast, SBP in C was still 14% above the baseline. C HR was unchanged, while in H it was 29% increased in the first 48 hours. After two weeks there were no significant differences in SBP and HR in C while in H a 14% reduction in SBP was observed in comparison to the baseline. In this group HR remained 3% higher. We noticed that in H group the behavior of SBP and HR did not change through circadian cycle phases. After SAD the behavior of SPB was still unchanged in H group while HR presented an increase of 6% during the active period. However, SBP behavior in the C was different during light/dark periods, both before and after SAD, with an increase in HR in active period before (8%) and after (11%) SAD. These data suggest that SAD is able to reduce SBP of H and to change the behavior of HR in hypertensive and control rats. FAPESP 2011/04111–6
Thyroid hormones can increase astrocyte fatty acid oxidation and survival; a possible mechanism for decreased tissue necrosis after cerebral stroke
Authors: Sayre Naomi L. , Holstein Deborah , Lechleiter James D.
Published: June 2021

Enhancement of astrocyte metabolism in mice significantly decreases lesion size after stroke. This was achieved after purinergic stimulation, but we know that thyroid hormones (TH) can also increase mitochondrial metabolism. Acute treatment with triiodothyronine (T3) increases long‐chain fatty acid oxidation (FAO) in several tissues to improve metabolism. Because astrocytes also use fatty acids for energy, we hypothesized that THs increase astrocyte FAO and improve survival after oxidative stress or stroke.

Results

Nanomolar concentrations of either diiodothyronine (T2) or T3 increase astrocyte FAO of [3H]palmitate into 3H2O by over 30% (p<0.01). The astrocyte mitochondrial membrane potential was measured using the potential sensitive dye tetramethyl rhodamine methyl ester. Acute T2 treatment (15 minutes) tripled the dye intensity (p<0.05), indicating improved mitochondrial membrane potential. These data are consistent with increased energy production upon TH treatment. Astrocytes were stressed with hydrogen peroxide, and viability was measured using the vital dye calcien‐AM. T2 and T3 each increased astrocyte viability 25% (p<0.01). To test if TH is protective in vivo, we induced focal ischemia in mice. Administration of T2 decreased the necrotic volume in the brain by half (p=0.02) 24 hours post‐stroke. Future testing will determine if TH‐mediated survival of astrocytes is dependent on FAO.
Inhibition of endocannabinoid degradation reduces neurological damage and blood brain barrier disruption following traumatic brain injury
Authors: Katz Paige S , Impastato Renata , Rogers Emily , Molina Patricia
Published: June 2021

Traumatic brain injury (TBI) contributes to over a third of all injury‐related deaths in the United States. Initial damage following TBI involves acute neuroinflammation, oxidative stress and altered blood brain barrier (BBB) integrity. Recent studies suggest a neuroprotective role for the endocannabinoid (EC) system including 2‐arachidonoyl glycerol (2‐AG) and N‐arachidonoyl‐ethanolamine (AEA) following TBI. We hypothesized that reducing EC degradation would reduce inflammatory and oxidative tissue damage, improve neurological (NSS) and neurobehavioral (NBS) outcomes, and preserve blood brain barrier (BBB) integrity. Male Sprague‐Dawley rats underwent a 5mm craniotomy and female Leur lock placement over the left hemisphere and were recovered for 3 days. TBI induced by lateral fluid percussion (1.4 J; 30 ms) produced apnea of 15 ± 5 second duration and a 522 ± 83 second delay in righting reflex. The impact of pharmacological inhibitors of 2‐AG and AEA degradation (JZL184; 16 mg/kg and URB597 0.3 mg/kg, respectively; administered IP 30 min post‐TBI) was examined during the first 72 h post injury. TBI markedly impaired NSS and NBS function, and disrupted BBB integrity. Inhibition of 2‐AG degradation (JZL 184) effectively reduced NSS and NBS impairment and significantly attenuated BBB disruption. These findings provide additional support for the potential therapeutic benefit of EC modulation in attenuating the detrimental impact on both NSS and NBS function and BBB integrity following TBI. DOD‐W81XWH‐11–2‐0011, NIAAA‐7577, NIAA‐19587
Retinal Astrocytes Mediate Neuroprotective Effects Through p38 MAPK Dependent Activation
Authors: Sivak Jeremy M , Nahirny Adrian , Livne‐Bar Izhar
Published: June 2021

Astrocytes play a fundamental role in maintaining a delicate neuronal homeostasis. In response to injury and stress, astrocytes undergo a switch; becoming hypertrophic and migratory, secreting cytokines, coordinating tissue remodeling, and increasing production of antioxidants. However, the cellular signals regulating this switch are still not well understood, and its impact on neurons remains unclear. As a developmental outpocketing of the forebrain, the retina is a bellwether and model for damage to the central nervous system (CNS). In the rodent eye, astrocytes remain tightly restricted to a single layer on the inner retinal surface, beside a single layer of retinal ganglion cell neurons. This relatively simple system provides an accessible model of glial‐neuronal interactions. We have used the retina to develop and establish robust models of astrocyte activation in response to oxidative, metabolic, and hypoxic stresses. Characteristic molecular and biochemical changes in activated cells have implicated signals through the p38 MAPK (mitogen activated protein kinase) pathway as a key mediator of this process. We demonstrate that modulation of this pathway is necessary for the cells to mount an appropriate injury response. However, quiescent cells produce a neuroprotective effect on retinal neurons, while activated cells exacerbate injury.
Validation of submaximal oxygen consumption tests for individuals with spinal cord injury
Authors: McCulloch John P , Aslan Sevda , Lorenz Doulgas , de Paleville Daniela Terson
Published: June 2021

Spinal cord injury decreases total daily energy expenditure, placing this population at a much higher risk for cardiovascular disease, obesity, and diabetes. However, exercise training individuals with spinal cord injuries have seen improvements in cardiovascular health and overall quality of life. The goal of this study is to establish reliable submaximal oxygen consumption tests for individuals with spinal cord injury. Compared to maximal tests, submaximal oxygen consumption tests are much cheaper, shorter in duration, and pose less of a health risk to the subjects.

Participants for this study were individuals between the ages of 18 and 65 with chronic spinal cord injury (n=12). Each participant performed two maximal oxygen consumption tests and two submaximal oxygen consumption tests over the course of four sessions. The tests were performed on a SCIFIT Arm Ergometer and a NuStep T4. Both machines were used for one maximal and one submaximal test, the order of testing was random. Heart rate, blood pressure, rating of perceived exertion, and ECG were all recorded in addition to the metabolic data. We predicted the estimated oxygen consumption values from the submaximal tests would show a strong positive correlation with the oxygen consumption values obtained from the maximal graded tests. Pearson correlation and regression using ordinary least squares was used to find any correlations.
Does nitric oxide upregulate activity and expression of heme oxygenase‐1 in the rat locus coeruleus during endotoxemia?
Authors: Soriano Renato N , Kwiatkoski Marcelo , Oliveira‐Pelegrin Gabriela R , Rocha Maria JA , Branco Luiz GS , Carnio Evelin C
Published: June 2021

We investigated the effect of increased bioavailability of nitric oxide (NO) on the lipopolysaccharide (LPS)‐induced increase in activity and expression of heme oxygenase (HO)‐1 in the locus coeruleus (LC) during endotoxemia. Rats were implanted with a guide cannula toward the fourth ventricle and were microinjected with a NO donor (NOC12; 100 nmol/2μl) and injected with LPS (100 μg/kg, intraperitoneal). Two hours after, we evaluated activity and expression of HO‐1 protein in LC punches. Data are expressed as mg of bilirubin/mg of protein or as the HO‐1/β‐actin protein expression ratio. In endotoxemic rats, microinjection of NOC12 tended to augment the LPS‐induced increase in activity (0.227±0.032 vs. 0.261±0.036, n=9–11, P>;0.05), and did not affect the LPS‐induced increase in expression of HO‐1 (1.121 ± 0.2167 vs. 0.7011 ± 0.1901, n=9–12, P>;0.05). The present findings indicate that increased bioavailability of NO does not further upregulate either activity or expression of HO‐1 in the LC of endotoxemic rats. Financial support: FAPESP.
Motor training program in children with fetal alcohol syndrome
Authors: Pinner Audrey Anna , Robbins Bradley Wayne , Thompson Whitney Rose , Vaughn Josh Austin , Walter Tara Leigh , Raynes Ediberto Alcantara
Published: June 2021

Fetal alcohol syndrome (FAS) is manifested among children whose mothers consumed alcohol during gestation. Symptoms of this disorder are varied, specifically affecting neurodevelopmental delay. Several studies suggest that using task‐specific motor training interventions in children with motor development delays increased their ability to perform motor tasks. Using verbal, visual, and auditory cues in conjunction with motor training have been proven to improve motor skills among children with motor developmental delay. In FAS, the diminished motor control does not improve throughout life, which warrants early intervention. There has been no evidence‐based literature that evaluates the use of interventions to increase motor skills in this population. Using Oxford Level of Evidence, we decided to explore the literature on increasing the motor skills and thereby come up with a physical therapy intervention that will improve the quality of life of children affected with FAS.
Regulation of adipose thermogenesis by Epidermal Growth Factor and angiotensin AT2 receptor activation
Authors: Littlejohn Nicole K. , Keen Henry L. , Liu Xuebo , Sigmund Curt D. , Grobe Justin L.
Published: June 2021

Transgenic “sRA” mice with hyperactivity of the brain renin‐angiotensin system (RAS) express human renin via the synapsin promoter and human angiotensinogen via its own promoter. sRA mice exhibit increased resting metabolic rate (RMR) due to reduced activation of adipose angiotensin AT2 receptors. To identify downstream pathways, RNA‐Seq was performed using inguinal adipose from sRA and littermate controls, with or without infusion of the AT2 agonist CGP‐42112a (“CGP,” 50 ng/kg/min, s.c.). In sRA vs controls, there were significant expression changes in 123 genes, which were mostly normalized by CGP. 51 of these genes are associated with epidermal growth factor (EGF). To determine the sufficiency of EGF to increase RMR, male C57Bl/6J mice were infused with EGF (0.8 ug/hr, 2 weeks, n=8) or saline (n=9), resulting in increased RMR (+38±12 cal/hr vs control, P=0.006), and a trend toward reduced inguinal (176±21 vs 140±17 mg P=0.20) and increased brown (54±6 vs 68±4 mg, P=0.08) adipose. Co‐infusion of CGP (n=5) caused trends toward ameliorated RMR (+15±22 cal/hr vs control, P=0.31 vs control, P=0.13 vs EGF) and inguinal adipose (187±21 mg, P=0.15 vs EGF). Ongoing studies are aimed at optimizing doses of EGF and CGP in C57Bl/6J mice, and at quantifying EGF and evaluating the effects of chronic EGF blockade in sRA mice. Together these data support a role for EGF, and modulation of EGF signaling by AT2 receptors, in RMR control.
Deletion of p22phox‐dependent reactive oxygen species (ROS) in the paraventricular nucleus (PVN) protects against diet‐induced obesity (DIO) by increasing thermogenesis and lipolysis.
Authors: Lob Heinrich E , Song Jiunn , Mark Allyn L , Davisson Robin L
Published: June 2021

Oxidative stress is implicated in the onset of obesity. We reported that deletion of p22phox, the required subunit for NADPH oxidase activity, in the PVN, prevents DIO by increasing energy expenditure and brown adipose tissue (BAT). Here we tested the hypothesis that reducing p22phox‐dependent ROS in the PVN protects against DIO by increasing β3‐adrenoreceptor (β3‐AR) activity, thereby enhancing BAT thermogenesis and white adipose tissue (WAT) lipolysis. We performed PVN‐targeted injection of an adenovirus encoding Cre recombinase (AdCre) or control AdLacZ in 6 week old p22phox/flox mice. Two weeks later, mice were fed a high fat diet (HFD, 42%) for 10 weeks. In AdCre vs AdLacZ treated DIO mice, we observed increases in β3‐AR mRNA (+2.6±0.6 fold, n=4, p<0.05) and PGC‐1α mRNA (+1.6±0.17 fold, n=5, p<0.05) in BAT. Lipolysis of subcutaneous WAT was also increased in AdCre vs AdLacZ DIO mice as measured by hormone‐sensitive lipase (HSL, +2.3±0.5 fold, n=6, p<0.05) and β3‐AR mRNA (+2.7±0.7 fold n=4, p<0.05). Blockade of β3‐AR with SR59230A (i.p., 2 mg/kg/day) reversed the increases in energy expenditure, BAT mass and mRNA levels of PGC‐1α, β3‐AR and HSL in BAT and WAT to AdLacZ levels. These data indicate that disrupting oxidative stress in the PVN during DIO increases β3‐AR in BAT and WAT, thereby increasing thermogenic metabolism and lipolysis and reducing adiposity.

12SDG9160010, HL063887, HL084207
Cerebral artery resistance is directly related to sympathetic nerve activity in men
Authors: Hart Emma , Harris Ashley D , Wise Richard G , Warnert Esther A , Stewart Lesley , Connor Kimberly , Paton Julian FR
Published: June 2021

Cerebral arterial resistance may play a role in determining the level of sympathetic nerve activity (SNA) at rest. We hypothesised that resistance to blood flow in the internal carotid, vertebral and the basilar arteries (ICA, VA, BA) is directly related to the level of muscle SNA (MSNA). In 4 men aged 20–52 years, blood flow in the ICA, VA and BA was measured using MR phase contrast imaging at 2 locations (1 cm below the VA convergence and 1 cm above) with simultaneous brachial blood pressure measurements (automated cuff). MSNA was measured via peroneal microneurography. Resistance in each vessel was calculated as mean arterial pressure/blood flow. Resting MSNA (30–82 bursts/100 heart beats) was positively related to vascular resistance in the left and right ICA (r = 0.87, r = 0.75, P<0.05), right and left VA (r = 0.87, r = 0.85, P=0.05). There was a weak positive relationship between MSNA and vascular resistance in the BA (r = 0.40). In summary, men with high resistance to blood flow through the ICA and VA have high MSNA. Thus, cerebral arterial resistance might determine the set point of sympathetic activity. This is important since high levels of SNA observed in hypertensive patients may be due to elevated cerebral vascular resistance. British Heart Foundation funded.
Leptin increases lumbar sympathetic nerve activity (LSNA) in part via excitatory amino acid inputs into the hypothalamic paraventricular nucleus (PVN)
Authors: Li Baoxin , Brooks Virginia L.
Published: June 2021

Intracerebroventricular (ICV) leptin increases LSNA and LSNA baroreflex gain (BRG). Previously, we demonstrated that the PVN is required, since PVN inhibition with muscimol completely reversed the effects of leptin. Inhibition of PVN melanocortin 3/4 receptors with SHU9119 only partially reversed leptin's effects, suggesting that another excitatory input into PVN also contributes. Therefore, we tested the hypothesis that leptin enhances baroreflex control of LSNA in part via increased PVN excitatory amino acid drive. In α‐chloralose anesthetized male SD rats, basal LSNA and LSNA BRG were assessed at baseline, 1 hr after ICV leptin (3 μg) or aCSF, and then again 30 min later after microinjection of 60 nL of aCSF (n=5) or kynurenate (Kyn; 2.7 nmol; n=6). After 60 min, leptin increased (P<0.05) basal LSNA (to 158±9%) and LSNA BRG (from 3.2±0.4 to 5.7±0.2 %/mmHg). After PVN aCSF, LSNA (to 186±21%) and LSNA BRG (to 7.2±0.6 %/mmHg) continued to rise (P<0.05). In contrast, PVN Kyn prevented further increases such that LSNA [to 144±7% (leptin) then 137±7% (Kyn)] and LSNA BRG [in %/mmHg: from 3.4±0.3 to 5.3±0.2 (leptin) then 5.1±0.2 (Kyn)] were decreased (P<0.05) compared to PVN aCSF. However, both LSNA and LSNA BRG remained elevated (P<0.05) versus baseline. No change in LSNA was observed following PVN microinjection of Kyn after ICV aCSF infusion. In conclusion, excitatory amino acid inputs into the PVN contribute to the sympathoexcitatory effects of leptin. Supported by HL088552 and AHA.
Activation of angiotensin II type‐1 receptors stimulates microglial production of pro‐inflammatory cytokines in hypothalamic paraventricular nucleus of heart failure rats
Authors: Yu Yang , Zhang Zhi‐Hua , Wei Shun‐Guang , Weiss Robert M , Felder Robert B
Published: June 2021

Activated microglia produce pro‐inflammatory cytokines, which contribute to sympathetic activity in heart failure (HF). Microglia express angiotensin (ANG) II type‐1 receptors (AT1R), an important component of the renin‐angiotensin system. However, the relationships between the renin‐angiotensin system, activation of microglia and increased pro‐inflammatory cytokines in key cardiovascular regulatory centers of the brain has not been fully elucidated. We hypothesized that activation of AT1R mediate microglial release of pro‐inflammatory cytokines in the hypothalamic paraventricular nucleus (PVN) in HF. Rats underwent coronary ligation to induce HF or sham surgery. HF rats received a 4 week intracerebroventricular infusion of vehicle, the microglial inhibitor minocycline or the AT1R antagonist losartan. Compared with sham, vehicle‐treated HF rats had increased (P<0.05) microglial marker CD68 and Emr1 mRNA, and augmented tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), cyclooxygenase‐2 (COX‐2) and AT1R mRNA in PVN. Minocycline‐treated HF rats had less CD68 and Emr1 mRNA and reduced TNF‐α, IL‐1β and COX‐2 but not AT1R mRNA in PVN. Losartan‐treated HF rats also had less Emr1 mRNA and reduced TNF‐α, IL‐1β and COX‐2 in PVN, as well as reduced AT1R mRNA. These results suggest that activation of AT1R contributes to microglial production of pro‐inflammatory cytokines in the PVN of HF rats.